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Characterization of the human ceruloplasmin cDNA and gene Koschinsky, Marlys Laverne 1988

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CHARACTERIZATION OF THE HUMAN CERULOPLASMIN cDNA AND GENE  By  M a r l y s Laverne  B.Sc.  Koschinsky  (Hons), The U n i v e r s i t y o f Winnipeg, 1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE  FACULTY OF GRADUATE STUDIES  (Department o f B i o c h e m i s t r y )  We accept t h i s t h e s i s as conforming to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1988 ® M a r l y s Laverne Koschinsky, 1988  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his thesis  and  study.  scholarly  or for  her  of  financial  jgf QCh+£S\l <JTA Kf  T h e University of British C o l u m b i a 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  I  I further  purposes  gain  the  shall  requirements  agree  that  agree  may  representatives.  permission.  Department  of  be  It not  that  the  be  Library  an  advanced  shall  permission for  granted  is  for  by  understood allowed  the  make  extensive  head  that  without  it  of  copying my  my or  written  ii  ABSTRACT  A cDNA f o r human c e r u l o p l a s m i n was i d e n t i f i e d i n a human l i v e r cDNA l i b r a r y by s c r e e n i n g w i t h m i x t u r e s  of synthetic oligonucleotides  complementary t o two r e g i o n s o f t h e c e r u l o p l a s m i n mRNA.  The r e s u l t i n g  c l o n e (phCP-1) c o n t a i n e d DNA coding f o r amino a c i d r e s i d u e s 202 - 1046 o f the p r o t e i n , f o l l o w e d by a 3' u n t r a n s l a t e d r e g i o n o f 123 bp and a p o l y ( A ) tail.  To i s o l a t e a d d i t i o n a l c l o n e s e x t e n d i n g  i n a 5' d i r e c t i o n , two  randomly-primed human l i v e r cDNA l i b r a r i e s were c o n s t r u c t e d i n t h e bacteriophage  v e c t o r s XgtlO and X g t l l .  c l o n e was i s o l a t e d  From t h e former l i b r a r y , a  (XhCP-1) t h a t c o n t a i n e d DNA coding f o r a p u t a t i v e  s i g n a l p e p t i d e c o n s i s t i n g o f 19 amino a c i d r e s i d u e s , f o l l o w e d by DNA encoding  r e s i d u e s 1 - 380 o f plasma c e r u l o p l a s m i n .  From the X g t l l  l i b r a r y , s i x c e r u l o p l a s m i n cDNA c l o n e s were p u r i f i e d , two o f which were shown t o c o n t a i n 10 and 38 bp o f non-coding sequence e x t e n d i n g XhCP-1.  5'  to  B l o t h y b r i d i z a t i o n a n a l y s i s u s i n g cDNA probes showed t h a t  c e r u l o p l a s m i n mRNA from t h e human hepatoma c e l l  l i n e HepG2 i s 3700  n u c l e o t i d e s i n s i z e , w h i l e human l i v e r RNA c o n t a i n e d an a d d i t i o n a l h y b r i d i z i n g s p e c i e s o f 4500 n u c l e o t i d e s i n s i z e . Ceruloplasmin  genomic DNA c l o n e s  (spanning  a r e g i o n o f approximately  45 Kbp) were o b t a i n e d by t h e s c r e e n i n g o f s e v e r a l human genomic phage l i b r a r i e s u s i n g cDNA probes.  These c l o n e s were i n i t i a l l y c h a r a c t e r i z e d by  r e s t r i c t i o n endonuclease mapping. p o s i t i o n s o f i n t r o n / e x o n boundaries (average  U s i n g DNA sequence a n a l y s i s , t h e were determined.  s i z e o f 183 bp) have been i d e n t i f i e d  corresponding  To date, 14 exons  i n the c e r u l o p l a s m i n  t o n u c l e o t i d e r e s i d u e s 1 - 2565 o f t h e coding  gene,  sequence.  The  iii  m a j o r i t y of the 14 i n t r o n s l o c a l i z e d w i t h i n t h i s r e g i o n were l o c a t e d i n analogous p o s i t i o n s i n t h e f a c t o r V I I I gene, thereby s u g g e s t i n g two p r o t e i n s have e v o l v e d  t h a t these  from a common a n c e s t r a l gene.  At l e a s t 4 exons have been l o c a l i z e d w i t h i n the 5' u n t r a n s l a t e d r e g i o n of the human c e r u l o p l a s m i n  gene, a l t h o u g h t y p i c a l  promoter elements have n o t y e t been i d e n t i f i e d .  eukaryotic  The s i g n i f i c a n c e o f t h i s  n o v e l o r g a n i z a t i o n remains u n c l e a r a t p r e s e n t . In a d d i t i o n t o t h e w i l d - t y p e ceruloplasmin  was  gene, a p r o c e s s e d  i d e n t i f i e d and c o n t a i n e d  DNA  pseudogene f o r human  corresponding  f u n c t i o n a l gene sequence encoding the c a r b o x y - t e r m i n a l r e s i d u e s and t h e 3' u n t r a n s l a t e d a r i s e n from a p r o c e s s e d  RNA  region.  to the  563 amino a c i d  The pseudogene appears t o have  s p e c i e s , s i n c e i n t e r v e n i n g sequences  c o i n c i d e n t w i t h those of t h e f u n c t i o n a l gene have been removed w i t h the exception  o f a s h o r t segment o f i n t r o n i c sequence which denotes t h e 5'  boundary o f t h e pseudogene.  Based on genomic Southern b l o t a n l y s i s  performed under h i g h s t r i n g e n c y c o n d i t i o n s , the pseudogene seems to comprise the o n l y sequence i n the human genome t h a t i s c l o s e l y r e l a t e d t o the w i l d - t y p e  gene.  Using  somatic c e l l h y b r i d i z a t i o n , the pseudogene was  l o c a l i z e d t o human chromosome 8; t h i s d i f f e r s wild-type  ceruloplasmin  from the l o c a t i o n of the  gene, which has been mapped to chromosome 3.  iv  TABLE OF CONTENTS Page Abstract  i i  L i s t of Tables  x  L i s t of Figures  xi  Abbreviations  xiii  Acknowledgements  xiv  Dedication I.  xv  INTRODUCTION  1  I.A  PERSPECTIVES  1  I.B  PROPERTIES OF CERULOPLASMIN  . . . .  1  I.B.I  Structure of the P r o t e i n  1  I.B.2  S i t e s of Ceruloplasmin  9  I.B.3  Functions of Ceruloplasmin  I.B.3.1 I.B.3.2 I.B.3.3 I.B.3.4 I.B.4 I.B.4.1 I.B.4.2 I.B.4.3  Biosynthesis  Ferroxidase a c t i v i t y Serum a n t i o x i d a n t a c t i v i t y Amine o x i d a s e a c t i v i t y Role o f c e r u l o p l a s m i n i n copper t r a n s p o r t . . . Regulation of Ceruloplasmin Expression Hormonal r e g u l a t i o n o f c e r u l o p l a s m i n synthesis Copper i n d u c t i o n of c e r u l o p l a s m i n expression Regulation of ceruloplasmin synthesis during inflammation  10 10 10 11 11 12 12 13 14  I.C  ABNORMALITIES IN COPPER HOMEOSTASIS - WILSON'S DISEASE . .  15  I.D  CHARACTERIZATION OF RAT CERULOPLASMIN  17  I.E  THE RELATIONSHIP OF CERULOPLASMIN TO OTHER COPPERCONTAINING PROTEINS  19  THE RELATIONSHIP BETWEEN CERULOPLASMIN AND PROTEINS INVOLVED IN BLOOD COAGULATION  26  I.F  V  Page I.G.  CHARACTERIZATION OF THE HUMAN FACTOR VIII GENE  32  I.G.I  H i s t o r i c a l Perspectives  32  I.G.2  Organization of the Factor VIII Gene  33  I.G.3  Evolutionary Aspects of Intron Positions Within the Factor VIII Gene  I.H  I.I  I.J  I. K. II.  35  DYNAMICS OF PROTEIN AND GENE EVOLUTION  38  I.H.I  The Molecular Clock  38  MECHANISMS OF GENE EVOLUTION  39  1.1.1  Gene Duplication  39  1.1.2  Gene Fusion  39  1.1.3  Exon Shuffling  40  1.1.4  Intron Insertion and Intron S l i d i n g  40  PSEUDOGENES  41  I.J.I  Non-Processed Pseudogenes  41  I. J.2  Processed Pseudogenes  42  The Present Study  43  MATERIALS AND METHODS  45  II. A  BACTERIAL HOSTS AND MEDIA  45  II.B  HYBRIDIZATION PROBES  48  II. B.l  P u r i f i c a t i o n and L a b e l l i n g of Oligodeoxyribonucleotides  48  II.B.2  Nick Translation  49  II.B.3  Klenow Labelling  II.B.4  Preparation of M13 Probes  . . . .  49 50  vi  Page II.C  IDENTIFICATION OF cDNAS FOR HUMAN CERULOPLASMIN  51  II.C.l  S c r e e n i n g o f a Human L i v e r cDNA L i b r a r y  51  II.C.2  P r e p a r a t i o n and S c r e e n i n g o f Randomly-Primed Human L i v e r cDNA L i b r a r i e s  52  II.D  SCREENING OF HUMAN GENOMIC LIBRARIES  54  II.E  ISOLATION OF NUCLEIC ACIDS  55  II.E.l  P u r i f i c a t i o n o f P l a s m i d DNA  55  II.E.2  I s o l a t i o n o f Bacteriophage  57  II.E.3  P r e p a r a t i o n o f Human Genomic DNA  DNA  59  II.E.4 I s o l a t i o n o f RNA II.E.4.1 P r e p a r a t i o n o f human l i v e r p o l y ( A ) RNA . . . II.E.4.2 P r e p a r a t i o n o f t o t a l RNA from HepG2 c e l l s . . .  60 60 61  BASIC DNA TECHNIQUES  61  II.F.l  R e s t r i c t i o n Enzyme D i g e s t i o n  61  II.F.2  E n d - L a b e l l i n g o f DNA Fragments  62  II.F.3 E l e c t r o p h o r e s i s o f DNA II.F.3.1 Agarose g e l e l e c t r o p h o r e s i s  62 62  II.F.3.2  63  +  II.F  II.F.4 II.G  II.H  Polyacrylamide gel e l e c t r o p h o r e s i s Southern  Transfers  64  DNA CLONING  64  II.G.l  Fragment P r o d u c t i o n  64  II.G.2  Ligation  65  II.G.3  Transformations  o f DNA i n t o pUC o r M13 V e c t o r s  65  DNA SEQUENCE ANALYSIS  66  II.H.l  S c r e e n i n g of M13 Clones  66  II.H.2  I s o l a t i o n of M13 Template DNA  66  II.H.3  DNA Sequence A n a l y s i s  69  vii  Page II.I  RNA ANALYSIS  71  11.1.1  Northern B l o t A n a l y s i s  71  11.1.2  RNA Dot B l o t s  72  11.1.3  Nuclease SI Mapping  72  II.J  CHROMOSOME MAPPING  73  II. K  SUMMARY OF HYBRIDIZATION/WASHING CONDITIONS  73  II. K.l  Genomic Southern B l o t A n a l y s i s  73  U.K.2  H y b r i d i z a t i o n C o n d i t i o n s Other Than F o r Genomic Southern B l o t s  74  I I I . RESULTS I I I . A CHARACTERIZATION OF THE HUMAN PRECERULOPLASMIN cDNA III. A.l  . . .  76  I n i t i a l S c r e e n i n g o f a Human L i v e r cDNA Library I s o l a t i o n o f cDNA Clones Encoding t h e 5' End  76  of C e r u l o p l a s m i n  76  III.A.3  DNA Sequence A n a l y s i s o f Human P r e c e r u l o p l a s m i n .  82  III.A.4  Ceruloplasmin T r a n s c r i p t A n a l y s i s  87  III.A.2  I I I . B CHARACTERIZATION OF THE WILD-TYPE HUMAN CERULOPLASMIN GENE III.B.l  III.B.2  III.B.3  III.B.4  I s o l a t i o n and R e s t r i c t i o n Endonuclease of Genomic Clones  90  Mapping 90  L o c a l i z a t i o n o f Intron/Exon J u n c t i o n s Corresponding t o t h e C e r u l o p l a s m i n Coding Region  93  P a r t i a l N u c l e o t i d e Sequence A n a l y s i s o f t h e Human C e r u l o p l a s m i n Gene  96  O r g a n i z a t i o n of t h e 5' End o f the Human C e r u l o p l a s m i n Gene  98  viii  Page III.B.4.1 III.B.4.2 III.B.4.3 III.B.4.4 III.B.4.5  Comparison o f genomic and cDNA sequence d a t a SI mapping a n a l y s i s o f exon 1 Southern b l o t a n a l y s i s o f exon L4 Northern b l o t a n a l y s i s o f t h e 5* end o f t h e human c e r u l o p l a s m i n gene RNA d o t b l o t a n a l y s i s  .  105 105  I I I . C ISOLATION AND COMPLETE CHARACTERIZATION OF A PSEUDOGENE FOR HUMAN CERULOPLASMIN III.C.l  III.C.2  III.C.3  I I I . C.4  98 98 98  I l l  I s o l a t i o n o f Genomic Clones C o n t a i n i n g t h e Human C e r u l o p l a s m i n Pseudogene  I l l  DNA Sequence o f t h e Human C e r u l o p l a s m i n Pseudogene  114  Nuclease SI A n a l y s i s o f t h e Human C e r u l o p l a s m i n Pseudogene  118  Chromosome L o c a l i z a t i o n o f t h e Human C e r u l o p l a s m i n Pseudogene  124  I I I . D GENOMIC SOUTHERN BLOT ANALYSIS OF THE HUMAN CERULOPLASMIN GENE IV.  124  DISCUSSION IV. A  132  CHARACTERIZATION OF THE HUMAN PRECERULOPLASMIN cDNA IV. A.1  DNA Sequence A n a l y s i s o f t h e Human P r e c e r u l o p l a s m i n cDNA  IV.A.2  I n t e r n a l Homology W i t h i n t h e C e r u l o p l a s m i n  . . .  132  cDNA Sequence IV.A.3 IV.B  136  A n a l y s i s o f the Human C e r u l o p l a s m i n T r a n s c r i p t  CHARACTERIZATION OF THE WILD-TYPE HUMAN CERULOPLASMIN GENE IV.B.l  IV.B.2  IV.B.3  C e r u l o p l a s m i n Gene O r g a n i z a t i o n to t h e Coding Sequence  132  .  137  138  Corresponding 138  DNA Sequence A n a l y s i s of t h e Wild-Type C e r u l o p l a s m i n Gene  139  Intron P o s i t i o n s Within the T r i p l i c a t e d A Domain o f Human C e r u l o p l a s m i n  139  ix  Page IV.B.4  IV.B.5  IV.C  IV.C.2  IV.C.3  IV.E V.  145  C h a r a c t e r i z a t i o n of t h e 5' End o f t h e Human C e r u l o p l a s m i n Gene  150  CHARACTERIZATION OF A PSEUDOGENE FOR HUMAN CERULOPLASMIN . IV.Cl  IV.D  Comparison o f t h e Gene O r g a n i z a t i o n o f C e r u l o p l a s m i n and F a c t o r V I I I  153  DNA Sequence A n a l y s i s o f t h e Human C e r u l o p l a s m i n Pseudogene  153  Chromosome L o c a l i z a t i o n o f t h e Human C e r u l o p l a s m i n Pseudogene  156  S p e c u l a t i o n s on t h e E v o l u t i o n a r y O r i g i n o f t h e Human C e r u l o p l a s m i n Pseudogene  157  A MODEL FOR THE EVOLUTION OF CERULOPLASMIN, FACTOR V AND FACTOR V I I I  159  CONCLUDING REMARKS  163  REFERENCES  165  X  LIST OF TABLES Page I  Comparison o f A Domains i n F a c t o r V, F a c t o r V I I I and Ceruloplasmin  30  Summary o f t h e Genotypes o f B a c t e r i a l Hosts Used i n t h e P r e s e n t Study  46  S u b c l o n i n g S t r a t e g y f o r t h e Wild-Type Human C e r u l o p l a s m i n Gene  67  IV  Compositions  70  V  N u c l e o t i d e Sequence o f Intron/Exon C e r u l o p l a s m i n Gene  II  III  o f M13 DNA Sequencing  Mixes J u n c t i o n s i n t h e Human  94  VI  Frequency o f N u c l e o t i d e s a t Intron/Exon  VII  S i z e s and P o s i t i o n s o f I n t r o n s and Exons W i t h i n t h e C e r u l o p l a s m i n Gene  VIII  Junctions  S e g r e g a t i o n o f t h e Human C e r u l o p l a s m i n Pseudogene w i t h Human Chromosomes i n Human-Hamster Somatic H y b r i d s  95  97  123  xi  LIST OF FIGURES Page 1.  S t r u c t u r a l Model o f t h e Human C e r u l o p l a s m i n M o l e c u l e  2.  R e l a t i o n s h i p between C e r u l o p l a s m i n and Other Containing Proteins  3.  4.  5.  5  Copper21  Comparison o f t h e S t r u c t u r a l O r g a n i z a t i o n o f C e r u l o p l a s m i n , F a c t o r V and F a c t o r V I I I  28  I n t r o n P o s i t i o n s w i t h i n t h e A and C Domains o f Human Factor VIII  36  Schematic  Summary o f t h e C l o n i n g o f t h e Human  P r e c e r u l o p l a s m i n cDNA  77  6.  Sequencing  7.  N u c l e o t i d e Sequence o f Human P r e c e r u l o p l a s m i n cDNA  83  8.  Comparative A n a l y s i s o f cDNA Clones \hCP-2 t o XhCP-6  85  9.  B l o t H y b r i d i z a t i o n A n a l y s i s o f Human C e r u l o p l a s m i n mRNA  10. 11.  S t r a t e g y f o r t h e Human P r e c e r u l o p l a s m i n cDNA  P a r t i a l R e s t r i c t i o n Map and Intron/Exon Human C e r u l o p l a s m i n Gene  . . . .  . . . .  91  Comparison o f t h e Sequence o f \hCP-6 w i t h O v e r l a p p i n g Genomic Sequence D e r i v e d from XWT2 Nuclease  13.  Southern B l o t A n a l y s i s o f Exon L4  14.  N o r t h e r n B l o t A n a l y s i s o f t h e 5* End o f t h e C e r u l o p l a s m i n Gene  15.  RNA Dot B l o t A n a l y s i s o f t h e 5' U n t r a n s l a t e d Region C e r u l o p l a s m i n Gene  17.  18.  99  SI Mapping o f Exon 1  P a r t i a l R e s t r i c t i o n Map and Sequencing Human C e r u l o p l a s m i n Pseudogene  88  Organization of the  12.  16.  80  101 103 .  106  of the 109  Strategy f o r the 112  N u c l e o t i d e Sequence o f t h e Human C e r u l o p l a s m i n Pseudogene and Comparison w i t h t h e Corresponding Region o f t h e C e r u l o p l a s m i n cDNA Sequence  115  Nuclease  119  SI Mapping A n a l y s i s o f t h e C e r u l o p l a s m i n Pseudogene . .  xii  Page 19.  Chromosome Mapping o f t h e Human C e r u l o p l a s m i n Pseudogene U s i n g Somatic C e l l H y b r i d A n a l y s i s  121  20.  Genomic Southern B l o t A n a l y s i s o f t h e Human C e r u l o p l a s m i n Gene .  125  21.  Genomic Southern A n a l y s i s o f t h e Human C e r u l o p l a s m i n Pseudogene and R e l a t e d Sequences  128  Genomic Southern B l o t A n a l y s i s o f t h e 3' End o f t h e Human C e r u l o p l a s m i n Gene  130  I n t r o n P o s i t i o n s W i t h i n t h e Three Repeated U n i t s o f Human Ceruloplasmin  140  P o s i t i o n s o f I n t r o n s i n t h e A Domains o f C e r u l o p l a s m i n and Factor VIII  143  Comparative P o s i t i o n s o f I n t r o n s i n C e r u l o p l a s m i n w i t h Corresponding Introns i n F a c t o r VIII  147  A Proposed Model f o r t h e E v o l u t i o n o f C e r u l o p l a s m i n , F a c t o r V and F a c t o r V I I I  160  22.  23.  24.  25.  26.  xiii  ABBREVIATIONS A ATP bp C cDNA cpm dNTP ddNTP DNA DTT EDTA  6 HEPES Kb, Kbp Kda Krpm mRNA Mr N OD PIPES poly(A) RNA SDS T TEMED Tris tRNA U UV W  +  adenosine adenosine-5'-triphosphate base p a i r s cytosine complementary DNA counts p e r minute deoxyribonucleotidetriphosphate dideoxyribonucleotidetriphosphate deoxyribonucleic acid dithiothreitol ethylenediamine t e t r a a c e t i c a c i d guanosine N-2-hydroxyethylpiperazine-N-2-ethanesulfonic Kilobases, Kilobase pairs Kilodaltons thousand r e v o l u t i o n s p e r minute messenger RNA r e l a t i v e m o l e c u l a r weight adenine, c y t o s i n e , guanine o r thymine optical density piperazine-N,N'-bis (2-ethanesulfonic acid) polyadenylated ribonucleic acid sodium d o d e c y l s u l f a t e thymidine N,N,N',N'-Tetramethylethylenediamine Tri(hydroxymethyl)aminoethane t r a n s f e r RNA uridine ultraviolet watts  acid  xiv  ACKNOWLEDGEMENTS  I would l i k e t o thank my s u p e r v i s o r , Ross M a c G i l l i v r a y , f o r h i s e n t h u s i a s t i c support  o f my c a r e e r ( " g r e a t " ) , and h i s w i l l i n g n e s s t o a l l o w  each member o f t h e l a b o r a t o r y t o develop  an independent approach t o  s c i e n t i f i c research.  an atmosphere o f warmth and  Ross has generated  f r i e n d s h i p w i t h i n h i s r e s e a r c h group t h a t has f i l l e d my p a s t f i v e w i t h many happy memories.  I would a l s o l i k e t o thank W a l t e r  years  (Waltman)  Funk f o r t h e s p e c i a l camaraderie t h a t we have shared over t h e y e a r s .  In  a d d i t i o n , I would l i k e t o acknowledge Heather K i r k , f o r t e c h n i c a l a d v i c e d u r i n g s e v e r a l a s p e c t s o f t h i s p r o j e c t , and Susan Heming, f o r h e r p a t i e n c e i n t h e p r e p a r a t i o n o f t h e manuscript. A v e r y s p e c i a l thanks t o Roland Russnak f o r h i s r e l e n t l e s s optimism —  i t might be c o n t a g i o u s !  DEDICATION  To Mother and  Father  f o r t h e i r t i r e l e s s l o v e and belief  i n me t h a t has  ! t h e s t r e n g t h t o keep  given trying.  1  I.  INTRODUCTION A.  PERSPECTIVES  Copper i s r e q u i r e d f o r the f u n c t i o n of a number of metalloenzymes metalloproteins Therefore,  present  i n b o t h p r o k a r y o t i c and  eukaryotic  cells.  s t r i c t maintenance of copper homeostasis i s e s s e n t i a l f o r many  v i t a l processes.  On  t h i s b a s i s , i t i s not  i n i t i a l d i s c o v e r y by Holmberg i n 1944,  biochemical  study.  T h i s , i n t u r n , has  s u r p r i s i n g that since i t s  ceruloplasmin  t r a n s p o r t p r o t e i n i n v e r t e b r a t e plasma) has  (the p r i n c i p a l  been the focus of  multicopper oxidase.  r e c e i v e d a t t e n t i o n i n analyses i n aerobic  t r a c i n g the e v o l u t i o n of cells  PROPERTIES OF HUMAN CERULOPLASMIN  B.l  S t r u c t u r e of the  large  has  also  metal-containing  1974).  Protein  Ceruloplasmin i s a blue,  crystallizable  ( M o r e l l , 1969;  Nakagawa,  g l y c o p r o t e i n t h a t b i n d s 90 - 95% of plasma copper i n  vertebrates. ( H a r r i s and and  (e.g. F r i e d e n ,  B.  a-2  of  In a d d i t i o n to i t s m u l t i f u n c t i o n a l n a t u r e which  r e n d e r s i t an i n t e r e s t i n g p r o t e i n f o r study, c e r u l o p l a s m i n  enzymes and p r o t e i n s  copper  intensive  r e s u l t e d i n the g e n e r a t i o n  s e v e r a l thousand papers d e a l i n g w i t h the p r o p e r t i e s of t h i s  1972)  and  The  remaining copper e x i s t s i n complexes of amino a c i d s  Sass-Kortsak, 1967), serum albumin (Sarkar  and W i g f i e l d ,  1968)  a t r i p e p t i d e composed of g l y c i n e - h i s t i d i n e - l y s i n e ( P i c k a r t et a l . ,  1980) , a l l of which may  provide  a u x i l i a r y t r a n s p o r t mechanisms  (Frieden,  1981) . M u l t i p l e enzymatic f u n c t i o n s have been a s c r i b e d to c e r u l o p l a s m i n S e c t i o n I.B.3; f o r reviews d e a l i n g w i t h the f u n c t i o n s of  (see  ceruloplasmin,  2  see F r i e d e n , 1981;  Owens, 1982;  Cousins,  1985), a l l of which  i n v o l v e the p r e s e n c e of i n t r i n s i c a l l y - b o u n d c u p r i c i o n s . c e r u l o p l a s m i n m o l e c u l e (Mr = 132 copper-binding  sites  Kda)  Although  Each  contains at l e a s t 6 or 7  (Ryden and B j o r k , 1976), which can be c a t e g o r i z e d  based upon the p h y s i c a l p r o p e r t i e s of l i g h t absorbance and behavior.  likely  electromagnetic  the s t o i c h i o m e t r y of the d i f f e r e n t copper types  c e r u l o p l a s m i n i s not w e l l - e s t a b l i s h e d , the f o l l o w i n g c o m p o s i t i o n proposed:  two  type I and one  paramagnetic resonance (EPR)  has  measurements of Deinum and Vanngard, 1973], Type I copper i s  i n s m a l l b l u e e l e c t r o n - t r a n s f e r p r o t e i n s and b l u e copper  of which c e r u l o p l a s m i n i s the o n l y mammalian r e p r e s e n t a t i v e .  oxidases  Type I  copper c e n t r e s a r e c h a r a c t e r i z e d by s t r o n g absorbance around 600 nm l i g h t ) , and  t y p i c a l l y r e s u l t i n narrow EPR  c i r c u l a r d i c h r o i s m (CD)  been  type I I [based on q u a n t i t a t i v e e l e c t r o n  and t h r e e ( o r f o u r ) type I I I (Ryden and B j o r k , 1976). present  of  hyperfine s p l i t t i n g .  and magnetic c i r c u l a r d i c h r o i s m (MCD)  (red  Based  studies  (Dawson e t a l . , 1979), type I s i t e s i n c e r u l o p l a s m i n a r e proposed to c o o r d i n a t e d by a c y s t e i n e , m e t h i o n i n e , and d i s t o r t e d t e t r a h e d r a l geometry.  be  two h i s t i d i n e l i g a n d s , i n a  Type I copper has  a redox p o t e n t i a l  a p p r e c i a b l y h i g h e r than t h a t of the Cu  (II)/Cu (I) couple  solution  to the d i s t o r t i o n of the copper  (Fee, 1975).  T h i s may  be due  geometry which r e s u l t s i n decreased  on  i n aqueous  a c t i v a t i o n energy r e q u i r e d f o r  e l e c t r o n t r a n s f e r ( W i l l i a m s , 1971). Type I I copper absorbs weakly a t 600 h y p e r f i n e s t r u c t u r e s i m i l a r to t h a t found Type I I copper i s p r e s e n t  nm,  and  f e a t u r e s an  i n t e t r a g o n a l copper complexes.  i n large blue multicopper  l a c c a s e , cytochrome o x i d a s e ,  ascorbate  EPR  oxidase  and  oxidases,  such as  c e r u l o p l a s m i n , as w e l l  3  as i n non-blue o x i d a s e s , such as monoamine o x i d a s e and g a l a c t o s e o x i d a s e . S t u d i e s of the c o o r d i n a t i o n environment of type I I copper i n c e r u l o p l a s m i n (Dawson e t a l . , 1978) provide evidence  and g a l a c t o s e o x i d a s e  (Bereman and Kosman,  1977)  f o r h i s t i d i n e c o o r d i n a t i o n of the copper i n t h e s e type I I  centres. The  type I I I copper c e n t r e i s b i n u c l e a r , composed of two  i o n s a n t i f e r r o m a g n e t i c a l l y coupled and  t h e r e f o r e EPR  copper ( I I )  nondetectable.  These  copper atoms a r e a s s o c i a t e d w i t h an i n t e n s e a b s o r p t i o n band a t 330  nm.  Type I I I copper i s p r e s e n t i n copper o x i d a s e s t h a t c a t a l y z e the r e d u c t i o n of dioxygen  t o two water m o l e c u l e s ,  o x i d a s e and  laccase.  Very  little  such as c e r u l o p l a s m i n ,  ascorbate  i s known about the c o o r d i n a t i o n geometry  o r s p e c i f i c l i g a n d groups f o r type I I I c e n t r e s (Urbach,  1981).  An  EPR  s i g n a l has been d e t e c t e d i n the l a t t e r two p r o t e i n s which d i f f e r s from the s i g n a l s r e s u l t i n g from type I o r type I I Cu 1980).  T h i s new  s i g n a l has been a t t r i b u t e d t o one of the p a i r of Cu  i o n s e x i s t i n g i n the b i n u c l e a r c e n t r e , and observed  ( I I ) i o n s (Reinhammar e t a l . ,  i s comparable to s i g n a l s  i n h a l f - m e t hemocyanin (Himmelwright e t a l . , 1978)  dismutase ( F i e l d e n e t a l . , 1974). shown t o c o n t a i n c o p p e r - b i n d i n g  and  superoxide  Both of the l a t t e r p r o t e i n s have been  sites i n binuclear centres.  D e s p i t e p e r s i s t e n t study, the assignment of c o p p e r - b i n d i n g s p e c i f i c r e g i o n s of the c e r u l o p l a s m i n p o l y p e p t i d e c h a i n remains undetermined.  sites  t h a t 50% of the non-blue c o p p e r - b i n d i n g d e r i v e d from the c a r b o x y l terminus - 1046).  o c c u r s i n an 11 Kda  (1983)  fragment  of c e r u l o p l a s m i n ( c o r r e s p o n d i n g to A d d i t i o n a l l y , based on  to  largely  However, i t has been r e c e n t l y demonstrated by Raju  amino a c i d r e s i d u e s 885  (II)  sequence  4  s i m i l a r i t y w i t h known b l u e and non-blue copper b i n d i n g s i t e s , s e v e r a l p u t a t i v e copper-binding  c e n t r e s have been i d e n t i f i e d i n c e r u l o p l a s m i n (see  Section I.E). E a r l y s t u d i e s suggested  t h a t c e r u l o p l a s m i n had a s u b u n i t s t r u c t u r e  c o n s i s t i n g o f 2 - 8 p o l y p e p t i d e c h a i n s (Freeman and D a n i e l , 1973; P o u l i k and Weiss, 1975; McCombs and Bowman, 1976). however, t h a t t h e observed  Ryden (1972) demonstrated,  s u b u n i t s corresponded  to proteolytic  t h a t c o u l d be e l i m i n a t e d when f r e s h plasma, supplemented w i t h i n h i b i t o r s , was used f o r t h e i s o l a t i o n o f c e r u l o p l a s m i n . proposed s i n g l e c h a i n s t r u c t u r e f o r t h e molecule,  fragments  protease  This l e d to a  which was l a t e r  c o n f i r m e d by amino a c i d sequence a n a l y s i s (Takahashi e t a l . , 1984). In v i t r o and p o s s i b l y i n v i v o , spontaneous p r o t e o l y t i c c l e a v a g e  occurs  r a p i d l y a t two h i g h l y s e n s i t i v e s i t e s f o l l o w i n g b a s i c amino a c i d r e s i d u e s , producing  fragments w i t h m o l e c u l a r weights o f 67 Kda, 50 Kda, and 19 Kda,  corresponding  t o t h e amino-terminal,  of t h e p r o t e i n , r e s p e c t i v e l y 1981a; see F i g u r e 1 ) .  middle,  and c a r b o x y - t e r m i n a l p o r t i o n s  ( K i n g s t o n e t a l . , 1980; Dwulet and Putnam,  Limited tryptic digestion i n v i t r o results i n  c l e a v a g e a t t h e two l a b i l e s i t e s d e s c r i b e d above, and a d d i t i o n a l l y degrades t h e 50 Kda fragment, p r o d u c i n g  25- and 26 Kda fragments,  and  s l o w l y c l e a v e s t h e 67 Kda fragment, y i e l d i n g 49- and 18 Kda fagments (Takahashi  e t a l . , 1983; see F i g u r e 1 ) . The p h y s i o l o g i c a l s i g n i f i c a n c e o f  the l i m i t e d p r o t e o l y t i c c l e a v a g e  i s u n c l e a r , b u t may f u n c t i o n i n p r o t e i n  r e g u l a t i o n as i s proposed f o r the d e a c t i v a t i o n of a n a p h y l a t o x i n s and bradykinin The  (see below).  complete amino a c i d sequence o f human c e r u l o p l a s m i n ,  of 1046 r e s i d u e s , was determined  consisting  by a n a l y s i s of the 19 Kda ( K i n g s t o n  5  F i g u r e 1.  S t r u c t u r a l model of the human c e r u l o p l a s m i n molecule  p r o t e o l y t i c cleavage  s i t e s and  i n t e r n a l amino a c i d sequence  ( m o d i f i e d from O r t e l e t a l . , 1984;  fragments as shown.  a r e connected  identity  i n t o the 67, 50 and  In the i n t a c t p o l y p e p t i d e c h a i n , these  by s i n g l e amino a c i d r e s i d u e s a r g i n i n e (R) and  a r e g i v e n i n K i l o d a l t o n s (Kda).  The  lysine  s i z e s of p r o t e o l y t i c  (K). arrows  fragments  The p o s i t i o n s of glucosamine  o l i g o s a c c h a r i d e attachment s i t e s a r e i n d i c a t e d by s o l i d diamonds; the carbohydrate  moiety t h a t i s m i s s i n g i n the type I I c e r u l o p l a s m i n v a r i a n t  is starred.  The  shown.  e x t e n t of the t r i p l i c a t e d u n i t s A l , A2 and A3  19  fragments  T r y p t i c c l e a v a g e s i t e s are i n d i c a t e d by v e r t i c a l arrows, w i t h broad i d e n t i f y i n g major s i t e s of c l e a v a g e .  on  see t e x t f o r d e t a i l s ) .  The p o l y p e p t i d e c h a i n i s c l e a v e d a u t o l y t i c a l l y Kda  based  i s also  N  t1  f Ut  £  1  U  N.  1  25 67  50  350 "Ai  k  ~~^  2  6  I  ^  -C  A  I  710 A2  C  U  Proteolytic Fragments (Kda)  19  1046 AT  ~l  Triplicated Units (amino acids)  OS  7  e t a l . , 1979)  and  50 Kda  (Dwulet and Putnam , 1981a) p r o t e o l y t i c  fragments; the sequence of the 67 Kda was  fragment and o v e r l a p p i n g  r e p o r t e d by Takahashi e t a l . (1984).  The  presence of an a d d i t i o n a l amino a c i d r e s i d u e 50 Kda Kda  fragments, and  fragments.  peptides  l a t t e r strategy revealed (Arg 481)  a s i n g l e r e s i d u e (Lys 667)  the  between the 67-  connecting  the 50-  I t i s assumed t h a t d u r i n g p r e p a r a t i o n , these two  r e s i d u e s are e x c i s e d by an enzyme w i t h c a r b o x y p e p t i d a s e - l i k e  and  and  19  basic  specificity.  A s i m i l a r mechanism i s i n v o l v e d i n the carboxypeptidase-B c a t a l y z e d removal of c a r b o x y - t e r m i n a l  a r g i n i n e or l y s i n e r e s i d u e s t h a t r e s u l t s i n  i n a c t i v a t i o n of k i n i n s , and  the C3a  and C5a  analphylatoxins  (Putnam,  1984). Ceruloplasmin  p o s s e s s e s attachment s i t e s f o r f o u r glucosamine-1inked  (GlcN) o l i g o s a c c h a r i d e s ( T e t a e r t e t a l . , 1982; The  l o c a t i o n of these carbohydrate  s e p a r a t i o n of G l c N - c o n t a i n i n g  T a k a h a s k i e t a l . , 1984).  attachment p o i n t s was  determined  peptides using reverse-phase high  l i q u i d chromatography, f o l l o w e d by amino a c i d sequence a n a l y s i s . s i t e s are l o c a t e d i n the 6 7 Kda  fragment, w h i l e one  p r o t e o l y t i c fragment (see F i g u r e 1 ) .  Ceruloplasmin  are not g l y c o s y l a t e d .  pressure Three  r e s i d e s i n the 50  Kda  A l l f o u r GlcN o l i g o s a c c h a r i d e s are  l i n k e d i n the o b l i g a t o r y t r i p e p t i d e a c c e p t o r (Clamp, 1975).  by  a l s o has  This i s probably  sequence Asn-X-Ser/Thr  t h r e e GlcN a c c e p t o r  sequences t h a t  the r e s u l t of t h e i r l o c a t i o n i n  b u r i e d , hydrophobic r e g i o n s of the p r o t e i n ( O r t e l e t a l . , 1984). Two  v a r i a n t s of c e r u l o p l a s m i n have been i d e n t i f i e d based  carbohydrate  composition.  In type I c e r u l o p l a s m i n  f o u r o l i g o s a c c h a r i d e s are p r e s e n t , Asn-339) i s m i s s i n g  on  (predominant form), a l l  w h i l e the second o l i g o s a c c h a r i d e ( i . e .  i n the l e s s abundant type I I form (Takahashi  et a l . ,  8  1984)  (see F i g u r e 1 ) .  The p h y s i o l o g i c a l s i g n i f i c a n c e of these two v a r i a n t  forms i s unknown a t p r e s e n t . Based on computer a n a l y s i s of the amino a c i d sequence, the  entire  human c e r u l o p l a s m i n m o l e c u l e has been shown to e x h i b i t an i n t e r n a l 3 - f o l d homology, w i t h each homology u n i t c o n s i s t i n g of approximately a c i d s (Takahashi designated  e t a l . , 1984;  A l , A2  and A3  see F i g u r e 1.  These u n i t s  350  amino  (arbitrarily  from amino- t o carboxy-terminus) share n e a r l y  40%  sequence i d e n t i t y when compared p a i r w i s e (see T a b l e 1 ) , i n c l u d i n g a h i g h degree of c o n s e r v a t i o n of the f o u r l e a s t f r e q u e n t amino a c i d s i n p r o t e i n s : m e t h i o n i n e , h i s t i d i n e , tryptophan,  and  c y s t e i n e ( O r t e l e t a l . , 1984).  amino a c i d sequence c o n s e r v a t i o n has been i n t e r p r e t e d t o suggest l e v e l of s t r u c t u r a l c o n s e r v a t i o n between these r e l a t e d segments e t a l . , 1984).  (Ortel  t h a t each of these t h r e e r e g i o n s i s s u b d i v i d e d  2 o r 3 domains (see F i g u r e 1 ) . homology u n i t s do not correspond  The boundaries  between the  the 67-,  50- and  19 Kda  cleavage proteolytic  However, t r y p s i n c l e a v e s a t a s i t e between the A l and Due  t o a d d i t i o n a l s i t e s of c l e a v a g e ,  Differential  a t homologous s i t e s between proposed  domains has been a t t r i b u t e d to observed secondary s t r u c t u r e s a t interdomain  A3  these 2 u n i t s are f u r t h e r  d i v i d e d i n t o subdomains, as i s d e t a i l e d i n F i g u r e 1. s e n s i t i v i t y to p r o t e o l y t i c cleavage  d i f f e r e n c e s i n both primary  boundaries  determined based on measurements of CD  and  ( O r t e l e t §_1. , 1984).  L o c a l secondary s t r u c t u r e w i t h i n the c e r u l o p l a s m i n p o l y p e p t i d e was  into  individual  to the s i t e s of p r o t e o l y t i c  d e s c r i b e d p r e v i o u s l y , which generate  domains.  a high  The p r o t e o l y t i c cleavage p a t t e r n of non-denatured  c e r u l o p l a s m i n suggests  fragments.  The  (Noyer and  Putnam, 1981)  chain as  w e l l as the c a l c u l a t i o n of parameters p r e d i c t i v e of secondary s t r u c t u r e  9  O r t e l e t a l . , 1984).  On t h i s b a s i s , i t has been proposed t h a t  c e r u l o p l a s m i n c o n s i s t s of 33% B sheet o r g a n i z a t i o n , 33% B t u r n s and a-helices.  20%  Coupled w i t h a c a l c u l a t e d hydropathy p r o f i l e f o r the p r o t e i n  ( O r t e l e t a l . , 1984), these data are i n accordance w i t h the domain model presented  i n F i g u r e 1.  I t i s a t t r a c t i v e to s p e c u l a t e t h a t d i f f e r e n t  domains w i t h i n the c e r u l o p l a s m i n m o l e c u l e may biological activities  B.2  correspond  (see S e c t i o n I.B.3).  S i t e s of C e r u l o p l a s m i n  Biosynthesis  O r i g i n a l l y , c e r u l o p l a s m i n b i o s y n t h e s i s was e x c l u s i v e l y w i t h i n the parenchymal c e l l s of the l i v e r 1969).  to i t s various  However, i t has  thought to  occur  (Neifakh et a l . ,  s i n c e been demonstrated t h a t the c h o r o i d p l e x u s ,  y o l k sac, p l a c e n t a and t e s t i s r e p r e s e n t e x t r a h e p a t i c s i t e s of c e r u l o p l a s m i n s y n t h e s i s i n the r a t ( A l d r e d e t a l . , 1987).  I t has  proposed t h a t c e r u l o p l a s m i n e x p r e s s i o n by these t i s s u e s may  be  been  important  i n the t r a n s p o r t of copper a c r o s s n a t u r a l b a r r i e r s e x i s t i n g between  these  compartments ( i . e . b l o o d / c e r e b r o s p i n a l f l u i d , m a t e r n a l / f e t a l c i r c u l a t i o n , and b l o o d / t e s t i s b a r r i e r s ) ( A l d r e d e t a l . , 1987). t r a n s p o r t p r o t e i n s t r a n s f e r r i n and e t a l . 1985;  S c h r e i b e r , 1987)  S y n t h e s i s of  the  t r a n s t h y r e t i n i n these t i s s u e s  (Dickson  f u r t h e r i m p l i c a t e s the importance of  t r a n s p o r t p r o t e i n s a t the i n t e r f a c e between e x t r a c e l l u l a r  compartments.  Furthermore, the s y n t h e s i s of t r a n s f e r r i n i n these t i s s u e s , coupled the known f e r r o x i d a s e a c t i v i t y of c e r u l o p l a s m i n important  with  (see S e c t i o n I.B.3) may  be  i n the t r a n s p o r t of i r o n a c r o s s compartment b a r r i e r s .  S i m i l a r l y , the a b i l i t y of c e r u l o p l a s m i n to o x i d i z e s e r o t o n i n and catecholamines  (see S e c t i o n I.B.3) may  various  be of p h y s i o l o g i c a l s i g n i f i c a n c e i n  10  the r e g u l a t i o n of c e r e b r o s p i n a l f l u i d composition.  Using  i n s i t u h i s t o h y b r i d i z a t i o n , Yang e t a l . (1986) have a l s o c e r u l o p l a s m i n mRNA w i t h i n c i r c u l a t i n g macrophages and I n t e r e s t i n g l y , gene e x p r e s s i o n of t r a n s f e r r i n has lymphocytes (Lum  e t a l . , 1987)  r o l e s f o r these two  again suggesting  genetically-linked  the t e c h n i q u e  of  observed  lymphocytes.  a l s o been shown i n  coordinated f u n c t i o n a l  (Weitkamp, 1983;  Yang e t a l . , 1984)  plasma p r o t e i n s .  B.3  F u n c t i o n s of  Ceruloplasmin  Ceruloplasmin  possesses  a number of enzymatic a c t i v i t i e s which  cannot be a t t r i b u t e d to a s u b u n i t o r g a n i z a t i o n , s i n c e t h e r e i s c o n c l u s i v e evidence  t h a t c e r u l o p l a s m i n i s s y n t h e s i z e d as a s i n g l e p o l y p e p t i d e  (see S e c t i o n I . B . I ) .  chain  Rather, the m u l t i f u n c t i o n a l n a t u r e of the p r o t e i n  has been a s c r i b e d to the c a t a l y t i c a c t i v i t i e s of the bound c u p r i c i o n s ( F r i e d e n , 1981;  see S e c t i o n I . B . I ) .  The  v a r i o u s f u n c t i o n s of  c e r u l o p l a s m i n are d e t a i l e d below: B.3.1  Ferroxidase a c t i v i t y  and M o r e l l , 1973;  (Curzon  F r i e d e n and H s e i h ,  a v a r i e t y of s u b s t r a t e s i n v i t r o ,  and O ' R e i l l y , I960;  1976).  Although  capable  Scheinberg of  oxidizing  f e r r o u s [Fe ( I I ) ] i r o n has been proposed  as the p r i n c i p a l p h y s i o l o g i c a l s u b s t r a t e f o r c e r u l o p l a s m i n . c a p a c i t y , c e r u l o p l a s m i n o x i d i z e s Fe  In  this  ( I I ) r e l e a s e d from f e r r i t i n to the  ( I I I ) form, f o r subsequent b i n d i n g to a p o t r a n s f e r r i n [ i . e . Fe ferroxidase (II)-ferritin is directly B.3.2  :• Fe  (Ill)-transferrin].  Thus,  ceruloplasmin  i n v o l v e d i n the r e g u l a t i o n of h e p a t i c i r o n m o b i l i z a t i o n . Serum a n t i o x i d a n t a c t i v i t y  G o l d s t e i n e t a l . , 1979).  ( A l - T i m i m i and  Dormandy,  1977;  I t i s well established that ceruloplasmin  can  Fe  11  serve as a scavenger  of f r e e r a d i c a l s and  superoxide  ions.  F r i e d e n (1981)  has e s t i m a t e d t h a t the c o l l e c t i v e r a d i c a l - s c a v e n g i n g p o t e n t i a l of c e r u l o p l a s m i n i n serum i s l e s s than t h a t of superoxide  dismutase.  However, s i n c e c e r u l o p l a s m i n i s e x t r a c e l l u l a r i n l o c a t i o n w h i l e  superoxide  dismutase i s p r i m a r i l y i n t r a c e l l u l a r , c e r u l o p l a s m i n a c t s as the major scavenger  i n plasma, p a r t i c u l a r l y d u r i n g the acute phase response  (see  below) when c e r u l o p l a s m i n l e v e l s are c h a r a c t e r i s t i c a l l y i n c r e a s e d . B.3.3 1981).  Amine o x i d a s e a c t i v i t y  C e r u l o p l a s m i n possesses  numerous aromatic a c t i v i t y may  be important  The  s i g n i f i c a n t oxidase a c t i v i t y  amines and phenols  in vitro.  Frieden, toward  Physiologically,  catecholamines.  r o l e of c e r u l o p l a s m i n i n copper t r a n s p o r t .  The  t h a t c e r u l o p l a s m i n i s a copper t r a n s p o r t p r o t e i n i s strengthened experiments demonstrating  this  i n the r e g u l a t i o n of l e v e l s of b i o g e n i c amines  such as s e r o t o n i n and v a r i o u s B.3.4  ( P e i s a c h and L e v i n e , 1963;  t h a t a c e r u l o p l a s m i n molecule  b i n d up t o t e n c u p r i c i o n s , i n a d d i t i o n to the  will  proposal by  reversibly  intrinsically-bound,  c a t a l y t i c a l l y a c t i v e copper atoms (McKee and F r i e d e n , 1971). c o n s i s t e n t w i t h p r o p o s a l s t h a t c e r u l o p l a s m i n i s the primary  This i s source of  copper f o r i n t r a c e l l u l a r metalloenzymes p r e s e n t i n v a r i o u s e x t r a h e p a t i c tissues  (Owen, 1965;  L i n d e r and Moor, 1977;  t h i s r e g a r d , t h e r e i s evidence  Campbell e t a l . , 1981).  In  t h a t the copper i o n s of c e r u l o p l a s m i n are a  p r e - r e q u i s i t e f o r copper u t i l i z a t i o n i n the b i o s y n t h e s i s of cytochrome c oxidase  (Marceau and A s p i n , 1973a,b; H s i e h and F r i e d e n , 1975).  been proposed t h a t c e r u l o p l a s m i n Cu r e c e p t o r s and  t h a t Cu  ( I I ) i s reduced  (I) i s subsequently  i n t r a c e l l u l a r a c c e p t o r ( s ) ( F r i e d e n , 1981).  I t has  a t c e l l membrane  t r a n s f e r r e d to an u n i d e n t i f i e d Alternatively,  ceruloplasmin  12  may  be taken up by e n d o c y t o s i s , and Cu  (I) may  then be r e l e a s e d by  p r o t e o l y s i s , accompanied by r e c y c l i n g of the p r o t e i n to the plasma membrane f o r r e l e a s e ( C o u s i n s , 1985).  T h i s l a t t e r model p r e c l u d e s  n e c e s s i t y of an a c c e p t o r f o r i n t r a c e l l u l a r t r a n s p o r t .  the  The net r e s u l t of  e i t h e r r o u t e of copper e n t r y i n t o the c e l l s i s t h a t the l a b i l e Cu  (I) form  would be o x i d a t i v e l y t r a n s f e r r e d to i n t r a c e l l u l a r apoenzymes, where i t c o u l d then be f i x e d w i t h the a i d of oxygen i n t o holoenzyme-Cu ( I I ) form. Recently,  evidence has been p r e s e n t e d  f o r the p r e s e n c e of a  c e r u l o p l a s m i n r e c e p t o r i n membranes from c h i c k e n a o r t a and (Stevens  e t a l . , 1984).  specific cardiac tissues  In t h i s study, membrane fragments d e r i v e d from  125 the l a t t e r t i s s u e s bound [ I ] - l a b e l l e d chicken ceruloplasmin with d i s s o c i a t i o n constant  (K.) of approximately d  10  —8  M.  This i s  c o n s i s t e n t w i t h s t u d i e s showing t h a t the a c t i v a t i o n of a o r t i c oxidase  lysyl  i s c o r r e l a t e d w i t h e l e v a t e d plasma c e r u l o p l a s m i n l e v e l s ( H a r r i s  and D i S i l v e s t r o ,  B.4  1981).  R e g u l a t i o n of C e r u l o p l a s m i n  B.4.1  Expression  Hormonal r e g u l a t i o n of c e r u l o p l a s m i n s y n t h e s i s .  Hormonal  f a c t o r s have been shown to i n f l u e n c e c e r u l o p l a s m i n p r o d u c t i o n by the (see C o u s i n s ,  1985  that epinephrine  f o r a recent review).  and e s t r a d i o l  i n c r e a s e serum c e r u l o p l a s m i n l e v e l s i n the hydrocortisone  have been shown to i n c r e a s e c e r u l o p l a s m i n l e v e l s i n c h i c k e n s 1965).  Based on these o b s e r v a t i o n s ,  s t r e s s - r e l a t e d change i n c e r u l o p l a s m i n Although  liver  Meyer e t a l . (1958) have shown  r a t , w h i l e b o t h a d r e n o c o r t i c o t r o p h i c hormone (ACTH) and  Hill,  a  (Starcher  i t has been proposed t h a t  involves adrenal  and  any  steroids.  a d r e n a l hormones have h i s t o r i c a l l y r e c e i v e d p a r t i c u l a r a t t e n t i o n  13  r e g a r d i n g t h e i r e f f e c t on c e r u l o p l a s m i n l e v e l s ,  i t has been demonstrated  more r e c e n t l y t h a t l e u k o c y t e endogenous mediator ( i n t e r l e u k i n I) can a l s o e l e v a t e serum c e r u l o p l a s m i n l e v e l s  (Wannemacher e t a l . , 1975).  The  s t i m u l a t o r y e f f e c t s of l e u k o c y t e endogenous mediator on c e r u l o p l a s m i n  and  s e v e r a l o t h e r plasma p r o t e i n s have been proposed i n the r e g u l a t i o n of  the  acute phase inflammatory B.4.2  response (see below).  Copper i n d u c t i o n of c e r u l o p l a s m i n e x p r e s s i o n .  Linder et a l .  (1979) have r e p o r t e d t h a t copper d i r e c t l y c o n t r o l s the plasma c o n c e n t r a t i o n of c e r u l o p l a s m i n i n d i e t - i n d u c e d copper d e f i c i e n t r a t s by r e g u l a t i o n of i t s l e v e l of s y n t h e s i s .  In these s t u d i e s , the e f f e c t s of  o r a l a d m i n i s t r a t i o n of copper to c o p p e r - d e f i c i e n t r a t s was  assessed  by  3 monitoring  the i n c o r p o r a t i o n of a two  i n t o plasma p r o t e i n s .  No  hour p u l s e dose of  [ H]-leucine  e f f e c t of copper a d m i n i s t r a t i o n was  s y n t h e s i s of plasma p r o t e i n s i n g e n e r a l . c e r u l o p l a s m i n s y n t h e s i s was  observed,  observed  However, a marked e f f e c t  resulting in a  enhancement ( n e a r l y t h r e e - f o l d ) a f t e r 6 - 8  hours,  thereby  proposed t h a t the sudden i n f l u x of copper a s s o c i a t e d w i t h  and may may  doses may  on  significant resembling  e f f e c t of i r o n on f e r r i t i n s y n t h e s i s ( D r y s d a l e e t a l . , 1966).  administered  on  I t has  the been  large  be s u f f i c i e n t to bypass normal c o n t r o l mechanisms,  e i t h e r a c t i v a t e c e r u l o p l a s m i n gene t r a n s c r i p t i o n s p e c i f i c a l l y ,  a l t e r some a s p e c t of t r a n s l a t i o n a l r e g u l a t i o n .  c o n s i s t e n t w i t h the o b s e r v a t i o n s of Weiner and parenchymal c e l l s .  In the l a t t e r study,  f o r 12 hours o r more s i g n i f i c a n t l y s e c r e t i o n by the c e l l s s u g g e s t i n g  These data  Cousins  are  (1980) u s i n g r a t  i n c u b a t i o n w i t h 50 pM 3  increased  or  copper  [ H]-ceruloplasmin  t h a t when e x t r a c e l l u l a r copper  content  14  was s u f f i c i e n t l y h i g h ,  ceruloplasmin  gene e x p r e s s i o n may have been  enhanced. B.4.3  Regulation  of ceruloplasmin  the acute phase response.  synthesis during  inflammation -  The acute phase r e a c t a n t s comprise a group o f  m a i n l y g l y c o p r o t e i n s which show c h a r a c t e r i s t i c a l l y a l t e r e d r a t e s o f synthesis i n the l i v e r  ( S c h r e i b e r e t a l . , 1982) r e s u l t i n g i n changes i n  t h e i r plasma c o n c e n t r a t i o n s stimuli reactant by  i n response t o a wide v a r i e t y o f inflammatory  [see Koj (1974) f o r a r e v i e w ] . (Larson,  Ceruloplasmin  i s an a c u t e phase  1974) and as such, i t s serum l e v e l s can become i n c r e a s e d  2 t o 3 - f o l d (from t h e normal l e v e l o f 15 - 60 mg/dl serum; Owen, 1982)  i n response t o inflammation.  The p r o p e r t y  of ceruloplasmin  antioxidant i s a t t r a c t i v e i n t h i s respect, since increased  as a serum ceruloplasmin  l e v e l s would be u s e f u l i n t h e subsequent n e u t r a l i z a t i o n o f l i p i d p e r o x i d a t i o n p r o d u c t s r e l e a s e d i n t o the serum upon t i s s u e damage (Bonta, 1978). The (LEM)  observed s t i m u l a t o r y e f f e c t of l e u k o c y t e  endogenous m e d i a t o r  on h e p a t o c y t e s has been proposed t o r e s u l t i n i n c r e a s e d  ceruloplasmin  synthesis during  the inflammatory response ( F r i e d e n , 1981)  (see above).  T h i s p r o t e i n , which i s r e l e a s e d by l e u k o c y t e s ,  stimulates  the uptake o f i r o n , z i n c , and amino a c i d s by l i v e r c e l l s and a l s o enhances the s y n t h e s i s and r e l e a s e o f acute phase r e a c t a n t s ceruloplasmin. not  The mechanism u n d e r l y i n g  including  the s t i m u l a t o r y e f f e c t s o f LEM i s  understood. Ceruloplasmin  expression  during  the acute phase response has r e c e n t l y  been s t u d i e d by a n a l y s i s o f r a t l i v e r samples f o l l o w i n g i n d u c t i o n of inflammation by i n j e c t i o n of the animals w i t h  turpentine  (Aldred e t a l . ,  15  1987). 350%  Ceruloplasmin  mRNA l e v e l s i n c r e a s e d t o a peak c o r r e s p o n d i n g t o  o f t h e normal v a l u e by 36 hours.  c e r u l o p l a s m i n has decreased  By 60 hours  t o normal l e v e l s .  post-inflammation,  As has been t h e case f o r  o t h e r a c u t e phase r e a c t a n t s s t u d i e d ( t r a n s f e r r i n , a ^ - m a c r o g l o b u l i n ,  6  c h a i n o f f i b r i n o g e n , a ^ - a c i d g l y c o p r o t e i n , and m e t a l l o t h i o n e i n - I ) ( S c h r e i b e r e t a l . , 1986), t h e r e g u l a t i o n o f c e r u l o p l a s m i n s y n t h e s i s d u r i n g the inflammatory  response appears t o o c c u r a t t h e mRNA l e v e l ,  affecting  t h e r a t e o f t r a n s c r i p t i o n and/or mRNA s t a b i l i t y as opposed t o t h e r a t e o f protein  C.  translation.  ABNORMALITIES IN COPPER HOMEOSTASIS - WILSON'S DISEASE In t h i s autosomal r e c e s s i v e d i s o r d e r ( a l s o r e f e r r e d t o as  h e p a t o l e n t i c u l a r degeneration),  pathogenesis  i s r e l a t e d t o abnormal copper  d e p o s i t i o n i n body t i s s u e s , e s p e c i a l l y the b r a i n and l i v e r 1948). disease.  Ceruloplasmin  (Cumings,  l e v e l s a r e c h a r a c t e r i s t i c a l l y decreased  T h i s was i n i t i a l l y demonstrated by Scheinberg  i n Wilson's  and G i t l i n  (1952)  who i s o l a t e d c e r u l o p l a s m i n from normals and W i l s o n ' s d i s e a s e p a t i e n t s and q u a n t i f i e d i t s l e v e l s both  immunochemically, and by m o n i t o r i n g t h e  decrease  i n absorbance (610 nm) o f c e r u l o p l a s m i n f o l l o w i n g r e d u c t i o n .  observed  c o r r e l a t i o n between decreased  ceruloplasmin  d i s e a s e has s i n c e been i n t e r p r e t e d t o suggest  l e v e l s and W i l s o n ' s  a defect i n the rate of  c e r u l o p l a s m i n b i o s y n t h e s i s ( P o u l i k and Weiss, 1975).  Additionally,  have been r e p o r t s t h a t the d i s e a s e i s a s s o c i a t e d w i t h s t r u c t u r a l of t h e c e r u l o p l a s m i n p r o t e i n .  The  there  anomalies  I n t h i s r e g a r d , V e r b i n a and Puchkova (1985)  have r e p o r t e d t h e i s o l a t i o n o f c e r u l o p l a s m i n from a W i l s o n ' s p a t i e n t t h a t d i f f e r s from normal c e r u l o p l a s m i n  disease  i n physicochemical,  16  immunological, and c a t a l y t i c p r o p e r t i e s . anomalous c e r u l o p l a s m i n  They have p o s t u l a t e d  that  this  may be the r e s u l t o f i n c o r r e c t p o s t - t r a n s l a t i o n a l  m o d i f i c a t i o n o f the p r o t e i n . Gaitskhoki  e t al^. (1975) r e p o r t e d  comparative immunochemical a n a l y s i s  125 (using  [  I]-ceruloplasmin  antibodies)  polysomes i n l i v e r b i o p s i e s o b t a i n e d disease p a t i e n t s .  from c o n t r o l s u b j e c t s  and W i l s o n ' s  T h i s study c l e a r l y demonstrated t h a t the amount o f  ceruloplasmin-forming was  of ceruloplasmin-synthesizing  polysomes i n p a t i e n t s a f f e c t e d w i t h W i l s o n ' s  10 - 20 times lower than t h a t determined f o r normal c o n t r o l s .  disease On t h i s  b a s i s , i t was proposed t h a t a decreased l e v e l o f t r a n s l a t a b l e mRNA i s t h e l i k e l y cause o f the g e n e t i c b l o c k  i n ceruloplasmin  c h a r a c t e r i s t i c of Wilson's disease. the r e c e n t  findings of Czaja  ceruloplasmin  that i s  This hypothesis i s consistent  e t a l . (1987).  with  The l a t t e r study showed t h a t  mRNA l e v e l s i n f i v e W i l s o n ' s d i s e a s e p a t i e n t s were d e c r e a s e d  to 33% t h a t o f c o n t r o l mRNA l e v e l s . (also synthesized  i n the  In contrast,  l i v e r ) were e l e v a t e d  the former group was not  l e v e l s o f albumin mRNA  t o 161%  p a t i e n t s compared w i t h normal l e v e l s , s u g g e s t i n g  nuclear  synthesis  i n Wilson's  disease  that l i v e r function i n  compromised due t o the d i s e a s e  state.  Using  run-on assays t o a n a l y z e t r a n s c r i p t i o n a l r a t e s , i t was found  the amount o f c e r u l o p l a s m i n  gene t r a n s c r i p t i o n i n the W i l s o n ' s  p a t i e n t s was decreased t o 44% t h a t o f c o n t r o l l e v e l s .  decreased c e r u l o p l a s m i n rate of protein  disease  T h i s has been  i n t e r p r e t e d t o i n d i c a t e t h a t i n a t l e a s t some cases o f W i l s o n ' s observed r e d u c t i o n o f plasma c e r u l o p l a s m i n  disease,  i n l e v e l s may be due t o  gene t r a n s c r i p t i o n , as opposed t o a d e f e c t  synthesis.  that  i n the  17  The  c a u s a l r e l a t i o n s h i p between c e r u l o p l a s m i n and W i l s o n ' s d i s e a s e  has become c o m p l i c a t e d  by the r e c e n t s t u d i e s o f Frydman e t a l . (1985),  showing l i n k a g e between the gene f o r W i l s o n ' s d i s e a s e and the e s t e r a s e D l o c u s on chromosome 13.  These d a t a have been f u r t h e r s u b s t a n t i a t e d by t h e  more r e c e n t assignment o f the W i l s o n ' s d i s e a s e l o c u s t o chromosome 13q (Yuzbasiyan-Gurkan e t a l . , 1987).  S i n c e the w i l d - t y p e c e r u l o p l a s m i n gene  has been u n e q u i v o c a l l y mapped t o chromosome 3 u s i n g b o t h somatic i n s i t u h y b r i d i z a t i o n techniques it  seems l i k e l y t h a t reduced  (Yang e t a l . , 1986;  c e l l and  R o y l e e t al_., 1987),  ceruloplasmin-specific transcription  observed  i n W i l s o n ' s d i s e a s e p a t i e n t s s t u d i e d by C z a j a e t a l . (1987) i s not t h e r e s u l t o f d e f e c t ( s ) w i t h i n the s t r u c t u r a l gene, but may i n v o l v e a t r a n s - a c t i n g f a c t o r ( s ) mapping t o chromsome 13.  D.  CHARACTERIZATION OF RAT CERULOPLASMIN G a i t s k h o k i e t a l . (1980) have p r e v i o u s l y r e p o r t e d the  of h i g h l y - p u r i f i e d r a t c e r u l o p l a s m i n mRNA by i n d i r e c t of c e r u l o p l a s m i n - s y n t h e s i z i n g r a t l i v e r polysomes. then t r a n s l a t e d i n a h e t e r o l o g o u s  gel electrophoresis. product  was 84 Kda.  immunoprecipitation  C e r u l o p l a s m i n mRNA was  wheat germ system; p r o d u c t s  t r a n s l a t i o n were immunoprecipitated  The r e s u l t s i n d i c a t e d t h a t the primary A d d i t i o n o f r a t l i v e r membranes t o the  f o l l o w i n g immunoprecipitation.  of c e l l - f r e e  and s u b j e c t e d t o SDS-polyacrylamide  system r e s u l t e d i n the v i s u a l i z a t i o n o f two p o l y p e p t i d e s Kda)  isolation  translation translation  (80 Kda and 65  The 65 Kda product was i d e n t i c a l t o  the s e c r e t e d form o f c e r u l o p l a s m i n i s o l a t e d from the G o l g i complex f o l l o w i n g i n v i v o p u l s e - l a b e l l i n g and s u b c e l l u l a r f r a c t i o n a t i o n  (Neifakh  e t a l . , 1979).  step,  On t h i s b a s i s i t was proposed t h a t a m a t u r a t i o n  18  r e s u l t i n g i n t h e appearance o f t h e 130 Kda plasma form o f c e r u l o p l a s m i n , i n v o l v e d l i g a t i o n o f two 65 Kda p o l y p e p t i d e s support  (Puchkova e t al.,  1981).  In  o f t h i s t h e o r y , P r o z o r o v s k i e t a l . (1982) have emphasized t h e  e x i s t e n c e o f sequence homology w i t h i n each h a l f of human h o l o c e r u l o p l a s m i n 125 based on s t r u c t u r a l s t u d i e s u s i n g  [  I ] - p e p t i d e mapping.  However  T a k a h a s h i e t a l . (1984) have c o n c l u s i v e l y demonstrated a t r i p l i c a t e d s t r u c t u r e f o r t h e p r o t e i n (see S e c t i o n I . B . I ) .  More r e c e n t l y , i t has been  shown t h a t a s i n g l e mRNA s p e c i e s o f approximately  3.8 Kb c o r r e s p o n d s t o  the c e r u l o p l a s m i n t r a n s c r i p t from r a t l i v e r , which i s more than s u f f i c i e n t i n s i z e t o encode t h e e n t i r e p r o t e i n ( A l d r e d e t a l . , 1987). A l d r e d e t a l . (1987) have a l s o r e p o r t e d t h e i s o l a t i o n o f a r a t c e r u l o p l a s m i n cDNA c l o n e .  U s i n g a p a r t i a l human c e r u l o p l a s m i n cDNA c l o n e  to s c r e e n a r a t l i v e r cDNA l i b r a r y , a c l o n e was i s o l a t e d t h a t  contained  DNA c o d i n g f o r t h e e q u i v a l e n t o f r e s i d u e s 194 - 879 o f t h e human c e r u l o p l a s m i n amino a c i d sequence (Takahashi  e t a l . , 1984).  The p r e d i c t e d  amino a c i d sequence d e r i v e d from t h e r a t cDNA shows c l o s e sequence identity  ( n e a r l y 75%) w i t h t h e determined amino a c i d sequence o f human  c e r u l o p l a s m i n from amino a c i d r e s i d u e s 194 - 276 ( i . e . w i t h i n t h e A l domain).  There i s s t r i k i n g  (approximately  i d e n t i t y i n the carboxy-terminal compared  98%) amino a c i d  sequence  r e g i o n o f t h i s l a t t e r sequence when  w i t h t h e human c e r u l o p l a s m i n p r o t e i n sequence, showing complete  c o n s e r v a t i o n from r e s i d u e s 227 - 276 w i t h the e x c e p t i o n o f one c o n s e r v a t i v e amino a c i d change o c c u r r i n g a t r e s i d u e 243. r a t c e r u l o p l a s m i n amino a c i d sequence a l s o c l o s e l y matches  The p r e d i c t e d t h a t determined  f o r human c e r u l o p l a s m i n beteen amino a c i d s 810 - 879 ( c o r r e s p o n d i n g A3 domain), showing 83% sequence i d e n t i t y  i n this region.  t o the  19  E.  THE  RELATIONSHIP OF CERULOPLASMIN TO  OTHER COPPER-CONTAINING  PROTEINS The e t a l . , 1984)  amino a c i d sequence of human c e r u l o p l a s m i n was  analyzed  (Takahashi  f o r segments s i m i l a r t o known type I  copper-binding s i t e s i n small blue e l e c t r o n t r a n s f e r p r o t e i n s and  p l a s t o c y a n i n ) , and  (e.g.  azurin  i n some l a r g e m u l t i c o p p e r o x i d a s e s whose  c r y s t a l l o g r a p h i c s t r u c t u r e s and/or amino a c i d sequences have been determined. The  three-dimensional  s t r u c t u r e of a z u r i n (a b a c t e r i a l  electron o  t r a n s p o r t p r o t e i n w i t h Mr  = 14 Kda)  r e s o l u t i o n e l e c t r o n d e n s i t y map e t a l . , 1978).  has  his-117 r e s i d u e s .  the s i n g l e type I copper i o n  coordinated  n e a r - i n f r a r e d absorption,  CD,  Like azurin, plastocyanin  blue  Cu  photosynthetic  to cys-112, met-121, h i s - 4 6  and MCD  been proposed based studies  and  on  (Solomon e t a l . , 1976).  (Mr = 10,500) a l s o b e l o n g s to the group  e l e c t r o n t r a n s f e r p r o t e i n s and  (II) ion.  present  This i s consistent with a d i s t o r t e d tetrahedral  geometry f o r t h a t type I s i t e t h a t has  of s m a l l b l u e  A  u s i n g X-ray d i f f r a c t i o n methods (Adman  Based on t h e s e d a t a ,  i n a z u r i n i s thought to be  been determined from a 3.0  P l a s t o c y a n i n has  p o s s e s s e s a s i n g l e type I  been i d e n t i f i e d as a component i n the  c h a i n of a number of green p l a n t s and  algae.  I t s X-ray  o  c r y s t a l s t r u c t u r e a t 2.7 (1978). I Cu  As  A r e s o l u t i o n has  been r e p o r t e d  by Colman e t a l .  i s the case f o r a z u r i n , the c o o r d i n a t i o n geometry of the  (II) i s consistent with a d i s t o r t e d tetrahedron,  c y s t e i n e t h i o l group ( c y s - 8 4 ) ,  l i g a n d e d by  a  a methionine t h i o e t h e r group (met-92)  histidine  imidazole  groups (his-37  I centres  proposed f o r a z u r i n and  and  his-87).  plastocyanin,  When compared to the the type I copper  type  and type  present  20  i n s t e l l a c y a n i n (a s m a l l b l u e p r o t e i n w i t h proposed e l e c t r o n t r a n s f e r f u n c t i o n ) d i f f e r s w i t h r e s p e c t to a t l e a s t one  coordination position.  T h i s i s based on the r e p o r t e d  amino a c i d sequence of s t e l l a c y a n i n (Bergman  e t a l . , 1977)  of methionine r e s i d u e s .  which i s d e v o i d  observed d i f f e r e n c e s i n the type I c e n t r e s p l a s t o c y a n i n as determined by EPR  T h i s may  explain  i n s t e l l a c y a n i n and  a n a l y s i s ( P e i s a c h e t a l . , 1967)  and  a  comparison of redox p o t e n t i a l s . On  the b a s i s of homology w i t h the i d e n t i f i e d l i g a n d s to the s i n g l e  type I copper i n a z u r i n and p l a s t o c y a n i n  (see above), i t has  suggested t h a t a t y p e I copper c e n t r e i s p r e s e n t 19 Kda  p r o t e o l y t i c fragment of c e r u l o p l a s m i n  F i g u r e s 2A and  2B).  been  i n the carboxy  (Kingston  terminal  e t a l . , 1979;  see  Three of the proposed l i g a n d s are the c l u s t e r e d  cys-1021, his-1026, and met-1031 r e s i d u e s ; the f o u r t h type I l i g a n d suggested by K i n g s t o n  e t a l . (1979) i s h i s - 9 5 6 , which i s 65 r e s i d u e s  t e r m i n a l t o the cys-1021 r e s i d u e .  The  amino  predicted cysteine, h i s t i d i n e  m e t h i o n i n e l i g a n d s are i n agreement w i t h the t h r e e d i m e n s i o n a l the copper a c t i v e s i t e i n human c e r u l o p l a s m i n  presented  and  model of  by Ryden (1982).  However, the l a t t e r study i n d i c a t e s t h a t the f o u r t h l i g a n d i s h i s - 9 7 5 which i s more l i k e l y , s i n c e t h i s p o s i t i o n corresponds to the l o c a t i o n of type I h i s t i d i n e l i g a n d s i n b o t h p l a s t o c y a n i n and I t has  been p o s t u l a t e d  may  l o c a t e d i n the 50 Kda  be  a z u r i n (see F i g u r e  f u r t h e r t h a t a second type I copper b i n d i n g p r o t e o l y t i c fragment (Dwulet and  2A).  site  Putnam,  1981b), where the l i g a n d s cys-680, his-685 and met-690 are l o c a t e d i n homologous p o s i t i o n s to the proposed type I s i t e i n the 19 Kda The  f o u r t h r e s i d u e comprising  to be his-637 (Dwulet and  fragment.  the type I s i t e i n t h i s fragment i s proposed  Putnam, 1981b), which a l s o corresponds to  the  21  F i g u r e 2.  The  crassa laccase plastocyanin  r e l a t i o n s h i p between human c e r u l o p l a s m i n ( L a c ) , Pseudomonas a e r u g i n o s a  a z u r i n (Azn),  poplar  ( P I ) , and b o v i n e s u p e r o x i d e dismutase (B.SOD) i n two  ( F i g u r e s 2A and  2B)  c o n t a i n i n g proposed copper l i g a n d s  and Putnam, 1981b; Germann and residues  (Cp), Neurospora  Lerch,  1986).  (taken from Dwulet  I d e n t i c a l amino a c i d  are boxed, and p o t e n t i a l l i g a n d s to the t h r e e t y p e s of copper  c e n t e r s are i n d i c a t e d by *1, *2  and  *3  respectively.  known type I l i g a n d s i n a z u r i n and p l a s t o c y a n i n H i s t i d i n e ligands implicated i n copper-binding dismutase are c i r c l e d .  The  ( i . e . Met-169) i s e n c l o s e d  Arrows  in  i n bovine superoxide  f o u r t h p o t e n t i a l type I l i g a n d i n l a c c a s e i n a diamond.  F o r human c e r u l o p l a s m i n ,  brackets.  identify  (see t e x t f o r d e t a i l s ) .  Numbers on the l e f t of each  sequence i d e n t i f y p o s i t i o n s w i t h i n the p r o t e i n s of the f i r s t given.  regions  the c o r r e s p o n d i n g  residues  homology u n i t i s g i v e n  2A.  Cp 276 ( A l ) Cp 637 (A2) Cp 975 (A3) Lac 99 B.SOD 39 Azn 46 PI 37  *1 H *1 H *1 H *1 H  A  A  F  F  N  G  •Q  A  L  G  I  F  Y  F  S  F  P  •I  D  F  G *1 H *1 H  D  H  G  F  ®  N *3 H *3 H *3  T  V  S *3 H *3 H *3  G  T  Y *3 H *3 H  V  Q  F  N  W  V  L  s  ®  T  A  A  D  N  I  V  P  G  E  W  M  L  S  T  L  G G  2B.  Cp 312 ( A l ) Cp 673 (A2) Cp 1014 (A3)  E P  G G  T I  —  -  F U  N L  V L A  Lac 164 Azn 104 PI 77  P E  G G K G  S E E  —  W  Q  Y  _  Y  L N  <3> F  E *3 H *3 H F  *1 C *1 C  *1 Q  N  L  L  *2  C  H  T  F  Y  H  r  D  H  T  G  *1  *2 H  G  D  H  A  W  H  *1 *2  C  G  T  *1  V  G  *1 P  *1 S  K  H  *1  *1  C *1 C  N  G  H  L  *1 P  H  T  G  L *1 M *1 M L *1 M *1 M  t  23  p o s i t i o n o f type I h i s t i d i n e l i g a n d s i d e n t i f i e d i n a z u r i n and p l a s t o c y a n i n (see F i g u r e 2A). A l t h o u g h t h e 67 Kda fragment, r e p r e s e n t i n g t h e amino t e r m i n a l  portion  of c e r u l o p l a s m i n has c y s t e i n e and h i s t i d i n e r e s i d u e s i n i d e n t i c a l p o s i t i o n s t o t h o s e p r e s e n t i n t h e 19 and 50 Kda fragments, t h e r e i s no c o r r e s p o n d i n g m e t h i o n i n e r e s i d u e (see F i g u r e 2B), which may p r e c l u d e t h e b i n d i n g o f a type I copper i o n i n t h i s r e g i o n (Takahashi e t a l . ,  1983).  However, t h e type I copper c e n t e r s i n c e r u l o p l a s m i n have been d i f f e r e n t i a t e d i n t o two subtypes, based on t h e k i n e t i c s o f r e o x i d a t i o n (Herve e t a l . ,  1978).  I n t h i s r e g a r d , i t has been shown more r e c e n t l y  t h a t t h e type I " f a s t " copper c e n t e r does n o t r e q u i r e m e t h i o n i n e as a c o o r d i n a t i o n l i g a n d , w h i l e t h e type I "slow" c e n t e r does 1981).  (Herve e t a l . ,  Thus, t h e 67 Kda p r o t e o l y t i c fragment a l s o p o s s e s s e s a p u t a t i v e  type I copper b i n d i n g s i t e which may resemble t h e b l u e copper c e n t e r i n s t e l l a c y a n i n which a l s o l a c k s methionine as a l i g a n d  ( s e e above).  A second r e g i o n w i t h i n the 19 Kda p r o t e o l y t i c fragment o f c e r u l o p l a s m i n i s homologous t o a known non-blue copper b i n d i n g s i t e i n b o v i n e and human s u p e r o x i d e dismutase et a l . ,  1980) and human cytochrome  (Richardson e t a l . ,  1975; Jabusch  c oxidase ( B a r r e l l e t a l . ,  of which c o n t a i n copper i n b i n u c l e a r c e n t r e s .  1979), a l l  The X-ray c r y s t a l  structure  o  of b o v i n e s u p e r o x i d e dismutase has been determined a t 3 A r e s o l u t i o n .  The  o  two  copper i o n s on o p p o s i t e s u b u n i t s w i t h i n the dimer a r e 34 A a p a r t .  The  o  copper and z i n c i n each s u b u n i t a r e a p p r o x i m a t e l y 6 A a p a r t , and they both form l i g a n d s t o t h e i m i d a z o l e r i n g o f h i s - 6 1 .  The p r o t e i n l i g a n d s t o the  copper a r e proposed t o be h i s - 4 4 , h i s - 4 6 , h i s - 6 1 and h i s - 1 1 8 ( i . e . h i s - X - h i s m o t i f ) , arranged i n a s l i g h t l y d i s t o r t e d square p l a n e and  24  thereby has  resembling  a type I I c e n t r e .  The 19 Kda fragment o f c e r u l o p l a s m i n  a h i s t i d i n e - r i c h sequence element (see F i g u r e 2A) t h a t i s homologous  to t h e non-blue copper c e n t r e d e s c r i b e d i n bovine dismutase ( O r t e l e t a l . , 1984).  and human  superoxide  Based on a t h r e e - d i m e n s i o n a l  model o f the  copper a c t i v e s i t e o f t h e human c e r u l o p l a s m i n 19 Kda p r o t e o l y t i c  fragment  (Ryden, 1982), i t has been proposed t h a t these h i s t i d i n e r e s i d u e s ( i . e . h i s - 9 8 0 and h i s - 9 8 2 , and  i n a d d i t i o n t o h i s - 9 7 8 and his-1020) (see F i g u r e s 2A  2B) may f u n c t i o n as type I I I l i g a n d s i n c e r u l o p l a s m i n .  The l a t t e r  model i d e n t i f i e s human c e r u l o p l a s m i n r e s i d u e s his-1022 and his-1028 as p o t e n t i a l type I I l i g a n d s ( s e e F i g u r e 2B). A c o r r e s p o n d i n g c l u s t e r resembling i s absent  t h a t i d e n t i f i e d i n t h e 19 Kda fragment o f c e r u l o p l a s m i n  from t h e 50 Kda p r o t e o l y t i c p e p t i d e  F i g u r e 2A).  histidine-rich  ( O r t e l e t a l . , 1984)  As a g e n e r a l o b s e r v a t i o n , t h e r e i s an u n u s u a l l y  (see  l a r g e number  of h i s - X - h i s sequences i n c e r u l o p l a s m i n , some o f which may be i m p l i c a t e d i n non-blue copper b i n d i n g . Fungal  l a c c a s e (Mr = 6 2  Kda) i s a b l u e oxidase  copper ions and a unique c y s t e i n e r e s i d u e .  containing four  I n i t i a l l y , peptides  containing  the s i n g l e s u l f h y d r y l group were i s o l a t e d and c h a r a c t e r i z e d ( B r i v i n g e t a l . , 1980). provided  A d d i t i o n a l amino a c i d sequence i n f o r m a t i o n has been  from p a r t i a l n u c l e o t i d e sequence a n a l y s i s of t h e l a c c a s e gene  (Germann and L e r c h , 1986).  Comparison o f the a v a i l a b l e amino a c i d  sequence f o r l a c c a s e w i t h t h a t of human c e r u l o p l a s m i n has r e v e a l e d the presence of s e v e r a l highly-conserved multicopper  oxidases  sequence elements i n these two  (see F i g u r e s 2A and 2B).  Due t o i t s conserved  p o s i t i o n w i t h proposed type I c y s t e i n e l i g a n d s i n c e r u l o p l a s m i n , a z u r i n , and p l a s t o c y a n i n (see F i g u r e 2B), the unique c y s t e i n e r e s i d u e i n l a c c a s e  25  i s thought t o c o o r d i n a t e type I copper.  A d d i t i o n a l l y , laccase contains  two h i s t i d i n e r e s i d u e s i n s i m i l a r p o s i t i o n s t o the proposed histidine  l i g a n d s i n the above c o p p e r - c o n t a i n i n g p r o t e i n s  and 2B).  However, the methionine  type I  (see F i g u r e s 2A  l i g a n d which i s conserved i n the  p r o t e i n s i s absent i n l a c c a s e , as i s the case f o r s t e l l a c y a n i n (Germann and L e r c h , 1986).  I t has been proposed  t h a t met-169 i n l a c c a s e (see F i g u r e 2B) may coordination.  latter  (see above)  (Germann and L e r c h ,  1986)  be i n v o l v e d i n type I copper  I n a d d i t i o n t o s i m i l a r i t y observed w i t h r e s p e c t t o type I  l i g a n d s , h i s t i d i n e r e s i d u e s have been i d e n t i f i e d i n l a c c a s e t h a t o c c u r i n i d e n t i c a l p o s i t i o n s t o proposed non-blue (see F i g u r e s 2A and Based  copper l i g a n d s i n c e r u l o p l a s m i n  2B).  on e a r l i e r s t u d i e s ( R i c h a r d s o n e t a l . ,  1975;  Ryden, 1982), i t  has been suggested t h a t the conserved sequence elements  i n ceruloplasmin  and l a c c a s e may  c o o r d i n a t e the b i n d i n g of type I I and/or type I I I copper,  i n a d d i t i o n t o t h e i r proposed (Briving et a l . , may  1980;  involvement i n type I copper c e n t r e s  Germann and L e r c h , 1986).  Thus, t h e s e  sequences  form a l i n k between type I , I I and I I I copper c e n t r e s p r e s e n t i n  multicopper oxidases. Taken t o g e t h e r , the above d a t a suggest t h a t the type I copper b i n d i n g site  i s s i m i l a r i n b o t h the s m a l l b l u e e l e c t r o n t r a n s f e r p r o t e i n s as w e l l  as l a r g e m u l t i c o p p e r o x i d a s e s (e.g. l a c c a s e and c e r u l o p l a s m i n ) .  I t also  seems t h a t i n a d d i t i o n t o the presence of a p u t a t i v e type I copper c e n t r e , the 19 Kda  fragment  of c e r u l o p l a s m i n i s s t r u c t u r a l l y r e l a t e d to  copper b i n d i n g s i t e s i d e n t i f i e d  i n o t h e r copper o x i d a s e s .  non-blue  It is likely,  t h e r e f o r e , t h a t these p r o t e i n s share a common e v o l u t i o n a r y o r i g i n ,  perhaps  26  d e r i v i n g from a p r i m o r d i a l gene encoding a s m a l l b l u e p r o t e i n  possessing  e i t h e r e l e c t r o n t r a n s f e r or o x i d a s e f u n c t i o n . There are s e v e r a l s i t e s w i t h i n the c e r u l o p l a s m i n  molecule that  c h a r a c t e r i z e d by the p o s i t i o n i n g of h i s t i d i n e s a d j a c e n t acid residues.  These s i t e s might be  are  t o b a s i c amino  i n v o l v e d i n copper b i n d i n g  as i s the  case f o r serum albumin, o r the plasma t r i p e p t i d e g l y - h i s - l y s ( P i c k a r t e t a l . , 1980). the b i n d i n g  The  f i r s t two  residues  of the l a t t e r m o t i f  are  of copper, w h i l e the s i d e c h a i n of the l y s y l r e s i d u e  proposed t o be n e c e s s a r y f o r the r e c o g n i t i o n by c e l l s u r f a c e T h i s i s analogous t o plasma albumin or a - f e t o p r o t e i n , h i s t i d i n e residue  (Aoyagi e t a l . , 1980).  of a h i s t i d i n e r e s i d u e next to a b a s i c r e s i d u e may a c t i v e s t r u c t u r e f o r copper uptake. ceruloplasmin  may  i n copper t r a n s p o r t  THE  the  to e i t h e r  Thus, the p o s i t i o n i n g be a b i o l o g i c a l l y  p r e s e n c e of such sequences i n  correspond to observed s i t e s of r e v e r s i b l e copper  (McKee and  F.  The  is  receptors.  i n which  t h a t b i n d s copper i s immediately a d j a c e n t  l y s i n e or a r g i n i n e r e s i d u e s  binding  involved i n  Frieden,  1971)  which may  (see S e c t i o n  I.B.3.4).  i n t u r n p l a y an e s s e n t i a l r o l e  RELATIONSHIP BETWEEN CERULOPLASMIN AND  PROTEINS INVOLVED IN  BLOOD COAGULATION An  i n t e r e s t i n g s t r u c t u r a l r e l a t i o n s h i p has  between c e r u l o p l a s m i n l a t t e r two  proteins  s t r u c t u r a l and 1984).  the b l o o d  (both w i t h Mr  coagulation  > 300  functional similarity  Both p r o t e i n s  (Jackson and  and  f u n c t i o n i n the  Nemerson, 1980)  Kda)  (Suzuki  f a c t o r s V and  VIII.  These  share a h i g h degree of e t a l . , 1982;  i n t r i n s i c blood  in conjunction  been shown to e x i s t  Nesheim e t a l . ,  clotting  cascade  w i t h an a c t i v a t e d , v i t a m i n - K  27  dependent c l o t t i n g f a c t o r respectively).  ( f a c t o r s IXa and Xa f o r f a c t o r s V I I I and V,  Both complexes r e q u i r e a p h o s p h o l i p i d s u r f a c e and c a l c i u m  i o n s , and subsequently  r e s u l t i n t h e s p e c i f i c a c t i v a t i o n o f a second  v i t a m i n K-dependent c o a g u l a t i o n p r o t e i n ( f a c t o r X and prothrombin f o r f a c t o r s V I I I and V, r e s p e c t i v e l y ) .  A n a l y s i s o f t h e complete amino a c i d  sequences o f human f a c t o r V (Jenny e t a l . , 1987) and human f a c t o r V I I I (Wood e t a l . , 1984; T o o l e e t a l . , 1984) p r e d i c t e d from t h e c o r r e s p o n d i n g cDNAs r e v e a l e d t h e e x i s t e n c e o f 3 types o f domains w i t h i n t h e two proteins:  a triplicated  "A" domain (approximately  320 - 380 amino a c i d  r e s i d u e s ) , a unique "B" domain (925 and 886 amino a c i d r e s i d u e s i n f a c t o r s V I I I and V, r e s p e c t i v e l y ) , and a d u p l i c a t e d "C" domain c o n s i s t i n g o f approximately  100 - 150 amino a c i d r e s i d u e s (see F i g u r e 3 ) .  Organization  of these u n i t s from amino-to c a r b o x y l t e r m i n a l w i t h i n t h e p r o t e i n s a r e as follows:  A l - A2 - B - A3 - C l - C2.  The "A" domains o f f a c t o r s V and  V I I I show a h i g h l e v e l o f s i m i l a r i t y w i t h t h e t r i p l i c a t e d u n i t s i n t h e human c e r u l o p l a s m i n molecule,  s h a r i n g approximately  i d e n t i t y when compared p a i r w i s e (see T a b l e I ) .  30 - 40% sequence  Of p a r t i c u l a r note i s t h e  c l u s t e r i n g of c y s t e i n e residues at s i m i l a r p o s i t i o n s w i t h i n the triplicated  "A" domains o f f a c t o r V I I I  ceruloplasmin  (Takahashi  (Vehar e t ad., 1984) and  e t a l . , 1983), i n d i c a t i n g a h i g h degree of  s t r u c t u r a l c o n s e r v a t i o n between these repeated u n i t s . domain p r e s e n t  The d u p l i c a t e d "C"  i n f a c t o r s V and V I I I i s u n r e l a t e d t o c e r u l o p l a s m i n , b u t  shows approximately  20% sequence i d e n t i t y when compared w i t h  which a r e p h o s p h o l i p i d - b i n d i n g (Poole e t a l . , 1981).  l e c t i n s from D i c t y o s t e l i u m  discoidins,  discoideum  The "B" domains i n f a c t o r s V and V I I I a r e each  28  F i g u r e 3.  Comparison o f t h e s t r u c t u r a l o r g a n i z a t i o n of c e r u l o p l a s m i n ,  f a c t o r V and f a c t o r V I I I . The t r i p l i c a t e d  A domain ( d e s i g n a t e d A l , A2 and A3 i n an amino-to  carboxyl d i r e c t i o n ) i s i d e n t i f i e d bars.  i n t h e 3 m o l e c u l e s by  cross-hatched  The B domain ( p r e s e n t i n f a c t o r s V and V I I I o n l y ) i s r e p r e s e n t e d  an open b a r .  The d u p l i c a t e d C domain ( a l s o p r e s e n t o n l y i n f a c t o r s V and  V I I I ) i s shown by s t i p p l e d b a r s . acid residues.  by  S i z e s o f t h e domains c o r r e s p o n d  t o amino  Comparison of the structures of factor VIII, factor V and ceruloplasmin  Factor VIII KWWWWWM:---  350  1000  350  amino acids  350 150  Ceruloplasmin  150  l\\\\\\\\\\l\\\\\\\\\\\K\\\\VW^  350  350  350  Factor V K \ \ \ \ \ \ \ \ \ \ l - - : ; l ;;;•;;••] :  350  350  1000  350 150  150  Table I.  Comparison  of "A" domains i n f a c t o r V, f a c t o r V I I I  and c e r u l o p l a s m i n (from Jenny et a l . , 1987).  Matches/Length  (%)  V-A2  V-A3  VIII-A1  VIII-A2  VIII-A3  Cer-Al  Cer-A2  Cer-A3  V-Al  31  32  36  30  27  31  32  21  V-A2  -  29  29  44  31  35  34  35  V-A3  -  -  33  31  39  32  34  39  VIII-A1  -  -  -  30  32  34  32  33  -  -  -  33  38  35  36  VIII-A3  -  -  -  -  -  28  34  36  Cer-Al  -  -  -  -  -  -  37  39  -  -  -  -  -  -  40  Domain  VIII-A2  Cer-A2  -  V a l u e s (expressed as percentages) represent t o t a l I d e n t i c a l amino a c i d matches d i v i d e d by o v e r l a p p i n g l e n g t h s ( i n c l u d i n g gaps).  31  s t r u c t u r a l l y unique when compared w i t h the r e s t of the molecule,  and  in  b o t h cases are removed upon a c t i v a t i o n of the p r o t e i n s . The seemingly  f u n c t i o n a l r e l a t i o n s h i p between c e r u l o p l a s m i n and these unrelated blood c l o t t i n g f a c t o r s i s unclear.  two  However, i t has  been proposed p r e v i o u s l y t h a t f a c t o r V i s a m e t a l l o p r o t e i n ( G r e e n q u i s t Colman, 1975) of a c t i v i t y atomic  r e q u i r i n g c a l c i u m and a v a r i e t y of metal  (Esmon, 1979;  Hibbard  e m i s s i o n and atomic  and Mann, 1980).  ions f o r expression  Recently, using  absorption spectroscopy,  f a c t o r V has  shown t o c o n t a i n copper i n the r a t i o of 1 copper i o n p e r mol (Mann e t a l . , 1984).  and  both  been  of f a c t o r V  S i n c e f a c t o r V e x h i b i t s an a b s o r p t i o n spectrum w i t h  no maximum a t e i t h e r 310 nm  (type I I I copper) o r 610 nm  (type I c o p p e r ) ,  the copper b i n d i n g i s assumed to be the e q u i v a l e n t of the type I I copper center present i n ceruloplasmin.  Although  a s s e s s e d w i t h r e s p e c t to i t s m e t a l - b i n d i n g  f a c t o r V I I I has not y e t been c o n t e n t , the p e p t i d e c h a i n s  n o n - c o v a l e n t l y a s s o c i a t e d i n a p r o c e s s t h a t i s EDTA s e n s i t i v e e t a l . , 1982).  (Fass  Thus, the a s s o c i a t i o n of the p e p t i d e c h a i n s i n the  V I I I m o l e c u l e may  be dependent upon bound metal  are  factor  and/or c a l c i u m i o n s .  The  f o u r amino a c i d s proposed as l i g a n d s f o r type I copper b i n d i n g i n the c e r u l o p l a s m i n molecule  (see S e c t i o n I-E)  are l o c a t e d i n analogous  p o s i t i o n s i n the A l and A3 u n i t s of f a c t o r V I I I (Vehar  e t a l . , 1984).  C o n s e r v a t i o n of these copper l i g a n d r e s i d u e s may  imply s i m i l a r  b i n d i n g c h a r a c t e r i s t i c s f o r b o t h f a c t o r V I I I and  ceruloplasmin.  In terms of p h y s i o l o g i c a l s i g n i f i c a n c e , the demonstrated r e l a t i o n s h i p between f a c t o r s V and V I I I and p o s s i b l e involvement  metal  structural  c e r u l o p l a s m i n suggests  of copper and/or o t h e r metal  c a l c i u m , i n the f u n c t i o n of f a c t o r s V and V I I I .  the  i o n s , i n a d d i t i o n to The p o s s i b l e r o l e of  32  metal b i n d i n g i n t h i s p r o c e s s  i s i m p l i e d by s t u d i e s showing the b i n d i n g  c a p a c i t y of y - c a r b o x y g l u t a m i c  a c i d residues f o r lanthanide ions  ( S p e r l i n g e t a l . , 1978).  These m o d i f i e d g l u t a m i c a c i d r e s i d u e s ( p r e s e n t  i n b l o o d - c l o t t i n g f a c t o r s I I , V I I , IX, X, p r o t e i n Z, p r o t e i n S and p r o t e i n C) a r e thought  t o be i n v o l v e d i n c a l c i u m b i n d i n g , which i s i n t u r n  proposed to mediate the i n t e r a c t i o n o f these p r o t e i n s w i t h surfaces i n vivo.  platelet  I t i s a l s o c o n c e i v a b l e t h a t p o t e n t i a l metal  ligands  p r e s e n t i n f a c t o r V and p o s s i b l y f a c t o r V I I I can i n t e r a c t w i t h a d i f f e r e n t metal  i o n j o i n t l y bound by the y - c a r b o x y g l u t a m i c  a c i d r e g i o n s of  factors  IX and X, t h e r e b y promoting complex f o r m a t i o n . C e r u l o p l a s m i n has a number of enzymatic  f u n c t i o n s a s c r i b e d to i t i n  a d d i t i o n to i t s r o l e i n copper t r a n s p o r t and homeostasis (see S e c t i o n I.B.3).  I t i s unknown a t t h i s time whether any of t h e s e  catalytic  a c t i v i t i e s a r e a l s o a s s o c i a t e d w i t h f a c t o r s V and V I I I .  G.  CHARACTERIZATION OF THE  G.l  H i s t o r i c a l Perspective  HUMAN FACTOR V I I I GENE  In the m a j o r i t y of c a s e s , the b l e e d i n g d i s o r d e r h e m o p h i l i a A (or c l a s s i c hemophilia)  r e s u l t s from mutations  ( e i t h e r s i n g l e base changes o r  g r o s s rearrangements) w i t h i n the s t r u c t u r a l gene c o d i n g f o r f a c t o r V I I I . S i n c e the f a c t o r V I I I gene i s l o c a t e d on the X chromosome, t h e r e i s a h i g h frequency  of h e m o p h i l i a A r e l a t i v e to autosomal c l o t t i n g d i s o r d e r s  (Haldane,  1935).  hemophilia  H i s t o r i c a l l y , s t u d i e s a d d r e s s i n g the n a t u r e  of  A have been s e r i o u s l y hampered by d i f f i c u l t i e s encountered  in  the p u r i f i c a t i o n of f a c t o r V I I I s i n c e i t i s an u n u s u a l l y l a r g e (Mr =  330  Kda), u n s t a b l e p r o t e i n , p r e s e n t i n low c o n c e n t r a t i o n s i n plasma (100  -  33  200 ng/ml) (Wood e t a l . , 1984).  S t u d i e s i n v o l v i n g the c h a r a c t e r i z a t i o n of  the f a c t o r V I I I gene were i n i t i a t e d to f a c i l i t a t e an u n d e r s t a n d i n g molecular  b a s i s of h e m o p h i l i a  A.  In a d d i t i o n , e x p r e s s i o n of the  of  cloned  gene i n v i t r o would p r o v i d e a v i r u s - f r e e p r e p a r a t i o n of recombinant V I I I f o r treatment G.2  factor  hemophiliacs.  O r g a n i z a t i o n of the F a c t o r V I I I Gene The  was  of  the  complete o r g a n i z a t i o n of the 186  r e p o r t e d by G i t s c h i e r e t a l . (1984).  Kbp  human f a c t o r V I I I gene  Initially,  factor VIII  clones  were i s o l a t e d from a genomic l i b r a r y c o n s t r u c t e d u s i n g a lymphoblast l i n e which was 49, XXXY).  d e r i v e d from an i n d i v i d u a l w i t h 4X chromosomes  T h i s l i b r a r y was  screened  cell  (Karyotype  u s i n g a unique 36-base s y n t h e t i c  o l i g o n u c l e o t i d e probe c o n s t r u c t e d on the b a s i s of a p r e v i o u s l y characterized factor VIII t r y p t i c peptide chromosome w a l k i n g ( n e a r l y 0.1%)  to extend  the i n i t i a l l y  of the X chromosome was  (Vehar e t a l . , 1984).  Using  i d e n t i f i e d c l o n e s , 200  Kbp  c h a r a c t e r i z e d t h a t encompassed  the  complete f a c t o r V I I I gene. DNA  sequence a n a l y s i s of i n t r o n / e x o n boundaries  of 26 exons i n the f a c t o r V I I I gene.  Although  consistent with reported d i s t r i b u t i o n s the exons are u n u s u a l l y corresponds  to the 100  large. Kda  100  Kda  l a r g e s t exon i s 3106  carboxy-terminal  T h i s exon corresponds  most exon s i z e s were  (Naora and Deacon, 1982), two  connecting peptide  F i g u r e 3) j o i n i n g amino- and respectively.  The  r e v e a l e d the p r e s e n c e  which correspond  The  i n length,  ( i . e . the B domain; fragments of 90-  and  see  and  80  Kda,  to a p h y s i o l o g i c a l u n i t , s i n c e the  p e p t i d e i s p r o t e o l y t i c a l l y e x c i s e d upon thrombin  ( F u l c h e r e t a l . , 1983).  bp  of  o t h e r l a r g e exon i s 1958  bp  activation l o n g , 1805  to the 3' u n t r a n s l a t e d r e g i o n of the gene.  Intron  bp  of  sizes  34  i n the f a c t o r V I I I gene were found t o be h i g h l y v a r i a b l e (Naora and Deacon, 1982) w i t h the l a r g e s t i n t r o n spanning 32.4 Kbp.  O v e r a l l , the  f a c t o r V I I I gene c o n s i s t s o f 9 Kbp o f exon sequence i n t e r r u p t e d by 177 Kbp of i n t e r v e n i n g sequence, s u g g e s t i n g  a lack of s e l e c t i v e pressure to  decrease i n t r o n s i z e . RNAse p r o t e c t i o n experiments u s i n g mRNA d e r i v e d from e i t h e r the AL-7 T c e l l hybridoma l i n e o r human l i v e r i n d i c a t e t h a t t h e t r a n s c r i p t i o n i n i t i a t i o n s i t e i n t h e f a c t o r V I I I gene i s p o s i t i o n e d a t -170 o r -172 respectively residue).  (+1 denotes the p o s i t i o n o f t h e i n i t a t i o r m e t h i o n i n e  A t 30 bp 5' t o t h e p r e d i c t e d mRNA s t a r t s i t e i s l o c a t e d t h e  sequence "GATAAA", which c l o s e l y resembles the Goldberg-Hogness consensus sequence ( i . e . the "TATA" b o x ) , proposed t o be r e q u i r e d f o r p r e c i s e i n i t i a t i o n o f t r a n s c r i p t i o n by e u k a r y o t i c RNA polymerase I I (Goldberg, 1979; B r e a t h n a c h and Chambon, 1981).  No "CAT"  sequence element  (Breathnach and Chambon, 1981) was observed upstream t o the "ATA" i n the f a c t o r VIII Following s i g n a l peptide  sequence  gene.  the 5* u n t r a n s l a t e d r e g i o n i s a t y p i c a l 19 amino a c i d (von H e i j n e ,  1982) c o n t a i n i n g two charged r e s i d u e s f l a n k i n g  a c o r e o f h y d r o p h o b i c amino a c i d s .  This s e c r e t o r y s i g n a l precedes the  mature p r o t e i n sequence o f 2332 amino a c i d r e s i d u e s , which i s f o l l o w e d by a "TGA" stop codon and a subsequent 3' u n t r a n s l a t e d r e g i o n o f 1802 bp. The conserved p o l y a d e n y l a t i o n s i g n a l "AATAAA" (Proudfoot 1976)  i s contained  p o s i t i o n of poly  and Brownlee,  i n the l a t t e r sequence, o c c u r r i n g 19 bp p r i o r t o t h e  (A) a d d i t i o n .  35  G.3  E v o l u t i o n a r y A s p e c t s of I n t r o n P o s i t i o n s w i t h i n t h e F a c t o r  VIII  Gene As d i s c u s s e d p r e v i o u s l y (see S e c t i o n I . F ) , the f a c t o r V I I I p r o t e i n i s composed of t h r e e d i f f e r e n t domains; the o r d e r of the domains i n t h e p r o t e i n i s A l - A l - B - A3 - C l - C2 (see F i g u r e s 3 and 4 ) . tandem gene d u p l i c a t i o n events (see S e c t i o n I . I . I ) have o c c u r r e d  If  i n the  e v o l u t i o n o f t h e f a c t o r V I I I gene, as i s s t r o n g l y suggested by the repeated  domain o r g a n i z a t i o n , c o n s e r v a t i o n of i n t r o n b o u n d a r i e s w i t h i n the  A and C r e p e a t s would be p r e d i c t e d ( D o o l i t t l e , 1985). d u p l i c a t i o n , intron/exon  F o r the C  boundaries occur p r e c i s e l y at the borders  of the  C1/C2 r e p e a t u n i t s , as would be expected i f a gene d u p l i c a t i o n event has occurred  (see F i g u r e 4 ) .  Again,  t h e r e i s an i n t r o n a t t h e boundary o f the  A3 and C l u n i t s (see F i g u r e 4) thus a l s o s u p p o r t i n g intron joining.  However, t h e A1/A2  a mechanism i n v o l v i n g  and A2/A3 j u n c t i o n s a r e each  contained  on one exon (see F i g u r e 4 ) . Within  the A and C repeated  are c o n s e r v e d , s u g g e s t i n g  u n i t s , o n l y some o f the i n t r o n b o u n d a r i e s  t h a t these  i n t r o n s were p r e s e n t  gene p r i o r t o d u p l i c a t i o n (see F i g u r e 4 ) . w i t h i n each o f the r e p e a t s  i n the a n c e s t r a l  The d i f f e r i n g number of exons  i s r e f l e c t i v e of e i t h e r i n t r o n l o s s o r i n t r o n  i n s e r t i o n f o l l o w i n g the i n i t i a l d u p l i c a t i o n events. The o r i g i n of the unique B domain i s h i g h l y s p e c u l a t i v e . i s contained  region  almost e n t i r e l y w i t h i n a 3106 bp exon as d e s c r i b e d  p r e v i o u s l y , where the end of the A2 r e p e a t repeat  This  a r e a l s o found.  and the b e g i n n i n g  Due t o i t s anomalous s i z e ,  of the A3  i t has been p o s t u l a t e d  t h a t the B domain may have a r i s e n by i n s e r t i o n of a p r o c e s s e d  gene  (mRNA-derived; see S e c t i o n I . J ) i n t o a s h o r t exon c o n t a i n i n g the A2/A3  36  F i g u r e 4.  L o c a t i o n of i n t r o n s  and d u p l i c a t e d  ( v e r t i c a l lines) within  C domains of human f a c t o r V I I I  the t r i p l i c a t e d A  (from G i t s c h i e r e t a l . ,  1984). For first  the A and C r e p e a t e d u n i t s , numbers i d e n t i f y the p o s i t i o n o f the  amino a c i d r e s i d u e i n each l i n e .  domain i s i n d i c a t e d ; numbered  The l o c a t i o n and e x t e n t of the B  numbers r e p r e s e n t amino a c i d r e s i d u e s .  consecutively.  The exons a r e  37  —I  1 330  8  1649  14  2020 '2173  i 20 i  i  i !  2  1  9  I  i 21 24  1  i  1  25  i  3 LJQ  15  22  100 Amino Acids  1  I  i  93  i  26  1  I 5 J  4  1 16  11  1 l  17.  12  i i I  iB  7  13 18  i 14 u_19  •B(712-1649) i  38  boundary.  Although the exon c o n t a i n i n g  the B domain corresponds to a  f u n c t i o n a l u n i t , which i s e x c i s e d upon thrombin a c t i v a t i o n of f a c t o r V I I I , correspondence of o t h e r exons to f u n c t i o n a l u n i t s of the p r o t e i n i s unclear  at t h i s  time.  H.  THE  DYNAMICS OF  PROTEIN AND  H.l  The  Molecular Clock  I n i t i a l studies  GENE EVOLUTION  i n v o l v i n g the comparison of p r o t e i n sequences  from s p e c i e s whose times of e v o l u t i o n a r y  d i v e r g e n c e are e s t a b l i s h e d  has  a l l o w e d an e s t i m a t e of the r a t e a t which mutations have been accumulating i n genomes.  On  t h i s b a s i s , i t was  suggested t h a t random m u t a t i o n of  o c c u r s a t a n e a r l y c o n s t a n t r a t e , which i s i n t u r n m a n i f e s t e d as c o n s t a n t r a t e of amino a c i d s u b s t i t u t i o n (Zuckerkandl and W i l s o n e t a l . , 1977). molecular clocks. construction  Thus, p r o t e i n sequences can  This empirical  of p h y l o g e n e t i c  trees  a l l o w i n g major c o n t r i b u t i o n s  (as can  events),  i t s evolutionary  L i e_t a l . , 1985),  evolutionary f u n c t i o n of a p r o t e i n i s  l i k e l y change due  to the d i f f e r e n t s e t  Even i f a p r o t e i n m a i n t a i n s a c o n s t a n t still  be  subject  the organism's c e l l u l a r  Observed d i f f e r e n c e s  i n the  l i k e l y depend upon d i f f e r e n c e s protein function  (Wilson e t §1^., 1977;  However, i f the  rate w i l l  r a t e of e v o l u t i o n may  variation within  approximate  occur w i t h independent p r o d u c t s of gene d u p l i c a t i o n  of s e l e c t i v e p r e s s u r e s . the  1965;  been u s e f u l i n the  to our knowledge of  r e l a t i o n s h i p s among organisms. modified  f i n d i n g has  a  Pauling,  s e r v e as  DNA  (Afinsen,  1959;  function,  to change i n response to  environment.  evolutionary  r a t e s of d i f f e r e n t p r o t e i n s  i n p r o b a b i l i t y t h a t mutations w i l l Ohno, 1970).  T h i s concept of  retain  "functional  39  c o n s t r a i n t " a l s o i l l u s t r a t e s why t h e r a t e o f e v o l u t i o n  i s variable at  different sites within  upon which areas  a p a r t i c u l a r p r o t e i n , depending  can t o l e r a t e v a r i a t i o n w i t h o u t r e s u l t i n g i n l o s s o f f u n c t i o n e t a l . , 1977). within  (Wilson  Thus, t h e i d e n t i f i c a t i o n o f h i g h l y conserved r e g i o n s  a p r o t e i n a l l o w s t h e d e t e c t i o n o f p o t e n t i a l f u n c t i o n a l domains  w i t h i n a molecule.  I.  MECHANISMS OF GENE EVOLUTION  1.1  Gene D u p l i c a t i o n The p r o c e s s o f gene d u p l i c a t i o n has been used t o e x p l a i n t h e  occurrence of multigene f a m i l i e s e i t h e r having s i m i l a r f u n c t i o n s , allowing  thereby  them t o a c t s y n e r g i s t i c a l l y (e.g. t h e g l o b i n gene f a m i l y ;  e t a l . , 1983) o r d i f f e r i n g  i n function  Edgell  (e.g. t h e l y s o z y m e - l a c t a l b u m i n  f a m i l y ; H a l l e t a l . 1982). I n c r e a s e d p r o t e i n s i z e compared w i t h a n c e s t r a l forms can be the r e s u l t o f i n t e r n a l gene d u p l i c a t i o n ( D o o l i t t l e , 1985; L i , 1983). e v e n t s , t h e e n t i r e a n c e s t r a l m o l e c u l e can be d u p l i c a t e d  I n such  o r t r i p l i c a t e d as  i s t h e case f o r t r a n s f e r r i n ( M a c G i l l i v r a y e t a l . , 1982) o r c e r u l o p l a s m i n (Takahashi e t a l . , 1983), r e s p e c t i v e l y . proteins  can be d u p l i c a t e d  ( D o o l i t t l e , 1985).  A l t e r n a t i v e l y , portions of  t o generate l a r g e r , more complex forms  I n t e r n a l gene d u p l i c a t i o n s a r e r e f l e c t e d by homologous  amino a c i d sequences and/or s i m i l a r t h r e e - d i m e n s i o n a l s t r u c t u r e s between duplicated 1.2  r e g i o n s (McLachlan,  1979).  Gene F u s i o n By t h e p r o c e s s of gene f u s i o n , new p r o t e i n s  j o i n i n g o f p r o t e i n domains d e r i v e d  are created  from d i f f e r e n t sources  by the  (Doolittle,  40  1985).  Coupled w i t h gene d u p l i c a t i o n s , gene f u s i o n events have been  proposed i n the f o r m a t i o n factors  (Doolittle,  1.3  coagulation  1985).  Exon S h u f f l i n g I t has  be  of complex m o l e c u l e s , such as the  been proposed t h a t exons are s t r u c t u r a l m o t i f s which  a s s o r t e d by r e c o m b i n a t i o n w i t h i n i n t e r v e n i n g sequences to y i e l d  proteins with d i f f e r e n t functions 1985).  ( G i l b e r t , 1978;  can  novel  B l a k e , 1983a,b; Rogers,  T h i s phenomenon of modular u n i t s h u f f l i n g i s thought t o have been  a dominant f o r c e i n the e v o l u t i o n of the s e r i n e p r o t e a s e gene (Rogers, 1985;  Neurath, 1985).  plasminogen a c t i v a t o r p r o v i d e s otherwise unrelated  1.4  superfamily  F o r example, the gene f o r t i s s u e the f i r s t example of exon t r a n s f e r between  genes (Ny e t a l . , 1984).  I n t r o n I n s e r t i o n and  Intron  Intron  been commonly observed between r e l a t e d  i n s e r t i o n has  Sliding  genes, such as w i t h i n the p r o t e a s e domain of the t r y p s i n gene f a m i l y (Rogers, 1985).  Some r e g u l a r i t y w i t h r e s p e c t  has  For example, i t seems t h a t new  been shown.  to the i n s e r t i o n of  i n s e r t i o n s tend to o c c u r  near the m i d d l e of p r e - e x i s t i n g exons, thus a l l o w i n g consistency  with respect  G i l b e r t , 1985).  to exon s i z e (Naora and  Secondly, i t has  e v o l u t i o n toward a  Deacon, 1982;  been suggested t h a t s m a l l  l e n g t h v a r i a t i o n s between members of p r o t e i n f a m i l i e s can be s u b s t i t u t i o n of a l t e r n a t i v e i n t r o n s p l i c e s i t e s , extension  thereby  or c o n t r a c t i o n of exons at i n t r o n j u n c t i o n s  1982a,b; C r a i k et a l . , 1983).  Resulting  amino a c i d r e s i d u e s )  to the s u r f a c e  o f t e n map  small  introns  Lonberg  and  observed the r e s u l t of  permitting  ( C r a i k et a l . ,  i n s e r t i o n s ( u s u a l l y 2 to of m o l e c u l e s , where  17  41  r e s u l t a n t s t r u c t u r a l m o d i f i c a t i o n s a r e t h e l e a s t d i s r u p t i v e t o the o v e r a l l t e r t i a r y structure (Craik et a l . , process of intron/exon s l i d i n g ,  1982a,b; C r a i k e t a l . ,  1983).  In t h i s  i t i s expected t h a t o n l y those v a r i a t i o n s  which r e s u l t i n m a i n t a i n a n c e o f t h e t r a n s l a t i o n a l r e a d i n g frame can be tolerated.  The mechanism  o f i n t r o n s l i d i n g has been p o s t u l a t e d t o e x p l a i n  s m a l l v a r i a t i o n s i n t h e l e n g t h o f r e l a t e d gene p r o d u c t s i n b o t h t h e d i h y d r o f o l a t e r e d u c t a s e (DHFR) and s e r i n e p r o t e a s e gene f a m i l i e s et a l . ,  (Craik  1983).  J.  PSEUDOGENES H i g h e r e u k a r y o t i c genes ( i n c l u d i n g human c e r u l o p l a s m i n ) commonly  e x i s t i n m u l t i g e n e f a m i l i e s t h a t c o n t a i n b o t h f u n c t i o n a l genes, as w e l l as c l o s e l y r e l a t e d sequences t h a t have l o s t t h e a b i l i t y t o produce a f u n c t i o n a l p r o d u c t due t o m u t a t i o n a l changes. been termed "pseudogenes" ( J a c q e t a l . , Pseudogenes f a l l  i n t o two c a t e g o r i e s :  These l a t t e r sequences have  1977; P r o u d f o o t , 1980). 1) non-processed pseudogenes and,  2) p r o c e s s e d pseudogenes; t h e c h a r a c t e r i s t i c s o f each c a t e g o r y a r e summarized 1.  below. Non-Processed Pseudosenes  T h i s group (mainly composed  o f t h e g l o b i n pseudogenes from a v a r i e t y  of s p e c i e s ; V a n i n , 1983) i n c l u d e s those psuedogenes t h a t have r e t a i n e d the i n t e r v e n i n g sequences found i n t h e i r f u n c t i o n a l c o u n t e r p a r t s . m a j o r i t y o f cases, t h e chromosomal  In the  l o c a t i o n o f these pseudogenes i s  a d j a c e n t t o the r e s p e c t i v e w i l d - t y p e gene, s u g g e s t i n g t h a t the pseudogene sequences have a r i s e n from gene d u p l i c a t i o n events (Vanin, 1983).  42  2.  Processed  Pseudogenes  T h i s second, more abundant c a t e g o r y o f pseudogenes  i s represented i n  many d i f f e r e n t gene f a m i l i e s from a number o f mammalian s p e c i e s (see V a n i n , 1985 f o r a r e c e n t r e v i e w ) .  Most p r o c e s s e d pseudogenes c o n t a i n  g e n e t i c l e s i o n s t h a t p r e c l u d e t h e p r o d u c t i o n o f a f u n c t i o n a l gene product.  These l e s i o n s i n c l u d e :  1) t h e presence  o f in-frame t e r m i n a t i o n  codons as a r e s u l t o f s i n g l e base s u b s t i t u t i o n s and 2) t h e i n s e r t i o n o r d e l e t i o n o f n o n - i n t e g r a l n u c l e o t i d e t r i p l e t s t h a t cause f r a m e s h i f t mutations,  thereby r e s u l t i n g i n premature t e r m i n a t i o n o f t r a n s l a t i o n .  There a r e f o u r examples, however, o f p r o c e s s e d pseudogenes d e l e t e r i o u s mutations:  t h e human m e t a l l o t h i o n e i n I I pseudogene  and Gedamu, 1984), t h e r a t RC9 cytochrome c pseudogene the mouse L32 r i b o s o m a l p r o t e i n pseudogene 1984), and t h e DHFRtyl pseudogene  rpL32-4A  (Chen e t a l . ,  I n a d d i t i o n t o v a r i o u s g e n e t i c mutations, possess  t h a t c o n t a i n no (Varshney  ( S c a r p u l l a , 1984),  (Dudov and P e r r y ,  1982). processed  pseudogenes  a number o f c h a r a c t e r i s t i c f e a t u r e s , t h e most d i s t i n g u i s h i n g o f  which i s t h e l o s s o f i n t r o n s r e l a t i v e t o t h e i r f u n c t i o n a l c o u n t e r p a r t s . The absence o f i n t r o n s i s u s u a l l y p r e c i s e - i . e . sequences 5' and 3' t o the i n t r o n s a r e c o n t i g u o u s . by t h e observed  P r o c e s s e d pseudogenes a r e a l s o c h a r a c t e r i z e d  d i v e r g e n c e o f sequence homology w i t h the w i l d - t y p e  c o u n t e r p a r t s a t p o i n t s c o r r e s p o n d i n g t o t h e b e g i n n i n g and end o f t h e f u n c t i o n a l genes.  N o t a b l e e x c e p t i o n s t o t h i s i n c l u d e the human  immunoglobulin e (Ueda e t a l . ,  1982) and Xtyl pseudogenes  (Hollis  e t a l . , 1982) and t h e mouse c o r t i c o t r o p i n B - l i p o t r o p i n p r e c u r s o r pseudogene  (Notake e t a l . ,  1983), which appear t o be DNA c o p i e s o f o n l y a  p o r t i o n of the w i l d - t y p e mRNA t r a n s c r i p t s , as w e l l as the mouse i\><x3  43  pseudogenes which c o n t a i n a d d i t i o n a l sequences compared t o t h e f u n c t i o n a l transcripts  (Vanin e t a l . ,  1980).  Many p r o c e s s e d pseudogenes have a  p o l y ( A ) t r a c t l o c a t e d immediately 3* t o t h e p o i n t a t which homology between t h e pseudogene and w i l d - t y p e gene ceases, and a r e o f t e n c h a r a c t e r i z e d by s h o r t (7 - 17 bp) d i r e c t r e p e a t s f l a n k i n g t h e pseudogene sequence.  L a s t l y , almost w i t h o u t e x c e p t i o n , p r o c e s s e d pseudogenes do n o t  share t h e same chromosomal l o c a t i o n as t h e c o r r e s p o n d i n g f u n c t i o n a l genes (Battey e t a l . ,  1982; Czosnek e t a l . ,  1984).  A number o f mechanisms were o r i g i n a l l y proposed f o r t h e o r i g i n o f p r o c e s s e d pseudogenes (Vanin, 1985).  However, based on t h e  c h a r a c t e r i s t i c s summarized above f o r a number o f compiled p r o c e s s e d pseudogene sequences, i t i s now g e n e r a l l y a c c e p t e d t h a t p r o c e s s e d pseudogenes have a r i s e n from r e v e r s e t r a n s c r i p t i o n o f mature mRNA species.  I n t h i s model, cDNA c o p i e s o f c o r r e s p o n d i n g mRNAs a r e then  randomly i n t e g r a t e d i n t o t h e genome, as has been p o s t u l a t e d f o r the d i s p e r s i o n o f t h e human snRNA pseudogenes (Denison e t a l . , Arsdell et al.,  1981) and t h e human A l u l f a m i l y o f r e p e t i t i v e sequences  (Jagadeeswaran e t a l . ,  K.  1982; Van  1981).  THE PRESENT STUDY The a n a l y s i s o f t h e s t r u c t u r e of a number o f r e p r e s e n t a t i v e s of  d i f f e r e n t gene f a m i l i e s has l e d t o an enhanced u n d e r s t a n d i n g o f the n a t u r e of p r o t e i n and gene e v o l u t i o n  [e.g. t h e g l o b i n gene f a m i l y  1983), t h e s e r i n e p r o t e a s e supergene f a m i l y immunoglobulin supergene f a m i l y  (Edgell et a l . ,  (Rogers, 1985), and the  (Hood e t a l . ,  1985)].  Based on t h e i r  shared s t r u c t u r a l s i m i l a r i t i e s as demonstrated by amino a c i d sequence  4A  determination  ( s e e S e c t i o n I . F ) , i t has been proposed t h a t c e r u l o p l a s m i n  and b l o o d c l o t t i n g f a c t o r s V and V I I I c o n s t i t u t e a gene f a m i l y 1985).  I n 1984, t h e p r e s e n t i n v e s t i g a t i o n was i n i t i a t e d  i n order to  c h a r a c t e r i z e t h e human c e r u l o p l a s m i n cDNA and gene, t h e r e b y comparison o f t h e gene o r g a n i z a t i o n o f t h i s m u l t i c o p p e r  (Doolittle,  facilitating a  oxidase to that  r e p o r t e d f o r human f a c t o r V I I I ( G i t s c h i e r e t a l . , 1984; see S e c t i o n I . G ) . S i n c e 1984, s e v e r a l p a r t i a l human c e r u l o p l a s m i n cDNA c l o n e s have been r e p o r t e d by o t h e r groups (Mercer and Grimes, 1986; Yang e t a l . , 1986). D e t a i l s c o n c e r n i n g t h e r e l e v a n c e o f t h e s e l a t t e r s t u d i e s w i l l be addressed i n t h e c o n t e x t o f subsequent s e c t i o n s .  45  II.  MATERIALS AND A.  METHODS  BACTERIAL HOSTS AND  MEDIA  The media used f o r b o t h growth of a p p r o p r i a t e b a c t e r i a l h o s t s and s c r e e n i n g of X phage c l o n e s was NZ  NZYC ( M a n i a t i s e t aJL.,  1982)  (10 g  amine type A, 2 g MgCl^, 5 g NaCI, 5 g Y e a s t E x t r a c t , 1 g Casamino  Acids per l i t r e ,  a d j u s t e d t o pH 7.5  were p l a t e d on NZYC-agar (1.5% w/v) NZYC-agarose (0.7% w/v). transformed w i t h pUC  by NaOH a d d i t i o n ) .  Phage l i b r a r i e s  p l a t e s , w i t h an o v e r l a y o f  The media used f o r the growth o f b a c t e r i a  p l a s m i d s was  L u r i a b r o t h (LB) ( M a n i a t i s e t a l . ,  (5 g Y e a s t E x t r a c t , 10 g B a c t o t r y p t o n e and 10 g NaCI p e r l i t r e ) . s e l e c t i o n o f b a c t e r i a c o n t a i n i n g pUC agar (1.5% w/v) medium was  yg/ml a m p i c i l l i n .  LB LB  a l s o used f o r s c r e e n i n g the human l i v e r cDNA l i b r a r y  c o n s t r u c t e d i n the pKT218 v e c t o r , except t h a t t e t r a c y c l i n e r e p l a c e d a m p i c i l l i n as the a n t i b i o t i c .  8 g B a c t o t r y p t o n e , 5 g NaCI p e r l i t r e ) ; (1.5% w/v)  (12.5  yg/ml)  B a c t e r i a c o n t a i n i n g phage  c l o n e s were grown i n YT medium ( M a n i a t i s e t a l . ,  YT agar  F o r the  p l a s m i d s , c l o n e s were p l a t e d on  p l a t e s supplemented w i t h 50 - 100  1982)  M13  1982)  (5 g Y e a s t  M13 Extract,  t r a n s f o r m a n t s were p l a t e d  p l a t e s o v e r l a y e d w i t h YT c o n t a i n i n g 0.75%  (w/v)  on  agar.  E. c o l i s t r a i n s JM101  and JM103 were m a i n t a i n e d on minimal media p l a t e s ,  p r e p a r e d as f o l l o w s :  3 g of agar i n a t o t a l of 160 ml dH^O  a u t o c l a v e d , c o o l e d t o 55°C, and mixed w i t h 40 ml o f 5X s a l t s K HP0 , 0.9 2 4  g KH P O .  A  2  4  0.2  g (NH, ) SO,,, 4  2  4  0.1  MgSO^, • 7H^0,  and 0.1 ml 10 mg/ml t h i a m i n e .  [2.1 g  g Na  C i t r a t e « 7 H 0 per 40 m l ] , 2 ml 20% g l u c o s e , 0.2 2  was  ml  20%  Bacteria for large-scale  p l a s m i d p r e p a r a t i o n s were grown e i t h e r i n LB medium, supplemented w i t h the a p p r o p r i a t e a n t i b i o t i c , o r i n M9 minimal media ( M a n i a t i s e t a l . ,  1982)  Table I I .  Summary of the genotypes of b a c t e r i a l h o s t s used i n t h e p r e s e n t  Vectors u t i l i z e d  Bacterial Strain  a)  b)  E_. c o l i MC1061  E. c o l i K802  i n c o n j u n c t i o n w i t h each host a r e a l s o  Genotype  Reference  _E. c o l i LE392  given.  Compatible V e c t o r System  araD139, A ( a r a , leu)7697 Alacx74, g a l l T , galK~ h s r ~ , hsm , s t r A  Casadaban and Cohen, 1980  human l i v e r cDNA l i b r a r y i n KT218 (Prochownik et a l . 1983)  hsdR , hsdM*, g a l ~ , met , SupE  M a n i a t i s _et _ a l . , 1982  human genomic l i b r a r y i n Charon 4A (Lawn et a l . , 1978)  CF~, hsdR514(i£nt£) supE44, supF58, l a c Y l , A ( l a c I Z Y ) 6 , galK2, galT22, metBl, trpR55, lambda  M a n i a t i s e t a l . , 1982  human genomic l i b r a r y i n EMBL 3 ( F r i s c h a u f et a l . , 1983)  P2 l y s o g e n of LE392  M a n i a t i s e t a l . , 1982  human genomic l i b r a r y i n EMBL 3 ( F r i s c h a u f et a l . , 1983)  +  -  c)  study.  P  -  d)  _E. c o l i P2 392  Bacterial  e)  Strain  E. c o l i RY1088  Genotype  Reference  A l a c U169, supE, supF, hsdR , hsdM*, met B, trpR, tonA21, proC::Tn5 (pmc9)  Young and D a v i s , 1983a,b  human l i v e r cDNA l i b r a r y in Xgtll. (Young and D a v i s , 1983a,b)  F~, t h i - 1 , t h r - 1 , leuB6, l a c Y l , tonA21, supE44, X~, Hfl+  A p p l e y a r d , 1954 Winnacker, 1987  human l i v e r cDNA l i b r a r y i n XgtlO  -  f)  _E. c o l i C600 H f l  +  Compatible V e c t o r System  g)  E. c o l i JM101  A l a c p r o , supE, t h i ~ , F', traD36, proAB, l a c I Q , lacZAM15  Messing, 1983  pUC v e c t o r s ( V i e i r a and M e s s i n g , 1982) M13 v e c t o r s (Messing, 1983)  h)  E. c o l i JM103  A l a c p r o , supE, t h i , s t r A , sbcB15, endA, hsdR", F', traD36, proAB, l a c I Q , lacZAM15  Messing, 1983  pUC v e c t o r s ( V i e i r a and M e s s i n g , 1982) M13 v e c t o r s (Messing, 1983)  -  48  c o n t a i n i n g 840 ml dH 0, 100 ml 10X s a l t s 2  K H  2 °4' P  0  -  5  s  N a C l  »  1  6  N  H  C 4  1  P  e r  1  0  0  m l  >»  20 ml 20% g l u c o s e , 10 ml 0.01 M C a C l 10 mg/ml thiamine  2 >  (7 g Na^PO^, 3 g  10'ml M g S 0 « 7 H 0 , 4  2  20 ml 20% Casamino A c i d s , 0.2 ml  and 0.2 g u r i d i n e .  A summary o f t h e genotypes o f v a r i o u s b a c t e r i a l s t r a i n s used i n t h i s study i s g i v e n i n T a b l e I I .  B.  HYBRIDIZATION PROBES  B.l  P u r i f i c a t i o n and L a b e l l i n g o f O l i g o d e o x y r i b o n u c l e o t i d e s Oligodeoxyribonucleotide mixtures  Atkinson  were s y n t h e s i z e d by Tom  i n t h e l a b o r a t o r y o f Dr. M. Smith, U n i v e r s i t y o f B r i t i s h  Columbia, u s i n g an A p p l i e d Biosystems 380 A DNA s y n t h e s i z e r .  Crude  o l i g o n u c l e o t i d e p r e p a r a t i o n s were p u r i f i e d through 20% d e n a t u r i n g ( c o n t a i n i n g 8.3 M urea) and i s o l a t e d by reverse-phase  gels  chromatography u s i n g  a C^g SEP-PAK ( M i l l i p o r e ) column as d e s c r i b e d by A t k i n s o n and Smith 32 (1984).  The o l i g o n u c l e o t i d e s were l a b e l l e d u s i n g  polynucleotide kinase unincorporated  [ y - P]-ATP and T4  (Chaconas and van de Sande, 1980), and  ATP was subsequently  removed by chromatography on G25  Sephadex. Three p o o l s o f h e p t a d e c a d e o x y r i b o n u c l e o t i d e s  were used as  h y b r i d i z a t i o n probes f o r t h e s c r e e n i n g o f human l i v e r cDNA l i b r a r i e s : POOL I :  5'd(TARTARTGYTTYTCYTT)3'  POOL I I :  5'd(ATNGCRTGCATYTTRTT)3 *  POOL I I I :  5'd(CCCATNARRTACCARTT)3'  where "R" r e p r e s e n t s both G and A, "Y" r e p r e s e n t s T and C, and "N" r e p r e s e n t s G,A,T and C.  The t h r e e n u c l e o t i d e p o o l s a r e complementary t o  49  the mRNA encoding amino a c i d r e s i d u e s 1 - 6 ,  937-942, and 962-967 o f  c e r u l o p l a s m i n , r e s p e c t i v e l y , as p r e d i c t e d from the amino a c i d (Takahashi e t a l . , B.2  1984).  Nick T r a n s l a t i o n P u r i f i e d DNA  32  sequence  fragments o r e n t i r e p l a s m i d s were l a b e l e d w i t h  P by n i c k - t r a n s l a t i o n as d e s c r i b e d by M a n i a t i s e t a l . (1975).  Approximately 200 - 500 ng o f DNA m i x t u r e , c o n t a i n i n g 50 mM 10 mM  was  l a b e l l e d i n 50 y l o f r e a c t i o n  T r i s - H C l pH 7.5,  5 mM  MgCl  2 >  0.05  B-mercaptoethanol, 20 yM dGTP, 20 yM dTTP, 1.4 mM  dCTP, 1.4  yCi/yl  [a P]-dCTP 32  [<x P]-dATP (3000 Ci/mMole), 32  (3000 Ci/mMole),  and 0.4 u / y l E. c o l i DNA (Boehringer-Mannheim). a t 15°C, and was 1% SDS/10 mM a t 68°C.  CaCl  2 >  dATP, 1.4  yCi/yl  1 p g / y l DNAse I  polymerase I (Romberg The above r e a c t i o n was  mM  (Sigma)  fragment) i n c u b a t e d f o r 60 - 120  s u b s e q u e n t l y t e r m i n a t e d by the a d d i t i o n of 3 volumes  min of  EDTA c o n t a i n i n g 25 yg tRNA, f o l l o w e d by h e a t i n g f o r 10 min  F r e e t r i p h o s p h a t e s were s e p a r a t e d from the l a b e l l e d s t r a n d s by  chromatography 1982).  0.2 mM  1.4  mg/ml BSA,  on 1.0 ml spun columns  of Sephadex G-50  (Maniatis et a l . ,  S p e c i f i c a c t i v i t i e s of r e s u l t a n t probes ranged from 0.5  - 1.0  x  8 10  cpm/yg. B.3  Klenow L a b e l l i n g DNA  was  V o g e l s t e i n (1983). DNA  a l s o l a b e l l e d u s i n g the method d e s c r i b e d by F e i n b e r g and R e a c t i o n m i x t u r e s t y p i c a l l y c o n t a i n e d 50 - 200 ng of  ( e i t h e r p u r i f i e d r e s t r i c t i o n fragments o r e n t i r e p l a s m i d s ) i n a t o t a l  volume of 50 y l . boiling  The DNA  sample  ( i n 30 y l of dH^O)  was  denatured by  f o r 3 minutes f o l l o w e d by r a p i d c o o l i n g on i c e f o r 5 minutes.  l a b e l l i n g r e a c t i o n was  s u b s e q u e n t l y c a r r i e d out i n 50 y l t o t a l volume,  The  50  c o n t a i n i n g 50 mM T r i s - H C l pH 8.0, 10 mM M g C l  lOmM B-mercaptoethanol,  2 >  32 20 yM dCTP, 20 yM dGTP, 20 yM dTTP, 1 y C i / y l (3000 Ci/mMole),  [a  P]-dATP  200 mM Hepes pH 6.6, 60 OD 260 nm/ml random  hexadeoxyribonucleotides  [p(dN6)] (P-L B i o c h e m i c a l s ) , 0.4 mg/ml BSA and  0.1 u / y l E. c o l i DNA polymerase I (Klenow fragment) L a b o r a t o r i e s (BRL) o r P-L B i o c h e m i c a l s ] . c o n t i n u e a t 37°C f o r e i t h e r 3 - 4  [Bethesda Research  E x t e n s i o n was a l l o w e d t o  hours o r o v e r n i g h t .  The r e a c t i o n was  t e r m i n a t e d by h e a t i n g t h e sample a t 68°C f o r 10 minutes, and u n i n c o r p o r a t e d n u c l e o t i d e s were s e p a r a t e d as d e s c r i b e d f o r n i c k - t r a n s l a t i o n (see above).  T y p i c a l l y , s p e c i f i c a c t i v i t i e s of resultant 8  probes ranged from 2 - 5 x 10 B.4  cpm/yg.  P r e p a r a t i o n o f M13 Probes M13 templates c o n t a i n i n g c e r u l o p l a s m i n DNA fragments were a l s o  used t o generate probes w i t h h i g h s p e c i f i c a c t i v i t i e s Candido, 1985).  (Russnak and  An a n n e a l i n g m i x t u r e c o n t a i n i n g 3.0 y l template (0.5 -  1 y g ) , 2.0 y l u n i v e r s a l p r i m e r (P-L B i o c h e m i c a l s ; 0.03 A 260 u n i t s / m l ) and 2.0 y l o f lOx a n n e a l i n g b u f f e r (100 mM T r i s - H C l pH 7.5, o 600 mM NaCI, 70 mM M g C l ) , was i n c u b a t e d a t 65 C f o r 15 minutes i n a 2  1.5 ml m i c r o f u g e tube.  A f t e r c o o l i n g t o room temperature, 1.0 y l of  20 mM DTT, 2.0 y l o f 0.5 mM dGTP, 2.0 y l o f 0.5 mM dTTP, 2.5 y l each of  [  of  E. c o l i DNA Polymerase I (Klenow fragment) were added.  was  a 3 2  P ] - d A T P and [  a 3 2  P ] - d C T P (25 y C i ; 3000 Ci/mMol) and 0.5 U The r e a c t i o n  a l l o w e d t o proceed f o r 10 minutes a t room temperature, and was then  f o l l o w e d by a 5 minute chase i n i t i a t e d by t h e a d d i t i o n o f 2.0 y l o f 0.5 mM dGTP and 2.0 y l o f 0.5 mM dCTP b e f o r e t e r m i n a t i o n of t h e r e a c t i o n by  h e a t i n g a t 68 C f o r 10 minutes.  Unincorporated  dNTPs were s e p a r a t e d  from l a b e l l e d s t r a n d s as d e s c r i b e d f o r n i c k t r a n s l a t i o n  (see above).  C.  IDENTIFICATION OF cDNAS FOR HUMAN CERULOPLASMIN  C l  S c r e e n i n g of a Human L i v e r cDNA L i b r a r y An a d u l t l i v e r cDNA l i b r a r y  (Prochownik e t a l . ,  1983) was k i n d l y  p r o v i d e d by Dr.S.H. O r k i n ( C h i l d r e n ' s H o s p i t a l M e d i c a l Center,  Boston).  T h i s l i b r a r y c o n t a i n s cDNA i n s e r t s o f > 500 bp i n s e r t e d i n t o the P s t 1 s i t e o f pKT218 by homopolymeric dG»dC t a i l i n g .  The cDNA l i b r a r y  was  screened u s i n g the c o l o n y h y b r i d i z a t i o n method o f G r u n s t e i n and Hogness (1975).  A m p i c i l l i n - r e s i s t a n t c o l o n i e s ( a p p r o x i m a t e l y 5,000 p e r 100 x  15 mm p e t r i p l a t e ) were t r a n s f e r r e d t o n i t r o c e l l u l o s e f i l t e r s  (82  BA-85, S c h l e i c h e r and S c h u e l l ) .  prepared  from the o r i g i n a l f i l t e r  lifts,  A second  s e t o f r e p l i c a s was  and b a c t e r i a on b o t h s e t s of f i l t e r s  a l l o w e d t o grow a t 37°C on LB p l a t e s c o n t a i n i n g t e t r a c y c l i n e c o l o n i e s were 1 - 2 to  mm  mm;  i n diameter.  until  F i l t e r s were s u b s e q u e n t l y  LB p l a t e s c o n t a i n i n g 170 yg/ml c h l o r a m p h e n i c o l ,  were  transferred  and t h e p l a s m i d s were  a l l o w e d t o a m p l i f y o v e r n i g h t a t 37°C. C e l l l y s i s was c a r r i e d out by p l a c i n g f i l t e r s  onto Whatman 3MM  paper  soaked w i t h 0.5 N NaOH, f o l l o w e d by i n c u b a t i o n a t room temperature  f o r 20  minutes.  again  In a s i m i l a r manner, f i l t e r s  w i t h NaOH f o r 20 minutes, minutes, minutes.  were subsequently  denatured  n e u t r a l i z e d w i t h 1 M T r i s - H C l pH 7.5 f o r 20  and f i n a l l y t r e a t e d w i t h 0.5 M T r i s - H C l pH 7.5/1.5 M NaCI f o r 20 The f i l t e r s  were then a i r - d r i e d and baked a t 68°C o v e r n i g h t .  P r i o r t o h y b r i d i z a t i o n , f i l t e r s were washed 3 times  i n 2X SSC b u f f e r (IX  SSC i s 0.15 M NaCI, 0.015 M Na C i t r a t e pH 7) i n o r d e r to remove  cellular  52  debris.  The r e p l i c a f i l t e r s were then screened  by u s i n g P o o l I I and P o o l  I I I o l i g o n u c l e o t i d e m i x t u r e s (see S e c t i o n I I . B . l ) as h y b r i d i z a t i o n probes.  H y b r i d i z a t i o n and washing c o n d i t i o n s were e s s e n t i a l l y  d e s c r i b e d by Fung e t a l . (1984) and a r e summarized i n S e c t i o n  those U.K.  P u t a t i v e p o s i t i v e c l o n e s were p u r i f i e d from t h e master p l a t e s , and t h e recombinant p l a s m i d s were a n a l y z e d obtain a d d i t i o n a l ceruloplasmin  (see S e c t i o n I I . E . l ) .  In order to  c l o n e s , t h e l i b r a r y was r e s c r e e n e d  n i c k - t r a n s l a t e d r e s t r i c t i o n fragment as a probe.  using a  H y b r i d i z a t i o n and  washing c o n d i t i o n s f o r the l a t t e r s c r e e n were e s s e n t i a l l y as d e s c r i b e d by M a n i a t i s e t a l . (1982), and a r e summarized i n S e c t i o n C.2  U.K.  P r e p a r a t i o n and Screening; o f Randomly-Primed Human L i v e r cDNA Libraries To  i s o l a t e cDNAs encoding t h e 5' end o f t h e c e r u l o p l a s m i n  t r a n s c r i p t , s e v e r a l randomly-primed human l i v e r cDNA l i b r a r i e s were constructed  i n t h e v e c t o r s XgtlO  or X g t l l  (Huynh e t a l . , 1984) by  W a l t e r Funk ( B i o c h e m i s t r y  Department, U n i v e r s i t y o f B r i t i s h  B r i e f l y , human l i v e r p o l y  (A)  +  RNA ( f o r i s o l a t i o n p r o c e d u r e , see S e c t i o n  II.E.4) was used as a template f o r f i r s t transcriptase.  Columbia).  s t r a n d cDNA s y n t h e s i s by r e v e r s e  DNase I - d i g e s t e d r a t thymus DNA (average l e n g t h 20  n u c l e o t i d e s ) was used as a p r i m e r ( G o e l e t and Karn, 1984).  Second s t r a n d  s y n t h e s i s was performed as d e s c r i b e d by Gubler and Hoffman (1983), u s i n g r i b o n u c l e a s e H (BRL), DNA polymerase I (BRL) and E. c o l i DNA l i g a s e (P-L Biochemicals).  A f t e r SI n u c l e a s e  r e s u l t a n t double-stranded  treatment t o g e n e r a t e b l u n t - e n d s , t h e  cDNA was methylated u s i n g EcoRI methylase and  S-adenosylmethionine (BRL), EcoRI l i n k e r s  (P-L B i o c h e m i c a l s )  to the ends, and the l i n k e r s were subsequently  were l i g a t e d  d i g e s t e d w i t h EcoRI.  The  53  cDNA was then chromatographed on a column (30 x 0.2 cm) o f B i o - G e l A-50 m (Bio-Rad), e q u i l i b r a t e d w i t h 0.01 M T r i s - H C l pH 7.5/0.3 M NaCl/0.001 M EDTA.  F r a c t i o n s c o m p r i s i n g t h e l e a d i n g edge of the cDNA peak  ( c o r r e s p o n d i n g t o cDNA fragments > 1 Kb) were p o o l e d and the DNA (~ 50 ng) was l i g a t e d w i t h 1 yg o f E c o R I - d i g e s t e d , dephosphorylated X g t l l DNA ( V e c t o r C l o n i n g  XgtlO o r  Systems).  H a l f o f t h e r e s u l t i n g DNA was packaged i n t o phage p a r t i c l e s i n v i t r o u s i n g a Gigapak ( V e c t o r C l o n i n g Systems).  The l i b r a r i e s c o n s t r u c t e d i n  X g t l O and X g t l l c o n t a i n e d 400,000 and 200,000 independent  recombinant  c l o n e s r e s p e c t i v e l y , and were subsequently p l a t e d a t h i g h d e n s i t y f o r 4 s c r e e n i n g (4 x 10  4 o r 2 x 10  X g t l O and X g t l l l i b r a r i e s ,  plaques p e r 150 mm p e t r i p l a t e f o r  respectively).  F o r p l a t i n g , a p p r o p r i a t e d i l u t i o n s o f phage p r e p a r e d  i n SM b u f f e r  (5.8 g NaCI, 2 g MgS0 , 50 ml 1 M T r i s - H C l pH 7.5, 5 ml 2% g e l a t i n p e r 4  l i t r e ) were i n c u b a t e d w i t h o v e r n i g h t c u l t u r e s o f a p p r o p r i a t e h o s t c e l l s a t 37°C f o r 10 minutes i n o r d e r t o a l l o w phage attachment.  p l a t e d on NZYC agarose.  P l a t e s were subsequently  u n t i l p l a q u e s were v i s i b l e b u t n o t c o n f l u e n t .  i n c u b a t e d a t 37 C  R e p l i c a s (2 s e t s ) o f the  p l a q u e s were then t r a n s f e r r e d t o n i t r o c e l l u l o s e c i r c l e s S c h l e i c h e r and S c h u e l l ) .  DNA was denatured  Phage were then o  by treatment  (132 mm; o f the  n i t r o c e l l u l o s e f i l t e r s w i t h 0.5 N Na0H/1.5 m NaCI f o r 5 minutes. filters minutes, minutes.  The  were then n e u t r a l i z e d by treatment w i t h 1 M T r i s - H C l pH 7.5 f o r 5 f o l l o w e d by treatment w i t h 0.5 M T r i s - H C l pH 7.5/1.5 M NaCI f o r 5 C e r u l o p l a s m i n cDNA c l o n e s were i d e n t i f i e d by  plaque-hybridization  (Benton and D a v i s , 1977) t o a p p r o p r i a t e r e s t r i c t i o n 32 fragments l a b e l l e d w i t h P by e i t h e r n i c k - t r a n s l a t i o n o r Klenow  54  extension  (see above).  autoradiography r e p l a t i n g and  Recombinant c l o n e s of i n t e r e s t were d e t e c t e d  and p u r i f i e d t o homogeneity by r e s c r e e n i n g a t decreased  by  s u c c e s s i v e rounds of  phage d e n s i t i e s .  l i b r a r y , p u r i f i e d phage c l o n e s were f u r t h e r screened  F o r the  XgtlO  u s i n g the P o o l  I  o l i g o n u c l e o t i d e m i x t u r e (see S e c t i o n I I . B . l ) i n o r d e r to i d e n t i f y cDNAs extending  f u r t h e s t i n a 5'  direction.  H y b r i d i z a t i o n and washing c o n d i t i o n s f o r the above screens summarized i n S e c t i o n  D.  U.K.  SCREENING OF HUMAN GENOMIC LIBRARIES Two  study.  One  constructed was  d i f f e r e n t human genomic DNA  was  i n t h e X Charon 4A v e c t o r  generously  X d e r i v a t i v e EMBL 3.  The  library  described  a p a r t i a l Sau  3A  c l o n e d i n t o the BamHl s i t e of  the  c o n s t r u c t e d by V a l Geddes (1987)  U n i v e r s i t y of B r i t i s h Columbia) and  was  p r i o r to a m p l i f i c a t i o n .  For both l i b r a r i e s , 1 x 10** p l a q u e s ,  genomic DNA.  initial  s c r e e n i n g was  representing 2 . 5 - 5  conservative estimate  c a r r i e d out on 5 x 10"*  genome e q u i v a l e n t s , based on  t h a t each c l o n e c o n t a i n e d  approximately  Phage were p l a t e d at a d e n s i t y of 3 - 5 x 10  petri dish.  The  p l a t i n g and  10 Kbp  plaques  -  the of per  s c r e e n i n g p r o c e d u r e s were e s s e n t i a l l y  performed as p r e v i o u s l y d e s c r i b e d f o r the XgtlO libraries  DNA,  This  a m p l i f i e d as  o t h e r l i b r a r y used was  T h i s l i b r a r y was  (Department of B i o c h e m i s t r y ,  mm  (Lawn e t a l . , 1978).  p r o v i d e d by Dr. T. M a n i a t i s and  d i g e s t of human lymphocyte genomic DNA,  screened  l i b r a r i e s were used i n t h i s  a p a r t i a l A l u l / H a e l l l d i g e s t of human genomic  ( M a n i a t i s e t a l . , 1982).  150  are  ( S e c t i o n II.C.2) w i t h the e x c e p t i o n  and  X g t l l cDNA  t h a t r e p l i c a s of the plaques  55  were t r a n s f e r r e d t o n i t r o c e l l u l a s e f i l t e r s and then i n c u b a t e d on NZYC p l a t e s a t 37°C t o a l l o w a m p l i f i c a t i o n of the phage (Woo, F o r s c r e e n s o t h e r than the i n i t i a l h i g h d e n s i t y s c r e e n , t h i s s t e p was  E.  ISOLATION OF NUCLEIC ACIDS  E.l  P u r i f i c a t i o n of Plasmid  amplification  DNA were r o u t i n e l y p u r i f i e d u s i n g a  m o d i f i e d a l k a l i n e l y s i s procedure o f B i r n b o i m and D o l y d e s c r i b e d by M a n i a t i s e t a l . (1982).  B r i e f l y , a 1.5  o v e r n i g h t b a c t e r i a l c u l t u r e c o n t a i n i n g a recombinant  (1979),  as  ml a l i q u o t o f an plasmid of  interest  p l a c e d i n a m i c r o f u g e tube, and b a c t e r i a were h a r v e s t e d by  c e n t r i f u g a t i o n i n an Eppendorf resuspended mM  1980).  omitted.  Small amounts o f p l a s m i d DNA  was  fresh  T r i s - H C l pH 8.0  of 200 y l of a s o l u t i o n c o n t a i n i n g 0.2 i n c u b a t e d a t 4°C f o r 5 minutes,  28.5  (60 ml 5 M potassium  ml dH^O;  f o r 5 minutes,  pH 4.8)  was  The p e l l e t glucose,  and 4 mg/ml lysozyme (Sigma).  i n c u b a t e d f o r 5 minutes a t room temperature,  solution  minutes.  i n 100 y l o f i c e - c o l d s o l u t i o n c o n t a i n i n g 50 mM  EDTA, 25 mM  was  c e n t r i f u g e f o r 2.5  a c e t a t e , 11.5  added.  10  This mixture  f o l l o w e d by the a d d i t i o n  N Na0H/l% SDS.  and then 150  This solution  y l of p o t a s s i u m  acetate  ml g l a c i a l a c e t i c  acid,  T h i s s u s p e n s i o n was  f o l l o w i n g which the p r e c i p i t a t e was  was  i n c u b a t e d on i c e  removed by  o c e n t r i f u g a t i o n a t 4 C f o r 5 minutes.  The r e s u l t a n t s u p e r n a t a n t  removed and e x t r a c t e d w i t h an equal volume of p h e n o l : c h l o r o f o r m v/v).  was (1:1  N u c l e i c a c i d s were p r e c i p i t a t e d by the a d d i t i o n of 2 volumes of  e t h a n o l (room temperature)  was  and were r e c o v e r e d by c e n t r i f u g a t i o n f o r 5  56  minutes.  The p e l l e t was washed w i t h 70% e t h a n o l , a i r - d r i e d , and  resuspended i n 50 y l o f TE b u f f e r  (10 mM T r i s - H C l pH 8.0, 1 mM EDTA).  Two d i f f e r e n t procedures were used f o r l a r g e - s c a l e isolations. for An  The T r i t o n l y s i s p r o c e d u r e (Katz e t a l . , 1973, 1977) was used  t h e l a r g e s c a l e p r e p a r a t i o n o f p l a s m i d s i n t h e pKT218 c l o n i n g aliquot  When t h e 0D,.  rt  of the culture  reached  - 0.7, 250 mg o f c h l o r a m p h e n i c o l was added and t h e c u l t u r e was  i n c u b a t e d a f u r t h e r 12 - 16 hours a t 37°C. centrifugation  C e l l s were h a r v e s t e d by  a t 6 K RPM i n a GS-3 r o t o r f o r 20 minutes, f o l l o w e d by  f r e e z i n g o f t h e p e l l e t s a t -20°C f o r 2 h o u r s . resuspended i n 6.25 ml o f s o l u t i o n c o n t a i n i n g T r i s - H C l pH 8.0. the  vector.  (5 ml) o f an o v e r n i g h t b a c t e r i a l c u l t u r e was used t o i n n o c u l a t e  1 l i t r e o f M9 medium a t 37°C. 0.6  plasmid  The c e l l s were then 25% w/v s u c r o s e and 50 mM  Lysozyme (1.5 ml o f 10 mg/ml s o l u t i o n )  s o l u t i o n was mixed by s w i r l i n g on i c e f o r 5 minutes.  was added, and EDTA (1.25 ml o f  a 0.5 M s o l u t i o n , pH 8.0) was added and mixed on i c e by s w i r l i n g f o r an a d d i t i o n a l 5 minutes.  Triton solution  [10 ml o f a s o l u t i o n  containing  10 ml o f 10% (w/v) T r i t o n X-100, 125 ml 0.5 M EDTA pH 8.0, 50 ml 1 M Tris-HCl  pH 8.0, 800 ml dH^O] was then added, and t h e s o l u t i o n was mixed  a g a i n f o r 5 minutes on i c e .  Following l y s i s , c e l l debris  was removed by o  centrifugation  a t 19 K RPM i n an SS-34 r o t o r f o r 30 minutes (4 C ) .  P l a s m i d DNA was s u b s e q u e n t l y i s o l a t e d by i s o p y c n i c  centrifugation  r o t o r / 2 0 hours/20°C) u s i n g C s C l - e t h i d i u m bromide d e n s i t y The  large-scale  as d e s c r i b e d  ( T i 70.1  gradients.  a l k a l i n e l y s i s procedure o f B i r n b o i m and Doly  (1979)  by M a n i a t i s e t aJL. (1982) was used f o r t h e i s o l a t i o n o f  recombinant pUC plasmids and M13 r e p l i c a t i v e form (RF) DNA (Messing, 1983).  I n t h e former case, 500 ml a l i q u o t s  of L b r o t h were  innoculated  57  w i t h 10 ml of the a p p r o p r i a t e b a c t e r i a l c u l t u r e (grown i n the presence  of  o a s e l e c t i v e a n t i b i o t i c ) and chloramphenicol DNA, 300  5.0  i n c u b a t e d a t 37 C f o r 12 - 16 hours w i t h o u t  amplification.  F o r the l a r g e - s c a l e i s o l a t i o n of M13  ml of an e x p o n e n t i a l l y - g r o w i n g JM103 o r JM101  y l of an M13  i n f e c t i o u s phage s u p e r n a t a n t  added t o 500 ml of YT b r o t h and  culture  s o l u t i o n , c e l l d e b r i s was  hours.  (see  above),  F o l l o w i n g a d d i t i o n of the p o t a s s i u m  acetate  removed by c e n t r i f u g a t i o n a t 19 K RPM  SS-34 r o t o r f o r 30 minutes. volumes of i s o p r o p a n o l , and  In  a l a r g e - s c a l e v e r s i o n of t h a t  d e s c r i b e d f o r the i s o l a t i o n o f s m a l l amounts of p l a s m i d DNA with several modifications.  and  (see S e c t i o n II.H.2) were  i n c u b a t e d a t 37°C f o r 8 - 1 0  b o t h c a s e s , the p u r i f i c a t i o n procedure was  RF  The  supernatant was  i n an  mixed d i r e c t l y w i t h  i n c u b a t e d a t room temperature  f o r 15  0.6  minutes.  N u c l e i c a c i d s were p e l l e t e d by c e n t r i f u g a t i o n f o r 20 minutes a t 10 K i n an SS-3A r o t o r . 11 g C s C l and 600 T h i s m i x t u r e was centrifugation  The p e l l e t s were resuspended  i n 11.0  y l e t h i d i u m bromide (10 mg/ml i n H 0) 2  i n c u b a t e d on i c e f o r 60 minutes,  (4 K RPM  f o r 5 minutes i n an HB-4  the f l o c c u l a n t p r e c i p i t a t e .  The  supernatant was  Q u i c k - S e a l Tubes (Beckman), and the DNA  was  RPM  ml TE, t o which were added.  f o l l o w e d by  low-speed  r o t o r ) i n o r d e r t o remove loaded i n t o 13  ml  banded by i s o p y c n i c d e n s i t y  g r a d i e n t c e n t r i f u g a t i o n under the c o n d i t i o n s d e s c r i b e d above. E.2  I s o l a t i o n of B a c t e r i o p h a g e Phage DNA  was  p u r i f i e d r o u t i n e l y from 20 ml  4A phage i s o l a t e s , c o n s i s t e n t l y t i c following conditions: phage was  DNA  200  F o r Charon  i n f e c t i o n s were o b t a i n e d u s i n g the  y l of SM b u f f e r c o n t a i n i n g 3.0  i n c u b a t e d a t 37°C f o r 20 minutes w i t h 100  host b a c t e r i a l culture.  lysates.  T h i s mixture was  - 4.0  x  10  6  y l of an o v e r n i g h t  added t o 20 ml of a p p r o p r i a t e  58  growth media i n a 125 ml Erlenmeyer vigorous shaking. post-innoculum.  f l a s k , and i n c u b a t e d a t 37°C w i t h  L y s i s was u s u a l l y observed w i t h i n 4 . 5 - 7  hours  F o r phage i s o l a t e s from e i t h e r t h e EMBL 3 genomic  library  or  X g t l l and XgtlO cDNA l i b r a r i e s , a s i n g l e plaque was added d i r e c t l y  to  a s o l u t i o n c o n t a i n i n g 100 y l o f s t a t i o n a r y - p h a s e b a c t e r i a l h o s t  cells  and 100 y l o f SM b u f f e r , and i n c u b a t e d a t 37°C f o r 20 minutes t o a l l o w attachment.  The m i x t u r e  ( c o n t a i n i n g t h e phage p l u g ) was then added t o  20 ml o f a p p r o p r i a t e growth medium i n a 125 ml Erlenmeyer i n c u b a t e d a t 37°C w i t h v i g o r o u s s h a k i n g . was  u s u a l l y observed w i t h i n 3 - 4 A t t h e time o f c e l l  lysis,  c u l t u r e , which was then l e f t  f l a s k , and  Under these c o n d i t i o n s ,  lysis  hours.  c h l o r o f o r m (3 mL) was added t o t h e  shaking slowly f o r a f u r t h e r 5 - 1 0  minutes.  A t t h a t time, t h e c o n t e n t s were c a r e f u l l y t r a n s f e r r e d t o a 30 ml Corex tube, such t h a t most o f t h e c h l o r o f o r m was l e f t b e h i n d , and t h e sample was c e n t r i f u g e d a t 10 K RPM f o r 10 minutes i n an SS-34 r o t o r .  To t h e  r e s u l t i n g supernatant, 3 ml o f 5 M NaCI and 3 g o f p o l y e t h y l e n e g l y c o l (PEG)  8000 (average m o l e c u l a r weight  c o n t e n t s were mixed, and l e f t phage p a r t i c l e s .  7000 - 9000) were added.  The  a t 4°C o v e r n i g h t i n o r d e r t o p r e c i p i t a t e  F o l l o w i n g c e n t r i f u g a t i o n o f t h e s u s p e n s i o n f o r 10  minutes a t 10 K RPM i n an HB-4 r o t o r , t h e phage p e l l e t was resuspended i n 500  y l o f DNase I b u f f e r  (50 mM HEPES pH 7.5, 5.0 mM M g C l , and 0.5 mM  C a C l ) , and t r a n s f e r r e d t o a microfuge 2  of  2  tube.  RNAse A (Sigma) (10 y l  a 5 mg/ml s t o c k ) and DNase I ( B o e h r i n g e r Mannheim) (5 y l of a 10  mg/ml s t o c k ) were added, and t h e s o l u t i o n was i n c u b a t e d a t 37°C f o r 1 hour.  F o l l o w i n g d i g e s t i o n , t h e s o l u t i o n was c e n t r i f u g e d f o r 5 minutes i n  a microfuge  i n o r d e r t o remove any remaining c e l l u l a r d e b r i s .  To t h e  59  s u p e r n a t a n t , 50  y l o f 10X  and  added p r i o r to d i g e s t i o n w i t h 4 y l of p r o t e i n a s e K  5%  SDS)  was  SET  buffer  (0.1  M T r i s - H C l pH  7.5,  0.2  M EDTA  o (40 mg/ml s t o c k s o l u t i o n ) The  s o l u t i o n was  (3:1  v/v  extracted  f o l l o w e d by  chloroform.  The  DNA  of 2 volumes of 95% c o l l e c t e d by  and  was  p r e c i p i t a t e d from the aqueous phase by  once w i t h an e q u a l volume of  e t h a n o l a t room temperature f o r 2 minutes,  centrifugation.  The  DNA  p e l l e t was  resuspended i n 50  by B l i n and  Stafford  in a buffer  0.5%  and  SDS,  100  hours a t 50°C. phenol and  The  and  buffer  EDTA) u n t i l the ^ 70  °  2  f  was  t l i e  The  The  d  i  DNA  TE.  Insoluble  material  was  M EDTA pH  was 8.0,  16  3 times w i t h equal volumes of  a  l  y  s  Tris-HCl a  t  e  w  a  s  yg/ml and  removed by  pH  8.0,  10  < °- the  v/v)  mM  RNAse A  0 5  s o l u t i o n was  times w i t h e q u a l volumes of p h e n o l : c h l o r o f o r m (1:1 against  r e s u l t i n g powder  i n c u b a t e d f o r 12 -  (50 mM  added t o a f i n a l c o n c e n t r a t i o n of 100  i n c u b a t e d at 37°C f o r 60 minutes.  70%  ground t o a f i n e  c o n s i s t i n g of 0.5  extracted  against  of  buffer.  L i v e r t i s s u e was  tissue)  s o l u t i o n was  then d i a l y z e d  NaCI, 10 mM  (1976).  yg/ml p r o t e i n a s e K,  ml  p r e p a r e d e s s e n t i a l l y as  i n a Waring b l e n d e r .  (10 ml/g  and  DNA  from human l i v e r was  dissolved  addition  washed w i t h 1.0  y l of TE  Genomic DNA  powder i n l i q u i d n i t r o g e n  C.  phenol:chloroform  v/v)  P r e p a r a t i o n of Human Genomic  described  was  t w i c e w i t h an e q u a l volume of  1:1  e t h a n o l , a i r - d r i e d , and E.3  ( B o e h r i n g e r Mannheim) f o r 30 minutes a t 68  solution  was  extracted  3  and  then  centrifugation  dialyzed  at 10 K  o i n an SS-34 r o t o r a t 4 C f o r 15 minutes. by  the  addition  MgCl^ and addition  1 mM  of G i l b e r t S a l t s  (5X  Salts  Genomic DNA i s 2.5  ethanol.  precipitated  M OT^OAc, 100  EDTA) to a f i n a l c o n c e n t r a t i o n of IX,  of 2 volumes of 95%  was  f o l l o w e d by  F o l l o w i n g c o l l e c t i o n by  mM the  RPM  60  c e n t r i f u g a t i o n , t h e DNA p e l l e t was allowed At t h i s time, any remaining  to rehydrate  f o r s e v e r a l days.  i n s o l u b l e m a t e r i a l was removed by  c e n t r i f u g a t i o n as p r e v i o u s l y d e s c r i b e d .  The f i n a l DNA p e l l e t was  resuspended i n TE, a t a c o n c e n t r a t i o n o f 0.5 - 1.0 mg/ml. Human genomic DNA was a l s o prepared  from white b l o o d c e l l s by Heather  K i r k (Department o f B i o c h e m i s t r y , U n i v e r s i t y o f B r i t i s h  Columbia)  a c c o r d i n g t o a m o d i f i e d p r o c e d u r e o f Kunkel e t a l . (1979), and was generously E.4  s u p p l i e d by Ms. K i r k f o r v a r i o u s a s p e c t s o f t h i s  study.  I s o l a t i o n o f RNA  E.4.1  P r e p a r a t i o n o f human l i v e r p o l y ( A )  +  RNA.  A l l glassware,  u t e n s i l s , and s o l u t i o n s were a u t o c l a v e d p r i o r t o u s e i n o r d e r t o i n a c t i v a t e contaminating  ribonucleases.  RNA was i s o l a t e d from human l i v e r  by t h e g u a n i d i n e h y d r o c h l o r i d e method (Chirgwin e t a l . , 1979). human l i v e r  ( o b t a i n e d from b r a i n - d e a d  donors and immediately  Powdered  frozen i n  l i q u i d n i t r o g e n ) was added t o b u f f e r (10 ml/10 mg o f t i s s u e ) c o n s i s t i n g o f 7.5 M g u a n i d i n e h y d r o c h l o r i d e (GuHCl) pH 7.5, 25 mM sodium c i t r a t e pH 7.0 and  0.1 M DTT.  The suspension  was d i s r u p t e d u s i n g a P o l y t r o n homogenizer.  N - l a u r y l s a r c o s i n e was added t o 0.5% (w/v) and the i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n (10 K RPM f o r 15 minutes a t 4°C i n an SS-34 rotor).  F o l l o w i n g a d d i t i o n o f e t h a n o l t o a f i n a l c o n c e n t r a t i o n o f 33%,  RNA was p r e c i p i t a t e d o v e r n i g h t a t -20°C.  The p r e c i p i t a t e was c o l l e c t e d  by c e n t r i f u g a t i o n under the c o n d i t i o n s d e s c r i b e d above, and d i s s o l v e d i n one h a l f o f t h e s t a r t i n g volume w i t h t h e GuHCl b u f f e r . m a t e r i a l was removed by c e n t r i f u g a t i o n .  Again, i n s o l u b l e  RNA was p r e c i p i t a t e d as d e s c r i b e d  above, resuspended i n one q u a r t e r the s t a r t i n g volume w i t h t h e GuHCl b u f f e r , and r e p r e c i p i t a t e d . The f i n a l RNA p e l l e t was resuspended i n  61  s t e r i l e dH^O,  and  any  f o r 15 minutes a t 4°C Human p o l y ( A ) o l i g o d(T) 1972).  +  p r e c i p i t a t e removed by  RNA  was  i s o l a t e d by  chromatography on a column of  c e l l u l o s e (Sigma) (Edmonds e t a l . , 1971;  4 mM  T r i s - H C l pH  7.5,  unbound RNA  f r a c t i o n was  EDTA, 0.5  Poly(A)  w i t h s t e r i l e dH^O;  r e a p p l i e d t o the column t w i c e ,  RNA  +  4.8  resuspended i n s t e r i l e dH^O  10  mM  was  and  and  The  the column  above u n t i l the 0D„,„ of the e l u a t e 260 s u b s e q u e n t l y e l u t e d from the  and p o o l e d .  V of 3 M NaOAc pH  Leder,  samples ( i n b u f f e r c o n t a i n i n g  f r a c t i o n s c o n t a i n i n g RNA  spectrophotometrically  A v i v and  M NaCI) were a p p l i e d t o the column.  washed w i t h the b u f f e r d e s c r i b e d l e s s than 0.05.  RPM  i n the SS-34 r o t o r ) .  T o t a l human or b o v i n e RNA  of 0.1  c e n t r i f u g a t i o n (10 K  RNA  was  was  was  column  were i d e n t i f i e d p r e c i p i t a t e d by  2 volumes of e t h a n o l .  at a concentration  of 0.6  the  Poly(A)  addition RNA  +  was  - 1. 0 mg/ml and  o s t o r e d i n a l i q u o t s at -70 E. 4.2  Preparation  from HepG2 c e l l s van  C.  of t o t a l RNA  (Knowles, 1980)  Oost e t a l . (1985) f o r RNA  was  from HepG2 c e l l s .  Total cellular  p u r i f i e d as p r e v i o u s l y d e s c r i b e d  i s o l a t i o n from the h y b r i d e n d o t h e l i a l  RNA by  cell 2  l i n e EA.hy926.  C e l l s grown t o c o n f l u e n c y  were d i s s o l v e d i n 135 t h i s s o l u t i o n by  2 yg)  ml  of GuHCl b u f f e r , and  successive  ethanol  BASIC DNA  F. 1  R e s t r i c t i o n Enzyme D i g e s t i o n  a  s  t o t a l RNA  was  cm  purified  p r e c i p i t a t i o n s as d e s c r i b e d  F.  w  on a t o t a l a r e a of 5225  from  above.  TECHNIQUES  With the e x c e p t i o n  of genomic d i g e s t s , DNA  r o u t i n e l y digested  i n a t o t a l volume of 20 y l , u s i n g  b u f f e r system d e s c r i b e d  by M a n i a t i s  e t §_1.  (1982).  ( u s u a l l y 0.5  BSA  (BRL)  -  the was  added to  62  a f i n a l concentration restriction plasmid  of 100  yg/ml.  In most cases,  enzyme were used per r e a c t i o n .  and phage DNA  Genomic DNA  preparations,  (5 - 10 yg) was  New  u n i t s of  F o r d i g e s t s of  5 yg of RNase A was  usually digested  y l u s i n g 30 - 50 u n i t s of a p p r o p r i a t e p u r c h a s e d from BRL,  1-5  small-scale  included.  i n a t o t a l volume of  r e s t r i c t i o n enzyme.  30  Enzymes were  England B i o l a b s , B o e h r i n g e r Mannheim, and  P-L  Biochemicals. R e s t r i c t i o n enzyme d i g e s t i o n m i x t u r e s were a n a l y z e d e l e c t r o p h o r e s i s i n agarose o r p o l y a c r y l a m i d e  gels  by  (see below), f o l l o w i n g  the a d d i t i o n of 0.1  volumes of l o a d i n g b u f f e r (0.25% bromophenol b l u e ,  0. 25% x y l e n e c y a n o l  and  F.2  25%  ficoll).  E n d - l a b e l l i n g , of DNA  Fragments  Where n e c e s s a r y , c e r u l o p l a s m i n EcoRI s i t e i n X g t l O or X g t l l v e c t o r s v i s u a l i z e d by  labelling  cDNA i n s e r t s , c l o n e d  (see S e c t i o n I I . C . 2 ) , were  the 5' overhang generated by EcoRI d i g e s t i o n .  These r e a c t i o n s were c a r r i e d out a c c o r d i n g using 1.  [  a 3 2  P ] - d A T P and  Reaction  to M a n i a t i s  e t a l . (1982),  the Klenow fragment of E. c o l i .  p r o d u c t s were analyzed  by p o l y a c r y l a m i d e  DNA  polymerase  gel electrophoresis  f o l l o w e d by a u t o r a d i o g r a p h y , as d e s c r i b e d below (see S e c t i o n F.3  E l e c t r o p h o r e s i s of  F.3.1  DNA  endonuclease d i g e s t i o n m i x t u r e s were a n a l y z e d  pH  8.3,  buffer et  89 mM  borate  samples of on 0.7  2.0  mM  restriction  - 1.5%  run i n e i t h e r 1XTBE b u f f e r (89 mM and  agarose g e l s ,  Tris-borate  EDTA) ( M a n i a t i s e t a l . , 1982)  (50XTAE i s 2 M T r i s base, 1 M g l a c i a l a c e t i c a c i d )  a l . , 1982), c o n t a i n i n g 0.5  F.3.I.).  DNA  Agarose R e l e l e c t r o p h o r e s i s .  which were poured and  i n t o the  yg/ml e t h i d i u m  bromide.  DNA  or 1XTAE  (Maniatis fragments  63  were v i s u a l i z e d under UV l i g h t  (260 nm) and photographs were taken w i t h a  P o l a r o i d camera u s i n g Type 57 f i l m . F.3.2  Polyacrylamide  polyacrylamide acrylamide)  Rel electrophoresis.  g e l s (5 - 10% prepared  Non-denaturing  from a s t o c k o f 29:1 a c r y l a m i d e : b i s -  were poured and r u n i n 1XTBE b u f f e r .  P o l y m e r i z a t i o n was  i n i t i a t e d by t h e a d d i t i o n o f ammonium p e r s u l f a t e and TEMED t o f i n a l c o n c e n t r a t i o n s o f 0.066% (w/v) and 0.04% (w/v) r e s p e c t i v e l y .  DNA  fragments were v i s u a l i z e d e i t h e r by s t a i n i n g o f t h e g e l s w i t h 10 ug/ml e t h i d i u m bromide i n 0.5XTBE f o l l o w e d by UV i r r a d i a t i o n o r by autoradiography  i f t h e fragments were e n d - l a b e l l e d .  In the l a t t e r  case,  o g e l were d r i e d under vaccuum a t 80 C f o r 40 minutes u s i n g a Bio-Rad g e l d r i e r and exposed t o X-ray f i l m i n t e n s i f y i n g screens  (Kodak XK-1).  Where r e q u i r e d ,  ( L i g h t n i n g P l u s , Dupont) were used a t -70°C.  For denaturing polyacrylamide  g e l s (6 - 20%, p r e p a r e d  38:2 a c r y l a m i d e : b i s - a c r y l a m i d e ) , u r e a added as a denaturant,  from a s t o c k o f  ( f i n a l c o n c e n t r a t i o n of 8.3 M) was  and t h e g e l s were poured and r u n i n 1XTBE b u f f e r .  P o l y m e r i z a t i o n o f t h e g e l s was i n i t i a t e d by t h e a d d i t i o n o f ammonium p e r s u l f a t e and TEMED t o f i n a l c o n c e n t r a t i o n s o f 0.066% (w/v) and 0.024% (w/v) r e s p e c t i v e l y . autoradiography F.4  G e l s were d r i e d , and t h e DNA was v i s u a l i z e d by  as d e s c r i b e d above.  Southern T r a n s f e r s DNA separated by e l e c t r o p h o r e s i s i n agarose g e l s was t r a n s f e r r e d  to n i t r o c e l l u l o s e  ( S c h l e i c h e r and S c h u e l l ) , Nytran  ( S c h l e i c h e r and  S c h u e l l ) o r Zetaprobe (Bio-Rad) e s s e n t i a l l y as d e s c r i b e d by Southern (1975), except  t h a t the a c i d d e p u r i n a t i o n step was r o u t i n e l y o m i t t e d .  fragments i n the g e l s were denatured  f o r 45 - 60 minutes i n 0.5 N NaOH,  DNA  64  1.5 M NaCI, and then n e u t r a l i z e d by t r e a t i n g t w i c e f o r 20 minutes w i t h 0.5 M T r i s - H C l pH 7.5/1.5 M NaCI. was  DNA  each  t r a n s f e r t o v a r i o u s membranes  c a r r i e d out o v e r n i g h t i n e i t h e r 10XSSC o r i n 1.0 M ammonium a c e t a t e  (pH 7 ) , the l a t t e r b e i n g more e f f i c i e n t . baked a t 68°C f o r 4 - 1 6  and  hours p r i o r t o h y b r i d i z a t i o n .  6.  DNA  G.l  Fragment P r o d u c t i o n DNA  F i l t e r s were then a i r d r i e d  CLONING  fragments f o r l i g a t i o n i n t o e i t h e r pUC  o r M13  v e c t o r s were  produced by s e v e r a l methods, i n c l u d i n g s o n i c a t i o n ( D e i n i n g e r , 1983) r e s t r i c t i o n enzyme d i g e s t i o n s and subsequent fragment i s o l a t i o n . l a t t e r case, DNA  o r by  In the  fragments were r e c o v e r e d from agarose g e l s by  e l e c t r o e l u t i o n i n t o d i a l y s i s t u b i n g c o n t a i n i n g e i t h e r 0.5XTBE o r 0.5XTAE buffer.  The DNA  cartridges  was  (BRL).  then p u r i f i e d by chromatography  I n t h i s p r o c e d u r e , DNA  c o n t a i n i n g 0.2 M NaCI. 2.0 M NaCI.  The DNA  was  was  through NACS PREPAC  l o a d e d and washed i n TE  e l u t e d i n 500 y l of TE c o n t a i n i n g  A f t e r a d d i t i o n o f 0.1 volume of 3.0 M Na a c e t a t e (pH 4.8)  fragment was p r e c i p i t a t e d w i t h 2 volumes  the  of 95% e t h a n o l and resuspended i n  a s m a l l volume of s t e r i l e TE b u f f e r . Random DNA  fragments were produced by s o n i c a t i o n  u s i n g a Heat Systems  Sonifier.  P l a s m i d DNA  0.5 M NaCI, 0.1 M T r i s - H C l pH 7.4, b u r s t s of 5 seconds each.  10 mM  (Deininger,  1983)  (10 - 20 yg i n 500 y l of  EDTA) was  sheared by 5 power  F o l l o w i n g e t h a n o l p r e c i p i t a t i o n , DNA  fragments  of 300 - 600 bp were s e p a r a t e d by e l e c t r o p h o r e s i s i n a 5% n o n - d e n a t u r i n g p o l y a c r y l a m i d e g e l and i s o l a t e d by e l e c t r o e l u t i o n .  The DNA  fragments were  made b l u n t - e n d e d by i n c u b a t i o n a t 37°C f o r 90 - 120 minutes w i t h 33  mM  65  T r i s - a c e t a t e pH 7.8, 66 mM potassium  a c e t a t e , 10 mM Mg a c e t a t e , 100 mg/ml  BSA, and 0.2 mM of each d e o x y r i b o n u c l e o t i d e  t r i p h o s p h a t e i n 50 y l t o t a l  volume, c o n t a i n i n g 6 u n i t s of T4 DNA polymerase (BRL). phenol/chloroform  Following  (1:1 v/v) e x t r a c t i o n , the DNA was p r e c i p i t a t e d  with  e t h a n o l and resuspended a t a c o n c e n t r a t i o n of 10 n g / y l o f TE b u f f e r . G.2  L i g a t i o n o f DNA i n t o pUC o r M13  Vectors  F o r r e s t r i c t i o n endonuclease mapping a n a l y s i s o r s o n i c a t i o n , DNA fragments were subcloned 1982) (pUC p l a s m i d s of Biochemistry,  i n t o pUC12 o r pUC13 v e c t o r s ( V i e i r a and Messing,  were k i n d l y p r o v i d e d by Dr. Roland Russnak, Department  U n i v e r s i t y of B r i t i s h Columbia).  F o r DNA  sequence  a n a l y s i s , the M13 v e c t o r s mp8, mp9, mpl8 and mpl9 were used. M13  l i g a t i o n s were u s u a l l y c a r r i e d out w i t h 10 ng o f v e c t o r DNA and  20 - 60 ng i n s e r t DNA i n 20 y l o f 50 mM T r i s - H C l pH 7.4, 10 mM 10 mM DTT, 1.0 mM  spermidine,  1.0 mM ATP and 100 yg/ml BSA.  MgCl  2 >  For  l i g a t i o n s u s i n g pUC v e c t o r s , 100 ng of v e c t o r DNA was l i g a t e d t o a 3 - f o l d molar excess  o f i n s e r t DNA, u s i n g the l i g a t i o n b u f f e r d e s c r i b e d above.  Phage T4 DNA  l i g a s e (BRL o r P-L B i o c h e m i c a l s )  c o h e s i v e o r blunt-ended  was added (0.5 o r 1 u n i t f o r  l i g a t i o n s r e s p e c t i v e l y ) and the r e a c t i o n was o  allowed  t o proceed  a t 15 C o v e r n i g h t f o r blunt-ended  l i g a t i o n s or at  o 4 C o v e r n i g h t f o r cohesive-ended G.3  ligations.  Transformations E. c o l i s t r a i n s JM103 o r JM101 were used as b a c t e r i a l h o s t s f o r  a l l pUC and M13 t r a n s f o r m a t i o n s competent by treatment  (see T a b l e I I ) .  w i t h 50 mM  A l i q u o t s o f competent c e l l s 5 y l o f l i g a t i o n mixture  B a c t e r i a l c e l l s were made  c a l c i u m c h l o r i d e (Messing,  1983).  (0.3 ml) were t y p i c a l l y transformed  (see p r e c e d i n g  section).  C e l l s were  with 3 incubated  66  w i t h DNA a t 4°C f o r 40 - 60 minutes 3 minutes p r i o r t o p l a t i n g .  and then heat-shocked a t 42°C f o r  B a c t e r i a i n f e c t e d w i t h recombinant  M13 phage  were assayed f o r t h e i r i n a b i l i t y t o c l e a v e 5 - b r o m o 4 - c h l o r o 3 - i n d o l y l g a l a c t o s i d e (X-Gal) as d e s c r i b e d by Messing appearance  of c l e a r plaques.  (1983), r e s u l t i n g i n t h e  The same c o l o u r assay was used t o d e t e c t  b a c t e r i a l c o l o n i e s c o n t a i n i n g pUC p l a s m i d s . For the  c l a r i t y , a summary o f t h e d i f f e r e n t s u b c l o n e s used i n a n a l y s i s o f  w i l d - t y p e human c e r u l o p l a s m i n gene i s p r e s e n t e d i n T a b l e I I I , and w i l l  be r e f e r r e d t o i n subsequent  sections.  H.  DNA SEQUENCE ANALYSIS  H.l  S c r e e n i n g o f M13 C l o n e s In  t h e case o f s o n i c a t i o n , m i x t u r e s o f randomly-sheared  fragments were c l o n e d i n t o M13 v e c t o r s (see S e c t i o n I I . G . l ) . i d e n t i f y recombinant  DNA  In order to  c l o n e s c a r r y i n g e i t h e r cDNA o r exon-encoding  sequences, M13 p l a q u e s were s c r e e n e d by p l a q u e h y b r i d i z a t i o n as p r e v i o u s l y described  (Benton and D a v i s , 1977) (see S e c t i o n I I . C . 2 ) .  Hybridization  and washing c o n d i t i o n s v a r i e d , depending o f t h e n a t u r e o f t h e probe ( s e e S e c t i o n I I . K f o r a summary o f h y b r i d i z a t i o n and washing c o n d i t i o n s used i n the  present study). H.2  I s o l a t i o n o f M13 Template  DNA  S i n g l e - s t r a n d e d M13 phage DNA from c l o n e s o f i n t e r e s t was p r e p a r e d as d e s c r i b e d by Messing  (1983).  Aliquots  (2 ml) o f YT c o n t a i n i n g  20 y l o f h o s t b a c t e r i a (JM101 o r JM103) were each i n n o c u l a t e d w i t h a o s i n g l e plaque, and i n c u b a t e d a t 37 C f o r 8 - 1 0  hours.  Bacterial  were p e l l e t e d by c e n t r i f u g a t i o n i n a 1.5 ml m i c r o f u g e tube.  Phage  cells  Table I I I .  Cloning S t r a t e g y f o r the Wild-Type Human Ceruloplasmin Gene.  V a r i o u s r e s t r i c t i o n fragments containing exon sequences were subcloned from c e r u l o p l a s m i n genomic c l o n e s ( F i g u r e 10) i n t o a p p r o p r i a t e pUC and/or HI3 v e c t o r s . A s t e r i s k s f o l l o w those pUC subclones t h a t were analyzed by s o n i c a t i o n . Crosses i d e n t i f y subclones that were used l n Northern b l o t a n a l y s i s o f the 5' end o f the gene. L I - L4 designate exons present i n the 5' untranslated r e g i o n of the gene.  CORRESPONDING EX0N(S)  RESTRICTION FRAGMENT  L1.L2 '  2.6 Kbp EcoRl (pUC13)  700 bp Xbal/EcoRI  Jt 85 bp Sau3A/EcoRI (mpl8) (pUC13)^^' '190 bp Sau3A (mpl8)  LI  —-»70 bp H i n f l / E c o R I (mpl8) »220 bp H i n f l (mpl8)  L2  PHAGE CLONE DERIVED FROM XWT7  XWT2  1.5 Kbp EcoRIt (pUC13) 800 bp Xba/EcoRI (pUC13)  420 bp EcoRIt (pUC13)  mpl8,19 2.7 Kbp HindlH/BcoRI ( UC13)t  L2  XWT7  L3  XWT7  -  XWT7  L4,l  XWT2  P  4.0 Kbp EcoRl (pUC13)t  1  \ rfcj. (pUC13)  JMnrfTTT/P^BT-r  "  »160 bp Ddel (mpl8) ,450 bp Ddel (mpl8)  \  1.4 Kbp EcoRl (pUC13)t 3.1 Kbp EcoRl (pUCL2)*t 1  CORRESPONDING EXON(S)  RESTRICTION FRAGMENT  PHAGE CLONE DERIVED FROM  4.6 Kbp EcoRI (pUC12)*  2  XWT2  800 bp EcoRI/Sall (apl8,19)  3  XWT6  4  XWT5  .^250 bp KpnI/EcoRI (mpl9) 3.2 Kbp EcoRI/Sall (pUCl2)^_~»800 bp Kpnl (opl9) -*1.9 Kbp KpnI/Sall (pUC18)»  5 6  XWT5  2.6 Kbp H i n d l l l (pUC12)«  7  XWT1  1.6 Kbp BamHl/Hlndlll (pDC12)*  8,9  XWT1  670 bp BamHl/Sstl (mpl8)  9  XWT1  10  XWT1  10  XWT3  10,11,12,13  XWT3  ",14  XWT3  ^,350. bp Xbal/EcoRI (mpl8) 1.8 Kbp EcoRI (pUC12)' " "" "  -*950 bp Xbal (apl8)  450 bp EcoRI (pOC12, npl8, mpl9) 1  1.2 Kbp EcoRI (pUC12)  » 490 bp BamHI/EcoRI (mpl9)  4.8 Kbp EcoRI (pUC13)* 2.1 Kbp EcoRI/Sall (pUa3*, np!9)  |  69  particles  i n 1.3 ml of s u p e r n a t a n t were p r e c i p i t a t e d by the a d d i t i o n of  0.3 ml of a s o l u t i o n c o n t a i n i n g 20% PEG, i n c u b a t i o n a t room temperature  f o r 15 minutes.  by c e n t r i f u g a t i o n f o r 5 minutes. 200 y l of low t r i s b u f f e r EDTA).  DNA  was  (50 mM  NaCI, 10 mM  then resuspended i n  T r i s - H C l , pH 7.5,  e x t r a c t i o n with chloroform.  volumes o f e t h a n o l .  H.3  phage were c o l l e c t e d  The phage p e l l e t was  p r e c i p i t a t e d by the a d d i t i o n of 0.1  low t r i s  M13  1  mM  p u r i f i e d by e x t r a c t i o n w i t h p h e n o l / c h l o r o f o r m (1:1 v / v ) ,  f o l l o w e d by a 1:1  of  2.5 M NaCI, f o l l o w e d by  F i n a l M13  The DNA  was  then  volumes o f 3 M Na A c e t a t e and 2  phage p e l l e t s were resuspended  i n 50 y l  buffer.  DNA  Sequence A n a l y s i s  A l l DNA  sequence was  determined u s i n g the d i d e o x y n u c l e o t i d e  c h a i n t e r m i n a t i o n t e c h n i q u e of Sanger e t a l . (1977) as m o d i f i e d f o r M13 phage templates (Messing e t a l . ,  1981).  Sequencing r e a c t i o n s were  performed u s i n g the d i d e o x y / d e o x y r i b o n u c l e o t i d e c o n c e n t r a t i o n s g i v e n i n Table I I I . M13 OD  DNA  template DNA  sequence  a n a l y s i s was  c a r r i e d out by h y b r i d i z i n g 4 y l of  w i t h 1 y l o f u n i v e r s a l sequencing p r i m e r (17mer;  260 u n i t s / m l ) , 1 y l dH 0, and 2 y l 10 x H i n b u f f e r  100 mM  T r i s - H C l pH 7.5,  70 mM  (600 mM  0.03  NaCI,  M g C l ) a t 68°C f o r 10 minutes. 2  F o l l o w i n g slow c o o l i n g t o room temperature, 1 y l of 15 yM dATP and  1.5  32 y l of [a of  P]-dATP (10 y C i / y l ; 3000 Ci/mMole) was  t h i s m i x t u r e was  then added to 1.5  d i d e o x y / d e o x y r i b o n u c l e o t i d e mix r e a c t i o n , 0.4  u n i t s DNA  added t o each tube. temperature,  y l of the a p p r o p r i a t e  (see T a b l e I V ) .  polymerase  added; 2 y l  To i n i t i a t e the e x t e n s i o n  1 Klenow fragment  (BRL, Pharmacia)  F o l l o w i n g i n c u b a t i o n f o r 15 minutes  1 y l of 0.5 mM  dATP was  a t room  added to each r e a c t i o n , and  the  was  T a b l e IV.  Compositions o f M13 DNA Sequencing Mixes.  Composition o f Sequencing Mixes  Nucleotide  d/ddG  d/ddA  d/ddT  d/ddC  dG  7.9  109.4  158.7  157.9  dT  157.6  109.4  7.9  157.9  dC  157.4  109.4  158.7  10.5  ddG  157.4  ddA  -  116.7  ddT  -  -  ddC  -  550.3 191.6  The numbers r e f e r t o c o n c e n t r a t i o n s (yM) o f d i d e o x y - and d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s used f o r t h e p r e p a r a t i o n o f M13 DNA sequencing mixes. The v a l u e s g i v e n were determined e m p i r i c a l l y by Dr. Joan McPherson, Department o f P l a n t S c i e n c e s , U n i v e r s i t y o f B r i t i s h Columbia.  71  c o l d "chase" was a l l o w e d t o proceed temperature.  f o r an a d d i t i o n a l 15 minutes a t room  A t t h i s time, 5 y l of dye mix (98% formamide, 10" mM EDTA  pH 8.0, 0.02% x y l e n e c y a n o l , 0.02% bromophenol b l u e ) was added. p r o d u c t s were denatured  by h e a t i n g a t 90°C f o r 3 minutes,  Reaction  and 2 y l of  each r e a c t i o n was a n a l y z e d on 6% d e n a t u r i n g p o l y a c r y l a m i d e g e l s (see Section II.F.3.2).  A f t e r e l e c t r o p h o r e s i s (0.9 W/cm), g e l s were d r i e d and  exposed o v e r n i g h t t o Kodak XK-1 f i l m a t room  temperature.  I n o r d e r t o a v o i d redundancy, most M13 c l o n e s generated by s o n i c a t i o n were f i r s t  a n a l y z e d u s i n g o n l y ddTTP r e a c t i o n s (Sanger e t a l . ,  Where n e c e s s a r y , fragments c l o n e d i n t o t h e M13  1980).  i n opposite o r i e n t a t i o n  were i d e n t i f i e d a c c o r d i n g t o t h e i r a b i l i t y t o form f i g u r e  8-like  c o n f i g u r a t i o n s , which m i g r a t e more s l o w l y i n agarose  (Messing,  gels  1983).  I.  RNA  ANALYSIS  1.1  Northern B l o t A n a l y s i s RNA samples (10 yg p o l y ( A )  t o t a l c e l l u l a r RNA) were denatured c o n t a i n i n g formaldehyde, formaldehyde-containing et a l . ,  1982).  +  human l i v e r RNA o r 20 yg HepG2  by a d d i t i o n of l o a d i n g b u f f e r  and subsequently agarose  s e p a r a t e d by e l e c t r o p h o r e s i s i n  g e l s (Lehrach e t a l . , 1977; M a n i a t i s  P r i o r to t r a n s f e r , g e l s were t r e a t e d w i t h 50 mM NaOH,  10 mM NaCI f o r A5 minutes,  n e u t r a l i z e d by treatment  f o r 45 minutes w i t h  0.1 M T r i s - H C l pH 7.5, and f i n a l l y soaked i n 20XSSC f o r 60 minutes.  RNA  was t r a n s f e r r e d t o n i t r o c e l l u l o s e i n 20XSSC b u f f e r , by the method of Southern 4-16  (1975).  F o l l o w i n g t r a n s f e r , b l o t s were a i r - d r i e d and baked f o r  hours a t 68°C.  C e r u l o p l a s m i n mRNA s p e c i e s were d e t e c t e d u s i n g  72  h y b r i d i z a t i o n and washing c o n d i t i o n s d e s c r i b e d f o r d o u b l e - s t r a n d e d probes  DNA  (see S e c t i o n I I . K ) .  1.2  RNA  Dot  Blots  Dot b l o t s f o r r a p i d a n a l y s e s were p r e p a r e d by s p o t t i n g 7.5 10 yg of human l i v e r mRNA i n s t e r i l e 10XSSC onto filters. hours.  h y b r i d i z a t i o n and washing  4-16  subsequently w i t h l a b e l l e d M13  c o n t a i n i n g c e r u l o p l a s m i n genomic DNA  1.3  nitrocellulose  F o l l o w i n g a i r d r y i n g , f i l t e r s were baked a t 68°C f o r The dot b l o t s were probed  fragments  -  templates  (see S e c t i o n II.K f o r  conditions).  N u c l e a s e SI Mapping S i n g l e - s t r a n d e d probes were p r e p a r e d from recombinant  templates as d e s c r i b e d p r e v i o u s l y  (see S e c t i o n I I . B . 4 ) .  p r o t e c t i o n a s s a y s (as d e s c r i b e d by Kay e t a l . ,  1986),  M13  F o r n u c l e a s e SI  100,000 - 150,000 8  cpm  o f s i n g l e - s t r a n d e d DNA  was  mixed w i t h 0.35  - 1.0  probe  ( s p e c i f i c a c t i v i t y = 10  yg of human l i v e r p o l y ( A )  volume of 30 y l h y b r i d i z a t i o n b u f f e r 400 mM  NaCI, 1 mM  EDTA).  80°C f o r 15 minutes  RNA  in a  (50% formamide, 10 mM  The h y b r i d i z a t i o n m i x t u r e was  NaCI, 2.5 mM  A f t e r d i g e s t i o n (60 minutes  ZnS0  A  final  PIPES pH  6.9,  then h e a t e d t o  and s u b s e q u e n t l y i n c u b a t e d a t 42°C f o r 12  F o l l o w i n g a n n e a l i n g , a s o l u t i o n (200 y l ) was Na A c e t a t e , 600 mM  +  cpm/yg)  hours.  added t h a t c o n t a i n e d 70  mM  and 150 - 200 U of n u c l e a s e SI.  a t 37°C), i n t a c t probe DNA  was  by the a d d i t i o n o f 30 y l of a s o l u t i o n c o n t a i n i n g 100 mM  precipitated  EDTA, 4 M  ammonium a c e t a t e and 100 yg of tRNA p e r ml, f o l l o w e d by 230 y l isopropanol.  R e a c t i o n p r o d u c t s were s e p a r a t e d by e l e c t r o p h o r e s i s on a  6%  d e n a t u r i n g p o l y a c r y l a m i d e g e l (see S e c t i o n II.F.3.2) and were v i s u a l i z e d  73  by exposure t o Kodak XK-1 f i l m o v e r n i g h t a t -70 C w i t h an i n t e n s i f y i n g screen.  J.  CHROMOSOME MAPPING Chromosome l o c a l i z a t i o n s t u d i e s  f o r the human c e r u l o p l a s m i n  pseudogene were performed i n c o l l a b o r a t i o n w i t h Dr. John Hamerton (Department o f Human G e n e t i c s , U n i v e r s i t y o f Manitoba) u s i n g somatic c e l l h y b r i d s which had been c h a r a c t e r i z e d cytogenetic 1985).  and isozyme a n a l y s i s  DNA from c u l t u r e d c e l l  e t a l . (1986).  human-hamster  p r e v i o u s l y by  (Donald e t a l . , 1983; R i d d e l l e t a l . ,  l i n e s was i s o l a t e d as d e s c r i b e d  DNA (5 yg) from each h y b r i d  by R i d d e l l  l i n e , as w e l l as c o n t r o l  human p l a c e n t a l and hamster DNA was d i g e s t e d  wth EcoRI, e l e c t r o p h o r e s e d on  1% agarose g e l s and t r a n s f e r r e d t o Zetaprobe (Bio-Rad) a c c o r d i n g t o t h e 32 manufacturer's s p e c i f i c a t i o n s .  B l o t s were probed w i t h a  P-labeled  r e s t r i c t i o n fragment s p e c i f i c f o r the human c e r u l o p l a s m i n pseudogene (see Section  I I . K f o r h y b r i d i z a t i o n and washing  conditions).  K.  SUMMARY OF HYBRIDIZATION/WASHING CONDITIONS  K.1  Genomic Southern B l o t  Hybridization 32  DNA DNA  fragments were d e t e c t e d by h y b r i d i z a t i o n t o  probes as d e s c r i b e d  by Kan and Dozy (1978).  wetted w i t h 3XSSC and then p r e h y b r i d i z e d solution containing  50% formamide,  T r i s - H C l pH 7.5, 10X Denhardts ficoll,  Membranes were f i r s t  f o r 2 - 4 hours a t 37°C i n a  6XSSC, 1 mM EDTA, 0.1% SDS, 10 mM  (DH) s o l u t i o n (1XDH i s 0.02% BSA, 0.02%  0.02% p o l y v i n y l p y r r o l i d o n e ) ,  yg/ml denatured h e r r i n g  P-labelled  0.05% sodium pyrophosphate, 100  sperm DNA, and 25 yg/ml p o l y ( A )  +  RNA.  74  Hybridizations  were c a r r i e d out i n the above b u f f e r , w i t h the a d d i t i o n of  denatured probe.  A f t e r h y b r i d i z a t i o n , which was a l l o w e d t o p r o c e e d f o r 24  - 36 hours a t 37°C, b l o t s were washed i n 2XSSC, 1XDH, and then washed t w i c e f o r 45 minutes each a t 50°C i n 0.1XSSC, 0.1% SDS.  Following  a  f i n a l room temperature r i n s e i n 0.1XSSC, f i l t e r s were a i r d r i e d and exposed t o f i l m  (see below).  F o r t h e chromosome l o c a l i z a t i o n  human-hamster h y b r i d p a n e l s were p r e h y b r i d i z e d formamide, 3XSSPE, (1XSSPE i s 0.1 mM  study,  o v e r n i g h t a t 42°C i n 50%  EDTA, 10 mM N a H P 0 2  4  pH 7.0 and  0.18 M NaCI), 1% SDS, 0.5% n o n - f a t powdered m i l k , 10% d e x t r a n s u l f a t e and 200 yg/ml salmon sperm DNA.  The h y b r i d i z a t i o n r e a c t i o n was c a r r i e d out  o v e r n i g h t i n the same b u f f e r w i t h the a d d i t i o n of n i c k - t r a n s l a t e d probe a t a concentration  o f 20 ng/ml.  Following  h y b r i d i z a t i o n , b l o t s were washed  t w i c e i n 2XSSC a t room temperature f o r a t o t a l of 10 minutes.  Filters  were then washed f o r 15 minutes i n 0.2XSSC, 0.1% SDS a t 55°C, and f i n a l l y f o r 15 minutes i n 0.2XSSC, 0.1% SDS a t 55°C. K.2  Hybridization For  C o n d i t i o n s Other Than f o r Genomic Southern B l o t s  a l l other h y b r i d i z a t i o n s  f i l t e r s were p r e h y b r i d i z e d a t 68°C f o r 1 - 4  hours.  68°C i n 6XSSC, 2XDH, 1 mM ( a t l e a s t 1 x 10 cpm/yg).  6  using  d o u b l e - s t r a n d e d DNA  i n a solution containing  6XSSC, 2XDH s o l u t i o n  F i l t e r s were then h y b r i d i z e d  overnight at  EDTA, 0.5% SDS. and the denatured probe  cpm/ml, w i t h s p e c i f i c a c t i v i t y > 0.5 x 10  Following  probes,  DNA  8  h y b r i d i z a t i o n , f i l t e r s were r i n s e d once a t room o  temperature i n 2XSSC, and then washed t h r e e times a t 68 C i n 1XSSC, 0.5% SDS f o r 30 - 40 minutes each, and f i n a l l y r i n s e d i n 2XSSC a t room temperature.  75  For 5 ' - l a b e l l e d oligodeoxyribonucleotide c a r r i e d out a t 37°C f o r 2 - 1 6 F i l t e r s were then h y b r i d i z e d oligonucleotide  probes, p r e h y b r i d i z a t i o n  was  hours i n 6XSSC, 2XDH, and 0.2% SDS.  a t 68°C w i t h t h e a d d i t i o n o f 5'-end l a b e l e d  ( a t l e a s t 1 x 10^ cpm/ml, w i t h s p e c i f i c a c t i v i t y  10^  cpm/pmole).  out  a t room temperature f o r 15 minutes, and then t w i c e f o r 15 minutes each  a t 37°C.  A l l washes c o n t a i n e d  ^  T h i s was f o l l o w e d  6XSSC, and were u s u a l l y c a r r i e d  by h i g h e r temperature washings a t 40 -  55°C, depending on t h e base c o m p o s i t i o n o f t h e o l i g o n u c l e o t i d e . A f t e r washing, n i t r o c e l l u l o s e f i l t e r s described  from a l l h y b r i d i z a t i o n s  above were a i r - d r i e d and exposed o v e r n i g h t  or Kodak X-Omat AR f i l m  t o e i t h e r Kodak XK-1  ( t h e l a t t e r b e i n g a p p r o x i m a t e l y 5 - f o l d more  s e n s i t i v e ) a t -70°C w i t h i n t e n s i f y i n g s c r e e n s .  76  I I I . RESULTS A.  CHARACTERIZATION OF THE HUMAN PRECERULOPLASMIN  A.1  I n i t i a l Screening  cDNA  o f a Human L i v e r cDNA L i b r a r y  Two hundred thousand recombinant c l o n e s from a human l i v e r library  cDNA  ( p r o v i d e d by Dr. S t u a r t O r k i n a t C h i l d r e n ' s H o s p i t a l , Harvard  U n i v e r s i t y ) were screened o l i g o n u c l e o t i d e mixtures  a t h i g h d e n s i t y by u s i n g p o o l I I and p o o l I I I (corresponding  t o amino a c i d r e s i d u e s 937 - 942  and 962 - 967 o f t h e c e r u l o p l a s m i n p r o t e i n sequence r e s p e c t i v e l y ( s e e F i g u r e s 5 and 7) as h y b r i d i z a t i o n probes.  One recombinant  clone  ( d e s i g n a t e d phCP-1) h y b r i d i z e d s p e c i f i c a l l y t o b o t h o l i g o n u c l e o t i d e mixtures.  R e s t r i c t i o n endonuclease mapping o f t h e p u r i f i e d p l a s m i d showed  t h a t phCP-1 c o n t a i n e d an i n s e r t o f approximately P s t l s i t e o f t h e pKT218 v e c t o r .  2.7 Kbp c l o n e d  into the  Subsequent DNA sequence a n a l y s i s (see  S e c t i o n I I I . A . 3 ) showed t h a t the phCP-1 i n s e r t c o n t a i n e d DNA encoding amino a c i d r e s i d u e s 202 - 1046 o f plasma c e r u l o p l a s m i n  (Takahashi  et a l . ,  1984), i n a d d i t i o n t o a 123 bp 3' u n t r a n s l a t e d r e g i o n and a p o l y ( A ) (see F i g u r e s 5 and 6 ) .  tract  I n o r d e r t o i s o l a t e a c l o n e ( s ) c o d i n g f o r t h e 5'  r e g i o n o f t h e c e r u l o p l a s m i n mRNA, t h e cDNA l i b r a r y was r e s c r e e n e d u s i n g a 322 bp H a e l l l - P s t l fragment as h y b r i d i z a t i o n probe (Probe A; see F i g u r e 6).  T h i s fragment was d e r i v e d from t h e 5' end o f t h e phCP-1 i n s e r t .  s i n g u l a r c l o n e was i d e n t i f i e d and found  A  t o c o n t a i n DNA c o r r e s p o n d i n g t o  amino a c i d r e s i d u e s 202 - 432 o f c e r u l o p l a s m i n .  Therefore,  the l a t t e r  c l o n e extended no f u r t h e r 5* than phCP-1. A.2  I s o l a t i o n o f cDNA Clones  Encoding the 5' End o f Human  Ceruloplasmin  F o r t h e i s o l a t i o n o f a c l o n e ( s ) c o n t a i n i n g cDNA sequence corresponding  t o the remainder o f the c e r u l o p l a s m i n mRNA, two  77  F i g u r e 5.  Schematic summary o f t h e c l o n i n g o f t h e human p r e c e r u l o p l a s m i n  cDNA. The phCP-1 c l o n e ( d e r i v e d from t h e S t u a r t O r k i n human l i v e r cDNA l i b r a r y ) encodes amino a c i d r e s i d u e s 202 - 1046 o f c e r u l o p l a s m i n , f o l l o w e d by a 123 bp 3* u n t r a n s l a t e d r e g i o n ( c r o s s - h a t c h e d bar) and a p o l y ( A ) tail.  The XhCP-1 c l o n e ( i s o l a t e d from a randomly-primed human l i v e r  cDNA l i b r a r y ) was found t o c o n t a i n sequence c o r r e s p o n d i n g  t o amino a c i d  r e s i d u e s 1 - 380 o f plasma c e r u l o p l a s m i n , preceded by a 19 amino a c i d signal peptide peptide ( s o l i d bar). s i g n a l peptide cleavage.  An arrow i d e n t i f i e s  the s i t e of  Stars i n d i c a t e the p o s i t i o n s of s y n t h e t i c  o l i g o n u c l e o t i d e probes used t o s c r e e n t h e r e s p e c t i v e cDNA l i b r a r i e s .  long  OLIGO dT PRIMER USED IN FIRST STRAND CDNA SYNTHESIS  RANDOM PRIMER USED IN FIRST STRAND CDNA SYNTHESIS  • DOUBLE-STRANDED CDNA  I  DOUBLE-STRANDED CDNA  CLONED INTO pKT 218 PLASMID  t  CLONED INTO XgtlO ECjORI  Ec,oR1  •  Xhaf  J  l__l_  ZrMpolyA  •  phCP-1  XhCP-1 3"NON-CODIN(jl -SEQUENCE /  SIGNAL SEQUENCE -57-  EqoRI  600  1200  1800  2400  3000  200  400  600  800  1000  i NUCLEOTIDES  3321  SIGNAL PEPTIDE I  -19'  J  AMINO ACIDS  1046  oo  79  randomly-primed cDNA l i b r a r i e s were c o n s t r u c t e d first was  of t h e s e , c l o n e d i n t o the EcoRI s i t e of the phage v e c t o r  screened  isolated  by plaque h y b r i d i z a t i o n u s i n g a 1071  h y b r i d i z e d to the 322 the phCP-1 cDNA i n s e r t one was  found  mixture,  fragment positive 13  fragment d e r i v e d from the 5' end  (Probe A, F i g u r e 6 ) .  of  Of these l a t t e r c l o n e s , o n l y  t o h y b r i d i z e s p e c i f i c a l l y t o the p o o l I o l i g o n u c l e o t i d e  which corresponds  ceruloplasmin  Of the 16  Southern b l o t a n a l y s i s showed t h a t  bp H a e l l l - P s t l  The  XgtlO,  bp P s t l - E c o R I  from phCP-1 (Probe B, F i g u r e 6) as a probe.  c l o n e s t h a t were i d e n t i f i e d ,  t o the amino-terminal  (see F i g u r e s 4 and  F i g u r e s 5 and 6) was DNA  (see S e c t i o n I I . C . 2 ) .  6).  T h i s c l o n e ( d e s i g n a t e d XhCP-1;  see  c h a r a c t e r i z e d by r e s t r i c t i o n endonuclease mapping  sequence a n a l y s i s (see below) and was  of a p p r o x i m a t e l y  6 amino a c i d s of plasma  1.2  Kbp,  corresponding  the mature c e r u l o p l a s m i n p r o t e i n .  found  t o c o n t a i n an EcoRI  insert  to amino a c i d r e s i d u e s 1 - 380  T h i s sequence was  and  of  preceded by a s i g n a l  p e p t i d e of 19 amino a c i d r e s i d u e s b e g i n n i n g w i t h a p u t a t i v e i n i t i a t o r m e t h i o n i n e (see F i g u r e s 5 and  7).  A second randomly-primed human l i v e r cDNA l i b r a r y was the EcoRI s i t e of the phage v e c t o r X g t l l and identify  screened  s c r e e n i n g was  a 150  c l o n e s were i d e n t i f i e d subsequently analysis,  purified  i t was  920  bp,  from the 5' end  of  F i v e p o s i t i v e cDNA  ( d e s i g n a t e d XhCP-2 to XhCP-6) and were to homogeneity.  From r e s t r i c t i o n  determined t h a t these c l o n e s c o n t a i n e d 1.1  5'  The probe used f o r l i b r a r y  bp E c o R I - H i n d l l fragment o b t a i n e d  the XhCP-1 c l o n e ( d e s i g n a t e d Probe C, F i g u r e 6 ) .  bp,  i n o r d e r to  a c l o n e ( s ) c o n t a i n i n g the n u c l e o t i d e sequence of the  u n t r a n s l a t e d r e g i o n of the c e r u l o p l a s m i n mRNA.  780  constructed i n  Kbp,  730,  and  1.0  kbp,  endonuclease EcoRI i n s e r t s of  f o r XhCP-2 to XhCP-6  80  F i g u r e 6.  R e s t r i c t i o n Map and Sequencing S t r a t e g y  Preceruloplasmin The  cDNA  Clones.  l o n g e r b a r s below the r e s t r i c t i o n map r e p r e s e n t  and XhCP-1 t h a t t o g e t h e r (hatched  f o r Human  i n c l u d e r e g i n s coding  t h e c l o n e s phCP-1  f o r the leader  peptide  b a r ) , t h e plasma p r o t e i n (open b a r ) , and t h e 3' u n t r a n s l a t e d  sequence ( s o l i d b a r ) .  Arrows i n d i c a t e t h e e x t e n t and d i r e c t i o n o f  n u c l e o t i d e sequence o b t a i n e d  from v a r i o u s M13 c l o n e s .  Restriction  fragment probes A, B, and C, which were used i n l i b r a r y s c r e e n i n g ( s e e t e x t f o r d e t a i l s ) a r e i n d i c a t e d d i r e c t l y below t h e r e s t r i c t i o n map bars).  The P s t l and EcoRl  (solid  s i t e s i n parentheses r e s u l t from t h e c l o n i n g  p r o c e d u r e s used i n t h e c o n s t r u c t i o n o f t h e cDNA l i b r a r i e s .  Kb, K i l o b a s e s .  CO n X  r.  (EcoRI)  c a  c Q.  C  a  rr o o  o o  LU  LU  rr o o  LU  Hindi  PROBE C (Pstl) (Pstl)  EcoRI  PROBE B  Haelll  PROBE A IphCPI  iXhCPI  o  0.6  1.2  1.8  2.4  3.0  3.6  Nucleotides (kb) CO  I-  1  82  respectively.  Subsequent DNA sequence a n a l y s i s (see below) showed t h a t  XhCP-2 and \hCP-6 extended an a d d i t i o n a l 19 and 38 bp r e s p e c t i v e l y , 5' to t h e p r e v i o u s l y c h a r a c t e r i z e d XhCP-1 c l o n e A.3  DNA Sequence A n a l y s i s o f Human  (see Figure 8 ) .  Preceruloplasmin  The complete n u c l e o t i d e sequence o f t h e phCP-1 and X.hCP-1 cDNA i n s e r t s was determined u s i n g t h e s t r a t e g y shown i n F i g u r e 6.  The m a j o r i t y  of t h e sequence o f phCP-1 was o b t a i n e d by a n a l y s i s o f randomly-sheared fragments c l o n e d i n t o M13.  The remainder o f t h e phCP-1 sequence, as w e l l  as t h e e n t i r e XhCP-1 n u c l e o t i d e sequence was determined by a n a l y s i s o f s p e c i f i c r e s t r i c t i o n endonuclease fragments c l o n e d  i n t o M13 v e c t o r s .  complete n u c l e o t i d e sequence o f these two cDNA c l o n e s t h a t t o g e t h e r human p r e c e r u l o p l a s m i n  i s shown i n F i g u r e 7.  on b o t h s t r a n d s .  encode  The p o s i t i o n o f each  n u c l e o t i d e was determined an average o f 3.4 times, was o b t a i n e d  The  and 62% o f t h e sequence  In t h e r e g i o n where they  overlap  ( n u c l e o t i d e r e s i d u e s 648 t o 1197), the sequences o f phCP-1 and XhCP-1 were found t o be i d e n t i c a l . Nucleotide  residues 1 - 5 7  code f o r an amino t e r m i n a l  leader  sequence, which i s removed p r i o r t o the appearance o f c e r u l o p l a s m i n i n plasma (Takahaski  e t a l . , 1984).  Nucleotides  58 - 3195 o f the cDNA  sequence encode the plasma form o f c e r u l o p l a s m i n 1984).  (Takahaski  et a l . ,  The open r e a d i n g frame i s f o l l o w e d by a 'TGA' stop codon (encoded  by n u c l e o t i d e s 3196 - 3198), a 3* u n t r a n s l a t e d r e g i o n o f 123 bp ( n u c l e o t i d e r e s i d u e s 3199 - 3321) and a p o l y ( A )  tail.  sequence c o n t a i n s a p u t a t i v e p o l y a d e n y l a t i o n s i g n a l , s i t u a t e d 14 n u c l e o t i d e s upstream of the p o l y ( A )  The 3' u n t r a n s l a t e d 'ATTAAA', which i s  tract.  83  F i g u r e 7.  N u c l e o t i d e Sequence o f Human P r e c e r u l o p l a s m i n  cDNA.  The sequence was determined by a n a l y s i s o f the o v e r l a p p i n g shown i n F i g u r e 6 (see t e x t f o r d e t a i l s ) . sequence o f human p r e c e r u l o p l a s m i n  s o l i d diamonds.  sites  (Takahashi  s i t e i s shown by a s o l i d arrow. e t a l . , 1984) a r e r e p r e s e n t e d by  Boxed sequences a r e complementary t o o l i g o n u c l e o t i d e  probes u s e d t o s c r e e n cDNA l i b r a r i e s . is underlined.  acid  i s i n d i c a t e d above t h e DNA sequence.  The p u t a t i v e s i g n a l p e p t i d a s e cleavage P o t e n t i a l carbohydrate  The p r e d i c t e d amino  clones  The p o l y a d e n y l a t i o n s i g n a l ATTAAA  84  Lya Glu Ly» Hit Tyr Tyr l i e G CIAAA CAA k i d CAT W r  60  tit  ATT G  75  H i s Sar Asn l i s Tyr Lau G i n Asn Gly Pro Aap Arg H e Gly Arg Leu Tyr Ly» Lya A l a Lau Tyr Lau Gin Tyr Thr Aap G l u Thr Phe Arg Thr Thr 11a G l u Lya Pro V a l Trp Leu G l y Pha Lau G l y Pro I l a l i e Lya A l a G l u CAT TCC AAT ATC TAT CTT CAA AAT GGC CCA GAT ACA ATT CGG AGA CTA TAT AAG AAG GCC CTT TAT CTT CAG TAC ACA GAT GAA ACC TTT AGG ACA ACT ATA GAA AAA CCG GTC TGG CTT GGG TTT TTA CCC OCT ATT ATC AAA GCT GAA 165 1B0 1M 110 225 240 255 270 285 300  A  90 100 110 120 130 Thr Gly Aap Lya V a l Tyr V a l Hia Leu Lya Asn Lau A l a Ear Arg Pro Tyr Thr Pha Mia Sar Hia G l y I l a Thr Tyr Tyr Lya G l u H i s G l u Gly A l a H e Tyr Pro Aap Asn Thr Thr Aap Pha G i n Arg A l a Aap Aap Lya V a l Tyr ACT GGA GAT AAA GTT TAT GTA CAC TTA AAA AAC CTT GCC TCT AGG CCC TAC ACC TTT CAT TCA CAT GGA ATA ACT TAC TAT AAG GAA CAT GAG GGG GCC ATC TAC CCT GAT AAC ACC ACA GAT TTT CAA AGA GCA GAT GAC AAA GTA TAT 315 330 345 360 175 390 405 420 *35 450 140 150 160 170 180 Pro Gly Glu G i n Tyr Thr Tyr Nat Lau Lau A l a Thr G l u G l u G i n Sar Pro Gly G l u Gly Aap G l y Aan Cya V a l Thr Arg l i e Tyr Hia Ear Hia I l a Asp A l a Pro Lys Aap H e A l a Ear Gly Lau I l a Gly Pro teu I l a I l a Cya CCA GGA GAG CAG TAT ACA TAC ATG TTG CTT GCC ACT GAA GAA CAA AGT CCT GGG GAA GGA GAT GGC AAT TGT GTG ACT AGG ATT TAC CAT TCC CAC ATT GAT GCT CCA AAA GAT ATT GCC TCA GGA CTC ATC GGA CCT TTA ATA ATC TGT  G l u Aap Phe Gin G l u Ser Aan Arg Met Tyr Ear V a l Asn Gly Tyr Thr Pha G l y Ear Leu Pro Gly Leu Ear Net Cya A l a G l u Aap Arg Val Lya Trp Tyr Leu Phe Gly Het G l y Aan G l u V a l Aap V a l H i s A l a A l a Pha Phe Hia GAA GAC TTC CAG GAG ACT AAC AGA ATG TAT TCT GTG AAT GGA TAC ACT TTT GGA AGT CTC CCA GGA CTC TCC ATG TGT GCT GAA GAC AGA GTA AAA TGG TAC CTT TTT GGT ATG GGT AAT GAA GTT GAT GTG CAC GCA GCT TTC TTT CAC  C l y G i n A l a Leu Thr Aan Lya Aan Tyr Arg H e Aap Thr H e Aan Leu Pha Pro A l a Thr Lau Pha Asp A l a Tyr Hat V a l A l a G i n Aan Pro G l y G l u Trp Het Leu Ser Cya G i n Asn Leu Asn Hia Leu Lya A l a Gly Leu G i n A l a GGG CAA GCA CTC ACT AAC AAG AAC TAC CGT ATT GAC ACA ATC AAC CTC TTT CCT GCT ACC CTG TTT GAT GCT TAT ATG GTG GCC CAG AAC CCT GGA GAA TGG ATG CTC AGC TGT CAG AAT CTA AAC CAT CTC AAA GCC GGT TTG CAA GCC 915 930 945 960 975 990 1.005 1,020 1,035 1,050  4  340 350 Pha Pha G i n V a l G i n G l u Cya ABn Lys Ser Ser Ser Lya Aap Aan H e Arg G l y Lys H i s V a l Arg fi TTT TTC CAC CTC CAG GAG TGT AAC AAG TCT TCA TCA AAG CAT AAT ATC CGT GGC AAG CAT GTT AGA C 1,065 1,080 1.095 1,110  360 370 4^ 380 :"yr Tyr H e A l a A l a Glu G l u H e l i e Trp Aan Tyr Ala Pro Ser G l y H e Aap H a Phe Thr Lya C l u Aan Lau Thr A l a TAC TAC ATT GCC GCT GAG GAA ATC ATC TGG AAC TAT GCT CCC TCT GGT ATA GAC ATC TTC ACT AAA GAA AAC TTA ACA GCA 1,125 1,140 1,155 1,170 1,185 1,200  390 400 410 420 430 Pro Gly Sar Asp Sar A l a Vel Phe Pha G l u G i n G l y Thr Thr Arg H e G l y Gly Sar Tyr Lya Lya Leu Val Tyr Arq G l u Tyr Thr Asp Ala Ser Phe Thr Asn Arg Lys G l u Arg G l y Pro G l u G l u G l u H i s Leu Gly H e Leu Gly CCT GGA AGT GAC TCA C C C GTG T T T TTT GAA CAA GCT ACC AC* AGA ATT GGA GGC TCT TAT AAA AAC CTG CTT TAT CCT CAC TAC ACA GAT GCC TCC TTC ACA AAT CCA AAG CAC ACA GGC CCT GAA GAA GAC CAT CTT GGC ATC CTG GGT 1,215 1,230 1,245 1,260 1,175 1,290 1,305 1,320 1,335 1,350  Pro Val H e Trp A l a G l u V a l G l y Asp Thr H e Arg Val Thr Phe His Aan Lya Gly A l a Tyr Pro Leu Ser H e G l u Pro H e Gly Val Arg Phe Asn Lys Asn Asn G l u G l y Thr Tyr Tyr Ser Pro Asn Tyr Aan Fro G i n Ser Arg CCT GTC ATT TGG GCA GAG GTG GGA GAC ACC ATC AGA GTA ACC TTC CAT AAC AAA GGA GCA TAT CCC CTC AGT ATT GAG CCG ATT GGG CTG AGA TTC AAT AAG AAC AAC GAG GGC ACA TAC TAT TCC CCA AAT TAC AAC CCC CAG AGC AGA 1,365 1,380 1,395 1,410 1,425 1,440 1,455 1,470 1,485 1,500 490 500 510 520 530 Sar V«l Pro Pro Sor A l a Ser Hia Val A l a Pro Thr G l u Thr Pha Thr Tyr G l u Trp Thr Val Pro Lya G l u V a l Gly Pro Thr Aan A l a Aap Pro Val Cys Leu A l a Lys Met Tyr Tyr Ser A l a V a l Aap Pro Thr Lyo Asp H e Phe AGT GTG CCT CCT TCA GCC TCC CAT GTG GCA CCC ACA GAA ACA TTC ACC TAT GAA TGG ACT GTC CCC AAA GAA GTA GGA CCC ACT AAT GCA GAT CCT GTG TGT CTA GCT AAG ATG TAT TAT TCT GCT GTG GAT CCC ACT AAA GAT ATA TTC 1.S1S 1,530 1,545 1,560 ' 1,575 1.S90 1,605 1,620 1,635 1,650 540 550 560 570 560 Thr Gly Leu H e G l y Pro Met Lys H e Cys Lya Lys Gly Ser Leu Hia A l a Aan Gly Arg G i n Lya Aap V a l Aap Lys C l u Phe Tyr Leu Phe Pro Thr Val Phe Asp G l u Asn G l u Ser Leu Leu Leu G l u Asp Asn H e Arg Met Phe ACT GGG CTT ATT GGG CCA ATG AAA ATA TGC AAG AAA GGA AGT TTA CAT GCA AAT GGG AGA CAG AAA GAT GTA GAC AAG GAA TTC TAT TTG TTT CCT ACA GTA TTT GAT GAG AAT GAG AGT TTA CTC CTG GAA GAT AAT ATT AGA ATG TTT 1,665 1,680 1,695 1,710 1,725 1,740 1,755 1,770 1,7B5 1,800 590 600 610 620 630 Thr Thr A l a Pro Asp G i n V a l Asp Lya G l u Aap G l u Aap Phe G i n Glu Sar Aan Lya Met H i s Ser Met Asn Gly Pha Het Tyr Gly Aan Gin Pro Gly Leu Thr Met Cys Lys Gly Asp Ser Val Val Trp Tyr Leu Phe Ser A l a G l y ACA ACT GCA CCT GAT CAG GTG GAT AAG GAA GAT GAA GAC TTT CAG CAA TCT AAT AAA ATG CAC TCC ATG AAT GGA TTC ATG TAT GGG AAT CAG CCG GGT CTC ACT ATG TGC AAA GGA GAT TCC GTC GTC TGG TAC TTA TTC AGC GCC GGA 1,815 1,830 1,845 1,860 1,875 1,890 1,905 1,920 1,935 1,950 640 650 660 670 680 Aan G)u A l a Asp V a l H i s Gly H e Tyr Phe Ser Gly Aan Thr Tyr Leu Trp Arg C l y C l u Arg Arg Asp Thr A l a Asn Leu Phe Pro Gin Thr Ser Leu Thr Leu H i s Met Trp Pro Aap Thr G l u C l y Thr Phe Asn Val C l u Cys Leu AAT GAG GCC GAT CTA CAT GGA ATA TAC TTT TCA GGA AAC ACA TAT CTC TCC AGA CCA GAA CCC AGA GAC ACA CCA AAC CTC TTC CCT CAA ACA AGT CTT ACG CTC CAC ATG TGG CCT GAC ACA GAG CGG ACT TTT AAT GTT CAA TGC CTT 1,965 1,980 1,995 2,010 2,025 2,040 2,055 2.070 2,085 2,100 690 700 710 720 730 Thr Thr Asp Hia Tyr Thr C l y G l y Met Lys G i n Lys Tyr Thr Val Kan G i n Cya Arg Arg Gin Ser C l u Aap Sar Thr Pha Tyr Leu Gly G l u Arg Thr Tyr Tyr l i e A l a Ala Val G l u Val G l u Trp Aap Tyr Ser Pro C l n Arg G l u AC* ACT GAT CAT TAC ACA CGC GCC ATG AAG CAA AAA TAT ACT GTG AAC CAA TGC AGC CCG CAG TCT GAG GAT TCC ACC TTC TAC CTG GGA GAG ACG ACA TAC TAT ATC GCA GCA GTG GAG GTG GAA TGG GAT TAT TCC CCA CAA AGG GAG 2,115 2,130 2,145 2,160 2.17S 2,190 2,205 2,220 2,235 2,250 Trp C l u Lys G l u Leu H i s H i s Leu G i n G l u G i n Asn Val Ser Aan A l a Phe Lau Aap Lys C l y G l u Phe Tyr H e Gly Ser Lys Tyr Lys Lya Val V a l Tyr Arg G i n Tyr Thr Asp Ser Thr Phe Arg V a l Pro Val G l u Arg Lys A l a TGG CAA AAC CAG CTG CAT CAT TTA CAA GAG CAG AAT GTT TCA AAT CCA TTT TTA GAT AAG GGA GAG TTT TAC ATA GGC TCA AAG TAC AAG AAA GTT GTG TAT CGG CAG TAT ACT GAT AGC ACA TTC CGT GTT CCA CTG GAG AGA AAA CCT 2,265 2,280 2,295 2,310 2,325 2,540 2,355 2,370 2,385 2,400 790 800 810 820 830 C l u C l u G l u Hia Leu C l y H e Lou Gly Pro G i n Leu Hia A l a Aap Val Gly Asp Lys V a l Lya H e H e Phe Lya Aan Met A l a Thr Arg Pro Tyr Ser H e H i s A l a H i s C l y Val G i n Thr G l u Ser Ser Thr V a l Thr Pro Thr Leu CAA CAA GAA CAT CTG GGA ATT CTA GGT CCA CAA CTT CAT GCA GAT GTT GGA GAC AAA GTC AAA ATT ATC TTT AAA AAC ATG GCC ACA AGG CCC TAC TCA ATA CAT GCC CAT GGG GTA CAA ACA GAG AGT TCT ACA GTT ACT CCA ACA TTA 2,415 2,430 2,445 2,460 2,475 2,490 2,SOS 2,520 2,535 2,550 840 850 860 870 880 Pro C l y G l u Thr Leu Thr Tyr V o l Trp Lys H e Pro G l u Arg Ser G l y A l a Gly Thr G l u Aap Ser A l a Cys l i e Pro Trp A l a Tyr Tyr Ser Thr V a l Asp G i n Val Lys Aap Leu Tyr Ser Gly Leu H e Gly Pro Leu H e V a l Cys CCA GCT GAA ACT CTC ACT TAC GTA TGG AAA ATC CCA GAA AGA TCT CCA GCT GCA ACA GAG GAT TCT CCT TGT ATT CCA TGG GCT TAT TAT TCA ACT GTG GAT CAA GTT AAG GAC CTC TAC AGT CCA TTA ATT GGC CCC CTG ATT GTT TGT 2,565 2,580 2,595 2,610 2,625 2,640 2,655 2,670 2,685 2,700 890 900 910 920 930 Arg Arg Pro Tyr Leu Lya Val Phe Asn Pro Arg Arg Lya Lau C l u Phe A l a Lau Leu Pha Lau V a l Pha Aap G l u Aan G l u Sar Trp Tyr Leu Asp Asp Asn H e Lys Thr Tyr Ser Asp Hia Pro C l u Lys Val Asn Lya Asp Asp G l u GGA AGA CCT TAC TTG AAA GTA TTC AAT CCC AGA AGG AAG CTG GAA TTT CCC CTT CTG TTT CTA GTT TTT GAT GAC AAT GAA TCT TCC TAC TTA GAT GAC AAC ATC AAA ACA TAC TCT GAT CAC CCC GAG AAA GTA AAC AAA GAT GAT GAG 2.715 2,730 2,745 2,760 2,775 2,790 2,805 2,820 2,835 2,B50 940 950 960 970 980 Glu Phe H e C l u Sar Asn Lya Met Hia A l a l i e Asn Gly Arg Met Ph* G l y Asn Lsu G i n Gly Lau Thr Met Hia V a l C l y Aap G l u Val Asn Trp Tyr Leu Het Gly Met Gly Aan Glu I l a Aap Leu Hia Thr Val Hia Phe H i s Gly GAA TTC ATA CAA AGC |AAT AAA ATG CAT GCT ATfr AAT GGA AGA ATG TTT GCA AAC CTA CAA GGC CTC ACA ATG CAC GTC GGA GAT GAA GTC |AAC TCC TAT CTG ATC*GG> ATG CGC AAT GAA ATA CAC TTA CAC ACT GTA CAT TTT CAC GGC 2.865 2,880 2,895 2,910 2,925 2,940 2,955 2,970 2,985 3,000  i  | (  |  990 1,000 1,010 1,020 1,030 Ear Pha Gin Tyr Lys Hia Arg Gly V a l Tyr Sar Ser Aap Val Phe Aap I l a Phe Pro Gly Thr Tyr G i n Thr Lau G l u Mat Pha Pro Arg Thr Pro C l y H e Trp Leu Leu Hia Cys H i s V a l Thr Asp H i s H e Hia A l a Gly Met CAT AGC TTC CAA TAC AAG CAC AGG GGA GTT TAT AGT TCT GAT CTC TTT GAC ATT TTC CCT GGA ACA TAC CAA ACC CTA GAA ATG TTT CCA ACA ACA CCT GGA ATT TGG TTA CTC CAC TGC CAT GTG ACC CAC CAC ATT CAT GCT GGA ATG 3,015 3,030 3,045 3,060 3,075 3,090 3,105 3,120 3,135 3,150 1,040 1,046 G l u Thr Thr Tyr Thr V a l Leu G i n Aan G l u Aap Thr Lya 8«r C l y STOP CAA ACC ACT TAC ACC GTT CTA CAA AAT GAA GAC ACC AAA TCT GCC TCA ATG AAA TAA ATT GGT GAT AAG TGC AAA AAA GAG AAA AAC CAA TCA TTC ATA ACA ATC TAT GTG AAA GTG TAA AAT AGA ATG TTA CTT TGG AAT GAC TAT AAA 3,165 3,180 3,195 3,210 3,225 3,240 3.25S 3,270 3,285 3,300  85  Figure  8.  Comparative a n a l y s i s o f cDNA c l o n e s XhCP-2 t o XhCP-6.  E c o R l i n s e r t s i s o l a t e d from t h e above c l o n e s were s u b c l o n e d i n t o M13 and  characterized  previously  by T - t r a c k i n g  characterized  Probe C (an E c o R I - H i n d l l  analysis.  Their positions  XhCP-1 cDNA c l o n e a r e shown.  r e l a t i v e to the  The l o c a t i o n of  r e s t r i c t i o n fragment used as a h y b r i d i z a t i o n  probe f o r l i b r a r y s c r e e n i n g ) i s i d e n t i f i e d by a s o l i d b a r .  Arrowheads  i n d i c a t e t h a t t h e c l o n e s extend 3' t o Probe C ( s e e t e x t f o r d e t a i l s ) . XhCP-2 and XhCP-6 were found t o extend 19 and 38 bp r e s p e c t i v e l y 5' t o XhCP-1.  The a d d i t i o n a l n u c l e o t i d e  clones i s given.  sequence c o n t a i n e d i n t h e s e two  5 -»~XhCP-1  ;  (EcoRi)  -38  + 1  _ _ _  Probe C  Hindi l j y  •XhCP-2 •XhCP-S •XhCP 4 •XhCP-5 ••XhCP 6  CO as  87  E c o R l i n s e r t s i s o l a t e d from XhCP-2, XhCP-3, XhCP-4, XhCP-5, and  XhCP-6 c l o n e s were subcloned i n t o M13 and i n i t i a l l y c h a r a c t e r i z e d by  T - t r a c k i n g a n a l y s i s (Sanger e t a l . , 1980) i n o r d e r p o s i t i o n s r e l a t i v e t o t h e XhCP-1 cDNA c l o n e these c l o n e s  (see F i g u r e 8 ) .  Two o f  (XhCP-2 and XhCP-6) were found t o extend 19 and 38  n u c l e o t i d e s r e s p e c t i v e l y f u r t h e r 5' than XhCP-1. sequence c o n t a i n e d A.4  t o determine t h e i r  i n these two c l o n e s  Ceruloplasmin  The a d d i t i o n a l 5*  i s a l s o presented  i n F i g u r e 8.  Transcript Analysis  A N o r t h e r n b l o t p r e p a r e d u s i n g samples o f b o t h p o l y ( A )  +  RNA  from human l i v e r and t o t a l RNA p u r i f i e d from HepG2 c e l l s was h y b r i d i z e d t o 32 the  P - l a b e l l e d cDNA i n s e r t i s o l a t e d from phCP-1.  I n b o t h l i v e r and  HepG2 RNA samples, t h e cDNA h y b r i d i z e d t o an mRNA s p e c i e s t h a t i s 3700 + 200 n u c l e o t i d e s  i n size (Figure 9).  s p e c i e s o f 4500 + 250 n u c l e o t i d e s ( F i g u r e 9, lane 1 ) .  The cDNA h y b r i d i z e d t o an a d d i t i o n a l  i n t h e human l i v e r p o l y ( A )  +  RNA  sample  Given t h a t e u k a r y o t i c mRNAs u s u a l l y c o n t a i n p o l y ( A )  t a i l s o f 180 - 200 n u c l e o t i d e s  ( P e r r y , 1976), b o t h o f t h e c e r u l o p l a s m i n  t r a n s c r i p t s a r e much l a r g e r i n s i z e than t h a t r e q u i r e d t o encode t h e e n t i r e coding et  region of the ceruloplasmin  polypeptide  chain  (Takahashi  a l . , 1984). A N o r t h e r n b l o t c o n t a i n i n g samples o f b o v i n e l i v e r p o l y ( A ) 32  probed a t h i g h s t r i n g e n c y u s i n g t h e phCP-1 ( r e s u l t s n o t shown). s p e c i e s was d e t e c t e d  +  P-labelled insert isolated  RNA was  from  I n t e r e s t i n g l y , only a s i n g l e h y b r i d i z i n g  which was 3800 + 200 n u c l e o t i d e s  in size.  88  Figure  9.  B l o t h y b r i d i z a t i o n a n a l y s i s o f human c e r u l o p l a s m i n mRNA.  RNA was s e p a r a t e d by e l e c t r o p h o r e s i s  i n a denaturing  agarose/formaldehyde g e l and t r a n s f e r r e d t o n i t r o c e l l u l o s e .  The f i l t e r  32 was h y b r i d i z e d  with  P - l a b e l l e d phCP-1.  The f i l t e r was exposed t o  X-ray f i l m f o r 18 hours a t -70°C w i t h i n t e n s i f y i n g s c r e e n s . 10 yg o f human l i v e r p o l y ( A ) RNA.  +  Lane 1,  RNA; l a n e 2, 20 yg o f t o t a l HepG2  cell  The p o s i t i o n s o f H i n d l l l fragments o f X phage DNA u s e d as s i z e  markers a r e shown.  Sizes are given i n Kilobases (Kb).  Kb 9.5 6.7  4.3 2.2 2.0  0.59  90  B.  CHARACTERIZATION OF THE WILD-TYPE HUMAN CERULOPLASMIN GENE  B.l  I s o l a t i o n and R e s t r i c t i o n Endonuclease Mapping o f Genomic Initally,  units  f i v e genomic e q u i v a l e n t s  [1 x 1 0  6  Clones  plaque-forming  ( p f u ) ] o f t h e M a n i a t i s human genomic phage l i b r a r y , c o n s t r u c t e d i n  the phage lambda v e c t o r Charon 4A, were screened by u s i n g t h e phCP-1 cDNA c l o n e as a h y b r i d i z a t i o n probe.  As d e s c r i b e d p r e v i o u s l y , t h i s cDNA  encodes amino a c i d r e s i d u e s 202 - 1046 o f c e r u l o p l a s m i n , and a l s o  contains  a 3* u n t r a n s l a t e d r e g i o n o f 123 bp, t e r m i n a t i n g w i t h a p o l y ( A ) t a i l .  The  f o u r t e e n p o s i t i v e c l o n e s i d e n t i f i e d from t h i s s c r e e n were p u r i f i e d t o homogeneity, and a n a l y z e d by r e s t r i c t i o n endonuclease mapping.  On t h i s  b a s i s , t h e c l o n e s were r e p r e s e n t a t i v e o f o n l y two i n d e p e n d e n t l y - d e r i v e d genomic c l o n e s .  One o f these c l o n e s  pseudogene f o r human c e r u l o p l a s m i n  ( d e s i g n a t e d 3^10) corresponded  (see S e c t i o n I I I . C ) .  ( d e s i g n a t e d XWT1; see F i g u r e 10) corresponded  The o t h e r  to a clone  t o the wild-type  c e r u l o p l a s m i n gene, as was determined i n i t i a l l y by r e s t r i c t i o n endonuclease mapping and Southern b l o t a n a l y s i s , and subsequently confirmed  by u s i n g DNA sequence a n a l y s i s  phage c l o n e s c o r r e s p o n d i n g Maniatis  (encoding  r e s c r e e n e d u s i n g t h e 1.2 Kbp EcoRI i n s e r t  t h e s i g n a l p e p t i d e and amino a c i d s 1 - 380 o f t h e  mature p r o t e i n ) as a h y b r i d i z a t i o n probe. clone  In order to obtain  t o t h e 5' end o f t h e c e r u l o p l a s m i n gene, t h e  l i b r a r y was s u b s e q e n t l y  from \hCP-l  (see below).  From t h i s s c r e e n , one p o s i t i v e  ( d e s i g n a t e d XWT2; see F i g u r e 10) was i d e n t i f i e d . To i s o l a t e a d d i t i o n a l c e r u l o p l a s m i n genomic c l o n e s , 1 x 10^ p f u  from a second genomic l i b r a r y  ( c o n s t r u c t e d i n t h e lambda phage v e c t o r EMBL  32 3) were screened,  using  P - l a b e l l e d cDNA i n s e r t s from both phCP-1 and  X.hCP-1 as h y b r i d i z a t i o n probes.  From t h i s s c r e e n , 10 d i f f e r e n t  91  F i g u r e 10.  Partial  r e s t r i c t i o n map  human c e r u l o p l a s m i n A complete map (S), H i n d l l l Xbal  ( X ) , Kpn  and i n t r o n / e x o n o r g a n i z a t i o n of the  gene.  of the 45 Kbp r e g i o n u s i n g the enzymes BamHl (B) , S a i l  (H), and EcoRI (E) i s shown; an incomplete (K), S s t l  ( T ) , and B a l l  (L) i s a l s o g i v e n .  map  f o r AccI ( A ) ,  Exons l o c a t e d  w i t h i n the c o d i n g r e g i o n a r e shown as b l a c k boxes i n a b a r above the r e s t r i c t i o n map labelled  and a r e numbered  (A - N).  ( L l - L4) p r e s e n t  1-14.  Corresponding  Approximate p o s i t i o n s o f i n t r o n s (LA - LD) and exons i n the 5* u n t r a n s l a t e d r e g i o n of the gene a r e i n d i c a t e d ;  l e n g t h s of these exons and i n t r o n s a r e undetermined. XWT1  i n t r o n s are a l s o  - XWT9 a r e shown below t h e r e s t r i c t i o n map.  Genomic phage  clones  Open c i r c l e s a t the  ends o f EMBL 3 phage c l o n e s r e p r e s e n t Sau3A s i t e s , w h i l e s o l i d diamonds a t the ends o f c l o n e s i s o l a t e d from the M a n i a t i s l i b r a r y linkers.  The r e l a t i v e  fragments i d e n t i f i e d corresponding  r e p r e s e n t EcoRI  p o s i t i o n s of the 4.6 Kbp and 2.4 Kbp EcoRI  from genomic Southern a n a l y s i s a r e a l s o shown,  t o the 3' end of the gene (see t e x t f o r d e t a i l s ) .  represents 1 Kilobase p a i r .  The s c a l e  93  recombinant c l o n e s were o b t a i n e d .  Seven o f these were found  t o correspond  to t h e w i l d - t y p e c e r u l o p l a s m i n l o c u s ( d e s i g n a t e d XWT3 t o XWT9; see F i g u r e 1 0 ) , w h i l e t h e remaining and  three clones  ( d e s i g n a t e d 3iJ;21, 34*29,  3»|/9) were i d e n t i f i e d as human c e r u l o p l a s m i n pseudogene c l o n e s (see  Section III.C). DNA was prepared and  from s m a l l s c a l e l y s a t e s o f w i l d - t y p e phage c l o n e s ,  a n a l y z e d by m u l t i p l e r e s t r i c t i o n enzyme d i g e s t i o n s and Southern b l o t  a n a l y s e s u s i n g a p p r o p r i a t e h y b r i d i z a t i o n probes d e r i v e d from cDNA c l o n e s . On t h i s b a s i s , a p a r t i a l r e s t r i c t i o n enzyme map o f c l o n e d genomic DNA was c o n s t r u c t e d clones i d e n t i f i e d genomic DNA.  ( F i g u r e 10).  ceruloplasmin  The n i n e w i l d - t y p e  ( F i g u r e 10) span a r e g i o n o f approximately  ceruloplasmin 45 Kbp of  Southern b l o t a n a l y s i s u s i n g M13 probes d e r i v e d from t h e 3'  end o f t h e c e r u l o p l a s m i n cDNA ( i . e . , c o n t a i n i n g sequences d e r i v e d from n u c l e o t i d e r e s i d u e s 2565 - 3321, F i g u r e 7) i n d i c a t e d t h a t t h e 3' end of the c e r u l o p l a s m i n gene was n o t r e p r e s e n t e d B.2  L o c a l i z a t i o n o f Intron/Exon Ceruloplasmin  i n t h e above phage c l o n e s .  J u n c t i o n s Corresponding  to the  Coding Region  In order t o i d e n t i f y e x o n - c o n t a i n i n g to t h e cDNA sequence of human p r e c e r u l o p l a s m i n ,  DNA sequences  corresponding  either specific  r e s t r i c t i o n endonuclease fragments h y b r i d i z i n g t o cDNA-derived probes o r fragments generated ligated  by s o n i c a t i o n of a p p r o p r i a t e genomic c l o n e s were  i n t o M13 v e c t o r s f o r a n a l y s i s (see T a b l e 3, s e c t i o n I I . G . 2 ) .  In  the l a t t e r case, coding sequences were i d e n t i f i e d by s c r e e n i n g M13 32 subclones  with  P - l a b e l l e d cDNA fragments as h y b r i d i z a t i o n probes.  i n t r o n / e x o n boundary sequences f o r exons 1 - 1 4 a r e p r e s e n t e d All  The  i n T a b l e V.  i n t r o n s c h a r a c t e r i z e d t o date a r e s p l i c e d a c c o r d i n g t o the GT..AG r u l e  T a b l e V.  N u c l e o t i d e S e q u e n c e o f I n t r o n / E x o n J u n c t i o n s i n t h e Human C e r u l o p l a s m i n G e n e .  3'  5' S P L I C E DONOR EXON  U  not  INTRON LD  determined  g  c  t  t  t  c  t  c  c  c  t  t  c  8  8  a  a  a  8  A  A  G  G  8  t  c  t  t  8  t  t  t  t  t  c  t  t  t  g  c  a  g  G  G  A  A  II  a  g  G  G  G  C  I  a  g  A  T  T  C  I I I  A  C  A  C  G  A  G  G  g  t  a  a  3  A  A  A  G  8  t  a  c  8  t  a  a  g  a  D  a  t  a  8  g  a  E  c  4  C  T  C  C  5  A  A  A  G  8  a  a  c  2  t  a  A  t  g  t  B  c  a  t  t  8  c  a  t  8  t  t  g  c  t  t  c  c  t  a  t  C  a  a  t  a  8  t  a  a  c  t  t  t  a  a  c  t  c  c  c  t  c  t  g  c  t  c  t  t  g  a  c  t  t  a  c  a  g  C  T  G  T  t  c  t  t  t  c  t  g  t  t  t  c  a  t  t  t  c  a  8  C  C  G  G  t  g  t  t  t  t  t  c  c  c  c  c  t  8  a  g  T  G  A  C  II  c  t  c  t  t  t  g  t  t  c  c  c  a  8  G  T  C  C  I  t  8  a  c  c  t  t  t  c  t  c  a  c  a  8  G  T  G  T  I  t  t  t  8  t  t  t  t  a  t  t  t '•. a c  a  g  G  A  T  G  0  t  t  t  c  c  c  a  a  c  t  t  t  t  8  C  C  A  T  I  t  c  t  t  a  t  t  t  c  c  a  c  g  G  G  A  C  II  c  a  c  t  t  t  t  8  G  T  C  C  I  6  G  A  A  G  8  t  a  a  t  t  F  a  a  c  a  c  7  C  T  G  G  g  t  g  a  g  t  G  a  a  t  t  c  8  A  G  A  A  g  t  g  a  g  t  H  t  a  a  t  8  9  A  C  A  G  8  t  a  a  8  t  I  t  c  a  t  10  C  A  C  T  g  t  a  a  g  t  J  t  t  a  K  t  t  t  11  not : d e t e r m i n e d  12  A  G  A  A  13  C  T  A  14  C  C  A  t  t  L  t  g  t  M  c  t  c  N  8  t  a  a  G  8  t  a  G  g  t  a  ACCEPTOR CODON PHASE  1  8  SPLICE  not t  t  c  c  t  a  c  a  t  c  c  a  g  c  c  a  I  determined t  c  t  t  E x o n s e q u e n c e i s shown i n u p p e r c a s e , w h i l e i n t r o n s e q u e n c e i s g i v e n i n l o w e r c a s e . The codon phase r e f e r s t o t h e p o s i t i o n o f t h e i n t r o n r e l a t i v e t o t h e codon t r i p l e t (Sharp, 1981), i . e . 0 : i n t r o n o c c u r s between codons, I : i n t r o n o c c u r s a f t e r t h e f i r s t n u c l e o t i d e o f t h e codon, and I I : i n t r o n o c c u r s f o l l o w i n g t h e second n u c l e o t i d e o f t h e codon. " L " d e s i g n a t e s i n t r o n s o r e x o n s o c c u r r i n g w i t h i n t h e 5' u n t r a n s l a t e d r e g i o n .  Table V I .  Frequencies  of N u c l e o t i d e s a t Intron/Exon  Junctions.  DONOR FREQUENCIES  +4  +3  +2  +1  -1  -2  -3  -4  -5  -6  C  2 6 0 5  2 5 3 3  2 9 0 2  8 2 1 2  13 0 0 0  0 0 13 0  2 11 0 0  1 8 0 2  9 1 3 0  0 3 9 1  CON  N  A C  A  G  G  T  R  A  G  T  G  A T  ACCEPTOR FREQUENCIES  -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 -10  -9  -8  -7  -6  -5  -4  -3  -2  -1  +1  +2  +3  +4  0 13 0 0  13 0 0 0  7 2 1 3  4 2 5 2  5 4 2 2  3 1 3 6  A  G  G  N  N  N  A T  2 5 4 2  0 3 6 4  0 2 8 3  3 0 7 3  1 0 10 2  2 2 4 5  1 3 8 1  2 0 4 7  0 2 9 2  0 1 10 2  2 0 8 3  1 3 4 5  0 3 6 3  1 0 9 3  2 0 7 4  1  C  0 4 6 3  5  1 1 1 10  CON  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  Y  N  Y  G  5  2  The f r e q u e n c i e s of n u c l e o t i d e s o c c u r r i n g a t i n t r o n / e x t r o n boundaries i n the human c e r u l o p l a s m i n gene are compared to the consensus sequence (CON) of Mount (1982). "N" r e p r e s e n t s G, A, T o r C, w h i l e "Y" denotes p y r i m i d i n e r e s i d u e s ( i . e . C o r T ) . S p l i c e j u n c t i o n s a r e l o c a t e d between -1 and +1.  96  (Breathnach  and Chambon, 1981;  Cech, 1983).  The  s u r r o u n d i n g s p l i c e j u n c t i o n s i n e u k a r y o t i c RNA genes (Breathnach  and Chambon, 1981;  consensus sequences  polymerase  Mount, 1982)  II-transcribed  were a l s o found  t o be i n  agreement w i t h those c h a r a c t e r i z e d i n the human c e r u l o p l a s m i n gene (see Table V I ) . determined  Although  the s p l i c e donor and a c c e p t o r sequences were not  f o r i n t r o n s K and L r e s p e c t i v e l y , i n t r o n p o s i t i o n s i n b o t h  cases c o u l d be a s s i g n e d u n e q u i v o c a l l y from sequences o b t a i n e d on side.  one  I n t r o n s p r e s e n t i n t h e c o d i n g sequence c o r r e s p o n d i n g t o t h e 3*  end  of the gene ( i . e . the r e g i o n c o n t a i n i n g n u c l e o t i d e r e s i d u e s 2656 - 3321 the cDNA sequence) were not determined,  of  s i n c e phage c l o n e s c o n t a i n i n g t h i s  a r e a were not o b t a i n e d from genomic s c r e e n i n g (see F i g u r e 10). 'B.3  P a r t i a l N u c l e o t i d e Sequence A n a l y s i s of the Human C e r u l o p l a s m i n Gene P a r t i a l DNA  the c o d i n g r e g i o n was Approximately DNA  sequence of the c e r u l o p l a s m i n gene c o r r e s p o n d i n g t o determined  by a n a l y s i s of M13  40% of the sequence d a t a was  subclones.  determined  sequence o b t a i n e d on one s t r a n d o n l y was  on b o t h s t r a n d s , and  determined  at l e a s t  twice.  S i z e s and r e l a t i v e p o s i t i o n s of i n t r o n s and exons i n the gene a r e summarized i n T a b l e V I I and shown s c h e m a t i c a l l y i n F i g u r e 10.  Intron  s i z e s were determined  mapping,  i n a l l cases by r e s t r i c t i o n endonuclease  and t h e r e f o r e r e p r e s e n t c l o s e approximations i n s i z e from 129  to a c t u a l s i z e s .  - 255 bp, w i t h a c a l c u l a t e d average  l e n g t h of 183  I n t r o n s t y p i c a l l y were found to be v a r i a b l e i n s i z e , r a n g i n g approximately determined  800 bp to 9.5  Kbp.  The p a r t i a l amino a c i d  from the c h a r a c t e r i z a t i o n of exons 1 - 1 4  Exons range  was  bp.  from  sequence i d e n t i c a l to that  97  Table V I I .  S i z e s and p o s i t i o n s o f i n t r o n s and exons w i t h i n the ceruloplasmin  Exon  Nucleotide Position  Length (bp)  Intron  gene.  Nucleotide Position  Length (bp)*  1  -12  146  158  A  159  -  2459  2300  2  2460  2708  248  B  2709  -  4759  2050  3  4760  4973  213  C  4974  -  5774  800  4  5775  5949  174  D  5950  -  7400  1450  5  7401  7656  255  E  7657  -  8957  1300  6  8958  9130  172  F  9131  - 12231  3100  7  12232  12372  140  G  12373  - 14273  1900  8  14274  14427  153  H  14428  - 15578  1150  9  15579  15791  212  I  15792  - 25192  9400  10  25193  25344  151  J  25345  - 26645  1300  11  26646  26859  213  K  26860  - 28110  1250  12  28111  28319  208  L  28320  - 29920  1600  13  29921  30061  140  M  30062  - 31311  1250  14  31312  31441  129  N  31442  ->32242  * S i z e s o f a l l i n t r o n s were e s t i m a t e d from r e s t r i c t i o n  enzyme a n a l y s i s .  800+  98  determined f o r t h e cDNA sequence c o r r e s p o n d i n g  to nucleotide residues 1 -  2555.  B.4  O r g a n i z a t i o n o f t h e 5' End o f t h e Human C e r u l o p l a s m i n  B.4.1  Comparison o f genomic and cDNA sequence d a t a .  n u c l e o t i d e sequence o f t h e XhCP-6 cDNA c l o n e  Gene  I n i t i a l l y , the  ( F i g u r e 8) was compared  w i t h o v e r l a p p i n g genomic DNA sequence ( c o r r e s p o n d i n g  to nucleotide  r e s i d u e s -288 - +412) d e r i v e d from s o n i c a t i o n o f a 3.1 Kbp EcoRI fragment (see T a b l e I I I ) . F i g u r e 11.  Alignment o f t h e n u c l e o t i d e sequences i s p r e s e n t e d i n  The observed d i v e r g e n c e  o f t h e genomic sequence w i t h t h a t o f  the cDNA sequence c o r r e l a t e d w i t h t h e p r e s e n c e o f a 3* consensus acceptor  splice  s i t e a t n u c l e o t i d e p o s i t i o n -13 i n t h e 5' u n t r a n s l a t e d r e g i o n o f  t h e human c e r u l o p l a s m i n subsequently B.4.2  confirmed  gene.  L o c a t i o n o f t h i s s p l i c e s i t e was  using nuclease  SI mapping a n a l y s i s (see below).  N u c l e a s e SI mapping a n a l y s i s o f exon 1.  s p l i c e acceptor  s i t e a t -13 was confirmed  The presence o f a  u s i n g the 700 bp genomic c l o n e  d e s c r i b e d above as a h y b r i d i z a t i o n probe f o r n u c l e a s e (see F i g u r e 12).  SI mapping a n a l y s i s  The observed p r o t e c t e d fragment o f 158 bp i n d i c a t e s the  p r e s e n c e o f an i n t r o n s p l i c e s i t e a t -13 bp, s i n c e a s p l i c e j u n c t i o n had been i d e n t i f i e d f o l l o w i n g n u c l e o t i d e r e s i d u e 146 i n t h e coding T h i s r e s u l t i s i n agreement w i t h DNA sequence data confirms  ( F i g u r e 11), and  t h a t t h e XhCP-6 cDNA c l o n e c o n t a i n s 26 bp c o r r e s p o n d i n g  exon i n t h e 5* u n t r a n s l a t e d B.4.3  region.  t o an  region.  Southern b l o t a n a l y s i s .  u n t r a n s l a t e d region (designated  The l o c a t i o n of an exon i n t h e 5'  L4; see F i g u r e s 8 and 10) was determined  by Southern b l o t a n a l y s i s o f an AccI/EcoRI d i g e s t of the 3.1 Kbp EcoRI  99  F i g u r e 11.  Comparison o f the sequence o f t h e \hCP-6 cDNA c l o n e w i t h  o v e r l a p p i n g genomic DNA sequence d e r i v e d from t h e phage c l o n e +1 i n d i c a t e s t h e f i r s t n u c l e o t i d e o f t h e i n i t i a t o r residue.  \WT2.  methionine  The p o i n t o f sequence d i v e r g e n c e between genomic and cDNA  sequences c o i n c i d e s w i t h t h e l o c a t i o n o f a 3' s p l i c e a c c e p t o r s i t e i n t h e genomic sequence ( v e r t i c a l arrow).  Sequences c o r r e s p o n d i n g t o exon L4  (from t h e cDNA) o r i n t r o n LD (from t h e genomic c l o n e ) a r e i n d i c a t e d .  100  ^  Intron LP  38  (  1  +  Q T C C G C C G C T T T C T C C C T T C G G A" A A G A A G G G G A A A A A A A  GENOMIC  CACTTCATTTCTTCTCAGGCTCCAAGAAGGGGAAAAAAA  cDNA  ~*  Exon L4  101  Figure  12.  Nuclease SI mapping a n a l y s i s o f exon 1.  A 700 bp fragment ( c o n t a i n i n g n u c l e o t i d e r e s i d u e s  -288 - +412 o f the  genomic DNA sequence) was used as a probe f o r SI n u c l e a s e p r o t e c t i o n analysis.  Following  h y b r i d i z a t i o n t o 0.5 yg o f human l i v e r  poly(A)  +  RNA and n u c l e a s e SI d i g e s t i o n , S l - r e s i s t a n t p r o d u c t s were s e p a r a t e d denaturing protected  polyacrylamide  g e l and v i s u a l i z e d by a u t o r a d i o g r a p h y .  band o f 158 bp was d e t e c t e d  A  ( l a n e 2 ) , c o r r e s p o n d i n g t o exon 1.  A H i n f l d i g e s t o f pBR322 was used as markers ( l a n e 1 ) . r e s u l t i n g fragments a r e g i v e n  on a  i n base p a i r s ( b p ) .  The s i z e s o f  102  bp 1631  506/517 396 344 298  220/221  154  103  F i g u r e 13.  Southern b l o t a n a l y s i s l o c a l i z i n g exon L4  i n the  5*  u n t r a n s l a t e d r e g i o n of human c e r u l o p l a s m i n . AccI/EcoRI ( p a n e l A, 3.1  Kbp  l a n e 1) or AccI  (panel A,  lane 2) d i g e s t s of  genomic EcoRI fragment c o n t a i n i n g exon 1 were t r a n s f e r r e d t o  n i t r o c e l l u l o s e and probed w i t h the XhCP-6 cDNA c l o n e .  In a d d i t i o n to  the pUC  v e c t o r band ( d e s i g n a t e d " P " ) , h y b r i d i z i n g s p e c i e s c o r r e s p o n d i n g  1.7  and  Kbp  the  0.85  Kbp  AccI fragments were d e t e c t e d .  fragments of phage X DNA  P o s i t i o n s of  used as s i z e markers are shown.  are g i v e n i n K i l o b a s e p a i r s (Kbp).  Hindlll  Fragment s i z e s  R e s t r i c t i o n a n a l y s i s of the 3.1  EcoRI (E) genomic fragment u s i n g AccI  to  (A) i s shown i n p a n e l B.  Kbp  The  r e l a t i v e p o s i t i o n s of exon L4 and exon 1 a r e i n d i c a t e d ; the l a t t e r exon i s d e s i g n a t e d by a s o l i d bar.  The p r e c i s e l o c a t i o n of exon L4 i s u n c e r t a i n ,  as i n d i c a t e d by the dashed l i n e s . transcription.  An arrow i d e n t i f i e s the d i r e c t i o n of  o 4>  105  genomic fragment ( F i g u r e 13; see above), u s i n g X.hCp-6 c l o n e d g e n e r a t e a h y b r i d i z a t i o n probe.  i n t o M13 t o  The h y b r i d i z a t i o n and washing  used were s i m i l a r t o those employed f o r o l i g o n u c l e o t i d e probes Section I I . B . l ) . detected, previous Kbp  (see  H y b r i d i z i n g DNA fragments o f 1.7 Kbp and 0.85 Kbp were  t h e l a t t e r o f which c o n t a i n s Southern a n a l y s i s (data not  EcoRI genomic fragment c o n t a i n s B.4.4  conditions  Exon 1 (see F i g u r e  shown).  10) based on  T h i s i n d i c a t e s t h a t t h e 3.0  a t l e a s t two exons (see F i g u r e  10).  N o r t h e r n b l o t a n a l y s i s o f the 5' end o f the human  ceruloplasmin  gene.  F o r f u r t h e r a n a l y s i s o f the 5' end gene o r g a n i z a t i o n ,  EcoRI fragments d e r i v e d  from X.WT7 and X.WT2 phage c l o n e s were s u b c l o n e d  (see T a b l e I I I ) and used as h y b r i d i z a t i o n probes f o r N o r t h e r n b l o t analysis of p o l y ( A )  +  RNA.  H y b r i d i z a t i o n t o b o t h 3.7 Kb and 4.5 Kb  c e r u l o p l a s m i n - s p e c i f i c t r a n s c r i p t s (see S e c t i o n II.A.4) was d e t e c t e d all  with  fragments t e s t e d (see F i g . 14) except the 1.4 Kbp EcoRI fragment.  h y b r i d i z a t i o n s i g n a l obtained  using  The  the 420 bp EcoRI probe was observed t o  be v e r y weak f o l l o w i n g a 5-day exposure, s u g g e s t i n g s h o r t exon sequence w i t h i n t h i s fragment.  For more p r e c i s e assignment o f  mRNA-encoding sequences i n the 5' u n t r a n s l a t e d fragment was subcloned f u r t h e r u t i l i z i n g  the p r e s e n c e o f a  segment, the 4.0 Kbp EcoRI  an i n t e r n a l H i n d l l l  site  (see  T a b l e I I I and F i g u r e 10), and the r e s u l t i n g two fragments were s u b s e q u e n t l y used as h y b r i d i z a t i o n probes f o r N o r t h e r n b l o t a n a l y s i s . shown i n F i g u r e Hindlll/EcoRI B.4.5  14A, h y b r i d i z a t i o n was d e t e c t e d  As  w i t h o n l y t h e 1.3 Kbp  fragment.  RNA dot b l o t a n a l y s i s .  w i t h i n the 5' u n t r a n s l a t e d  In o r d e r  t o l o c a l i z e f u r t h e r the exons  r e g i o n of the human c e r u l o p l a s m i n  genomic fragments i n pUC13 were subcloned i n t o M13 v e c t o r s  gene, EcoRI (see  Table  106  F i g u r e 14.  Northern  b l o t a n a l y s i s of the 5' end of the c e r u l o p l a s m i n gene.  Samples of human l i v e r p o l y ( A ) electrophoresed i n a denaturing  +  RNA  (10 ug each) were  agarose-formaldehyde g e l and t r a n s f e r r e d 32  to n i t r o c e l l u l o s e .  F i l t e r s were h y b r i d i z e d w i t h  and H i n d l l l / E c o R I (HE) the gene ( p a n e l A ) .  P - l a b e l l e d EcoRI  r e s t r i c t i o n fragments d e r i v e d from the 5' end  S i z e s of the two h y b r i d i z i n g RNA 32  designates  P - l a b e l l e d H i n d l l l fragments of X DNA.  a p o s i t i v e c o n t r o l , s i n c e t h i s 3.1  t o 3' d i r e c t i o n from l e f t  EcoRI (E) and H i n d l l l B;  to r i g h t .  (H) c o r r e s p o n d i n g  Kbp  transcription.  "C"  genomic fragment  B l o t s a r e shown s e q u e n t i a l l y i n a For c l a r i t y , a r e s t r i c t i o n map  in  to t h i s r e g i o n i s shown i n p a n e l  fragment s i z e s a r e g i v e n i n K i l o b a s e p a i r s (Kbp).  i d e n t i f i e s an EcoRI l i n k e r .  i n days.  species detected are given i n K i l o b a s e s ,  c o n t a i n s exon 1 (see t e x t f o r d e t a i l s ) . 5*  of  Fragment s i z e s of probes ( K i l o b a s e s ) a r e shown above  each b l o t ; b r a c k e t e d numbers r e p r e s e n t l e n g t h s of exposure times  based on p o s i t i o n s of  (E)  The b r a c k e t e d  An arrow i n d i c a t e s the d i r e c t i o n of  E  108  UJ-  ui-  iii-  10  a  t  109  F i g u r e 15.  RNA d o t b l o t a n a l y s i s o f the 5' u n t r a n s l a t e d r e g i o n o f the  c e r u l o p l a s m i n gene. Human l i v e r p o l y ( A )  +  RNA  (7.5 yg) was s p o t t e d onto n i t r o c e l l u l o s e  and probed w i t h M13 c l o n e s d e r i v e d from a p p r o p r i a t e r e s t r i c t i o n (see T a b l e I I I ) .  fragments  A p a r t i a l map o f t h e r e g i o n i s shown; a b b r e v i a t i o n s used  a r e E - EcoRI; S - Sau3A; F - H i n f l ; D - Ddel; H - H i n d l l l . E r e p r e s e n t s an EcoRI l i n k e r .  The b r a c k e t e d  The e x t e n t and d i r e c t i o n o f sequence  a n a l y s i s o f t h e M13 c l o n e s used f o r d o t b l o t h y b r i d i z a t i o n s a r e shown by t h i n arrows below t h e r e s t r i c t i o n map. d i r e c t i o n of t r a n s c r i p t i o n .  Corresponding  by d o t b l o t a n a l y s i s a r e shown. (Kbp).  The t h i c k arrow d e s i g n a t e s the exons L l , L2, and L3 i d e n t i f i e d  The s c a l e r e p r e s e n t s 0.1 K i l o b a s e p a i r s  Ill  III).  M13  c l o n e s were a n a l y z e d  were s u b s e q u e n t l y  initially  used to generate  The  results  probes f o r dot  blot  i n d i c a t e the p r e s e n c e of  l e a s t 4 exons i n the 5' u n t r a n s l a t e d sequence of the gene, w i t h  minimum of 2 s e p a r a t e exons w i t h i n the 1.5 Ll  sequence a n a l y s i s and  double-stranded  h y b r i d i z a t i o n a n a l y s i s ( F i g u r e 15). at  by DNA  and  L2;  see F i g u r e 10), one  fragment ( d e s i g n a t e d L3;  C.  ISOLATION AND  Kbp  EcoRI fragment  exon w i t h i n the 1.3  see F i g u r e 10)  p r e v i o u s l y w i t h i n the 1.7  Kbp  AccI  and  one  Kbp  a  (designated  Hindlll/EcoRI  exon (L4)  determined  fragment (see above).  COMPLETE CHARACTERIZATION OF A PSEUDOGENE  FOR  HUMAN CERULOPLASMIN C l  I s o l a t i o n of Genomic DNA Ceruloplasmin  Clones  C o n t a i n i n g the Human  Pseudogene  As d e s c r i b e d p r e v i o u s l y (see S e c t i o n I I I . B . l ) , one pseudogene c l o n e  ( d e s i g n a t e d 3v|»10) was  obtained  ceruloplasmin  from the s c r e e n i n g of a  human genomic l i b r a r y c o n s t r u c t e d i n the Charon 4A v e c t o r , u s i n g phCP-1 cDNA c l o n e as a h y b r i d i z a t i o n probe.  The  c l o n e as a pseudogene f o r human c e r u l o p l a s m i n was endonuclease mapping and was  subsequently  analysis  library  i d e n t i t y of t h i s based on  u s i n g DNA  (see f o l l o w i n g s e c t i o n s ) .  restriction  and  3^29)  sequence a n a l y s i s and n u c l e a s e Three a d d i t i o n a l pseudogene  were i s o l a t e d  i n the phage v e c t o r X EMBL 3 ,  cDNA c l o n e s as h y b r i d i z a t i o n probes.  A t o t a l of n e a r l y 21 Kbp  This SI  clones  from a human genomic  u s i n g b o t h phCP-1 and  XhCP-1  R e s t r i c t i o n enzyme mapping a n a l y s i s  i n d i c a t e d t h a t the l a t t e r t h r e e phage c l o n e s o v e r l a p p e d ( F i g u r e 16).  initial  Southern b l o t a n a l y s i s ( r e s u l t s not shown).  confirmed  ( d e s i g n a t e d 3<J/9, 3^21,  the  of contiguous  the 3iJ/10 c l o n e  genomic DNA  is  112  F i g u r e 16.  P a r t i a l R e s t r i c t i o n Map  Ceruloplasmin  Pseudogene.  The complete r e s t r i c t i o n map the r e s t r i c t i o n map 3*1*21, 3«|;29 ( i s o l a t e d Maniatis l i b r a r y ) .  and Sequencing S t r a t e g y f o r t h e Human  f o r EcoRI (E) i s shown.  The l i n e s  above  r e p r e s e n t the f o u r o v e r l a p p i n g phage c l o n e s 3»|/9, from the Geddes l i b r a r y ) , and 3i|/10 (from t h e The s o l i d b a r r e p r e s e n t s t h e r e g i o n o f the  c e r u l o p l a s m i n pseudogene t h a t i s homologous t o t h e c e r u l o p l a s m i n cDNA sequence.  Solid  c i r c l e s a t t h e ends o f EMBL 3 phage c l o n e s  represent  Sau3A s i t e s , w h i l e s o l i d squares a t t h e end o f the 3i|»10 phage c l o n e isolated  from t h e M a n i a t i s l i b r a r y r e p r e s e n t EcoRI l i n k e r s .  The  locations  of BamHl (B) and H i n d l l l (H) s i t e s used f o r s u b c l o n i n g and sequence a n a l y s i s a r e shown w i t h i n the 3»|/10 c l o n e ; the remainder of the map i s incomplete f o r t h e s e two enzymes.  The r e g i o n c o n t a i n i n g the B a m H l / H i n d l l l  fragment has been expanded below the r e s t r i c t i o n map.  Arrows below t h i s  r e g i o n i n d i c a t e the d i r e c t i o n and e x t e n t of n u c l e o t i d e sequence o b t a i n e d from v a r i o u s M13  clones.  chromosomal l o c a l i z a t i o n r e p r e s e n t s 1 Kbp.  Probe A was used as a h y b r i d i z a t i o n probe f o r s t u d i e s (see t e x t f o r d e t a i l s ) .  The s c a l e  ate  3^21 • 3^29 •  ' 3^10 •  1  1  L.  1—L  ' JJ  1  #-J—i  1  B  —  1  1—I  •  1  H  4  1  ,1 kbp, 1 Probe A  E ^ I ^- I  E I  1 kbp  E T  •  114  represented  by these  f o u r recombinant c l o n e s , w i t h t h e human  pseudogene mapping t o approximately  ceruloplasmin  1.7 Kbp w i t h i n t h i s r e g i o n  (see F i g u r e  16) . C.2  DNA Sequence A n a l y s i s o f t h e Human C e r u l o p l a s m i n  Pseudogene  The n u c l e o t i d e sequence o f t h e c e r u l o p l a s m i n pseudogene ( F i g u r e 17) was determined by u s i n g t h e s t r a t e g y o u t l i n e d i n F i g u r e 16. n u c l e o t i d e was determined an average o f 3.4 times, was o b t a i n e d  on b o t h s t r a n d s .  Nucleotides  Each  and 54% o f t h e sequence  53 - 1644 o f t h e pseudogene  sequence a r e v e r y s i m i l a r t o r e s i d u e s 1502 - 3318 o f t h e c e r u l o p l a s m i n cDNA ( n e a r l y 97% i d e n t i c a l ; see F i g u r e 1 7 ) , extending segment c o r r e s p o n d i n g clone  t o t h e 3' u n t r a n s l a t e d r e g i o n o f t h e phCP-1 cDNA  (see Section I I I . A ) .  by a p o l y ( A )  through t h e 123 bp  The pseudogene, however, i s n o t c h a r a c t e r i z e d  t r a c t a t t h e expected p o l y a d e n y l a t i o n s i t e .  DNA sequence  a n a l y s i s i n a 3' d i r e c t i o n r e v e a l e d t h e p r e s e n c e o f an u n u s u a l 54 bp segment, composed p r i m a r i l y o f repeated  CT d i n u c l e o t i d e s ( n u c l e o t i d e  r e s i d u e s 1867 - 1920; see F i g u r e 17). Following  t h e sequence c o r r e s p o n d i n g  t o t h e 3' u n t r a n s l a t e d r e g i o n o f  phCP-1, t h e next 43 bp o f pseudogene sequence ( n u c l e o t i d e s 1645 - 1687) correspond (designated  t o t h e 3' u n t r a n s l a t e d sequence o f a c e r u l o p l a s m i n Cp-1) c h a r a c t e r i z e d by Yang e t a l . (1986).  cDNA c l o n e  This clone  differs  from phCP-1 i n t h a t the 3' u n t r a n s l a t e d r e g i o n extends f o r an a d d i t i o n a l 120  bp p r i o r t o t h e p o i n t o f p o l y ( A )  addition.  F o l l o w i n g t h i s 43 bp  segment, t h e remainder of t h e pseudogene sequence p r i o r t o t h e p o l y ( C T ) segment shares  little  s i m i l a r i t y w i t h the 3* u n t r a n s l a t e d r e g i o n from the  cDNA c l o n e d e s c r i b e d by Yang e t a l . (1986).  115  F i g u r e 17.  N u c l e o t i d e sequence of the human c e r u l o p l a s m i n pseudogene and  comparison w i t h the c o r r e s p o n d i n g  r e g i o n of the c e r u l o p l a s m i n cDNA  sequence. The pseudogene sequence was determined by a n a l y s i s of the o v e r l a p p i n g c l o n e s shown i n F i g u r e 16.  I/E denotes p o s i t i o n s of  b o u n d a r i e s i n the w i l d - t y p e gene (see S e c t i o n I I I . B ) .  intron-exon The p o s i t i o n of the  AGCT i n s e r t i o n t h a t r e s u l t s i n a f r a m e s h i f t m u t a t i o n i s e n c l o s e d  i n a box.  The s i z e s and p o s i t i o n s of d e l e t i o n s (A) r e l a t i v e t o the c e r u l o p l a s m i n cDNA sequence a r e a l s o shown; the r e g i o n of the cDNA c o r r e s p o n d i n g 213 bp d e l e t i o n has been o m i t t e d .  The p l a c e s  (5* and 3') where the  pseudogene sequence d i v e r g e s from the w i l d - t y p e arrowheads (see t e x t f o r d e t a i l s ) .  sequence a r e i n d i c a t e d by  The cDNA sequence  n u c l e o t i d e s 1502 - 1864 and 2078 - 3318 of phCP-1. i d e n t i c a l n u c l e o t i d e s i n corresponding  t o the  represents  Asterisks indicate  p o s i t i o n s i n t h e two sequences.  Dashes were i n t r o d u c e d a t p o i n t s of i n s e r t i o n o r d e l e t i o n i n o r d e r t o maximize  homology.  1  20  ^  40  i  ATTCTGACATTAGAAAGCACACTTCA CCTCTCTAATGTGA C C T T T C T C A C A G *  60  80  100  120  140  G TG TG CCTC CTTC AG CTTCCCATG TGG CA CCCACAG A A A CATTCACCTATGAATGGA CTG TCC CCAAA GAAGTAGGA CCCACTAATG CAG A TCCTC TA TG G T G T G C C T C CTTCAG C C T C C C A T G T G G C A C C C A C A G A A A C A T T C A C C T A T G A A T G G A C T G T C C C C A A A G A A G T A G G A C C C A C T A A T G C A G A T C C T G T G T G  1520  160  1540  180  1560  1580  200  1600  220  240  T C T A G C T A A G A T G T A T T A T T C T G C T G T G G A T C C C A C T A A A G A T A T A T T C A C T G G G C T T A T T G G G C C A A T G A A A A T A T G C A A G A A A G G A A G T T T A CA T G C A T C T A G C T AAG ATG T A T T A T T C T G C T G T G G A T C C C ACT A A A G A T A T A T T C A C T G G G C T T A T T G G G C C A A T G A A A A T A T G C A A G A A A G G A A G T T T A C A T G C A  1620  260  1640  2B0  1660  1680  300  1700  320  340  A A T T G G A G A C A G A A A G A T G T A G A C A A G G A G T T C T A T T T G T T T C C T A T A G T A T T T A A T G A G AA T G A G G G T T T A C T C C T G G A A G A T A A T A T C A G A A T G T T T A  A A T G G G A G A C A G A A A G A T G T A G A C A A G G A A T T C T A T T T G T T T C C T A C A G T A T T T G A T G A G AA T G A G A G T T T A C T C C T G G A A G A T A A T A T T A G A A T G T T T A  1720  1740  1760  l/E;A 360  380  213 bp  1780  1B0O  1 420  400  440  C A A C T G C A C C T CftTCAGGTGGA T A A G G C A G A T G A A G A C T T T C A G G A A T C T A A T A A A A T G C A C T G G A C T T T T A A T G T T G A A T G C C T T A CAGCTJAG Clfc A T C CAACTGCACCTGATCAGGTGGATAAGGAAG ATGAAGA C T T T C A G GA A T C T A A T A A A A T G C A C T G GAC T T T T A A T G T T G A A T GCCTTAC A A C T  1820  460  1840  480  1860  500  2080  GATC  2100  520  540  A T T A C A C A G G C G G C A T G A A G C A A A A A T A T A C T G T G A A C C A A T G C A G G T G G CAGTCTGAGGATrcCACCTTCTACCTGGGAGAGAGG  ACATACTAT A T C G C  ATTACACAGGCWJCATGAAGCAAAAATATACTGTGAACCAATGCAGGCGGCAGT^  2120  560  2140  2160  SeO  600  2180  620  2200  640  AG C A G T G G A A G T G G A A T G G G A T T A T T C C C C A C A A A G G G A G T G G G A A A A G G A G C T G C A T C A T T T A C A A G A G C A G A A T G T T T C A A A T G C A T T T T T A G A T A A G A G C A G T G G A G G T G G A A T G G G A T T A T T C C C C A C A A A G GG A G T G G G A A A A G G A G C T G C A T C A T T T A C A A G A G C A G A A T G T T T C A A A T G C A T T T T T A G A T A A G  2220  660  2240  2260  680  2280  700  720  2300  740  G G A G A G T T T T A C A T A G G C T C A A A G T A C A A G A A A G T T G T G T A T C G G C A G T A T A C T G A T T G C A C G T T C C G T A T T C C A G T G G AG AG A A A A G C T G A A G A A G A A C G G A G A G T T T T A C A T A G G C T C A A A G T A C A A G A A A G T T G T G T A T C G G C A G T A T A C T G A T A G C A C A T T C C G T G T T C C A G T G G A G A G A A A A G C T G A A G A A G AA C  2320  760  2340  2360  7B0  23B0  800  820  2400  840  A T C T G G GA A T T C T A G G T C T A C A A C T T C A T G C A G A T G T T G G A G A C A A A G T C A A A A T T A T C T T T A A A A A C A T G A C C A C A A G G C C C T A C T C A A T A C A T G C C C A A T C T G G G A A T T C T A G G T C C A C A A C T T C A T G C A G A T G T T G G AGA C A A A G T C A A A A T T A T C T T T A A A A ACA T G G C C A C A A G G C C C T A C T C A A T A C A T G C C C A  2420  860  2440  2460  880  2480  900  920  2500  940  TGGGGTACAAACGGA G A G T T C T A C A T T T A T T C C A G C A T T A C C A G G T G A A A C T C T C AC T T AC C T ATGGAAAATCCCAGAAAGAT C T G G A GCTGGAACAGAG T G G G G T A C A A A C A G A G A G T T C TA C A G T T A C T C C A A C A T T A C C A G G T G A A A C T C T C A C T T A C G T A T G G A A A A T C C C A G A A A G A T C T G G A G C T G G A A C A G A G  2520  960  2540  2560  980  2580  1000  1020  2600 1040  G A T T C T G C T T G T A T T C C A T G G G C T T A C T A T T C A A C T G T G G A T C A A G T T A A G G A T C T C T A C A G T G GA T T A A T T G G C C C C C T G A T T G T T T G TCGAA G A CA T T GATTCTCCTTCTATTCCATGGGCTTATTATTCAACTG  2620 1060  2640  T G G A T C A A G T TA A G G A C C T C T A C A G T G G A T T A A T T G G C C C C C T G A T T G T T T G T O G A A G A C C T T  2660  1080  2680  1100  1120  2700 1140  A C T T G A A A G T A T T C A A T C C C A G A A A G A A A C T G G A A T T T A C C C T T C T G T T T C T A G T T T T T G A T G AG A A T G A A T C T T G G T A T T T A G A T G A C A A C A T C A A A A C  A C T T G A A A G T A T T C A A T C C C A G A A G G A A G C T G G AATTTq C C C T T C T G T T T C T A G T T T T T G ATGAGAATG AA T C T T G G T A C T T A G A T G A C A A C A T C A A A A C  2720 1160  2740  2760  1180  2780  1200  1220  2800 1240  A A A C T C T G A T C A C C C C A A G A A A G T A A A C A A A G A T G A T G A G G A A T T C A T A G A A A G C A A T A A A A T G C A T G C T G T T A A T G G A A G AA T G T T T G G A A A C C C A C A A A T A C T C T G A T C A C C C C G A G A A A G T A A A C A A A G A T G A T G A G G A A T T C A T A G A A A G C A A T A A A A T G C A T G C T A T T A A T G G A A G A A T G T T T G G A A A C C TA C A A  2820  2840  2860  2880  2900  Al7bp 1260  1280  1  1300  G G C C T C A C A A T G C A C A T G G GA GA T G A A G C C A A  1320  T G G G C G A T G A A A T AG A C T T A C A C A C T G T A C A T T T T C A C G G C C A T A G C T T C T  GGCCTCACAATGCACGTGGGAGATGAAGT C A A C T G G T A T C T G ATGGGAATGGGCAATGAAATAGA C T T A C A C A C T G T A C A T T T T C A C G G C C A TAGC T T C C  2920 1340  2940 1360  2960  2980  1380  1400  3000 1420  A A T A C A A G C A C A G G G C T C T T T A T A G T T C T G A T G T C T T T G A C A T T T T C C C T G G A A C A T A C C A A A C C C T A G A A A T G T T T C C A A G A A C A C C T G G AA T T T G G T T A A T A C A A G C A C A G G G G A G T T T A T A G T T C T G A T G T C T T T G A C A T T T T C C C T G G A A C A T A C C A A A C C C T A G A A A T G T T T C C A A G AA C A C C T G G A A T T T G G T T  3020 1440  3040 1460  3060  3080  1480  1500  3100 1520  A C T C C A C T G C C A T G T G A C T G A C C A C A T T C A T G C T G G E A T G G A A A C C A C T T A C A T T G TTCTAC A A A A T G A A G A C A C C A A G T C T G G CTGA A T G A A A T A C A T T ACTCCACTGCCATGTGACCGACCACATTCATGCTGGAATCGAAACCACTTACACCGTTCT  3120 1540  3140 1560  3160 1580  3180 1600  3200 1620  G G T G A T A A G T G G A A A A A A G A G A A A A A C C A A T G A T T C A T A A C A A T G T A T G T G A A A G T G T A A A A T A G A A TG T T A C T T T G G A A T G A C T A T A A A C A 1 T G AAAG A G G T G A T A A G T G G A A A A A A G AG A A A A A C C A A T G A T T C A T A A C A A T G T A T G T G A A A G T G T A A A A TAG AA T G T T A C T T T G G AA T G A CTA T A A A CATTAAAAG A  3220 1640  3240 1660  3260 1680  3280 •  1700  3300 1720  AG A C T G G A A A C A T A C A A C T T T G T G C A T T T G T G G G G G A A A A C T A T T A A T T T T T T C A G AG CA C T G T A G C G G T A G CCACAAAG C CAG C T G CCCATG TCCTTGG -GACTGGAG  3318 1740  1760  1780  CTCTACACACGGCCCACTCACTCACATGCCATCAGTGTAGGCTCTCA  1B40  I860  1800  1820  T T G T G T G T G T C C C C T T C T G C T T T C A T A G G C C C G G G C C A T T A G C A TTG T T T AGT  1B80  1900  1920  GTGAGAGAGGCAGGAGCTGCCGGGTTCCCCCCTCTCTCTCTCTCTCC CTCTCTCTCTCTCTCCXTrCTCTCTCTCrCTCTCT C T C T  117  The  5' end of the pseudogene sequence ( n u c l e o t i d e r e s i d u e s 10 -  see F i g u r e 17)  corresponds  to the sequence of 3' end  the w i l d - t y p e gene ( d a t a not shown). sequence (Breathnach  and Chambon, 1981;  p r i o r to n u c l e o t i d e r e s i d u e 53. corresponding Table V).  On the b a s i s of DNA  a n a l y s i s confirms Kbp  Cech, 1983)  t h a t the 2.6  acceptor  i s located  immediately  i n the w i l d - t y p e  from the w i l d - t y p e gene. Kbp  EcoRI fragment l o c a t e d d i r e c t l y 5' does not h y b r i d i z e t o  pseudogenes c h a r a c t e r i z e d t o date  p o i n t s of d i v e r g e n c e  the  (Vanin, 1985), the  a t the c e r u l o p l a s m i n pseudogene a r e not f l a n k e d by  repeats.  c e r u l o p l a s m i n cDNA i s a l s o shown i n F i g u r e 17.  2077 of the human c e r u l o p l a s m i n cDNA sequence.  and  A d e l e t i o n of 213  i n the pseudogene sequence, c o r r e s p o n d i n g  bp  was  to n u c l e o t i d e s 1865  amino a c i d r e s i d u e s 483  - 602  of the c e r u l o p l a s i n coding  of amino a c i d s u b s t i t u t i o n s ( F i g u r e 17).  continues  f o r an a d d i t i o n a l 9 amino a c i d r e s i d u e s .  see F i g u r e 17)  213  i n the bp d e l e t i o n  t h i s open r e a d i n g frame, which  i n s e r t i o n of 4 d u p l i c a t e d n u c l e o t i d e r e s i d u e s 448;  The  to  sequence.  O c c a s i o n a l base changes w i t h i n t h i s open r e a d i n g frame r e s u l t  i n the pseudogene sequence m a i n t a i n s  -  P r i o r to t h i s d e l e t i o n ,  the pseudogene sequence c o n t a i n s an open r e a d i n g frame c o r r e s p o n d i n g  occurrence  to  In c o n t r a s t  A comparison of the n u c l e o t i d e sequences of the pseudogene  observed  gene (see  Southern b l o t  a r e a of the w i l d - t y p e gene ( r e s u l t s not shown).  to most p r o c e s s e d  short d i r e c t  splice  sequence a n a l y s i s , n u c l e o t i d e r e s i d u e 9  EcoRI fragment (see F i g u r e 16)  corresponding  of i n t r o n p r e s e n t i n  T h i s s p l i c e j u n c t i o n i s i n the  p o s i t i o n to a s p l i c e s i t e present  marks the p o i n t o f d i v e r g e n c e  the 0.8  A consensus 3*  52;  then  At t h i s p o i n t , an  (AGCT) ( n u c l e o t i d e s 445  causes a f r a m e s h i f t mutation,  such t h a t a  TGA  -  118  t e r m i n a t i o n codon occurs  immediately f o l l o w i n g t h i s i n s e r t i o n .  The  remainder o f t h e pseudogene sequence i s s i m i l a r t o t h e phCP-1 cDNA c l o n e and  t h e cDNA c l o n e s r e p o r t e d by Yang e t a l . (1986) b u t c o n t a i n s a number  of base s u b s t i t u t i o n s compared w i t h the cDNA sequence. s m a l l d e l e t i o n o f 17 bp c o r r e s p o n d i n g of t h e cDNA ( s e e F i g u r e 17).  There i s a l s o a  t o n u c l e o t i d e r e s i d u e s 2942 - 2958  However, t h i s d e l e t i o n does n o t r e s u l t i n  t h e resumption o f an open r e a d i n g frame i n t h e pseudogene sequence. C.3  N u c l e a s e SI A n a l y s i s o f t h e Human C e r u l o p l a s m i n  Pseudogene  The p r e s e n c e o f t h e 213 bp d e l e t i o n i n t h e pseudogene sequence as compared t o t h e cDNA sequence was u t i l i z e d t o a n a l y z e pseudogene-specific  transcripts.  the presence of  A s i n g l e - s t r a n d e d DNA fragment (286  n u c l e o t i d e s i n l e n g t h ) d e r i v e d from t h e human c e r u l o p l a s m i n was used as a h y b r i d i z a t i o n probe f o r n u c l e a s e  SI a n a l y s i s .  cDNA sequence T h i s probe  corresponds t o a r e g i o n spanning n u c l e o t i d e r e s i d u e s 1927 - 2213 o f t h e cDNA sequence, thereby  c o n t a i n i n g 150 bp w i t h i n t h e d e l e t e d  i d e n t i f i e d i n t h e pseudogene sequence (see F i g u r e 18B). human l i v e r p o l y ( A )  +  region  P r o t e c t i o n of  RNA w i t h t h i s probe r e s u l t e d i n a s i n g l e p r o t e c t e d  band o f 286 bp, c o r r e s p o n d i n g  t o the wild-type  A d d i t i o n a l l y , a band c o r r e s p o n d i n g  transcript  ( F i g u r e 18A).  to t h e f u l l - l e n g t h probe was observed  ( F i g u r e 18A) which c o n t a i n s M13 sequences i n a d d i t i o n t o c e r u l o p l a s m i n sequences. nuclease  I f t h e pseudogene was t r a n s c r i b e d i n l i v e r , and assuming t h a t  SI c l e a v e s a l l s i n g l e - b a s e mismatches between t h e probe and the  p u t a t i v e t r a n s c r i p t , p r o t e c t e d DNA fragments of 47bpand 55 bp would be expected  (corresponding  t o n u c l e o t i d e s 453 - 499 and 501 - 555  r e s p e c t i v e l y ; see F i g u r e 17). s i z e , which would correspond  However, no p r o t e c t e d fragment o f a s m a l l e r t o an RNA s p e c i e s c o n t a i n i n g the 213 bp  119  F i g u r e 18.  Nuclease  SI t r a n s c r i p t a n a l y s i s f o r the c e r u l o p l a s m i n  pseudogene. P a n e l b shows the l o c a t i o n of the 286 bp probe used f o r SI n u c l e a s e p r o t e c t i o n a n a l y s i s r e l a t i v e t o the 213 bp d e l e t i o n observed pseudogene sequence.  i n the  P a r t o f t h i s probe (150 bp) i s w i t h i n the d e l e t e d  r e g i o n ( c o r r e s p o n d i n g t o n u c l e o t i d e s 1865 - 2077 o f the cDNA), w h i l e 136 bp o f t h e probe a r e 3' t o t h e d e l e t i o n . ( l a n e A) o r 0.35 yg ( l a n e B) of p o l y ( A )  F o l l o w i n g h y b r i d i z a t i o n t o 1 yg +  mRNA and n u c l e a s e  S l - d i g e s t i o n , n u c l e a s e - r e s i s t a n t p r o d u c t s were s e p a r a t e d on a d e n a t u r i n g p o l y a c r y l a m i d e g e l and v i s u a l i z e d by a u t o r a d i o g r a p h y corresponding observed,  t o t h e s i z e of the u n d i g e s t e d  (panel a ) .  f u l l - l e n g t h probe (FLP) was  as w e l l as a p r o t e c t e d band of 286 bp c o r r e s p o n d i n g  wild-type transcript.  A band  to the  A H i n f l d i g e s t of pBR322 was used f o r markers.  s i z e s of r e s u l t i n g fragments a r e g i v e n i n base p a i r s .  The  a  b A B 506/517 mm  | probe(286 bp)  1  150 bp  396 mm 344 2 9 8  —  *  —-FLP — — 2 8 6 bp  18^65  136 bp  "  2077 'wild-type transcript  ^ w v *  220/221 Oft  A  2 1 3 bp  -putative pseudogene transcript  121  F i g u r e 19.  Chromosome Mapping of the Human C e r u l o p l a s m i n Pseudogene U s i n g  Somatic C e l l H y b r i d A n a l y s i s . A 1.1  Kbp  p s e u d o g e n e - s p e c i f i c probe (Probe A; see F i g u r e 16)  h y b r i d i z e d t o E c o R I - d i g e s t e d DNA ( l a n e s 1-22). T a b l e 8.  from human-hamster h y b r i d c e l l  Numbering of the c e l l  E c o R I - d i g e s t e d hamster DNA  l i n e s corresponds  was  lines  t o t h a t shown i n  ( l a n e H) and human p l a c e n t a l  DNA  32 ( l a n e HP)  were i n c l u d e d as c o n t r o l s .  H i n d l l l - E c o R I fragments of X DNA  The p o s i t i o n s of  P-labelled  used as s i z e markers a r e shown.  Fragment s i z e s a r e g i v e n i n K i l o b a s e p a i r s  (Kbp).  T-  21.2-  5.2. 5.0^ 4-3as-  20. 1-9—I 1.61.40.95H 0-83' 0.56-I  W  W  W  (D  N  00  CD  CD  -r-  CM  W  v  lO  CD  CO —  CD I" CM -i— CM CM CM -0  1  CL  I  Table VIII.  Segregation Of The Human Ceruloplasmin Pseudogene With Human Chromosomes In Human - Hamster Somatic Hybrids  I d e n t i f i a b l e , Intact Human Chromosomes  8  C e l l Line  Response t o Probe A  1  41.06  ' .-•  2  45.01  -  .3 4  5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  45.43 76.14 76.31 76.33 + 79.05b . 80.05d 80.14c 80.17a .82.82a 85.16a .+ 89.27a 100.02b 102.05b 103.04 - • 111.02a 112.10a + 120.33 120.35 + 133.05 + 134.02a + Z Discordancy i  D  1  2  3  - -  -  4  + -  5  -  6  + -  -  7  8  - -  9  - -  -  10  11  + -  + - - - - - - + + - - + - + + - - - - - - - - - - + - + + + - + - + - - + - - - - - - - - - - - + + - - - ' - - + + + + - - + - - - ' + + - - - + - - - - - - - - - + + + + - - + - + - - - - + - - - + - - + - - - - - + - - + + - - - - - - + + - - - - + - - + + - - - + - + + - - + - - - - - + + - - - - - + + + - + - - - - - - + - + - + - + + - + + - + 27 18 32 23 45 45 32 0 41 27  12  -  -  13  +  14  +  -  -  -  -  + + + + + + 27  + + + + + + + 41  + + + + + + + + + 27  + + + + + 45  15  + -  16  + -  17  + -  + + - + • + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + 36 64 50  18  19  + ' -  -  20  21  + -  22  -  + + , + ' + + + + + + + + + + + + + + + + - '+ + + + + + + + + + + + + - ' + + + + - + + + + + 50 32 55 55  X T  +  + -  -  + - + - + + - + + - + + + + + +'+ + + - - + - - + - + + + + + + + - + 45 41 41  Presence (+) or absence (-) of human chromosomes as determined by cytogenetic analysis and confirmed by isoryme analysis (Donald et al.„ 1983; R i d d e l l et a l . , 1985). a  ^Presence (+) or absence (-) of human EcoRI-digested sequences homologous to the human ceruloplasmin pseudogene probe (Probe A).  M  124  d e l e t i o n was (Figure  Chromosomal L o c a l i z a t i o n of the Human C e r u l o p l a s m i n Pseudogene The  determined by Kbp  c o n c e n t r a t i o n s used  18A).  C. 4  1.1  observed at e i t h e r of the RNA  chromosomal l o c a t i o n of the c e r u l o p l a s m i n pseudogene somatic c e l l h y b r i d  analysis.  EcoRI r e s t r i c t i o n fragment (Probe A,  pseudogene, was  For  E c o R I - d i g e s t e d human-hamster somatic c e l l h y b r i d t h a t a l l 22 h y b r i d  cell  l i n e s tested  the p r e s e n c e or absence of the 1.1 VIII).  Control  ( l a n e HP,  Figure  lanes containing 19)  t h i s purpose, an i s o l a t e d  Figure  used as a h y b r i d i z a t i o n probe.  band and  DNA  ( l a n e H,  Figure  No  lane containing  w h i l e the expected 1.1  D.  19)  19),  THE  of  (Table  Kbp  detected i n  DNA in  the  EcoRI band DNA.  HUMAN CERULOPLASMIN GENE  i s o l a t e d from l i v e r t i s s u e  w i t h s e v e r a l r e s t r i c t i o n endonucleases, and  the  agarose g e l e l e c t r o p h o r e s i s .  t r a n s f e r to n i t r o c e l l u l o s e , DNA  the  showed  were i n c l u d e d  observed i n the human p l a c e n t a l  I n i t i a l l y , human genomic DNA  fragments were s e p a r a t e d by  to  were concordant f o r  c r o s s - h y b r i d i z a t i o n was  GENOMIC SOUTHERN BLOT ANALYSIS OF  digested  (Figure  chromosome 8  the Southern b l o t a n a l y s i s .  c o r r e s p o n d i n g to Probe A was  l o c a t e d 3*  e i t h e r E c o R I - d i g e s t e d human p l a c e n t a l  or hamster DNA  hamster DNA,  16),  Southern b l o t a n a l y s i s  ( l a n e s 1 - 22)  Kbp  was  was  resulting Following  fragments were a n a l y z e d w i t h  32 P - l a b e l l e d h y b r i d i z a t i o n probes d e r i v e d characterized Section  previously  human c e r u l o p l a s m i n cDNA c l o n e s phCP-1 and  III.A).  VhCP-1  Using the XhCP-1 h y b r i d i z a t i o n probe ( F i g u r e  l a r g e s i n g l e band (> 23 Kbp) predicted  from the  based on  was  d e t e c t e d w i t h a BamHl d i g e s t ;  (see  20B),  a  this is  r e s t r i c t i o n endonuclease mapping of the w i l d - t y p e gene  125  F i g u r e 20.  Genomic Southern B l o t A n a l y s i s o f t h e Human C e r u l o p l a s m i n  Human l i v e r DNA (10 yg) was d i g e s t e d w i t h v a r i o u s  restriction  enzymes (BamHl, EcoRI, P s t l , and H i n d l l l ) and e l e c t r o p h o r e s e d on 1.0% agarose g e l s .  F o l l o w i n g t r a n s f e r t o n i t r o c e l l u l o s e , t h e b l o t was 32  hybridized using  probes.  P - l a b e l l e d phCP-1 ( p a n e l A) o r \hCP-l 32  Positions of  ( p a n e l B) as  P - l a b e l l e d H i n d l l l fragments o f phage X. DNA  used as s i z e markers a r e g i v e n i n K i l o b a s e p a i r s  (Kbp).  Gene.  126  00  #* LU  > I CO CM  (J) CO  I  CMi CM CM  in b  00  CM6 CM  CM CM  in 6  127  (see F i g u r e 10) t h i s probe. Kbp,  3.1  and  suggests  t h a t a s i n g l e gene i s b e i n g d e t e c t e d  Other h y b r i d i z i n g fragments p r e s e n t  Kbp,  1.8  Kbp),  Pstl  (9.5 Kbp)  genomic d i g e s t s a l l correspond restriction  enzyme map  with  i n EcoRI (15 Kbp,  and H i n d l l l  (5.7 Kbp,  to fragments i d e n t i f i e d  4.5  i n the  of the w i l d - t y p e gene (see F i g u r e  4.6 Kbp)  partial  10).  U s i n g the phCP-1 cDNA c l o n e as a h y b r i d i z a t i o n probe f o r EcoRI, BamHl. H i n d l l l and P s t l human genomic d i g e s t s ( F i g u r e 20A), p a t t e r n i s more complex.  subsequently  EcoRI bands which c o u l d n o t be  identified  of the w i l d - t y p e gene (see F i g u r e 10) were a s s i g n e d  t o e i t h e r a pseudogene f o r human c e r u l o p l a s m i n  to n u c l e o t i d e r e s i d u e s 1502  - 3318  of the cDNA sequence; see  I I I . C ) o r to the 3*  end of the w i l d - t y p e gene (see below).  case, the 0.45  0.8  Kbp,  resulting  M u l t i p l e bands were d e t e c t e d w i t h each  r e s t r i c t i o n endonuclease used. i n t h e r e s t r i c t i o n map  the  Kbp,  and  1.5  Kbp  (corresponding Section In the  former  EcoRI fragments ( i n d i c a t e d by  arrows i n F i g u r e 21)  correspond  and F i g u r e 16).  remainder of the EcoRI fragments i n F i g u r e 20A,  The  to the pseudogene l o c u s (see S e c t i o n I I I . C as  w e l l as a l l of those d e t e c t e d u s i n g the XhCP-1 h y b r i d i z a t i o n probe ( F i g u r e 20B)  have been a s s i g n e d unambiguously t o the  c e r u l o p l a s m i n gene (see F i g u r e s 10 and The  specific 22B).  c h a r a c t e r i z e d by  human genomic DNA  ( F i g u r e 22A)  Southern using  two  h y b r i d i z a t i o n probes d e r i v e d from the cDNA sequence ( F i g u r e  Two  bands were d e t e c t e d w i t h each h y b r i d i z a t i o n probe, one  i n each case corresponds remaining  22).  3' end of the c e r u l o p l a s m i n gene was  b l o t a n a l y s i s of E c o R I - d i g e s t e d  wild-type  two  to the pseudogene l o c u s (see F i g u r e 21).  fragments (4.6  Kbp  and  2.4  Kbp)  were thus  of which The  unequivocally  128  F i g u r e 21.  Genomic Southern A n a l y s i s of the Human C e r u l o p l a s m i n  Pseudogene  and R e l a t e d Sequences. EcoRI-digested  human l i v e r genomic DNA was h y b r i d i z e d w i t h e i t h e r the  phCP-1 ( p a n e l A) o r XhCP-1 ( p a n e l B) c e r u l o p l a s m i n cDNA c l o n e s , under c o n d i t i o n s of h i g h s t r i n g e n c y .  Arrows i n d i c a t e bands t h a t have been  a s s i g n e d t o t h e human c e r u l o p l a s m i n pseudogene (see F i g u r e 16). remaining  bands correspond  t o the w i l d - t y p e  The  l o c u s (see t e x t f o r d e t a i l s ) .  32 P o s i t i o n s of  P - l a b e l l e d H i n d l l l fragments o f phage X DNA u s e d as  s i z e markers a r e shown. (Kbp).  Fragment s i z e s a r e g i v e n i n K i l o b a s e p a i r s  129  A 23.723.7-  9.5-  67-  9567-  4.5-  V  V  2.2-  20-  059-  130  F i g u r e 22.  Genomic Southern b l o t A n a l y s i s o f t h e 3' End o f t h e Human  Ceruloplasmin EcoRI-digested  Gene. human lymphocyte genomic DNA was h y b r i d i z e d u s i n g  e i t h e r Probe 1 o r Probe 2 ( p a n e l A ) , d e r i v e d from t h e 3' end o f t h e human c e r u l o p l a s m i n cDNA sequence ( p a n e l B ) .  Arrows i n d i c a t e bands t h a t have  been a s s i g n e d t o t h e c e r u l o p l a s m i n pseudogene l o c u s ( s e e t e x t f o r d e t a i l s ) ; t h e remaining  bands correspond  t o t h e 3' end o f t h e w i l d - t y p e  32 c e r u l o p l a s m i n gene.  P o s i t i o n s of  P-labelled Hindlll  phage X DNA used as s i z e markers a r e shown. Kilobase p a i r s  (Kbp).  fragments o f  Fragment s i z e s a r e g i v e n i n  Kbp  23.7  -  9.5  -  6.7  -  4.3  _  B  E 2417  2.2 2.0  _  0.59  _  Poly(A)  2851 PROBE 1  3321 E 2  132  assigned  t o t h e w i l d - t y p e gene (see F i g u r e 10), w i t h t h e 2.4 Kbp EcoRI  h y b r i d i z i n g s p e c i e s r e p r e s e n t i n g t h e 3'-most fragment.  IV.  DISCUSSION A.  C h a r a c t e r i z a t i o n o f t h e Human P r e c e r u l o p l a s m i n  cDNA  A.1  DNA Sequence A n a l y s i s o f t h e Human P r e c e r u l o p l a s m i n  cDNA  I n i t i a l l y , a human c e r u l o p l a s m i n cDNA c l o n e o f 2.7 Kbp (phCP-1) was i s o l a t e d from a cDNA l i b r a r y p r e p a r e d mixtures  from human l i v e r mRNA, by u s i n g  o f s y n t h e t i c o l i g o n u c l e o t i d e s as h y b r i d i z a t i o n probes.  c l o n e was found  T h i s cDNA  t o encode amino a c i d r e s i d u e s 202 - 1046 o f t h e  c e r u l o p l a s m i n p r o t e i n sequence, f o l l o w e d by 123 bp o f 3' u n t r a n s l a t e d sequence (preceded tract.  by a TGA stop codon) and t e r m i n a t i n g w i t h a p o l y (A)  Two randomly-primed human l i v e r cDNA l i b r a r i e s were  subsequently  32 screened  using appropriate  P - l a b e l l e d r e s t r i c t i o n fragments.  c l o n e s o f 1.2 Kbp and 1.0 Kbp ( d e s i g n a t e d \hCP-l  Two  and XhCP-6,  r e s p e c t i v e l y ) were i d e n t i f i e d t h a t t o g e t h e r c o n t a i n e d cDNA sequence encoding  amino a c i d r e s i d u e s 1 - 380 o f t h e mature p r o t e i n preceded by a  p u t a t i v e 19 amino a c i d l o n g s i g n a l p e p t i d e and 38 bp o f 5' u n t r a n s l a t e d sequence. N u c l e o t i d e s 58 - 3195 o f t h e cDNA sequence code f o r the plasma form of c e r u l o p l a s m i n ; t h e p r e d i c t e d amino a c i d sequence agrees c o m p l e t e l y  with  t h a t r e p o r t e d p r e v i o u s l y by Takahashi e t a l . (1984) who used p r o t e i n chemistry  techniques.  i s somewhat A + U r i c h  O v e r a l l , t h e base c o m p o s i t i o n  o f c e r u l o p l a s m i n mRNA  ( 3 3 % A, 26% U, 22% G, 19% C ) , r e f l e c t i v e o f t h e  c o d i n g r e g i o n i n which 60% o f t h e codons end i n e i t h e r A o r U. o b s e r v a t i o n i s i n c o n t r a s t t o the codon usage i n o t h e r  This  l i v e r mRNAs, such  133  as those  f o r prothrombin ( M a c G i l l i v r a y and  e t a l . , 1985), or f a c t o r XII 90% of the codons end  ( C o o l e t aJL., 1985), i n which  i n G or C.  One  r e g i o n of the c e r u l o p l a s m i n mRNA (CGC rarely  Davie, 1984), f a c t o r X  (2 of 51 a l a n i n e r e s i d u e s a r e encoded by  The  approximately  codon i s not used i n the f o r a r g i n i n e ) and  3' u n t r a n s l a t e d r e g i o n (spanning  (Fung  coding  o t h e r s a r e used  GCG).  n u c l e o t i d e r e s i d u e s 3199  -  c o n t a i n s a p u t a t i v e p o l y a d e n y l a t i o n s i g n a l ATTAAA (Proudfoot  and  1976)  tail.  t h a t i s l o c a t e d 14 n u c l e o t i d e s upstream of the p o l y ( A )  3321)  Brownlee, This  p o l y a d e n y l a t i o n s i g n a l i s observed i n 12% of such 3*  t e r m i n a l sequences  from v e r t e b r a t e s  i s a v a r i a n t of  (Wickens and  Stephenson, 1984), and  the  more commonly observed s i g n a l AATAAA ( B i r n s t i e l e t a l . , 1985). Nucleotide residues 1 - 5 7  code f o r an amino t e r m i n a l e x t e n s i o n of  amino a c i d s t h a t i s removed p r i o r to the appearance of c e r u l o p l a s m i n plasma.  A m e t h i o n i n e r e s i d u e occurs  sequence and may  a t r e s i d u e -19  f u n c t i o n as the i n i t i a t o r methionine.  i s r i c h i n hydrophobic amino a c i d s , and peptide  in this  (von H e i j n e ,  i n i t i a t i o n of export  1982;  The  leader  peptide  signal  Such sequences f u n c t i o n i n the  of nascent p o l y p e p t i d e  endoplasmic r e t i c u l u m ( B l o b e l e t a l . , 1979).  c h a i n s a c r o s s the rough The  ceruloplasmin  cDNA  sequence p r e d i c t s t h a t an A l a - L y s bond (encoded by n u c l e o t i d e s 55 see F i g u r e 7) i s c l e a v e d d u r i n g removal of t h i s l e a d e r p e p t i d e . c o n s i s t e n t w i t h the s p e c i f i c i t y of s i g n a l p e p t i d a s e 1983)  which t y p i c a l l y occurs  cleavage  (von  60;  This i s Heijne,  f o l l o w i n g b a s i c r e s i d u e s t h a t are preceded  s m a l l h y d r o p h o b i c amino a c i d s . synthesized  in  peptide  thus resembles a t y p i c a l  Watson, 1984).  T h i s suggests t h a t c e r u l o p l a s m i n  i n l i v e r as a t y p i c a l p r e p r o t e i n , c o n t a i n i n g a s i g n a l  sequence of a t l e a s t 19 amino a c i d s .  19  by  is peptide  134  Mercer and Grimes (1986) have r e p o r t e d t h e c h a r a c t e r i z a t i o n o f a p a r t i a l human c e r u l o p l a s m i n  cDNA c l o n e t h a t was i s o l a t e d from a human  l i v e r cDNA l i b r a r y c o n s t r u c t e d  i n t h e phage v e c t o r X g t l O .  DNA sequence  a n a l y s i s showed t h a t t h e c l o n e extended from the amino t e r m i n a l sequence t o 114 amino a c i d s s h o r t o f t h e carboxy-terminus. s i g n a l p e p t i d e sequence o b t a i n e d  Grimes (1986).  The proposed  from DNA sequence a n a l y s i s o f X.hCP-1  (see F i g u r e 7) was found t o agree c o m p l e t e l y and  leader  w i t h t h a t r e p o r t e d by Mercer  However, t h e 38 bp o f 5' u n t r a n s l a t e d sequence  determined by a n a l y s i s o f b o t h XhCP-2 and XhCP-6 cDNA c l o n e s ( s e e F i g u r e 8) d i f f e r s c o m p l e t e l y  from t h e 14 bp o f 5' u n t r a n s l a t e d  determined by Mercer and Grimes (1986).  sequence  The sequence d i v e r g e n c e  occurs  immediately 5* t o t h e proposed i n i t i a t o r methionine r e s i d u e and does n o t appear t o o c c u r as t h e r e s u l t o f a f r a m e s h i f t mutation. t h i s discrepancy  i s unclear a t present,  The reason f o r  s i n c e the n u c l e o t i d e sequences o f  XhCP-2 and XhCP-6 a r e i n complete agreement w i t h t h a t determined from the a n a l y s i s o f the c o r r e s p o n d i n g  genomic r e g i o n (see F i g u r e 11).  The  p o s s i b i l i t y t h a t t h e observed d i f f e r e n c e i s the r e s u l t o f t h e u t i l i z a t i o n of a l t e r n a t i v e s p l i c i n g p a t t e r n s i s u n l i k e l y , s i n c e t h e p o i n t o f sequence divergence two  ( i . e . immediately 5' t o t h e i n i t i a t o r methionine) between t h e  cDNA sequences does n o t correspond  wild-type  gene; an i n t r o n / e x o n  t o t h e l o c a t i o n o f an i n t r o n i n the  j u n c t i o n was i d e n t i f i e d a t n u c l e o t i d e  p o s i t i o n -13 on the b a s i s o f comparative DNA sequence a n a l y s i s and nuclease  SI mapping (see S e c t i o n I I I . B . 4 . 2 ) .  More p r o b a b l y ,  r e p o r t e d by Mercer and Grimes (1986) r e p r e s e n t s The  a cloning a r t i f a c t .  n u c l e o t i d e sequence o f two p a r t i a l cDNA c l o n e s  ceruloplasmin  have been r e p o r t e d  independently  t h e sequence  f o r human  by Yang e t §_1.  (1986).  135  These c l o n e s  ( i s o l a t e d from the S t u a r t O r k i n human l i v e r cDNA l i b r a r y )  n e a r l y i d e n t i c a l to the p r e v i o u s l y d e s c r i b e d 2.7 Section III.A).  However, one  ( d e s i g n a t e d CP-1)  Kbp  phCP-1 c l o n e  ( o c c u r r i n g between r e s i d u e s 1041  and  1042  sequence) as w e l l as an a d d i t i o n a l 121 The  (see  of the c l o n e s a n a l y z e d by Yang e t a l . (1986)  c o n t a i n s an i n s e r t i o n of f o u r amino a c i d  3' u n t r a n s l a t e d r e g i o n .  are  bp  residues  of the c e r u l o p l a s m i n p r o t e i n (extending 3' t o phCP-1) i n the  commonly used p o l y a d e n y l a t i o n s i g n a l AATAAA  i s l o c a t e d 16 bp upstream from the p o l y ( A ) t r a c t i n the CP-1  clone.  The  o r i g i n of t h e s e d i f f e r e n t cDNA c l o n e s i s u n c e r t a i n a t p r e s e n t ,  although  these d a t a c l e a r l y suggest  transcripts  some h e t e r o g e n e i t y  i n ceruloplasmin  w i t h r e s p e c t to the p o i n t of p o l y a d e n y l a t i o n .  Although  the  corresponding  r e g i o n of t h e w i l d - t y p e c e r u l o p l a s m i n gene has not y e t been c h a r a c t e r i z e d (see S e c t i o n I I I . B ) , s e v e r a l e x p l a n a t i o n s i n s e r t i o n i n the CP-1 postulated. e t a l . , 1986)  c l o n e d e s c r i b e d by Yang e t a l . (1986) can  be  (Gitschier  r e v e a l s the p r e s e n c e of an i n t r o n between exons 19 and  a c i d r e s i d u e s 1041 i n the CP-1  to the c e r u l o p l a s m i n cDNA sequence encoding  - 1042,  Vehar e t a l . , 1984).  c l o n e may  20  amino  A d d i t i o n a l amino a c i d s  have a r i s e n from a l t e r n a t i v e s p l i c i n g of  i n t r o n i n some l i v e r c e l l s d u r i n g RNA explanations  amino a c i d  Examination of the f a c t o r V I I I genomic sequence  ( i . e . , corresponding  present  f o r the observed  processing.  c o u l d i n c l u d e the presence of two  the donor's genome, o r the e x i s t e n c e of two  an  Other p l a u s i b l e  ceruloplasmin a l l e l e s i n  ceruloplasmin  loci.  The  l a t t e r assumption i s somewhat d o u b t f u l , s i n c e a second w i l d - t y p e gene not d e t e c t a b l e by e i t h e r genomic Southern b l o t a n a l y s i s (see S e c t i o n III.D) o r chromosome mapping (Yang e t a l . , 1986;  Royle e t a l . , 1987).  was  136  A.2  I n t e r n a l Homology W i t h i n t h e C e r u l o p l a s m i n  cDNA Sequence  E x t e n s i v e amino a c i d sequence i d e n t i t y has been r e p o r t e d p r e v i o u s l y w i t h i n t h e t h r e e r e p e a t e d A domains o f c e r u l o p l a s m i n et a l . , 1984).  As expected,  (Takahashi  t h i s homology extends t o t h e n u c l e o t i d e  sequences o f t h e r e p e a t e d u n i t s when they a r e a l i g n e d . of c e r u l o p l a s m i n e x h i b i t approximately p a i r w i s e (Yang e t a l . , 1986; Koschinsky  The t h r e e domains  46 - 51% i d e n t i t y when compared e t a l . , 1986).  Within these  three  domains, s p e c i f i c r e g i o n s show h i g h e r l e v e l s o f sequence c o n s e r v a t i o n (Yang e t a l . , 1986).  A comparison o f t h e n u c l e o t i d e sequence  amino a c i d r e s i d u e s 204 - 276 w i t h t h a t encoding  r e s i d u e s 903 - 975  r e v e a l s 66.2% i d e n t i t y , w h i l e r e s i d u e s 204 - 276 compared w i t h 565  encoding  residues  - 637 shows 61.2% i d e n t i t y , and r e s i d u e s 565 - 637 a l i g n e d w i t h  r e s i d u e s 903 - 975 shows 59.4% n u c l e o t i d e i d e n t i t y  (Yang e t a l . , 1986).  I n t e r e s t i n g l y , a s i m i l a r r e g i o n from t h e d e r i v e d amino a c i d sequence o f a p a r t i a l r a t c e r u l o p l a s m i n cDNA c l o n e ( A l d r e d e t a l . , 1987) (see S e c t i o n I.D)  shows remarkable s i m i l a r i t y w i t h t h e p u b l i s h e d human amino a c i d  sequence from r e s i d u e s 194 - 276 ( 7 5 % i d e n t i t y ) , w i t h almost complete conservation  (98%) from r e s i d u e s 227 - 276, except  amino a c i d change a t r e s i d u e 243.  f o r a conservative  T h i s h i g h l y conserved  region,  present  i n each o f t h e t r i p l i c a t e d u n i t s i n the human c e r u l o p l a s m i n m o l e c u l e may represent a region subjected to f u n c t i o n a l c o n s t r a i n t . I n t e r e s t i n g l y , these r e g i o n s a r e n o t c o i n c i d e n t w i t h proposed c o p p e r - b i n d i n g ceruloplasmin  (see S e c t i o n I . E ) .  These h i g h l y  centers i n  conserved,  s t r u c t u r a l l y - r e l a t e d areas may thus be i n d i c a t i v e of t h e e v o l u t i o n a r y m a i n t a i n e n c e o f an a l t e r n a t i v e c e r u l o p l a s m i n f u n c t i o n (Yang e t a l . , 1986) .  137  A.3  A n a l y s i s o f t h e Human C e r u l o p l a s m i n T r a n s c r i p t Northern  b l o t a n a l y s i s o f t h e human c e r u l o p l a s m i n  transcript  r e v e a l e d t h e p r e s e n c e o f two h y b r i d i z i n g s p e c i e s i n human l i v e r p o l y ( A )  +  RNA, (3.7 + 200 Kb and 4.5 + 250 Kb), u s i n g t h e phCP-1 cDNA c l o n e as a h y b r i d i z a t i o n probe. hepatoma c e l l  I n c o n t r a s t , t o t a l RNA i s o l a t e d from t h e human  l i n e HepG2 (Knowles, 1980) ( a l s o probed w i t h phCP-1)  c o n t a i n e d o n l y t h e s m a l l e r mRNA s p e c i e s . s e c r e t e d by HepG2 c e l l s  Ceruloplasmin  i s s y n t h e s i z e d and  (Knowles e t a l . , 1980), s u g g e s t i n g  RNA s p e c i e s encodes a f u n c t i o n a l c e r u l o p l a s m i n t r a n s c r i p t .  t h a t t h e 3.7 Kb The  c e r u l o p l a s m i n cDNA c l o n e s p r e v i o u s l y d e s c r i b e d i n t h i s study c o n t a i n a t o t a l o f 3359 bp o f sequence i n a d d i t i o n t o a p o l y ( A )  t r a c t which i s  u s u a l l y 180 - 200 bp l o n g i n e u k a r y o t i c mRNAs ( P e r r y , 1976).  On t h i s  b a s i s , b o t h s p e c i e s appear t o be l a r g e r than t h a t r e q u i r e d t o encode t h e p r e c e r u l o p l a s m i n mRNA. These t r a n s c r i p t s may r e f l e c t t h e p r e s e n c e o f l o n g 5' o r 3' u n t r a n s l a t e d segments which a r e d i f f e r e n t i a l l y p r o c e s s e d , g e n e r a t i o n o f two RNA s p e c i e s .  r e s u l t i n g i n the  The p o s s i b i l i t y o f a l t e r n a t i v e  splicing  p a t t e r n s o c c u r r i n g a t the 3' end o f t h e c e r u l o p l a s m i n t r a n s c r i p t has been by Yang e t §J^. (1986) t o e x p l a i n an i n s e r t i o n o f 4 amino a c i d s  suggested  i n t h e p r o t e i n coding sequence (see S e c t i o n IV.A.1). identified  The e x t r a sequence  i n t h e l a t t e r c l o n e when compared t o phCP-1 ( i n c l u d i n g t h e  a d d i t i o n a l 121 bp observed  i n the 3' u n t r a n s l a t e d segment) does n o t ,  however, account f o r the s i z e d i f f e r e n c e (approximately observed  800 n u c l e o t i d e s )  between the two h y b r i d i z i n g RNA s p e c i e s d e t e c t e d u s i n g  blot analysis.  Itis still  Northern  p o s s i b l e t h a t an as y e t u n i d e n t i f i e d cDNA  c l o n e may c o n t a i n a d d i t i o n a l sequence i n t h e 3' u n t r a n s l a t e d segment,  138  thereby d i f f e r i n g  from p r e v i o u s l y c h a r a c t e r i z e d cDNA c l o n e s w i t h  t o the p o i n t o f p o l y a d e n y l a t i o n .  This proposal  is feasible,  respect  since  h e t e r o g e n e i t y w i t h r e s p e c t t o the p o s i t i o n o f p o l y a d e n y l a t i o n i n the phCP-1 c l o n e compared t o the cDNA c l o n e d e s c r i b e d by Yang e t a l . (1986) has  a l r e a d y been demonstrated.  t h i s study,  However, based on the r e s u l t s p r e s e n t e d i n  i t i s most l i k e l y t h a t the two d i f f e r e n t c e r u l o p l a s m i n mRNA  s p e c i e s a r i s e from d i f f e r e n t i a l p r o c e s s i n g and/or promoter f u n c t i o n a t t h e 5' end o f the gene (see S e c t i o n IV.B.5). I n c o n t r a s t t o the r e s u l t s d e s c r i b e d above u s i n g human l i v e r poly(A)  +  RNA, i t i s i n t e r e s t i n g t o n o t e t h a t a s i n g l e  ceruloplasmin  t r a n s c r i p t o f 3.8 kb i s d e t e c t e d u s i n g r a t l i v e r mRNA, and t h a t comparable r e s u l t s have been o b t a i n e d u s i n g RNA d e r i v e d from r a t t e s t i s , y o l k p l a c e n t a , and c h o r o i d p l e x u s  ( A l d r e d e t ajL., 1987).  sac,  S i m i l a r l y , o n l y one  c e r u l o p l a s m i n mRNA s p e c i e s i s d e t e c t e d u s i n g bovine p o l y ( A )  +  RNA.  This  r e i n f o r c e s the p r o p o s a l t h a t the 3.7 kb human c e r u l o p l a s m i n RNA s p e c i e s i s l i k e l y s u f f i c i e n t t o encode a f u n c t i o n a l c e r u l o p l a s m i n p r o t e i n .  B.  CHARACTERIZATION OF THE WILD-TYPE HUMAN CERULOPLASMIN GENE  B.1  Ceruloplasmin  Gene O r g a n i z a t i o n Corresponding  t o the Coding  Sequence Seven recombinant phage c o n t a i n i n g w i l d - t y p e human c e r u l o p l a s m i n genomic sequences were i s o l a t e d from human genomic X phage  libraries.  From r e s t r i c t i o n endonuclease mapping a n a l y s i s coupled w i t h Southern b l o t a n a l y s i s , the c e r u l o p l a s m i n gene was found length.  t o span a t l e a s t 50 kbp i n  The l o c a t i o n o f 14 exons was determined  (corresponding to  n u c l e o t i d e s 1 - 2555 o f the cDNA sequence); the 3' end o f the gene ( i . e .  139  the r e g i o n c o r r e s p o n d i n g t o n u c l e o t i d e r e s i d u e s 2556 - 3321 sequence) was screening.  not r e p r e s e n t e d i n the phage c l o n e s o b t a i n e d from  library  However, genomic Southern b l o t a n a l y s i s a l l o w e d the  i d e n t i f i c a t i o n of two genomic EcoRI fragments encompassing B.2  of the cDNA  (4.6 kbp and 2.4  kbp)  this region.  DNA  Sequence A n a l y s i s o f the Wild-Type  A l l s p l i c e d o n e r / a c c e p t o r sequences  C e r u l o p l a s m i n Gene  conform t o the GT..AG r u l e  f o r n u c l e o t i d e s immediately f l a n k i n g exon boundaries Chambon, 1981;  Cech, 1983).  (Breathnach and  F u r t h e r f l a n k i n g sequences  are i n general  accordance w i t h compiled n u c l e o t i d e f r e q u e n c i e s (Breathnach and Chambon, 1981;  Mount, 1982).  The sequences  o f the exons were found t o be  t o t h e p r e v i o u s l y determined cDNA sequence.  identical  The s i z e s o f c h a r a c t e r i z e d  exons range from 129 bp - 255 bp, w i t h a c a l c u l a t e d median s i z e of bp.  T h i s i s c o n s i s t e n t w i t h p u b l i s h e d exon s i z e d i s t r i b u t i o n s  Deacon, 1982).  As found i n o t h e r e u k a r y o t i c genes,  c e r u l o p l a s m i n gene a r e h i g h l y v a r i a b l e The t o t a l s i z e o f i n t r o n s determined  183  (Naora  and  i n t r o n s i z e s i n the  (0.8 kbp - a p p r o x i m a t e l y 9.5  kbp).  to date i s a p p r o x i m a t e l y 30 kbp  i s a l r e a d y i n excess of t h a t p r e d i c t e d by Naora and Deacon  which  (1982),  a c c o r d i n g t o the observed v a r i a b i l i t y of t o t a l i n t r o n s i z e as a f u n c t i o n of t o t a l exon l e n g t h . B.3  I n t r o n P o s i t i o n s W i t h i n the T r i p l i c a t e d A Domains of Human Ceruloplasmin As has been d e s c r i b e d p r e v i o u s l y  (see S e c t i o n I.A), the e n t i r e  human c e r u l o p l a s m i n molecule p o s s e s s e s an i n t e r n a l t r i p l i c a t e d which  structure,  l i k e l y r e p r e s e n t s the p r o d u c t of s u c c e s s i v e gene d u p l i c a t i o n  (Dwulet and Putnam, 1981b: D o o l i t t l e , 1984;  see S e c t i o n IV.D).  If  events  140  F i g u r e 23.  I n t r o n p o s i t i o n s w i t h i n the t h r e e r e p e a t e d u n i t s of human  ceruloplasmin. The  top, m i d d l e and bottom l i n e s correspond  sequence of the A l , A2  and A3  to the amino a c i d  t r i p l i c a t e d regions r e s p e c t i v e l y .  Except  f o r gaps i n t r o d u c e d i n o r d e r to maximize homology, the complete amino a c i d sequence i s g i v e n c o n t i n u o u s l y i n s i n g l e l e t t e r code.  Residues boxed a t a  g i v e n p o s i t i o n a r e i d e n t i c a l ; n o n - i d e n t i c a l r e s i d u e s i n the m i d d l e sequence a r e e n c l o s e d intron positions. asterisks. circles.  i n dashed boxes.  T r i a n g l e s i n d i c a t e determined  Carbohydrate attachment s i t e s a r e d e s i g n a t e d  P u t a t i v e type I copper l i g a n d s a r e i d e n t i f i e d by  1984) .  solid  Homologous p a i r s of c y s t e i n e r e s i d u e s a r e shaded and  arrows i d e n t i f y f o u r s i t e s of t r p t i c c l e a v a g e  by  vertical  ( m o d i f i e d from O r t e l e t a l . ,  141  1  K(E KJH Y Y I G I I  W 0 Y AS D H G - EKK L ISV 0 TE H  351  HYRHYYIAA  WjKjY A P S G I O I F T K E ^ L T A P G S DSIA V F F E Q T T R I G G S  711  G[TR[T]Y Y I A A Y  E  w b Y[S|P Q R E V(FK]E[I]H H  E|T F RJT T I ik  P 0 R I GR L  S_N]A£L]O 0 IFV  Q E  K G E F Y|I G S K  p v w — [ L ~ S | F [ L G P I T K ATJTJG D K VII V A E Y G 0T" I R  IETEHLGILT  R  HL K H  .1  L0  T F H!N K G  Q L H A ID Y G D K K V I I F K N H0  T T D F Q R A D D K Y Y P G E QYYT YM I Y H P Q S  R^Tv  F T YE W P P S A S H YP£ET A  T E S S T Y T P T L P G E TL T Y V w  141  L A T E E Q S[P~G]E G  G M  S V T R I Y[HJS  A PK D I  501  P(T THA T Y P;K;E Y G P  P Y | I A K HY Y S  P TK 0 1  841  K 1 P E R S GJAJGJT E  S A « I P W AY Y S  QY K DIL'YIS G L I G P L I|Y  190  K E K H I 0 R E F Y Y H[?JS T[vJ0 E | [ F J S W YLI E D N I K T Y C S E P E K V 0 K OjNjE 0 F Q E S N[R  551  R Q  891  PRRKL  V  0 Y 0  - -  E F  F P T Y F D E-JJfjE Sjl LjL E ON I R M F T.T Ap;o Q]Y D K[E:P E D F Q E S N K  E F  L 0 L Y F 0 E-JE SHY ifblo H IK T YJS 0 H P E K vftillC D D E[EIF[T1E S H K  D Y HA A FF H G Q A L T. N|KJH  240  ^ Y | Y  Y K WYLFGHGHE  601  MHSH  Y Y W Y I F S"A]G N E 0 V HG I YF[S;G N T Y L W - -  939  H H[A I  YN  D TI N I  290 649  - E  R0TA M L  989  Y[Y  D V F0I  N I sfs~sl I 0 M I R*G K R R'QlSlE 0 S T F Y L  OJN E D T K SJG  L F0 A Y  S LT L Y < 3T L  IGHGN E  Y A Q NP G  0H0S  DILIH T V HF H G  Q H L_MJH  EHI T F K V E I T T P G ifwlLftlH  H VT D H  H s F g Y[KJH  142  segmental DNA d u p l i c a t i o n has p l a y e d a r o l e i n t h e e v o l u t i o n o f t h e c e r u l o p l a s m i n gene, one may expect  the o c c u r r e n c e  of i n t r o n  boundaries  between t h e A domains w i t h i n the c e r u l o p l a s m i n gene ( D o o l i t t l e , The  comparative alignment  of c e r u l o p l a s m i n  o f i n t r o n boundaries  1985).  w i t h i n the repeated A u n i t s  ( F i g u r e 23) i s f a c i l i t a t e d by t h e r e l a t i v e l y h i g h degree  of s i m i l a r i t y between the r e l a t e d segments. F i g u r e 21, t h e boundaries  As i s c l e a r l y  illustrated in  o f t h e A1/A2 and A2/A3 r e p e a t s a r e each  c o n t a i n e d on one exon (exons 6 and 12, r e s p e c t i v e l y ) , which i s i n c o n s i s t e n t w i t h a DNA d u p l i c a t i o n mechanism where t h e boundaries o f r e p e a t e d u n i t s would p r e d i c t a b l y f a l l w i t h i n i n t r o n s .  This i s reminiscent  of t h e f a c t o r V I I I gene o r g a n i z a t i o n ( s e e F i g u r e 4, S e c t i o n I.G.3) i n which t h e A1/A2 and A2/A3 j u n c t i o n s a r e each c o n t a i n e d on one exon.  This  o b s e r v a t i o n i s l i k e l y t h e r e s u l t o f i n t r o n l o s s w i t h i n these two genes subsequent t o t h e i r i n i t i a l  formation,  such t h a t t h e present-day  gene  o r g a n i z a t i o n s a r e no l o n g e r r e f l e c t i v e o f t h e o r i g i n a l gene d u p l i c a t i o n events. As I.G),  i s a l s o t h e case f o r t h e f a c t o r V I I I gene (see F i g u r e 4, S e c t i o n  o n l y some o f t h e i n t r o n boundaries  conserved  w i t h i n the A repeats are  i n t h e c e r u l o p l a s m i n gene (see F i g u r e 23). Furthermore, each o f  the t h r e e A domains w i t h i n t h e f a c t o r V I I I and c e r u l o p l a s m i n genes c o n t a i n a d i f f e r e n t number o f exons (see F i g u r e 24). The p r e c i s e alignment of some i n t r o n boundaries t h a t these  w i t h i n the repeated u n i t s of c e r u l o p l a s m i n  suggests  i n t r o n s may have a r i s e n f o l l o w i n g the s u c c e s s i v e gene  d u p l i c a t i o n events,  e i t h e r by l o s s o f one member of a d u p l i c a t e d i n t r o n ,  o r by i n s e r t i o n of new i n t r o n s . indistinguishable.  These l a t t e r two events  are e s s e n t i a l l y  143  Figure  24.  and  P o s i t i o n s of i n t r o n s  ceruloplasmin  factor VIII.  The numbers represent  ( t ) i n the A domains of  i d e n t i f y i n g the e x t e n t of the t r i p l i c a t e d A u n i t s  nucleotide  residues.  Codon phases ( i . e . ,  I, I I , or 0 i n d i c a t i n g  the p o s i t i o n of each i n t r o n r e l a t i v e t o the codon t r i p l e t ) a r e shown. C e r u l o p l a s m i n i n t r o n s a r e l a b e l l e d A - N, w h i l e i n t r o n s i n f a c t o r V I I I a r e designated a - r .  Dashed l i n e s d e s i g n a t e areas i n which i n t r o n s have n o t  y e t been l o c a l i z e d i n t h e c e r u l o p l a s m i n the B domain i n f a c t o r V I I I  gene.  i s indicated.  The r e l a t i v e p o s i t i o n of  A1 CERULOPLASMIN REPEAT UNITS (Nucleotides) INTRON P O S I T I O N S - C P  INTRON POSITIONS- F.VIII  A2  1053  57 A  B  II  I  T  r  111  A3 3138  2133  C  D  E  F  G  I  H  K  I  I  II  I  I  -f—I—I—r-H  T  til  i  T  1 H  t \ 1;  M  N  I I I  1!  B Domain  A-  145  In an analogous s i t u a t i o n , a - f e t o p r o t e i n (Eiferman e t a l . , 1981) and  albumin (Sargent e t a l . , 1981) each have a t r i p l i c a t e d gene s t r u c t u r e  ( i . e . composed of 3 s i m i l a r s e t s of 4 exons). proposed t h a t these genes have a r i s e n through a n c e s t r a l gene a p p r o x i m a t e l y 1981).  On t h i s b a s i s , i t has been the d u p l i c a t i o n of an  300 - 500 m i l l i o n y e a r s ago (Eiferman e t a l . ,  U n l i k e c e r u l o p l a s m i n o r f a c t o r V I I I , a l l i n t r o n boundaries  the r e p e a t e d u n i t s i n a - f e t o p r o t e i n and albumin a r e almost conserved.  S i n c e i t has been observed  within  precisely  t h a t i n a number of genes, i n t r o n s  s e p a r a t e p r o t e i n domains ( B l a k e , 1983a, b; G i l b e r t , 1978; Go, 1981, 1983), the o c c u r r e n c e o f i n t r o n s i n a - f e t o p r o t e i n and albumin a t the b o r d e r s o f the t h r e e g e n e t i c domains may r e f l e c t d i v i s i o n s between f u n c t i o n a l u n i t s . Indeed, a l t h o u g h a c t i v e b i n d i n g s i t e s i n mammalian a - f e t o p r o t e i n s a r e p o o r l y c h a r a c t e r i z e d , s p e c i f i c f u n c t i o n s have been a s s i g n e d t o t h e 3 g e n e t i c u n i t s i n albumin ( P e t e r s and R e i d , 1977) (Domain 1: f a t t y a c i d b i n d i n g , Domain 2: binding).  However,  long-chain  b i l i r u b i n b i n d i n g , and Domain 3:  indole  f o r b o t h the c e r u l o p l a s m i n and f a c t o r V I I I genes,  c o r r e l a t i o n s between exons and f u n c t i o n a l domains a r e u n c l e a r a t p r e s e n t . Thus, t h e c o n s e r v a t i o n of some i n t r o n boundaries  w i t h i n the r e p e a t e d u n i t s  of t h e s e l a t t e r two genes cannot be c o r r e l a t e d w i t h the i n c i d e n c e of functional units. B.4  Comparison of the Gene O r g a n i z a t i o n s of C e r u l o p l a s m i n  and F a c t o r  VIII Comparative a n a l y s i s of the gene o r g a n i z a t i o n s of c e r u l o p l a s m i n and b l o o d c o a g u l a t i o n f a c t o r V I I I ( G i t s c h i e r e t a l . , 1984) i s u s e f u l i n t r a c i n g the e v o l u t i o n a r y h i s t o r y of these two p r o t e i n s .  As can be c l e a r l y  seen,  closely i n  i n t r o n s A - L i n the c e r u l o p l a s m i n gene correspond  146  p o s i t i o n t o i n t r o n s p r e s e n t i n the f a c t o r V I I I gene, and except f o r i n t r o n H, i n t e r r u p t t h e r e a d i n g  frame i n the same phase ( F i g u r e 24). When the  p r o t e i n sequences o f c e r u l o p l a s m i n  and f a c t o r V I I I  a r e a l i g n e d t o maximize  i d e n t i t y , some o f these i n t r o n s i n t e r r u p t e i t h e r the same amino a c i d i n the two p r o t e i n s acids  (e.g.,  ceruloplasmin  i n identical positions  Figure  25).  progenitor  i n t r o n s B, I , J , and K) o r amino  (.e.g, c e r u l o p l a s m i n  i n t r o n s C and E) (see  T h i s s t r o n g l y suggests t h a t these i n t r o n s were p r e s e n t i n t h e  gene, p r i o r t o d u p l i c a t i o n .  the c e r u l o p l a s m i n ceruloplasmin  I n two cases ( i n t r o n s A and L i n  gene), i n t r o n p o s i t i o n s v a r y i n t h e f a c t o r V I I I and  genes by one amino a c i d (3 n u c l e o t i d e  residues).  These  i n t r o n s a l s o may have been p r e s e n t i n t h e a n c e s t r a l gene ( i . e . p r i o r t o d u p l i c a t i o n ) , and d i f f e r s l i g h t l y i n p o s i t i o n i n t h e p r e s e n t - d a y genes due t o i n s e r t i o n o r d e l e t i o n o f s i n g l e amino a c i d s w i t h i n t h e c o d i n g sequences of e i t h e r f a c t o r VIII or ceruloplasmin.  I t i s also possible that  s m a l l v a r i a t i o n s may be t h e r e s u l t o f i n t r o n s l i d i n g been p o s t u l a t e d triphosphate  these  (see below) , as has  t o explain s i m i l a r d i f f e r e n c e s i n the organizations  isomerase genes from A s p e r g i l l u s n i d u l a n s  of the  and maize (McKnight  e t a l . , 1986). Introns  D, F, and G i n t h e c e r u l o p l a s m i n  gene d i f f e r  i n p o s i t i o n by  10, 7, and 12 amino a c i d s r e s p e c t i v e l y i n the f a c t o r V I I I gene (see Figure  25), a l t h o u g h t h e phases o f these t h r e e  are t h e same (see F i g u r e  24).  i n t r o n s i n the two genes  F o r i n t r o n s D and G, a model  involving  i n t r o n s l i d i n g seems u n l i k e l y , s i n c e no c o r r e s p o n d i n g i n s e r t i o n s o r d e l e t i o n s a r e observed a t these p o s i t i o n s i n t h e two genes ( F i g u r e 25). This  suggests t h a t independent i n t r o n i n s e r t i o n has o c c u r r e d  l o c a t i o n s i n the c e r u l o p l a s m i n  and f a c t o r V I I I  a t these  genes and these  introns  147  F i g u r e 25.  Comparative p o s i t i o n s of i n t r o n s i n c e r u l o p l a s m i n  corresponding The  (A)  with  introns i n factor VIII (?).  f a c t o r V I I I amino a c i d sequence i s a l i g n e d w i t h t h a t of  c e r u l o p l a s m i n i n the r e g i o n of the A domain. designates  The  r e s i d u e s which are i d e n t i c a l i n the two  consensus proteins.  line The  numbering above the sequences corresponds to t h a t of f a c t o r V I I I .  The  numbers p r e c e d i n g  the  first Figure  the c e r u l o p l a s m i n l i n e s r e p r e s e n t s the number of  amino a c i d i n the l i n e . 24.  I n t r o n s a r e l a b e l l e d as d e s c r i b e d i n  148  FACTOR  -19 -10 MOIELSTCFFICILRFCFS  VIM  1 10 20 30 HO 50 60 70 80 90 ATRRmGAV^LSVDYM--0SDLGELPVDARFPPRVPKSFPFNTSVmnLFVEFTDHLFNIAKPRPPHMGLL6PTI0AEVYDTVVITLI<NMASHPVSL 1 KF.KHYYIG11ETTWDYASDHGEKKl 1SVOTEHSNIYLONGPDR1 GRIYKKAIYIGYTDETFRTT ]EKPVWLGFIGP11 KA-TGDKVYVHLKNIASRPYTF YY-G—c-£WDY VD P YKK-L TD--F P-W-G-LGP-I-AE-^-V—LKN-AS-P— T  FACTOS V!)! CERULOPLASMIN CONSENSUS  A  100 HO 120 130 110 150 160 170 lgT 190 HAVGVSYVKA5EGAEYDD0TS0REKEDDKVFPGGSHTYVW0VUENGPMASDPLCLTYSYLSHVDLVKDLN5GLIGALLVCREGSLAKEKT0TLHKFILL HSHGITYYKEKE6AIYPDNTTDFORAD0KVYPGEOYTYP1LLATEEOSPGEGDGNCVTRIYHSH1DAPKDIASGLIGPL11CKKDSLDKEKEKH1DREFVV K--G-Y-K—EGA-Y-D-T DDKV-PG—TY E—P—D—C-T—Y-SH-D—KD—SGL1G-L--C—iSL-KEK  FACTOR VIII CIRULOPLASMIN CONSENSUS  101  FACTOR VIH CERULOPLASMIN CONSENSUS  200 210 220 230 210 • 250 260 270 280 290 -FAVFDEGKSWHSETKNSL MGDRDAASARAWPKMHTVNGYVNRSLPGl!GCHRKSVYWHVIGMGTTPEVHS! FLEGHTFLVRNHROASLEI SP I 201 ff SWDENFSHYLEDNIKTYCSEPEKV0K0NEDFOESNRMYSVNGYTFGSLPGI.SMCAEDRVKWYLFGMGNEVDVHAAFFHGOALTNKNYR IDTINLFPA -F-Y-DE—SV—E D-D M-TVNGY—SUPGL—C V-W—GMG VH--F—G N-R P-  FACTOR VIM CERULOPLASMIN CONSENSUS  300 310 • 320 330 310 350 360 370 380 390 TFLTAOTLLMDLGOFLLFCHlSSHOHDGnEAYVKVDSCPEEPOLRMXNNEEAEDYDDDLTDSEKDWRFDDDNSPSFlOIRSVAK.KHPKTWVHYIAAEEE 301 TLFDAYMVAONPGEWniSCONlNHUAGLQAFFOVQECMCSSSKD — NIRGKHVRHYYIAAEEi T—A G—L-C H—jG—A—V-C YIAAEE-  FACTOR VI II CERULOPLASMIN CONSENSUS  100 • 110 120 130 110 150 160 170 180 DWDYAPLVLAPDDRSYXSOYLN NGPOR1GRKYXKVRFMAYTDETFKTREA1OHE—SGILGPLLYGEVGDTLLI! FKNQASRPYNIYPHGIT 363 IWNYAPSGIDIFTKENLTAPGSDSAVFFE0GTTR16GSYKKLVYREYTDASFTNRKERGPEEEHLG1LGPV1WAEVGDTIRVTFHNKGAYPLSIEPIGVR -W-YAP i G--R1G—YKK YTD--F—R E GILGP EVGDT F-N P—1-P-G—  I  B  g  h  C  D  E  I  190 500 510 520 530 510 550 560 • 570 DVRP LYSR--RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLJGPULICYKE5VDGrGriQIMSDKRNVIL FNKNNEGTYYSPNYNPQSRSVPP5ASHVAPTETETYEWTVPKEVGPTNADPVCLAKMYYSAVDPTKDIFTGUIGPMKICKKGSLHA'IGRQKDVDKE-FYL Y5 P-E-F-Y-WTV—E-GPT—DP-CL—Y-S-V D—GLIGP—1C-K-S G-0---DK L T  FACTOR V I l l CERULOPLASMIN ;ONSENSUS  163  H FACTOR vin CERULOPLASMIN CONSENSUS  I  f  580 590600 610 • 620 630 610 650 660 670 F-5VFDENRSWYLTEN IORFLPNPAGVOLEDPEFOASNIf.HSINGYVFD-SLOUSVCLHEVAYWYlLSlGAOTDFLSVFFSGYTFKHKMVYEDTLTLFPF 562 FPTVFDENESLLLEDN1RMFTTAPD0VDKEDEDFOESNKMH5HNGFMYGN0PGLTMCKGDSVWYLFSAGNEADVHGIYFSGNTYI.WRGERRDTANLFPO F—VFDEN-S—L—Nl—F—P—V—ED—FO-SN-flHS-NG L--C WY—S-G—D FSG-T DT—LFP•  m  •  i  FACTOR VIII CERULOPLASMIN CONSENSUS  680 T 690 700 710 720 730. 710 750 760 770 SGETVFttSMENPGlWILGCHNSDFRNRGMTAllKVSSCDKNTGDYYEDSYEDISAYLLSKNNA IEPRSF S0NSRHPSTRQK0FNATT1PENDIEKTDPWF 662 TSLTLHMWPDTEGTFNVECLTTDHYTGGMKQKYTVNOCRROSEDST (707) —T—M G C—D 6M V—C D—  FACTOR VI11 CERULOPLASMIN CONSENSUS  K 1690 1730 1710 1750 1760 1720 1700 1710 1680 FKKVVFQEFTDGSFTQPLYRGEINEHLGILG KEDFDIYDEDENOSPRSFQKKTRHYF1AAVERLWDYGKSSSP ...„.,. -HVLRNRAOSGSVPO708 FYLGERTYYIAAVEVEWDYSPOREWEXELHHLQEONVSNAFLDKGEFYIGSKYKKWYROYTDSTFRVPVERKAEEEHLGILG KKW TD—F—P—R EHLG-LG F R-Y-1AAVE—WDY H-L—A-S  L  O  17^_  w  M  FACTOR VIII CERULOPLASMIN CONSENSUS  1780 1790 1800 1810 1820 1830 1810 1850 1860 PY1RAEVEDN1MVTFRN0ASRPYSFYSSLISYEED0R0GAEPRKNFVKPNETKTYFWKV0HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHT 791 POIHADVGDKVK! 1FKNMATRPYSIHAHGV0TESSTVTPTLPGETL TYWK 1PF.RSGAGTEDSACIPWAYYSTVDOVKDLYSGL1 GPL 1VCRR p-.-A-V-D F-N-A-RPYS E PjTY-WK C—WAY-S-VD—KD—S6L1GPL-VC —  FACTOR VI II CERULOPLASMIN CONSENSUS  1870 1880 1890 1900 1910 1920 1930 1910 1950 1960 NTLNPAHGROVTVOEFALFFTIFDETKSWYFTENMERNCRAPCNIOMEDPTFKENYRFHAINGYIMDTLPGLVMAQDORIRWYLLSMGSNENIHSIHFSG PYLKVFNPRRKl—EFALLFIVFDENESWYIDDN1KTYSDHPEKVNKDDEEF1ESNKMHAINGRMFGNLQGITMHVGDEVNWYIMGMGNE1DLHTVHFHG -L R EFAL-F-FDE-SWY—N P D-F-E HALNG L-Gl-5 WYL—MG H—Hr-G  N  FACTOR VIII CERULOPLASMIN CONSENSUS  881  1970 1980 1990 2000 2010 2020 2030 2010 2050 2060 HVFTVRKKEEYKMALYHLYPGVFETVEMLPSKAG1WRVECLIGEHLHAGMSTLFLVYSNKC0TPLGMASGH1RDF0ITASGQYGOWAPKLARLHYSGSIN 982 HSFOYXHRGVYS5DVFD1FPGTYQTLEMFPRTPGIHLLHCHVTDHIHAGMETTYTVL0NEDTKSG . H-F Y PG—T-EM-P—G1W—C-—H-HAGM-T---V--N  149  have c o i n c i d e n t a l l y i n t e r r u p t e d  amino a c i d s  i n the same codon phase.  A l t e r n a t i v e l y , i n t r o n s may have o r i g i n a l l y been p r e s e n t i n i d e n t i c a l positions lost. and  i n f a c t o r V I I I and c e r u l o p l a s m i n , and may have been subsequently  T h i s model, however, d i c t a t e s the p r e s e n c e of v e r y s m a l l exons (10  7 bp, r e s p e c t i v e l y )  i n the a n c e s t r a l  gene, t h e r e b y making the former  t h e o r y more a t t r a c t i v e . The d i f f e r e n c e  i n the p o s i t i o n of i n t r o n h i n the f a c t o r V I I I gene  c o r r e s p o n d i n g t o i n t r o n F i n c e r u l o p l a s m i n may be t h e r e s u l t o f i n t r o n sliding.  Amino a c i d sequence comparison i n t h e r e g i o n  o f these  junctions  i n f a c t o r V I I I and c e r u l o p l a s m i n shows a low degree of  s i m i l a r i t y , w i t h a d e l e t i o n observed i n t h e f a c t o r V I I I p r o t e i n r e l a t i v e to that of ceruloplasmin (Figure deletions possible  25).  intron  sequence  Since i n s e r t i o n s or  a r e a consequence of the i n t r o n s l i d i n g mechanism, i t i s that j u n c t i o n a l s l i d i n g  (due t o the f o r m a t i o n and u t i l i z a t i o n of  a l t e r n a t i v e s p l i c e donor/acceptor s i t e s w i t h i n  exons o r i n t r o n s )  may  account f o r t h i s observed v a r i a t i o n i n i n t r o n p o s i t i o n , w h i l e m a i n t a i n i n g the  c o d i n g phase of the i n t r o n . The remainder of the i n t r o n s  date ( i . e . ,  i d e n t i f i e d i n the c e r u l o p l a s m i n gene to  i n t r o n s M and N, c o n t a i n e d w i t h the A3 r e p e a t e d u n i t ; see  F i g u r e 24) do n o t seem to c o r r e l a t e w i t h the p o s i t i o n s f a c t o r V I I I gene.  I t i s thus l i k e l y t h a t  of the two genes have r e s u l t e d deletion  of i n t r o n s  i n the  introns present i n t h i s  region  from independent i n t r o n i n s e r t i o n or  events.  F i g u r e 24 c l e a r l y shows that  i n several  f a c t o r V I I I gene possesses more i n t r o n s As has been s t a t e d p r e v i o u s l y ,  corresponding regions,  the  than does the c e r u l o p l a s m i n gene.  however, i t i s not p o s s i b l e  to d i s t i n g u i s h  150  whether t h i s r e f l e c t s i n t r o n i n s e r t i o n i n the f a c t o r V I I I gene, o r corresponding B.5  i n t r o n l o s s i n the c e r u l o p l a s m i n  gene.  C h a r a c t e r i z a t i o n of the 5' End of the Human C e r u l o p l a s m i n  Gene  The i n t e r r u p t i o n o f t h e 5' u n t r a n s l a t e d r e g i o n by a t l e a s t i n t r o n s renders  the o r g a n i z a t i o n of t h e 5 * end of the human  gene somewhat u n u s u a l .  A l t h o u g h n o t a common o c c u r r e n c e ,  four  ceruloplasmin  the p r e s e n c e of  one i n t r o n i n t h e 5' u n t r a n s l a t e d sequence o f a number of genes from d i v e r s e organisms has been r e p o r t e d  [e.g., t h e human and c h i c k e n  insulin  gene ( P e r l e r e t a l . , 1980; B e l l e t a l . , 1980), t h e heat shock p r o t e i n 83 gene from D r o s o p h i l i a (Hackett of y e a s t  (Schneider  (Irminger  and L i s , 1983), and t h e n u c l e a r COX4 gene  and Guarente, 1987).  However, i t has been r e p o r t e d  e t a l . , 1987) t h a t t h e s i n g l e - c o p y , human i n s u l i n - l i k e growth  f a c t o r I I (IGFII) gene p o s s e s s e s a t l e a s t 3 exons ( d e s i g n a t e d i n a 5' t o 3' d i r e c t i o n ) i n the 5' u n t r a n s l a t e d r e g i o n . encode a l t e r n a t i v e 5* u n t r a n s l a t e d r e g i o n s  These exons  i n a t i s s u e - s p e c i f i c manner  r e s u l t i n g i n I G F - I I mRNA s p e c i e s of v a r i a b l e l e n g t h s . and  1, 2, and 3,  Brain, placenta,  a d r e n a l g l a n d c o n t a i n a 6.0 kbp IGF-II t r a n s c r i p t , u t i l i z i n g exon 3 i n  the 5' u n t r a n s l a t e d r e g i o n , w h i l e  l i v e r c o n t a i n s a 5.3 kbp I G F - I I  t r a n s c r i p t , w i t h the 5' u n t r a n s l a t e d segment d e r i v e d from exons 1 and 2. The n o v e l o r g a n i z a t i o n of these  t r a n s c r i p t s was  f i r s t d e t e c t e d when i t was  found t h a t the 5' u n t r a n s l a t e d r e g i o n s of c h a r a c t e r i z e d cDNA c l o n e s d i f f e r e d from the 5* sequence r e p o r t e d 1984).  i n the genomic c l o n e  (Dull et a l . ,  I t has n o t been e s t a b l i s h e d whether the heterogenous t r a n s c r i p t s  are due t o the e x i s t e n c e of two t i s s u e - s p e c i f i c promoters ( i . e . one upstream of exon 1 and one upstream of exon 3) as i s the case f o r the mouse a-amylase gene (Young e t a l . , 1981; S c h i b l e r e t a l . , 1983) (see  151  below), o r whether d i f f e r e n t t r a n s c r i p t s patterns  i n various tissues.  been determined, thereby the 5' u n t r a n s l a t e d  a r i s e from a l t e r n a t i v e  splicing  The 5' e x t e n t o f the IGF-II gene has not  rendering  the p o s s i b i l i t y o f a d d i t i o n a l  yet  exons i n  segment.  The a-amylase t r a n s c r i p t s accumulate i n two d i f f e r e n t t i s s u e s o f the mouse - the s a l i v a r y g l a n d and the l i v e r .  The n u c l e o t i d e sequences o f  the two mRNA s p e c i e s are i d e n t i c a l , w i t h the e x c e p t i o n non-translated  regions.  The 5* t e r m i n a l 158 n u c l e o t i d e s o f the major  l i v e r species are separated a 4.5 Kbp i n t r o n .  Kbp.  from the f i r s t  exon i n the c o d i n g  sequence by  T h i s l e a d e r sequence i s u n r e l a t e d t o the 5' t e r m i n a l 47  nucleotides present from t h e f i r s t  o f the 5'  i n the s a l i v a r y g l a n d c o u n t e r p a r t  exon i n the coding  and i s  sequence by an a d d i t i o n a l  separated i n t r o n o f 2.6  The two d i s t i n c t mRNA s p e c i e s a r i s e due t o the p r e s e n c e o f a  s p e c i f i c promoter f o r each t r a n s c r i p t  (Schibler  e t a l . , 1983); t h e  r e l a t i v e a c t i v i t y o f these two promoters i s t i s s u e - s p e c i f i c . Interestingly, for  the use o f a l t e r n a t i v e promoters has a l s o been d e s c r i b e d  the g e n e r a t i o n  forms o f y e a s t It  o f two t r a n s c r i p t s encoding i n t r a c e l l u l a r and s e c r e t e d  i n v e r t a s e ( C a r l s o n and B o t s t e i n , 1982) .  i s i n t e r e s t i n g t o s p e c u l a t e as t o the b i o l o g i c a l s i g n i f i c a n c e o f  the heterogenous 5' n o n - t r a n s l a t e d  l e a d e r s observed i n the I F G - I I and  a-amylase genes.  transcripts  Perhaps v a r i a b l e  result in different  l e v e l s o f t r a n s l a t i o n a l e f f i c i e n c i e s , thereby  selectively  protein  I t has been r e p o r t e d  RNA  concentrations  in different tissues.  s p e c i e s c o n t a i n i n g g r e a t e r than two hundred r e s i d u e s  non-coding r e g i o n are t r a n s l a t e d  regulating that  i n the 5'  l e s s e f f i c i e n t l y than are s h o r t e r  sequences by e u k a r y o t i c ribosomes i n v i t r o (Young e t a l . , 1981).  leader Thus,  152  the l e n g t h o f t h e 5* n o n - t r a n s l a t e d segment may r e g u l a t e t h e e f f i c i e n c y o f t r a n s l a t i o n o r perhaps a f f e c t mRNA s t a b i l i t y .  The s i g n i f i c a n c e o f  m u l t i p l e exons i n t h e 5' u n t r a n s l a t e d r e g i o n o f t h e c e r u l o p l a s m i n gene i s u n c l e a r a t p r e s e n t s i n c e both t h e 3.7 Kb and 4.5 Kb mRNA s p e c i e s a r e d e t e c t a b l e w i t h 5' end probes t e s t e d t o d a t e . if  T h i s would n o t be expected  a l t e r n a t i v e s p l i c i n g p a t t e r n s g i v e r i s e t o these two t r a n s c r i p t s .  U t i l i z a t i o n o f two d i f f e r e n t promoters (as has been d e s c r i b e d f o r t h e mouse a-amylase gene and t h e y e a s t i n v e r t a s e gene) cannot be r u l e d out at present.  I t i s p o s s i b l e , however, t h a t t h i s unusual  o r g a n i z a t i o n may  be i m p l i c a t e d i n t h e r e g u l a t i o n o f t i s s u e - s p e c i f i c e x p r e s s i o n o f t h e c e r u l o p l a s m i n gene, as d e s c r i b e d above f o r t h e a-amylase and IGF-II genes.  U n f o r t u n a t e l y , d a t a have n o t y e t been o b t a i n e d r e g a r d i n g t h e  synthesis of ceruloplasmin i n extrahepatic t i s s u e s .  Alternatively, i t i s  p o s s i b l e t h a t t h e n o v e l o r g a n i z a t i o n o f t h e c e r u l o p l a s m i n gene may be r e q u i r e d f o r t h e r e g u l a t i o n o f c e r u l o p l a s m i n l e v e l s by exogenous copper, o r d u r i n g t h e acute phase inflammatory  response ( s e e S e c t i o n I.B.4).  With  r e f e r e n c e t o t h e l a t t e r p r o p o s a l , t h e complete gene o r g a n i z a t i o n s o f s e v e r a l acute phase r e a c t a n t s have been determined  [e.g.  transferrin  (Lucero e t a l . , 1986) and f i b r i n o g e n ( C r a b t r e e e t a l . , 1985)]. b a s i s o f DNA sequence d e t e r m i n a t i o n s , unusual  these l a t t e r genes do n o t possess  o r g a n i z a t i o n s i n t h e i r r e s p e c t i v e 5' ends.  the 5' end o f t h e c e r u l o p l a s m i n mRNA u s i n g primer SI mapping techniques  On the  Further a n a l y s i s of  e x t e n s i o n , and n u c l e a s e  t o e s t i m a t e exon s i z e s s h o u l d a l l o w d e t e r m i n a t i o n o f  the s i z e o f t h e 5' u n t r a n s l a t e d l e a d e r , and i d e n t i f i c a t i o n o f t h e transcriptional start site.  These r e s u l t s , coupled w i t h a n a l y s i s o f  e x t r a h e p a t i c human c e r u l o p l a s m i n b i o s y n t h e s i s and complete  153  c h a r a c t e r i z a t i o n o f the 5' end o f t h e gene may r e s o l v e the e x i s t e n c e o f the two c e r u l o p l a s m i n mRNA s p e c i e s d e t e c t a b l e w i t h N o r t h e r n b l o t analysis.  Thus f a r , DNA sequence a n a l y s i s o f the 5' u n t r a n s l a t e d  region  has n o t r e v e a l e d t h e p r e s e n c e o f any t y p i c a l e u k a r y o t i c promoter elements, strongly suggesting  t h a t t h e 5' most end o f the c e r u l o p l a s m i n  transcript  has n o t y e t been i d e n t i f i e d .  C.  CHARACTERIZATION OF A PSEUDOGENE FOR HUMAN CERULOPLASMIN  C l  DNA Sequence A n a l y s i s o f t h e Human C e r u l o p l a s m i n  Pseudogene  U s i n g p r e v i o u s l y c h a r a c t e r i z e d cDNA c l o n e s f o r human ceruloplasmin  as h y b r i d i z a t i o n probes f o r human genomic l i b r a r i e s ,  o v e r l a p p i n g recombinant phage c l o n e s encoding approximately contiguous  genomic DNA were o b t a i n e d .  f o r human c e r u l o p l a s m i n was i d e n t i f i e d  21 kbp o f  W i t h i n t h i s r e g i o n , a pseudogene ( n e a r l y 2 Kbp i n l e n g t h ) ,  corresponding  t o n u c l e o t i d e r e s i d u e s 1502 - 3198 o f t h e human  ceruloplasmin  cDNA sequence.  A d d i t i o n a l l y , t h e pseudogene extends through  the 123 bp o f t h e 3' u n t r a n s l a t e d sequence t h a t i s p r e s e n t cDNA c l o n e , and c o n t i n u e s  four  i n t h e phCP-1  f o r a f u r t h e r 40 bp, t h e sequence o f which  corresponds t o t h e 3' u n t r a n s l a t e d sequence o f a c e r u l o p l a s m i n d e s c r i b e d by Yang e t a l . (1986).  cDNA c l o n e  On t h i s b a s i s , i t appears t h a t the  pseudogene has been d e r i v e d from an mRNA s p e c i e s c o r r e s p o n d i n g  t o the cDNA  c l o n e d e s c r i b e d by Yang e t a l . (1986), as opposed t o the s h o r t e r mRNA species represented  by phCP-1.  The d e r i v a t i o n o f the c e r u l o p l a s m i n  pseudogene from t h i s p a r t i c u l a r mRNA s p e c i e s i s i n t e r e s t i n g , s i n c e i t has been shown t h a t f o r d i f f e r e n t r a t cytochrome c mRNA s p e c i e s o c c u r r i n g a t the same i n t r a c e l l u l a r c o n c e n t r a t i o n , m u l t i p l e pseudogenes have a r i s e n  154  p r e f e r e n t i a l l y from one mRNA ( S c a r p u l l a , 1984). t h a t t h i s may be due t o l o c a l secondary  I t has been p o s t u l a t e d  s t r u c t u r e i n the 3' end o f t h e  mRNA, which f a c i l i t a t e s b i n d i n g o f enzymes i n v o l v e d i n r e v e r s e t r a n s c r i p t i o n o r subsequent i n t e g r a t i o n i n t o t h e genome ( S c a r p u l l a ,  1984).  As i s c h a r a c t e r i s t i c o f p r o c e s s e d pseuodgenes, complete DNA sequence a n a l y s i s r e v e a l e d t h a t t h e human c e r u l o p l a s m i n pseudogene l a c k s i n t r o n s p r e s e n t i n t h e w i l d - t y p e gene.  I n c o n t r a s t t o numerous o t h e r examples o f  p r o c e s s e d pseudogenes i n which t h e i n t e r v e n i n g sequences a r e p r e c i s e l y removed (e.g., V a n i n e t a l . , 1980), t h e c e r u l o p l a s m i n pseudogene c o n t a i n s a 213 bp d e l e t i o n ( c o r r e s p o n d i n g t o n u c l e o t i d e s 1865 - 2077 o f t h e cDNA sequence) t h a t o c c u r s e x a c t l y a t t h e boundaries w i l d - t y p e gene.  o f exon 11 i n t h e  There i s a l s o a s m a l l 17 bp d e l e t i o n i n t h e pseudogene  sequence, b e g i n n i n g a t n u c l e o t i d e 2943 o f t h e cDNA sequence. been determined  whether t h i s d e l e t i o n a l s o corresponds  i n t r o n / e x o n boundaries d e l e t i o n observed  i n t h e w i l d - t y p e gene.  I t has n o t  to the l o c a t i o n of  I t i s u n c l e a r i f t h e 213 bp  i n t h e pseudogene o c c u r r e d a t t h e time o f i n t r o n  p r o c e s s i n g , o r whether t h e d e l e t i o n i s t h e r e s u l t o f a subsequent  mutation  event. The  5' boundary o f t h e pseudogene i s c h a r a c t e r i z e d by a s h o r t  sequence t h a t i s homologous t o t h e 3' end o f i n t r o n H i n t h e w i l d - t y p e gene.  The presence  o f t h i s i n t r o n i c segment i s expected,  since the  pseudogene d i v e r g e s from t h e w i l d - t y p e gene p r i o r t o t h e 5'end o f t h i s intron.  T h e r e f o r e , t h e a p p r o p r i a t e 5' s l i c e r donor s i t e  (Breathnach and  Chambon, 1981; Cech, 1983), r e q u i r e d f o r i n t r o n removal i s absent. Although  t h e pseudogene appears t o have been d e r i v e d from a p r o c e s s e d  RNA s p e c i e s ( i . e . l a c k i n g i n t e r v e n i n g sequences),  t h e r e i s no p o l y ( A )  155  t r a c t present  i n t h e sequence.  pseudogenes have a p o l y ( A )  tail,  While the m a j o r i t y o f p r o c e s s e d s e v e r a l exceptions  (Vanin e t a l . , 1980; Notake e t a l . , 1983). absence o f a p o l y ( A )  have been r e p o r t e d  I t i s possible that the  t r a c t i n t h e human c e r u l o p l a s m i n pseudogene sequence  may be t h e r e s u l t o f t h e mechanism o f i t s f o r m a t i o n , base p a i r i n g between t h e p o l y ( A ) 3* u n t r a n s l a t e d r e g i o n .  tail  possibly involving  o f t h e mRNA and U - r i c h r e g i o n i n t h e  I n t h i s case, t h e s i x T r e s i d u e s  f o l l o w i n g t h e 3' u n t r a n s l a t e d segment c o r r e s p o n d i n g Yang e t a l . (1986) may r e p r e s e n t  observed  t o t h a t d e s c r i b e d by  a s i t e o f mRNA s e l f - p r i m i n g .  e x p l a n a t i o n may a l s o account f o r t h e d i v e r g e n c e  t h a t occurs  This  at this point  between t h e pseudogene sequence and t h e remainder o f the 3' u n t r a n s l a t e d r e g i o n r e p o r t e d by Yang e t a l . (1986). The p r e s e n c e o f a repeated  CT d i n u c l e o t i d e segment a t the 3' end o f  the c e r u l o p l a s m i n pseudogene sequence c o r r e s p o n d i n g is  interesting.  t o the coding  A 116 bp segment, composed m a i n l y o f repeated GA  d i n u c l e o t i d e s corresponding  t o t h e coding  s t r a n d has been r e p o r t e d a t t h e  3' end o f t h e mouse c o r t i c o t r o p i n B - l i p o t r o p i n p r e c u r s o r (Notake e t a l . , 1983).  I n t h e l a t t e r case,  t h i s repeated  pseudogene segment  immediately f o l l o w i n g t h e p o i n t a t which t h e pseudogene d i v e r g e s wild-type  gene.  strand  occurs from the  I n t h e human c e r u l o p l a s m i n pseudogene, the r e p e a t e d TC  r e g i o n occurs  172 bp 3' t o t h e p o i n t a t which the pseudogene d i v e r g e s  the w i l d - t y p e  gene.  from  The r a t m e t a l l o t h i o n e i n pseudogene 14b, which has  been c h a r a c t e r i z e d by Andersen e t a l . (1986), c o n t a i n s a 42 bp p o l y ( C A ) tract,  l o c a t e d approximately  300 bp 3' t o the s i t e o f p o l y a d e n y l a t i o n .  While s t r e t c h e s o f r e p e a t i n g CA r e s i d u e s have been found i n e u k a r y o t i c DNA at  the s i t e o f recombination  events such as gene c o n v e r s i o n  (Shen e t a l . ,  156  1981), and  are thought to induce Z-DNA c o n f o r m a t i o n a l  and R i c h , 1983), the f u n c t i o n of s i g n i f i c a n t l y s t r e t c h e s remains u n c l e a r .  T r a c t s of d(GA)  changes (Nordheim  long p u r i n e o r p y r i m i d i n e  •d(TC) n  have been  a t many s i t e s i n e u k a r y o t i c genomes [e.g. human U l RNA 1984)  and  Interestingly, a d(GA)  . d(TC)  2 ?  demonstrated i n a p o l y o m a v i r u s - t r a n s f o r m e d h o s t DNA process  genes (Htun e t a l . ,  the murine immunoglobulin u-c heavy c h a i n gene  e t a l . , 1983).  cell  2 7  (Richards  t r a c t has  that t h i s repeated  been  l i n e near the end  segment t h a t i s r e s p o n s i b l e f o r a r r e s t of the v i r a l (Baran e t a l . , 1987).  found  n  In the l a t t e r case,  of a  replication  i t has been p o s t u l a t e d  sequence, i n c o n j u n c t i o n w i t h an i n v e r t e d r e p e a t ,  s e r v e as an a r r e s t s i t e f o r chromatin  replication in vivo.  In  pseudogenes, i t i s a t t r a c t i v e to s p e c u l a t e t h a t repeated p y r i m i d i n e p u r i n e s t r e t c h e s may  be  i n v o l v e d i n the p r o c e s s  i n t o the genome, s i n c e v e r y l i t t l e mediating C.2  t h i s event  (Vanin,  or  of pseudogene i n t e g r a t i o n  i s known about the mechanism(s)  1985).  Chromosomal L o c a t i o n of the Human C e r u l o p l a s m i n A l l processed  may  Pseudogene  pseudogenes s t u d i e d to date a r e l o c a t e d on  d i f f e r e n t chromosomes than t h e i r f u n c t i o n a l c o u n t e r p a r t s .  This i s i n  c o n t r a s t to non-processed pseudogenes, which have l i k e l y a r i s e n from gene d u p l i c a t i o n events  and  are t h e r e f o r e on the same chromosome as  r e s p e c t i v e w i l d - t y p e gene (Vanin, 1985). human-hamster h y b r i d c e l l  lines  (Donald  Using p r e v i o u s l y c h a r a c t e r i z e d e t a l . , 1983), the human  c e r u l o p l a s m i n pseudogene has been a s s i g n e d to chromosome 8. has been r e c e n t l y v e r i f i e d , u s i n g the technique (Wang e t a l . , 1987).  This d i f f e r s  functional ceruloplasmin  the  This  result  of i n s i t u h y b r i d i z a t i o n  from the u n e q u i v o c a l  assignment of  l o c u s to chromosome 3q25 (Yang e t a l . , 1986;  the  157  Royle e t a l . , 1987).  I t has been r e p o r t e d p r e v i o u s l y by Yang e t a l .  (1986) t h a t a 0.8 kbp EcoRI fragment t h a t can be i d e n t i f i e d i n genomic Southern b l o t s probed w i t h t h e c e r u l o p l a s m i n cDNA segregates chromosome 11.  However, t h e p r e s e n t  study suggests  w i t h human  t h a t t h i s 0.8 kbp  EcoRI fragment i s p a r t o f t h e human c e r u l o p l a s m i n pseudogene, and maps t o chromsome 8 ( s e e S e c t i o n I I I . C ) . unclear a t present,  The reason  f o r t h i s discrepancy i s  s i n c e genomic Southern b l o t a n a l y s i s i n d i c a t e s t h a t  t h e r e i s o n l y one pseudogene f o r human c e r u l o p l a s m i n .  However, s i n c e t h e  mapping a n a l y s i s r e p o r t e d by Yang e t a l . (1986) was performed u s i n g the c e r u l o p l a s m i n cDNA t o probe human-mouse h y b r i d c e l l l i n e s , t h e 0.8 kbp band d e t e c t e d may r e p r e s e n t a c r o s s - r e a c t i n g s p e c i e s i n t h e mouse genome. T h i s i s i n agreement w i t h p r e v i o u s d i f f i c u l t i e s  encountered i n chromosome  mapping when u s i n g cDNA fragments as h y b r i d i z a t i o n probes ( J . Hamerton, personal C.3  communication). S p e c u l a t i o n s on t h e E v o l u t i o n a r y O r i g i n o f t h e Human Ceruloplasmin  Pseudogene  The human c e r u l o p l a s m i n pseudogene shares a p p r o x i m a t e l y n u c l e o t i d e sequence i d e n t i t y compared t o t h e w i l d - t y p e coding sequence, s u g g e s t i n g e v o l u t i o n a r y time. analyzed  t o date  ceruloplasmin  t h a t i t has been formed r e l a t i v e l y r e c e n t l y i n  This i s c h a r a c t e r i s t i c of processed  pseudogenes  [e.g. L i e t a l . , 1981; F r e y t a g e t a l . , 1984), a l l of  which have a r i s e n f o l l o w i n g mammalian r a d i a t i o n years ago) (Vanin, 1985)].  (approximately  80 m i l l i o n  I n t e r e s t i n g l y , t h e e x i s t e n c e of a p r o c e s s e d  pseudogene f o r r a t c e r u l o p l a s m i n has been suggested et  97%  r e c e n t l y by Shvartsman  a l . (1985) based on p r e l i m i n a r y r e s t r i c t i o n endonuclease mapping  a n a l y s i s of r a t c e r u l o p l a s m i n genomic c l o n e s .  T h i s suggests  t h a t the  158  formation  of the ceruloplasmin  of r a t s and humans.  pseudogene o c c u r r e d  p r i o r t o the divergence  This i s consistent with the notion that  processed  pseudogenes have a r i s e n i n genomes f o l l o w i n g t h e widespread appearance o f mammals on e a r t h . Presumably pseudogenes a r e n o t s u b j e c t t o f u n c t i o n a l c o n s t r a i n t s . T h i s has been i n t e r p r e t e d t o suggest t h a t t h e p a t t e r n o f n u c l e o t i d e s u b s t i t u t i o n s i n pseudogenes should ( L i , 1983).  Thus, t r a n s v e r s i o n s  pyrimidine-purine transitions  r e f l e c t patterns  ( i . e . purine-pyrimidine or  base s u b s t i t u t i o n s ) s h o u l d  ( i . e . purine-purine  o f i n t r i n s i c mutation  occur  t w i c e as f r e q u e n t l y as  or pyrimidine-pyrimidine  mutations occur randomly ( L i , 1983). s u b s t i t u t i o n p a t t e r n a t the f i r s t  base changes), i f  Compared w i t h t h e n u c l e o t i d e  and second p o s i t i o n s o f codons i n  f u n c t i o n a l genes, i t has been shown f o r pseudogenes t h a t t h e r e l a t i v e f r e q u e n c y o f t r a n s i t i o n s i s much g r e a t e r than t h a t o f t r a n s v e r s i o n s ( G o j o b o r i , 1982).  T h i s suggests t h a t a sequence under no f u n c t i o n a l  c o n s t r a i n t w i l l become A - T r i c h , due t o t h e spontaneous deamination o f c y t o s i n e and 5 - m e t h y l c y t o s i n e .  Of t h e 44 n u c l e o t i d e s u b s t i t u t i o n s  observed i n t h e human c e r u l o p l a s m i n are t r a n s i t i o n mutations.  ;  pseudogene sequence, 34 o f these (7 7%)  .  Genomic Southern b l o t a n a l y s i s i n d i c a t e s t h a t t h e r e a r e no human ceruloplasmin wild-type aberrant  pseudogene sequences c o r r e s p o n d i n g  gene.  t o t h e 5' end o f the  T h i s suggests t h a t the pseudogene may have a r i s e n from an  t r a n s c r i p t , as the r e s u l t of i n i t i a t i o n w i t h i n the gene.  Such a  model has been proposed f o r t h e mouse c o r t i c o t r o p i n B - l i p o t r o p i n pseudogene (Notake e t a l . , 1983). ceruloplasmin  T h i s pseudogene i s s i m i l a r t o the human  pseudogene, i n t h a t i t i s o n l y a p a r t i a l copy of the  159  f u n c t i o n a l gene, encoding  the c a r b o x y - t e r m i n a l  143 amino a c i d r e s i d u e s and  the 3' u n t r a n s l a t e d r e g i o n (Notake e t a l . , 1983). two  In the case o f these  pseudogenes, RNA polymerase I I I i n i t i a t i o n c o u l d have o c c u r r e d w i t h i n  the r e s p e c t i v e genes.  I n v i t r o e x p r e s s i o n s t u d i e s suggest  that  aberrant  i n i t i a t i o n o f t r a n s c r i p t i o n of t h e human c o r t i c o t r o p i n B - l i p o t r o p i n p r e c u r s o r can occur a t a s i t e w i t h i n t h e gene c o r r e s p o n d i n g  t o t h e 5' end  of t h e homologous pseudogene sequence ( M i s h i n a e t a l . , 1982). A d d i t i o n a l l y , i t has been shown t h a t RNA polymerase I I I a c t i v i t y can r e s u l t i n t r a n s c r i p t s i n i t i a t i n g upstream o f t h e human B g l o b i n gene, which i s n o r m a l l y 1983).  t r a n s c r i b e d by RNA polymerase I I ( C a r l s o n and Ross,  Furthermore, these  be b o t h p o l y a d e n y l a t e d  l a t t e r a b e r r a n t t r a n s c r i p t s have been shown t o  and s p l i c e d  ( C a r l s o n and Ross, 1983).  Therefore,  the p o s s i b l e g e n e r a t i o n o f t h e human c e r u l o p l a s m i n pseudogene from an a b e r r a n t RNA polymerase I I I event or the observed  D.  does n o t account f o r i n c o r r e c t  splicing  l a c k of p o l y a d e n y l a t i o n .  A MODEL FOR THE EVOLUTION OF CERULOPLASMIN, FACTOR V AND FACTOR VIII Based on i t s i n t e r n a l 3 - f o l d repeated  and  comparison t o known c o p p e r - b i n d i n g  proteins  s t r u c t u r e ( S e c t i o n I.B.I)  c e n t e r s i n s e v e r a l b l u e copper  ( S e c t i o n I . E ) , Dwulet and Putnam (1981b) have proposed a model  f o r t h e e v o l u t i o n of t h e c e r u l o p l a s m i n gene. c e r u l o p l a s m i n has evolved (approximately  T h i s model suggests  from a s m a l l p r i m o r d i a l c o p p e r - b i n d i n g  350 amino a c i d s i n l e n g t h ) .  t h r e e repeated  (see F i g u r e 26).  protein  Tandem d u p l i c a t i o n s of t h i s  a n c e s t r a l gene c o u l d then have g i v e n r i s e t o the present-day ceruloplasmin  that  gene f o r  Because the p a i r w i s e comparison of the  u n i t s i n the human c e r u l o p l a s m i n molecule shows very  160  F i g u r e 26.  A proposed model f o r the e v o l u t i o n of c e r u l o p l a s m i n (CP),  f a c t o r V (FV) and f a c t o r V I I I ( F V I I I ) (see t e x t f o r d e t a i l s ) . The t r i p l i c a t e d  A domain i s shown by c r o s s - h a t c h e d b a r s w h i l e the B  and C domains ( p r e s e n t i n f a c t o r s V and V I I I ) a r e i d e n t i f i e d s t i p p l e d bars, respectively. by a s o l i d b a r .  by open and  The c e r u l o p l a s m i n pseudogene i s r e p r e s e n t e d  MY d e s i g n a t e s m i l l i o n  years.  IWWNM  ANCESTRAL  GENE  TRIPLICATION (400 MY)  IWWWlWWWlWWWM  DUPLICATION  FUSION  • EUDc iwwwlvv.-:!-: DUPLICATION  I\\\\\NJ:-,::.-|-::.V.:1  IWWWIWWWI  | \ \ \ \ \ \ M -• T^TI  |\\\\\\M\\\\\\\|  * FORMATION (<80MY)  PRESENT  CP  FV  FVIII  162  s i m i l a r values determined;  (see T a b l e 1 ) , the o r d e r of the t r i p l i c a t i o n cannot  two  be  consecutive elongations l i k e l y occurred c l o s e together  an e v o l u t i o n a r y time s c a l e ( D o o l i t t l e ,  1984).  Without comparative  on  amino  a c i d sequence d a t a and/or m o l e c u l a r weights f o r c e r u l o p l a s m i n from more p r i m i t i v e s p e c i e s , the time of the f i r s t d u p l i c a t i o n event estimate.  f o r the d u p l i c a t e d h a l v e s o f t r a n s f e r r i n r a t e of change of c e r u l o p l a s m i n was s e t s of d u p l i c a t i o n s may ( D o o l i t t l e , 1984).  the same as t h a t  ( D o o l i t t l e , 1984).  have o c c u r r e d w i t h i n the same time  Thus, i f the  t h i s b a s i s , i t would seem r e a s o n a b l e  grown by d u p l i c a t i o n a t the same time.  that ceruloplasmin  suggested  t h a t the  event r e s u l t i n g i n the f o r m a t i o n of c e r u l o p l a s m i n was  has the  triplication  coincident with  ( i . e . 500 m i l l i o n y e a r s ago),  a c l o s e d v a s c u l a r system and a u r o g e n i t a l system.  the  (Doolittle,  T h i s i s i n agreement w i t h  p r o p o s a l of Dwulet and Putnam (1981b) who  both  frame  S i n c e lampreys have a f u l l - s i z e d t r a n s f e r r i n ,  appearance of v e r t e b r a t e animals  observed  s i m i l a r t o t h a t of t r a n s f e r r i n ,  l a t t e r event has been p l a c e d a t > 400 m i l l i o n y e a r s ago On  to  I n t e r e s t i n g l y , the degree of s i m i l a r i t y between the t h r e e  repeated u n i t s i n ceruloplasmin i s approximately  1984).  is difficult  The  latter  the  possessing anatomical  developments n e c e s s i t a t e d t h a t plasma p r o t e i n s have m o l e c u l a r weights of > 60 Kda  to avoid r e n a l e x c r e t i o n .  As has been p r e v i o u s l y d i s c u s s e d , f a c t o r V I I I was  shown to be  s t r u c t u r a l l y r e l a t e d to human c e r u l o p l a s m i n on the b a s i s of amino a c i d similarity  (Wood e t a l . , 1984;  Vehar e t a l . , 1984).  Although  initially  i n d i c a t e d on the b a s i s of l i m i t e d amino a c i d sequence d e r i v e d from f a c t o r V (Church f a c t o r V (Jenny  bovine  e t a l . , 1984), the complete amino a c i d sequence of human e t a l . , 1987)  has  shown c l e a r l y t h a t t h i s p r o t e i n i s  163  s t r u c t u r a l l y r e l a t e d t o both  f a c t o r V I I I and c e r u l o p l a s m i n .  I t has  t h e r e f o r e been proposed t h a t c e r u l o p l a s m i n , f a c t o r V and f a c t o r V I I I form a gene f a m i l y d e r i v e d from a common a n c e s t r a l gene (see F i g u r e 26).  In  t h i s scheme, t h e e n t i r e t r i p l i c a t e d gene f o r human c e r u l o p l a s m i n would have undergone d u p l i c a t i o n , thereby f a c t o r s V and V I I I .  forming  the progenitor species f o r  As i s d e p i c t e d s c h e m a t i c a l l y i n F i g u r e 26, t h i s  event  l i k e l y o c c u r r e d r e l a t i v e l y c l o s e i n e v o l u t i o n a r y time t o t h e f o r m a t i o n o f the i n i t i a l  t r i p l i c a t e d s t r u c t u r e , s i n c e when compared p a i r w i s e , t h e A  r e p e a t e d u n i t s i n c e r u l o p l a s m i n , f a c t o r V and f a c t o r V I I I share s i m i l a r l e v e l s o f amino a c i d i d e n t i t y  ( T a b l e 1 ) , i m p l y i n g t h a t they have  been e v o l v i n g f o r a s i m i l a r l e n g t h o f time. d u p l i c a t i o n event  very  P r i o r to the t h i r d  gene  l e a d i n g t o t h e f o r m a t i o n o f f a c t o r s V and V I I I , t h e B  and C domains were l i k e l y i n s e r t e d by independent gene f u s i o n events ( F i g u r e 26), t h e r e b y VIII molecules. provides  c r e a t i n g t h e l a r g e r and more complex f a c t o r V and  The e v o l u t i o n o f c e r u l o p l a s m i n and f a c t o r s V and V I I I  an example o f d i v e r g e n t e v o l u t i o n i n which f a c t o r s V and V I I I  have adopted d i v e r s e f u n c t i o n s compared t o c e r u l o p l a s m i n .  E.  CONCLUDING REMARKS Comparative a n a l y s e s  o f t h e gene o r g a n i z a t i o n s of s t r u c t u r a l l y  r e l a t e d p r o t e i n s c o n t r i b u t e g r e a t l y t o our knowledge of gene e v o l u t i o n , a i d i n g i n t h e unambiguous families.  c l a s s i f i c a t i o n o f genes i n t o v a r i o u s  F o r example, a l t h o u g h  multigene  the sequence of a n g i o t e n s i n o g e n  i s only  20% i d e n t i c a l t o t h a t of a - a n t i t r y p s i n , t h e d i s t r i b u t i o n of i n t r o n s i n these two genes i s p r e c i s e l y conserved t h e i r unequivocal  (Tanaka e t §_1. , 1984) thus  assignment t o the same gene f a m i l y ( D o o l i t t l e ,  allowing 1985).  164  Examination o f the gene o r g a n i z a t i o n o f c e r u l o p l a s m i n w i t h  that  p r e v i o u s l y r e p o r t e d f o r f a c t o r V I I I ( G i t s c h i e r e t §_1. , 1984) c o n f i r m s these genes a r e members of t h e same gene f a m i l y , and as such have a r i s e n from a common a n c e s t r a l gene.  likely  C h a r a c t e r i z a t i o n o f the f a c t o r V  gene (which as a l s o been proposed as a member of the l a t t e r f a m i l y , based on shared  that  s t r u c t u r a l p r o p e r t i e s ) w i l l prove  multigene  interesting  s i n c e one might p r e d i c t t h a t i t w i l l e x h i b i t a more s i m i l a r o r g a n i z a t i o n to  f a c t o r V I I I than does c e r u l o p l a s m i n , based on s i m i l a r f u n c t i o n a l  c o n s t r a i n t s shared by t h e two b l o o d c o a g u l a t i o n f a c t o r s .  As has been  d i s c u s s e d p r e v i o u s l y , development o f a more complete u n d e r s t a n d i n g  of the  e v o l u t i o n a r y h i s t o r y o f c e r u l o p l a s m i n i t s e l f w i l l r e q u i r e comparative p h y l o g e n e t i c d a t a , i n v o l v i n g t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f c e r u l o p l a s m i n from p r i m i t i v e v e r t e b r a t e s such as h a g f i s h o r lampreys. J u s t as c e r u l o p l a s m i n has been rendered chemical  study,  the molecular  b i o l o g y of t h i s multicopper  p r o v i n g t o be e q u a l l y i n t r i g u i n g . a s p e c t s o f t h i s study, p o s s e s s i n g an unusual  a fascinating protein f o r oxidase i s  As has been shown i n t h e v a r i o u s  t h e c e r u l o p l a s m i n gene i s h i g h l y complex i n n a t u r e , o r g a n i z a t i o n a t the 5' end w i t h m u l t i p l e i n t r o n s  i n t e r r u p t i n g t h e 5* u n t r a n s l a t e d l e a d e r segment, and h e t e r o g e n e i t y r e s p e c t t o t h e s i t e o f p o l y a d e n y l a t i o n a t t h e 3' end.  Therefore,  with complete  knowledge of t h e o r g a n i z a t i o n o f t h i s gene w i l l enhance our u n d e r s t a n d i n g of  gene r e g u l a t i o n i n e u k a r y o t i c systems.  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