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The DNA sequence of a rat RT1 Esub β DNA : assessment of homology within class 11β chains Robertson, Katherine Anne 1985

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THE DNA SEQUENCE OF A RAT RT1 Eg cDNA; ASSESSMENT OF HOMOLOGY WITHIN CLASS II g CHAINS. by KATHERINE ANNE ROBERTSON B.Sc B i o l o gy , U n i v e r s i t y of V i c t o r i a , 1982 A Thes is Submitted i n P a r t i a l F u l f i l l m e n t of the Requirements f o r the Degree of Master of Sc ience i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MEDICAL GENETICS We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA December 1985 (c) Katherine Anne Robertson, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of OeJApMc.S  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Dflr.q.jqgr ABSTRACT The Major H i s t o c o m p a t i b i l i t y Complex (MHC) i s a group of l o c i encoding c e l l su r face g l y cop ro te i n s i nvo l ved i n the r e gu l a t i o n of the immune response. The l o c i w i t h i n the complex d i s p l a y an unusua l ly h igh degree of polymorphism i n the two spec ies s tud ied to date; mice and humans. In order to determine i f t h i s polymorphism i s found across other spec i e s , and i f the encoded molecules show cons i s t en t s i m i l a r i t i e s across the spec i e s , the MHC of a t h i r d mammal, the r a t , was s t ud i ed . The MHC of the r a t , the RT1 Complex, codes f o r two types of C lass I I mo lecu les , A and E, each composed of an a and g c ha i n . A cDNA l i b r a r y made from Wistar r a t sp leen po ly(A) RNA was screened w i th a DNA fragment from an RT1 Ag gene to determine the coded message of a C lass I I 0 gene. A s i n g l e cDNA c lone was i s o l a t e d and when sequenced was found to encode the complete RT1 Eg cha in on the bas i s of comparison t o the sequence of the C lass II g cha ins of mice, as t h i s cha in had not been p r ev i ou s l y sequenced i n the r a t . The p red i c ted p r o t e i n sequence of the RT1 Eg cha in was found to con ta in g rea te r amino a c i d sequence i d e n t i t y to the equ iva len t genes i n mice and humans (82% and 73%) than to the RT1 Ag gene (58%). Comparison of the p red i c t ed p ro te i n s of the A and E g genes of r a t s , mice and humans showed 105 out of 278 res idues were i d e n t i c a l across a l l s i x cha i n s . In a l l s i x cha ins a number of reg ions con ta i n i ng s t r e t ches of i d e n t i c a l res idues were found which may be res idues i nvo l ved i n s t r u c t u r e and f u n c t i o n . The d ivergence of the Ag and Eg chains across th ree spec ies was examined by comparing the number of replacement and s i l e n t nuc l eo t i de changes between the p r o t e i n coding sequences. Th is comparison showed tha t whereas the number of s i l e n t s i t e s u b s t i t u t i o n s increased w i t h i n c r ea s i ng spec ies d ive rgence , the number of replacement s i t e s u b s t i t u t i o n s d i d not , suggest ing tha t these s u b s t i t u t i o n s are under s e l e c t i v e p res su re . TABLE OF CONTENTS Cal) page INTRODUCTION The Major H i s t o c o m p a t i b i l i t y Complex 1 I . C e l l u l a r B i o l ogy - H i s to ry 2 I I . The Three C lasses of Products 3 I I I . Genet ics of the Major H i s t o c o m p a t i b i l i t y Complex 5 A. The Rat RT1 System 5 B. The Mouse H-2 System 7 C. The Human HLA System 8 IV. S t ru c tu re of the MHC Molecules 9 A. C lass II Molecules 10 B. C lass I Molecules 12 V. Funct ion of the MHC Products 13 A. C lass I and C lass I I C e l l Sur face P ro t e i n s 13 B. Assoc ia ted Recogn i t i on by T Lymphocytes 14 V I . Importance of Studying the MHC 16 METHODS AND MATERIALS Stock So l u t i on s 18 I . Plasmids pRTlE^.l and pRT1.2 19 1. cDMA L i b r a i r i e s 19 2. I s o l a t i o n of an RT1 Ae Gene 20 3. Nick T r an s l a t i o n of Ag Gene 20 4. I s o l a t i o n of Clones H y b r i d i z i n g to the As Gene 22 5. I s o l a t i o n of P lasmid DNA 23 a. Plasmid P repara t i on b. P u r i f i c a t i o n of Closed C i r c u l a r P lasmid 6. Cha r a c t e r i z a t i o n of the Plasmids 26 I I . M13 Sequencing 27 1. Sequencing Strategy 27 2. Son i ca t i on of P lasmid 28 3. Po lymer i za t i on 28 4. L i g a t i o n 29 5. Transformat ion of Host C e l l s 30 a. Competent Ce l I s b. Transformat ion of E . c o l i 6. Dot B l o t H y b r i d i z a t i o n 33 a. F i l t e r P repara t i on b. H y b r i d i z a t i o n 7. Template P repara t i on 34 8. P repara t ion of Terminat ion Mixes 35 9. P repara t ion of Acry lamide Gels 36 10. Dideoxy Sequencing Reac t ion 37 11. Compi la t ion of Data 39 R E S U L T S Gv) • I . C h a r a c t e r i z a t i o n o f t h e p R T l E g . l cDNA a n d P r e d i c t e d P r o t e i n 4 0 A . p R T l E g . l cDNA I n s e r t 4 0 B . P r e d i c t e d P r o t e i n S e q u e n c e o f p R T l E g . l 4 3 C . C o m p a r i s o n o f p R T l E g . l t o E q u i v a l e n t DMA i n D i f f e r e n t S p e c i e s 4 6 D. P r e d i c t e d P r o t e i n D o m a i n s o f p R T l E g . l 4 6 E . C o m p a r i s o n o f p R T l E g . l P r e d i c t e d P r o t e i n S e q u e n c e t o C l a s s I I P r o t e i n s 4 8 I I . C o m p a r i s o n o f N u c l e o t i d e C h a n g e s W i t h i n C l a s s I I g C h a i n s 5 3 I I I . C h a r a c t e r i z a t i o n o f p R T 1 . 2 5 3 G E N E R A L D I S C U S S I O N I . E v o l u t i o n 5 8 I I . S i g n i f i c a n c e o f t h e P o l y m o r p h i s m 64 I I I . F u t u r e R e s e a r c h 6 5 C O N C L U S I O N S 66 R E F E R E N C E S 67 LIST OF TABLES page Table 1 Comparison of the cDNA Sequence of pRT lEg. l 44 to C lass II & Genes Table 2 P red i c ted P r o t e i n Sequence Homology of C l a s s 49 II 8 Chains Table 3 Sequence Divergence of S i m i l a r Genes Across D i f f e r en t Species 54 LIST OF FIGURES page F igu re 1 Models of Immunoglobulin Super fami ly P ro te i n s Showing 4 Homology F igu re 2 Genet ic Organ i za t i on of the Major H i s t o c o m p a t i b i l i t y 6 Complex i n Man, Mouse and Rat F igure 3 Sequencing Strategy of pRT lEg. l cDNA Inser t 41 F igure 4 DNA Sequence of pRT lEg. l i n comparison to H-2 Eg and 42 RT1 Ag F igu re 5 P red i c ted Amino Ac id Sequence of pRT lEg . l i n Comparison 45 to HLA-DRg and H-2 Eg F igure 6 Amino Ac id Residues Conserved Across A and E g Chain 50 P ro te ins F igure 7 Sequencing St rategy of pRT1.2 cDNA Inser t 56 F igure 8 DNA Sequence of the Three Fragments of pRT1.2 57 F igure 9 Proposed Evo lu t i ona ry Tree of Immunoglobulin Family 59 From Typ ica l P recursor F i gu re 10 Divergence of Replacement and S i l e n t Changes i n C lass 62 II e Genes as a Funct ion of Time INTRODUCTION 1 The Major H i s t o c o m p a t i b i l i t y Complex The major h i s t o c o m p a t i b i l i t y complex (MHC) i s a unique group o f l o c i i n which are found genes encoding c e l l su r f a ce molecules i nvo lved i n the r e g u l a t i o n of the immune response. The MHC complex i s i nvo lved i n the r e c ogn i t i o n of s e l f versus nonse l f and bestows the a b i l i t y o r i n a b i l i t y of the organism to react to d i f f e r e n t an t i gen i c s t i m u l i . A l l ve r tebra tes s tud ied so f a r have a s i m i l a r complex w i t h c l o s e l y l i n k e d c l a s s I and c l a s s II reg ions (Hood, 1983). A unique f e a t u r e of c e r t a i n genes of the MHC i s a high degree of polymorphism. S i x t y percent of a l l non-MHC l o c i i n mice are monomorphic and the remaining polymorphic l o c i u sua l l y have one or two a l l e l e s which are more f r equen t , and l e s s than ten percent are heterozygous at these l o c i ( K l e i n , 1979). The most f r equen t l y o c cu r r i ng a l l e l e of C lass I l o c i was found i n on ly twe lve percent of w i l d o r inbred mice. Most others were of l e s s than two percent f requency, w i th n inety percent of the mice heterozygous. The polymorphisms i nvo l ved many amino ac id s u b s t i t u t i o n s between a l l e l e s , not on ly a s i n g l e base change in the DNA. The polymorphic nature of the MHC makes an idea l system t o study the d ivergence of f unc t i ona l p ro te ins and the a f f e c t of s e l e c t i o n on p a r t i c u l a r reg ions w i t h i n a p r o t e i n . By comparing the p ro te i n s from two s i m i l a r s pe c i e s , and two more d i s t a n t l y r e l a t ed spe c i e s , the amount of d ivergence between s i m i l a r MHC encoded molecules can be c o r r e l a t e d t o the amount of e vo l u t i ona ry t ime s ince the two spec ies o r i g i n a l l y separa ted . Th is study looks at the DNA sequence f o r one MHC-encoded c e l l su r face molecu le in the r a t and compares i t t o the equ iva len t sequence i n the mouse, which d iverged ten m i l l i o n years ago, and to t ha t of the human, which d iverged about seventy mi 11 ion years ago. 2 I . C e l l u l a r B io logy - H i s to ry The genes coding f o r " s e l f " molecules were f i r s t d i scovered i n mice through the phenomenon of tumor r e j e c t i o n . C. Jensen (1902) found tha t a l i v e tumor t ransp lan ted from one mouse to another was accepted or r e j e c t ed i n the r e c i p i e n t animal depending on how c l o s e l y r e l a t ed i t was t o the donor an ima l . By c ros s i ng inbred l i n e s of mice, the phenomenon was found to f o l l ow Mendel ian pat terns of genet i c con t ro l and more than one gene was i n v o l v ed . By using an t i bod i e s , P. Gorer (1937a, 1937b) descr ibed an ant igen II found on the c e l l s of one mouse s t r a i n A but not another. S t r a i n A der i ved tumors were t r ansp l an ted i n t o s t r a i n A, s t r a i n B, t h e i r FI progeny, and backcross progeny mice and the s u s c e p t i b i l i t y t o tumor fo rmat ion c o r r e l a t e d w i th the ant igen II a n t i b od i e s . Gorer found a c o r r e l a t i o n between t r a n s p l a n t i b i 1 i t y and t h i s blood group ant igen which l a t e r became known as part of the mouse H-2 system. In p a r a l l e l s tud i e s Gibson and Medawar (1943) found sk i n g r a f t i n g i n humans to be regu la ted by a s i m i l a r immune phenomenon and l a t e r concluded tha t t r a n s p l a n t a t i o n ant igens were on normal c e l l s as we l l s as tumors (Medawar, 1946). Although these ant igens were not c o r r e l a t e d w i th the blood ant igens d i scovered i n mice, i t was concluded tha t t r a n s p l a n t a t i o n ant igens were a l so found on white blood c e l l s . G. Sne l l (1948) used the term h i s t o c o m p a t i b i l i t y ant igens f o r the su r face molecules r e spons i b l e f o r determin ing t i s s u e r e j e c t i o n . Congenic s t r a i n s of mice were developed which had d i f f e r e n t h i s t o c o m p a t i b i l i t y genes on s i m i l a r genet i c backgrounds, which a l lowed the i s o l a t i o n of recombinants to c ha r a c t e r i z e the complex. A second type of immune response was shown to be under the con t ro l of the MHC. Genet ic con t ro l of t h i s immune response was f i r s t s tud ied i n guinea p igs w i th the use of s yn the t i c po l ypep t i d e s . I t was found t ha t i n o c u l a t i o n of these ant igens , eg . (T,G)-A-L or (H,G)-A-L, i n t o two d i f f e r e n t s t r a i n s caused a high ant ibody t i t e r response in one s t r a i n and a low response in another . When the two s t r a i n s were crossed and back-crossed, a n a l y s i s of the segregat ing popu lat ions showed each response to be under the con t r o l of a s i n g l e dominant gene, c a l l e d the Immune Response ( I r ) gene. Th is I r gene was a l s o mapped to the MHC r eg i on . I I . The Three C lasses of Products The molecules encoded by the MHC are d i v i ded i n t o th ree c l a s se s accord ing t o s t r u c t u r a l and- func t i ona l s i m i l a r i t i e s . F igure 1 compares the general s t r u c t u r e of the C lass I and C lass I I molecules and demonstrates the s i m i l a r i t y to other molecules of the immunoglobulin f am i l y encoded ou t s i de the MHC. The C lass I molecules show s t r u c t u r a l s i m i l a r i t i e s i n being c e l l su r face g l y cop ro te i n s composed of a cha in encoded w i t h i n the MHC, a s soc i a t ed noncova lent ly w i th a ^ m i c r o g l o b u l i n p ro t e i n encoded elsewhere i n the genome (chromosome 2 i n mice, 15 i n m a n ) ( f i g . l ) . The MHC encoded cha i n , which i s anchored in the membrane, conta ins the b i o l o g i c a l a c t i v i t y and a n t i g e n i c i t y of the molecule but the f unc t i on of ^ m i c r o g l o b u l i n i s unknown. The C l a s s II molecules are grouped together as they are b i o chem i ca l l y s i m i l a r and share o r gan i z a t i ona l s i m i l a r i t i e s i n being composed of an a cha in and B cha i n , noncova lent ly bound t oge the r . C lass I I molecules show r e s t r i c t e d exp ress i on , mainly on B lymphocytes of macrophage, an t igen p resen t ing c e l l s , d e n d r i t i c c e l l s , and some a c t i v a t ed T c e l l s , but a l s o have been found i n smal l amounts on the c a p i l l a r y wa l l endo the l i a l c e l l s , rena l tubu le c e l l s , tumour c e l l s of melanomas and g l iomas, e p i t h e l i a l c e l l s of va r i ous organs and pos s i b l y on some secre ted f u n c t i o n a l l y a c t i v e f a c t o r s (Kaufman, 1984). There have been a number of d i f f e r e n t genes found w i t h i n the MHC complex, e a r l i e r c l a s s i f i e d as C l a s s I I I genes. These i n c l ude the complement Areas showing high degree (from Hood, 19S3) of homology are shaded. components C2, C4, Fac to r B, cytochrome p450, and s t e r o i d 21-hydroxy lase . They are grouped under the MHC by l o c a t i o n ra the r than f u n c t i o n , and w i l l not be d i s cussed f u r t h e r . I I I . Genet ics of the MHC The genet i c map of the MHC i n a v a r i e t y of spec ies has been developed by l ook ing f o r recombinat ion w i t h i n the MHC, then c o r r e l a t i n g the s e r o l o g i c a l l y determined c e l l su r f a ce markers tha t are d i f f e r e n t . By measuring the frequency of recombinat ion between two l o c i , gene t i c d i s tances can be c a l c u l a t e d in comparison t o the e n t i r e genome, assuming tha t recombinat ion occurs randomly throughout the genome. Although t h i s was o r i g i n a l l y the best method of model ing the MHC, molecu lar c l o n i ng and DNA sequencing have demonstrated tha t the r e l a t i v e d i s tances have been est imated t o be too long due to h igh recombinat iona l hot spots and t ha t some of the o r i g i n a l l y mapped l o c i are a r t i f a c t s of MHC i n t e r a c t i o n s and may not p h y s i c a l l y e x i s t ( K l e i n , 1983). The gene t i c o r gan i z a t i on of the major h i s t o c o m p a t i b i l i t y complex of the human, mouse, and ra t i s compared in f i g u r e 2. A. The Rat RT1 System The r a t MHC, c a l l e d RT1 complex, has been shown t o encode C lass I and II l o c i . F igure 2 shows the s t r u c t u r e of the RT1 complex determined so f a r . Because l e s s i s known about the segregat ing l i nkage groups (chromosomes) i n the r a t , the p o s i t i o n in the genome r e l a t i v e to other genes i s not known, but the complex appears to be arranged in a manner s i m i l a r to the murine H-2. The RT1.A and C l o c i appear t o encode C lass I an t igens , but RT1.C has a more l i m i t e d t i s s u e d i s t r i b u t i o n (Gunther, 1985). Between ten and twenty C lass I genes i n t o t a l have been found i n r a t s when us ing mouse C lass I genes as probes f o r Southern h y b r i d i z a t i o n s of r a t genomic DNA. Only two have been FIGURE 2 Genetic Organization of the Major H i s t o c o m p a t i b i l i t y Complex i n Man, Mouse and Rat. HUMAN 11LA COMPLEX Chromosome 6 cent romere Class II Class I I I Cl a s s 1 (GLO) D UP DO UP _£2_p.L.g4. X t 1 _ C l a s s Peg ion Subregion S UP(SB) 2 ff 2 a 1 i\ II nx n o i D O ) no ! a (i | UP l Ji 2 J i _ .1 fi __a a ( B o d m e r , 1 9 8 4 T r o w s d a l e , 1 9 8 4 ) Mouse H-2 COMPLEX Chromosome 17 centromere Class I Class [ I Class I I I Class I K C :0 ' L ^ i ^ c ^ s j p D / l_ Qa Tl< i 'i n L ..i.Qa2__Qa3_ijria-i)a1«_. C l a s s Pegion Subregion 8 A P2 A (J A a !• ^ Efi2 !•: i i . — . ( L a r h a m m a r , 1 9 8 3 a K l e i n , 1 9 8 3 ) RAT RTl COMPLEX Chromosome Unknown Class I A F Class 11 B D C l a s s I E C CJ ass Region (GLO) A E V - I . . An An2.. „ _ U j E„ \ # recombination hot spots —*• d i r e c t i o n of t r a n s c r i p t i o n 5' to 3' ( G i l , 1 9 8 3 D i a m o n d , 1 9 8 5 ) i d e n t i f i e d so f a r on the c e l l su r face (Gunther, 1985). Of a l l the C lass I c lones mapped, e igh ty to n inety percent mapped to the C r eg i on , wh i l e l e s s than twenty percent mapped to the A r eg i on , as would be p red i c ted i f the A reg ion was a c t u a l l y equ iva len t to the H-2 D/K reg ion of the mouse. The RT1.B and RT1.D were found to encode the A - l i k e and E - l i k e molecules r e s p e c t i v e l y , by us ing mouse H-2 C lass I I cDNA probes. A recombinant w i t h i n the RT1.B reg ion was found in the f i r s t generat ion of an inbred s t r a i n c rossed w i th a w i l d s t r a i n . Th is enabled the determinat ion of the order of genes i n the RT1.B reg ion to be RT1.A, RT1.B 3 a, RT1.D (Blankenhorn, 1985). Both RT1.B and RT1.D reg ions have been found to code f o r two p and one a cha ins by Southern h y b r i d i z a t i o n ana l y s i s us ing the equ iva l en t mouse H-2 genes. The order and d i s tance between genes was p red i c ted to be s i m i l a r to the mouse a l so as shown by over lapp ing c lones (Diamond, 1985). While polymorphic RT1 l o c i have been found i n w i l d popu la t i ons of r a t s , t he re appears t o be l e s s polymorphism than i n mice (Cramer, 1978, Wagener, 1979). In the C lass I I molecules i s o l a t e d from th ree hap lo types , th ree a sequences i s o l a t e d were i d e n t i c a l wh i l e the & cha ins showed the ex i s t ence of two or more d i s t i n c t sequences by N-terminal sequencing^ (Cecka, 1980, Cook, 1979). B. The Mouse H-2 System Most of the ana l y s i s has been done on the murine system because of the l a r ge number of inbred s t r a i n s made a v a i l a b l e and the oppor tun i ty f o r man ipu la t i on i n breeding and s e r o l o g i c a l t e s t i n g . F igure 2 shows a mo lecu la r map o f the H-2 reg ion and the accompanying Qa/Tla reg ion on chromosome 17, w i t h the genes i s o l a t e d so f a r drawn i n . By comparing the frequency o f recombinat ion w i t h i n the complex t o the l eng th of the MHC i n base p a i r s , the ex i s t ence of two recombinat iona l hot spots between the E and A subregions was demonstrated. W i th in the C lass I l o c i the re has been evidence so f a r f o r two K genes, two D genes, at l e a s t ten Qa 2,3 genes, and up to t h i r t e e n genes i n the T l a reg ion (Weiss, 1984). The I reg ion has been shown to con ta in an Aa, Ag2 pseudogene, A g l , Eo, and two Eg genes (Ste inmetz , 1982). Th is appears to be the t o t a l number of genes i n the I r eg i on , as determined by southern b l o t s of c r o s s - h y b r i d i z i n g probes. In the murine inbred and w i l d popu l a t i on , the re appear t o be over f i f t y a l l e l e s from the K, D/L l o c i of the C lass I reg ion (Weiss, 1984). The Qa 2 ,3/T la reg ions show l e s s polymorphism, a l though there has been l e s s work on them f o r comparison. The C lass II 8 cha ins of both H-2 E and A types are a l s o polymorphic but the a cha ins show l e s s polymorphism (Ste inmetz , 1982). C. The Human HLA System Wi th in the l a s t few y ea r s , the o r gan i z a t i o n of the human MHC, the HLA complex on chromosome 6, has been determined. The HLA A, B, and C reg ions are the murine H-2 K and D equ iva len t C lass I r eg i ons , wh i l e the HLA D/Dr reg ion i s the homologue to the I reg ion in mice. Po s s i b l e human homologues t o the mouse Qa and T la ant igens have been s e r o l o g i c a l l y and b i ochemica l l y c ha r a c t e r i z ed but t h e i r genes have not been mapped to the MHC ye t (Ste inmetz and Hood, 1983). More i s known about the D reg ion which has been c loned from the centromer ic border of the G lyoxy lase - I (GLO) reg ion to the t e l omer i c border at the C4 gene. Another d i f f e r en c e from the mouse i s the ex i s t ence of the unique DP subregion w i t h i n the D reg ion which shows d i f f e r en ce s i n sequence from the other subc lasses and was found t o e l i c i t a d i f f e r e n t p r o l i f e r a t i v e response demonstrat ing a s l i g h t l y d i f f e r e n t f un c t i on (Kaufman, 1984). I t has no counterpar t i n the mouse (Hur ley , 1982). Through the use of a l l o a n t i s e r a and monoclonal an t i bod ies to type c e l l s , the C lass I molecules have shown the h ighest number of a l l e l e s , a l though C lass I I molecules have not been s tud ied as ex t ens i v e l y y e t . At a human hap lo typ ing workshop i n 1984 a t o t a l of 24 HLA-A a l l e l e s , 47 HLA-B a l l e l e s , and 8 HLA-C a l l e l e s have been s e r o l o g i c a l l y de f ined so f a r . In the HLA-D r eg i on , 16 DR a l l e l e s , 3 DQ(OC), and 6 DP(SB) a l l e l e s have been determined (Bodmer,1984). A few years ago, the only method of i d e n t i f y i n g and comparing MHC encoded molecules was s e r o l o g i c a l l y or by pept ide mapping. While the polymorphic nature of the molecules was apparent, i t was the development of cDNA and genomic sequencing which showed the actua l extent of the v a r i a t i o n and conse rva t i on . Sequence ana l y s i s has shown ex tens ive polymorphism i n the HLA C lass I I a cha ins and some polymorphism i n the DQ 8 cha ins (Schenning et a l , 1984) but the DRg cha in appears t o be i n v a r i a n t (Larhammar, 1982). R e s t r i c t i o n s i t e polymorphisms show the DPa cha in t o con ta in l e s s a l l e l i c polymorphism than the DQ and DRa (Go r sk i , 1984). IV. S t r u c t u r e of the MHC Molecules Extens ive work has been c a r r i e d out to determine the DNA sequence and general p ro te i n s t r u c t u r e of the C lass I I 8 cha ins i n the mouse and human. The equ iva len t molecules show homology but i t i s important to look at another spec ies to p red i c t whether these s i m i l a r i t i e s hold i n general f o r C lass II mo lecu les . Not much in fo rmat ion has been obta ined f o r the ra t MHC. This study looks at the ra t RT1 C lass II 8 cha ins by c h a r a c t e r i z i n g a cDNA encoding one of the C lass I I 8 c ha i n s . The DNA sequence i s used to p red i c t the p r o t e i n sequence and compared at the nuc l eo t i de l e ve l t o s i m i l a r p ro te ins i n the mouse and human, to look f o r p o s s i b l e s t r u c t u r a l and f unc t i ona l reg ions of the mo lecu les . . O r i g i n a l s t ud i e s on the e l u c i d a t i o n of MHC encoded molecules i nvo l ved d i s r u p t i n g the c e l l membrane w i th detergents and i s o l a t i n g the MHC molecule w i th an t i b od i e s . Degradat ion of noncovalent bonds by SDS treatment and sepa ra t i on by two dimensional gel e l e c t r o pho r e s i s e xh i b i t e d two cha ins of d i f f e r e n t s i z e and charge. Another method used papain d i g e s t i o n to chemica l l y c l eave the p ro t e i n away from the c e l l membrane to determine the s i z e of the e x t r a c e l l u l a r p o r t i o n . The C lass I molecules were found to be composed of a heavy cha in of about 43 to 46 Kd noncova lent ly bound to a 12 Kd l i g h t c ha i n , wh i l e the C lass I I molecules were made up of a heavy a c i d i c cha in of 33 t o 35 Kd, a ssoc i a ted w i th a l i g h t bas i c cha in of 27 t o 29 Kd (Cunningham, 1977). A number of N- l inked carbohydrate mo ie t i e s were found on the exposed sur faces which were of s i m i l a r compos i t ion between the two c l a s s e s but d i f f e r e n t from those of other g l y c op r o t e i n s . N-terminal amino a c i d sequencing was then used to obta in l i m i t e d sequence f o r the membrane d i s t a l domains of the products from immunoprec ip i t a t i on . The development of mo lecu la r b io logy techniques expanded r a p i d l y the amount of i n f o rmat i on obta ined by making pos s i b l e the i s o l a t i o n of messenger RNA, cDNA c l o n i n g , gene h y b r i d i z a t i o n and DNA sequenc ing. A. C lass II Molecu les By l ook i ng mainly a t the HLA-encoded molecu les , i t has been determined tha t the c l a s s I I molecules are composed of an a cha in assoc ia ted noncova lent ly w i th a g c h a i n . Each cha in i s composed of two e x t r a c e l l u l a r domains, a membrane d i s t a l a l or g l domain, and a proximal a2 or g2 domain, which are connected by a short h yd r oph i l i c pept ide to the hydrophobic transmembrane reg ion and a short h yd r oph i l i c C-termina l cy top lasmic t a i l (Kaufman, 1984). Both chains are g l y c o s y l a t e d , but the a cha in has more carbohydrate mo i e t i e s , account ing f o r i t s s l i g h t l y heav ie r weight (Hood, 1983). The f i r s t domain of the l i g h t cha in i s about 95 amino ac ids l ong , w i t h two c y s t e i n e res idues at about p o s i t i o n s 15 and 79 forming a d i s u l f i d e loop of 64 amino a c i d s . The s i n g l e complex N- l inked carbohydrate moiety of the g cha in i s attached at res idue 19. Human C lass I I g l domains (HLA DQg, DRg, DPg) show homology t o murine c l a s s I I g l domains (H-2 A and E) but a l s o a low 1 1 homology to human C lass I a l and a.2 domains (Larhammar, 1982). The heavy cha in f i r s t domain ( a l ) i s sho r te r i n l eng th , only 85 to 88 amino a c i d s , and has no c y s t e i n s f o r a d i s u l f i d e l oop . A l l the a cha ins are g l y c o sy l a t ed at a homologous res i due , and some of the types bear two s i t e s (eg. DR). The three dimensional s t r u c tu r e s of a l and p i are unknown. The second domain of C lass II B cha ins (&2) i s about 95 amino ac ids long w i th a d i s u l f i d e loop o f 56 amino ac ids from res idues 117 to 173. The a cha in second domain i s o f the same length but has the c y s t e i n res idues at 107 and 163. Th is domain has a number of i n v a r i a n t areas t ha t have homology to immunoglobul ins, e s p e c i a l l y around the c y s t e i n s and t r yp tophans . One area has a l t e r n a t i n g hydrophobic amino a c i d s , which i n immunoglobulins f i l l the i n t e r n a l space between two immunoglobulin sheets . The c l a s s I I domains are the most homologous to the constant reg ions of the Gamma 3 and Mu 4 immunoglobulins and are as r e l a t e d t o these constant domains as they are to one another. The connect ing pept ide j o i n i n g the e x t r a c e l l u l a r domains t o the transmembrane reg ion i s 11 to 13 amino ac ids in l eng th . The h yd r oph i l i c nature of the pept ide i s due to a number of se r i ne res idues on the s cha in and g lutamic ac id and p r o l i n e r e s i de s on the a c ha i n . The hydrophobic membrane spanning reg ion i s about 30 amino ac ids long and shows l i t t l e conse rva t i on between the two cha i n s . There does, however, appear to be conserva t i on between d i f f e r e n t & cha in hydrophobic sequences and between a cha in sequences, w i th l e s s conse rva t i on surrounding these r eg i ons . Th is suggests a s e l e c t i v e pressure i s mainta ined by the i n t e r a c t i o n of the a and B cha ins (or other p r o t e i n s ) i n the hydrophobic r eg i on . I t has been suggested tha t the transmembrane reg ions of C lass I I ant igens are packed toge the r as two a - h e l i c e s , as t h i s a l lows the maximum amount of H bonding po t en t i a l to be f i l l e d w i th g l y c i n e s on the faces of both a and B cha ins (T ravers , 1984). The end of the transmembrane reg ion conta ins p o s i t i v e l y charged res idues which are thought to react w i th the negat ive charges of the phospho l i p id group on the i n s i d e edge of the membrane. The cy top lasmic t a i l of charged and h y d r o p h i l i c res idues f o l l ows t o become the C-termina l end. B. C lass I Molecu les The C lass I molecules i n c lude the h i gh l y polymorphic T ransp l an ta t i on Ant igens of the H-2 K and D/L reg ions i n mice and HLA A, B and C i n man, as we l l as the l e s s polymorphic Qa-2 , 3 and T l a r eg i ons , which so f a r show no equ i va l en t reg ion in man. In the r a t , the C lass I molecules are encoded by the RT1 A, E and C reg ions w i th the A and E genes poss i b l y corresponding to the Qa/Tla regions (Blankenhorn, 1985). Whi le the C lass I K and D ant igens are ub i qu i t ous , the molecules encoded by the Qa/Tla reg i ons , p r ev i ous l y des ignated Hematopoiet ic D i f f e r e n t i a t i o n Ant igens , show t i s s u e s p e c i f i c exp res s i on ; Qa on some lymphocyte and e ry th rocy te subpopulat ions (Kaufman, 1984) and T la on immature lymphocytes ( T i z a r d , 1984). Most of the research on C lass I molecules has been done i n mice on the K and D/L equ iva len t an t i gens , but the Qa/Tla reg ion ant igens show a s i m i l a r s t r u c t u r e . Ana l y s i s of these molecules by r e cen t l y developed techn iques revea led only the heavy cha in to be encoded by genes of the MHC. The l i g h t cha in was determined to be the g2mic rog lobu l in p r o t e i n , a smal l p r o t e i n w i th a d i s u l ph i de br idge i n the ex te rna l domain d i s p l a y i n g s t r u c t u r a l homology to the immunoglobulin constant domain a l so but encoded on.another chromosome. The heavy cha in i s d i v i d ed i n t o f i v e s t r u c t u r a l domains, th ree of which are exposed on the su r f a c e . The externa l domains are each approx imate ly 90 res idues i n length w i th the a 2 and ct3 membrane proximal domains con ta i n i ng i n t e r n a l d i s u l f i d e br idges spanning 60 r e s i due s . There have been up to th ree N- l inked carbohydrate mo ie t i es of 3 Kd bound to the e x t r a c e l l u l a r p o r t i o n , depending on the type and a l l e l e of the ant igen ( K l e i n , 1982). The transmembrane reg ion i s about 40 res idues i n length and conta ins an uncharged core of 25 mainly hydrophobic r e s i dues . Th is i s fo l l owed by a cytop lasmic domain of about 30 res idues i n l eng th . The $2mic rog lobu l in has been shown by b ind ing s tud ies of pept ide f ragments, t o be assoc ia ted w i th the a3 domain. P r e l im ina ry X-ray c r y s t a l l o g r aphy has demonstrated a two - f o l d ax i s of symmetry composed of the a3 and a2 domains adjacent to the g2mic rog lobu l in and g l domains, which i s s i m i l a r to tha t formed i n ant ibody domains (Hood, 1983). V. Funct ion of the MHC Products A. C lass I and C lass II C e l l Sur face P ro t e i n s The c e l l su r face molecules encoded by the MHC are important to study because they p lay an important r o l e i n an organism's immune response to f o r e i g n an t i gens . Th is a l lows the organism t o destroy both v i r a l l y i n f e c t e d c e l l s and aberrant c e l l s d i s p l a y i n g new an t i gens . Not much i s known on the mechanisms of ant igen r e cogn i t i on i n the system of an organism and a l though a number of immune phenomena have been a t t r i b u t e d t o both C lass I and C lass II molecules of the MHC, the ma jo r i t y of these are de f ined us ing exper imental systems. These phenomena, f o r example a l l o g r a f t r e j e c t i o n or mixed lymphocyte r e a c t i o n , a l l have the common denominator r e f l e c t i n g a l l o gene i c T c e l l r e c ogn i t i o n . Wi th in an i n d i v i d u a l , the MHC C lass I and C l a s s I I molecules are i nvo l ved w i th the ant igen r e cogn i t i o n by T c e l l s . The T c e l l recognizes ant igen only i n a s s o c i a t i o n w i th C lass I or C l a s s I I molecules from the MHC of i t s own hap lo type . Th is phenomenum i s termed MHC r e s t r i c t i o n . The C lass I I molecules are i nvo l ved i n ant igen p r e s en t a t i o n , and s t imu l a t e predominant ly T lymphocytes programmed to become he lper or suppressor regu la to ry c e l l s which r egu l a te the immune lymphocytes. The r e c ogn i t i o n of f o r e i gn ant igens i n context w i th which molecules c o n s t i t u t e s e l f , i s determined i n the thymus ep i t h e l i um . The s igna l given by the C lass II ant igen probably causes a s p e c i f i c p r o l i f e r a t i v e s igna l which causes the e f f e c t o r lymphocyte popu la t i on t o expand. The C lass I molecules are predominant ly recogn ized by T c e l l s s t imu la ted to become c y t o t o x i c T c e l l s aga ins t f o r e i gn ant igens such as v i r a l an t i gens . These c y t o t o x i c or k i l l e r T c e l l s are ab le to recogn ize and destroy i n f e c t e d host c e l l s . The c y t o t o x i c T c e l l recogn izes the f o r e i gn ant igen i n a s s o c i a t i o n w i th the C lass I molecule of i t s own haplotype and w i l l not recogn ize the same ant igen on the sur face of a c e l l w i th a d i f f e r e n t C lass I hap lo type . A p a r t i c u l a r v i r u s i s o f t en recogn ized by a c e r t a i n C lass I K or D/L ant igen but the system i s f l e x i b l e , as mice w i th mutat ions i n t ha t p a r t i c u l a r a l l e l e w i l l use a d i f f e r e n t C lass I gene as a r e s t r i c t i o n element (Hood, 1983). The r e s t r i c t i o n i s determined i n the thymus ep i t he l i um where the immature lymphocyte recognizes s e l f as the ant igens present on t h i s t i s s u e . The matur ing lymphocyte recogn izes the v i r a l ant igens i n a s s o c i a t i o n w i th the C lass I T ransp l an ta t i on Ant igens (H-2 K and D) on the s e n s i t i z i n g or ant igen present ing c e l l ( K l e i n , 1979). B. Assoc ia ted Recogn i t ion by T Lymphocytes The T c e l l recepto r which recogn izes t h i s dual s p e c i f i c i t y cou ld be two separate receptors w i th one recogn i z ing the MHC molecule and one r e cogn i z i ng the ant igen (Blanden et a l , 1978), o r the MHC molecule cou ld a s soc i a t e w i th the ant igen on the c e l l su r face and be recogn ized as a s i n g l e e n t i t y (Metz inger , 1981). This l a s t model seems u n l i k e l y as the re does not appear to be any f un c t i o na l areas on the C lass II molecules capable of becoming a r e cep to r ( K l e i n , 1981). A po s s i b l e exp l ana t i on f o r t h i s a s s o c i a t i v e r e c ogn i t i o n i n add i t i o n to the d i r e c t ant igen r e cogn i t i o n found i n B c e l l s cou ld be tha t i t prov ides a l i n kage between the r e c ogn i t i o n and e f f e c t o r mechanism to enable con t ro l over the s p e c i f i c i t y of the e f f e c t o r f u n c t i o n . The regu la to ry c e l l would recogn ize ant igen i n context w i th the C lass II molecules of he lper c e l l s and C lass I molecules of the c y t o t o x i c T c e l l l y t i c s i gna l ( K l e i n , 1979). The immune response was s tud ied by examining what c o n s t i t u t e s nonresponse to an ant igen i n an animal and i n f e r r i n g back t o de f ine response. The po s s i b l e de fec t s i n a nonresponding animal could be at the l e ve l of the T c e l l r ecep to r o r the MHC-antigen combinat ion. One theory s t a t e s the gene encoding the T c e l l recepto r cou ld be nonfunct iona l or de le ted but t h i s idea can be d i scarded because nonresponse cou ld not be mapped to a locus on another chromosome where the T c e l l r ecep to r genes would be l o ca ted as we l l as to the MHC ( K l e i n , 1984). Nonresponse cou ld a l so be due to suppress ion of c e r t a i n T c e l l r e cep to r s , however, a l though i n a few documented cases suppress ion was the cause of nonresponse, i t does not exp l a i n the ma jo r i t y of the cases ( K l e i n , 1983). Another model desc r ibes the ma l func t i on to be at the l e v e l of the MHC-antigen r e c o g n i t i o n , and a l though the T c e l l recepto r i s present i n the popu la t i on of c e l l s , i t cannot be used. Two d i f f e r e n t hypothes i s , the Determinant S e l e c t i o n Hypothes is and the B l i n d Spot Hypothes i s , have been suggested t o p red i c how t h i s model works. Determinant S e l e c t i o n Hypothesis s t a tes tha t the ant igen f a i l s to i n t e r a c t w i th the " s e l f " molecule of the nonresponder i n d i v i d u a l , and as a r e s u l t the ant igen p resen t ing c e l l f a i l s t o present the ant igen i n a recogn i zab le form t o the T c e l l recepto r (Rosentha l , 1980). The a l t e r n a t i v e and favoured t heo ry , the B l i n d Spot Hypothes i s , s t a tes t ha t nonresponse i s due to the T c e l l recepto r r e p e r t o i r e being incomple te , so f un c t i o na l T c e l l s r e cogn i z i ng c e r t a i n antigen-MHC molecule combinat ions are l a c k i n g ( k l e i n , 1984). These b l i n d spots are produced s oma t i c a l l y e i t h e r by p o s i t i v e s e l e c t i o n of c e r t a i n c lones by expans ion, o r by negat ive s e l e c t i o n by f un c t i o na l i n a c t i v a t i o n of c e r t a i n T c e l l s , at the t ime when the immune system " l e a r n s " s e l f t o l e r a n c e , t o avo id r e a c t i ng 16 aga ins t the s e l f component. The cross r e a c t i v i t y between s e l f and some n o n s e l f / s e l f r e cogn i t i on causes the b l i n d spots r e s u l t i n g i n nonresponse. ' A l though not much i s known about the exact i n t e r a c t i o n of the C lass II or C lass I c e l l sur face ant igens i n the f un c t i on i ng of the immune response, i t must be a cen t ra l r o l e . Tolerance i s a demonstrat ion of nonresponse which cou ld be det r imenta l t o an organism i f the t o l e rance was to a pathogenic v i r u s . The determinat ion of the secondary and t e r t i a r y s t r u c t u r e of these important molecules may even tua l l y l ead to c lues on how the molecules i n t e r a c t and t he r e f o r e f un c t i on at the c e l l su r face and w i t h i n the cytoplasm on a l l c e l l t ypes . V. Importance of Studying the MHC The C lass I I 6 cha ins tha t have been s tud ied to date show a high degree of c onse rva t i on , but they have only been looked at i n mice and humans. To determine i f t h i s conserva t i on extends ou t s i de these two spec i e s , a C l a s s I I 8 cha in from the ra t i s examined i n t h i s s tudy . The ana l y s i s of t h i s molecule w i l l determine whether the f i r s t comparison showed h igh homology by chance or whether the trends can be gene ra l i z ed t o C lass II s c ha i n s . By l i n i n g up the 3 cha ins of the three spec i e s , i n f e rences about necessary res idues i n the p ro t e i n can be made. Amino ac ids t ha t are conserved, and groups of s i m i l a r res idues tha t remain the same across a l l t h ree spec ies extend observat ions on f unc t i ona l areas w i t h i n the domains of the mo lecu les . L i kew i se , areas d i s p l a y i n g a s t r e t c h of complete ly d i f f e r e n t amino ac ids may form part of the determinant recognized by T c e l l r e cep to r s , o r i n t e r a c t w i th f o r e i gn an t i gen . The s t r u c t u r e of a r a t C lass I I 3 cha in w i l l add t o the s t r u c t u r e i n general of mammalian s pe c i e s . The l o c a t i o n of reg ions d i s p l a y i n g l i t t l e homology can suggest where polymorphic reg ions may occur and supply add i t i o na l i n f o rmat i on on po t en t i a l s i t e s between a l l e l e s . With a number of sequences l i n e d up, the po t en t i a l number of changes w i t h i n a l l e l e s and the r e s t r i c t e d areas of polymorphism may be suggested. By s tudy ing genes of such a polymorphic nature, r e s t r i c t i o n s of s e l e c t i o n on p ro t e i n e vo l u t i o n can be looked a t . The sequence of a ra t c l a s s II B cha in inc reases the amount of i n fo rmat ion to base conc lus ions on. In t h i s study a ra t cDNA l i b r a r y i s screened w i th fragments from a r a t AB gene of another haplotype ( s t r a i n ) , t o i s o l a t e add i t i ona l C lass I I B cDNAs. The only sequences of RT1 C l a s s I I genes pub l i shed to date are a RT1 Ag gene ( E c c l e s , 1985) and a RT1 Act cDNA ( W a l l i s , 1984). Comparison of the DNA sequence of a dd i t i o na l chains to other equ iva len t molecules i n mice and humans w i l l extend the cur ren t i n f o rmat i on known about C lass II mo lecu les , and determine where the polymorphic and conserved reg ions of the molecules are po t en t i a l y l o c a t e d . METHODS AND MATERIALS Stock So l u t i on s 50X TAE Bu f f e r 2 mM Tris(hydroxymethyl)aminomethane Hydroch lor ide ( T r i s -HC l ) 1 M Hydroch lo r ide Acetate (HAc) .1 M E thy l ened i am ine te t r a -a ce t i c a c i d , disodium (EDTA) 20X SSC 3 M Sodium Ch l o r i de (NaCl) .3 M Sodium C i t r a t e 100X Denhardt S o l u t i o n 4% Fi co l 1 type 400 4% Po l y v i ny l Py r r o l i done 4% Bovine Serum Albumen (BSA) TEN 8 Bu f f e r 10 mM T r i s -HC l pH 8.0 1 mM EDTA 100 mM NaCl TE Bu f f e r 10 mM T r i s -HC l pH 8.0 1 mM EDTA 10X TBE Bu f f e r 10 mM Tr is(hydroxymethyl)aminomethane Hydroxide (Tr is-OH) pH 8.3 .9 M Bo r i c a c i d .025 M EDTA I. Plasmids pRT lEg. l and pRT1.2 1. cDNA L i b r a r i e s 19 A C lass II Ag gene was used to screen two cDNA l i b r a r i e s made from Wis ta r r a t s of the haplotype RT l . u . P lasmid pRTlEg. l was i s o l a t e d from an Aa enr i ched l i b r a r y made by McMaster (1984). In b r i e f , po lyadeny la ted (poly A) RNA was separated from t o t a l sp leen c e l l RNA by o l i g o ( d T ) - c e l l u l o s e a f f i n i t y chromatography. Po ly(A) RNA was enr iched f o r C lass I I Aa mRNA by p repa ra t i ve sucrose g rad ien t c e n t r i f u g a t i o n and a l i q u o t s of each f r a c t i o n t r a n s l a t e d i n a c e l l - f r e e assay i n the presence of r a d i o a c t i v e amino ac ids and i d e n t i f i e d by immuno p r e c i p i t a t i o n . The f r a c t i o n s con ta i n i ng the s p e c i f i c mRNA f o r ra t Aa cha ins were used as templates f o r cDNA s y n t h e s i s . F i r s t s t rand cDNA was synthes i zed us ing AMV (Avian Mye l ob l a s t o s i s V i r u s ) reverse t r a n s c r i p t a s e , separated, C - t a i l e d w i th te rmina l t r a n s f e r a s e , and the second s t rand synthes i zed us ing o l igo(dG) pr imer and reverse t r a n s c r i p t a s e . Double stranded cDNA of g rea te r than 600 base pa i r s was e l u ted by agarose gel e l e c t r o p h o r e s i s . The plasmid vec to r pBR322, d iges ted to complet ion w i th Pst I, was t a i l e d w i th (dG) t a i l s of f i v e to ten nuc leo t ides and annealed t o equimolar amounts of C - t a i l e d double stranded cDNA by heat ing then c o o l i n g . Calc ium c h l o r i d e t r ea ted E s che r i c h i a c o l i ( s t r a i n RR1), was then transformed w i th the annealed DNA, p l a t ed out on t e t r a c y c l i n e LB Broth agar p l a t e s , and incubated ove rn i gh t . Co lon ies con ta i n i ng recombinant plasmids were t e t r a c y c l i n e r e s i s t a n t and a m p i c i l l i n s e n s i t i v e . A second cDNA l i b r a r y was made us ing t h i s same method w i th t o t a l po ly(A) mRNA f o r cDNA templates ins tead of C lass II Aa cha in enr iched po ly(A) RNA. 2. I s o l a t i o n of an RT1 Ag Gene The Ag gene used f o r screen ing the cDNA l i b r a r i e s was obta ined from a Sprague Dawley (RTl .b) r a t genomic l i b r a r y by S. Ecc les ( E c c l e s , 1985). B r i e f l y , a lambda Charon 4A l i b r a r y made from ra t l i v e r genomic DNA was screened w i th a human HLA-DQg 1.1 k i l o ba se cDNA, ( p l l - g l ) obta ined from D. Larhammar (1982a). A p o s i t i v e p l asmid , Ag io , was i s o l a t e d from the l i b r a r y , d iges ted w i th r e s t r i c t i o n enzymes and subcloned i n t o M13 v e c t o r s . By r e h y b r i d i z i n g wi th the HLA-DQg cDNA probe, subclones con ta in ing the coding reg ion of the gene were i s o l a t e d , sequenced, and a l i gned to the mouse H-2 Ag gene (Larhammar, 1983a). Fo l l ow ing t h i s method, a 3.5 Kb EcoRI fragment was determined to code f o r the g l and g2 domains of the Ag gene and subsequent ly used t o screen the cDNA l i b r a r i e s . 3. Nick T r an s l a t i o n of Ag Gene 10 X Nick T r an s l a t i o n (NT) Bu f f e r 500 mM Tr is-HCL 50 mM Magnesium Ch l o r i d e ( MgC12) DNase A c t i v a t i o n Bu f f e r 10 mM Tr i s -HC l 5 mM MgC12 .1 mg/ml Bovine Serum Albumin (BSA) 3 X Nick T r an s l a t i o n Mix 100 u l s 10X NT Bu f f e r 20 u l s 50 mM D i t h i o t h r e i t o l (DTT) 40 u l s [2mg/ml] BSA 40 u l s 500 uM deoxyguanosine (dGTP) (Sigma Chem Co,St Lou is ,Mo.) 40 u l s 500 uM deoxythymidine (dTTP) 40 u l s 35 uM deoxyadenosine (dATP) 40 u l s 35 uM deoxycy t i d ine (dCTP) 20 u l s 10 mM Calcium Ch l o r i d e (CaC12) 21 Sample Bu f f e r 2.0% G lycero l i n d i s t i l l e d water (dH20) 1% Bromophenol Blue (BPB) The 3.5 Kb EcoRI fragment was i s o l a t e d from the Agio c lone by the f o l l o w i n g method. AglO was d iges ted t o complet ion overn ight w i th EcoRI. One f i f t h volume of sample bu f f e r was added and the DNA run on a 1% agarose gel (.25 g. agarose, .5 ml 50X TAE Bu f f e r i n 25 mis dH20, and Eth id ium Bromide (EtBr) added to a f i n a l concen t ra t i on of .8 ug/ml) i n IX TAE at 150 v o l t s . Standards made from EcoRI-BamHI cut phage lambda were run concu r ren t l y to i d e n t i f y the 3.5 Kb band. A shor t wave u l t r a v i o l e t i l l u m i n a t o r was used to v i s u a l i z e the DNA to cut the requ i red band out w i th a r a zo r b lade . The agarose s lab con ta in ing the i n s e r t was s l i pped i n t o 25mm wide d i a l y s i s t u b i ng , and the DNA f u r t h e r e lec t rophoresed i n t o the enclosed b u f f e r . Once the bu f f e r was c o l l e c t e d , the DNA was p r e c i p i t a t e d f o r one hour at -70 degrees C w i th one t en th volume 5 M Sodium Ch l o r i d e (NaCl) and three volumes 95% E thano l . The p r e c i p i t a t e d DMA was recovered by c e n t r i f u g a t i o n f o r ten minutes at 10,000 RPM i n an Eppendorf M i c ro fuge . The DNA was n ick t r a n s l a t e d w i th P32 r a d i o l a b e l e d deoxyr iboadenosine t r i phospha te (dATP) and deoxyr ibocy tos ine t r i phospha te (dCTP) by the f o l l o w i n g r ea c t i on (Rigby et a l , 1977). Ten u l s of Deoxyr ibonuc lease I (1 mg/ml DNase i n dH20, Boehr inger Mannheim, Do rva l , Quebec) was a c t i v a t e d w i t h 90 ul of DNase A c t i v a t i o n Bu f f e r f o r 30 minutes on i c e , then d i l u t e d one thousand f o l d . In an eppendorf microfuge tube placed on i c e were p i pe t ed : 17 u l s NT Mix 24 ul DNA d i s s o l v ed i n dH20 (about .1 ug DNA) 2.5 u l s a32P-dATP (New England Nuc l ea r ,On ta r i o ) 2.5 u l s a32P-dCTP (NEN) 2.5 u l s a c t i v a t ed DNase (.25 ng t o t a l ) 2 u l s DNA Polymerase I (BI0RAD) at [10 u n i t s / u l ] The r e a c t i o n was incubated f o r one hour at 15 degrees Z then the enzymes stopped immediately by heat ing to 68 degrees f o r f i v e minutes. Three u l s of [10 mg/ml] tRNA were added to the s o l u t i o n and the probe separated from f r e e nuc l eo t i des by e l u t i o n o f f an a f f i n i t y chromatography column packed w i th Sephadex G-50 beads (Pharmacia, Do rva l , Quebec). The probe e l u t i o n was monitored w i t h a ge iger counter and the f i r s t peak of r a d i o a c t i v i t y c o l l e c t e d i n t o microfuge tubes f o r ethanol p r e c i p i t a t i o n . Upon resuspens ion, 100 ug of pBR322 DNA were added t o the probe and the probe bo i l e d f o r ten minutes f o r dena tu ra t i on j u s t p r i o r t o being added to the h y b r i d i z a t i o n s o l u t i o n and f i l t e r s . 4. I s o l a t i o n of Clones H y b r i d i z i n g to the A& Gene H y b r i d i z a t i o n So l u t i o n 6X SSC IX Denhardt s o l u t i o n 1 mM EDTA .5% Sodium Dodecyl Su l f a t e (SDS) SDS So l u t i o n IX SSC s o l u t i o n .1% SDS The two l i b r a r i e s were screened by the colony h y b r i d i z a t i o n method of Gruns te in and Hogness (1975). P lasmid i n f e c t ed E . c o l i s t r a i n RR1 from the two l i b r a r i e s were d i l u t e d w i th LB broth (D i f co Lab Co . , Madison, Wiscons in , 20 g / l i t e r dH20), and i n d i v i d u a l l y p l a ted out on f r e sh LB broth 1.5% agar p l a t e s con ta i n i ng 20 ug/ml t e t r a c y c l i n e . S t e r i l e 82 mm c i r c u l a r n i t r o c e l l u l o s e f i l t e r s were numbered w i th p e n c i l , p laced number s ide down on top of a p l a t e and gent ly pushed onto the p l a t e . The f i l t e r s were l e f t to s i t f o r f i v e minutes, wh i l e the numbers were t r aced onto the bottom of the p l a t e , and then removed c a r e f u l l y on to new LB 1.5% agar p l a t e s con ta i n i ng [100 mg/ml] ch lo ramphen i co l . These were l e f t to incubate overn ight f o r plasmid a m p l i f i c a t i o n . The f i l t e r s were then removed and the DNA immobi l i zed by p l a c i ng each f i l t e r on 3MM Whatman paper soaked i n .5 N Sodium Hydroxide (NaOH), 1.5 M Sodium Ch lo r i de (NaCl) f o r 20 minutes. The f i l t e r s were removed and a i r d r i ed f o r f i v e minutes. The procedure was repeated us ing 1.0 M Tr i s -HC l at pH 7.4 to wash, then 1.5 M NaC l , .5 M T r i s -HC l pH 7.4 t o n e u t r a l i z e . The f i l t e r s were a i r d r i ed f o r 30 minutes then the DNA baked on f o r two hours at 68 degrees C. The f i l t e r s were submerged i n .02% Denhardt s o l u t i o n i n a Pyrex g lass baking d i s h , covered w i th saran wrap and l e f t p r ehyb r i d i z i n g overn ight at 68 degrees C. The f i l t e r s were hyb r i d i zed overn ight at 68 degrees i n a p e t r i e d i sh con ta i n i ng the h y b r i d i z a t i o n s o l u t i o n and the DNA probe at one m i l l i o n CPM /ug DNA per ml of s o l u t i o n per f i l t e r . The f i l t e r s were then washed th ree t imes i n 2X SSC at room temperature, then at 68 degrees C i n .1% SDS s o l u t i o n f o r 45 minutes , and two more t imes i n f r e sh 2X SSC, .1% SDS f o r 30 minutes. A f t e r r i n s i n g two t imes at room temperature i n 2X SSC , the f i l t e r s were a i r d r i e d , and exposed to X-ray f i l m (Kodak X-Omat XPR-1) w i th i n t e n s i f i c a t i o n at -20 degrees C. The co l on i e s d i s p l a y i n g a p o s i t i v e s i gna l were t r aced back t o t h e i r r e spe c t i v e p l a t e s and the o r i g i n a l co lony r ep l a t ed on LB agar p l a t e s us ing s t e r i l e t o o t h p i c k s . To reduce the number of f a l s e pos i t i v e s^ the second set of agar p l a t e s was rescreened us ing f i l t e r papers. 5. I s o l a t i o n of Plasmid DNA The two co l on i e s showing up as p o s i t i v e were each used to i n o cu l a t e ten mis of LB broth con ta in i ng 20 ug/ml t e t r a c y c l i n e a n t i b i o t i c . Th is was grown up f o r th ree hours in a 37 degree shaking i n cuba to r , then t r a n s f e r r e d to two l i t e r Er lenmeyer f l a s k s con ta in i ng one l i t e r of s t e r i l e LB broth w i th 20 ug/ml t e t r a c y c l i n e . These l a rge c u l t u r e s were incubated w i th shaking at 37 degrees u n t i l the absorbance at 600nm wavelength was between .6 and .8 (about th ree hou rs ) , then chloramphenicol ( d i s so l ved i n 95% Ethanol) was added to a f i n a l concen t ra t i on of 250 ug/ml f o r p lasmid a m p l i f i c a t i o n , and the cu l t u r e s l e f t i n cuba t i ng ove rn i gh t . a . P lasmid P repara t i on Sucrose So l u t i o n 50 mM Glucose (Dextrose, D i f co Co.) 25 mM Tr i s -HC l ph 8.0 10 mM EDTA Lysozyme added f r e sh to f i n a l concen t ra t i on [4 mg/ml] So l u t i o n I I ( f r e s h l y made) .2 M NaOH 1% SDS So l u t i o n I I I (3 M Potass ium, 5 M Acetate) 60 mis 5 M Potassium Acetate 11.5 mis concentrated g l a c i a l a c e t i c a c i d dH20 to 100 mis R ibonuc lease A (Sigma, type I-A) at 5 mg/ml i n dH20, p r ev i ou s l y b o i l e d f o r ten mi nutes) C e l l s from one l i t e r of c u l t u r e were cen t r i f uged i n t o two p e l l e t s i n 300 ml po lycarbonate b o t t l e s at 5,000 RPM f o r 15 minutes at 4 degrees C i n a Sorval RC-5B c en t r i f u ge w i th a GSA Rotor . The supernatant was poured o f f , and each p e l l e t resuspended i n 8 mis of the sucrose s o l u t i o n us ing a s t e r i l e 10 ml pi pet . Each suspension was t r an s f e r ed t o a 50 ml s t e r i l e po lycarbonate tube then the lysozyme added to a f i n a l concent ra t i on of 4 mg/ml. The tubes were gent ly i nve r t ed and l e f t at room temperature f o r f i v e minutes. S ix teen mis of i c e co ld s o l u t i o n I I were added, the tubes i nve r t ed g en t l y , and l e f t on i c e f o r ten minutes. F i n a l l y 12 mis of So l u t i o n I I I were added, the tubes i n ve r t ed and l e f t on i c e another ten minutes . The l y sed c e l l deb r i s was then removed by c e n t r i f u g a t i o n at 10,000 RPM f o r f o r t y minutes at 4 degrees C. The supernatant from each tube was d i v i ded i n t o two 50 ml po lycarbonate tubes and .6 volume i c e co l d Isoproponol used to p r e c i p i t a t e the DNA. A f t e r a minimum of f i f t e e n minutes at room temperature, the DNA was recovered by c e n t r i f u g a t i o n at 10,000 RPM f o r f i f t e e n minutes, and the p e l l e t d r i e d under vacuum. The p e l l e t was then resuspended i n 10 mis of TEN 8 Bu f f e r and 150 ul of [5 mg/ml] R ibonuclease A added and l e f t at room temperature f o r f i f t e e n minutes. The DNA was ex t ra c ted from the enzymes and p ro t e i n s by phenol e x t r a c t i o n . Ten mis of phenol ( sa tu ra ted i n TEN 8) were vortexed i n , c en t r i f uged f o r f i v e minutes and the bottom phenol l a ye r removed. The upper i no rgan i c l a y e r was vortexed w i th 10 mis ch loro form and c e n t r i f u g e d . The top aqueous l a y e r was p i pe t t ed o f f i n t o c l ean 50 ml po lycarbonate tubes and p r e c i p i t a t e d w i th 1 ml 3 M Sodium Acetate (NaAc), and 30 mis i c e co ld 95% ethanol f o r one hour at -70 degrees C. b. P u r i f i c a t i o n of C losed C i r c u l a r P lasmid Ten grams of cesium c h l o r i d e and 1 mg eth id ium bromide were added to the DNA and made up to a f i n a l volume of 9 mis w i th TE b u f f e r . The tube was thoroughly vor texed and the dens i t y ad jus ted t o a sucrose dens i t y o f 35 t o 36% w i th a r e f r a c t omete r . The s o l u t i o n was t r an s f e r ed t o 10 ml quick sea l c en t r i f u ge tubes , and the g rad ien t s formed i n a Beckman L8-M U l t r a f uge w i th a Type 70 . IT i v e r t i c a l r o t o r at 60,000 RPM, a t 15 degrees f o r 16 hours . By i l l u m i n a t i o n of the separated bands w i th u l t r a v i o l e t l i g h t , the bottom band was removed w i th an 18 gauge needle and s y r i n g e . The recovered DNA was d i l u t e d up to 10 mis w i th TE bu f f e r and the eth id ium bromide removed by e x t r a c t i o n three t imes w i th N-Butanol, f o l l owed by p r e c i p i t a t i o n . 6. Cha r a c t e r i z a t i o n of the Plasmids The quan t i t y of plasmid obta ined and t he r e f o r e the DNA concen t r a t i on , was determined by a n a l y s i s of the absorbance at 260nm wavelength by spectrophotometry. The Molar E x t i n c t i o n C o e f f i c i e n t f o r double stranded DNA i s 20 (Maniatus, 1975) and when the product of the absorbance at 260nm and the d i l u t i o n f a c t o r i s d i v i d ed by the Molar E x t i n c t i o n C o e f f i c i e n t , the r e s u l t equals the concen t ra t i on of the DNA i n ug per m l . Th is concen t ra t i on was conf i rmed by e l e c t o rpho res i ng a smal l sample on an agarose gel w i th a s tanda rd . 10X S a l t Bu f f e r s 10X Low: 10X Medium: 10X High: 100 mM T r i s 100 mM T r i s 500 mM T r i s 100 mM Magnesium Sulphate (MgS04) 100 mM MgS04 100 mM MgS04 10 mM D i t h i o t h r e i t o l (DTT) 10 mM DTT 10 mM DTT 500 mM NaCl 1 M NaCl To determine the s i z e of the cDNA i n s e r t s i n the pBR322 p lasmid, each p lasmid was d iges ted t o complet ion w i th 2 un i t s per ug of Pst I i n a r e a c t i o n mixture w i th IX High S a l t bu f f e r and 100 ug/ml Bovine Serum Albumen f o r two hours at 37 degrees C. .5 ug of DNA was then e lec t rophoresed on a 1% agarose gel w i t h standards made from Taq I r e s t r i c t e d pBR322 p l asm id . R e s t r i c t i o n maps o f the plasmids were made by a s e r i e s of two ug a l i q u o t s i n g l e and double d i ges t r eac t i ons us ing the enzymes BamHI, TaqI, Smal, A v a l , EcoRI, H i n d l l l , N r u l , PvuII and S a i l . Three t o f i v e un i t s of enzyme per ug of DNA were added to the r e a c t i o n mixes w i th .1 volume of the appropr i a te 10X s a l t bu f f e r (as recommended by Bethesda Research Labs) and nuc lease f r e e BSA t o a f i n a l concen t ra t i on of 100 ug/ml. A l l of the enzymes had only s i n g l e or a few s i t e s i n pBR322 and comparison of the r e s u l t i n g fragment s i z e s to a map of pBR322 enabled the determinat ion of r e s t r i c t i o n s i t e s i n the i n s e r t and i t s o r i e n t a t i o n w i t h i n the p lasmid . I I . M13 Sequencing 1. Sequencing St rategy The c l on ing vectors M13mp8 and M13mp9 (Messing, 1983) were used f o r sequencing the cDNA. These were r e s t r i c t e d w i th a number of enzymes producing 5' and 3' overhang and b lunt ended i n s e r t s i t e s . To ob ta in subclones of a good s i z e f o r the sequencing r e a c t i o n s , two methods of DNA f ragmentat ion were used. One method i nvo l ved us ing f r equen t l y c u t t i n g r e s t r i c t i o n enzymes to d i v i d e the DNA i n t o over lapp ing fragments t ha t cou ld p a r t i a l l y be o r i en ted on the i n s e r t by comparison to the r e s t r i c t i o n map. The cDNA i n s e r t s were i s o l a t e d from the plasmid by d i g e s t i o n w i th Ps t I and e l e c t r o e l u t e d as desc r ibed above. These fragments were e i t h e r c loned d i r e c t l y i n t o the M13mp9 vec tor r e s t r i c t e d w i th the same enzyme, o r two ug a l i q u o t s f u r t h e r d iges ted w i th the f r equen t l y c u t t i n g enzymes H a e l l l or R s a l . A f t e r denatura t ion of the enzymes by heat ing t o 68 degrees f o r ten minutes, and a d d i t i o n of EDTA to a concen t ra t i on of lOmM, the DNA was d i r e c t l y l i g a t e d t o the v e c t o r s . The a l t e r n a t e method of generat ing random over l app ing fragments i nvo l ved s on i c a t i n g the complete p lasmid con ta in i ng the i n s e r t , and l i g a t i n g a l l of the fragments i n t o l i n e a r b lunt-ended v e c t o r s , as desc r i bed by De in inger (1983) . 2. Son i ca t i on of P lasmid 28 In a 1.5 ml s i l i c o n i z e d eppendorf tube, the f o l l ow i ng was p i p e t t ed : 10 ug of the plasmid DNA i n 390 ul of dH20 50 ul 5 M NaCl 50 ul 1 M T r i s pH 7.4 10 ul 500 mM EDTA then vortexed and p laced on i c e . The f i n e t i p of a B ioson ik IIA s on i c a t o r B r onw i l l S c i e n t i f i c ) was immersed i n the s o l u t i o n , and the DNA son i ca ted f o r f i v e second i n t e r v a l s at a power s e t t i n g of 60, f o r f i v e separate b u r s t s , vo r t ex i ng and r ep l a c i ng the tubes on i c e i n between bu r s t s . The DNA was then p r e c i p i t a t e d w i th 1 ml of 95% ethanol at -70 degrees C f o r one hour, and resuspended i n 25 ul ImM T r i s pH 8 .0 . The son i ca ted DNA was s i z e f r a c t i o n a t e d on a 1% agarose gel by e l e c t r o p h o r e s i s . .5 mg of DNA was loaded i n t o 25 ul w e l l s , w i th Bromophenol Blue sample bu f f e r and e lec t rophoresed a long s i de s i z e standards f o r two hours at 100 v o l t s . The DNA determined to be between 300 and 600 base p a i r s long was cut out w i th a razor b lade, i s o l a t e d i n d i a l y s i s tub ing and phenol ex t r a c t ed before p r e c i p i t a t i o n w i t h 1 volume of 4M ammonium aceta te (NH3Ac) and 3 volumes of 95% e thano l . The DNA was resuspended and r e p r e c i p i t a t e d overn ight w i th .1 volume 1 M NaCl at -20 degrees. 3. Po l ymer i z a t i on 10X Po l ymer i z a t i on Bu f f e r 330 mM Tr is-OH 660 mM Potassium Acetate 100 mm Magnesium Acetate pH 7.8 The p r e c i p i t a t e d DNA was c e n t r i f u g e d , r i n s ed w i th 70% e thano l , and the p e l l e t resuspended i n 25 ul ImM T r i s pH 8.0 The po l ymer i za t i on r e a c t i o n was incubated f o r two hours at 37 degrees C: 25 ul son ica ted DNA 5 ul 10X po l ymer i za t i on bu f f e r 4 ul 2.5 mM dNTP 2 ul BSA [2mg/ml] 14 ul dH20 1 ul T4 DNA Polymerase [3 u n i t s / u l ] (BIOLABS) The DNA was ex t rac ted two t imes w i th phenol then the concen t ra t i on determined. Th is was accomplished by s po t t i ng 1 ul of the DNA mixed w i th 1 ul o f [2 mg/ml] eth id ium bromide on t o a p iece of saran wrap placed over a UV lamp, and the dens i t y of the f l ou rescence compared to standards of known concen t ra t i on s i m i l a r l y spo t t ed . Once the DNA had been p r e c i p i t a t e d , i t was resuspended to a concen t ra t i on of 10 ng/ul f o r l i g a t i o n i n t o the b lunt end r e s t r i c t e d (Smal) M13mp9 ve c t o r s . 4. L i g a t i o n 10X L igase Bu f f e r 500 mM Tr i s -HC l pH 7.8 100 mM MgC12 The fragments of DNA were mainly c loned i n t o M13mp9 p rev i ous l y r e s t r i c t e d w i th Smal, or Ps t I and Smal. A number of l i g a t i o n s were t i t r a t e d out w i th the amount of DNA added between 10 ng, 20 ng, 50 ng, and 100 ng. The lowest c oncen t r a t i on g i v i n g p o s i t i v e plaques was u sua l l y used f o r making templates t o get as few double i n s e r t i o n s as p o s s i b l e . For r e s t r i c t i o n enzyme der i ved f ragments, 10 ng of DNA gave a s u f f i c i e n t r a t e of l i g a t i o n of i n s e r t but f o r son i ca ted f ragments, 50 t o 75 ng of DNA were r e qu i r e d . The r eac t i on was set up i n 1.5 ml microfuge tubes as f o l l ows : v 10 ul DNA and dH20 2 ul l i n e a r vec to r [10 ng/u l ] cut w i th the appropr ia te enzyme 2 ul 10X l i g a s e bu f f e r 2 ul 10 mM dATP 2 ul 50 mM DTT 2 u l T4 DNA l i g a s e (BIOLABS) d i l u t e d : 1.5 ul of [400 u n i t s / u l ] l i g a s e 1.5 ul 10X l i g a s e bu f f e r 12 ul dH20 and l e f t overn ight at 4 degrees C. When l i g a t i n g son i ca ted f ragments, 2 un i t s of T4 Po l ynuc l eo t i de Kinase (BIOLABS) were added to the mix ture and incubated at 37 degrees C f o r twenty minutes before the a dd i t i o n of l i g a s e , t o ensure the DNA had a 5' phosphate and 3' hydroxyl end. 5. Trans format ion of Host C e l l s a . Competent C e l l s •• Minimal Glucose P l a t e s 5X S a l t s 5.25 g Potassium Phosphate,monobasic 2.25 g Potassium Phosphate ,d ibas i c .50 g Ammonium Sulphate .25 g Sodium C i t r a t e i n 100 mis dH20, autoc laved f o r 200 mis t o make 1.5% agar p l a t e s : 40 mis 5X S a l t s 3 g Bactoagar (D i f co) 160 mis dH20 were autoc laved, when cool the f o l l ow i ng added: .2 mis 20% MgS04 (autoc laved) .1 ml Thiamine [10 mg/ml] ( m i l l i p o r e f i l t e r e d ) 1 ml 40% Glucose (Dextrose) i n dH20 (autoc laved) 2X YT Medium 16 g Bactot ryptone (D i f co) 10 g Bacto Yeast Ex t r a c t (D i f co) 5 g NaCl dH20 to one l i t e r , autoc laved 2X YT Sof t Agar .75% Bactoagar i n 2X YT Medium 2X YT P l a t e s 1.5% Bactoagar i n 2X YT Medium E . co l i c e l l s of the s t r a i n JM103 were made competent f o r t r ans fo rmat i on w i th ca l c ium ch l o r i d e as descr ibed by Lederberg and Cohen(1974). A colony scraped o f f a f r e sh overn ight Minimal Glucose Agar p l a t e of JM103 was used to i n o cu l a t e a s t e r i l e 125 ml Erlenmeyer f l a s k con ta i n i ng 40 mis of 2X YT Medium. The c e l l s were grown i n a shaking i ncuba to r at 37 degrees u n t i l the absorbance at 600nm wavelength was between .4 and .6 (about th ree hours ) . The c e l l s were immediately t r a n s f e r r e d t o s t e r i l e 50 ml tubes on i c e , and c en t r i f uged at 3,000 RPM f o r f i v e minutes at 4 degrees i n a precoo led SS-34 Roto r . The supernatant was c a r e f u l l y poured o f f the p e l l e t e d eel I s , then 20 mis of i c e co l d lOOmM Calc ium Ch l o r i d e (CaC12)was added and the c e l l s gent ly resuspended w i th a s t e r i l e pasteur p i p e t t e . The c e l l s were l e f t on i c e f o r t h i r t y minutes to one hour, then c en t r i f uged down aga in . A f t e r the supernatant was removed, the c e l l s were gent ly resuspended i n 4 mis of lOOmM CaC12 and l e f t two to twenty- four hours at 4 degrees C. b. Transformat ion of E . co l i 32 Into a s t e r i l e 1.5 ml microfuge tube s i t t i n g on i c e , were p ipe ted: 200 u l s competent JM103 c e l l s 7 u l s l i g a t i o n mix (about 7 ng vec to r ) 43 u l s TEN 8 bu f f e r and l e f t f o r f o r t y minutes. The c e l l s were then heat shocked at 37 degrees f o r ten minutes. Into 3 ml d i sposab le t e s t tubes, 20 ul lOOmM I sop ropy l t h i o B -D - ga l a c t o s i de (IPTG) and 200 ul of growing JM103 c e l l s ( i n l og phase) were p i pe t t ed and 125 ul of the transformed c e l l s added. Three m i l l i l i t e r s of 2X YT s o f t agar (at 42 degrees C) were p i pe t t ed i n t o the tube , 40 ul of 2% 5-Bromo-4-ch lo ro-3- indo ly l -B L D - g a l a c t o s i d e (X-Gal) i n Dimethyl Formamide (DMF) added, the contents mixed by i n v e r s i o n th ree t imes , and then poured out over 2X YT agar p l a t e s , t o be incubated at 37 degrees ove rn i gh t . Th is method r e su l t ed i n about 500 plaques per p l a t e , and wh i l e us ing the r e p l i c a t i o n form (RF) of uncut mp9 vec to r as a c o n t r o l , r e su l t e d i n a t r ans fo rmat i on ra te of about one m i l l i o n co l on i e s per ug of DNA. The f o l l o w i n g morning, 2 mis of 2X YT broth and 20 ul of overn ight JM103 c u l t u r e were p laced i n l abe led 15 ml s t e r i l e d i sposab le tubes and each i no cu l a t ed w i th one c l e a r plaque from the agar p l a t e s , beg inn ing w i th the p l a t e of lowest DNA c oncen t r a t i on , and us ing s t e r i l i z e d pasteur p i pe t s to p u l l up the p l u g . The tubes were put i n the shaking i n cuba to r at 37 degrees f o r about e ighteen hours . The c e l l s were then c en t r i f uged f o r ten minutes at 1500 RPM i n t o a p e l l e t , l e av i ng the phage i n suspens ion . The supernatant w i t h the c e l l s was s tored i n t e s t tubes at 4 degrees u n t i l the templates were made. The transformed c e l l s and phage obta ined from c lones w i th r e s t r i c t i o n enzyme de r i ved fragments were used d i r e c t l y f o r making templates but those der i ved from c lones w i th son ica ted DNA were screened f o r the presence of cDNA i n s e r t . 6. Dot B l o t H y b r i d i z a t i o n a . F i l t e r P repara t i on The son ica ted DNA c lones were screened f o r those con ta i n i ng the cDNA i n s e r t by us ing probes made from the o r i g i n a l i n s e r t s to h y b r i d i z e to f i l t e r s con ta i n i ng supernatant from the i s o l a t e d p o s i t i v e p laques . N i t r o c e l l u l o s e f i l t e r s were c a r e f u l l y marked w i th evenly spaced numbers (about 50 spots per f i l t e r ) w i th p e n c i l , and th ree u l s of each phage supernatant were spotted on a number. For c o n t r o l s , two u l s of the i s o l a t e d p lasmid i n s e r t were spotted on each f i l t e r as p o s i t i v e and 2 u l s of supernatant obta ined from growing up a blue plaque were spot ted on as nega t i ve . The f i l t e r papers were l e f t to a i r dry f o r ten minutes , then the DNA was denatured by the f o l l ow i n g method. 3MM Whatmann Chromatography paper was placed over a g l a s s sheet and soaked i n denatur ing s o l u t i o n (.5N NaOH,1.5M NaC l ) . The paper was soaked aga in , then the f i l t e r s l a i d on top so tha t the s o l u t i o n was absorbed everywhere on the f i l t e r . The f i l t e r s were l e f t i n p lace f o r t h i r t y minutes then al lowed to a i r dry f o r f i v e minutes wh i l e new paper was p laced on the g lass sheet . The procedure was repeated w i th 1.0 M T r i s -HC l pH 7.4 and f i n a l l y w i th .5M T r i s -HC l ,1 .5M NaCl pH 7.4. A f t e r a l l ow ing the f i l t e r s t o a i r dry f o r t h i r t y minutes, they were baked i n a 68 degrees C oven between two sheets of 3MM Chromatography paper f o r two hours . b. H y b r i d i z a t i o n H y b r i d i z a t i o n So l u t i o n 6 mis 20X SSC .2 ml 100X Denhardt So l u t i o n .1 ml .2M EDTA 1 ml 10% SDS 12.7 mis dH20 Washing So lu t i ons 34 I . 300 mM NaCl I I . 300 mM NaCl 60 mM Tr i s -HC l pH 8.0 60 mM Tr i s -HC l pH 8.0 2 mM EDTA 2 mM EDTA .5% SDS I I I . 3 mM Tr is-OH Twenty mis of the f r e s h l y made h y b r i d i z a t i o n s o l u t i o n was warmed to 68 degrees i n a square p l a s t i c p e t r i e d i sh and the f i l t e r s were dropped i n t o t h i s s o l u t i o n to p rehyb r i d i ze f o r one hour at 65 degrees C. 100 ug pBR322 and 50 ug E . co l i DNA were added to the n ick t r a n s l a t e d cDNA i n s e r t and bo i l e d f o r ten minutes. Th is was added to 3 mis of prewarmed h y b r i d i z a t i o n s o l u t i o n and poured i n t o a Seal-a-meal bag con ta in ing the f i l t e r s . The sea led bag was l e f t overn ight at 65 degrees between two g lass p l a t e s . The f i l t e r s were p laced i n t o a tupperware sandwich con ta ine r and washed tw i ce f o r f i v e minutes at room temperature i n Washing So l u t i o n I , tw ice f o r t h i r t y minutes at 60 degrees i n S o l u t i o n I I , and tw i ce at room temperature f o r 30 minutes i n So l u t i o n I I I . A f t e r a i r d ry ing f o r t h i r t y minutes, the f i l t e r s were set up t o autoradiograph ove rn i gh t . Supernatants from which a p o s i t i v e spot was obta ined were then used f o r template p r epa ra t i on . 7. Template P repara t i on The method of M13 template p repara t i on used was as f o l l o w s . 1.5 ml microfuge tubes were f i l l e d w i th c e l l suspens ion from each e ighteen hour c u l t u r e grown from the p o s i t i v e p laques. The c e l l s were p e l l e t e d out by c e n t r i f u g a t i o n f o r f i v e minutes, then one ml of supernatant removed to a f r e sh tube us ing a P1000 pipetman w i t h a s t e r i l e b lue t i p . 250 u l s of a 20% Po lye thy lene G l y co l ( PEG) , 2.5M NaCl s o l u t i o n were added and vor texed, then the tubes l e f t t o stand at room temperature f o r twenty t o t h i r t y minutes . The phage DNA was cen t r i f uged down f o r f i v e minutes i n t o a small whi te p e l l e t , and the supernatant poured o f f , w i th any remaining supernatant removed by a P200 pipetman a f t e r r e c e n t r i f u g a t i o n . The p e l l e t was d i s s o l v ed i n 200ul TE bu f f e r w i th v o r t e x i ng , then s equen t i a l l y phenol ex t r a c ted w i th 170 ul phenol , then 75 ul phenol , 75 ul ch lo ro fo rm, and f i n a l l y 50 ul phenol , 50 ul ch loro form as descr ibed above. A f t e r the f i n a l c e n t r i f u g a t i o n s t ep , 175 ul of the upper aqueous l a y e r were t r a n s f e r r e d t o a new tube to be p r e c i p i t a t e d w i th 3M NaAc and 95% ethanol f o r one hour at -70 degrees C. The p r e c i p i t a t e d DNA was resuspended i n TE bu f f e r and r e p r e c i p i t a t e d overn ight at -20 degrees C. The template DNA was r i n sed w i t h 200 ul 70% ethanol and f i n a l l y resuspended i n 40 ul TE b u f f e r . 8. P repara t i on of Terminat ion Mixes The nuc l eo t i de t e rm ina t i on mixes were mixed and t i t r a t e d by the f o l l o w i n method. The stock s o l u t i on s were made up i n the f o l l ow i n g order: 100 mM Deoxynuc leot ide s tocks: 2 1Deoxyguanosine 5 1 - T r i phosphate,sodium 2'Deoxyadenosi ne 5 ' -T r iphosphate ,sod i um 2 1 Deoxythymidine 5 ' -Tr iphosphate,sod ium 2 'Deoxycy t id ine 5 ' -Tr iphosphate,sod ium (dGTP) (dATP) (dTTP) (dCTP) were a l1 d i1uted t o .5mM a l i q u o t s w i th dH20 lOmM Dideoxynuc leot ide s tocks: were d i l u t e d w i th dH20 t o : 2 ' ,3 ' ,D ideoxyguanos ine 5 ' -Tr iphosphate,sod ium (2XddGTP) 1.6mM 2 ' ,3 ' ,D ideoxyadenos ine 5 1 -Tr iphosphate ,sod ium (2XddATP) .2mM 2 ' , 3 ' ,D i deocy thym id i ne 5 ' -Tr iphosphate,sod ium (2XddTTP) 2.0mM 2 ' , 3 ' , D i d e o x y c y t i d i n e 5 ' -Tr iphosphate,sod ium (2XddCTP) .2mM dNTP mixes were made as f o l l ows . Add:/dNTP dT dC dG dA .5mM dGTP 100 ul 100 ul 5 ul 100 ul .5mM dTTP 5 ul 100 ul 100 ul 100 ul .5mM dCTP 100 ul 5 ul 100 ul 100 ul dH20 95 ul 95 ul 95 ul 0 ul dd/dNTP Terminat ion mixes were made as f o l l o w s . Reagents:/cone: 2X IX .5X 2XddNTP s tocks 10 ul 5 ul 2.5 ul dNTP s tocks 10 ul 10 ul 10 ul dH20 0 ul 5 u l 7.5 ul Using these t e rm ina t i on mixes t o sequence a template of known sequence, the concen t ra t i ons d i s p l a y i n g the best t e rm ina t i on r e s u l t s evenly across a l l nuc l eo t i des were determined. 200ml s tocks of the co r r e c t concen t ra t i on of ddNTP s tocks were made and mixed w i t h equal volumes of the dNTP s t o c k s . 9. P repa ra t i on of Acry lamide Gels 40% Acry lamide (38:2) 38% Acry lamide (98.5% Pure Acry lamide from BI0RAD),de ion ized and f i l t e r e d 2% N,N 1 -Methy lene-b i s -ac ry lamide (Bis)(BI0RAD) dH20 Acry lamide Gels Urea (BI0RAD) 10X TBE 40% Acry lamide dH20 8% 6% . 25 g 25 g 2.5 mis 2.5 mis 10 mis 7.5 mis 20 mis 22.5 mla 37 The urea was d i s s o l v ed i n the s o l u t i o n by warning i t up and the s o l u t i o n deaerated under vacuum, then the f o l l ow i n g added: 10% Ammonium Pe r su l f a t e i n dH20 .33 mis .33 mis and f i n a l l y , immediately preceding the pour ing of the g e l : N,N,N 1 ,N ' -Tet ramethy le thy lened iamine (TEMED) 15 u l s 15 u l s Two 38X20 cm g lass p l a t e s , one con ta in i ng r abb i t ear notches, were coated on one s ide w i th one ml of 1% S i l i c o n (1% S i l ane i n T r i ch l o roe thane) and a l lowed to d r y . The p l a t e s were r i n sed w i th water , then washed w i t h 95% e t hano l , and al lowed to d r y . The two p l a t e s were taped together along the s ides and bottom, s i l i c o n i z e d faces t oge the r , w i th .75 mm p l a s t i c spacers separa t ing them. The p la tes were clamped together w i t h bu l l dog c l i p s and t i l t e d at a t h i r t y degree angle to pour the prepared acry lamide i n between the p l a t e s . A p l a s t i c we l l forming comb con ta in i ng e ighteen .68X1.1 cm we l l s was p laced i n the top and the gel l e f t to set f o r t h i r t y minutes. Once the gel had s e t , the tape was cut from the bottom, the comb removed and the we l l s r i n sed w i th bu f f e r . The p l a tes were pos i t i oned on the e l e c t r o p ho r e s i s apparatus w i th metal p l a t e s clamped on top and prerun i n .5X TBE bu f f e r f o r ten minutes at 35 Watts per g e l . The we l l s were r i n sed f r e e of urea again p r i o r to l o ad i ng . 10. Dideoxy Sequencing React ion 10X HinF Bu f f e r 100 mM T r i s -HC l pH 8.0 70 mM MgC12 600 mM NaCl Klenow D i l u t i o n Bu f f e r 100 mM T r i s -HC l pH 8.0 100 mg/ml BSA (BRL) 10 mM DTT Gel Sample Bu f f e r 3 98% Formamide (de ion ized) .1% Bromophenol Blue .1% Xylene Cyanol dyes were d i s s o l v ed i n formamide then lOmM EDTA added The M13 sequencing reac t i ons were c a r r i e d out as desc r i bed by Sanger (1977). Into each 1.5 ml microfuge tube 2 u l s HinF bu f f e r and 1 ul M13 Sequencing Pr imer (17mer from Pharmacia B iochemica ls ) were added to 5 u l s of template and vo r texed . The tubes were p laced i n a heat ing b lock at 75 degrees C which was a l lowed to cool t o room temperature f o r 45 to 60 minutes . Meanwhile, 1.5 ul a l i q u o t s of each dd/dNTP t e rm ina t i on mix were p ipeted i n t o sepa ra te , l a b e l l e d microfuge tubes; one of each of the 4 dd/dNTP nuc l eo t i des f o r each template sequenced. When the template r eac t i ons had f a l l e n t o about 32 degrees C, 1 ul of 15uM P32-2'Deoxyadenosine 5 'Tr iphosphate (dATP from New England Nuc lea r , Lach ine, Quebec) was vortexed i n t o each tube . From each template r e a c t i o n , 2 u l s were dispensed i n t o the p r ev i ou s l y a l i quo t ed microfuge tubes con ta in i ng dd/dATP and dd/dTTP, and 2.5 u l s were dispensed i n t o those con ta in i ng dd/dGTP and dd/dCTP. These were spun b r i e f l y and 1 ul (.2 u n i t s ) of Klenow fragment (DNA Polymerase I l a rge fragment from BIOLABS, d i l u t e d i n IX Klenow d i l u t i o n bu f f e r ) was added and mixed w i th a pipetman severa l t imes . The reac t i ons were incubated f o r exac t l y f i f t e e n minutes at room temperature, then chased w i t h 1 ul of .5mM dATP and incubated a f u r t h e r f i f t e e n minutes . The reac t i ons were terminated w i th 5 ul of the sample b u f f e r . P r i o r t o l oad ing the samples on the g e l , the DNA was denatured at 90 degrees f o r th ree minutes and plunged i n t o an i c e water bath f o r th ree minutes . Two u l s of each r eac t i on mixture were loaded onto the g e l s , i n the order 1 G, A, T, C, w i th four templates per g e l . To ob ta in the maximum amount of sequence per template , two gels w i th the same load ing order were e lec t rophoresed at a t ime, one long 6% gel f o r about 2.5 hours and one short 8% gel f o r s i x t y to e ighty minutes, at 35 Watts per g e l . The gel was t r an s f e r ed from the p l a tes to 3MM Chromatography paper and d r i ed down under vacuum at 80 degrees wi th a gel d r i e r , and se t up to develop Kodak XRP-1 f i l m overn ight at room temperature. 11. Compi la t ion of Data The computer program of Staden (1980) was used to compi le the DNA sequence d a t a . The sequence data was t r a n s l a t e d i n t o p red i c t ed amino ac id sequence and compared to other sequences us ing programs by Delaney (1982) and Wi lbur and Lipman (1983). The d ivergence of the g cha ins was c a l c u l a t e d by the number of nuc l eo t i de changes between two sequences caus ing e i t h e r an amino ac id change (replacement s i t e ) o r no change ( s i l e n t s i t e ) . Th i s was adjusted f o r m u l t i p l e events by c a l c u l a t i n g the percentage co r rec ted d ivergence as desc r i bed by P e r l e r (1980). RESULTS 40 I . C h a r a c t e r i z a t i o n of the pRT lEg . l cDNA and P red i c ted P ro t e i n A. pRT lEg . l cDNA Inser t Two cDNA l i b r a r i e s made from Wis ta r r a t spleen po ly(A) RNA were screened w i t h a DNA fragment from a C lass I I A3 gene. A s i n g l e p o s i t i v e c lone was i s o l a t e d from screen ing 1000 c lones of the a-cha in enr i ched l i b r a r y , and named pRT l Eg . l . R e s t r i c t i o n enzyme ana l y s i s showed pRTlEp.1 was approx imate ly 900 nuc l eo t i des l ong , w i th an i n t e r na l Ps t I and TaqI r e s t r i c t i o n endonuclease s i t e . F igure 3 shows a r e s t r i c t i o n map of the i n s e r t w i t h i n pBR322 and the c l on i ng s t ra tegy used to ob ta i n ove r l app ing f ragments . Random DNA fragments, 300 t o 500 basepa i rs i n l e ng th , were generated by s on i c a t i ng pRT lEg . l and c loned i n t o the M13mp9 vec to r and screened w i th the cDNA i n s e r t of t h i s p lasmid . R e s t r i c t i o n enzyme fragments were a l so prepared to o r i e n t the fragments and ob ta in any sequence missed by the son i ca ted f ragments. The average length of sequence determined per subc lone was about 250 nuc l eo t i des when son icated fragments were subc loned, wh i l e the r e s t r i c t i o n fragment subclones were l i m i t e d by the s i z e of f ragments. The sequence of 24 templates were over lapped t o ob ta in the complete sequence of both st rands of the cDNA i n s e r t , through the po ly(C) t a i l s t o the Ps t I i n s e r t s i t e s of the pBR322 p lasmid . The cDNA conta ined 830 nuc l eo t i des and was f l anked by a 10 base 5' and a 15 base 3' po ly(C) t a i l . F igure 4 shows the sequence of pRT lEg. l compared to the coding reg ions of the RT1 Ag gene (E c c l e s , 1985) and the H-2 E3 cDNA ( S a i t o , 1983). By l i n i n g up homologous nuc l eo t i de s , the cDNA was found to con ta i n 37 base p a i r s of the l eader sequence, 800 bp of the message, and 93 base p a i r s past the stop codon of the 3' un t r ans l a t ed reg ion but was m i s s i ng the pu t a t i v e po ly (A) adeny l a t i on s i g n a l , AATAAA. P R T I E 3 . I shows a h igher degree of sequence i d e n t i t y t o the H-2 E3 cha in FIGURE 3 Sequencing str a t e g y of pRT.l 1 cDNA Insert R e s t r i c t i o n Enzymes 5' AAA! H = Ilae III. P = I'st 1 R = Rsa T. T = Taq I P .. H RT R II R PR R p ,, - - p 3' i « i 1^  R.E. Fragments Sonicated pOR322 Sequence 4 2 FIGURE 4 DNA Sequence of pRTlE/3.1 i n Comuaris on K-2 ^ and RT1 h/i * y < < < < — * y • y — < « < < < 4 < • < < y * y < « < < < y < — < y — < « < < y — « — y * 'J y * ,w < • < y u < u u — 'J — « y < < * < y < < < * < — y y — •J • Stop codon i s boxed, 3 sequences to l i n e up wi T.M = transmembrane , CyT region, L s = i e a d e r sequ (H-2 E/2» from Saito,1983 ep gap i s inserted inro K-2 and pRTl th the RT1 A sequence = cytop:ismic t a i l , 3'UT =3' untrans ence CP=Connecting Peptide RT1 A/S from Eccles,1985) -A-o < • c a • < - < < • * < 3 £ « < < « < * < y « y < < y y • < < • < < < • < • < < y « y y < • < * < • y < * < __ < y < y — y y y y < « < < < y * y y < • < * < < u • y < < y < y * y c « y * y * y y < < — o y * * H < £ < y < • < y - » — < u u < < > — » < • < * < * —< < • < • < • w * < — » — * — — — • — * • — • — *  * * — • — H ' H * • X • — • < -< < • < -< < • < • < •J < • < * < < • <  • < * < * - < 2 c 5 Hi < € 2 < * < < * < w < * c * — c • < u 2 r' « • o c < * < » < < • < 'Z < - < 'j < w • • < * < • < < • < * < • < * < < < < < < * * w u * u * * < • '•Z * < * V * < « < u *r * • y * " • w w < * < W < * < w w * < * < * < < • A A * < < £ < u — £ < < • w < • < * w < < < « < u 12 u • u £ < • < * o < • < < • < * < c * < « < r- < • < • < < < • < £ < • — • < < - < c * — <• « < V < 4 < W * < < < :^ « — — < £ " £ CJ -M -< » < — * < * < < < < 5 r z -^ < < y y • y • < . < r: * y * — • w * y < * < y * y < y < < — * _ • < y y * y • y y • y y * • y < * < <r * < y • y * w * y y * y * y < • <• < < y u • • y < •• < y < < • < < <• y * * y y • • y < • < w • < • < <. « < y y y • y * y < — 4 • < • •_. y < y * * y < * < < * < y y • y y • y < y tr •• y * y y • y y 'S * c o • y < y y • y < « < y o y y y y < < « < < < y y • £ y * y < < < * < — • < 4 < • < ** _ * •m. y y e < * < y y < * < y * < • < « < * < y * u * y • u • y u • y u • < • < y * y < • < y u « o u • y y y * • ~^ * «— y * y • y u • y u * y ^« o * w — — than the RT1 Ag c ha i n . These d i f f e r en ce s are summarized i n Table 1 which compares the l e v e l of DNA sequence i d e n t i t y between a number of C lass I I g cha ins i n the r a t , mouse and human across the d i f f e r e n t s t r u c t u r a l domains. The o v e r a l l homology to the H-2 Eg cDNA (88.5%) i s cons ide rab ly h igher than when compared to the RT1 Ag gene (69.5%). Even the 3' un t rans l a ted reg ion a f t e r the stop codon i s h i gh l y conserved i n the RT1 Eg DNA wh i l e tha t of the RT1 Ag cou ld not be l i n e d up. From t h i s degree of homology, i t was concluded t ha t the cDNA i n s e r t of the RT lEg . l p lasmid encoded the RT1 Eg c ha i n . B. P red i c t ed P ro t e i n Sequence of pRT lEg . l F igure 5 shows the p red i c ted amino ac id sequence of pRT lEg . l , and comparison t o the equ iva len t chains of the mouse, H-2 Eg and human, HLA DRg. A l l t h r ee p ro t e i n s are 238 amino ac ids l o ng . Al ignment w i th the 264 res idue H-2 Eg cDNA showed the cDNA s t a r t e d 19 res idues a f t e r the methionine i n i t i a t i o n codon of the mouse Eg p r o t e i n . Comparison of the p red i c ted p ro te i n sequence of pRT lEg. l t o the f i r s t twenty-seven res idues of the s e r o l o g i c a l l y i s o l a t e d Ag and Eg cha ins of mice determined by N-terminal sequencing ( K l e i n , 1979), showed e ight of e leven known res idues were i d e n t i c a l t o the Eg cha ins wh i l e none of the f i v e known i n Ag were i d e n t i c a l . Th is i s f u r t h e r support t ha t pRT lEg . l encodes an RT1 Eg c h a i n . Cecka (1980) sequenced the f i r s t twenty res idues of C l a s s II molecules i s o l a t e d from th ree r a t haplotypes (RT1.1, a, and n) but cou ld not d i s t i n g u i s h between RT1 E and A molecu les . In s i x of the twelve known res idues pRT lEg . l cou ld be matched to one of the two po s s i b l e amino ac ids at t h a t p o s i t i o n . The cDNA of pRTlEg. l i s from the haplotype RT l .u so would be expected t o show some d i f f e r e n c e s , but i n many cases some of the po s i t i o n s were not ob ta inab le across a l l the hap lo types , so the f u l l sequence would not demonstrate as l i t t l e c onse rva t i on . Nine out of t h i r t e e n were i d e n t i c a l t o the Egk mouse gene. TABLE 1 Comparison of the cDNA Sequence of pRTlEg. l to C lass II g Genes ( i n percent of homologous n u c l e o t i d e s ) . FUNCTIONAL DOMAINS: pRT lEg . l t o SPECIES and LS g l g2 CP TM CytT 3'UT TOTAL GENE: RAT *RT1 Ag - 68.4 72.7 66.7 71.7 61.4 - 69.5 MOUSE H-2 Eg 85.7 85.6 89.7 87.9 90.0 94.5 87.0 88.5 HUMAN HLA-DRg 71.4 77.2 84.8 69.7 88.3 89.5 30.4 81.4 HLA-DQg 71.4 69.7 74.1 60.6 70.0 50.0 25.9 69.2 HLA-DPg - 66.3 78.7 66.7 66.7 61.4 20.4 71.0 * References- RT1 Ag (Ecc l e s ,1985) , H-2 Eg (Sa i t o , 1983 ) , HLA-DRg (Gusta fsson,1984) , HLA-DQg (Larhammar,1983a), and HLA-DPg (Go r sk i , 1984 ) . FIGURE 5 Predicted Amino A c i d Sequence of pKTlE^. 1 i n Comparison to IILA -DRyj and H-2 Fy£ Cysteine residues and Asn-G.ly-'J'lir y l y c o s y l a t i o n s i t e are boxed, and boundaries of domains marked. (H-2 E£ from Saito,1983. HLA-DRA from Gustafson,1984) U S 11-2 */» t au Sar Pro Pro Val Ala t on Val Aig Aafi Hir Aiu Pro Aig Plia 1 all Ulll lyr Val llir Sar i:yi. Ilia Pile lyr Aan Oly Un- Gill R I 1 Val Sor • Pro Pro • Val Ala • I I I I ! • Vul Al u A|<|| I'ro llir I'm Al II « I'lio 1 Ull • Oly lyr • 1 ail 1 ya I ' l l . Gin • I.ys • Ilia I'l.a lyi Aan • Uly • til! • Uln III A l>K/t lati Sur Sur Pro 1 «u Ala 1 U l l Ala (ily Al.p llir Aiu I ' I I I Al'U I'liu 1 m l Gin 1 yr Sor llir Sar Gin <-yi- Ilia I'liu PI in Aan Uly I I . I Qlii Ilia • Arg Pl.a 1 ail aiu * , U I'lia 1 lo lyr Aon A I U Olu Ulll Aan 1 oil A i U Pi>« Aap Sur Asp Val Illy llln lyr Arg Ala Val llir 53 Gin *. u Val • Aig Ian 1 Ull Ala Al (I 1 on 1 Is lyr Ai.il Al U ulu Ulu lyr Ala A I U I'llU As,. Sur Asp Vol Illy (11 I I 1 yr Aiu Ala Val llir Glii • IB Val Aig lyr 1 UU Asp Arg lyr I'liu Ilia A911 llln llln (.111 A B U Val Aru I'l.o Asp Sur Asp Val lily Ulll I'll* Al U Ata Val llir Ulll lull Gly Arg Pro Aap Ala 0111 Aan I rp Aau Sar Uln I'ro Olu l la l«u Glu Aap Ala Aig Ala Sal- Val Asp llu* lyi « • • » • • « « « • Lou Gly Arg Pro Sur Ala Uln lyr Aru Asn I ya uln I ya Uln I'liu Mul Uln Aiu A i u Aig Ala Ala Val Asp llu- lyi 83 Arg Ilia Asn Aru Ula Asn • • • Arg Ilia Aan Leu Gly Arg Pro AS|> Ala Uln lyr I rp Ami Sur Gin Lya Asp Luu I uu Ulll Uln I ya Ai g Uly Arg Vol Abp Aatl lyi DOMAIN ,„ Val Oln I'm llu- Val llu- Val lyr Pro llir lya llu- Uln Pro I ail Oln Ilia Ilia tyr Glu l l a Sar Aa|> Lya Plia I ail Val Arg Arg Arti • » . » • lyr Glu l l a Pliu Asp Arg Plia luu Val I'ro Ai g Ai p « . . . . Tyr Gly Val Val Ulu Sur I'lia II.r Val uln Arg Ai u Asn I au Lau Val Asn I on Leu Val Cys Cya Vul Ulu I'i u lya Val llir Vul lyr Pro Sur lya llir Mil Pi o luu Ulu Ilia Ilia . . • • • * • • • » . « • » « • Vul Ilia I'i u lya Val ll.r Val lyr Pro Sur lya llu- Ulu Pro luu Uln Ilia Ilia I I ] Sur Val Sur A»|i I'liu lyr Pro uly Ann l la Ulu Val Arg l ip Plia Arg Aau Uly lya Glu (llu Glu llir Gly I lo • • « • • • • • • * • • * » » • • • « • • Sur V.i I Sur Asp. I'liu lyr Pro Uly Sur l lu Ulu Val Arg lip I'lia Ai g Asn lily I yu Ulu Ulu lya Aap Gly I ail Aan leu leu Val Cya Sur Vul Sur Uly I'liu lyr Pro Uly Sur l lu Ulu Val Arg lip I'lia Arg Ami Uly Ulll Ulu Ulu lya llir Gly Val * * * * * * * I7J Val Sar Ihr Oly lau Val Aru Aan Gly Asp l ip llir I'l.a Glu llir luu Vat Mul luu Glu llu- Val Pro Uln Sor Oly Glu Val lyr llir Val Sur llir Gly luu l l a Aig Uly Asp lip llu I'l.u Uln luu luu Val Mul luu Ulu llir Val Pro Uln Uly Uly Ulu Vnl lyr llir Val Sur Ihr Uly tun l ie Ilia Asn lily Asp l ip 1 ' ' ' " > •<'•' 1 "" Vul Mul luu Ulu llir Vnl Pro Aru Sur Gly Ulu Vnl lyr llir C P T M * 3 0 3 lya Ala Uln Sur llir Sur Ala Uln Aan lyalMul leu Sur Gly I * * I ya A1 it Ulu Sor Ihr Stir Ala CI it Akit lya tyolMut Sur Gly * » • • • » . I » • Aru **• *»U Sor Olti Sur Ala Uln Sur lysJMut Lau Sur Gly ClT T.4IL 233 lyr I'lia Aru *»•» <• >" l.ya'Uly 41 In Sor Gly loll Gin I'ro Cyt aln Val Olu Ilia Pro Sur 1 oil 11.1 Aap Ulu Val Glu Ilia Pro Sur 1 uu I'ro Sur Cya Glu Val Ulu Ills Pro Sur Val 11,1 Sur Val Gly Oly Plia Val L en Gly 1 uu 1 uu I'liu Val Gly Oly t la Val I O ' l Uly 1 uu 1 Ull I'liu Val Gly Oly Plia Val 3 IB t oil Uly 1 U U 1 Ull I'liu Tlir Oly leu Leu Sur • llir Gly (au Leu Aau Arg Oly Plia Leu Sor lyr I'liu Arg Sur liln lyti Uly Uln Sur Gly luu Gin Pro lyi- I'liu Aru Asn Uln lya uly Ilia Sur Uly luu Gin Pro * Conserved r e s i d u e s i n a l l Ui l i k e p r o t e i n s - Conserved r e s i d u e s in a l l MHC p r o t e i n s LS - LeaderSeAuei^M. T M - - Ti»«»i»ai>0'-ji ,C' CP sCon-MXi^ P^'+ile OyfrTciii- GfU»>io-*n\!£, Ta'i\ C. Comparison of pRT lEg. l to Equ iva lent DNA i n D i f f e r e n t Species Comparison of the equ iva len t gene products of the three spec ies shows no i n s e r t i o n s or d e l e t i o n s are needed to l i n e up the nuc l eo t i de sequence f o r the maximum amount of homology (F igure 5 ) . This i s not t r ue f o r the n o n - a l l e l i c comparisons. The RT1 Ag gene has an add i t i on of th ree nuc l eo t i des at the end of the g l domain r e l a t i v e to pRT lEg . l which meant a gap had t o be in t roduced i n the two Eg sequences f o r proper a l ignment . S ince t h i s a dd i t i o n i s not found i n any of the human C lass I I a l l e l e s , but i s found' i n the H-2 Ag genes (Larhammar, 1983a), i t appears to be a mutat ion developed a f t e r rodents d iverged but before ra t s d iverged from mice. S i m i l a r l y , a gap of 24 nuc l eo t i des i s needed i n the cy top lasmic reg ion of the DQg cDNA (Larhammar, 1983b) t o l i n e i t up to DRg (Gustafsson, 1984), DPg (Rous-Dosseto, 1983), r a t Ag ( E c c l e s , 1985) and pRT l Eg . l . D. P red i c t ed P r o t e i n Domains of pRT lEg. l The domain s t r u c t u r e of the C lass II chains f o l l ows the i n t ron-exon s t r u c t u r e of the corresponding genes (Hood, 1983). The s t r u c t u r e of the H-2 Eg gene ( Sa i t o , 1983) was used to p r ed i c t the f un c t i ona l domains of the p r o t e i n coded f o r by pRTEg. l . The f i r s t 37 nuc leo t ides of pRT lEg. l code f o r the f i n a l 7 amino ac ids of the leader sequence, w i th s i x res idues being hydrophobic as observed i n t y p i c a l l eader sequences. The s i gna l sequence i s needed f o r the i n s e r t i o n of the p recursor i n t o the rough endoplasmic r e t i c u l um membrane where some of the g l y c o s y l a t i o n occurs , and i s c leaved o f f a f t e r i n s e r t i o n . K r e i l (1981) found archetypa l s i gna l sequences to end i n a res idue w i th no s i de cha in (Gly) but wh i l e i t i s observed i n Ag molecules (Larhammar, 1983a, Ma l i s s en , 1983), and DCg, i t i s not found i n pRT l E g . l , or any of the other g cha ins compared here , so i t i s probably on ly c o i n c i d e n t a l l y conserved i n some C lass I I equ iva len t p r o t e i n s . The f i r s t 94 amino ac ids of the mature p ro te i n code f o r the d i s t a l e x t r a c e l l u l a r domain, g l . Cyste ine res idues at pos i t i on s 16 and 80 form a pu t a t i v e d i s u l f i d e loop and the c h a r a c t e r i s t i c N -g l y cosy l a t i on s i t e of Asparag ine-G lyc ine-Threon ine at p o s i t i o n s 20 t o 22 a t tach the only carbohydrate moiety found on the g c ha i n . The second e x t r a c e l l u l a r domain, 32, found proximal t o the membrane, i s comprised of amino ac ids 96 t o 189. The cys te i ne res idues l o ca ted at p o s i t i o n s 118 and 174 form the pu t a t i v e d i s u l f i d e loops c h a r a c t e r i s t i c of molecules i n the immunoglobulin f a m i l i e s . A shor t connect ing pept ide of e leven h yd r oph i l i c res idues (eg. asparagine l y s i n e , t h reon ine , s e r i n e , g lu tamine) , j o i n s the g2 domain to the twenty amino ac i d long hydrophobic transmembrane r eg i on . The amino a c i d a l i g n i n g to the f i r s t res idue (methionine) of the transmembrane reg ion of the Eg, Ag and human equ iva len t g cha ins has a s i n g l e base mutat ion i n pRTlE2.1 and DPg r e s u l t i n g i n the p o s i t i v e l y charged l y s i n e and po la r th reon ine res idues r e s p e c t i v e l y . S ince t h i s would not i n t e r a c t w i th the hydrophobic l i p i d s i n the membrane, the actua l reg ion spanning the membrane i s probably sho r te r i n the other molecules i f such a change i s t o l e r a t e d . S i x g l y c i n e s are found i n the transmembrane reg ion which are conserved across a l l the g cha ins compared here . These are thought to c on t r i bu t e to the a h e l i c a l f o rmat ion of t h i s reg ion (Travers et a l , 1984) and i n t e r a c t s w i th s i m i l a r h e l i c e s i n the a cha in or other membrane p ro te ins (Beno i s t , 1983). Th is reg ion i s fo l l owed by a h y d r o p h i l i c cy top lasmic t a i l of 18 res idues of which the f i r s t ha l f are p o s i t i v e l y charged. The rodent C l a s s I I Eg cha ins appear to con ta in only two p o s i t i v e charges wh i l e other cha ins are more h igh l y charged ( four on DRg and RT1 Ag, s i x on DPg, and f i v e on DCg and H-2 Ag) . The p o s i t i v e charges are thought to i n t e r a c t w i t h the negat ive charges on the phospho l i p id l a y e r of the membrane, t o s t a b l i z e the p r o t e i n i n the membrane (Ma l i s sen , 1983). pRT l Eg . l , l i k e the HLA DR and H-2 E g cha i n s , conta ins a unique pheny la lan ine res idue two amino ac ids i n from the beginning of the t a i l where other chains are h y d r o p h i l i c through the i n i t i a l part of t h i s r e g i on . This may impart a bend i n the s t r u c t u r e important i n the i n t e r a c t i o n w i th the a c ha i n . E. Comparison of pRTlEg. l P red i c ted P r o t e i n Sequence to C lass II P ro te ins A t a b u l a t i o n of the t o t a l number of conserved res idues i n each domain between the r a t C lass I I g cha ins and those of the mouse and human are shown i n Table 2 . The sequences are d i v i ded i n t o domains f o r comparison by f un c t i o na l reg ion as s e l e c t i o n would be a c t i ng a t t h i s l e v e l . As seen in the DNA compar isons, the t o t a l p r o t e i n homology i s h igher i n equ iva l en t molecules (81%) than between molecules w i t h i n the one spec ies (58%). The t o t a l homology between the RT1 Eg and HLA DRg, and between RT1 Ag and HLA DQg i s h igher than between RT1 Eg and HLA DQg, and RT1 Ag and HLA DRg . Th is supports prev ious s tud ies conc lud ing t ha t the H-2 E molecule i s equ iva len t to the HLA DR wh i l e the H-2 A molecule i s equ iva len t to HLA DQ (Bono,1982). The DPg p r o t e i n shows no more sequence i d e n t i t y o v e r a l l to DQg (62%) or DRg (60%) than to RT1 Eg (61%) or RT1 Ag (60.8%), a l though Long (1984) c la ims t ha t i t d i s p l a y s s l i g h t l y more sequence i d e n t i t y t o the Eg2 gene. A l so pRT lEg . l i s equa l l y homologous to DQg (63%) and DPg (61%). These comparisons a l l suggest tha t none of the three human genes have d iverged any l a t e r than the two rodent genes f o r the C lass I I mo lecu les . S ince ex tens ive genomic prob ing has f a i l e d t o reveal an equ iva len t DP gene i n the mouse (Ste inmetz , 1982), i t appears t h i s equ iva len t has been l o s t i n r a t s and mice. F i gu re 6 shows the al ignment of the Eg and Ag equ iva len t cha ins between the th ree s pe c i e s . The conserved amino ac ids w i t h i n the A o r E molecule can be compared t o the conserved amino ac ids across a l l C l ass II mo lecu les , t o look a t s p e c i f i c f un c t i ona l or s t r u c t u r a l r eg i ons . TABLE 2 P red i c ted P ro te i n Sequence Homology of C lass I I g Chains i n Rat , Mouse, and Human ( i n percent conserved amino a c i d s ) . DOMAINS COMPARISONS RAT RAT TO MOUSE RAT TO HUMAN RTl Eg *RT1 Ag RTl Eg H-2 Eg RTl Ag H-2 Ag RTl Eg HLA-DRg RTl Ag HLA-DQg RTl Eg HLA-DQg RTl Eg HLA-DP| LS - 85.7 - 57.1 - 57.1 -ei 50.0 73.7 70.0 61.1 63.3 61.1 53.5 B2 67.0 87.2 84.0 85.1 82.0 71.3 74.5 CP 63.6 90.9 90.0 60.0 100.0 70.0 70.0 TM 65.0 85.0 95.2 85.0 85.7 65.0 50.0 CytT 42.1 89.5 84.2 73.7 26.3 42.1 42.1 Tota l 58.1 82.1 79.9 72.8 71.4 63.8 61.3 * Re ferences- RTl Ag (Ecc l e s ,1985) , H-2 Eg (Sa i t o , 1983 ) , H-2 Ag (Larhammar, 1983), HLA-DRg (Gusta fsson, 1984), HLA DQg (Larhammar, 1983b), and HLA-DPg (Go r sk i , 1984) . FIGURE 6 Amino A c i d Residues Conserved A c r o s s A and E > Chains (H-2 E/s from Saito,1983. 11LA-DR/3 from Gustafsson,1984. PT1 A* from Eccles,1985. H-2 k* from Larhammar,1983a. HLA-DQ/3 from Larhammar,1983b.) R T l E / i H-2 E4 HLA-DR4 R T l A/?> H-2 A <3 HLA-DQ4 RT 1 E /J H-2 E -0 HLA-DR-3 RT 1 A ^ H-2 A/3 HLA-D03 R T l E<i H-2 E 6 HLA-DR3 R T l A H-2 A 4 HLA-UQ3 & 1 DOMAIN # ««V # * * * * * * **** * * » * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * RDPTPRFLGYI.KFECI IF YNG I ORVRI.L ARE IYNRF E YARf DSDVGE YRAV 11" I.GRPSAF YRNKOKE FMERRRAAVDI YCR RDTRPRFLEYVISECHFYNGI 01 IVRFI.FRF I YNRF Fill. RTDSDVGE YR A V I E LGRPDA ENWIISUPF. I I.EDARASVD T YCR GDTRPRFLE YSTSECIIFFNGIERVRYLDRYFHNOE E NVRFIJ5IJVGE FRAV I ELGRPDAE YWNSOKDLLEOKRGRVDNYCR i r in i i i I I i mil i I I i I I I I I I I i n DFVYOFKGLCYYTNG TOR I RSVDRRFYNQE E F l.R YDSDVGE F RAI. I E I. GRSWADDWNSOKE I I.EOKRA EMDI VCR GDSERI IF VYOFMGECYF TNG I OR I RYV I R Y I YNRF E Y VR YDSDVGEI IRA V I E I.GRPDAI; YWNSOPE I I.ER I RAE I.DI VCR RDSPEDFVY0FKGMCYF1NUI ERVRLVSRSI YNREEVVRFDSDVGEFRAVI1.LGI.PAAE YWNSUKU I LERKRAAVORVCR * * * * * A * * * * * * ** '80 C> 2 DOMAIN • *** * * *** * ***** ***** ********** **** ********* ** * ***** ******* ** HNY - E I FDRF LVPRR V E P K V I V YPSK'I OP I. El II INI. I. V C S V S D F YI'GS I E VRWT RNGKF EKDGI. VS I Gl. I RNGDW I F O I L V H N Y - E I S D K F L V R R R V F . P l VI V Y P I KTOPLEII I INI.I. V C S V S D F Y P G N I EVRWF RNGK E E F I G I VS I Gl. VRNGDW I FD I I. V HNY-GVVESF1V0RR|VHPKV1 V Y P S K I OPI.Ol II IN I .LVCSVSGF Y P G S I EVRWI RNGOEEK I G V V S I GL IHNGDW F FQ1 L V ' ' ' ERPNI /A I SI.SRI'EAI'NI'IIU/LI'icsiii)l "YPAo.Kvri i lrRNGOFJ; ridvvfli'oi'iKhldilvlliUi lU E O P H W I S L S I U E A L N I I I IN 11 VCS V IDF YPAK 1KVRWF RNGOE E I V G V S S 1 01. I RNGDW 1 FOVL V V E P I VI I S P S R I E A L N I I I I N L L V C S V I DF YPAOIKVRWFRNDOE E I A G V V S I P L I RNGDW I F01 LV * * * * * * * ** * * ** * * * * * * * ** 160 YNYEETEVPlSLRRl IINY EGPE 11ITSLRRI IINY-OLELRT'f LORF * * * * * * * * * * * * * * * * * * * * * * * * * * * * • CP ****** ************ * * * , * * *** i MLETVP0GGEVY1C0VEIIPSI PSPVRVEVKAOSTSAONI MLE TVPOSGEVYT COVE I IPS 1.1 DP VI VE H< AOS I S AONI' mlVPRSGEVYlCOVEHPSVISPLI VEW I I I I I I I I I I ill **• TRANSMEMBRANE ***** ************ KMSGVGG1 Vt.GILT LGAGLF V|Y FNSOKGOSGLOI MI.SGVGGFVLGLI.F I GAGI.F I RARSESAOSklMLSGVGGF VLGLLF LGAGI.F I ' " I I I I I I I M l I I I I T I CYTOPLASMIC *** ***** * * 1 GLI.N YFiRNOKGOSGLOP IGLI.S Y PftNOKGI ISGLOPRGFLS MLEMTPORGDVYTCIIVDIIASLESPVTVEWRAOSFSAOSI' Ml. EM I PRRGE VY I CHVEHPSI.KSP I T VEWRAOSE S A W5I' MLEM1P0RGUVY1CHVEIIPSLOSPI IVEWRAOSESAOSI MLSGIGGLVI GV I F LGLGLF I RI IKROKGPOGPPPAGl LO Ml.SGI GGCVI.GV 1 F I.GI.GI.F I //I IRSOKGPRGPPPAGI 1.0 Ml.SGI GGF VLGL I FLGLGL I IHIIRSOK Ll.H * * * * * * ** * * * * * * * ** ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 238 * I d e n t i c a l r e s i d u e s between A and E c h a i n s | I d e n t i c a l r e s i d u e s between a l l c h a i n s CP =Connect:i ri() p e p t i d e 0 C y s t e i n e s i n v o l v e d i n d i s u l f i d e bond ^ N-y.lycosy l a t i o n s i t e o The g l domains a l l show c l ose to 60% homology i n terms of sequence i d e n t i t y between r a t and human and c l o se to 70% homology between ra t and mouse i n both the Eg chains and the Ag cha i n s . The g l domain i s cons idered t o con ta in the an t i g en i c determinant of the C lass I I molecule as s tud ies demonstrat ing exon s h u f f l i n g of Eg domains l o c a l i z e d C lass I I MHC r e s t r i c t i o n of ant igen p resen ta t i on to a T he lper c e l l l i n e to t h i s r e g i o n . The fewer d i f f e r en ce s between rodent than between human and r a t comparisons would r e f l e c t the evo l u t i ona ry d i s tance s ince the two spec ies d i ve rged , which i s seventy m i l l i o n years f o r mammals and ten m i l l i o n f o r rodents (Young, 1950). S p e c i f i c a l l y , the areas of h ighest conservat ion across a l l molecules appear t o be i n the middle of t h i s domain at res idues 40 to 55. Th is area may be i nvo l ved i n forming the back bone s t r u c t u r e of t h i s domain. The areas of l e a s t conse rva t i on w i t h i n each molecule type appears on e i t h e r s i de of t h i s conserved area and a l s o a f t e r res idue 83 to the end of the domain. Th is reg ion may t he r e f o r e be i nvo l ved i n fo rmat ion of the a l l o t y p i c determinant . Mengle-Graw and McDevit t (1983) compared a l l e l e s of the Eg gene, and found the re were fou r major polymorphic r eg i ons , res idues 1 to 13, 22 t o 38, 61 to 74, and 85 t o 93. Th is imp l i e s the a l l e l i c d i f f e r en ce s between RT1 Eg cha ins are r e s t r i c t e d to these areas . The g2 domains show more conservat ion than the g l domains; g rea te r than 80% both between rodents and between rodents and humans. In f a c t , between Ag and Eg molecules t h i s domain showed the h ighest degree of c onse r va t i on , suggest ing a common s t r u c t u r a l f u n c t i o n i s mainta ined i n t h i s domain. The beg inn ing of the g2 domain conta ins the most v a r i a t i o n between the two molecule types wh i l e the re i s s t i l l many conserved res idues w i th the s i n g l e molecule between spe c i e s , suggest ing a f un c t i ona l reg ion s p e c i f i c f o r the type of molecule i n v o l v e d . There are a number of s t r e t ches of f i v e or more consecut ive conserved res idues throughout t h i s domain i n a l l the C lass I I molecules which are probably i nvo l ved i n ma in ta in ing the shape of the mo lecu le . The cy top lasmic t a i l and transmembrane reg ions show the h ighest amount of sequence i d e n t i t y i n equ iva lent chains across the th ree spe c i e s , i n Eg and Ag p r o t e i n s . These reg ions , however, are l e s s conserved between the A and E mo lecu les . The high degree of conservat ion i n these reg ions i s unusual i n o ther g l y cop ro te i n s l i k e the C lass I molecules ( W a l l i s , 1984). Th is suggests a f u r t h e r f u n c t i o n to the transmembrane reg ions bes ides i n t e r a c t i o n w i th the membrane. The transmembrane reg ion cou ld be invo lved i n a and 3 i n t e r a c t i o n , and keeping AaE8 or EaAg hybr ids from fo rm ing . The i d e n t i f i c a t i o n of pRTlEg. l as an Eg chain was determined by the r e l a t i v e l y h igher l e v e l of conservat ion found when pRT lEg . l i s compared to an Eg cDNA as opposed to an Ag gene. Gustafsson (1984) found tha t i n humans, the h ighest l e v e l of conserva t ion w i t h i n the C lass I I g subc lasses occurs i n the areas coding f o r the transmembrane and cy top lasmic r eg i ons , t o the extent t ha t molecules can be c l a s sed as a l l e l e s of DR, DP, o r DQ by the sequence homology w i t h i n these r eg i ons . When compared to the three human C lass I I a l l e l e s , pRT lEg . l shows the h ighest homology o v e r a l l t o DRg (81.4%), s p e c i f i c a l l y i n the transmembrane and cy top lasmic coding reg ions (89%). Th i s i s i n agreement w i th previous s tud ies tha t conf i rm the H-2 E genes to be the equ iva l en t of the human HLA DR genes. The 3' un t r ans l a t ed regions of the Eg cha ins i n r a t and mouse had 87% homology which was even h igher than the l eade r sequence and g l domains. Whi le more v a r i a t i o n would be expected i n the l eader sequence than i n other domains due to d i f f e r e n t process ing s i g na l s needed i n the d i f f e r e n t s pe c i e s , i t would be expected t o be under more c on s t r a i n t s than the 3' un t rans l a ted r e g i o n . The s p e c i f i c amino ac ids h i gh l y conserved across a l l the molecules (eg. a l l the g l y c i n e s ) would be i nvo l ved i n forming the s t r u c t u r e t o embed i n the c e l l membrane as desc r ibed be fo re . The lack of conse rva t i on i n the cy top lasmic t a i l across a l l molecules may demonstrate the s p e c i f i c f un c t i o na l r o l e of the molecules i n i n t r a c e l l u l a r communicat ion. I I . Comparison of Nuc leot ide Changes Wi th in C lass I I 3 Chains « The divergence i n the sequence of C lass II 3 cha ins was c a l c u l a t e d by comparing the number of nuc l eo t i de changes r e s u l t i n g i n an amino a c i d replacement (replacement s u b s t i t u t i o n ) t o the number r e s u l t i n g i n no replacement ( s i l e n t s u b s t i t u t i o n ) . Amino ac id changes g i v i n g r i s e t o replacement s u b s t i t u t i o n s w i l l tend to be acted upon by s e l e c t i o n wh i l e changes caus ing s i l e n t s u b s t i t u t i o n s w i l l be p u t a t i v e l y neu t ra l and can t he r e f o r e be used as a measure of the mutat ion ra te ( P e r l e r , 1980). The d ivergence i n the 3 cha in was c a l c u l a t e d by comparing the A and E 3 cha ins between mice and r a t s and rodents and humans, and f i n a l l y between the two 3 cha ins w i t h i n each of the th ree spec i e s . Par t A of Table 3 shows the percentage of the s i l e n t s u b s t i t u t i o n s and the replacement s ub s t i t u t i o n s i n C lass II 3 genes c a l c u l a t e d as the observed number of s u b s t i t u t i o n s d i v i ded by the po ten t i a l number of s u b s t i t u t i o n s . The percentage co r rec ted d ivergence i s an est imate of the mutat ion ra te co r re c ted f o r m u l t i p l e events ( P e r l e r , 1980). By comparing the changes between rodents and humans to those of r a t s and mice, the t a b l e demonstrates t ha t the s i l e n t s u b s t i t u t i o n s have accumulated dur ing e v o l u t i o n . The l a r g e s t d i f f e r en c e i s seen when the A and E molecules w i t h i n each spec ies are compared, f o r both s i l e n t and replacement s u b s t i t u t i o n s . Th is agrees w i th the theory t ha t the gene d u p l i c a t i o n event g i v i ng r i s e to the E 3 and A 3 genes was before the d ivergence of these mammals. I I I . C ha r a c t e r i z a t i o n Of pRT1.2 The screen ing of 10,000 c o l on i e s from the t o t a l mRNA l i b r a r y us ing r a d i o l a b e l e d DNA fragments of the RTl A 3 gene r e su l t e d i n one p o s i t i v e c l one , pRT1.2. Through the use of r e s t r i c t i o n enzyme d i g e s t i o n , pRT1.2 was determined to con ta in a c loned i n s e r t of about 1100 nuc l eo t i des l ong , and d i d TABLE 3 Sequence Divergence of S i m i l a r Genes Across D i f f e r e n t Spec ies . Species Compared Percentage Corrected Divergence Time of Di vergence S i l e n t S i t e s Replacement S i t e s ( i n M Years) A . C l ass II e Chain Genes RTl Eg: H-2 Eg 18.7 10.6 8 RTl Ag: H-2 Ag 20.6 13.3 8 RTl Eg: HLA-DRg 45.1 15.7 70 RTl Ag: HLA-DQg 47.8 16.6 70 H-2 Eg: HLA-DRg 44.1 12.6 70 H-2 Ag: HLA-DQg 46.5 17.0 70 RTl Eg: RTl Ag 60.5 34.6 110* H-2 Eg: H-2 Ag 65.6 28.2 110* HLA-DRg:HLA-DQg 59.1 26.3 110* Non-MHC Genes P r e p r o i n s u l i n Genes Rat: Rat 32.3 1.8 15* Rat: Human 76.0 5.2 70 Rat: Chicken 64.0 10.7 250 Human:Chicken 122.0 8.0 250 Growth Hormone Genes Rat: Human 71.0 19.4 70 a and g G lob in Genes a B a B Rabbit:Human 31.8 45.5 10.8 5.6 50 Mouse: Rabbi t 81.7 63.8 10.9 12.1 70 Mouse: Human 83.0 49.4 8.4 12.9 70 Mouse: Chicken 87.2 78.8 20.0 26.7 250 Rabb i t :Ch icken 63.9 80.8 22.8 23.3 250 Human: Chicken 74.6 70.1 20.9 22.9 250 a t o g G lob in Gene Mouse 120.0 51.1 500 Rabbit 91.7 48.5 500 Human 89.5 46.3 500 Chicken 87.0 51.0 500 * Time of d ivergence ex t rapo l a ted from s i l e n t and replacement s i t e s . Corrected d ivergence of non-MHC genes c a l c u l a t e d by P e r l e r (1980). not d i s p l a y any s i m i l a r r e s t r i c t i o n enzyme s i t e s to pRT lEg . l (F igure 7 ) . The i n i t i a l sequencing s t ra tegy i s shown below the i n s e r t , demonstrat ing tha t most of the sequence was determined, but not a l l the fragments were ove r l aped . The known r e s t r i c t i o n s i t e s found w i t h i n the sequenced segments were used to o r i e n t a t e the sequence w i th respect to the i n s e r t w i t h i n the p l a sm id . F igure 8 shows the sequence of the 3 separate f ragments. At the 3' end, a twenty base po ly(A) s t r i n g preceded the C t a i l s , and a pu t a t i v e po ly(A) adeny la t i on s i gna l (AATAAA) was found twenty nuc l eo t i des up stream from t h i s . The known regions were a l i gned t o a number of C lass II a and g cDNA and genomic sequences using computer a na l y s i s and no s i g n i f i c a n t l i neups cou ld be made. The sequences of pRT lEg. l were compared to the EMBL data bank (Cameron, 1983) and no s i g n i f i c a n t sequence homology was found. pRT lEg . l was not s tud ied f u r t h e r as there was no evidence of a comparable sequence i n any of the genes of the immunoglobulin f a m i l y , o r any o ther genes. FIGURE 7 Sequencing Strategy of pRTl.2 cDNA Insert Hi i T T H _ l _ H _1_ CcvJTlO I COUTIC- 3 I Cm • .'CO b fracments H = Hae I I I Hi = Hind I I I B = Bam HI P = Pst I T = Tac I FIGURE 8 DNA Sequence cf the Three Fragments of p R T 1 . 2 P u t a t i v e poly(A) adenylation s i t e i s b o x e d , r e s t r i c t i o n s i t e s marked. C O N T I G 1: 1 0 2 0 3 0 4 0 5 0 G C C T T T A A C A A A A T A A A A 7 T T C T G G A T G A A A C T T C T C C C T A G T A C T C T G C G O 7 0 8 0 9 0 ' 1 0 0 T A A C T A T A T C A A T A C A A T A A A A C A T A A T A A T G C G G G G T T A G G G A T T T A G C T o o l 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 T C A G T G G T A G A G C G C T T G C C T A G G A A G C G C A A G G C C C T G 3 G T T C G G T C C C 1 S 0 1 7 0 1 8 0 1 9 0 2 C 0 C A G C T C C A A A A A A A A G A A C C A A A A A A A A A A T A A T A T A T A T A T A T A T A A T A * 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 A T G C A A T G C A T A T T A A T A T A T A T G A T T A A C A G T T C A G T T T A T T A G G G G C T 2 = 0 2 7 0 2 8 0 2 S 0 3 C 0 T T A A T A A A G T A A A A C C T T A C A G T G A A A A A T A A C A T A A G C T T T T A T G A T T T 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 C C G T T C G A G C A G G A C A G A C A T G A G T G A G G A G C C C T A G T A A C A G T C A T A C G 3 5 0 3 7 0 T T G C G T G A G T A C G G G G G G ' 5 ' end C C N T I G 2 : 1 0 2 0 3 0 4 0 5 0 G A G G A T C C C C A G G C T A A C C A G A G A A A C C T G T C T C A A A C A A C A A A A C A A C A S O 7 0 8 0 9 0 1 0 0 A A C C A A C A A A C C A C C T C A G 7 A A T T T A T A C A G A A A T T T C C A C C C C A C C C T T 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 C T A G G C T T A C C C G A C T T T C C C A G G G A A G A C A T A C A C T G T T C T T C T G T T G A 1 S 0 1 7 0 1 8 0 1 9 0 2 C 0 C A T C A C T T G G G G T A C C A G A G A C G G A C G G A A G A C T C C A C C T A A G G C T A G C T 2 1 0 2 2 0 2 3 C 2 4 0 2 5 0 T A G T A A A C C A G G G A G T T T A C T G G G A T G G C C T C C A G G A G C G C T G C A A C A A C 2 S 0 2 7 0 2 3 0 2 9 0 3 0 0 A G C G G T A A G C T C A C A A A A G T C C A A T C T G C G T C T T T G G C G C T C C T G T T G G A 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 T T A A C A G A A G C T C C T C C A G A A A G C G C T T A A T G T C T G G A A C C T T G A G G A G G 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 G A C T T T G G G A T G G A G T T T C A T C A G T T T C C T G A G T G C C C C C T A A G A A T A G A TGGC CONTIG 3: 10 20 30 40 50 CC7TGAACTC AGAGATCCAC CCGTCTCTGC CTCCCAGGTG CTGGGTTCAA 60 70 80 90 100 AGGTTTGCAC TATCACCACC TGGCAAGTTA TTCATTCTTA ATTTTTACAA 110 120 130 140 150 GTACCCCTGA CACCTATCCT AGACACTGCT GGATCCTCJA A TAAAlGTCTTT 6a.r-.rJl. 160 170 180 190 ACTAAATTTA AAAAAAAAAA AAAAAACCCC CCCCCCCCCC CCC 3 ' end GENERAL DISCUSSION I. E vo l u t i on The DNA sequence of the cDNA encoding the ra t RTl C l ass II Eg gene determined i n t h i s study shows that the gene d i s p l a y s high sequence i d e n t i t y t o the equ iva len t molecules i n the mouse, H-2 Eg, and human, HLA DRg. The amino ac id sequence of pRT lEg. l shows tha t the RTl Eg molecule i s a member of the immunoglobulin super f a m i l y . Comparison of the amino ac i d sequence of PRT lEg . l and a v a r i e t y of immunoglobulin l i k e molecules has lead t o the de te rminat ion of a number of conserved res idues found throughout t h i s gene f a m i l y . By comparing g2mic rog lobu l i n , the C lass I a3 domain, the constant C3 reg ion of immunoglobul ins, the C lass II a2 and g2 domains and the Thy-1 mo lecu le , n ine amino ac ids of the membrane proximal e x t r a c e l l u l a r reg ions were determined to be i n v a r i a n t (T ravers , 1984). These reg ions desc r ibed on pRT lEg . l ( f i g . 5) extends the observat ions and strengthens the sugget ion t ha t they are i n v a r i a n t f o r s t r u c t u r a l reasons. S i m i l a r l y , when the molecules coded f o r w i t h i n the MHC are l i n e d up, f ou r add i t i ona l i n v a r i a n t res idues become apparent (T ravers , 1984), and these are a l so found i n pRT l Eg . l . These molecules a long w i th the T c e l l r ecep to r , T8 ant igen and other T c e l l markers a l l show cons ide rab le s t r u c t u r a l homology, w i th s i m i l a r l y p laced i n t e r n a l d i s u l f i d e bonds. I t has been p red i c t ed they form a s i m i l a r set of a h e l i c a l s t r e t che s t ha t form faces capable of i n t e r a c t i n g w i t h pa i red molecules of o ther membrane bound molecu les . The f i r s t ex te rna l domains ( a l ) of C lass I heavy cha ins and the g l domai of C lass I I g cha ins share a h igh ly conserved pept ide sequence of s i x amino ac ids (Ma l i s sen , 1983), of which f i v e are found i n p R T l E g . l . Lee (1982) found the DRa cha in more homologous t o C lass I (31%) than DRg (30%) was but DRg was c l o s e r to g2 m i c r og l obu l i n (32%) than DRa (29%). The exon- in t ron o r gan i z a t i ona l s t r u c t u r e of the genes encoding these chains ( f i g u r e 9) a l s o IGURE 9 Proposed E v o l u t i o n a r y Tree of Immunoglobulin Family from T y p i c a l Predecessor C l a ^ X 4 KI • W J I VX. ai .*i Tff ck 1 „ I C U a J i t o r M • _ : Q-= 3 ' U n t r a n s l a t e d <2y » C^-fejpUsi^'iC Ta'iJ Shown below are the homologous domains i n the structure and t y p i c a l gene orqanization of the d i f f e r e n t proteins, (from Hood,1985 and Malissen,1983. suggests a c l o s e r evo lu t i ona ry r e l a t i o n s h i p of C lass II 3 cha ins to C lass I a u (heavy) cha ins and C lass II a chains to 02 m i c r og l obu l i n (Hood, 1 9 3 3 ) . The s i m i l a r i t i e s between the C lass II a and 32 m i c r og l obu l i n genes and the C lass II 0 and C lass I genes suggests e i t h e r a pa t te rn of d ivergence desc r ibed i n f i g u r e 9 , or convergence of the chains due to s t r u c t u r a l c o n s t r a i n t s . The high degree of s i m i l a r i t y i n exon- in t ron gene s t r u c t u r e and DNA sequence between a number of immunog lobu l in- l i ke molecules has l ed to the proposal t ha t a s i n g l e p rogen i to r gene gave r i s e to t h i s supergene f am i l y (Hood, 1 9 8 5 ) . Th is p r imord ia l gene would con ta in a l eader sequence, an immunoglobul in domain u n i t , and a transmembrane reg i on , and would code f o r a c e l l su r f a ce p r o t e i n w i th the a b i l i t y to i n t e r a c t w i t h o ther s i m i l a r un i t s as a d imer. A f t e r p a r t i a l d u p l i c a t i o n i n t o v a r i a b l e and constant reg ions s i m i l a r to those found i n present day immunoglobulin r eg i ons , d ivergence of a second d u p l i c a t i o n of the constant reg ion would lead to the f am i l y of genes w i t h i n the major h i s t o c o m p a t i b i l i t y complex ( f i g u r e 9 ) . The imp l i ed sepa ra t i on of the d i f f e r e n t c l a s ses of MHC products by d u p l i c a t i o n must have occurred up to 5 0 0 m i l l i o n years ago and we l l before d ivergence of the mammals (Lee, 1 9 8 2 ) . I t has been suggested tha t t h i s immunoglobulin repeat ing u n i t could have dup l i c a t ed tw i ce to form a C lass I I type c ha i n . As the d i s t a l domain d iverged f u r t h e r w i th fewer s t r u c t u r a l c on s t r a i n t s put on i t , i t became dup l i c a t ed again i n one gene (poss i b l y by an uneven c rossover event) and was ab le to i n t e r a c t w i th a s i n g l e immunog lobu l in - l i ke un i t mainta ined elsewhere i n the genome, c r e a t i n g the i n i t i a l C lass I molecule (Kaufman, 1 9 8 4 ) . As the number of dup l i c a t ed genes i n c reased , the re were fewer r e s t r i c t i o n s r e s t r a i n i n g the d ivergence of the c e l l su r face markers, and recombinat ion would be al lowed to occu r . The h igher sequence i d e n t i t y o f the r a t C lass I I E3 gene t o equ iva len t E$ gene sequence i n mice and humans than t o the r a t C lass I I A3 gene suggests tha t the C lass II reg ion was formed by a s e r i e s of s e r i a l gene d u p l i c a t i o n s p r i o r . t o mammalian s p e c i a t i o n (80 m i l l i o n years ago). The Evo lu t i ona ry Clock Hypothes is (Zuckerkard l et a l , 1965) s t a tes tha t the accumulat ion of s u b s t i t u t i o n s may be used as a measure of the d ivergence . Using the method of P e r l e r (1980), e x t r a po l a t i o n from the p l o t of s i l e n t s u b s t i t u t i o n s i n f i g u r e 10 suggests d ivergence of the C lass II Ag and Eg genes was approx imate ly 25 t o 30 m i l l i o n years before the d ivergence of mammals, i e . 100 t o 110 m i l l i o n years ago. Th is may not be an accurate est imate as the accumulat ion of s i l e n t s u b s t i t u t i o n s i s not l i n e a r w i t h t ime . When the percentage co r rec ted d ivergence of the A and E g cha ins i n r a t s , mice and humans i s p l o t t ed aga ins t evo lu t i ona ry t ime, i t can be seen more c l e a r l y tha t the accumulat ion of s u b s t i t u t i o n s i s not l i n e a r w i th t ime and t ha t replacement s u b s t i t u t i o n s reach a p lateau a f t e r ten m i l l i o n years ( f i g u r e 10) . This n o n l i n e a r i t y cou ld be due to s e l e c t i o n a c t i ng t o ma in ta in f u n c t i o n , such tha t on ly a l i m i t e d number of replacements are a l lowed to occur and are f u l f i l l e d r ap i d l y dur ing e v o l u t i o n . Sec t i on B of Table 3 shows the number of changes i n some non-MHC genes when r a t genes are compared to equ iva len t genes i n the human, r a b b i t , mouse and ch i c ken . P e r l e r et al (1980) examined s i l e n t and replacement s u b s t i t u t i o n s i n p r e p r o i n s u l i n genes and found tha t wh i l e the unse lec ted reg ions ( i e i n t rons and DNA surrounding the gene) showed h igh accumulat ion of replacement and s i l e n t changes, the reg ions of the gene under s e l e c t i o n (exons) showed f i v e t imes s lower d ivergence. The accumulat ion of s i l e n t s i t e s , however, appeared t o sa tu ra te at about 85 M y ea r s , wh i l e changes i n replacement s i t e s cont inued t o be l i n e a r w i th t ime u n t i l 250 m i l l i o n years ( P e r l e r , 1980). I t was concluded from the study of replacement and s i l e n t changes in non-MHC genes tha t s i l e n t s i t e s may not prov ide a good e s t ima t i on of d ivergence extending over a long t ime s c a l e ( P e r l e r , 1980). FIGURE 10 Divergence of Replacement and S i l e n t Changes i n Class II Genes as a Function of Time 6CH MILLION YEARS 4 S i l e n t Substitutions 0, Replacement Substitutions In order to est imate the d ivergence of MHC C lass II genes, the comparison of MHC C lass II genes from spec ies more d i s t a n t l y r e l a t e d t o mammals w i l l be needed to examine the r e l i a b i l i t y of us ing the replacement changes f o r an e s t ima t e . For example, the ch icken MHC encodes two se t s of C lass II products (Crone, 1981) tha t i f homologous to C lass II A and E molecules of mammals, would p lace the gene d u p l i c a t i o n event p r i o r to 250 m i l l i o n years ago. The gene d u p l i c a t i o n event r e s u l t i n g i n the C lass I I reg ion cou ld i n vo l ve more than the s i n g l e gene and ins tead be a repeat ing u n i t con ta i n i ng an a and g gene w i th pos s i b l y a second g gene. In the rodents , both the C lass I I A and E subregions con ta in a g l , g2, and a gene, i n tha t order from the centromere, w i t h the coding s t rand of the a gene s i t u a t e d 3 1 to 5' and both g genes s i t u a t e d 5' to 3' i n mice, ( t h i s i s not known i n the r a t ) , so the two d i f f e r e n t genes are 5' to 3' fo l l owed by 3' to 5' ( t a i l to t a i l ) (Ste inmetz , 1982). In humans, the HLA l o c i appear to be more complex, and a l l the genes are not complete ly l o c a l i z e d but here the repeat ing u n i t appears to be predominant ly an a and a g gene on l y . The repeat ing u n i t s i n humans appear t o be arranged w i th the g gene 3' to 5' f o l l owed by the a gene 5' to 3' (head to head) moving from the centromere. This may have a f unc t i ona l exp lana t i on where the occurrence of adjacent i n i t i a t i o n s i t e s a l l ows r e gu l a t i o n of s i m i l a r amounts of p ro te i n chains f o r a s s o c i a t i o n (Go r s k i , 1984), but s ince the oppos i te o r i e n t a t i o n i s found i n rodents i t may not app ly . However, the arrangement tha t i s p a r a l l e l between these spec i e s , the oppos i te o r i e n t a t i o n of the a and g genes, suggests t h i s may p lay a r o l e i n assur ing the a genes can not recombine w i th the g genes and i n s t e a d , keep them as a u n i t . I t i s not known whether more than one g cha in i s t r a n s c r i b ed t o combine w i t h the o cha ins i n each subreg ion. In many of the subregions the ex t r a g cha ins have been found to be pseudogenes. In humans there has been a s s o c i a t i o n of d i f f e r e n t g cha ins to the same a cha in desc r i bed (Shacke l f o rd , 1982) and not a l l of the c e l l s express the same DR molecule in one i n d i v i d u a l . These observat ions suggest there i s add i t i ona l f l e x i b i l i t y of the system at the l e ve l of c i s or t r a n s - l i n k e d p ro t e i n a s s o c i a t i o n w i th the a cha in gene product (Roux-Dosseto, 1983) to generate f u r t h e r polymorphism. I I . S i g n i f i c a n c e of the Polymorphism Since the ex tens ive polymorphism demonstrated here has been mainta ined through e v o l u t i o n , the re must be an important reason f o r i t s e x i s t en ce . A s s o c i a t i v e r e cogn i t i on has been suggested as necessary f o r a l i n kage between r e c ogn i t i o n and the e f f e c t o r c e l l f u n c t i o n through regu la to ry c e l l s ( K l e i n , 1979). Given t h i s need f o r s e l f r egu la to ry mo lecu les , i t can be seen how a l a r ge v a r i e t y of MHC molecules would be needed to i n t e r a c t w i th f o r e i gn an t i gens , and tha t can be recognized by T c e l l r e cep to r s , whatever the mechanism. The v a r i a b i l i t y o f response l i n k ed to the MHC mo lecu les , i s due t o the range of the T c e l l r ecep to r r e p e r t o i r e (B l i nd Spot Hypo thes i s ) , and exp l a i n s the need f o r inc reased v a r i a b i l i t y by he te rozygos i t y at most MHC l o c i , where gene t rans-complementat ion may a l so occur (Charron, 1984). Th is non-response to c e r t a i n an t i g en i c determinants arose as a f un c t i on of t o l e r an ce , necessary to avo id autoaggress ion and t h i s was balanced w i th the need to cover as many of the a l t e r n a t i v e determinants to be found on present and f u tu re f o r e i gn pathogens. The presence of more than one C lass I and C lass II molecule may a l so be an e v o l u t i o n a r i l y de r i ved back up system f o r ma in ta in ing prev ious and t e s t i n g out new an t i gen i c determinant s i t e s . In humans, the DQ molecules are found on d i f f e r e n t c e l l s and at a d i f f e r e n t stage i n ontogeny than DR suggest ing the molecules have pos s i b l y been s p e c i a l i z e d f o r d i f f e r e n t f unc t i ons ( N a t a l i , 1984). S ince the cytop lasmic t a i l s o f membrane i n se r t ed p ro t e i n s have been proposed t o mediate the e f f e c t s of ex te rna l s t i m u l i to the c e l l i n t e r i o r (Larhammar, 1983) and s ince the cy top lasmic t a i l of Eg i s so d i f f e r e n t from t ha t on Ag mo lecu les , the i n t r a c e l l u l a r f un c t i on i s probably qu i t e d i f f e r e n t . The ex i s tence of polymorphism w i t h i n the MHC a l so f unc t i on s at the popu la t i on l e v e l of a spec ies to ensure each i n d i v i d u a l has a d i f f e r e n t immune response system. I f a new ant igen i s acqu i red by pathogens, the re i s a g rea te r chance there w i l l be some i n d i v i d u a l s capable of mounting a response. Th is means there w i l l be a lower chance of one o r i g i n a l harmful s t r a i n of v i r u s o r b a c t e r i a caus ing an epidemic w i t h i n the popu l a t i on . With the inc reased mutat ional ra te of change of pathogens over tha t of v e r t eb r a t e s , there has been a s e l e c t i v e f o r ce t o keep the popu la t i on as d i v e r s e as p o s s i b l e at t h i s complex. I I I . Future Research As more DNA sequence data i s obta ined on a l l the MHC systems s t ud i ed , the evo l u t i ona ry p i c t u r e w i l l f u r t h e r u n f o l d . I t i s important to i n c l ude another spec ies i n the i n t e r s p e c i f i c s tud i e s as i n one study comparing DNA and p r o t e i n sequences, nuc l eo t i de s u b s t i t u t i o n s were found to accumulate more r a p i d l y i n rodents than other mammals, which may be due to a sho r t e r generat ion t ime (Wu et a l , 1985). Another mammal, f o r example a pr imate , would be i n t e r e s t i n g t o compare to look f o r inc reased complex i ty over the r a t , and to see i f the conserva t ion and s u b s t i t u t i o n s were mainta ined w i t h i n the same s lope i n evo lu t i ona ry t ime . The search f o r s i m i l a r immunog lobu l in - l i ke genes i n animals f u r t h e r d iverged from mammals, l i k e b i r d s o r even r e p t i l e s , may e l u c i d a t e a gene s i m i l a r to the suggested p rogen i t o r of t h i s f am i l y of genes. CONCLUSIONS 66 1. Determinat ion of the DNA sequence of a ra t RTl C lass II Eg cDNA5 ( p rev i ous l y unknown), showed the p red i c ted p r o t e i n sequence encoded by the gene to be homologous to the RTl Ag gene (58%), but even more s i m i l a r t o the mouse H-2 Eg gene (82%) and the human HLA DRg gene (73%). 2. The coding sequence of the RTl Eg gene f u r t h e r de f ines the general f ea tu res of C lass II g genes. Comparison of the p r o t e i n sequences of the Eg and Ag equ iva len t molecules i n the r a t , mouse and human d i s p l a y s a number of s p e c i f i c res idues conserved across a l l spec ies which may be i nvo l ved i n s t r u c t u r e and f u n c t i o n . In a d d i t i o n , the second domain of the r a t RTl Eg gene conta ins nine conserved res idues found i n a l l immunog lobu l in - l i ke p r o t e i n s , p l a c i ng t h i s gene i n the immunoglobul in supergene f a m i l y . 3. The number of s i l e n t and replacement nuc l eo t i de s u b s t i t u t i o n s w i t h i n the p r o t e i n equence were determined between r a t , mice and human Ag and Eg equ i va l en t genes and p l o t t e d aga ins t the t ime s i n ce the two spec ies d i v e rged . The s i l e n t s u b s t i t u t i o n s were found to i n c rease l i n e a r l y w i th t ime of d ivergence wh i l e replacement s u b s t i t u t i o n s d i d not i n c rease a f t e r ten m i l l i o n y e a r s . Th is suggests t ha t once the replacement s u b s t i t u t i o n s were sa tu ra ted w i t h i n the mo lecu le , f u n c t i o na l r e s t r a i n t s acted t o s e l e c t aga ins t an inc rease i n the number of replacement s u b s t i t u t i o n s . An a l t e r n a t i v e exp l ana t i on would be tha t rodents evo lve f a s t e r than other spec ies due t o a sho r t e r generat ion t ime thereby showing more d ivergence f o r the number of years s ince s p e c i a t i o n . 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