Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The copper-zinc superoxide dismutase gene from Drosophila melanogaster : attempts to clone the gene using… Seto, Nina Oi Ling 1987

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1987_A6_7 S47.pdf [ 4.06MB ]
Metadata
JSON: 831-1.0097048.json
JSON-LD: 831-1.0097048-ld.json
RDF/XML (Pretty): 831-1.0097048-rdf.xml
RDF/JSON: 831-1.0097048-rdf.json
Turtle: 831-1.0097048-turtle.txt
N-Triples: 831-1.0097048-rdf-ntriples.txt
Original Record: 831-1.0097048-source.json
Full Text
831-1.0097048-fulltext.txt
Citation
831-1.0097048.ris

Full Text

The Copper-Zinc Superoxide Dismutase Gene from D r o s o p h i l a melanogaster; Attempts to c l o n e the gene using two mixed sequence oligonucleotide probes by Nina O.L. Seto B. Sc.(Hon) U n i v e r s i t y of Toronto, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Biochemistry We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1987 © Nina O.L. Seto, 1987. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biochemistry The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 18. 1987.  DE-6G/81) i i ABSTRACT Superoxide dismutase i s an enzyme which scavenges superoxide r a d i c a l s and i s thought to be a l o n g e v i t y determinant, as there e x i s t s a p o s i t i v e c o r r e l a t i o n between s u p e r o x i d e dismutase concentration and maximum l i f e span p o t e n t i a l . The c y t o s o l i c CuZn superoxide dismutase i n melanogaster has been p u r i f i e d and sequenced, but the gene has not been cloned. However, when i t i s a v a i l a b l e t h e CuZn SOD gene may be r e i n t r o d u c e d i n t o the D r o s o p h i l a genome v i a the P-element t r a n s f o r m a t i o n system so i t s e f f e c t s on the l i f e s p an p o t e n t i a l of D r o s o p h i l a may be stu d i e d . T h i s study d e s c r i b e s attempts to c l o n e the CuZn SOD g e n e f r o m D^ m e l a n o g a s t e r u s i n g two m i x e d s e q u e n c e oligonucleotide probes. The SI probe corresponds to amino acids 43-48 o f t h e p r o t e i n s e q u e n c e and c o n t a i n s 128 d i f f e r e n t o l i g o n u c l e o t i d e s e q u e n c e s r e p r e s e n t i n g a l l p o s s i b l e codon combinations p r e d i c t e d from the amino a c i d sequence. The GT3 probe i s t a r g e t e d to amino a c i d s 90-95 of the p r o t e i n . In t h i s probe, deoxyguanosine was p l a c e d i n p o s i t i o n s where a l l four n u c l e o t i d e s may occur to d e c r e a s e probe h e t e r o g e n e i t y . The probes were used to s c r e e n D_^  m e l a n o g a s t e r Canton-S and Oregon-R genomic lambda l i b r a r i e s . T h r e e p o s i t i v e c l o n e s i s o l a t e d from the Canton-S l i b r a r y had i d e n t i c a l n u c l e o t i d e sequence i n the GT3 probe b i n d i n g r e g i o n , and sequencing of the probe binding s i t e r evealed t h a t one member of the GT3 probe had formed a 15 bp duplex w i t h t h e phage DNA. S c r e e n i n g o f the i i i Oregon-R l i b r a r y produced four c l o n e s which hybridized with both GT3 and SI probes. When t h e s e phage DNA were h y b r i d i z e d to polytene chromosomes by ^in s i t u h y b r i d i z a t i o n , none mapped to 68AB on the t h i r d chromosome, the l o c a t i o n of the CuZn SOD gene. These r e s u l t s suggest that m o d i f i c a t i o n of the c l a s s i c a l strategy used i n t h i s study i s necessary, and implications on probe design are discussed. i y TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i v LIST OF FIGURES . v i ABBREVIATIONS v i i ACKNOWLEDGEMENTS i X INTRODUCTION 1 MATERIALS AND METHODS 12 Materials 12 B a c t e r i a l s t r a i n s 12 D. melanogaster genomic DNA p r e p a r a t i o n 13 Radioactive l a b e l l i n g of oligonucleotides.... 14 Screening genomic l i b r a r i e s by plaque h y b r i d i z a t i o n 15 I. Primary screen 15 I I . Secondary screen 16 Bacteriophage lambda DNA preparation 17 I. Large scale preparation 17 I I . Lambda phage DNA i s o l a t i o n from primary screen 18 I I I . Lambda phage DNA i s o l a t i o n from secondary screen 18 Agarose gel electrophoresis and Southern h y b r i d i z a t i o n 18 I. R e s t r i c t i o n enzyme digests 18 I I . Agarose g e l electrophoresis 19 I I I . Southern transfer and h y b r i d i z a t i o n 19 In s i t u h y b r i d i z a t i o n to p o l y t e n e chromosomes 20 I. L a b e l l i n g the probe 20 V I I . In s i t u h y b r i d i z a t i o n 21 Subcloning phage DNA into plasmids 21 I. L i g a t i o n reaction 21 I I . Plasmid and M13 transformations 21 I I I . Growth of transformants 22 DNA sequence determination 23 I. Template preparation 23 I I . DNA sequencing 23 RESULTS 26 The stringency of h y b r i d i z a t i o n 26 Primary screen of Canton-S lambda l i b r a r y 29 Secondary screen of Canton-S lambda l i b r a r y 29 DNA sequence analysis of GT3 binding s i t e (CS-1) 29 Primary screen of Oregon-R lambda l i b r a r y 37 Analysis of Oregon-R primary screen phages 37 Secondary screen of Oregon-R lambda l i b r a r y 38 Mapping of phage DNA to polytene chromosomes.. 43 The GT3 probe binding s i t e of phage 109 43 DISCUSSION 50 I. Oligonucleotide probe heterogeneity 50 I I . Oligonucleotide probe sequence 51 I I I . Oligonucleotide probe length 53 IV. Oligonucleotide probe design 54 REFERENCES 57 v i LIST OF FIGURES PAGE FIGURE 1. The amino a c i d sequence o f Cu/Zn Superoxide dismutase f o r melanogaster 5 FIGURE 2. The mixed sequence o l i g o n u c l e o t i d e probes SI and GT3 8 FIGURE 3. Oregon-R genomic and lambda l i b r a r y DNA Southern analysis 27 FIGURE 4. Southern a n a l y s i s of DNA from GT3 p o s i t i v e phage from the Canton-S genomic lambda l i b r a r y . . . 30 FIGURE 5. DNA sequencing s t r a t e g y o f the GT3 binding s i t e of the Canton-S l i b r a r y GT3 p o s i t i v e phage... 33 FIGURE 6. The nucleotide sequence and t r a n s l a t i o n of the GT3 binding s i t e of phage CS-1, 2, and 3 from the Canton-S l i b r a r y 35 FIGURE 7. Southern a n a l y s i s o f Oregon-R primary screen phages 39 FIGURE 8. Southern a n a l y s i s of phage from the Oregon-R l i b r a r y that hybridizes both GT3 and SI probes... 41 FIGURE 9. Determination of the chromosomal lo c a t i o n of phage 109 and phage CS-2 by iri s i t u h y b r i d i z a t i o n to polytene chromosomes. 44 FIGURE 10. Nucleotide sequence and t r a n s l a t i o n of the GT3 binding s i t e of phage 109 from the Oregon-R l i b r a r y 48 v i i LIST OF ABBREVIATIONS ATP adenosine-5' triphosphate bp base pair(s) BSA bovine serum albumin cpm counts per minute d deoxy dd dideoxy dNTP deoxynucleoside-5' triphosphate dATP deoxyadenosine-5 1 triphosphate dCTP deoxycytidine-5 1 triphosphate dGTP deoxyguanosine-5' triphosphate dTTP thymidine-5' triphosphate DNA deoxyribonucleic acid DNase deoxyribonuclease DTT d i t h i o t h r e i t o l EDTA ethylenediaminetetraacetate EtBr ethidium bromide IPTG iso p r o p y l - 8-D-thiogalactopyranoside kb kilobase pair(s) kDa kilodalton(s) LB L u r i a broth LBMgT LB media wit h 5 ug/ml thymidine, 10 mM MgSO^ mA milliamperes mM mil l i m o l a r v i i i PEG polyethylene g l y c o l PFU plaque forming u n i t s RF r e p l i c a t i v e form RNA ri b o n u c l e i c a c i d RNase ribonuclease SDS sodium dodecyl s u l f a t e ss s i n g l e stranded Southern the Southern transfer procedure:the transfer of DNA from an agarose g e l to a membrane TEMED N,N,N*,N'-tetramethylethylenediamine T r i s tris(hydroxymethyl)aminomethane U unit(s) uCi microcurie ug micrograms uM micromolar uv u l t r a v i o l e t V v o l t s Xgal 5-bromo-4-chloro-3-indolyl- $-D-galactopyranoside ix ACKNOWLEDGEMENTS I wish to thank my supervisor Dr. Gordon Tener for encouragement, support and guidance. I am indebted to Dr. S h i z u Hayashi for countless hours o f d i s c u s s i o n , h e l p , and guidance. I thank Dr. Hayashi f o r a l l her c o n t r i b u t i o n s t o t h i s work and f o r her inestimable p a t i e n c e . I thank J e f f Leung and C r a i g Newton for sharing t h e i r knowledge and ide a s w i t h me. Last but not l e a s t , I thank Dr. Ian Gillam for h i s discourses - so I may be "educated". 1 INTRODUCTION Superoxide d i s m u t a s e s a r e enzymes w h i c h c a t a l y t i c a l l y scavenge superoxide r a d i c a l s (0 2~) • They are thought to be an e s s e n t i a l component o f the b i o l o g i c a l defense against oxidative damage mediated by sup e r o x i d e r a d i c a l s , which are produced as a by-product of oxygen metabolism (1-4). Superoxide dismutases (SOD) are m e t a l l o p r o t e i n s with e i t h e r copper and z i n c , manganese, or i r o n as l i g a n d s . P r o k a r y o t e s p o s s e s s t h e two c l o s e l y r e l a t e d FeSOD and MnSOD, whereas e u k a r y o t e s have a t e t r a m e r i c MnSOD (MW 80-90kDa) i n t h e mit o c h o n d r i a l m a t r i x and an inde p e n d e n t l y e v o l v e d d i m e r i c (MW 31-33kDa) CuZn SOD in the cyt o s o l (5,6). Superoxide d i s m u t a s e c a t a l y z e s t h e d i s m u t a t i o n o f two superoxide r a d i c a l s into molecular oxygen and hydrogen peroxide: 2 0 2 ~ + 2 H + > 0 2 + H 2 0 2 Catalase and/or peroxidase removes hydrogen peroxide by changing i t to water: 2 H 2 ° 2 > 2 H 2 ° + ° 2 Any e x i s t i n g superoxide and hydrogen peroxide form the ve r y r e a c t i v e h y d r o x y l r a d i c a l («0H) v i a the i r o n - c a t a l y z e d Haber-Weiss reaction: ° 2 ~ + H 2 ° 2 > * 0 H + ° 2 + 0 H ~ The h y d r o x y l r a d i c a l may i n i t i a t e f r e e r a d i c a l r e a c t i o n s which r e s u l t i n l i p i d , p r o t e i n and DNA damage (7). Despite the 2 unique nature of the s u p e r o x i d e s u b s t r a t e , a m u l t i p l i c i t y o f assays for measurement o f s u p e r o x i d e dismutase a c t i v i t y has been devised (8-13). The b i o l o g i c a l f u n c t i o n o f sup e r o x i d e dismutase i s as an antioxidant and i t s r o l e as a p o s s i b l e l o n g e v i t y determinant has been proposed (14,15). Non-genetic t h e o r i e s o f aging a t t r i b u t e damage of s t r u c t u r a l and c e l l u l a r components of the c e l l to d a i l y "wear and t e a r " . The f r e e r a d i c a l t h e o r y o f aging p o s t u l a t e s that free r a d i c a l damage increases with age and contributes to the b i o l o g i c a l changes observed w i t h aging (16-19) . Free r a d i c a l s produced as tr a n s i e n t intermediates i n normal c e l l u l a r metabolism may attack otherwise s t a b l e m o l e c u l e s and thus contribute to the observed c e l l u l a r damage. I t has been proposed t h a t the aging process begins with d y s d i f f e r e n t i a t i o n or the l o s s of the proper d i f f e r e n t i a t e d s t a t e o f t h e c e l l s . The p r o c e s s o f aging represents the response o f the organism to these changes. There i s evidence that a n t i o x i d a n t s such as SOD protect against oxygen free r a d i c a l damage which can c o n t r i b u t e to d y s d i f f e r e n t i a t i o n and aging (14,15). There e x i s t s a r e l a t i o n s h i p between metabolic rate and aging i n both p o i k i l o t h e r m s and homeotherms. Mammals have s t a b l e metabolic rates and s p e c i e s - s p e c i f i c l i f e spans. L i f e spans of p o i k i l o t h e r m s a r e g e n e r a l l y v a r i a b l e and d e t e r m i n e d by environmental c o n d i t i o n s . F a c t o r s which reduce metabolic rate extend the l i f e span o f p o i k i l o t h e r m s . S p e c i e s w i t h h i g h e r 3 metabolic r a t e s (and i n c r e a s e d oxygen u t i l i z a t i o n ) have higher i n t r a c e l l u l a r c o n c e n t r a t i o n s o f s u p e r o x i d e r a d i c a l s . A small f r a c t i o n of superoxide r a d i c a l s escape quenching despite elaborate enzymatic and non-enzymatic defenses a g a i n s t them. Therefore, antioxidants may be important i n determining l i f e s p a n . In an attempt to determine i f a g e - r e l a t e d changes occur in antioxidant e f f i c i e n c y , s u p e r o x i d e dismutase concentration as a f u n c t i o n of age has been d e t e r m i n e d f o r v a r i o u s s p e c i e s and r e s u l t s have been v a r i e d ( 2 0 - 2 6 ) . In D. m e l a n o g a s t e r m i t o c h o n d r i a l MnSOD d e c l i n e d 21% between 5 and 58 days of age whereas c y t o s o l i c CuZn SOD remained r e l a t i v e l y constant (27-29). However, a p o s i t i v e c o r r e l a t i o n e x i s t s between the t i s s u e concentration of SOD per s p e c i f i c m e t a b o l i c r a t e (SMR) and l i f e span p o t e n t i a l (LSP) f o r the 12 primate and 2 rodent s p e c i e s s t u d i e d (30,31). T h e r e f o r e , s i n c e SOD/SMR=k(LSP), LSP i s d i r e c t l y related to the l e v e l of SOD i n the t i s s u e . The r a t i o of SOD per amount of oxygen consumed i s c o n s t a n t for each species, and longer l i v e d species must have a higher concentration of SOD. T h e r e a r e many a d v a n t a g e s t o u s i n g D r o s o p h i l a as an e x p e r i m e n t a l a n i m a l i n a g i n g s t u d i e s . D r o s o p h i l a grow vigorously i n the l a b o r a t o r y , are " o l d " at 40 days, and more i s known about i t s genetics than any other higher eukaryote. E l u c i d a t i n g the r o l e of SOD i n aging was the impetus behind cloning the CuZn and Mn SOD genes from D^ _ melanogaster. The 4 cloned SOD genes may be r e i n t r o d u c e d i n t o the Drosophila genome v i a the P-element t r a n s f o r m a t i o n system (32-34). P-transposable DNA i n j e c t e d into the e a r l y embryo i n t e g r a t e s into the genome of germ l i n e c e l l s o f the i n d i v i d u a l . P-element v e c t o r s c a r r y the neomycin resistance gene which a l l o w s s e l e c t i o n of transformants on the a n t i b i o t i c G418 (35). In order to clone the CuZn SOD gene a probe was needed and to synthesize the probe i t was n e c e s s a r y to know the amino acid sequence of the p r o t e i n . The e u k a r y o t i c CuZn SOD p r o t e i n s sequenced are: human, bov i n e , p o r c i n e , horse, yeast, swordfish, and D r o s o p h i l a ( 3 6 - 3 8 ) . C o m p a r i s o n o f t h e s e e u k a r y o t i c sequences shows conserved r e g i o n s . The D r o s o p h i l a CuZn SOD (32 kDa) c o n s t i t u t e s 0.4% o f the t o t a l s o l u b l e p r o t e i n and has a s p e c i f i c a c t i v i t y 1.5 times h i g h e r than any o t h e r p u r i f i e d SOD (39-41). The p r o t e i n c o n s i s t s o f 151 amino a c i d s and i s 57% homologous to the bovine protein (Figure 1). The strategy used to c l o n e CuZn SOD was to use mixtures of s y n t h e t i c o l i g o n u c l e o t i d e s r e p r e s e n t i n g a l l p o s s i b l e codon combinations predicted from a 6 amino acid segment of the protein as a probe to i d e n t i f y the cloned DNA. Complex probe mixtures are undesirable but o f t e n i n e v i t a b l e due to the redundancy of the genetic code. I d e a l l y , the amino a c i d sequence chosen should use the l e a s t number of codons p o s s i b l e . Methionine and tryptophan have unique codons, but are two o f the r a r e s t amino a c i d s i n proteins. A l l other amino a c i d s a r e coded f o r by 2,4 or even 6 5 FIGURE 1. The amino a c i d sequence o f c o p p e r / z i n c s uperoxide dismutase from D. m e l a n o g a s t e r ( 3 6 ) . The enzyme e x i s t s as a dimer, c o n s i s t i n g o f two i d e n t i c a l s u b u n i t s . Each s u b u n i t c o n s i s t s of 151 amino acids and the amino terminus of the pr o t e i n i s acetylated. Two e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e forms of SOD d i f f e r a t p o s i t i o n 96. The SOD ( f a s t ) v a r i a n t (shown here) has Asp-96, whereas the SOD(slow) v a r i a n t has Lys-96. (A=Ala, R=Arg, N=Asn, D=Asp, B=Asx, C=Cys, Q=Gln, E=Glu, Z=Glx, G=Gly, H=His, I=Ile, L=Leu, K=Lys, M=Met, F=Phe, P=Pro, S=Ser, T=Thr, W=Trp, Y=Tyr, V=Val). 6 10 20 30 V V K A V C V I N G 40 G E V C G L A K G L 70 H F N P Y G K E H G 100 C P T K V N I T D S 130 A D D L G Q G G H E D A K G T V F F E Q E S S G T P V K V S 50 60 H G F H V H E F G D N T N G C M S S G P 80 90 A P V D E N R H L G D L G N I E A T G D 110 120 K I T L F G A D S I I G R T V V V H A D 140 150 L S K S T G N A G A R I G C G V I G I A 151 K 7 codons. In p r i n c i p l e , each sequence i n a mixed oligonucleotide probe may e l i c i t a p o s i t i v e s i g n a l . T h e r e f o r e , i f two probes targeted to d i f f e r e n t r e g i o n s o f the p r o t e i n are c o n s t r u c t e d , then the p r o b a b i l i t y t h a t o n l y one DNA fragment w i l l be common to both oligonucleotide f a m i l i e s i s high. The f i r s t probe named SI i s t a r g e t e d to amino acids number 43-48 o f the CuZn SOD sequence ( F i g u r e 2 ) . The SI probe was designed b e f o r e the Cu/Zn SOD s e q u e n c e from D r o s o p h i l a was a v a i l a b l e . Sequence data from human, cow, horse and yeast show amino acids 42-48 are h i g h l y conserved, except for amino acid 47 which i s e i t h e r glutamic a c i d or glutamine (Figure 2). Therefore, both glutamic acid and glutamine codons had to be included i n the SI probe. L a t e r , the D. melanogaster sequence was found to have a Glu-47, which i s c o n s i s t e n t with what was predicted for the SI probe. Inc l u s i o n o f every p o s s i b l e codon for amino acids 42-48 resulted i n 128 d i f f e r e n t 17 base o l i g o n u c l e o t i d e s , which together form the SI probe. The s e c o n d p r o b e GT3 was d e s i g n e d w i t h t h e D.  melanogaster sequence a v a i l a b l e , and i s t a r g e t e d to amino acids 90-95, a segment u n i q u e t o t h e D r o s o p h i l a Cu/Zn SOD. To include every p o s s i b l e codon, t h i s segment would also require a family of 128 d i f f e r e n t o l i g o n u c l e o t i d e s . Therefore, to decrease the degeneracy of the probe, deoxyguanosine(dG) was placed i n p o s i t i o n s 9 and 12 where a l l f o u r nucleotides dA, dT, dC, dG are 8 FIGURE 2. The mixed sequence o l i g o n u c l e o t i d e probes SI and GT3. The SI probe c o r r e s p o n d s t o amino a c i d s 43-48 o f the CuZn superoxide dismutase p r o t e i n . This region was conserved with the exception of p o s i t i o n 47 where Glu-47 was found i n yeast, horse and D. melanogaster and Gln-47 was found f o r human and bovine sequences. The SI probe was designed to include both Glu and Gin a t p o s i t i o n 47, as t h e p r o b e was d e s i g n e d b e f o r e t h e D. melanogaster sequence was a v a i l a b l e . The SI probe c o n s i s t s of 128 d i f f e r e n t sequences (17 n u c l e o t i d e s i n length) and i s the complement o f the coding s t r a n d . The GT3 probe corresponds to r e s i d u e s 9 0 - 9 5 , a v a r i a b l e s e g m e n t u n i q u e t o t h e D. melanogaster p r o t e i n . The probe h e t e r o g e n e i t y was decreased by * p l a c i n g dG a t p o s i t i o n s 9 and 12 (G ) w h e r e a l l f o u r nucleotides may o c c u r . The GT3 probe c o n s i s t s of 8 variants (17 nucleotides i n length) and represents the coding strand. 9 SI PROBE 43 PHE 44 HIS 45 VAL 46 HIS 47 GLU GLN 48 PHE A l l p o s s i b l e c o d o n s UUU C CAU C GUG A U C CAU C CAA G G UUU C C o d i n g s t r a n d s 5 SI Probe 5 (Complementary S t r a n d ) TTT C 1 AA CAT C TTC C G GTG A T C ATG G CAT C GAC A T C CAA G G ATG G TT 17 AAA G GT3 PROBE 90 ASP 91 LYS 92 PRO 93 THR 94 LYS 95 VAL A l l p o s s i b l e codons GAU C UGU C CCG A U C ACG A U c AAA G GUG A U C GT3 P r o b e 1 GAT C TGT G CCG* ACG* AAA 1 7 GT 10 possible (Figure 2). The s u b s t i t u t i o n of dG into these p o s i t i o n s r e s t s on the assumption t h a t dG can base p a i r with dC, and that p a i r i n g w i t h dT and dA i s n o t d e s t a b i l i z i n g . W i t h t h i s m o d i f i c a t i o n , the degeneracy o f the GT3 probe was reduced to 8 d i f f e r e n t 17 base oligo n u c l e o t i d e s . These o l i g o n u c l e o t i d e s (GT3 and SI) may be r a d i o a c t i v e l y l a b e l l e d and used as d i r e c t h y b r i d i z a t i o n probes to s c r e e n genomic DNA which has been c l o n e d i n t o lambda phage l i b r a r i e s (42,43). In p r i n c i p l e , s e l e c t i v e h y b r i d i z a t i o n of the probes to t h e i r cognate targets i s p o s s i b l e as i n t e r n a l mismatches e x h i b i t a much lower melting temperature than p e r f e c t duplexes (44,45). These c o n d i t i o n s have t o be e m p i r i c a l l y d e t e r m i n e d as the d i s s o c i a t i o n temperature (T^) of the o l i g o n u c l e o t i d e duplexes are h i g h l y s e q u e n c e d e p e n d e n t and e m p i r i c a l l y d e t e r m i n e d e q u a t i o n s such as T d ( ° C ) = 4(G+C) + 2(A+T) can o n l y be used as an estimate of the actual d i s s o c i a t i o n temperature (46,47). The DNA o f p u t a t i v e p o s i t i v e s may be mapped to p o l y t e n e chromosomes to determine whether the sequence originates from the same chromosomal l o c a t i o n as the CuZn SOD gene. The presence of polymorphic v a r i a n t s of SOD i n s e v e r a l n a t u r a l p o p u l a t i o n s of Drosophila led to the g e n e t i c mapping o f the c y t o s o l i c SOD gene to 32.5 on the t h i r d chromosome (48). Production of c y t o s o l i c SOD de f e c t i v e mutants mapped the gene to 68AB on 3L (the l e f t arm of the t h i r d chromosome) on the p o l y t e n e chromosomes. C y t o s o l i c SOD mutant l e t h a l i t y c o r r e l a t e d w i t h i n c r e a s e d oxygen metabolism by 11 Drosophila a t the time o f e c l o s i o n (49). Attempts to c l o n e the D r o s o p h i l a CuZn SOD gene by using the two mixed o l i g o n u c l e o t i d e p r o b e s (GT3 and SI) to s c r e e n genomic lambda l i b r a r i e s i s the subject of t h i s t h e s i s . 12 MATERIALS AND METHODS MATERIALS E. c o l i JM101, LE392, and Q358 were p r o v i d e d by J . Leung. The D r o s o p h i l a m e l a n o g a s t e r Oregon-R genomic l i b r a r y was constructed i n lambda EMBL3 by J . Leung i n 1984. The Canton-S l i b r a r y was c o n s t r u c t e d i n lambda Charon 4 and ob t a i n e d from Maniatis (43) . M13mpl8 and mpl9 were a m p l i f i e d from o r i g i n a l P.L. Biochemical s t o c k s . M13mp8 and pUC13 were provided by S. Hayashi. O l i g o n u c l e o t i d e s GT3, S I , and M13 u n i v e r s a l primers were synthesized by T. A t k i n s o n (M. Smith Laboratory, UBC) . y - [ 3 2 P ] A T P (3000 Ci/mmol, 10 u C i / u l ) and a - [ 3 2 P ] d N T P (3000 Ci/mmol) were from Amersham Corp., and deoxy and dideoxyNTPs from P.L. Biochemicals. R e s t r i c t i o n enzymes, T4 l i g a s e , E. c o l i DNA polymerase I, E_^_ c o l i DNA polymerase (Klenow fragment) were purchased from New E n g l a n d B i o l a b s , Bethesda Research Laboratories, Boehringer Mannheim, or Promega Biotech, and used interchangeably u n l e s s s p e c i f i e d . E ^ c o l i RNA polymerase was from P.L. B i o c h e m i c a l s . C a l f i n t e s t i n a l phosphatase was from Boehringer Mannheim, BSA and l y s o z y m e from Sigma, agarose ( U l t r a p u r e ) from BRL, a c r y l a m i d e from Eastman Kodak Co., N,N'-Methylene bisacrylamide from Matheson Coleman and B e l l , and TEMED from BioRad. Hybond-N (nylon) membranes i n r o l l s and 82 mm d i s c s were from Amersham Corp. The X-ray f i l m (Curix RP1) was from Agfa-Gevaert. BACTERIAL STRAINS 13 The h o s t s f o r melanogaster Canton-S and Oregon-R genomic l i b r a r i e s were LE392 and Q358, re s p e c t i v e l y . Both stocks were kept frozen i n 15% g l y c e r o l . Q358 (in NZYM (56)) and LE392 (in LBMgT) were grown to stationary phase with 0.2% maltose to induce the lambda receptor (56). The c e l l s were p e l l e t e d by spinning at 4000g (5 min.). The p e l l e t was resuspended i n 0.5 volumes of 0.01M MgSO^ and s t o r e d a t 4°C ( v i a b l e f o r 1 week). D. MELANOGASTER GENOMIC DNA PREPARATION D. melanogaster h i g h m o l e c u l a r weight genomic DNA was p r e p a r e d e s s e n t i a l l y a s d e s c r i b e d w i t h t h e f o l l o w i n g modifications (42). One gram of f r o z e n D. melanogaster (Oregon R s t o c k from G. Tener) was p l a c e d i n the bottom o f a Dounce homogenizer and ground with 5 ml of s t e r i l e i c e cold s o l u t i o n (i0 mM Tris-HCl (pH 7.5), 60 mM NaCl, 10 mM EDTA, 0.15 mM spermine, 0.15 mM spermidine) and f i l t e r e d through f i n e gauze or 2 layers of Miracloth (Calbiochem-Behring Corp.) into a s t e r i l e centrifuge tube. The n u c l e i were p e l l e t e d by c e n t r i f u g a t i o n (12,000g, 10 minutes, 4°C) and the p e l l e t and tube r i n s e d with 0.1 M NaCl, 20 mM Tris-HCl (pH 8) , 10 mM EDTA and resuspended i n 5 ml of the same bu f f e r . Proteinase K (500 ug) was added and a l l the n u c l e i were lysed simultaneously by i n j e c t i n g 1 ml of 10% SDS into the s o l u t i o n . The s o l u t i o n was incubated f o r 3 hours at 50°C and 4 ml of the same buffer was added b e f o r e phenol extraction. A f t e r l y s i s , mechanical shearing (by m i x i n g , shaking, etc.) was kept to a minimum and s o l u t i o n s c o n t a i n e d no more than 1 mg DNA/20 mis 14 of nuclear lysate during phenol extractions. The DNA s o l u t i o n was e x t r a c t e d w i t h equal volumes o f phenol/CHCl^(1:1) g e n t l y by r o l l i n g on a wheel (15 min., room temperature). The tubes were centrifuged (10,000g, 5 minutes) to s e p a r a t e the phases. The e x t r a c t i o n was repeated with phenol/CHCl^ and then once with CHC1 3. The DNA was p r e c i p i t a t e d by adding c o l d 95% EtOH to the f i n a l aqueous phase and the DNA was removed with a g l a s s rod. The DNA was a i r dri e d b r i e f l y and r e d i s s o l v e d i n 500 u l of TE 8 (10 mM T r i s - H C l (pH 8 ) , 1 mM EDTA). When necessary, h e a t i n g at 65°C was used to r e d i s s o l v e the e n t i r e p e l l e t . RADIOACTIVE LABELLING OF OLIGONUCLEOTIDE PROBES The 5' OH of the oligodeoxyribonucleotides were l a b e l l e d with 32 [ p ] b y p o l y n u c l e o t i d e kinase which t r a n s f e r s the r a d i o a c t i v e 32 phosphorus from y - [ P]ATP. One hundred picomoles of GT3 or SI were reacted w i t h 250 uCi (80 pmoles) of y -[ 3 2P]ATP and 25 units of polynucleotide kinase i n 0.1 M T r i s - H C l (pH 7.5), 20 mM M g C l 2 , 0.2 mM EDTA , 0.2 mM s p e r m i d i n e , and 10 mM DTT f o r 45 minutes a t 37°C. The r e a c t i o n was t e r m i n a t e d by h e a t i n g a t 65°C f o r .10 minutes. c o l i tRNA (250ug) was added as a c a r r i e r and the sample d i l u t e d with 200 u l of TE 8. The reaction mixture was loaded onto a 0.5 ml column o f DEAE-cellulose and washed with 2 ml of TE 8. The u n i n c o r p o r a t e d l a b e l was eluted with 8 ml of 0.2 M NaCl/TE 8 and the oligonucleotides were eluted i n 3.0 ml of 1.0 M NaCl/TE 8. T o t a l i n c o r p o r a t e d counts were g r o u t i n e l y 2 x 1 0 cpm (30-40% i n c o r p o r a t i o n ) . The probe was 15 h y b r i d i z e d to f i l t e r s a t 40 ul / c m and no l e s s than 1 x 10 cpm/ml i n 6 x SSC ( 1 x SSC = 0.15 M NaCl, 0.015 M NaCitrate (pH 7.2)), 50 mM sodium phosphate (pH 6.8), 5 x Denhardt's reagent (lxDenhardt's reagent = 0.02% F i c o l l , p o l y v i n y l pyrrolidone, and BSA), 0.5% SDS, and 20 ug/ml E. c o l i tRNA. SCREENING GENOMIC LIBRARIES BY PLAQUE HYBRIDIZATION  I . P r i m a r y Screen The t i t e r of the Canton-S and Oregon-R lambda l i b r a r i e s was determined by p l a t i n g a s e r i e s o f 1 0 - f o l d d i l u t i o n s o f the o r i g i n a l phage s t o c k . The phage was added to 100 u l of 10 mM M g C l 2 / C a C l 2 and 100 u l o f r e s u s p e n d e d Q358. The phage and bacteria were incubated f o r 15 minutes a t 37°C, and plated with 3 ml of s o f t top agarose (0.7% agarose i n NZYM at 55°C) on NZYM plate s . A f t e r the agarose hardened the plates were inverted and incubated at 37°C f o r 10-14 hours. B o t h l i b r a r i e s were s c r e e n e d by t h e i r i s i t u p l a q u e h y b r i d i z a t i o n method developed by Benton and Davis (50). Each l i b r a r y was p l a t e d on s i x p l a t e s w i t h 10^ p l a q u e forming units(PFU) per 82mm p l a t e and incubated u n t i l the plaques were j u s t t o u c h i n g but not c o n f l u e n t . The p l a t e s were c h i l l e d a t 4°C f o r 1 hour b e f o r e b l o t t i n g onto n y l o n f i l t e r s (Hybond-N). The phage and DNA was adsorbed by p l a c i n g two f i l t e r s on each pl a t e s e q u e n t i a l l y , the f i r s t f o r 3 minutes and the second for 5 minutes. The f i l t e r s were o r i e n t a t e d by asymmetric ink spots, and a i r d r i e d . The phage and DNA were denatured and f i x e d by 16 placing the f i l t e r s DNA side up on b l o t t i n g paper soaked i n 1.5 M NaCl/0.5 M NaOH then n e u t r a l i z e d with 1.5 M NaCl/1.0 M Tris-HCl (pH 7.5) for 5 minutes each. A f t e r a i r d r y i n g the f i l t e r s , the DNA was covalently fixed to the nylon membrane by i r r a d i a t i o n with uv l i g h t (254nm, 4 minutes) . The f i l t e r s were washed i n 6xSSC by g e n t l y rubbing w i t h a g l o v e d f i n g e r and then p r e h y b r i d i z e d (6xSSC, 10xDenhardt's reagent, 0.2% SDS) f o r at l e a s t 3 hours before h y b r i d i z a t i o n with the GT3 probe f o r at l e a s t 12 hours at 37°C. The f i l t e r s were washed i n 6xSSC (2 changes of b u f f e r for 30 minutes) a t room temperature and then washed a t higher s t r i n g e n c y (52°C f o r Canton-S and 50°C f o r Oregon-R screens) twice for 15 minutes each. F i l t e r s were exposed to X-ray f i l m f o r 4 days a t -70°C wi t h an i n t e n s i f y i n g s c r e e n . Agar p l u g s containing plaques from the r e g i o n corresponding to a p o s i t i v e on the autoradiogram were p i c k e d (with the wide end of a s t e r i l e pasteur pipette) into 0.5 ml of SM b u f f e r with 5 u l of CHCl^. I I . Secondary Screen In order to i s o l a t e a s i n g l e p o s i t i v e phage from the p o s i t i v e agar plug picked i n the h i g h d e n s i t y screen, the plaques must be plated to a d e n s i t y of 200 to 400 PFU per plate and re-screened as d e s c r i b e d f o r the primary s c r e e n s . Plaques were allowed to grow bigger for the secondary screens and the autoradiogram exposure time was decreased to 2 days. P o s i t i v e spots aligned with a s i n g l e plaque. This was picked into 200 u l of SM b u f f e r w i t h 5 u l o f CHC1- ( u s i n g the narrow end of a 17 pasteur p i p e t t e ) . BACTERIOPHAGE LAMBDA DNA PREPARATION  I. Large s c a l e p r e p a r a t i o n To i s o l a t e DNA from lambda phage c l o n e s , 1.25 ml o f resuspended Q358 was i n f e c t e d w i t h about 1x10 PFU o f phage d i l u t e d i n 1.0 ml o f 10 mM M g C l 2 / C a C l 2 . The phage was adsorbed for 15 minutes a t 37°C and then used to inoculate 250 ml of prewarmed NZYM.' The c u l t u r e was shaken v i g o r o u s l y at 37°C u n t i l i t l y s e d c l e a r (3-8 h o u r s , depending on t i t e r ) . CHCl^ (1 ml) was added and s w i r l e d s l o w l y f o r 5 minutes before p e l l e t i n g c e l l debris by c e n t r i f u g a t i o n for 15 minutes at 8000 g. The supernatant was mixed w i t h 0.15 volumes 5 M NaCl/0.3 volumes 50% PEG6000 and the phage p r e c i p i t a t e d f o r a t l e a s t 12 hours at 4°C ( 5 1 ) . The P E G - p r e c i p i t a t e d phage was p e l l e t e d by c e n t r i f u g a t i o n (8000 g, 15 min., 4°C) . The supernatant was poured o f f and the tubes were spun f o r another minute and the supernatant removed as c l e a n l y as p o s s i b l e . The phage p e l l e t was resuspended i n 5 ml of DNasel b u f f e r (50 mM Tris-HCl (pH 7.5), 5 mM M g C l 2 , 0.5 mM C a C l 2 ) and i n c u b a t e d a t 37°C with 50 u l of DNasel (1 mg/ml i n 0.15 M NaCl, 50% g l y c e r o l ) and 50 u l RNase A (10 mg/ml). Aft e r incubation f o r 3 hours the debris was p e l l e t e d by c e n t r i f u g a t i o n a t 12,000 g f o r 5 m i n u t e s and the phage supernatant was t r e a t e d w i t h 75 u l P r o t e i n a s e K (10 mg/ml), 0.5 ml 10% SDS, 100 u l of 250 mM EDTA (pH 7.5) and incubated at 68°C f o r 2 h o u r s . The DNA was p u r i f i e d by two phenol/CHCl., (1:1) 18 extractions and one CHC1 3 e x t r a c t i o n . The DNA was concentrated by EtOH p r e c i p i t a t i o n and resuspended i n 250 u l TE 8 (Yield 200-250 ug). I I . Lambda phage DNA i s o l a t i o n from primary screen phages Preparation of phage DNA from the primary screen plugs was exactly as described above except 100 u l of host Q358 was infected with 50 u l o f t h e p r i m a r y p l u g phage s o l u t i o n and used to i n o c u l a t e a 20 ml NZYM c u l t u r e . The p r e c i p i t a t e d DNA was redissolved i n 50 u l of TE 8 and the concentration checked on an agarose g e l . I I I . Lambda phage DNA i s o l a t i o n from secondary screen phages Indi v i d u a l phages i s o l a t e d from the secondary screen were also grown i n 20 ml NZYM cu l t u r e s . These cultures were inoculated with 140 u l of phage suspension and 100 u l of Q358. The DNA was prepared as d e s c r i b e d above. AGAROSE GEL ELECTROPHORESIS AND SOUTHERN HYBRIDIZATION  I. R e s t r i c t i o n enzyme d i g e s t s R e s t r i c t i o n digests were performed using 10-50 mM Tris-HCl (pH 7.5), 10 mM M g C l 2 , 1 mM DTT, 100 ug/ml BSA, and 0, 50, or 100 mM NaCl as r e q u i r e d by the enzyme. The DNA was digested with 2-fold excess of enzyme for 3 hours i n a 100 ul volume and 5 u l was loaded onto a mini agarose g e l to check f o r completeness of d i g e s t i o n b e f o r e the r e s t of the digest was p r e c i p i t a t e d by EtOH, resuspended i n TE 8, and loaded onto a large g e l for transf e r onto membranes. 19 I I . Agarose g e l e l e c t r o p h o r e s i s Agarose gel e l e c t r o p h o r e s i s was i n 90 mM TBE (90mM T r i s , 90 mM b o r i c a c i d , 1 mM EDTA, pH 8.3) and g e l s v a r i e d from 0.5-1.2% agarose (with 1 ug/ml EtBr). The gels were usually run at 2.5V/cm for 12 to 14 hours. Digested genomic DNA (40 ug) was resuspended in 40 u l TE 8 and heated at 65°C f o r 5 minutes b e f o r e loading onto a g e l . Primary s c r e e n phage DNA (10 ug) and i n d i v i d u a l phage DNA (5 ug) were r e d i s s o l v e d i n 15 u l TE 8. Gel loading buffer (40% sucrose, 0.1% bromphenol b l u e , 0.05% xylene cyanol, 0.1% SDS, 1 mM EDTA) was added before loading (5 u l ) . I I I . Southern t r a n s f e r and h y b r i d i z a t i o n The agarose g e l s were d e n a t u r e d 2x10 m i n u t e s i n 1.5 M NaCl/0.5 M NaOH and n e u t r a l i z e d 2x10 minutes i n 1.5 M NaCl/1.0 M Tris-HCl (pH 7.5) at room temperature with shaking (52). The DNA was then t r a n s f e r r e d to n y l o n membranes (Hybond-N) with 20xSSC overnight. The membrane was a i r d r i e d before covalent linkage of the DNA by uv i r r a d i a t i o n (254 nm, 4 minutes) and washed in 6xSSC by gently rubbing w i t h a g l o v e d f i n g e r b e f o r e p r e h y b r i d i z a t i o n (6xSSC, 10xDenhardt's reagent, 0.2% SDS) i n heat sealed bags for at l e a s t 3 hours. The DNA on the f i l t e r s was h y b r i d i z e d with e i t h e r GT3 or SI o l i g o n u c l e o t i d e s (6xSSC, 50 mM sodium phosphate (pH 6.8), 5 x Denhardt's reagent, 0.5% SDS, 20 ug/ml E^ c o l i tRNA) a t 37°C. Membranes w i t h DNA from i n d i v i d u a l phage were hybridized f or at l e a s t 12 hours, whereas membranes with genomic DNA were h y b r i d i z e d f o r a t l e a s t 40 hours. The f i l t e r s were 20 washed at room temperature i n 6xSSC f o r 30 minutes and then a higher stringency wash was performed at e i t h e r 50 or 52°C (GT3) and 47°C (SI) i n 6xSSC. F i l t e r s were exposed f o r 2-7 days, depending on the counts retained. IN SITU HYBRIDIZATION TO DROSOPHILA POLYTENE CHROMOSOMES I. L a b e l l i n g the probe The DNA probe was l a b e l l e d w i t h [ 1 2 5 I ] - C T P ( [ 1 2 5 l ] -5-iodo CTP). This was done by in v i t r o t r a n s c r i p t i o n o f the phage DNA by c o l i RNA poly m e r a s e i n t h e presence o f [ I]-CTP (53-55). The phage DNA was t r e a t e d with RNase A (1 ug/ul) and phenol extracted twice and p r e c i p i t a t e d w i t h 0.6 volumes of 20% PEG/2.5 M NaCl and resuspended i n 20 ul TE 8. The t r a n s c r i p t i o n reaction consisted of 5 ug of phage DNA and 80 uCi [ i]-CTP i n 40 mM Tris-HCl (pH 7.5), 10 mM DTT, 10 mM MgCl 2, 20 uM ATP, GTP, UTP, and 5 U of E_^_ c o l i RNA polymerase. T h i s was incubated at 37°C for 2 hours f o l l o w e d by DNase I treatment (10 ug DNase I, 40 mM T r i s - H C l (pH 7.8), 100 ug E. c o l i tRNA) f o r 20 minutes a t room t e m p e r a t u r e . T h i s m i x t u r e was e x t r a c t e d w i t h p h e n o l / C H C l 3 (once) and w i t h C H C l ^ ( t h r e e t i m e s ) b e f o r e 2 loading the aqueous phase on a Sephadex G-25 column (0.196cm x 20 cm). The f i r s t r a d i o a c t i v e peak was c o l l e c t e d ( in 0.3 M NaOAC (pH 7.2), 50 uM EDTA, 0.01% SDS) and p r e c i p i t a t e d by 2.5 volumes of 95% EtOH and r e d i s s o l v e d i n an a p p r o p r i a t e volume of 70% formamide, 0.06 M K H 2 P 0 4 , 0.06 M K 2 H P 0 4 , 5 mM EDTA, 4mM KOH, and 0.5 M KCl. 21 I I . In s i t u h y b r i d i z a t i o n The probe (prepared above) was placed onto a s l i d e containing s a l i v a r y gland chromosomes a t 10^ cpm per s l i d e and hybridized a t 45°C f o r 2 d a y s . The s l i d e s were washed, c o a t e d w i t h emulsion, and developed e s s e n t i a l l y as described (55). 5UBCL0NING PHAGE DNA INTO PLASMIDS I . L i g a t i o n R e a c t i o n The plasmid (pUC13, M13mpl8 or mpl9 RF) was digested (2 ug i n 20 u l volume) with the d e s i r e d r e s t r i c t i o n enzymes and heated at 65°C for 10 minutes. C a l f i n t e s t i n a l phosphatase was added (CIP 1 u n i t i n 50 mM T r i s - H C l pH 9.0, 1 mM M g C l 2 , 0.1 mM ZnS0 4, 1 mM spermidine-total volume 50 ul) and r e a c t e d for 30 minutes at 50°C. To t e r m i n a t e the r e a c t i o n 40 u l H 20, 5 u l 10% SDS, and 10 u l 10xSTE buffer(10xSTE= 0.1 M Tris-HCl pH 8.0, 1 M NaCl, 10 mM EDTA) was added and heated at 68°C f o r 15 minutes. The mixture was phenol e x t r a c t e d twice (phenol/CHCl^ 1:1) and c h l o r o f o r m extracted once before EtOH p r e c i p i t a t i o n of DNA. The phage DNA to be s u b c l o n e d was d i g e s t e d and mixed i n v a r y i n g m o l a r r a t i o s w i t h 100 ng o f l i n e a r i z e d and dephosphorylated plasmid or RF DNA and l i g a t e d with 0.1 U T4 DNA ligase/10 u l i n 50 mM T r i s - H C l pH 7.5, 10 mM M g C l 2 , 1 mM DTT, and 0.8 mM ATP a t 15°C f o r 12-18 hours (56). I I . Plasmid and Ml3 Transformations Plasmid and M13 DNA was i n t r o d u c e d into JM101 that were made competent by the C a C l - method (56) . A c o l o n y o f JM101 from a 22 minimal glucose plate was grown in 2YT u n t i l OD^^ was 0.5-0.6. The c e l l s were pelleted (5 min. at 4000 g) and resuspended in half the o r i g i n a l culture volume of s t e r i l e 50 mM C a C l 2 . After incubation on ice for 20 minutes the ce l l s were pelleted and resuspended in 1/10 the o r i g i n a l culture volume. Aliquots (300 ul) of competent JM101 were incubated on ice with the aliquots of the ligation mixture for 40 minutes. For pUC plasmids, the ce l l s were heat shocked for 3 minutes at 42°C and plunged on ice; then 0.7 ml LB was added and the c e l l s shaken for 1 hour at 37°C. Aliquots (0.1-0.3 ml) were plated on LBAmp (100 ug/ml) plates with 50 ul Xgal(2%) and 10 ul IPTG (100mM) . For Ml3 transformations, the c e l l s were returned to room temperature after heat shock. Fi f t y ul Xgal(2%), 10 ul IPTG (100mM) , 200 ul fresh exponential JM101, and 3 ml of soft agar (0.7% in 2YT at 55°C) were added and the c e l l s plated on 2YT p l a t e s (57). After incubation overnight at 37°C, white transformants (plaques or colonies) were picked individually or screened by transfer onto Hybond-N. III. Growth of transformants Transformants containing pUC plasmids were picked into 2 mis of LBAmp (100 ug/ml) and grown to saturation. M13 transformants were grown in 2 ml of 2YT containing 20 ul of JM101 (grown overnight in minimal glucose) at 37°C for 5 hours. Plasmid and RF preparations were by the alkaline l y s i s method (56). Part of the cultures were frozen in 15% glycerol as stock and the supernatant from M13 cultures were stored as high t i t e r phage 23 stocks or used for ssDNA template preparations. DNA SEQUENCE DETERMINATION I. Template p r e p a r a t i o n S i n g l e stranded DNA template from M13 t r a n s f o r m a n t s was prepared by spinning 1.2 ml o f the Ml3 culture for 5 minutes i n a microfuge. The supernatant was c a r e f u l l y removed and 0.3 ml of 20% PEG6000/2.5 M NaCl was added and mixed. The phage were pr e c i p i t a t e d at room temperature f o r 15 minutes and p e l l e t e d by c e n t r i f u g a t i o n f o r 15 minutes i n the m i c r o f u g e . The supernatant was discarded and the p e l l e t was resuspended i n 0.2 ml of TE 8 and e x t r a c t e d twice w i t h an equal volume o f phenol/CHCl^(1:1) being c a r e f u l to l e a v e a l l o f the i n t e r f a c e . The aqueous layer was EtOH p r e c i p i t a t e d twice and redissolved i n 50 u l of TE 8. Double stranded DNA templates ( e i t h e r plasmids or M13 RFs) were prepared by taking 5 ug of plasmid and digesting with RNase A (1 ug/ul) , phenol e x t r a c t i n g once, and making the s o l u t i o n up to 0.5 M NaCl/TE 8 (100 u l t o t a l ) . The RNA was removed by passing the DNA through a spun column ( d e s k t o p c e n t r i f u g e , 1400rpm, approx. 5 min., c a l i b r a t e d to recover 100 u l ) of c r o s s l i n k e d Sepharose 4B. The r e c o v e r e d DNA was p r e c i p i t a t e d and rehydrated i n 40 u l of 0.2 M NaOH and denatured a t room temperature for 5 minutes. The s o l u t i o n was n e u t r a l i z e d with 1/10 v o l of 2 M NH4OAc (pH 4.5) and p r e c i p i t a t e d w i t h 2.5 volumes o f 95% EtOH. The DNA was dissolved i n 10 ul TE 8 j u s t before sequencing (58). I I . DNA Sequencing 24 DNA sequence was determined by the dideoxynucleotide chain ter m i n a t i o n method (59). The ssDNA template (5 ul) was mixed with 1 u l of M13 u n i v e r s a l forward or r e v e r s e primer (4 pmoles) and 2 u l of a n n e a l i n g b u f f e r (100 mM NaCl, 100 mM Tris-HCl (pH 7 . 5 ) ) . The m i x t u r e was h e a t e d a t 65°C f o r 10 minutes and c o o l e d s l o w l y to room t e m p e r a t u r e . F o r d o u b l e s t r a n d e d sequencing, 1-2 ug of template was annealed f o r 15 minutes at 37°C (58). In both c a s e s , 1 u l 15 uM dATP and 1.5 u l a - [ 3 2 p ] -dATP 3000 Ci/mmole) were added to the template/primer mix and 2.2 u l o f t h e f i n a l m i x t u r e was d i s t r i b u t e d t o tubes c o n t a i n i n g 1.5 u l of each o f the A,T,C,G deoxy/dideoxy mixes. The following nucleotide mixes were used (60): dG/ddG: 89 uM ddGTP, 7.9 uM dGTP, 158 uM dTTP, 158 uM dCTP dA/ddA: 116 uM ddATP, 111 uM dGTP, 111 uM dTTP, 111 uM dCTP dT/ddT: 547 uM ddTTP, 158 uM dGTP, 7.9 uM dTTP, 158 uM dCTP dC/ddC: 547 uM ddCTP, 158 uM dGTP, 158 uM dTTP, 10.5 uM dCTP The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 1 u l of DNA polymerase I Klenow f r a g m e n t ( 0 . 5 U / u l i n 80 mM p o t a s s i u m phosphate (pH 7.5), 0.8 mg/ml BSA, 40% g l y c e r o l , 10 mM DTT). The r e a c t i o n m i x t u r e s were i n c u b a t e d a t 37°C f o r 15 minutes and then chased by the a d d i t i o n of 1 u l dNTP (0.5 mM) to each tube with another 15 minute i n c u b a t i o n . The reactions were terminated by adding 5 u l of formamide/dye mix (98% deionized formamide, 10 mM EDTA, 0.2% bromophenol blue and xyl e n e c y a n o l ) . The samples were heated for 3 minutes a t 90-100°C and placed on i c e . 25 The samples (1 ul) were electrophoresed in 6% or 8% polyacrylamide gels (acrylamide : methylenebisacrylamide (19:1), 8 M urea, 0.06% ammonium persulfate, 20 ul TEMED, 50 mM TBE) at 1600-1800 V such that the current did not exceed 25mA. The gel was run until the bromophenol blue reached the bottom (40cmxl8cmx0.35cm gels) or 1.5 hr after the last loading with a maximum of 3 loadings (0, 1.5, 3 hr). The gels were dried onto Whatman 3MM paper using a vacuum gel drier (1.5 hr, 80°C) and were exposed to X-ray film at room temperature for hours or days depending on radioactivity incorporated. 26 RESULTS THE STRINGENCY OF HYBRIDIZATION The stringency of hybridization must be decided upon before embarking on screening of the l i b r a r y . Hybridization 5°C below the dissociation temperature(T^) allows perfect duplexes to be detected (61) . The hyb r i d i z a t i o n range for GT3 and SI was determined to be 45-53°C and 37-47°C, r e s p e c t i v e l y (an estimate from Td=4 (G+C) + 2(A+T) in 6xSSC (44,46)). Reactions should be performed under the most s t r i n g e n t conditions to eliminate hybridization to r e l a t e d sequences but with mixed sequence oligonucleotides the stringency must be a compromise i . e . the s t r i n g e n c y must be high enough f o r signals to be discernible over background but s t i l l allow the hybrization of possibly a l l sequences in the mix. The hybridization temperature was determined by probing genomic and library DNA and looking for the presence of d i s t i n c t bands above background at the chosen temperature (Figure 3). Hybridization of GT3 to digested Oregon-R genomic and l i b r a r y DNA at 50°C showed multiple bands of varying intensities. Due to i t s high degeneracy (128 variants) and low melting, bands were not v i s i b l e when the SI probe was used to probe membrane-bound genomic DNA at or below 47°C. Subsequent hyb r i d i z a t i o n of GT3 was at 52°C (high stringency) and 50°C (lower s t r i n g e n c y ) whereas SI was at 47°C. Lower temperatures were not feasible due to background interferences so i t i s possible that lower melting sequences i n the probe were 27 FIGURE 3. Oregon-R genomic and lambda l i b r a r y DNA Southern a n a l y s i s . Oregon-R genomic DNA (I) and lambda l i b r a r y DNA (II) were d i g e s t e d w i t h (a) EcoRI (b) E c o R I / S a l l (c) H i n d l l l (d) H i n d l l l / S a l l and transferred onto nylon membranes (Hybond-N). The membranes were h y b r i d i z e d w i t h [ 3 2 p ] l a b e l l e d GT3 a t 37°C and washed at i n c r e a s i n g l y h igher temperatures u n t i l p o s i t i v e bands were d i s c e r n i b l e above background. The membranes wit h (III) genomic DNA and (IV) lambda l i b r a r y DNA were washed at 50°C, i n GxSSC, and exposed to X - r a y f i l m f o r 4 days a t -70°C. The autoradiograms show GT3 h y b r i d i z e s to d i f f e r e n t fragments a t v a r y i n g i n t e n s i t i e s . The s i z e marker (m) was lambda DNA digested with H i n d l l l . 28 29 excluded. D i r e c t c o r r e l a t i o n o f bands i n the Oregon-R genomic and l i b r a r y DNA was n o t p o s s i b l e s i n c e t h e l i b r a r y was constructed as Mbol p a r t i a l d i g e s t s l i g a t e d i n t o the BamHI s i t e of the EMBL3 vector(42). PRIMARY SCREEN OF CANTON-S LAMBDA LIBRARY The GT3 probe was used t o s c r e e n t h e D^ melanogaster 4 Canton-S lambda l i b r a r y (43). The primary s c r e e n of 6x10 PFU (equivalent to 6 genomes) by GT3 a t 52°C r e s u l t e d i n 5 p o s i t i v e s i g n a l s c l e a r l y d i s c e r n i b l e above background. The SI probe could not be used for primary s c r e e n s as i t showed non-specific h y b r i d i z a t i o n to lambda arms or some component o f the l y s e d b a c t e r i a . SECONDARY SCREEN OF CANTON-S LAMBDA LIBRARY The i n d i v i d u a l phages r e s p o n s i b l e f o r the p o s i t i v e signals were i s o l a t e d and the DNA i n s e r t s were released from the Charon 4 v e c t o r by d i g e s t i o n w i t h EcoRI ( which produces 19 and 11 Kb vector arms). Analysis of the EcoRI fragments showed 3 d i f f e r e n t phages (CS-1, CS-2, and CS-3) wi t h common as well as unique s i z e fragments. Phage CS-3 had a 3.7 Kb EcoRI band which had been cleaved to smaller fragments i n CS-2 (Figure 4). Therefore, these three phages may have overlapping genomic i n s e r t s with polymorphic EcoRI s i t e s . Hybridization with GT3 showed that the 4.7 Kb (from CS-1) and 7.0 Kb (from CS-2, CS-3) EcoRI fragments hybridized GT3 (Figure 4). DNA SEQUENCE ANALYSIS OF GT3 BINDING SITE (CANTON-S LIBRARY) 30 FIGURE 4. Southern a n a l y s i s o f DNA from GT3 p o s i t i v e phage from the Canton-S genomic lambda l i b r a r y . The genomic i n s e r t was released from the phage v e c t o r arms (Charon 4) by d i g e s t i o n with EcoRI. The two highest m o l e c u l a r weight bands (19 and 11 Kbp) on the agarose g e l (I) are the v e c t o r arms. The EcoRI d i g e s t i o n patterns r e v e a l t h r e e d i f f e r e n t phage: CS-1 (a,b), CS-2 (c,d), and CS-3 ( e , f , g , h , i ) . The DNA sep a r a t e d on the agarose g e l was transferred to Hybond-N and h y b r i d i z e d to GT3. The f i l t e r was washed a t 52°C i n 6XSSC and t h e r e s u l t i n g autoradiogram i s shown ( I I ) . Phage CS-1 (a,b) has a 4.7 Kb p o s i t i v e EcoRI fragment whereas phage CS-2 and CS-3 ( c - i ) have a 7.0 Kb GT3 binding EcoRI fragment. 31 II a b c d e f g h i -< 7 .0 Kb - « 4 . 7 Kb 32 The 4.7 Kb (from CS-1) and 7.0 Kb (from CS-2 and 3) GT3 p o s i t i v e EcoRI fragments were c l o n e d i n t o pUC 13 and pEMBL8-, r e s p e c t i v e l y . The plasmid w i t h the 4.7 Kb i n s e r t (CS-1) was digested with PstI to produce a 500 bp GT3 p o s i t i v e fragment, which was subcloned into M13mp8 f o r Sanger sequencing. Sequencing the s i n g l e stranded template f o r 200 bp (from one end of the insert) did not reveal any homology to CuZn SOD when translated i n a l l 3 reading frames. T - t r a c k a n a l y s i s o f the 10 other clones c o n t a i n i n g the 500 bp P s t I i n s e r t showed t h a t the i n s e r t was cloned i n the same o r i e n t a t i o n i n a l l 10 clones. Sequencing from the other end of the i n s e r t by double stranded sequencing of the M13 RF showed the GT3 hy b r i d i z i n g s i t e s t a r t i n g one nucleotide a f t e r the PstI recognition s i t e (Figure 5). The GT3 h y b r i d i z i n g s i t e of the 7.0 Kb fragment from CS-2 and CS-3 was sequenced on both strands by the method of Maxam and G i l b e r t (S. Hayashi). The three p o s i t i v e phage (CS-1, 2 and 3) had i d e n t i c a l nucleotide sequence i n the GT3 p r o b e b i n d i n g r e g i o n and i d e n t i c a l GT3 binding s i t e s . The GT3 binding region was r e c o g n i z e d by t r a n s l a t i o n of the DNA s e q u e n c e r e s u l t i n g i n t h e a m i n o a c i d s e q u e n c e Asp-Cys-Pro-Thr-Lys-Ly_s whereas GT3 was o r i g i n a l l y targeted to Asp-Cys-Pro-Thr-Lys-Val i n CuZn SOD (F i g u r e 6 ) . The one GT3 sequence responsible for b i n d i n g t h i s phage i s 5'-GAT TGT CCG ACG AAG GT-3' which forms 15 p e r f e c t Watson-Crick base p a i r s and one dG:dT pair with the phage DNA ( F i g u r e 6). This member o f the GT3 33 FIGURE 5. DNA s e q u e n c i n g s t r a t e g y f o r t h e GT3 b i n d i n g s i t e of the Canton-S l i b r a r y GT3 p o s i t i v e phage. The three phage from the Canton-S l i b r a r y ( C S - 1 , CS-2, CS-3) had i d e n t i c a l n u c l e o t i d e sequence i n the GT3 probe h y b r i d i z i n g r e g i o n . The GT3 b i n d i n g s i t e (open box) was s e q u e n c e d by t h e Sanger c h a i n t e r m i n a t i o n method ( s o l i d arrows) on one s t r a n d o f CS-1 and on both strands of CS-2 and CS-3 by the Maxam and G i l b e r t chemical method (dotted arrows). 34 T 100 bp . 230 130 200 Sanger Sequencing Maxam and Gilbert Sequencing Oligonucleotide binding site 180 35 FIGURE 6. The nucleotide sequence and t r a n s l a t i o n of the GT3 binding site of phage CS-1, 2, and 3 from the Canton-S library. Translation of the nucleotide sequence around the GT3 binding s i t e produced a sequence of 38 amino acids. More nucleotide sequence upstream of the GT3 b i n d i n g s i t e i s required to determine the number of amino a c i d s that i s encoded by t h i s sequence. There exists a polymorphic s i t e six nucleotides 5' to * the s t a r t of the GT3 b i n d i n g s i t e (G ). The CS-2 and 3 sequence has GG at t h i s s i t e . The GT3 probe binding s i t e was recognized by the amino acid sequence Asp-Cys-Pro-Thr-Lys-Lys as opposed to the desired SOD sequence Asp-Cys-Pro-Thr-Lys-Val. A l l nucleotide sequences were translated in a l l three phases and no other homology to the SOD p r o t e i n was found. The phage sequence 3'-CTA ACA GGC TGC TTC TT-5' hybridized the GT3 sequence 5'-GAT TGT CCG ACG AAG GT-3'. 36 ATA GCC AAG GCG AAG ATA GCC I LE ALA LYS ALA LYS ILE ALA AAA TAC TTG GCG CGG GCT GAG LYS TYR LEU ALA ARG ALA GLU GAG ATT CAC GCC AAT TTC CTG GLU ILE HIS ALA ASN PHE LEU GCC AAC GAT TGT CCG ACG AAG ALA ASN ASP CYS PRO THR LYS P s H AAG CTG CAG TTC CAG CTC TCG LYS LEU GLN PHE GLN LEU SER CTG LEU 37 mixed o l i g o n u c l e o t i d e f a m i l y appears to be the h i g h e s t melting member to have hybridized to a genomic sequence from the Canton-S l i b r a r y . PRIMARY SCREEN OF OREGON-R GENOMIC LAMBDA LIBRARY Screening with GT3 a t a lower s t r i n g e n c y (to include lower m e l t i n g members o f GT3) was p e r f o r m e d a t 50°C i n a newly constructed Oregon-R genomic lambda l i b r a r y . The Oregon-R l i b r a r y has never been a m p l i f i e d ( i n c o n t r a s t to the much older Canton-S l i b r a r y ) and a l l c l o n a b l e sequences should be present. 4 S c r e e n i n g 6x10 PFU (6 genomes) o f t h e Oregon-R l i b r a r y resulted i n 97 primary s c r e e n p l u g s c o r r e s p o n d i n g to p o t e n t i a l p o s i t i v e s i g n a l s . ANALYSIS OF OREGON-R PRIMARY SCREEN PHAGES The DNA from the primary s c r e e n phage p l u g s ( c o n t a i n i n g approximately 100 i n d i v i d u a l phage species) was p u r i f i e d i n order to eliminate f a l s e p o s i t i v e s due to non-specific h y b r i d i z a t i o n of GT3 to b a c t e r i a l DNA and d e b r i s . An i n i t i a l strategy was to amplify and prepare DNA from the 97 primary plugs i n pools of ten and to determine which pool had both GT3 and SI h y b r i d i z i n g i n s e r t s . The r e s u l t s suggested that f i v e of the ten pools d i d not contain i n s e r t s which h y b r i d i z e d both probes. However, subsequent a n a l y s i s of DNA from i n d i v i d u a l primary plugs w i t h i n the pool showed t h i s was not the c a s e . T h i s may be due to over or under r e p r e s e n t a t i o n of c e r t a i n phage d u r i n g c o m p e t i t i v e growth. T h e r e f o r e , DNA from a l l 97 p r i m a r y s c r e e n p l u g s had to be 38 p u r i f i e d . H y b r i d i z a t i o n o f b o t h p r o b e s to primary p l u g DNAs r e v e a l e d t h a t : 75 h y b r i d i z e d SI probe wi t h 31 o f these a l s o h y b r i d i z i n g GT3, 3 were GT3 p o s i t i v e o n l y , and 19 d i d not bind either probe (Figure 7). SECONDARY SCREEN OF OREGON-R GENOMIC LAMBDA LIBRARY Low density screening of a l l 31 GT3/S1 p o s i t i v e primary plugs resulted in the i s o l a t i o n of s i n g l e phage species capable of binding GT3. Digestion with S a i l released the e n t i r e i n s e r t from the EMBL3 vector, but t h i s a n a l y s i s was d i f f i c u l t since S a i l cuts the genomic i n s e r t infrequently. Therefore, each phage was digested with H i n d l l l and hybridized with both probes. A l l phages showed d i f f e r e n t r e s t r i c t i o n fragment patterns (although some may be overlapping phages) wi t h the e x c e p t i o n o f 5 phage which were i d e n t i c a l . Four phages (109, 224, 253, 292) h y b r i d i z e d both probes ( F i g u r e 8 ) . The g e l s were d e l i b e r a t e l y o v e r l o a d e d to increase the i n t e n s i t y of the s i g n a l . D i f f e r e n t H i n d l l l fragments h y b r i d i z e d GT3 and SI i n phage 109, 224, and 253, whereas i n phage 292 both probes h y b r i d i z e d the same fragment. Hybridization of GT3 and SI on d i f f e r e n t H i n d l l l fragments i s p l a u s i b l e since the probe binding s i t e s may be separated by introns i n the gene. By analogy to the human CuZn SOD sequence (62) , t h e r e are two i n t r o n s between t h e s e q u e n c e s where t h e two probes sh o u l d h y b r i d i z e . Phage 253 appears to have a strong (2.9 Kb) and weak (1.3 Kb) SI h y b r i d i z i n g fragment. This may be due to two v a r i a n t s of SI binding d i f f e r e n t H i n d l l l fragments of the 39 FIGURE 7. Southern a n a l y s i s o f Oregon-R p r i m a r y screen phages. The phage DNA from each p r i m a r y s c r e e n p l u g was d i g e s t e d w i t h H i n d l l l and the fragments s e p a r a t e d on an agarose g e l ( I ) . There ar e many H i n d l l l f r a g m e n t s p e r l a n e , a s e a c h p r i m a r y p l u g c o n t a i n s a p p r o x i m a t e l y 100 phage s p e c i e s . The most prominent band i s the 4.4 Kb H i n d l l l fragment which i s the EMBL3 vector arm. The DNA f r a g m e n t s were t r a n s f e r r e d o nto Hybond-N and h y b r i d i z e d w i t h GT3 (50°C) or SI (47°C). The a u t o r a d i o g r a m of the GT3 probed f i l t e r ( I I ) shows t h a t a H i n d l l l fragment from primary plugs 1-3 (41-43) and 6 (46) s t r o n g l y h y b r i d i z e s to the GT3 probe whereas t he r e s t o f the phage weakly or qu e s t i o n a b l y h y b r i d i z e the GT3 probe. The a u t o r a d i o g r a m o f the SI probed f i l t e r ( I I I ) shows t h a t m u l t i p l e H i n d l l l f ragments from each primary screen p o o l h y b r i d i z e SI sequences. Therefore, a l l the p r i m a r y p l u g s (41-49) e x a m i n e d may c o n t a i n GT3 and SI probe bindi n g sequences and a l l were a n a l y z e d f u r t h e r . The s i z e marker (m) was lambda DNA digested w i t h H i n d l l l . 40 41 FIGURE 8 . Southern a n a l y s i s o f phage from the Oregon-R l i b r a r y that h y b r i d i z e both GT3 and SI probes. The r e s u l t of screening the Oregon-R genomic l i b r a r y was four phages ( (a)=109, (b)=224, (c)=253, (d)=292 ) that h y b r i d i z e both SI and GT3 probes. (I) The phage DNA was d i g e s t e d w i t h H i n d l l l and analyzed on an 0.7% agarose g e l . A l l four phage show d i f f e r e n t r e s t r i c t i o n patterns and a common 4.4 Kb EMBL3 v e c t o r arm. The DNA was transferred onto Hybond-N and h y b r i d i z e d w i t h GT3 and SI probes. The auto radiograms of the f i l t e r s probed with GT3 (II) and SI (III) show that d i f f e r e n t H i n d l l l fragments h y b r i d i z e d the two probes, except i n phage 292 where both probes h y b r i d i z e the same 7 Kb H i n d l l l fragment. Phage 253 has two SI p o s i t i v e fragments which may be due to h y b r i d i z a t i o n o f d i f f e r e n t SI sequences. A l l four phage were mapped on p o l y t e n e chromosomes to determine whether they o r i g i n a t e from the same chromosomal l o c a t i o n as the SOD gene. The s i z e marker (m) was lambda DNA digested with H i n d l l l . 42 m ) • 11 i n i i n i i I i 43 i n s e r t . Phage 253 i s a l s o the weakest GT3 binding phage as hy b r i d i z a t i o n o f GT3 does not occur a t 52°C. Phage 109, 224, and 292 s t i l l b i n d GT3 a t 54°C. MAPPING OF PHAGE DNA TO DROSOPHILA POLYTENE CHROMOSOMES The chromosomal l o c a t i o n of CuZn SOD has been determined (48,49) to be at 68AB on the t h i r d chromosome and a phage which maps to the same s i t e would be a good c a n d i d a t e f o r being the CuZn SOD c l o n e . Tn s i t u h y b r i d i z a t i o n o f phage 109, 224, 253, and 292 showed that none of these phage hybridized to 68AB. Phage 109, 224, and 292 mapped to 3A3-4 (X), 87D5-7 (3R) , and 77B1-2 (3L) , r e s p e c t i v e l y (Figure 9a) . The GT3 p o s i t i v e phage from the Canton-S l i b r a r y (CS-2) mapped to 90EF (3R) (Figure 9b). Phage 253 mapped to many chromosomal l o c a t i o n s . THE GT3 PROBE BINDING SITE OF PHAGE 109 The GT3 probe b i n d i n g r e g i o n of phage 109 was sequenced to determine whether the probe was h y b r i d i z i n g to related sequences (and p o s s i b l y pseudogenes) or v i a mismatched duplexes. A 2.6 Kb GT3 p o s i t i v e BamHI f r a g m e n t was s u b c l o n e d i n t o pUC13 t o f a c i l i t a t e s u b c l o n i n g s m a l l e r fragments i n t o M13 vectors. The BamHI clone was d i g e s t e d w i t h Sau3A and a l l the fragments were l i g a t e d i n t o M13mpl9 and s c r e e n e d w i t h GT3. A 190 bp GT3 p o s i t i v e Sau3A i n s e r t was sequenced on both s t r a n d s . The GT3 probe b i n d i n g s i t e was found to be 140 bp from one end of the i n s e r t and was r e c o g n i z e d by t r a n s l a t i o n i n one re a d i n g frame r e s u l t i n g i n A s p - C y s - P r o - T h r , f o u r o f t h e s i x amino a c i d s 44 FIGURE 9. D e t e r m i n a t i o n of the chromosomal l o c a t i o n o f phage 109 and phage CS-2 by _iri s i t u h y b r i d i z a t i o n t o p o l y t e n e chromosomes. Iri v i t r o t r a n s c r i p t i o n o f (a) phage CS-2 (the 7.0 Kb EcoRI fragment cloned into pEMBLS-) and (b) phage 109 i n the presence o f [ ^ 2 ^ l l -CTP p r o d u c e d l a b e l l e d RNA which was hybridized to p_^ _ melanogaster p o l y t e n e chromosomes. Phage CS-2 mapped to 90EF (3R) , and phage 109 mapped to 3A3-4 (X) . S i m i l a r l y phage 224, 253 and 292 were also mapped on the polytene chromosomes (figures not shown) , none of which hybridized to 68AB (on 3L) where the CuZn SOD gene i s l o c a t e d . T h i s f i g u r e i s courtesy of Dr. Shizu Hayashi. 45 46 47 s p e c i f i e d by GT3 ( A s p - C y s - P r o - T h r - L y s - V a l ) . The phage 109 seq u e n c e 3'-CTG ACG GGC TGC TAT AC-5' forms 13 p e r f e c t Watson-Crick base p a i r s with GT3 (Figure 10). The duplex ends at p o s i t i o n 14 with a dA:dA mismatch (r e a d i n g 5' — > 3' on GT3). This phage 109 sequence may h y b r i d i z e to 5'-GAC TGC CCG ACG AAA GT-3' and 5'-GAC TGC CCG ACG AAG GT-3' o f the GT3 probe. These GT3 sequences form a 13 base p a i r duplex with phage 109 which i s s t a b l e even at 54°C, p r o b a b l y due t o t h e h i g h GC c o n t e n t (9/13) of the f i r s t 13 n u c l e o t i d e s o f the sequence. T r a n s l a t i o n of the e n t i r e 190 bp i n s e r t i n a l l 3 phases d i d not show any other amino acid homology to the CuZn SOD sequence. 48 FIGURE 10. Nucleotide sequence and t r a n s l a t i o n of the GT3 probe binding s i t e of phage 109 from the Oregon-R l i b r a r y . T r a n s l a t i o n of the nucleotide sequence produced the sequence Asp-Cys-Pro-Thr, f o u r o f t h e s i x a m i n o a c i d s s p e c i f i e d b y G T 3 (Asp-Cys-Pro-Thr-Lys-Val). The phage 109 sequence 3'-CTG ACG GGC TGC TAT AC-5* forms 13 p e r f e c t Watson-Crick base pai r s with the co r r e s p o n d i n g GT3 sequences 5'-GAC TGC CCG ACG AAA GT-3' and 5'-GAC TGC CCG ACG AAG GT-3'. ( *** = amber codon ) 49 80 90 CTC CGC CAC TAG TCC ACT AGT LEU ARG HIS * * * SER THR SER 100 110 120 TAG TTG CCT CCT CTG CGA GCC * * * LEU PRO PRO LEU ARG ALA 130 140 ATC ACA CCT CAA TAC TGT TCA ILE THR PRO GLN TYR CYS SER 150 160 GAC TGC . CCG ACG ATA TGT TGT ASP CYS PRO THR ILE CYS CYS 170 180 CCG TGT GGC TGC CCA GGC GCG PRO CYS GLY CYS PRO GLY ALA 190 TCC TCT T SER SER 50 DISCUSSION To d a t e , mixed sequence o l i g o d e o x y r i b o n u c l e o t i d e probes 13-17 bases i n l e n g t h have been s u c c e s s f u l l y used to i s o l a t e p r o t e i n genes of unknown DNA sequence from l i b r a r i e s of low to moderate c o m p l e x i t y (61,63). The mixed sequence probes often lack the s p e c i f i c i t y required f o r probing sequences as complex as those i n a Drosophila or mammalian genomic l i b r a r y . Successful use of mixed sequence o l i g o n u c l e o t i d e probes r e s t s l a r g e l y on probe design i . e . heterogeneity, base sequence, and length of the probe. Each o f these f a c t o r s and t h e i r i m p l i c a t i o n s on probe design w i l l be d i s c u s s e d i n l i g h t o f the r e s u l t s presented i n t h i s study. I. OLIGONUCLEOTIDE PROBE HETEROGENEITY The redundancy o f the g e n e t i c code o f t e n l e a d s to complex mixtures of o l i g o n u c l e o t i d e probes. Genes have been i s o l a t e d using mixtures of great c o m p l e x i t y (>32 d i f f e r e n t sequences) but o n l y i n cDNA l i b r a r i e s (64). In mi x t u r e s o f g r e a t e r than 16 sequences, each i n d i v i d u a l sequence comprises l e s s than 6% of the t o t a l mixture, r e s u l t i n g i n u n f a v o u r a b l e s i g n a l to noise r a t i o s i n h y b r i d i z a t i o n experiments. For example, genomic DNA Southerns h y b r i d i z e d w i t h SI d i d n o t p r o d u c e d i s c e r n i b l e bands as the background was high. T h e r e f o r e , the g r e a t sequence complexity of the SI probe (128 v a r i a n t s ) r e s t r i c t e d i t s use to h y b r i d i z a t i o n with pure cloned DNA only. The greater the h e t e r o g e n e i t y o f the probe, the larger the 51 range of d i s s o c i a t i o n t e m p e r a t u r e s f o r o l i g o n u c l e o t i d e - D N A d u p l e x e s w i l l be. The d i s s o c i a t i o n t e m p e r a t u r e o f e a c h o l i g o n u c l e o t i d e d i f f e r s , making th e s e l e c t i o n o f a s u i t a b l y s t r i n g e n t and s e l e c t i v e h y b r i d i z a t i o n c o n d i t i o n for a l l members d i f f i c u l t or i m p o s s i b l e . T h e r e f o r e , a l a r g e number of p o s i t i v e s are picked up i n s c r e e n i n g complex l i b r a r i e s . The greater the complexity of the probe, the g r e a t e r the p r o b a b i l i t y of binding unrelated sequences w i l l be. T h i s was observed i n the primary screen of the Oregon-R l i b r a r y . In p r i n c i p l e , every sequence i n the SI probe may e l i c i t a p o s i t i v e s i g n a l and most of the phage (75/97) did h y b r i d i z e SI sequences. The four phages (109, 224, 253, 292) which h y b r i d i z e d to both GT3 and SI probes a l l mapped to d i f f e r e n t chromosomal l o c a t i o n s . T h e r e f o r e , i t i s v e r y important to keep t h e number o f s e q u e n c e s i n a probe to a minimum. Low degeneracy probes a r e p o s s i b l e i f an amino acid sequence contains methionine and tryptophan, both having only one codon. A s i n g l e codon may a l s o be chosen f o r an amino acid by examining codon preference data i n t h a t organism, as there e x i s t s a b i a s i n the usage of the s e v e r a l degenerate codons for an amino acid (65,66). However, t h i s i s o n l y p o s s i b l e i f extensive codon usage data for the organism are a v a i l a b l e and there e x i s t s the p o s s i b i l i t y t h a t the p r e f e r r e d c o d o n may not be used a t the sequence where the oligonucleotide probe i s targeted. I I . OLIGONUCLEOTIDE PROBE SEQUENCE The oligonucleotide sequence determines the s t a b i l i t y of the 52 oligonucleotide-DNA complex. The s t a b i l i t y depends not only on the nucleotide c o m p o s i t i o n , but a l s o on the nucleotide sequence of the probe (67-69). I t has been noted that i n a 17 nucleotide long mixed sequence probe, h y b r i d i z a t i o n o f the lowest melting probe (usually lowest GC content) w i l l a l s o allow h y b r i d i z a t i o n of 12 or 13 bp r e g i o n s (70). The GT3 b i n d i n g s i t e of phage 109 shows t h a t at the h y b r i d i z a t i o n s t r i n g e n c y chosen to i n c l u d e lower m e l t i n g sequences, a 13 bp d u p l e x was r e s p o n s i b l e f o r e l i c i t i n g the GT3 p o s i t i v e s i g n a l . The degeneracy o f the 17 base GT3 probe was decreased from 128 to 8 variants by the placement o f dG i n p o s i t i o n s 9 and 12, where a l l four nucleotides may o c c u r . P a i r i n g with dC i s normal, but p a i r i n g with dG i s c o n s i d e r e d u n f a v o r a b l e . I t was assumed t h a t dG forms a f a v o r a b l e wobble base p a i r w i t h dT and t h a t p a i r i n g to dA i s weakly s t a b i l i z i n g . This assumption was based on observations that the r i b o n u c l e o t i d e s rG:rU and rG:rT form stable base p a i r s . However, i t has been argued t h a t the assumption i s f a l s e s i n c e dG:dT i s l e s s s t a b l e than rG:rT (68,71). Recent s t u d i e s on t h e s t a b i l i t y o f m i s m a t c h e s i n o l i g o n u c l e o t i d e duplexes r e v e a l t h a t the s t a b i l i t y of dG:dT, dG:dG and dG:dA p a i r s are comparable and s t a b i l i z i n g (69). The placement o f dG as the t h i r d base o f the p r o l i n e and t h r e o n i n e c o d o n s may n o t a g r e e w i t h codon p r e f e r e n c e s i n D r o s o p h i l a . A l t h o u g h e x t e n s i v e c o d o n usage d a t a a r e n o t a v a i l a b l e f o r D r o s o p h i l a , t h e sequence a n a l y s i s o f 8 genes 53 reveals that f o r p r o l i n e the codon CCG i s the l e a s t used and that CCC i s the most used. S i m i l a r i l y f o r t h r e o n i n e , ACG i s r a r e l y used and ACC i s the most f r e q u e n t l y used (66). In both cases, the most favored codon would r e q u i r e a dG:dG p a i r i n g with the GT3 sequence. Although dG:dG i n t e r a c t i o n s a r e not d e s t a b i l i z i n g , i t i s important to remember that the d i f f e r e n c e i n s t a b i l i t y between dC:dG and the mismatched dG:dT, dG, dA i s large (69). Therefore, the placement of dG in these p o s i t i o n s i n GT3 would increase the tendency o f the probe to bind r e l a t e d but i n c o r r e c t sequences containing dC at the c o r r e s p o n d i n g p o s i t i o n on the target DNA. Both GT3 binding s i t e s sequenced i n t h i s study show dG:dC p a i r i n g at p o s i t i o n s 9 and 12 of the probe, and both strongly hybridize unrelated sequences. I I I . OLIGONUCLEOTIDE PROBE LENGTH The length of the o l i g o n u c l e o t i d e probe required to pr e d i c t an unique s e q u e n c e may be s t a t i s i c a l l y c a l c u l a t e d . For a g D r o s o p h i l a genome (1.65x10 b p ) , a probe 15 n u c l e o t i d e s long should p r e d i c t a unique sequence whereas for the human genome 9 (3x10 bp) a probe 17 n u c l e o t i d e s i n l e n g t h i s r e q u i r e d (71). Each member o f the mixed sequence probe may h y b r i d i z e a unique sequence s p e c i f y i n g the same amino a c i d s . The c l o n e i s o l a t e d from the Canton-S l i b r a r y had a 15 bp sequence capable of coding for f i v e of the amino acids i n CuZn SOD. This nucleotide sequence should be unique, and the sequence i n the SOD gene should be 54 s p e c i f i e d by another member o f GT3. The GT3 binding sequences i n the two clo n e s s t u d i e d are pr o b a b l y i n t e r g e n i c sequences since the p o t e n t i a l open r e a d i n g f r a m e s a r e not v e r y l o n g . These i n t e r g e n i c sequences would be e l i m i n a t e d i n a cDNA l i b r a r y . Screening lower c o m p l e x i t y l i b r a r i e s (such as cDNA l i b r a r i e s ) has not always been s u c c e s s f u l u s i n g two s h o r t oligonucleotide probes (72,73). T h i s has prompted the use o f lo n g e r unique probes (74). In long probes (>50 bp) , the u n c e r t a i n t y a t each codon i s l a r g e l y i g n o r e d , as the probe l e n g t h i s used to c o n f e r probe s p e c i f i c i t y . T h e o r e t i c a l l y , t h e s p e c i f i c i t y o f a two probe combination i s l e s s than t h a t o f a probe o f length equal to the combined length of the shorter probes (68). I s o l a t i o n of genomic clones using short mixed oligonucleotide probes has never been reported. An unique 52 base long probe was used to i s o l a t e a genomic c l o n e c o n t a i n i n g the human factor IX gene (74). The two other cases o f genomic clone i s o l a t i o n used even l o n g e r probes, a l t h o u g h t h e y may have been l o n g e r than necessary (75,76). T h e r e f o r e , l o n g e r unique probes may be used to overcome the problems encountered using short mixed probes. IV. OLIGONUCLEOTIDE PROBE DESIGN Theoretical and p r a c t i c a l considerations of probe design have r e c e n t l y been d e s c r i b e d i n g r e a t d e t a i l (68). The problems encountered i n s e a r c h i n g f o r g e n e s i n DNA banks o f g r e a t e r complexity has r e q u i r e d m o d i f i c a t i o n o f the c l a s s i c a l strategy 55 used in t h i s study. Longer probes have been used to i n c r e a s e probe s p e c i f i c i t y as short stretches o f p e r f e c t homology o c c u r r i n g by chance have produced f a l s e p o s i t i v e s . One approach used to obtain longer p r o b e s has been t o "snap t o g e t h e r " 10 o v e r l a p p i n g s h o r t o l i g o n u c l e o t i d e s and f i l l i n g i n the gaps to form a long double stranded probe (76). Another modification of probe d e s i g n uses deoxyinosine (dl) i n p o s i t i o n s of ambiguity where a l l 4 n u c l e o t i d e s are p o s s i b l e . Inosine i s a guanosine analog and o c c u r s n a t u r a l l y i n the wobble p o s i t i o n of the anticodon o f some t r a n s f e r RNAs where i t appears t o p a i r w i t h r C , rU and r A . The t h e r m a l s t a b i l i t y o f o l i g o n u c l e o t i d e d u p l e x e s c o n t a i n i n g d e o x y i n o s i n e has been studied. Independent of sequence e f f e c t s , the order of s t a b i l i t y i s dI:dC > dltdA > dI:dT, dI:dG (77). Deoxyinosine i s s u p e r i o r to dG o p p o s i t e dA/dC ambiguities since d l p a i r s l e s s s t r o n g l y w i t h dC and more s t r o n g l y with dA than dG. For p r o b i n g dG/dT a m b i g u i t i e s , both dG and d l are non-selective. Use of d l i n these cases i s also advantageous, as d l does not increase the tendency o f the probe to bind unrelated sequences by forming s t r o n g dG:dC p a i r s . With these f a c t o r s i n mind, one must d e c i d e whether the b e s t sequence f o r the probe corresponds to the coding or non-coding strand. •In l i g h t o f the r e s u l t s o f t h i s study, an improved probe might be a m o d i f i e d GT3 w h i c h u s e s d l i n p o s i t i o n s o f base 56 ambiguity 5'- ACC TTI GTI GGA CAA TCI CCI GTI GC -3' T G G The r e v i s e d GT3 probe i s 26 n u c l e o t i d e s i n l e n g t h and i s derived from the non-coding s t r a n d as opposed to GT3, which was from the coding strand. The new probe would also be 9 nucleotides longer and s p e c i f y 3 a d d i t i o n a l amino ac i d s . Despite the g u i d e l i n e s a v a i l a b l e f o r o l i g o n u c l e o t i d e probe design, d e c i s i o n s c o n c e r n i n g probe l e n g t h , h e t e r o g e n e i t y , and sequence are s t i l l a matter o f educated guesswork and whether any probe w i l l f i n d the d e s i r e d gene sequence can only be determined by experimentation. 57 REFERENCES 1. McCord, J.M., F r i d o v i c h , I . (1969) Superoxide dismutase - An enzymic f u n c t i o n f o r e r y t h r o c u p r e i n (hemocuprein), J . B i o l . Chem., 244, 6049-6055. 2. F r i d o v i c h , I . (1978) The b i o l o g y o f oxygen r a d i c a l s , Science, 201, 875-880. 3. F r i d o v i c h , I. (1982) T o x i c i t y i n prokaryotes: importance of superoxide dismutase, i n Superoxide Dismutases V o l . I, Oberley, L.W., Ed., CRC Press, Inc., Boca Raton, 79-88. 4. Brunori, M., R o t i l i o , G. (1984) Bi o c h e m i s t r y of oxygen r a d i c a l species, Meth. Enzymol., 105, 22-35. 5. W e i s i g e r , R.A., F r i d o v i c h , I . (1973) Superoxide dismutase - o r g a n e l l e s p e c i f i c i t y , J . B i o l . Chem., 248, 3582-3592. 5. Steinman, H.M. (1982) Superoxide dismutases: p r o t e i n c h e m i s t r y and s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s , i n Superoxide Dismutase V o l . I, Oberl e y , L.W., Ed., CRC Press, Inc., Boca Raton, 11-68. 7. H a l l i w e l l , B., Gutteridge, J.M.C. (1984) Role of ir o n i n oxygen r a d i c a l reactions, Meth. Enzymol., 105, 47-56. 8. F r i d o v i c h , I . (1982) M e a s u r i n g t h e a c t i v i t y o f sup e r o x i d e d i s m u t a s e s : an embarrassment o f r i c h e s , i n Superoxide Dismutase V o l . I, Oberl e y , L.W., Ed., CRC Press, Inc., Boca Raton, 69-77. 9. McCord, J.M., C r a p o , J.D., F r i d o v i c h , I . (1977) Superoxide dismutase a s s a y s : a review o f methodology, i n Superoxide and S u p e r o x i d e D i s m u t a s e s , M i c h e l s o n , A.M., McCord, J.M., F r i d o v i c h , I . , Eds., Academic Press, London, 11-17. 10. F l o h e , L., O t t i n g , F. (1984) Superoxide dismutase assays, Meth. Enzymol., 105, 88-93. 11. C r a p o , J.D., McCord, J.M., F r i d o v i c h , I . (1978) P r e p a r a t i o n and a s s a y o f s u p e r o x i d e d i s m u t a s e s , Meth. Enzymol., 53, 382-393. 12. K i r b y , T.W., F r i d o v i c h , I . (1982) A p i c o m o l a r s p e c t r o p h o t o m e t r i c assay f o r su p e r o x i d e dismutase, Anal. Biochem., 127, 435-440. 58 13. Beauchamp, C , F r i d o v i c h , I . (1971) S u p e r o x i d e dismutase: improved a s s a y s and an assay a p p l i c a b l e to acrylamide g e l s , Anal. Biochem., 44, 276-287. 14. C u t l e r , R.G. (1985) A n t i o x i d a n t s and l o n g e v i t y o f mammalian species, Basic L i f e Sciences, 35, 15-73. 15. Sohal, R.S., A l l e n , R.G. (1985) R e l a t i o n s h i p between metabolic rate, free r a d i c a l s , d i f f e r e n t i a t i o n and aging: a u n i f i e d theory, Basic L i f e Sciences, 35, 75-104. 16. Harman, D. (1981) The aging process, Proc. N a t l . Acad. S c i . U.S.A., 78, 7124-7128. 17. Harman, D. (1968) Free r a d i c a l theory of aging: e f f e c t of free r a d i c a l r e a c t i o n i n h i b i t o r s on the m o r t a l i t y rate of male LAF mice, J . Gerontology, 23, 476-482. 18. Harman, D. (1982) The f r e e r a d i c a l theory of aging, i n Free Radicals in B i o l o g y V o l . V, P r y o r , W.A., Ed., Academic Press, New York, 255-275. 19. Shock, N.W. (1981) B i o l o g i c a l theories of aging, i n CRC Handbook of B i o c h e m i s t r y i n Aging, F l o r i n i , J.R., Ed., CRC Press Inc., Boca Raton, 271-232. 20. Bartosz, G., Tannert, C., F r i e d , R., Leyko, W. (1978) Superoxide dismutase a c t i v i t y d e c r e a s e s during erythrocyte aging, Experientia, 34, 1464. 21. Paynter, D.I., C a p l e , I.W. (1984) Age-related changes i n a c t i v i t i e s of the s u p e r o x i d e dismutase enzymes i n t i s s u e of the sheep and the e f f e c t o f d i e t a r y copper and manganese on these changes, J . N u t r i t i o n , 114, 1090-1916. 22. R e i s s , U., Gershon, D. (1976) R a t - l i v e r superoxide dismutase, Eur. J . Biochem., 63, 617-623. 23. Kellogg, E.W., F r i d o v i c h , I. (1976) Superoxide dismutase i n the r a t and mouse as a f u n c t i o n o f age and longevity, J . Gerontology, 31(4), 405-408. 24. Reiss, U., Gershon, D. (1976) Comparison of cytoplasmic superoxide dismutase i n l i v e r , h e a r t and b r a i n of aging r a t s and mice, Biochem. Biophys. Res. Commun., 73, 255-262. 59-25. M a s s i e , H.R., A i e l l o , V.R., I o d i c e , A.A. (1979) Ghanges with age i n copper and s u p e r o x i d e dismutase l e v e l s i n brains o f C57BL/6J mice, Mech. Ageing Dev., 10, 93-99. 26. Lammi-Keffe, C.J., Swan, P.B., Hegarty, P.V.J. (1984) Copper-zinc and manganese sup e r o x i d e dismutase a c t i v i t i e s i n cardiac and s k e l e t a l muscles d u r i n g aging i n male r a t s , Gerontology, 30, 153-158. 27. N i c k l a , H., Anderson, J . , P a l z k i l l , T. (1983) Enzymes i n v o l v e d i n oxygen d e t o x i f i c a t i o n d u r i n g development o f Drosophila melanogaster, E x p e r i e n t i a , 39, 610-612. 28. Massie, H.R., A i e l l o , V.R. W i l l i a m s , T.R. (1980) Changes in superoxide dismutase a c t i v i t y and copper during development and a g e i n g i n t h e f r u i t f l y D r o s o p h i l a  melanogaster, Mech. Ageing Dev., 12, 279-286. 29. Bartosz, G., Leyko, W., F r i e d , R. (1979) Superoxide d i s m u t a s e and l i f e span o f D r o s o p h i l a m e l a n o g a s t e r , Experientia, 35, 1193. 30. T o l m a s o f f , J.M., Ono, T., C u t l e r , R.G. (1980) S u p e r o x i d e d i s m u t a s e : c o r r e l a t i o n w i t h l i f e - s p a n and s p e c i f i c m e t a b o l i c r a t e i n p r i m a t e s p e c i e s , Proc. N a t l . Acad. S c i . U.S.A., 77, 2777-2781. 31. C u t l e r , R.G. (1984) A n t i o x i d a n t s , aging, and longevity, i n Free R a d i c a l s i n B i o l o g y V o l . 6, P r y o r , W.A., Ed., Academic Press, New York, 371-428. 32. Rubin, G.M., S p r a d l i n g , A.C. (1983) V e c t o r s f o r P-element mediated gene t r a n s f e r i n D r o s o p h i l a , Nucleic Acids Res., 11(18), 6341-6351. 33. R u b i n , G.M., S p r a d l i n g , A.C. (1982) G e n e t i c t r a n s f o r m a t i o n o f D r o s o p h i l a w i t h t r a n s p o s a b l e element vectors, Science, 218, 348-353. 34. Spradling, A . C , Rubin, G.M., Transposition of cloned P-elements into D r o s o p h i l a Germ L i n e Chromosomes, Science, 218, 341-353, 1982. 35. S t e l l e r , H., P i r r o t t a , V., A transposable P vector that confers s e l e c t a b l e G418 r e s i s t a n c e to Drosophila larvae, EMBO J . , 4, 167-171, 1985. 60 36. L e e , Y.M., F r i e d m a n , D . J . , A y a l a , F . J . (1985) Superoxide dismutase: an e v o l u t i o n a r y p u z z l e , Proc. N a t l . Acad. S c i . U.S.A., 82, 824-828. 37. H e r i n g , K., Kim, S.A., M i c h e l s o n , A.M., O t t i n g , F., Puget, K., S t e f f e n s , G.J., Fl o h e , L. (1985) The primary structure of porcine Cu-Zn superoxide dismutase-evidence for a l l o t y p e s o f s u p e r o x i d e d i s m u t a s e i n p i g s , B i o l . Chem. Hoppe-Seyler, 366, 435-445. 38. Rocha, H.A., B a n n i s t e r , W.H., B a n n i s t e r , J.V. (1984) The amino-acid sequence o f c o p p e r / z i n c superoxide dismutase from swordfish l i v e r , Eur. J . Biochem., 145, 477-484. 39. Lee, Y.M., Ayala, F.J., Misra, H.P. (1985) P u r i f i c a t i o n and p r o p e r t i e s o f s u p e r o x i d e d i s m u t a s e from D r o s o p h i l a  melanogaster, J . B i o l . Chem., 260, 2212-2217. 40. Lee, Y.M., M i s r a , H.P., A y a l a , F . J . (1981) Superoxide dismutases i n D r o s o p h i l a m e l a n o g a s t e r : B i o c h e m i c a l and s t r u c t u r a l c h a r a c t e r i z a t i o n of allozyme v a r i a n t s , Proc. N a t l . Acad. S c i . U.S.A., 78, 7052-7055. 41. Lee, Y.M., A y a l a , F . J . (1985) Superoxide dismutase i n Drosophila melanogaster, mutation s i t e d i f f e r e n c e between two electromorphs, FEBS, 179, 115-119. 42. K a i s e r , K., Murray, N.E. (1985) The use o f phage lambda r e p l a c e m e n t v e c t o r s i n t h e c o n s t r u c t i o n o f representative genomic DNA l i b r a r i e s , i n DNA Cloning, V o l . I, Glover, D.M. (Ed.), IRL Press, Oxford, 1-47. 43. M a n i a t i s , T., H a r d i s o n , R . C , Lacy, E., Lauer, J . , O'Connell, C , Quon, D., Sim, G., E f s t r a t i a d i s , A. (1978) The i s o l a t i o n o f s t r u c t u r a l g e n e s from l i b r a r i e s o f eukaryotic DNA, C e l l , 15, 687-701. 44. Wallace, R.B., S h a f f e r , J . , Murphy, R.F., Bonner, J . , Hirose, T., I t a k u r a , K. (1979) H y b r i d i z a t i o n o f synthetic o l i g o d e o x y r i b o n u c l e o t i d e s to $ X174 DNA: the e f f e c t o f a s i n g l e base p a i r mismatch, Nucleic Acids Res., 6, 3543-3557. 45. Wallace, R.B., Johnson, M.J., H i r o s e , T., Miyake, T., Kawashima, E.H., I t a k u r a , K. (1981) The use o f synthetic o l i g o n u c l e o t i d e s as h y b r i d i z a t i o n probes I I . Hybridization of oligonucleotides o f mixed sequence to ra b b i t beta-globin DNA, Nucleic Acids Res., 9, 879-894. 61 46. Meinkoth, J . , Wahl, G. (1984) Hybridization of n u c l e i c a c i d s immobilized on s o l i d s u p p o r t s , A n a l . Biochem. 138, 267-284. 47. Smith, M. (1985) S y n t h e t i c oligodeoxyribonucleotides as to o l s i n molecular genetics: the c h a r a c t e r i z a t i o n of the CYC1 ( i s o - l - c y t o c h r o m e c e n c o d i n g ) l o c u s o f Saccharomyces  c e r e v i s i a e , B i o c h i m i e , 67, 717-723. 0 0 48. Jelnes, J.E. (1971) I d e n t i f i c a t i o n of hexokinases and l o c a l i s a t o n of a fructokinase and a tetrazolium oxidase locus i n D r o s o p h i l a melanogaster, H e r e d i t a s , 67, 291. 49. Campbell, S.D., H i l l i k e r , A . J . , P h i l l i p s , J.P. (1986) Cytogenetic a n a l y s i s o f the cSOD m i c r o r e g i o n i n Drosophila  melanogaster, G e n e t i c s , 112, 205-215. 50. Benton, W.D., D a v i s , R.W. (1977) Screening lambda-gt recombinant c l o n e s by h y b r i d i z a t i o n to s i n g l e plaques i n  s i t u , S c i e n c e , 196, 180-182. 51. Yamamoto, K.R., A l b e r t s , B.M. (1970) R a p i d bacteriophage sedimentation i n the presence of polyethylene g l y c o l and i t s a p p l i c a t i o n t o l a r g e s c a l e v i r u s p u r i f i c a t i o n , Virology, 40, 734-744. 52. Southern, E.M. (1975) D e t e c t i o n o f s p e c i f i c sequences among DNA fragments sepa r a t e d by g e l e l e c t r o p h o r e s i s , J . Mol. B i o l . , 98, 503-517. 53. G a l l , J.G., P a r d u e , M.L. (1971) N u c l e i c a c i d h y b r i d i z a t i o n i n c y t o l o g i c a l p r e p a r a t i o n s , Methods i n Enzymology, XXI, Grossman, L., Moldave, K. (Eds.), Academic Press, New York, 470-480. 54. Wensink, P.C., Finnegan, D.J., Donelson, J.E., Hogness, D.S. (1974) A system f o r mapping DNA sequences i n the chromosomes of D r o s o p h i l a melanogaster, C e l l , 3, 315-325. 55. Pardue, M.L. (1985) In s i t u h y b r i d i s a t i o n , i n Nucleic Acid H y b r i d i s a t i o n , Haines, B.D., Higgins, S.J. (Eds.), IRL Press, Oxford, 179-202. 56. M a n i a t i s , T. , F r i t s c h , E .F., Sambrook, J . (1982) Molecular C l o n i n g . A L a b o r a t o r y Manual, published by Cold Spring Harbor Laboratory Press, 1982. 62 57. Messing, J . (1983) New M13 v e c t o r s f o r c l o n i n g , Methods in Enzymology, 101, 20-78. 58. Chen, E.Y., Seeburg, P.H. (1985) Supercoil sequencing: a f a s t and simple method f o r sequencing plasmid DNA, DNA, 4, 165-170. 59. Sanger, F ., N i c k l e n , S. , Coul s o n , A.R. (1977) DNA sequencing w i t h c h a i n - t e r m i n a t i n g i n h i b i t o r s , Proc. N a t l . Acad. S c i . U.S.A., 74, 5463-5466. 60. Newton, C H . (1984) MSc. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia. 61. Suggs, S.V., Wallace, B., H i r o s e , T., Kawashima, E.H., I t a k u r a , K. (1981) Use o f s y n t h e t i c o l i g o n u c l e o t i d e s as h y b r i d i z a t i o n probes: I s o l a t i o n o f cl o n e d cDNA sequences f o r human b e t a ^ - m i c r o g l o b u l i n , P r o c . N a t l . Acad. S c i . U.S.A., 78, 6612F-6617. 62. Levanon, D., Lieman-Hurwitz, J . , D a f r i c , N., Widgerson, M., Sherman, L., B e r n s t e i n , Y., Laver-Rudich, Z., Danciger, E., S t e i n , 0., Groner, Y. (1985) A r c h i t e c t u r e and anatomy of the chromosomal l o c u s i n human chromosome 21 encoding the Cu/Zn superoxide dismutase, EMBO J . , 4, 77-84. 63. M i c h e l s o n , A.M., Markham, A . F . , O r k i n , S.H. (1983) I s o l a t i o n and DNA sequence o f a f u l l - l e n g t h cDNA clone for human X chromosome-encoded phosphoglycerate kinase, Proc. N a t l . Acad. S c i . U.S.A., 80, 472-476. 64. Balland, A., Courtney, M. , J a l l a t , S., T e s s i e r , L.H., Sondermeyer, P., de l a S a l l e , H., Harvey, R., Degryse, E., Tolstoshev, P. (1985) Use of s y n t h e t i c oligonucleotides i n gene i s o l a t i o n and manipulation, Biochemie, 67, 725-736. 65. Grantham, R. , G a u t i e r , C. , Gouy, M. (1980) Codon f r e q u e n c i e s i n 119 i n d i v i d u a l g e n e s c o n f i r m c o n s i s t e n t choices of degenerate bases a c c o r d i n g to genome type, Nucleic Acids Research, 8, 1893-1912. 66. O'Connell, P., Rosbash, M. (1984). Sequence, s t r u c t u r e , and codon preference o f the D r o s o p h i l a ribosomal protein 49 gene, Nucleic Acids Research, 12, 5495-5513. 63 67. B r e s l a u e r , K.J., Frank, R., B l o c k e r , H., Marky, L.A. (1986) P r e d i c t i n g DNA d u p l e x s t a b i l i t y from the base sequence, Proc. N a t l . Acad. S c i . U.S.A., 83, 3746-3750. 68. Lathe, R. (1985) S y n t h e t i c o l i g o n u c l e o t i d e probes deduced from amino a c i d s e quence d a t a : t h e o r e t i c a l and p r a c t i c a l considerations, J . Mol. B i o l . , 183, 1-12. 69. A b o u l - e l a , F., Koh, D., T i n o c o , I . (1985) Base-base mismatches. Thermodynamics o f double h e l i x formation for dCA^XA-.G + d C T Y T ->G (X , Y =A, C , G , T) , N u c l e i c A c i d s Research, 13, 4811-4824. J 70. Wood, W.I., G i t s c h i e r , J . , Lasky, L.A., Lawn, R.M. (1985) Base c o m p o s i t i o n - i n d e p e n d e n t h y b r i d i z a t i o n i n tetramethylammonium c h l o r i d e : A method f o r olig o n u c l e o t i d e s c r e e n i n g o f h i g h l y complex gene l i b r a r i e s , P roc. N a t l . Acad. S c i . U.S.A., 82, 1585-1588. 71. Smith, M. (1983) S y n t h e t i c oligodeoxyribonucleotides as probes f o r n u c l e i c a c i d s and as primers i n sequence determination i n Methods of DNA and RNA sequencing, Weissman, S.M. (Ed.), Praeger Publishers, New York, 23-68. 72. F u r u t a n i , Y., Morimoto, Y., Shibahara, S., Noda, M., Takahashi, H., Hirose, T., A s a i , M., Inayama, S., Hayashida, H., M i y a t a , T., Numa, S. (1983) C l o n i n g and sequence a n a l y s i s o f cDNA f o r ovi n e c o r t i o t r o p i n - r e l e a s i n g f a c t o r precursor, Nature, 301, 537-540. 73. Itoh, N., Slemmon, J.R., Hawke, D.H., Williamson, R., M o r i t a , E., I t a k u r a , K., R o b e r t s , E., S h i v e l y , J .E., C r a w f o r d , G.D., S a l v a t e r r a , P.M. (1986) C l o n i n g o f Drosophila c h o l i n e a c e t y l t r a n s f e r a s e cDNA, Proc. N a t l . Acad. S c i . U.S.A., 83, 408.1-4085. 74. Jaye, M., de l a S a l l e , H., Schamber, F., Balland, A., Ko h l i , V., F i n d e l i , A., T o l s t o s h e v , P., Lecocq, J . (1983) I s o l a t i o n of a human a n t i - h a e m o p h i l i c f a c t o r IX cDNA clone u s i n g a unique 52-base s y n t h e t i c o l i g o n u c l e o t i d e probe deduced from the amino a c i d sequence of bovine f a c t o r IX, Nucleic Acids Research, 11, 2325-2334. 64 75. U l r i c h , A., Berman, C.E., D u l l , T . J . , Gray, A., Lee, J.M. (1984) I s o l a t i o n o f t h e human i n s u l i n - l i k e growth f a c t o r I gene usi n g a s i n g l e s y n t h e t i c DNA probe, EMBO J . , 3, 361-364. 76. Anderson, S., K i n g s t o n , I.B. (1983) I s o l a t i o n o f a genomic c l o n e f o r bovine p a n c r e a t i c t r y p s i n i n h i b i t o r by using a unique-sequence s y n t h e t i c DNA probe, Proc. N a t l . Acad. S c i . U.S.A., 80, 6838-6842. 77. M a r t i n , G.H., C a s t r o , M.M. (1985) Base p a i r i n g involving deoxyinosine: i m p l i c a t i o n s f o r probe design, Nuc. Acids Research, 13, 8927-8938. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097048/manifest

Comment

Related Items