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Low level Tc1 transposition and Tc1-induced mutations in dpy-5 Babity, Joseph M. 1993

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LOW LEVEL TC1 TRANSPOSITION AND TC1-INDUCED MUTATIONS IN DPY-5 by JOSEPH MARK BABITY B.Sc., University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES GENETICS PROGRAM  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA February 1993 © Joseph Mark Babity, 1993  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.  (Signature)  Department of MEDICAL GENETICS The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  March 5, 1993  ABSTRACT  The transposable element Tcl is the most thoroughly studied transposon in the nematode Caenorhabditis elegans. In this thesis I have examined Tcl transposition in the Bristol strain of C. elegans and analyzed Tcl-induced mutations in the dpy-5 gene of chromosome I. In the Bristol strain of C. elegans, germline Tcl transposition has been undetectable; however, Bristol isolates were identified in which lowlevel Tcl transposition was observed. In the gene identified as dpy-5, one of many genes that affect overall worm morphology in C. elegans, Tcl-induced mutations were examined and used to clone the dpy-5 gene and a second gene in the dpy-5 region. A novel Tcl element from a Bristol isolate was cloned and the genomic insertion site was analyzed. The sequence of the insertion site was found to be similar to the consensus sequence for transposition insertion indicating that germline Tcl transposition had occurred. Further analysis identified a derivative Bristol strain in which a high level of germline Tcl transposition was observed.  Abstract  Six Tcl-induced alleles of dpy-5 were studied. From one of these alleles a novel 2.7 kilobase pair Tclhybridizing EcoRI fragment was isolated and used as a tag in cloning the dpy-5 gene. Examination of Tcl-induced mutations of dpy-5 and wildtype revertants revealed that the mutation was the result of Tcl transposition into a 1.1 kilobase pair genomic EcoRI fragment. The dpy-5 gene was sequenced and shown to encode a 254 amino acid protein. The gene has no known homologues or protein patterns; however, a potential secretion signal peptide has been identified. Northern analysis indicates that the gene is developmentally expressed, correlating well with the onset of the Dpy-5 phenotype. Analysis of DNA sequences upstream from the dpy-5 gene reveals the presence of a neighbouring gene with homology to casein kinase I.  iii  TABLE OF CONTENTS TITLE PAGE ^  i  ABSTRACT ^  ii  TABLE OF CONTENTS ^  iv  LIST OF FIGURES ^ LIST OF TABLES ^ ACKNOWLEDGEMENTS ^ DEDICATION ^  viii x xi xii  I.^INTRODUCTION ^  1  Section 1 1.1. Eukaryotic Transposable Elements ^ 3 1.2. The C. elegans Transposable Element Tc1 ^ 4 1.3. Tcl Transposition ^  6  1.4. The Target Site for Tcl Insertion ^ 8 1.5. Tcl Excision ^ 1.6. Evidence for Homologue Dependent Tc1 Reversion ^  8 10  1.7. Regulation of Tc1 Transposition ^ 11 1.8. Tissue-Specific Regulation of Tcl Activity ^  13  1.9. Tcl Activity in Bristol Strains of C. elegans ^  15  1.10. Tcl Transposition in an N2/B0 Hybrid Strain ^  16  Section 2 2.1. Genes Important in Organismal Morphology ^ 17  iv  Table of Contents 2.2. The Cuticle of Caenorhabditis elegans ^ 18 2.3. Cuticle of the Morphological Mutant dpy-5 ^  22  2.4. Genes Important in C. elegans Morphology. ^ 23 MATERIAL AND METHODS ^  26  1.1.  Nomenclature ^  1.2.  Nematode Culture Conditions ^ 27  1.3.  Nematode Strains ^  1.4.  Origin of the dpy-5(s1300) Allele ^ 29  1.5.  Strain Construction ^  1.6.  Preparation of Genomic DNA ^ 31  1.7.  Purification of Plasmid DNA ^ 33  1.8.  Isolation of Bacteriophage DNA ^ 33  1.9.  Lambda ZAP DNA Isolation ^  26  28  30  35  1.10. Isolation of Nematode RNA ^ 36 1.11. Restriction Enzyme Digests ^ 37 1.12. Agarose Gel Electrophoresis ^ 37 1.13. Electroelution ^  37  1.14. Construction of a Lambda Zap Library ^ 38 1.15. Screening Phage Libraries ^ 39 1.16. Purification of Bacteriophage Clones ^ 40 1.17. Labelling of DNA Probes ^  40  1.18. Southern Transfer and Hybridization ^ 41 1.19. Northern Transfer and Hybridization ^ 42 1.20. DNA Sequencing ^  44  Table of Contents  1.21. Sequence Analysis ^ III. RESULTS ^  46 47  Section 1 1.1. Altered Tcl hybridization in Bristol Strains ^  47  1.2. Isolation of a Tc1 Clone from KR579-D ^ 54 1.3. Further Tcl Mobilization and Mutator Activity ^  59  1.4. Isolation and Analysis of the Mutation dpy-5(s1300) ^  59  1.5. Isolation of a Tcl Clone from KR1163 ^ 63 Section 2 2.1. Examination of Putative Tcl-induced dpy-5 alleles ^ 67 2.2. Isolation of C. elegans Genomic Clones... ^ 67 2.3. Isolation of C. elegans cDNA clones ^ 70 2.4. Isolation of C. briggsae Genomic Clones ^  70  2.5. Sequence Analysis of the dpy-5 Gene ^ 74 2.6. Sequence Data for the C. briggsae dpy-5 Homologue ^  84  2.7. Identification of a Putative Secretory Signal Sequence ^  84  2.8. Expression of the dpy-5 Gene ^ 89 2.9. Analysis of the Cosmid B0342 ^ 96 2.10. Identification of Upstream Coding Region ^ 97  vi  Table of Contents IV. DISCUSSION ^  111  1.1. Analysis of Tcl Transposition in CB51 ^ 112 1.2. Analysis of a Tcl Insertion Site in CB51 ^ 113 1.3. The Observed Changes in Tcl Transposition ^  114  1.4. Analysis of Tcl-induced Mutations of dpy-5 ^  118  1.5. Identification of the Phage Clone KR#85 and LG I Cosmid Contig ^  120  1.6. Sequence Analysis of the dpy-5 Gene ^ 120 1.7. Northern Analysis of the dpy-5 Gene ^ 124 1.8. Developmental Northern Blot Analysis ^ 126 1.9. Injection Rescue of dpy-5 ^ 126 1.10. Identification of a Casein Kinase I homologue ^  130  1.11. Summary ^  130  1.12. Proposals for Future Research ^ 132 REFERENCES ^  134  APPENDIX 1 ^  142  APPENDIX 2 ^  144  APPENDIX 3 ^  158  vii  LIST OF FIGURES Figure 1. Diagrammatic sketch of cuticle structure. .20 Figure 2. Tcl hybridization pattern of Bristol strains ^  48  Figure 3. Tc1 hybridization pattern in Cambridge strains ^  50  Figure 4. Altered Tcl hybridization pattern in the strain KR579 ^  51  Figure 5. The relationship of CB51-derived strains ^  52  Figure 6. Isolation of an additional Tcl-hybridizing band from KR579-D ^ 55 Figure 7. N2 and KR579-D DNA hybridized with DNA flanking the Tcl insertion site ^ 56 Figure 8. Sequence of the Tc1 insertion site from KR579-D ^  57  Figure 9. Tcl hybridization pattern of CB51-derived strains ^ 60 Figure 10. Southern blot of N2 and unc-38 dpy-5(s1300) unc-87 DNA ^  64  Figure 11. Sequence of the Tcl insertion site from the strain KR1163 ^  65  Figure 12. Southern blot of DNA isolated from Dpy-5 and wildtype revertant strains ^ 68 Figure 13. Southern blot of DNA isolated from Dpy-5 and wildtype strains ^  69  Figure 14. Restriction enzyme map of the Charon4 genomic phage KR#85 ^  71  Figure 15. Cosmid contig identified by the genomic phage KR#85 ^  72  Figure 16. Restriction map of the dpy-5 region ^ 73  viii  List of Figures Figure 17. Alignment of dpy-5 genomic and cDNA sequences ^  75  Figure 18. Genomic sequence data from the dpy-5 gene ^  81  Figure 19. Comparison of C. elegans and C. briggsae DNA and amino acid sequences ^ 85 Figure 20. Detection curve for a eukaryotic signal sequence ^  90  Figure 21. Northern blot hybridization of dpy-5 message ^  91  Figure 22. Northern blot analysis of dpy-5 mutants ^  92  Figure 23. Northern blot analysis of dpy-5(e61) and dpy-5(e61) smg-4(ma116) RNA ^ 94 Figure 24. Northern hybridization of developmentally staged RNA ^ 95 Figure 25. Southern blot of C24H1 and B0342 Cosmids ^  98  Figure 26. Alignment of dpy-5 upstream genomic and cDNA sequences ^  99  Figure 27. C. elegans genomic sequence data from the dpy-5 upstream gene ^ 102 Figure 28. Comparison of C. elegans and C. briggsae DNA and amino acid sequences ^ 105  ix  LIST OF TABLES Table 1. dpy-5 and dpy-5 Revertant Alleles ^ 142 Table 2. Additional Strains Used in This Thesis ^ 143  x  ACKNOWLEDGMENTS  I would like to thank my research supervisor, Dr. Ann Rose, for her support and encouragement. I would like to thank my supervisory committee, Dr. David Baillie, Dr. David Holm, Dr. George Spiegelman, and Dr. Stephen Wood for reading my thesis. Sincere thanks to my colleagues in the Rose lab, Ann Marie Howell, Terry Starr, Shiv Prasad, Linda Harris, Ken Peters, Kim McKim, Monique Zetka, Jennifer McDowall, and Sheldon McKay for their instruction and helpful discussion. I would especially like to thank my parents, Mark and Stephanie and my wife, Debbie for their love and support. This research was financially supported by a Post Graduate Fellowship from the National Sciences and Engineering Research Council and a University Graduate Fellowship.  xi  DEDICATION  To Deb. For all your love and support.  xii  I. INTRODUCTION  Caenorhabditis elegans is a small free-living soil nematode that is well suited as a experimental model system (Brenner 1974). C. elegans has a life cycle of approximately three days under optimal growth conditions and can be easily cultured in the laboratory. The nematode has two sexes, hermaphrodites and males, which are both about 1 mm in length as adults. Hermaphrodites produce both oocytes and sperm and can reproduce by selffertilization. Males arise spontaneously via Xchromosome nondisjunction and can fertilize hermaphrodites. Hermaphrodites normally produce about 300 progeny during a reproductive life span. C. elegans is a simple organism both anatomically and genetically. The adult hermaphrodite has 959 somatic nuclei and the adult male has 1031 somatic nuclei. This simplicity has allowed the complete characterization of the anatomy at the electron microscope level and the characterization of the complete cell lineage. The small genome size (1 X 108 nucleotide pairs per haploid genome) is an asset in the study of the nematode at the molecular level (Sulston and Brenner 1974). A physical map of the  1  Introduction genome, an ordered cosmid library of genomic DNA fragments, has nearly been completed. The construction of this map has been aided by the isolation of specific genes using the method of transposon tagging. Transposable elements are mobile DNA sequences that can be utilized as tools for molecular analysis. The movement of these DNA sequences is under genetic control. However, the mechanism of genetic control is not well understood in all organisms. In Caenorhabditis elegans, the transposable element Tcl (Emmons et al. 1983) is regulated in a strain and tissue-specific manner, although the mechanism of this regulation is not known. In this thesis, Tc1 transposition is examined in the C. elegans Bristol strain in which germline Tcl transposition is not normally observed. Spontaneous Tclinduced mutations in the dpy-5 gene are also examined. In addition, the Tcl transposon is used as a tool to clone the dpy-5 gene and another gene in the dpy-5 region of chromosome I.  2  Introduction Section 1  1.1. Eukaryotic Transposable Elements Transposable elements are ubiquitous in both eukaryotic and prokaryotic organisms. Genetic methods have allowed the detection, identification, and analysis of these elements in several eukaryotic organisms such as yeast, worms, Drosophila, and humans. These elements are often characterized as dispersed repetitive DNA sequences capable of movement from one genomic location to another. The movement of these elements can result in spontaneous mutations and chromosomal rearrangements. Therefore, transposable elements affect the genome in many ways, ranging from gene mutation to genomic evolution. Transposable elements in higher eukaryotes have been identified by unstable mutations (McClintock 1950, 1951; Green 1967). The insertion and excision of these elements is under genetic control. P element mobility in Drosophila normally occurs only in the germline and only after specific interstrain crosses (reviewed by Engels 1989). In maize, the movement of Ds transposons is under the control of Ac elements (reviewed by Fedoroff 1989). In both cases, complete or autonomous elements encode  transposases required for their own movement. During  3  Introduction  transposition these elements can sustain internal deletions resulting in defective or non-autonomous elements that contain recessive defects. These elements are still able to transpose by using a functional transposase from a complete element, provided that the inverted repeats required for transposase function are intact. The regulation of transposase activity in turn regulates transposable element activity.  1.2. The C. elegans Transposable Element Tcl  The most thoroughly studied transposable element in Caenorhabditis elegans is the transposon Tcl  (Tc=transposon C. elegans) discovered by Emmons et al. 1983; reviewed in Moerman and Waterston 1989. This transposon was initially identified in restriction fragment length polymorphisms between the two common laboratory strains of C. elegans, variety Bristol, N2 strain (Brenner 1974) and variety Bergerac, BO strain (Nigon 1949). Southern blot analysis of the two strains revealed restriction fragments that were 1.6 kilobase pairs longer in BO DNA that in N2 DNA (Emmons, Klass, and Hirsh 1979). Analysis of the cloned fragments showed that the 1.6 kb length difference was due to the presence of a dispersed repeated sequence. This sequence was  4  Introduction present in N2 at approximately 30 copies and in BO at over 300 copies per haploid genome (Emmons et al. 1983; Liao, Rosenzweig, and Hirsh 1983). This sequence was subsequently found to be present in all strains of  C. elegans with most strains containing a "low copy number" similar to N2. The initial element was found to be structurally similar to insertion sequence elements. Due to its structure and its repeated nature, the sequence was assumed to be a transposable element and was named Tcl (Rosenzweig, Liao, and Hirsh 1983a). Tc1 is 1612 base pairs (bp) in length (Rosenzweig, Liao, and Hirsh 1983a; Moerman, Kiff, and Waterston 1991), with 55 bp terminal perfect inverted repeats (Moerman, Kiff, and Waterston 1991) and appears to encode a 343 amino acid polypeptide (Schukkink and Plasterk 1990; Prasad et a/. 1991). The protein Tc1A, encoded by Tcl, has been shown to bind specifically to the 55 bp inverted repeat of the Tcl transposon (R. Plasterk, unpublished results). Most Tcl elements in both strains are conserved in size and structure (Emmons et al. 1983; Eide and Anderson 1985a,b; Harris and Rose 1989). On Southern blots of N2 and BO DNA cut with EcoRV, which cuts within the inverted repeats of Tcl, the majority of the hybridizing signal is 1.6 kb in size (Mori, Moerman,  5  Introduction and Waterston 1988). This homogeneity in size is in contrast to the heterogeneity in length observed for P elements in Drosophila (O'Hare and Rubin 1983) and Ac/Ds elements in maize (Pohlman, Fedoroff, and Messing 1984). Although Tcl elements exhibit homogeneity in length, there is microheterogeneity among the Tcl elements in the BO strain of C. elegans. This heterogeneity has been detected primarily through restriction enzyme digest comparisons with PSI(Be)T1, the first cloned Tcl element from the BO strain. Approximately 10% of BO Tcl elements contain a HindIII site not found in PSI(Be)T1 and at least one copy of this variant is present in the N2 strain (Rose et al. 1985). Another subset of BO Tcl elements contains an EcoRI site (Eide and Anderson 1985b) of which an example is also present in the N2 strain (Harris and Rose 1989). An example of a Thai Tcl variant has also been observed that is present in the BO strain and makes up about 10% of the Tcl population (Moerman and Waterston 1989).  1.3. Tcl Transposition Conclusive evidence that Tcl is capable of transposition came from the genetic analysis of  6  Introduction  spontaneous mutants. In the examination of different C. elegans strains, including the N2 and BO strains, only  the BO strain was found to exhibit mutator activity (Moerman and Waterston 1984). The muscle gene unc-22 was used as an assay for mutator activity (Brenner 1974). The forward spontaneous mutation frequency of unc-22 in all strains except BO was less than or equal to 10-6 per gamete. However, for the BO strain the forward spontaneous mutation frequency was 10-4 per gamete. The mutant alleles reverted at a high frequency, and both the forward mutation rate and the frequencies were affected by the genetic background (Moerman and Waterston 1984). These characteristics are associated with previously identified transposable elements. Homozygous mutations in the unc-22 gene disrupt body wall musculature and are characterized by an easily detectable continuous fine twitch of the body wall muscles (Waterston, Thomson, and Brenner 1980; Moerman 1980; Moerman et al. 1982). Mutants that are heterozygous for unc-22 resemble the wildtype worm under normal culture conditions, but can readily be identified in the presence of a 1% solution of nicotine. In this solution heterozygotes for unc-22 twitch rapidly, while wildtype animals are rigidly paralyzed (Moerman and  7  Introduction Baillie 1979). Using twitching in the presence of nicotine as an assay, mutations may be detected at frequencies as low as 10-7 to 10-8 per gamete. Revertants of the unc-22 gene can be detected by an increase in mobility.  1.4. The Target Site for Tcl Insertion The Tcl transposon exhibits insertion site specificity. The analysis of Tcl insertion sites revealed that Tcl insertion occurs at a TA dinucleotide (Rosenzweig, Liao, and Hirsh 1983b; Eide and Anderson 1988; Mori et al. 1988). Sequence analysis of various Tcl insertion sites suggested a 9 bp consensus sequence for Tcl insertion (Mori et al. 1988; Eide and Anderson 1988 in Moerman and Waterston 1989): [GA(G/T)(A/G)TA(T/C)(G/C)T]. The only absolute requirement for Tcl insertion is the TA dinucleotide that is conserved in all examined insertion sites.  1.5. Tcl Excision Tcl elements undergo somatic excision from genomic sites at a high frequency in both N2 and BO strains of C. elegans (Emmons, Roberts, and Ruan 1986; Harris and Rose 1986; Moerman, Benian, and Waterston 1986).  8  Introduction  Evidence of somatic excision was first observed in Southern blots of restriction-enzyme-digested BO DNA hybridized with unique-sequence probes flanking Tcl insertion sites (Emmons  et  al. 1983). In these  instances, two bands are observed that differ 1.6 kb in  size. These represent one genomic fragment containing Tc1 and another containing the empty target site. Empty target sites normally accumulate to approximately 1% to 5% of the filled sites in populations of mixed ages. However, under certain conditions such as prolonged starvation, the fraction of certain sites can approach 50% (Emmons et al. 1985). In addition to somatic excision, high frequency germline excision is observed in the BO strain (Moerman and Waterston 1984). Germline reversion of unc-22 Tc1 alleles occurs at a frequency of about 2 X 10-3 and reversion of unc-54 Tcl alleles occurs at a frequency of 1O  ^10-7, depending upon their location in the gene  (Eide and Anderson 1985a; Eide and Anderson 1988). In these instances, the requirement for restoration of gene function places restraints on the type of reversion events observed. Therefore, the frequencies observed likely represent the lower limit of germline Tcl excision. Examples of germline reversion frequencies  9  Introduction approaching 10 -2 have been observed for some genes and are therefore similar to the somatic reversion rates seen in Southern blot analysis (Moerman and Waterston 1989).  1.6. Evidence for Homologue Dependent Tcl Reversion In heterozygotes for Tcl-induced alleles of unc-22, the frequency of spontaneous reversion can be greatly increased (Mori, Moerman, and Waterston 1990). This result is also seen for the P element in Drosophila (Engels et a/. 1990). Analysis of a P element insertion mutant of the white gene showed that the mutant reverted at an elevated frequency when the homologous chromosome had the wildtype sequence at that location. Engels proposed a model in which increased reversion frequency may be explained by transposon excision followed by DNA repair using the homologous chromosome as a template. The variety of rearrangements associated with reversion sites for Tcl-induced mutations of unc-22 may be the result of imprecise repair of the double-stranded gap by the host replication and repair machinery. (Moerman, Kiff, and Waterston 1991; Plasterk 1991). Imprecise repair of Tcl excision may also explain the chromosome rearrangements associated with spontaneous Tcl mutations (Clark et al. 1990).  10  Introduction 1.7. Regulation of Tcl Transposition The recovery of unstable unc-22 Tcl alleles allowed the study of Tcl transposition in the germline. Both the induction rate of unc-22 Tcl mutations and their stability are affected by the genetic background (Moerman and Waterston 1984). In the BO strain, the spontaneous forward mutation rate at the unc-22 gene is about 10-4, compared to 10-6 to 10-7 in the N2 strain. In a BO genetic background, the reversion frequency for an unc-22 Tc1 mutation is approximately 10-3, but when placed in an N2 background, they revert at a frequency of less than 10 -6 . The same mutations when introduced into an N2/B0 genetic background become unstable once more (Moerman and Waterston 1984). In addition, strains that cause unc-22 Tcl alleles to become unstable also increase the forward mutation rate at the unc-22 locus. These observations suggest that Tcl insertion and transposition activities are correlated (Mori, Moerman, and Waterston 1988). The effect of altering the genetic background also suggests that excision and transposition are regulated by transacting factors. Tcl copy number has little or no effect on the frequency of Tcl transposition. Nine strains were examined for the ability to generate unstable Tcl-induced  11  Introduction unc-22 mutations (Moerman and Waterston 1984). Amongst  these, three were high-copy-number strains for Tcl, yet only the BO strain was active for Tcl transposition. In another study, a spontaneous unc-22 Tcl mutation, originally derived from BO, was stabilized in an N2 genetic background and then crossed into BO. From this cross, isogenic N2/B0 lines containing the unc-22 Tcl mutation were established. These strains were then examined with respect to Tcl copy number and reversion frequency at the unc-22 site. It was found that the reversion frequency varied from 10-3 to less than 10-7 and was not correlated with the Tc1 copy number. In addition, strains have been constructed that contain less than 60 copies of Tcl yet have Tcl activity comparable to the BO strain (Mori, Moerman, and Waterston 1988). Study of the BO strain suggests that the Tcl activity in BO is due to factors that are polygenic and are present at multiple sites in the genome. Investigation into Tcl activity in BO failed to reveal a single major factor responsible for Tcl activity (Moerman and Waterston 1984). Single BO chromosomes were replaced by their N2 homologues without significantly affecting Tcl behaviour. Also it was determined that unstable  12  Introduction unc-22 Tcl mutations required several crosses to the N2 strain in order to become stabilized. The factors responsible for Tcl activity in the BO strain are termed mutators. Analysis of N2/B0 hybrids suggests that there may be relatively few mutator loci spread over a few chromosomes in the BO genome. Mixed N2/B0 hybrid lines were established and monitored for reversion of an unc-22 Tc1 allele (Mori, Moerman, and Waterston 1988). Three of eleven strains examined had greatly reduced mutator activity. If mutator activity was present at many different sites scattered throughout the BO genome, a large number of strains with no mutator activity would not be expected.  1.8. Tissue-Specific Regulation of Tcl Activity Regulation of Tcl activity is apparent in examination of Tcl excision in germline and somatic tissues. Germline Tcl excision is more sensitive to genetic background than somatic Tcl excision. The frequency of germline excision from unc-22 varies 1000fold between BO (10-3 excision frequency) and N2 (less than 10 -6 excision frequency) (Moerman, Benian, and Waterston 1986; Moerman and Waterston 1984). On the other hand, somatic Tcl excision varies little between BO  13  Introduction  and N2 strains in the ratio of excision to insertion bands. This was measured by Southern analysis for an unc-22 Tcl allele in BO and N2 genetic backgrounds  (Moerman, Benian, and Waterston 1986). Therefore somatic Tcl excision is constitutive while germline Tcl excision is regulated. In the N2 strain the position and number of Tcl elements in the genome remain constant. Further evidence that Tcl activity is regulated in a tissue-specific manner comes from the analysis of EMSinduced mutants that increase germline Tcl transposition and excision, but do not affect the soma (Collins, Saari, and Anderson 1987). These mutants exhibited a four to ten times higher reversion frequency for an unc-54 Tc1 mutant than their parents. Although the mutants were isolated in a screen for increased excision, a corresponding increase in Tcl transposition was also observed. The relationship between the EMS-induced mutants and the endogenous BO mutators is not known. While the mutator activity in BO is thought to be transposon-related, the EMS-induced mutators may be due to Tcl transposons or possibly host regulatory genes whose products interact with transposons (Moerman and Waterston 1989). While the BO mutator activity has only been shown to be associated with the Tcl transposon, one  14  Introduction EMS-induced mutant has been shown to cause transposition of other transposons in C. elegans (Collins, Saari, and Anderson 1987).  1.9. Tcl Activity in Bristol Strains of C. elegans In Bristol strains only two occurrences of germline Tcl movement have been reported. In one case a mutator strain occurred spontaneously in the strain CB30  (sma-1(e30]) (C. Trent in Moerman and Waterston 1989). It was noted that the strain contained about 50 additional copies of Tcl, in addition to the normal complement of 30 copies found in Bristol strains. The strain has exhibited mutator activity and spontaneous Tcl-induced alleles of unc-22 have been isolated (C. Trent and C. Link, unpublished results). The earliest frozen stock of CB30 (frozen in 1969) has a Tcl pattern on a Southern blot identical to the pattern normally observed in Bristol strains and does not possess mutator activity (J. Hodgkin, unpublished results). The onset of Tcl activation was estimated to have occurred between 1969 and 1975 (J. Hodgkin, unpublished results). The other occurrence of Tcl activity in a Bristol strain involved the strain BC313 (rec-1(s180]) (Rattray 1986). In this strain, the pattern of Tcl hybridization  15  Introduction  on a Southern blot was observed to differ in comparison to the pattern normally observed in Bristol strains. In BC313, several Tcl-hybridizing bands were present in addition to the 30 copies normally observed in Bristol strains. The origin of these additional Tcl-hybridizing bands is unknown. One possibility is that the bands corresponded to the presence of additional Tcl elements and that the additional elements were the result of Tcl transposition. Since Tcl transposition is normally undetectable in the germline of Bristol strains, an investigation of this result may be important in understanding how the regulation of Tcl activity is altered between mutator and non-mutator strains. By attempting to understand this regulation, it may be possible to gain an insight into evolutionary constraints placed on transposable element mobility.  1.10. Tcl Transposition in an N2/B0 Hybrid Strain  A spontaneous dumpy mutation in C. elegans was isolated after heat shock in an N2/B0 hybrid strain (L. Donati, personal communication). The mutation was found to be an allele of dpy-5 and given the allele designation s1300 (R. Rosenbluth, personal communication). In all, five additional Tcl-induced  16  Introduction alleles of the dpy-5 gene were obtained. Investigation of these dpy-5 alleles provided an opportunity to investigate Tcl transposition in an N2/B0 hybrid strain and to clone and analyze the gene with respect to its structure and function in C. elegans.  Section 2  2.1. Genes Important in Organismal Morphology In C. elegans, many genes have been identified that affect the gross morphology of the worm. Mutant phenotypes include dumpy, blister, long, small, roller, and squat (Brenner 1974; Cox et al. 1980). The most common class of these mutants is dumpy, of which 26 genes have been identified. Dumpy mutants are short and fat in comparison to wildtype worms, are easily scored and usually do not interfere with growth and reproduction and are therefore particularly useful as genetic markers in the study of growth and development (Brenner 1974). Examples of these genes are known to be important in the formation of extracellular cuticle in the nematode (Ouazana, Garrone, and Godet 1985; von Mende et a/. 1988).  17  Introduction  The cuticle of C. elegans is related to the extracellular matrix of vertebrates. Both structures consist of a complex arrangement of extracellular macromolecules. In C. elegans, the cuticle forms an extracellular structure that covers the outermost surfaces of the nematode. In vertebrates, the extracellular matrix surrounds cells, determining the physical properties of a tissue. Although the nematode cuticle is less complex than the extracellular matrix of vertebrates, the cuticle of C. elegans is amenable to a molecular genetic analysis of its assembly.  2.2. The Cuticle of Caenorhabditis elegans  The cuticle of C. elegans is synthesized and secreted by the underlying hypodermal cell layer. Prior to each postembryonic molt, a new cuticle is synthesized and the cuticle from the previous stage is shed. In some nematodes, a larger version of the same cuticle is secreted at each molt. However, in C. elegans, at least four different cuticle types are secreted, with each type differing substantially in ultrastructure and protein composition. Therefore, postembryonic development of the hypodermis requires both a pattern of discontinuous cellular activity and a program of genetic switches that  18  Introduction  allow the production of different cuticles at each molt (Cox, Staprans, and Edgar 1981). Some features of the extracellular cuticle are common to various stages of C. elegans development. The external cuticle is organized into two main layers: the cortical and basal layers. The surface features of the cuticle are created by the cortical layer. The surface of the cuticle is divided into regularly spaced circumferential ridges called annulae. In addition, elevated longitudinal ridges termed alae mark the lateral surface during Li, dauer, and adult stages (Singh and Sulston 1978). The greatest morphological differences between the various cuticles occur in the basal layer (see Figure 1). In the adult, the basal and cortical layers are separated by a fluid filled layer containing columnar structures called struts that separate the two major layers. The cortical layer consists of an electron-dense surface and an amorphous under layer. The basal layer is composed of two layers of highly organized fibers that spiral around the animal in opposite directions, each at an angle of 600-700 relative to the longitudinal axis of the worm (Cox, Staprans, and Edgar 1981).  19  Introduction  Adult  L4  Dauer  ,..b1 Ll  400  cl  ,b1  Figure 1. Diagrammatic sketch of adult and juvenile  cuticles. Cuticle structures indicated are: clay, cortical layer; blue, basal layer; st, struts. After Cox, Staprans, and Edgar (1981)  20  Introduction In the L4 cuticle, like other juvenile cuticles, the fluid filled layer found in the adult is absent. The external and internal cortical layers are similar to the corresponding structures in the adult cuticle. The basal layer of the L4 cuticle contains two fiber layers that are organized in a spiralling manner similar to the adult cuticle. Alae are not present (Cox, Staprans, and Edgar 1981). In the dauer cuticle, the cortical layer is similar to the cuticle of other stages except that the external cortical layer is thicker. The basal layer is characterized by a striated zone that is separated from the hypodermis by a loosely organized fibrillar material. Alae are present in this stage (Cox, Staprans, and Edgar 1981). In the Ll cuticle, the cortical layer is similar to the cuticle of other stages. The basal layer contains a striated zone that is less distinct than that seen in the dauer stage. There is no intermediate layer separating the hypodermis and the striated layer in the L1 cuticle. Alae are also present at this stage (Cox, Staprans, and Edgar 1981). The protein composition of the different cuticles is unique. Each cuticle contains a number of major protein  21  Introduction components in the 50K-200K molecular weight range and a number of minor components >200K molecular weight and <50K molecular weight. Most cuticle proteins are unique to an individual stage, while a few proteins are present in more than one, but not all cuticle stages (Cox, Staprans, and Edgar 1981). The stage-specific differences in cuticle structure and protein composition indicate that the same cuticle is not reiterated at each stage. Instead each cuticle stage appears to share certain components and also contain protein unique to that stage. Therefore, some genes appear to function at all molts while other genes function only at specific molts. This finding may explain some of the stage specificity observed in particular morphological mutants.  2.3. Cuticle of the Morphological Mutant dpy-5 Examination of dpy-5 shows alterations in cuticle morphology and modifications in the gel pattern of extractable cuticle proteins in comparison to the wildtype. The adult cuticle of the dpy-5 worm was examined and found to differ in cuticle morphology (Ouazana, Garrone, and Godet 1985). In the Dpy-5 mutant, the cuticle is thicker than in the wildtype (0.75 gm versus 0.5 gm) and the distance between annulae is  22  Introduction decreased from 0.8 gm-0.9 gm to 1.3 gm in the wildtype. The two fiber layers of the basal layer appear to be disorganized in the cuticle and the columnar struts are thicker in the cuticle of the mutant. The protein composition of the cuticle in dpy-5 is different from that in the wildtype. Examination of the major cuticle components of dpy-5 indicates the presence of three additional high molecular weight bands not found in the wildtype worm (Ouazana, Garrone, and Godet 1985). In addition, one of the major bands routinely observed in the wildtype is present in very minor quantities in the Dpy-5 mutant. These alterations in cuticle composition and morphology suggest that the dpy-5 gene product may be important cuticle morphogenesis.  2.4. Genes Important in C. elegans Morphology Of the 26 dumpy genes that exist, six have been cloned and the gene products of four have been identified. dpy-2, dpy-10 (A. Levy and J. Kramer, unpublished results), dpy-7 (I. Johnstone and J. Barry, unpublished results), and dpy-13 (von Mende et al. 1988) encode collagen genes. Since the cuticle is made of greater than 90% collagen proteins (Cox, Kusch, and Edgar 1981), it seems clear that some morphological mutants  23  Introduction would result from mutations in collagen genes. The dpy genes that are known to encode collagens likely play an important role in cuticle development and assembly. However, the recessive nature of these mutations suggests a function that is not replaced by other collagens of related structure. In C. elegans, there are approximately 50 to 100 collagen genes (Cox, Kramer, and Hirsh 1984). Due to the overall similarity of these genes, it is possible that many of the collagen gene products may substitute for each other during cuticle assembly. Evidence supporting this hypothesis comes from the study of two known collagen genes: sqt-1 and rol-6 (Kramer et al. 1988; Kramer et al. 1990). Worms that are homozygous for null mutations in either gene are essentially wildtype in appearance. This is not the case for dumpy collagen genes, since analysis of the dpy-13 gene reveals that it contains sequences unique to C. elegans that likely contribute to a unique function (von Mende et al. 1988). Other components of the cuticle must also play a role in proper cuticle formation. These include srf mutants that alter the expression of surface antigens on the cuticle of C. elegans (S. Politz, unpublished results) and the cut-1 gene that encodes a noncollagenous  24  Introduction  component of the dauer larva cuticle (Sebastiano, Lassandro, and Bazzicalupo 1991). The gene products of dpy-5 (this thesis) and dpy-20 (D. Suleman, D. Clark, and  D. Baillie, unpublished results) are not collagens and have no known homologues. Interactions with the bli-4 gene indicate that the dpy-5 gene may be important in normal cuticle formation (Peters, McDowall, and Rose 1991). The dpy-5 gene suppresses blistering of the cuticle in bli-4 worms both as a homozygote and a heterozygote, suggesting that the cuticle of C. elegans is altered in Dpy-5 worms that suppress blistering. The study of the dpy-5 gene will be useful in better understanding the role of non-collagen proteins in cuticle development and assembly. A better understanding of cuticle development in C. elegans in turn may provide insight into the formation of more complex extracellular structures such as those found in insects and vertebrates. The study of Tcl-induced alleles of dpy-5 affords an opportunity to study the alterations in gene expression caused by Tcl insertion and provides a tool with which to isolate the gene for molecular analysis. This analysis in turn provides information that allows a better understanding of the dpy-5 gene and its effect on worm morphology.  25  II. MATERIALS AND METHODS  1.1. Nomenclature  The nomenclature used in this thesis follows guidelines described by Horvitz et al. (1979). Gene names are italicized and are represented by a three letter name followed by a number indicating the order in which they were discovered. For example, dpy-5 is the fifth dumpy gene discovered. Allele names are given as one or two lower case letters followed by a number. The letters are a lab designation, while the numbers indicate the particular mutation. For example, for the allele h14, the letter h indicates that the mutation was  isolated in the Rose lab and the number 14 indicates that the mutation is the fourteenth mutation isolated in the Rose lab. Strain names contain one or two upper case letters followed by a number. The letters are a lab designation, while the numbers indicate the particular strain. For example, for the strain KR579, KR indicates that the strain was named in the Rose lab and the number 579 indicates that the strain is the 579th strain named in the Rose lab. Phenotypes for a given mutation are presented as the gene name with no italics and starting with a capital letter. For example, dpy-5 is a gene  26  Materials and Methods name, while Dpy-5 is a phenotype. Names that refer to DNA, such as gene names and allele names, are italicized. Names that refer to whole animals, such as strain names and phenotypes, are not italicized. In this study, plasmids are pCeh followed by a number, where p indicates plasmid, CEO indicates C. elegans, and h indicates the Rose lab. For example, pCehl48, is Rose lab C. elegans DNA plasmid 148.  1.2. Nematode Culture Conditions Nematode strains were grown at 16°C or 20°C on Nematode Growth Media (NGM) agar plates carrying a lawn of 0P50, a leaky uracil-requiring strain of Escherischia coli (Brenner 1974). Nematode Growth media is constituted as follows:  NGM media: 3g NaC1  After autoclave (20 minutes)  17g Agar  1 ml Cholesterol (5mg/m1 in Ethanol)  5g Bactotryptone  1 ml 1M CaC12  dH20 to 1 L  1 ml 1M MPG4 25 ml 1M CHG2P0I4 (pH 6.0)  27  Materials and Methods  Lines of the KR579 strain were maintained on plates at 20°C by transferring one or two hermaphrodites to a fresh plate every 1-2 weeks for a period of 12 months. Subsequently, DNA from these lines was isolated and examined with respect to the Tcl-hybridizing pattern. One of these lines was frozen and designated KR1082. The lines were maintained on plates for an additional period of 2 years, then DNA was isolated and Tcl-hybridizing patterns were re-examined.  1.3. Nematode Strains Two nematode species were used in this work, Caenorhabditis elegans and Caenorhabditis briggsae.  Wildtype C. elegans strains used in this work include the Bristol strain, N2 and the Bergerac strain, BO. The wildtype C. briggsae strain used was G16. The C. elegans var. Bristol strain CB51 (unc-13[e51]) was isolated at the beginning of 1968  (Brenner 1974) and maintained in liquid nitrogen at the Laboratory of Molecular Biology, Cambridge, England. CB1833 (dpy-5[e61] unc-13[e511) was constructed by S. Brenner at the end of 1968 (J. Hodgkin, personal communication) and is used as a working strain of unc-13. CB51 and CB1833 were obtained from A. Chisolm, Cambridge,  28  Materials and Methods  England. KR579, a strain derived from CB51, was obtained from the Caenorhabditis Genetics Center, Columbia, Missouri. The wildtype N2 strain and mutant strains designated "BC" were obtained from D. Baillie, Simon Fraser University, Burnaby, B.C. "KR" is the official strain designation for the Rose laboratory. The high recombination strain BC313 (rec-l[s180]) was spontaneously observed in a dpy-5 unc-15 +/+ + unc-13 heterozygote that was constructed from BC187 (dpy-5(e61] unc-15(e73] rec-1(1801) and BC82 (unc-13[e51]) (Rose and  Baillie 1979).  1.4. Origin of the dpy-5(s1300) Allele  The dpy-5(s1300) allele was isolated as a spontaneous mutation in a Bristol-Bergerac hybrid strain of C. elegans (R. Rosenbluth, Simon Fraser University). The mutation was mapped to chromosome I and subsequently identified as an allele of the dpy-5 gene, s1300 (R. Rosenbluth). The strain carrying the dpy-5(s1300) mutation was named BC1906. The spontaneous occurrence of the dpy-5(s1300) mutation in a Bristol-Bergerac hybrid background presented the possibility that the mutation may have been the result of a Tcl transposition event.  29  Materials and Methods 1.5. Strain Construction  The Bristol-Bergerac strain BC1906 was crossed to Bristol N2 worms to reduce the number of Bergerac-derived Tcl elements. BC1906 hermaphrodites were crossed to N2 males and wildtype (dpy-5(s1300)/+) Fl hermaphrodites were selected from the Fl generation. Individual hermaphrodites were allowed to self-cross, the homozygous dpy-5(s1300) hermaphrodites were selected and a strain  established. This procedure was repeated several times in order to replace high-copy-number Bergerac chromosomes with low-copy-number Bristol chromosomes. To further reduce the number of Bergerac-derived Tcl elements, the strain KR1163 (unc-38[e264] dpy-5[s1300] unc-87(e1459]) was constructed. The unc-38 gene maps approximately 0.5 map units (m.u.) to the left of dpy-5, while the unc-87 gene maps approximately 1.5 m.u. to the right of dpy-5. By constructing this strain, Bergerac-derived Tcl elements outside of the unc-38 unc-87 interval were removed. Homozygous unc-38 hermaphrodites were crossed to Bristol N2 males and heterozygous males (unc-38/+) were selected from the Fl generation. These males were crossed to dpy-5(s1300) hermaphrodites and wildtype hermaphrodites (unc-38 +/+ dpy-5) from the Fl generation were selected. These hermaphrodites were allowed to  30  Materials and Methods self-cross and Dpy hermaphrodites from the F2 generation were selected. These hermaphrodites were allowed to self-cross and homozygous unc-38 dpy-5(s1300) hermaphrodites from the F3 generation were selected. In addition, unc-87 hermaphrodites were crossed to Bristol N2 males and wildtype males (unc-87/+) were selected from the Fl generation. These males were crossed to the unc-38 dpy-5(s1300) hermaphrodites and wildtype hermaphrodites (unc-38 dpy-5(s1300) +/+ + unc-87) from the Fl generation were selected. These hermaphrodites were allowed to self-cross and DpyUnc hermaphrodites from the F2 generation were selected. These hermaphrodites were allowed to self-cross and homozygous unc-38 dpy-5(s1300) unc-87 hermaphrodites from the F3 generation were selected. The net result of this protocol was to eliminate all BO-derived Tcl elements outside of the area immediately surrounding the dpy-5 gene.  1.6. Preparation of Genomic DNA Nematode genomic DNA was prepared by a method modified by J. Curran and D. Baillie (personal communication) from that of Emmons, Klass, and Hirsh (1979). Caenorhabditis strains were grown on NGM agarose plates carrying a lawn of wildtype E. coli. Nematodes  31  Materials and Methods were grown until the plates became crowded, at which point they were washed from the plates with 0.5% NaCl and transferred to 50 ml Falcon tubes. The worms were then pelleted by centrifugation at 2000 RPM for five minutes, rinsed with 0.5% NaCl and pelleted again at 2000 RPM. The worms were rinsed and pelleted in this manner until the worms appeared to be free of contaminating bacteria. The worm pellet was resuspended in proteinase K buffer (100 mM Tris-HC1, pH 8.5, 50 mM EDTA, 200 mM NaCl, 1% SDS), then proteinase K (Pharmacia) was added to a final concentration of 0.05 mg/ml and the nematodes were placed at 65°C for 15 minutes. The sample was then gently extracted three times with TE (10 mM Tris, pH 8.0, 1 mM EDTA)-saturated phenol, once with 1:1 phenol-chloroform, and once with chloroform. Separation of the organic and aqueous phases was by centrifugation (2000 RPM for five minutes). The DNA was precipitated by adding 1/10th volume 8 M NH40Ac, two volumes of 95% ethanol. The DNA was wound out of solution on a glass rod, washed with 70% ethanol, and dissolved in 1 X TE containing 10 Ag/m1 RNase A. Samples were stored at 4°C.  32  Materials and Methods 1.7. Purification of Plasmid DNA Single colonies were picked from L broth plates and transferred to 2.5 ml of 2 X YT broth containing 50 Ag/ml of ampicillin and grown overnight at 37°C. 1.5 ml of the overnight culture was transferred to a microfuge tube and spun at 14,000 RPM for 30 seconds. The cells were resuspended in 300 Al of 10 mM Tris-HC1, 1 mM EDTA, 0.1 M NaOH, 0.5% SDS and vortexed for 2-5 seconds. 200 Al of 3 M Na0Ac (pH 5.2) was added and the mixture was vortexed for another 2-5 seconds. The microfuge tube was then spun 14,000 RPM for 2 minutes and the supernatant collected. The DNA was precipitated by adding 2 volumes of 100% ethanol and pelleted by centrifugation at 14,000 RPM for 15 minutes. The DNA pellet was rinsed with 70% ethanol, dried in a vacuum desiccator, and dissolved in 50 Al of 1 X TE containing 10 Ag/ml RNase A. Samples were stored at -20°C.  1.8. Isolation of Bacteriophage DNA Charon 4 bacteriophage were grown on the E. coli strain C600 as follows. A single colony of C600 cells was picked and transferred to 5 ml of L broth, which was grown overnight at 37°C. The cells were pelleted at 2,000 RPM for five minutes, the supernatant discarded,  33  Materials and Methods  and the cells resuspended in 2.5 ml of 10 mM MgC12. 1.0 ml of the resuspended cells was infected with 1 X 107 phage and incubated at 37°C for 10 minutes. 1/10th of the infected cells were mixed with 3 ml of top agarose, poured onto each of 10 L broth plates, and incubated at 37°C overnight. Five ml of SM buffer (50 mM Tris-HC1, pH 7.5, 100 mM NaC1, 10 mM MgC12, 0.01% gelatin) were overlaid on each confluently lysed plate and stored overnight at 4°C. The overlay was pooled and centrifuged at 10,000 RPM for 10 minutes to pellet bacterial debris. To 40 ml of the overlay, 4.0 g of polyethylene glycol (PEG) 8000 and 2.25 g of NaC1 was added. This solution was gently mixed at room temperature until the salt had dissolved and then incubated at 4°C for 3 hours. The resulting phage precipitate was centrifuged at 10,000 RPM for 10 minutes and the pellet dissolved in 9 ml of SM buffer. 6.75 g of CsC1 was added, the mixture transferred to a 12 ml heat sealable tube, and centrifuged at 60,000 RPM overnight. The resulting phage band was removed using a 1 ml syringe and stored at 4°C. DNA was extracted from the CsCl-purified phage by formamide extraction. 50 Al of the phage stock was mixed with 5 Al of 2 M Tris-HC1, 0.2 M EDTA, pH 8.0, 50 Al  34  Materials and Methods formamide, and incubated for 2 hours at room temperature. The bacteriophage DNA was precipitated by adding 50 Al of distilled H20 and 300 Al of 95% ethanol, and collected by centrifugation at 14,000 RPM for 5 seconds. The precipitated DNA was rinsed with 70% ethanol, air dried, and dissolved in 50 Al of 1 X TE.  1.9. Lambda ZAP DNA Isolation DNA from lambda ZAP bacteriophage was isolated as plasmid DNA according to the Stratagene protocol. 1 X 105 lambda ZAP phage and 1 X 107 R408 helper phage were mixed with 200 Al of the E. coli strain BB4 and incubated for 15 minutes at 37°C. This mixture was added to 5 ml of 2 X YT broth and incubated with shaking for 4 hours at 37°C to produce Fl phage particles containing the packaged plasmid. The mixture was heated for 20 minutes at 70°C and then centrifuged at 2,000 RPM for 5 minutes. 200 Al of the supernatant was mixed with 200 Al of BB4 cells and incubated at 37°C for 15 minutes. 10 Al of this mixture was then plated onto L broth plates containing 50 Al/ml of ampicillin and incubated overnight at 37°C. Single colonies from these plates were used to isolate plasmid DNA.  35  Materials and Methods 1.10. Isolation of Nematode RNA Nematodes were grown on plates in the same manner as described for genomic DNA isolation. Pelleted worms were frozen in liquid nitrogen and then powdered in a chilled mortar and pestle. 3 ml of homogenization buffer (7.5 M guanidinium hydrochloride, 25 mM sodium citrate, pH 7.0, 0.1 M B-mercaptoethanol) was added to the powdered worms. The homogenate was then passed through a 21 gauge needle five times to reduce the viscosity and shear genomic DNA. The homogenate was gently layered over a 1 ml cushion of CsC1 solution (5.7 M CsCl, 25 mM sodium citrate, pH 5.0 that had been treated with 0.1% diethylpyrocarbonate (DEPC) for 15 minutes and autoclaved) and centrifuged at 42,000 RPM for 16 hours at 22°C in a SW50.1 rotor. The RNA pellet was suspended in 300 Al of sterile, DEPCtreated dH20. The aqueous RNA was precipitated by adding sterile DEPC-treated 3 M sodium acetate (pH 5.2) to a final concentration of 0.25 M and 2.5 volumes of 95% ethanol. The RNA was pelleted by centrifugation at 14,000 RPM for 5 minutes, rinsed with 70% ethanol, airdried, and resuspended in sterile DEPC-treated dH20.  36  Materials and Methods 1.11. Restriction Enzyme Digests Restriction endonucleases were obtained from Pharmacia, Bethesda Research Laboratories, and Boehringer-Mannheim. Digestion of DNA with restriction enzymes was carried out under the conditions recommended by the manufacturer.  1.12. Agarose Gel Electrophoresis Restriction enzyme-digested DNA was size fractionated by agarose gel electrophoresis. DNA was loaded onto 0.5%-1.2% agarose gels prepared in 1 X TBE buffer (89 mM Tris-HC1, 89 mM boric acid, 1 mM EDTA, pH 8.0) containing 1 Ag/m1 ethidium bromide. The gels were electrophoresed in 1 X TBE at either 30 volts for large gels (15 cm X 20 cm) or at 90 volts for small gels (6 cm X 10 cm). DNA fragments were visualized and photographed using a 300 nm wavelength UV transilluminator.  1.13. Electroelution Electroelution was done to recover DNA fragments from agarose gels following electrophoresis. DNA fragments were visualized using the 300nm wavelength UV transilluminator. DNA was excised from the gel using a razor blade and placed into 10 mm diameter Spectra/poor  37  Materials and Methods standard dialysis tubing cut to a length of about 5 cm. The tube was sealed at one end with a plastic clip, filled with approximately 400 Al of 1 X TBE, air bubbles removed, and the tube sealed at the other end. The dialysis tube containing the gel slice was then placed in a small gel apparatus submerged in 1 X TBE and 90 volts was applied for about one hour to elute the DNA from the gel slice. The eluted DNA was recovered from the dialysis tubing using a micropipette. Preparation of dialysis tubing: Prior to use, dialysis tubing was boiled for 10 minutes in a 2% NaHCO3 solution, rinsed thoroughly in dH20, and then placed in dH20 and autoclaved for 10 minutes. Prepared tubing was stored in dH20 at 4°C.  1.14. Construction of a Lambda ZAP Library A lambda ZAP library was constructed according to Stratagene protocol from KR1163 DNA prepared as follows. EcoRI-digested genomic DNA was fractionated on a 0.7% Low Melting Point Agarose (Sigma) gel. An agarose gel slice containing DNA fragments 2.5 kb to 3.5 kb in size was cut out of the gel and melted at 65°C in 5 volumes of 20 mM Tris-HC1, 1 mM EDTA, pH 8.0 for 10 minutes, extracted once with TE-saturated phenol, once with 1:1 phenol-  38  Materials and Methods chloroform, and once with chloroform. The DNA was precipitated by adding 1/10th volume 8 M NH40Ac and one volume of isopropanol.  1.15. Screening Phage Libraries Phage were isolated from five different libraries: a partial EcoRI Charon 4 N2 genomic library constructed by T. Snutch (Simon Fraser University), a partial EcoRI Charon 4 C. briggsae genomic library constructed by T. Snutch (Simon Fraser University), a lambda ZAP N2 cDNA library constructed by R. Barstead and R. Waterston (Barstead and Waterston 1989), a lambda gt10 KR579-D genomic library constructed by R. Mancebo (our lab), and a lambda ZAP KR1163 genomic library described above. Bacteriophage were plated on NZY (1.0% NZ-amine, 0.5% yeast extract, 10 mM MgC12, 0.2% maltose, pH 7.0) agarose plates at a frequency of approximately 4000 plaques per plate. Plates were incubated overnight at 37°C and then allowed to cool at 4°C for at least one hour. Dry nitrocellulose filters (Schleicher and Schuell) were layered on top of the plates for 5 minutes at room temperature. The filters were removed and treated with 0.5 M NaOH, 1.5 M NaC1 for 5 minutes, then with NH40Ac for 5 minutes, and finally with 2 X SSPE for 5 minutes.  39  Materials and Methods The treated filters were air-dried and then baked at 80°C for one hour. The baked filters were hybridized with the appropriate radiolabelled probe, washed, and air-dried. Positive plaques identified by autoradiography were picked from plates by removing and transferring a small plug to a 1.5 ml tube containing SM buffer. The titre in this tube ranged from 1 X 103 to 1 X 106 plaque forming units per ml. Positive plaques were purified by successive rounds of plating and screening.  1.16. Purification of Bacteriophage Clones Bacteriophage clones which were identified in the initial screen were further purified in the following manner. A positive plaque was picked by removing a small plug approximately 2 mm in diameter and placed in 0.5 ml of sterile SM buffer. An agarose plug typically contained between 1 X 103 and 1 X 106 bacteriophage. Approximately 50 to 100 phage were plated and the plates were again screened. This procedure was repeated until all the plaques plated hybridized to the probe.  1.17. Labelling of DNA Probes DNA probes were labelled with 32P-dATP by the oligolabelling technique described by Feinberg and  40  Materials and Methods Vogelstein (1983). Probes with a specific activity of 1 X 10 8 to 1 X 10 9 counts per minute/pg were synthesized using this procedure. Probes were purified in a Sephadex G25 (Pharmacia) spin column. Immediately prior to use, the labelled probes were boiled for 10 minutes and then cooled on ice for 5 minutes.  1.18. Southern Transfer and Hybridization Following electrophoresis, the agarose gel was soaked in 0.25 M HC1 for 15 minutes. The gel was then rinsed in dH20 and soaked in 0.5 M NaOH, 1.5 M NaC1 for 30 minutes. Finally the gel was rinsed in dH20 and then soaked in 1 M NH40Ac for 30 minutes. The treated gel was placed on a 3 MM Whatman paper wick that was soaked in 10 X SSC. A Nytran filter (Schleicher and Schuell) was cut to the size of the gel, soaked in 1 M NH40Ac for 15 minutes and layered on top of the gel. Care was taken to remove any air bubbles that may have been trapped between the gel and the Nytran membrane. Two 3 MM Whatman filters cut to the size of the gel and soaked in 1 M NH40Ac were placed on top of the Nytran membrane. The DNA was allowed to transfer to the membrane by capillary action for two hours. The filter was removed, rinsed with 10 X SSC, air dried, and then baked at 80°C for one  41.  Materials and Methods hour. The baked filter was transferred to a heat sealable bag and approximately 1 ml of 5 X SSPE, 0.3% SDS was added for each 20 cm2 of Nytran membrane. Labelled probe was added and hybridization was allowed to take place overnight at 62°C. The filter was washed twice for 15 minutes in prewarmed wash solution (0.5 X SSC, 0.3% SDS) and once for 30 minutes. The filter was removed, air dried, sealed in plastic wrap and exposed to Kodak RP or AR film using Dupont Lightning Plus intensifying screens at -70°C.  1.19. Northern Transfer and Hybridization RNA isolated from the different larval stages of  C. elegans was provided by T. Snutch and D. L. Baillie (Simon Fraser University). Northern blot filters containing RNA from the different larval stages were prepared by S. Prasad (Simon Fraser University).  C. elegans RNA was denatured in 2.2 M formaldehyde; 50% deionized formamide; 0.2 M morpholinopropane sulfonic acid, pH 7.0, 5 mM Na0Ac, 1 mM EDTA (1 X MOPS) for 15 minutes at 60°C. RNA was electrophoresed in a 1.1% agarose gel containing 1 X MOPS; 2.2 M formaldehyde, pH 7.0) at 80 volts for 2 - 3 hours. The RNA samples used for marker lanes contained 1 pg of ethidium bromide.  42  Materials and Methods  RNA transfer was accomplished using a nylon based membrane, Gene Screen (New England Nuclear). In this method, the gel was rinsed once in distilled water for 15 minutes and then rinsed twice in 0.025 M potassium phosphate buffer. The nylon membrane was rinsed in dH20 and then soaked in 0.025 M potassium phosphate prior to use. This membrane was layered on top of the gel and care was taken to remove any air bubbles. Two 3 MM Whatman filters, cut to the appropriate size, were placed on top of the Gene Screen membrane. RNA was allowed to transfer to the membrane filter by capillary action overnight. The filter was removed, briefly soaked in potassium phosphate buffer, and placed on a glass plate with the RNA side up. The membrane was then exposed to a UV Transilluminator at a distance of 10 cm for a period of 5 minutes. This treatment was used to covalently crosslink the RNA to the Gene Screen membrane. After UV crosslinking, the membrane was baked at 80°C for one hour. The baked filter was transferred to a heat sealable bag and approximately 1 ml of 5 X SSPE, 0.3% SDS was added for each 20 cm2 of Nytran membrane. Labelled probe was added and hybridization was allowed to take place overnight at 68°C. The filter was washed twice for 15 minutes in prewarmed wash solution (2 X SSPE, 0.3%  43  Materials and Methods  SDS) and once for 30 minutes. The filter was removed, air dried, sealed in plastic wrap and exposed to Kodak RP or AR film using Dupont Lightning Plus intensifying screens at -70°C. A stage-specific Northern blot was provided by S. Prasad, Simon Fraser University. Each lane was loaded with 10 mg of RNA. The RNA filter had not been previously probed and then stripped.  1.20. DNA Sequencing  Plasmid DNA was isolated as previously described. Nested sets of plasmid insert deletions were constructed using the method of Henikoff (1984). DNA sequence was obtained using double-stranded dideoxy sequencing of plasmid DNA (Sanger, Nicklen, and Coulson 1977; Hattori and Sakaki 1986). Closed circular plasmid DNA was isolated by gel electrophoresis in low melting point agarose. Low melting point agarose containing the closed circular DNA was cut from the gel and placed in 500 Al of 20 mM Tris-HC1, 1 mM EDTA (pH 8.0). This sample was then heated at 70°C for 15 minutes to melt the agarose. The agarose was then extracted twice with TE-saturated phenol followed by one extraction with chloroform. 1/10th volume of 8 M NH40Ac and one volume of isopropanol was added to precipitate the DNA. The pellet was rinsed with  44  Materials and Methods  70% ethanol and vacuum dried. Approximately 1.0 jig of DNA in a volume of 16 Al (1 X TE) was mixed with 4 Al of 1 M NaOH; 1 mM EDTA and left at room temperature for five minutes. To this mixture, 2 Al of 2 M NH40Ac (pH 5.4) was added and mixed. The DNA was precipitated by adding 150 Al of 95% ethanol and pelleted for 10 minutes in a microcentrifuge. The pellet was rinsed twice with 70% ethanol, dried for 10 minutes in a vacuum desiccator, and dissolved in 10 Al distilled water. The denatured plasmid DNA was mixed with 1 Al M13 oligonucleotide primer (5 ng/A1), 1.5 Al Klenow buffer (70 mM Tris-HC1, pH 7.5, 200 mM NaCl, 70 mM MgCl2, 1 mM EDTA) and incubated for 15 minutes at 60°C. After cooling to room temperature for 15 minutes, 1 unit of DNA polymerase I (Klenow fragment; Pharmacia; 1 unit/A1) and 2.0 Al of 35 S-ATP (NEN; 1350 Ci/mM) were added. The mixture was  dispensed into four tubes, each containing 1.5 Al of the different dideoxynucleotide mixes (Pharmacia) and incubated for 15 minutes at 45°C. One Al of chase solution (0.125 mM for each dNTP) was added and incubated for 15 minutes at 45°C. To each sample 6.0 Al of loading buffer (95% formamide, 12.5 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol) was added and the samples stored on ice until required. Immediately prior to  45  Materials and Methods  loading, the samples were heated to 95°C for three minutes. Two Al of each sample was loaded on a 6% acrylamide-7 M urea gel and electrophoresed at 50 watts using 0.5 X TBE buffer for two to four hours. An aluminum plate was clamped onto the sequencing apparatus to maintain a constant heat distribution. After electrophoresis the gel was transferred to Whatman 3 MM paper and vacuum dried for 30 minutes at 80°C (Southern 1975). The filter was wrapped in plastic and exposed to Kodak RP film for one to three days.  1.21. Sequence Analysis  Sequence data was assembled using the Delaney Sequence program to identify overlaps and the Eye-ball Sequence Editor ESEE (written by E. Cabot, Simon Fraser University) to edit and assemble sequences. Potential eukaryotic signal sequence was identified using the PSignal program of PCGene. Protein and DNA database searches were done using the FASTA algorithm (Pearson and Lipman 1988).  46  III. RESULTS  Section 1  1.1. Altered Tcl hybridization in Bristol Strains The presence of additional Tcl-hybridizing bands was discovered during the molecular analysis of a high recombination strain BC313. A Southern blot of EcoRI-digested DNA was observed to differ from that of the N2 strain when hybridized with Tc1 sequences (Rattray 1986). At least six additional Tcl-hybridizing bands were present in addition to the 30 Tcl-hybridizing bands normally present in N2 strains. In order to investigate the source of the additional bands, strains used in construction of BC313 were examined. It was found that the additional Tcl-hybridizing bands were derived from the Unc-13 strain CB51 (Figure 2). Two Unc-13 strains derived from CB51 by out-crossing (BC82 and BC193) differed from CB51 and each other with respect to the number of Tcl-hybridizing bands. However, the original Unc-13 strain CB51 and CB1833 that were obtained from the Laboratory of Molecular Biology at Cambridge, England (see Materials and Methods) had a Tcl hybridization  47  Results  Figure 2. Tcl hybridization pattern of Bristol strains Genomic DNA was digested with EcoRI and probed with Tc1. Lane 1=N2, Lane 2=CB51, Lane 3=BC313. Arrows indicate additional bands not normally present in Bristol strains.  48  Results pattern identical to N2 (see Figure 3). These results suggested that the alteration in Tcl-hybridization pattern occurred after the original CB51 strain was frozen in Cambridge. Two separate isolates of CB51 were maintained at 20°C for a period of approximately two months in an attempt to observe additional changes in Tcl hybridization. DNA from these isolates was examined with respect to the Tcl hybridization pattern (see Figures 4 and 6). The two isolates were referred to as KR579 and KR579-D (KR579-D indicates that DNA was isolated although the worm stock no longer exists). In these substrains of CB51, the Tc1 patterns were again observed to differ from N2, CB51, and each other (see Figure 5 for relationships between these substrains of CB51 and N2). Several experiments were done in order to determine if the additional Tcl-hybridizing bands corresponded to the presence of additional Tcl elements (Babity, Starr, and Rose 1990). Digestion of N2 and KR579 DNA with several restriction enzymes showed that the additional bands were not the result of additional EcoRI sites or the conversion of Tcl elements to the Tcl(Eco) variant described by Harris and Rose (1989). The number of Tclhybridizing bands did not appear to be correlated to the  49  Results  Figure 3. Tcl hybridization pattern in Cambridge strains Genomic DNA was digested with EcoRI and probed with Tcl. 1=N2, Lane 2=CB51, Lane 3=CB1833. Strains obtained from the Laboratory of Molecular Biology at Cambridge, England have the Bristol pattern of Tcl hybridization.  50  Results  N2. KR579  Figure 4. Altered Tcl hybridization pattern in the strain KR579 Genomic DNA was digested with EcoRI and probed with Tcl. In the strain KR579, additional Tcl-hybridizing bands are present and are indicated by arrows.  51  Results  CB51 BC82  BC313 BC193  KR579 KR5794) KR1082  KR1787  Figure 5. The relationship of CB51-derived strains CB51 has the Bristol pattern of Tcl hybridization. The strain BC82 came from a CB51/Bristol heterozygote. BC313 and BC193 were derived from crosses involving BC82. KR579 is a descendant of the original CB51 strain. KR579-D refers to a preparation of DNA. BC82, BC313, BC193, KR579, and KR579-D have more Tcls than CB51 and the pattern is different for each strain. KR1082 has the same pattern of Tcls as KR579, but was cultured independently. The parentheses indicate no change in the Tcl Southern blot hybridization pattern. KR1787 has more Tcls than KR579.  52  Results  Unc-13 phenotype of CB51 or the high recombination phenotype of BC313 in which the presence of additional Tcl-hybridizing bands was originally detected. Strains containing the unc-13 alleles (e450) and (e1091) both show the normal N2 Tcl hybridization pattern. The highrecombination strains BC187 and BC196 also contained the normal N2 pattern of Tcl hybridization (Babity, Starr, and Rose 1990). The additional Tcl-hybridizing bands in KR579 also did not appear to be the result of unequal cross-over events between adjacent Tcl elements. Unequal crossingover would result in the duplication or deletion of sequences flanking the Tcl elements. By using unique sequences flanking Tcl elements in the N2 strain, it was possible to determine if sequences had been duplicated in the KR579 strain. Nine probes containing unique sequences flanking Tcl insertions in the N2 strain (Harris and Rose 1989) were hybridized to N2 and KR579 DNA (Babity, Starr, and Rose 1990). Eight of the nine probes did not detect a difference between the strains; however, in one case (probe pCeh44), an extra band was observed in the KR579 strain.  53  Results 1.2. Isolation of a Tc1 clone from KR579-D  In order to demonstrate that the presence of additional Tcl-hybridizing bands in CB51-derived strains was the result of Tcl transposition, a novel Tclhybridizing EcoRI fragment from KR579-D was identified (6th band from the bottom, see Figure 6, R. Mancebo, unpublished data). Unique sequence DNA flanking the Tcl element (pCehl40) was used to probe EcoRI-digested genomic DNA from N2 and KR579-D. This probe detected a 0.9 kb band in N2 and a 2.5 kb band in KR579-D (see Figure 7). The 1.6 kb restriction fragment length difference between the two strains corresponds to the presence of an additional Tcl element in the KR579-D isolate not present in the N2 strain. Part of the cloned Tcl element and the genomic insertion site were sequenced. The sequence of the insertion site is CATGTAAGT (see Figure 8). This sequence is similar to the published consensus sequence [GA(G/T)(A/G)TA(T/C)(G/C)T] derived for Tcl insertion sites from strains in which Tcl is mobile (Mori et al. 1988; Eide and Anderson 1988 in Moerman and Waterston 1989). The sequenced insertion site shares identity with seven of the nine nucleotides in the consensus sequence.  54  Results  2  Figure 6. Isolation of an additional Tcl-hybridizing  band from KR579-D Genomic DNA was digested with EcoRI and probed with Tc1. Lane 1=N2, Lane 2=KR579-D. The arrow indicates an additional 2500 bp Tc1-hybridizing band.  55  Results  I  OM^2.5  am^0.9 1^2  Figure 7. N2 and KR579-D DNA hybridized with DNA  flanking the Tcl insertion site Genomic DNA was digested with EcoRI and probed with pCehl40. Unique sequence DNA detects a 900 bp band in Bristol and a 2500 bp band in KR579-D. Lane 1=KR579-D, Lane 2=N2. The 1600 bp difference in size demonstrates the presence of a Tcl element in KR579-D that is absent in Bristol.  56  Results  Figure 8. Sequence of the Tcl insertion site from KR579-D Raw sequence data from both sides of the cloned Tcl element are shown. 1. Sequence shown includes 5 bp of genomic DNA and 23 bp of Tcl sequence ending at the EcoRV site (GATATC) within the inverted repeat. The 5' portion of the Tcl insertion site is indicated with asterisks. 2. Sequence shown includes 23 bp of Tcl sequence starting at the EcoRV site (GATATC) within the inverted repeat and 4 bp of genomic sequence. The 3' portion of the Tcl insertion site is indicated with asterisks. 3. The cloned Tcl insertion site and the published Tcl insertion site consensus sequence are shown. The cloned Tcl insertion site matches at 7 out of 9 nucleotides as indicated by vertical lines.  57  Results  GA GCTT GTACAT GT CA CGACC 33 II ITT CT AT AG  CCAAAAGTGGATATCT TT T TGGCCAGCACTGT AAG-TG AG/IAG AT ATCAT  CCAAAAGTGGATATCT TTT 1GC4CCAGCACI31 AAnTG AG AG ATATCAT  1.  3'^ 5 ..GTACATGTCACGACCGGTTITTCTATAG .CATGTCACGAGCTGGCCAAAAAGATATC •'5 *****^ 3*  2  ****3 • 5'^ GATATCTITTIGGCCAGCACTGTAAGT CTATAGAAAAACCGGICGAGCACTICA 3'^ 5•  3  CATGTAAGT  Ill^I  GATGTATGT GA CC  58  Results 1.3. Further Tc1 Mobilization and Mutator Activity  Eight lines of the CB51 strain were maintained on plates for a period of one year in order to determine if Tcl accumulation would continue. DNA was isolated from these lines; however, no additional Tcl elements could be detected by Tcl hybridization to Southern blots. One of the lines was frozen as KR1082. These lines were maintained on plates for another two years and the DNA re-examined. Additional Tcl elements were observed in one of the eight lines maintained (KR1787). The Tcl pattern of KR1787 is shown in Figure 9. In the KR1787 strain at least ten additional Tcl elements had accumulated with respect to the parental stock. Using an assay developed by Moerman and Waterston (1984) mutator activity in KR1787 was examined. The forward mutation rate of the unc-22(1V) gene was 17 twitchers per 105 individuals screened from the KR1787 strain whereas no twitchers were observed in 105 individuals from the CB51 strain. Twitcher strains have been established and revertants observed.  1.4. Isolation and Analysis of the Mutation dpy-5(s1300) The dpy-5(s1300) mutation was isolated as a spontaneous dumpy mutation in a Bristol-Bergerac hybrid  59  Figure 9. Tcl hybridization pattern of CB51-derived strains Genomic DNA was digested with EcoRI and probed with Tcl. Lane 1=N2, Lane 2=KR579, Lane 3=KR579-D, Lane 4=KR1787. Additional Tc1 elements are present in KR579, KR579-D, and KR1787 in comparison to Bristol. While the pattern is different in each strain, KR1787 has many additional Tcl elements.  60  Results  strain, BC1906 (R. Rosenbluth, Simon Fraser University). This mutation was subsequently shown by R. Rosenbluth to be an allele of the dpy-5 gene through genetic mapping and complementation analysis. This allele was proposed to be a transposon-induced mutation because it was isolated in a Bergerac-derived strain C. elegans in which Tcl is mobile and causes mutations at a high frequency (Moerman and Waterston 1984; Eide and Anderson 1985a). In order to determine if dpy-5(s1300) was a Tc1 transposon-induced allele, a molecular analysis of the mutation was undertaken. The first step in the analysis was to cross the BC1906 strain to Bristol worms in order to reduce the number of Tcl elements present. Bristol strains of C. elegans normally contain approximately 30 copies of  Tcl, while Bergerac strains contain approximately 300 Tc1 elements (Emmons et a/. 1983). This difference in Tcl copy number can be visualized in Southern blot hybridizations using Tcl as a probe. In Bristol strains, Tcl hybridization produces a discrete pattern of bands, while in Bergerac strains the higher number of Tcl elements produces a smear of Tcl-hybridizing bands. Since BC1906 is a Bristol-Bergerac hybrid strain, the number of Tcl elements is too great to determine if a  61  Results  single Tcl-hybridizing band correlates with a Tcl-induced mutation in the dpy-5 gene. Two methods were used to reduce the number of Bergerac-derived Tcl elements. In the first, BC1906 was crossed to the Bristol N2 males, the Fl generation hermaphrodites isolated and allowed to self-cross, followed by selection of dumpy hermaphrodites in the F2 generation. By repeatedly crossing the strain carrying the dpy-5(s1300) mutation to the Bristol N2 strain, the number of Tcl elements derived from the Bergerac genome was greatly reduced. However, closely linked Tc1 elements may not have been removed by this method. Therefore, a second method was used in which closely linked genetic markers were crossed onto the chromosome carrying the dpy-5(s1300) mutation. A strain with the genotype unc-38(e264) dpy-5(s1300) unc-87(e1459), KR1163, was  constructed (see Materials and Methods). By crossing on these markers, all Tc1 elements except those immediately flanking the dpy-5 gene were removed. Genomic DNA from the KR1163 strain was isolated. This DNA was cut with the restriction enzyme EcoRI and electrophoresed on a 0.7% agarose gel. The gel was then used to make a Southern blot which was hybridized with a radiolabelled Tcl probe. Autoradiography of this  62  Results  Southern blot revealed a Tc1-hybridizing pattern similar to that of Bristol strains (see Figure 10). However, an additional Tcl-hybridizing band corresponding to an EcoRI fragment approximately 2.7 kb in size was observed. This additional Tcl element must be tightly linked to the dpy-5 gene.  1.5. Isolation of a Tcl Clone from KR1163  A lambda ZAP library was constructed from EcoRIdigested DNA from the strain KR1163 and screened using Tcl as a probe. The 2.7 kb Tcl-hybridizing fragment associated with the dpy-5(s1300) mutation was isolated and the Tcl insertion site examined. A portion of the cloned Tcl element and the genomic site were sequenced. The sequence of the insertion site is similar to the published consensus sequence derived for Tcl insertion sites (Mori et al. 1988; Eide and Anderson 1988 in Moerman and Waterston 1989). The sequenced insertion site shares identity with seven of the nine nucleotides in the consensus sequence (see Figure 11).  63  Results  Figure 10. Southern blot of N2 and unc-38 dpy-5(s1300) unc-87 DNA Genomic DNA was digested with EcoRI and probed with Tcl. Lane 1=N2, Lane 2=KR1163. The arrow beside Lane 2 indicates the presence of an additional 2700 bp Tclhybridizing band that is absent in Bristol.  64  Results  Figure 11. Sequence of the Tcl insertion site from the strain KR1163  Raw sequence data from both sides of the cloned Tcl element are shown. 1. Sequence shown includes 5 bp of genomic DNA and 23 bp of Tcl sequence ending at the EcoRV site (GATATC) within the inverted repeat. The 5' portion of the Tcl insertion site is indicated with asterisks. 2.  Sequence shown includes 23 bp of Tcl sequence  starting at the EcoRV site (GATATC) within the inverted repeat and 4 bp of genomic sequence. The 3' portion of the Tcl insertion site is indicated with asterisks. 3.  The cloned Tcl insertion site and the published Tcl  insertion site consensus sequence are shown. The cloned Tcl insertion site matches at 7 out of 9 nucleotides as indicated by vertical lines.  65  Results  GGTATOGATAAGCTTGATA TeTT-TTTGGCCAGCACTG/ACTCCTGCAA GTGCTC1 001ATCGATAAGCT1GA1A 7011TTTGGCCAGCACTGTACTCCTGCAATGTGCTC1 -  GGTATOGATAAGCTT ATA GOTATCGATAAGCTTGATA  CTTTTTGGCCAGCACTGTATOTOTGGCATTCACAAA CTITTIGGCCAOCACTGTATGTGTGGCATTCACAAA  5 • 1^3 ' ..CCICATGICACGACCGGITTTTCTATAG 1.GGAGTCACGAGCTGGCCAAAAAGATATC 5.***** 3'  2 3.  5 GATATCTITTTGGCCAGCACTGTAIGT CTATAGAAAAACCGGICGAGCACTACA 3' 5• GGAGTATGT I HUM GATGTATGT GA CC  66  Results  Section 2  2.1. Examination of Putative Tc1-Induced dpy-5 Alleles  Unique sequence flanking the Tcl insertion site cloned from the strain KR1163 was used to probe DNA from six strains carrying putative Tcl-induced mutations if dpy-5: dpy-5(s1300), dpy-5(h14), dpy-5(mn303), dpy-5(m476), dpy-5(bx9), and dpy-5(bx10). In addition,  wildtype revertants of the mutations dpy-5(h14) and dpy-5(mn303) were examined. For the mutations dpy-5(s1300), dpy-5(h14), dpy-5(mn303), dpy-5(bx9), and dpy-5(bx10), a 1.6 kb restriction fragment length  difference (RFLD) was detected in comparison to the wildtype and wildtype revertants (see Figures 12 and 13). For the dpy-5(m476) mutation, no RFLD was detected. Therefore, a 1.6 kb insertion is present in five of the six putative Tcl-induced dpy-5 alleles. In the wildtype and wildtype revertantsthe 1.6 kb insertion is absent.  2.2. Isolation of C. elegans Genomic Clones  Unique DNA sequences flanking the Tcl insertion site from KR1163 were used to screen a genomic Charon4 bacteriophage library (constructed by T. Snutch, Simon Fraser University). The genomic phage KR#85 was  67  Results  Figure 12. Southern blot of DNA isolated from Dpy-5 and wildtype revertant strains  Genomic DNA was digested with EcoRI and probed with Tcl flanking sequences. Lane 1=dpy-5(h14), Lane 2=dpy-5(mn303), Lane 3=dpy-5(h14) wildtype revertant,  Lane 4=dpy-5(mn303) wildtype revertant, Lane 5=dpy-5(s1300), Lane 6=dpy-5(m476). A 2700 bp band is  detected for dpy-5(s1300), dpy-5(h14), and dpy-5(mn303) mutations. An 1100 bp band is detected for dpy-5(m476) and the wildtype revertants of dpy-5(h14) and dpy-5(mn303). The 1600 bp difference corresponds to the  presence/absence of Tcl.  68  Results  Figure 13. Southern blot of DNA isolated from Dpy-5 and wildtype strains  Genomic DNA was digested with EcoRI and HindIII and probed with a dpy-5 cDNA. Lane1=dpy-5(bx9), Lane 2=dpy-5(bx10), Lane 3-N2. The probe hybridizes to three  fragments in the N2 strain measuring 3.0 kb, 0.35 bp, and 0.25 bp in size. In the two Tc1-induced dpy-5 alleles, the probe hybridizes to three fragments measuring 3.0 kb, 1.95 kb, and 0.25 kb in size. The 1.6 kb RFLD corresponds to the insertion of Tcl. Minor bands at 1.1 kb and 0.35 kb represent incomplete HindIII digestion and Tcl excision respectively.  69  Results identified and purified. A restriction enzyme map of KR#85 is shown (see Figure 14). The KR#85 phage contains approximately 11 kb of genomic DNA. This phage was sent to John Sulston and Alan Coulson (Laboratory of Molecular Biology at Cambridge, England) where it was used to map a 350 kb cosmid contig to chromosome I in the C. elegans genome (Figure 15).  2.3. Isolation of C. elegans cDNA Clones The 1.1 kb genomic fragment from KR#85 was used to screen a lambda ZAP cDNA library constructed by Robert Barstead (Barstead and Waterston 1989). In this screen seven cDNAs were identified from approximately 3 X 104 phage clones. The clones that hybridized to the 1.1 kb genomic EcoRI fragment were purified and the plasmids excised according to Stratagene protocol. Two cDNA clones measuring 392 bp and 890 bp in size were selected for further analysis (see Figure 16).  2.4. Isolation of C. briggsae Genomic Clones A cDNA clone was used to screen a lambda gt10 C. briggsae genomic library (constructed by T. Snutch, Simon Fraser University). The clones that hybridized to the probe were purified and used in restriction enzyme  70  ^ ^  Results  KR#85 RESTRICTION MAP  B  EE^EE E I^It^I  EH E  CC^EEHH E XE  I^1^I  1  'Eh^m 411111^i^1  E  ^  B S B I^i^i  BHB  ^  B  H SE 1^II  3.0^0.3 1.1 1 KB  E•EcoRI^X•Xhol^B•BamHI H•HindlIl^S•Sall^C•Clal  Figure 14. Restriction enzyme map of the Charon4 genomic  phage KR#85 The dpy-5 gene is contained on three EcoRI fragments measuring 3.0 kb, 0.3 kb, and 1.1 kb in length. Exons are depicted as thicker lines.  71  ^  Results  C30H7^ F52B8^ C08D6^(Y48A1 2)^ C25G7 C24C1 CO4F9  M01E11  a2661 02032^F40012 (Y1 3C7)  K01810^  F56A3 K6  Cl 6G8 ^  KR#85  MO1A1 2 C29C1 2  Cl 2H4 CO9G2  AD7 C 34G6 MO1E4^ --' CO3E6 CO4F1^C30A1 1 B0414 CO8C1 1^JF8 C25B5  80290 (Y26A2)  B0342^C3086 RO1H1 1  F27C1 C24H1  C32E7  F56H1  R49 R0789^ (Y53F1 0) CO1 G8^ C28A7 (Y51 G8) W0881 1^ C24E5 F27E10^ Cl 2H1 C5589^ T07G5 F22D9^ DA8 C40G4 C25G3^ MO2A5^ (Y4607) C4008 WO4A6  C30B6  F42A5 (Y41H11)^ (Y2587) ^ F55F8 C32F1 0 WO6A6  MO1A10 .SaLa —. C24A1 1^T^ (Y3 8 HI)  COOF3^  C53A1 1  CO7F1 0  Y53H 1 1  &a.y_H.a  R13B6 DDDG7  dpy-5 J.BabitRose  10 Hindi(' sites  Figure 15. Cosmid contig identified by the genomic phage  KR#85 The figure shows a cosmid contig identified by the genomic phage KR#85. Both cosmids and yeast artificial chromosomes (YACs) are shown. YACs are shown in parentheses. The length of both cosmids and YACs lines correspond to the number of HindIII sites. The position of the dpy-5 gene is shown relative to physical map represented by the cosmid contig.  72  B0207  Results  .  1  CC^E E HH^E  E  1.8^  I  I  *ink^.9II^  4 .11 I .^  11^.7^  Tc 1 250 bp  cDNA 2 mismilisymi  cDNA 1  Figure 16. Restriction Map of dpy-5 Region The figure shows three EcoRI genomic fragments that contain the dpy-5 gene. The six exons are depicted as thick line segments. The dpy-5 gene is transcribed from left to right. The Tcl insertion into the fifth exon corresponds to the insertion site in dpy-5(s1300). Two cDNAs are shown. cDNA 2 is a full length cDNA.  73  Results and genomic blot analysis. A 9.0 kb EcoRI fragment was identified which was homologous to the C. elegans probe. Further analysis revealed that the homologous region was contained within a smaller 3.1 kb EcoRI-BglII fragment. This fragment was subcloned and sequence data was obtained.  2.5. Sequence Analysis of the dpy-5 Gene Sequence data was obtained from both genomic subclones and cDNA subclones (see Figure 17). DNA sequence was obtained from three genomic EcoRI fragments measuring 3.0 kb, 0.3 kb, and 1.1 kb in length. DNA sequence was also obtained from two cDNA clones measuring approximately 392 bp and 890 bp in length. By utilizing the sequence data from these two sources it was possible to infer the size of the dpy-5 gene. The dpy-5 gene encodes a 254 amino acid protein. The gene contains six exons measuring 268 bp, 105 bp, 105 bp, 90 bp, 83 bp, and 114 bp in length. The five introns are 416 bp, 170 bp, 54 bp, 233 bp, and 47 bp in length (see Figure 18). Sequence obtained from the dpy-5 gene was used to search the EMBL nucleotide and protein databases. Searches of the database did not reveal any informative similarities. At present the dpy-5 gene does not appear  74  Results  Figure 17. Alignment of dpy-5 Genomic and cDNA Sequences Genomic and cDNA sequences are aligned. Genomic DNA=C.eleg, 392 bp cDNA=cDNA1, and 890 bp cDNA=cDNA2. Dashes indicate cDNA gaps corresponding to introns.  75  76  Results  C.ELEG^TCTCCCTTTTCATTCGTCCAAATCTTCATTTCTTTTCCAAAATGCACCTCTTTCTTTCTG 60 III^111^1111111111111111111111111111 cDNA2^CGCGGAATCCGTTTTTTTTTTTTTTTTTTCCAAAATGCACCTCTTTCTTTCTG 53 cDNA1 C.ELEG^TTTTTCTTCTGCTTATCCTACCGTTAATTTCCACCTCAGCAGTTGAGAATAATCAAAATA 120 111111111111111111111111111111111111111111111111111111111111 cDNA2^TTTTTCTTCTGCTTATCCTACCGTTAATTTCCACCTCAGCAGTTGAGAATAATCAAAATA 113 cDNA1 C.ELEG^TTGTGGATTTTTCTGTGAAAGATTTCAACAAAAATATTAATTCAATCGATATTCTCAACA 180 111111111111111111111111111111111111111111111111111111111111 cDNA2^TTGTGGATTTTTCTGTGAAAGATTTCAACAAAAATATTAATTCAATCGATATTCTCAACA 173 cDNA1 C.ELEG^AAAAATGGGATATTGTTTCGAAATATGTGAAATTTAATGGAAACTCATCGAAATTGAATG 240 111111111111111111111111111111111111111111111111111111111111 cDNA2^AAAAATGGGATATTGTTTCGAAATATGTGAAATTTAATGGAAACTCATCGAAATTGAATG 233 cDNA1 C.ELEG^GAAGACTCCGATTATCGGAAAAAATTGACAAAATCATTTTTAAATTGGGAGACGATGGAG 300 111111111111111111111111111111111111111111111111111111111111 cDNA2^GAAGACTCCGATTATCGGAAAAAATTGACAAAATCATTTTTAAATTGGGAGACGATGGAG 293 cDNA1 C.ELEG^CTAATCAGAGTAAGTTTTTTGAAATTTAAAAGTGGATTAGCGCCCTATGGTATGACTCCT 360 cDNA2^CIIIICIII^  302  cDNA1 C.ELEG^ATGATTCTAAAATGATAAATTTTCACAGTAAAACTTTGCGAAACTGGTTTGATATTTTTA 420 cDNA2 cDNA1 C.ELEG^ATAACATTTAAAAATGGTATTGATTCAGTTCTCAACTGTTATAATTTTGGAATATTCGAA 480 cDNA2 cDNA1  77  Results C.ELEG  TGTTCCAAAAAATTTATCTGAAAAAGTTCTGAAATCAAAAAAAAATTCTTCTACCAAATA 540  cDNA2 cDNA1 C.ELEG  GGCAAAATGTTTTCTACGACTTTTATATTTTAATCAAGTTAAGAAATATTTTTTGTTAAG 600  cDNA2 cDNA1 C.ELEG  AAAAATAGTGCAAGAACGTTCAAAATTTCCGAAAAAAAAACCGAGTTTATCGAAAATTTG 660  cDNA2 cDNA1 C.ELEG  GCAATTTGCCAAACTCTTACTGACTAGCAGCAATCCTCTTGTTTTCTAAAAATTTTAATT 720  cDNA2 cDNA1 C.ELEG cDNA2  TTCAGATGAAATAATAGTTTCCTTGGGTAATAGAAATACAACTCATATGACTGTGCTCTC 780  1111111111111111111111111111111111111111111111111111111  ATGAAATAATAGTTTCCTTGGGTAATAGAAATACAACTCATATGACTGTGCTCTC 357  cDNA1 C.ELEG  GGATTCATACAAAATGAAAGTATCCACTTCGAAGCAAGTTGTTCGGAAAGGTCTGTTGAA 840  cDNA2  GGATTCATACAAAATGAAAGTATCCACTTCGAAGCAAGTTGTTCGGAAAG  11111111111111111111111111111111111111111111111111  ^  407  cDNA1 C.ELEG  AAGGTTTTGAAAATTGTATTCTGTTTTACGTTGTTAACAAAGACAATTGCTCAAGCAGCC 900  cDNA2 cDNA1 C.ELEG  TCAAGCCAGCAAGCCAAAACAAAACTTTAAAAAACTGAAATTCGGAATGAGAAAAAAAAA 960  cDNA2 cDNA1  78  Results  C.ELEG  ATACATAAAGGCTAATTAAAATTGTAATAAACCTTTCCAGAACTGTCTCTGAGAACTGGT 1020  I III I I I 1 1 1 1 1 I III I I I I  cDNA2  AACTGTCTCTGAGAACTGGT 427  cDNA1 C.ELEG  TCATCAAACGAGAACGGATGTGCTTGTGTACACGGAAATTGTGCTTGCTGCCTAGAAATT 1080  cDNA1  111111111111111111111111111111111111111111111111111111111111 TcATcAAA7iiiiiiiTiiiiiiiiiiTITilliTilliiTITTIIiiiiiiiiiiiiii 487 CGCGAGAACGGATGTGCTTGTGTACACGGAAATTGTGCTTGCTGCCTAGAAATT 54  C.ELEG  TCTGTTCCAGAATTCAGACATTCAGGTAATAGTTTTCCTTAAATTTTGTTTTCTTAATTT 1140  cDNA2  cDNA2 cDNA1 C.ELEG  TCTGITIIIIIIT'ICIGICITT Ill ^ 1111111111111111111111111 TCTGTTCCAGAATTCAGACATTCAG^  512 79  cDNA1  TCCAATACTTTTAATTCAGTTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCTTG 1200 11111111111111111111111111111111111111111 AGTTTCCATTGGCTTG 553 11171717TUTMITITT7771111111111111111 TTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCTTG 120  C.ELEG  GATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTTGTATGTAAAGA 1260  cDNA2  cDNA2  111=11111.111112=11111111111111^  cDNA1  602 1111111111111111111111111111111111111111111111111 GATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTT ^ 169  C.ELEG  ACTTTAAGATGGAATACGCGGTCAAATGGGTTCCATATGAATATGTTTAAATAATTCTCA 1320  cDNA2 cDNA1 C.ELEG  AATTCAGATATCCCACAAAAGGAATTCAGAAAAAGCGAGTTGAAACTACTAATCCAACTT 1380  cDNA2 cDNA1 C.ELEG  TCAAACTCGCGAATTTCTAACTCTGCTAATTTTTTGCTATTTAAATATAAATTTCACAGA 1440  cDNA2 cDNA1  79  Results  C.ELEG^ACTTTTGTTTCACATAAAAAAACAATTGTTAATAATTTTCAGTGAGAAACCCCCCACCAG 1500 111111111111111111 ^ TGAGAAACCCCCCACCAG 620 cDNA2 111111111111111111 cDNA1 ^ TGAGAAACCCCCCACCAG 187 C.ELEG^TCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATTCA 1560 111111111111111111111111111111111111111111111111111111111111 cDNA2  1111111 111111111 111111 I I 1111111 1111111111 11111 11111111 111111  68°  cDNA1^TCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATTCA 247  C.ELEG^CAAAGGTAAAATTGTTTCTGGTAACACGAAAATTCCAATTTTATTTATTCAGTTGGACTT 1620 11111 ^ 11111111 cDNA2^CAAAG^ TTGGACTT 688 11111 ^ 11111111 cDNA1^CAAAG^ TTGGACTT 255 C.ELEG^GGACAAGAAGGAGAAAATTCTTTCCGGATGCATGGATTTCGAAGTGGAATTAATTCATTT 1680 111111111111111111111111111111111111111111111111111111111111 ^748 cDNA2 cDNA1^GGACAAGAAGGAGAAAATTCTTTCCGGATGCATGGATTTCGAAGTGGAATTAATTCATTT 315 C.ELEG^AAGAGTTCTTACTTTCAAGCTTGGATGTTTTAGAATGCCAATCTGAAATTAAACAAATAT 1740 111111111111111111111111111111111111111111111111111111111111 cDNA2 808  cDNA1^AAGAGTTCTTACTTTCAAGCTTGGATGTTTTAGAATGCCAATCTGAAATTAAACAAATAT 375  C.ELEG^GTTATGTTGTAACAGTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAGTTATGTT 1800 111111111111111111111111111111111111111111111111111111111111 cDNA2^111111::::::CAGTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAGTTATGTT 868 cDNA1^  392  C.ELEG^TTTTGGTTTTGCTCTTTGCTTGATTTTCAGAGACGAAGCTTCTCGTTCGTCTGCATTC 1858 11111 cDNA2^TTTTGAAAAAAAAAACG^ 890 cDNA1  80  Results  Figure 18. Genomic sequence data from the dpy-5 gene Genomic dpy-5 sequence is shown. Sequence data was derived from cDNA and genomic sequences. Upper case letters indicate coding sequence, while lower case letters indicate non-coding sequence. The inferred amino acids are shown below each codon. The sequenced Tcl insertion site for dpy-5(s1300) mutation is underlined.  81  Results CTTTCAGCATTGGAAACCTATTTATTCAAGGAAATTAATAAACAAATTATTGTAATTTA -180 TGTTTACCGCATCAATCTTTACCATCGATTCAACGTCATCATTCTAGCCAGTTGTCAAA -120 AAAAACATATCCGCAGATGTTATCTTTGATTTATTTATTGGGAGAACATCTGGTTGTGT^-60 AACAGAGTCAACTGACCTTCTCCCTTTTCATTCGTCCAAATCTTCATTTCTTTTCCAAA ^-1 ATG CAC CTC TTT CTT TCT GTT TTT CTT CTG CTT ATC CTA CCG TTA ^45  Net His Leu Phe Leu Ser Val Phe Leu Leu Leu Ile Leu Pro Leu ^15 ATT TCC ACC TCA GCA GTT GAG AAT AAT CAA AAT ATT GTG GAT TTT ^90  Ile Ser Thr Ser Ala Val Glu Asn Asn Gin Asn Ile Val Asp Phe ^30 TCT GTG AAA GAT TTC AAC AAA AAT ATT AAT TCA ATC GAT ATT CTC ^135  Ser Val Lys Asp Phe Asn Lys Asn Ile Asn Ser Ile Asp Ile Leu ^45 AAC AAA AAA TGG GAT ATT GTT TCG AAA TAT GTG AAA TTT AAT GGA ^180  Asn Lys Lys Trp Asp Ile Val Ser Lys Tyr Val Lys Phe Asn Gly ^60 AAC TCA TCG AAA TTG AAT GGA AGA CTC CGA TTA TCG GAA AAA ATT ^225  Asn Ser Ser Lys Leu Asn Gly Arg Leu Arg Leu Ser Glu Lys Ile ^75 GAC AAA ATC ATT TTT AAA TTG GGA GAC GAT GGA GCT AAT CAG A gt 270  Asp Lys Ile Ile Phe Lys Leu Gly Asp Asp Gly Ala Asn Gin A ^89 aagttttttgaaatttaaaagtggattagcgccctatggtatgactcctatgattctaaa 330 atgataaattttcacagtaaaactttgcgaaactggtttgatatttttaataacatttaa 390 aaatggtattgattcagttctcaactgttataattttggaatattcgaatgttccaaaaa 450 atttatctgaaaaagttctgaaatcaaaaaaaaattcttctaccaaataggcaaaatgtt 510 ttctacgacttttatattttaatcaagttaagaaatattttttgttaagaaaaatagtgc 570 aagaacgttcaaaatttccgaaaaaaaaaccgagtttatcgaaaatttggcaatttgcca 630 aactcttactgactagcagcaatcctcttgttttctaaaaattttaattttcag AT^686  sn^90 GAA ATA ATA GTT TCC TTG GGT AAT AGA AAT ACA ACT CAT ATG ACT^731  Glu Ile Ile Val Ser Leu Gly Asn Arg Asn Thr Thr His Met Thr ^105 GTG CTC TCG GAT TCA TAC AAA ATG AAA GTA TCC ACT TCG AAG CAA ^776  Val Leu Ser Asp Ser Tyr Lys Met Lys Val Ser Thr Ser Lys Gin ^120  GTT GTT CGG AAA G gtctgttgaaaaggttttgaaaattgtattctgttttacgtt 831  Val Val Arg Lys G^  124  gttaacaaagacaattgctcaagcagcctcaagccagcaagccaaaacaaaactttaaaa 891 aactgaaattcggaatgagaaaaaaaaaatacataaaggctaattaaaattgtaataaac 951  82  Results  ctttccag AA CTG TCT CTG AGA ACT GOT TCA TCA AAC GAG AAC GGA ^997 ^lu Leu Ser Leu Arg Thr Gly Ser Ser Asn Glu Asn Gly^137 TGT GCT TGT GTA CAC GGA AAT TGT GCT TGC TGC CTA GAA ATT TCT 1042 Cys Ala Cys Val His Gly Asn Cys Ala Cys Cys Leu Glu Ile Ser^152 GTT CCA GAA TTC AGA CAT TCA G gtaatagttttccttaaattttgttttctt 1094 159 Val Pro Glu Phe Arg His Ser V^  aattttccaatacttttaattcag TT TOT GTC AAT GCA ACC TAT AAC CCA 1144 ^al Cys Val Asn Ala Thr Tyr Asn Pro ^168 GTT TCC ATT GGC TTG GAT TTG TCG ATT GGA GTT GAT GGA CAT TAT 1189 Val Ser Ile Gly Leu Asp Leu Ser Ile Gly Val Asp Gly His Tyr ^183 TTC AGT GAA GAA ATA TCT T gtatgtaaagaactttaagatggaatacgcggtc 1242 189 Phe Ser Glu Glu Ile Ser L^  aaatgggttccatatgaatatgtttaaataattctcaaattcagatatcccacaaaagga 1302 attcagaaaaagcgagttgaaactactaatccaactttcaaactcgcgaatttctaactc 1362 tgctaattttttgctatttaaatataaatttcacagaacttttgtttcacataaaaaaac 1422 aattgttaataattttcag TO AGA AAC CCC CCA CCA GTC TGT TTT TCT 1470 ^eu Arg Asn Pro Pro Pro Val Cys Phe Ser ^199 CTT CCG ATT CCC GGC GCA GAG CAC ATT GCA GGA GTA TGT GTG GCA 1515 Leu Pro Ile Pro Gly Ala Glu His Ile Ala Gly Val Cys Val Ala ^214 TTC ACA AAG gtaaaattgtttctggtaacacgaaaattccaattttatttattcag 1571 217 Phe Thr Lys^  TTG GAC TTG GAC AAG AAG GAG AAA ATT CTT TCC GGA TGC ATG GAT 1616 Leu Asp Leu Asp Lys Lys Glu Lys Ile Leu Ser Gly Cys Met Asp^232 TTC GAA GTG GAA TTA ATT CAT TTA AGA OTT CTT ACT TTC AAG CTT 1661 Phe Glu Val Glu Leu Ile His Leu Arg Val Leu Thr Phe Lys Leu ^247  GGA TGT TTT AGA ATG CCA ATC TGA AATTAAACAAATATGTTATGTTGTAACA 1713 254 Gly Cys Phe Arg Met Pro Ile * ^ GTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAGTTATGTTTTTTGGTTTTGCTC 1773 TTTGCTTGATTTTCAGAGACGAAGCTTCTCGTTCGTCTGCATTCGCTTATTTCATCGCCC 1833 ATCTACACTCCCAATAGTGGTTTTTCATTTCTTCTACTGGAGATGATTCTGATAGACTTT 1893  83  Results  to be similar to any of the published sequences in the EMBL database.  2.6. Sequence Data for the C. briggsae dpy-5 Homologue  Partial sequence data was obtained for the C. briggsae dpy-5 gene homologue (see Figure 19).  Analysis of the C. briggsae sequence reveals a high degree of conservation between the dpy-5 gene and its homologue in C. briggsae. Amino acid sequence conservation between exons 4, 5, and 6 is high measuring 93%, 96%, and 89% respectively. In addition, amino acid substitutions are almost all conservative changes. Alignment of the two genomic sequences shows that the intron/exon boundaries are maintained between the species. The introns in C. briggsae differ in size in comparison to those of C. elegans, with introns 4 and 5 measuring 46 bp and 52 bp compared to  233  bp and 47 bp in  C. elegans.  2.7. Identification of a Putative Secretory Signal Sequence  The inferred amino acid sequence of dpy-5 was examined using the PC/Gene 5.0 programs in an attempt to identify patterns of potentially biological significance.  84  Results  Figure 19. Comparison of C. elegans and C. briggsae DNA and amino acid sequences The figure contains genomic sequence data from C. elegans (C.eleg) and C. briggsae (C. brig), cDNA sequence from C. elegans (cDNA), and inferred amino acid sequences from C. elegans (CePROT) and C. briggsae (CbPROT).  85  Results  C. brig^CTTGGAAGCTCTGTCCCAGAATTTAGACATTCAGGATTTTTGAAAAATTGTTTTGTAGAG 60 C.eleg^TTTCTGTTCCAGAATTCAGACATTCAGGTAATAGTTTTCCTTAAATTTTGTTTTCTTAAT 60 111111111111111111111111111 cDNA^TTTCTGTTCCAGAATTCAGACATTCAG - -Intron 3 ^ 27 CePROT^ISVPEFRHS^ 9 CbPROT C. brig^TCATACAAAAACGTATTTCAGTTTGTGTCAATGCAACTTACAACCCAGTTTCCATTGGAT 120 1111111111111111^11^11111111111111111^1 C.eleg^TTTCCAATACTTTTAATTCAGTTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCT 120 ^ 111111111111111111111111111111111111111 cDNA^ TTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCT 66 CePROT^ VCVNATYNPVSIG 22 1^1^1^1^1^1^1^1^1^1^1^1 CbPROT^ CVNATYNPVSIG 12 C. brig^TGGATTTATCAGTCGGAGTGGATGGACACTACTTTACGGAAGAAGTTTCTCGTAAGGTTT 180 1111111^11^1^11111^WIWI^11^11^1^111111^1^111 C.eleg^TGGATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTTGTATGTAAA 180 111111111111111111111111111111111111111111111111111 cDNA^TGGATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTT-Intron 4 117 CePROT^LDLSIGVDGHYFSEEIS^39 ^1^1^1^1^1^1^1^II^1 1111 CbPROT^LDLSVGVDGHYFTEEVS^29 C. brig^CTTTTTTTTGCCGAAACGTAGTGTG^  205  C.eleg^GAACTTTAAGATGGAATACGCGGTCAAATGGGTTCCATATGAATATGTTTAAATAATTCT 240 cDNA CePROT CbPROT C.brig C.eleg^CAAATTCAGATATCCCACAAAAGGAATTCAGAAAAAGCGAGTTGAAACTACTAATCCAAC 300 cDNA CePROT CbPROT C.brig C.eleg^TTTCAAACTCGCGAATTTCTAACTCTGCTAATTTTTTGCTATTTAAATATAAATTTCACA 360 cDNA CePROT CbPROT  86  Results  C. brig ^  CTTTATTTGCAGICCTTITWATTT 233  C.eleg^GAACTTTTGTTTCACATAAAAAAACAATTGTTAATAATTTTCAGTGAGAAACCCCCCACC 420 1111111111111111 cDNA TGAGAAACCCCCCACC 133 LRNPPP 45 CePROT CbPROT  LRNPPP  35  C.brig  AATCTGTTTCTCTCTTCCAATTCCTGGCGCTGAACATATTGCAGGTGTTTGTGTTGCTTT 293  C.eleg  AGTCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATT 480  I^I I I I I I I^I I I I I I I I^I I I I I^I I I I I^I I^I I^I I I I I I I I^I I^I I I I I^I I^I I 111111111111111111111 111111111111111111111111111111111111111 cDNA^AGTCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATT 193  CePROT^VCFSLPIPGAEHIAGVCVAF  65  11111111111111111 11 CbPROT^ICFSLPIPGAEHIAGVCVAF 55  C. brig^CACGAAAGTGAGTGACTAGTTCCATGAATTAAATCTTATTCCGATTTAAATGTTTTCAGT 353 111^11^ 1 C.eleg^CACAAAGGTAAAATTGTTTCTGGTAACACGAAAA ^TTCCAATTTTATTTATTCAGT 535 1111111^ 1 cDNA^CACAAAG--Intron 5 ^ T 201 CePROT^T K^ 67 1^1 CbPROT^T K^ 57 C. brig^TAGATTTGGACAAGAAAGCCAAGATTCTATCAGGATGCATGGATTTCGAAGTTGAATTGA 413 C.eleg IGIACIIIIIIIIIIIGIAGIAAWATTLIIIIIIIIIIIIIIIIIIIIGRATTAI 595 111111111111111111111111111111111111111111111111111111111111 cDNA^TGGACTTGGACAAGAAGGAGAAAATTCTTTCCGGATGCATGGATTTCGAAGTGGAATTAA 261 CePROT^LDLDKKEKILSGCMDFEVEL 87  1111 11^11111111111 1177  CbPROT^LDLDKKAKILSGCMDFEVEL  C. brig^TTCATTTGAGAGCTCTCAGTTTCCACTTGGGATGCTTCAGAATGCCAATTTGAGTGTTTG 473 C.eleg 6" cDNA^TTCATTTAAGAGTTCTTACTTTCAAGCTTGGATGTTTTAGAATGCCAATCTGAAATTAAA 321 CePROT^IHLRVLTFKLGCFRMPI *^104  1111 ^1^1^111111111 *^94  CbPROT^IHLRALSFHLGCFRMPI  C. brig^AATAGCTGTTTTGCAAAAAATATTTTGATAAAGTTTTAATCTTTTGACTTCATTGTTTAC 533 C.eleg^CAAATATGTTATGTTGTAACAGTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAG 715 111111111111111111111111111111111111111111111111111111111111 cDNA^CAAATATGTTATGTTGTAACAGTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAG 381 CePROT CbPROT  87  Results  C. brig^ACTTCAATGCATCCATGTGCATCCATGAGTGTACATGGAAACCAATACAGCGTTTAACCG 593 C.eleg^TTATGTTTTTTGGTTTTGCTCTTTGCTTGATTTTCAGAGACGAAGCTTCTCGTTCGTCTG 775 111111 111 111 405 cDNA^TTATGTTTTTTGAAAAAAAAAACG ^ CePROT CbPROT  88  Results In this analysis, the PSignal program was used to predict a eukaryotic signal sequence with a cleavage site between positions acids 22 and 23 in the amino acid sequence (see Figure 20).  2.8. Expression of the dpy-5 Gene The 1.1 kb EcoRI fragment was used in Northern analysis experiments. This fragment was used to probe RNA isolated from different adult populations. Northern analysis revealed that the 1.1 kb fragment identified a 950 bp message in the N2 strain (Figure 21). The message could be readily observed with an overnight exposure. RNA was also isolated from strains carrying dpy-5(e61), dpy-5(s1300), and dpy-5(m476) mutations. In all cases the message was the same size as in the wildtype N2 strain (see Figure 22). Gene expression of the dpy-5(e61) allele was further examined. The dpy-5(e61) allele is a sing suppressible allele of the dpy-5 gene (Hodgkin et al. 1989). Smg suppression takes the form of allele specific, gene general information suppression. Sing suppression is recessive and incomplete and may be involved with mRNA processing, transport, or stability (Hodgkin et al. 1989). Northern blot analysis was done on RNA from  89  Results  -20 7  -25  7  -30 H -36: -40^IiiiiiiiiiiiiiiiiiiiiiiIIIIIIIIIIIIIIIIIIIIIIIIIII 1^50^100^158^200^258 Sequence:Mhlflsvfl 1 lilplistalVEN  Figure 20. Detection curve for a eukaryotic signal  sequence Analysis of the inferred dpy-5 amino acid sequence using the PSignal program detects a potential eukaryotic signal sequence.  90  Results  Figure 21. Northern blot hybridization of dpy-5 message 10 pg of total RNA from C. elegans was fractionated by electrophoresis in a 1.2% denaturing agarose gel. The blot was probed with the plasmid pCehl40. The probe detects a message 950 bp in size. The arrows indicate the position of 28S and 16S RNA.  91  Results  Figure 22. Northern blot analysis of dpy-5 mutants 10 Ag of total RNA from C. elegans was fractionated by electrophoresis in a 1.2% denaturing agarose gel. The blot was probed with the plasmid pCehl40. Lane 1=dpy-5(s1300), Lane 2=dpy-5(m476) wildtype revertant, Lane 3=dpy-5(m476), Lane 4=N2.  92  Results dpy-5(e61) and dpy-5(e61) smg-4(mall6) worms (see Figure  23). Examination of the hybridization pattern shows that there is no apparent difference in the size of the dpy-5 message from both worms. The difference in RNA quantity is due to uneven loading of RNA samples. The 1.1 kb EcoRI fragment was also used to probe stage-specific RNA populations. In this experiment a stage-specific Northern blot (provided by S. Prasad, Simon Fraser University) containing embryonic, Li and L2, L3, L4, and adult RNA was probed. It was observed that the message was less abundant in embryos, Li, and L2 worms in comparison to L3, L4, and adult worms (see Figure 24). The pattern of expression is such that the message is present at low levels in the embryo and the L1 stage, but increases in L2 worms through to the adult stage of development. The message is most abundant in the L4 and adult stages. These results suggest that expression of the dpy-5 message is developmentally regulated. The pattern of dpy-5 gene expression correlates well with the onset of the Dpy-5 phenotype. Previous analysis by Garth Duncan (this lab) demonstrated that the onset of the Dpy-5 phenotype appears to begin around the L2 stage in development. Experiments were done in which the  93  Results  Figure 23. Northern blot analysis of dpy 5(e61) and dpy-  5(e61) smg 4(ma116) RNA -  Lane 1=dpy 5(e61) and Lane 2=dpy 5(e61) -  -  smg 4(ma116). Although there appears to be a difference -  in the quantity of dpy 5 message, ethidium staining -  reveals that the lanes are not evenly loaded.  94  Results  Figure 24. Northern hybridization of developmentally  staged RNA C. elegans RNA from different developmental stages was fractionated by electrophoresis in a 1.2% denaturing agarose gel. The blot was probed with the plasmid pCehl40. Lane 1=embryonic RNA, Lane 2=L1 and L2 RNA, Lane 3=L3 RNA, Lane 4=L4 RNA, Lane 5=adult RNA, Lane 6=polye RNA. Increased dpy-5 gene expression is  observed later in development. The markers shown on the left indicate 28S and 16S RNA.  95  Results  length of dpy-5(e61) unc-13(e450) worms was measured. In this experiment, a dpy-5 unc-13 double mutant was constructed to facilitate measuring: the double mutant in this instance is paralyzed and can therefore be easily measured. The severity of the dumpy phenotype increases from the L2 larval stage through to the adult. The pattern of dpy-5 gene expression and the onset of the Dpy-5 phenotype suggest that the dpy-5 gene may play an increasingly important role in cuticle formation later in development.  2.9. Analysis of the Cosmid B0342  Transformation experiments using the cosmid B0342 were found to be successful in rescuing worms homozygous for the dpy-5(e61) mutation (H. Browning, unpublished results). However, analysis of B0342 DNA suggests that the cosmid does not contain the entire dpy-5 gene. Southern blot analysis was done on DNA isolated from two cosmids, C24H1 and B0342, using a full length dpy-5 cDNA as a probe. The cosmid B0342 is located to the left of the cosmid C24H1 (see Figure 15). The two cosmids overlap, although the extent of the overlap is not known. The cDNA probe hybridizes to three EcoRI fragments in the C24H1 cosmid measuring 3.0 kb, 0.3 kb, and 1.1 kb in  96  Results  length. The cDNA probe hybridizes to only the 3.0 kb EcoRI fragment in the B0342 cosmid (see Figure 25).  These results suggest that the B0342 cosmid contains only the 5' portion of the dpy-5 gene. Specifically, the first three exons of the dpy-5 gene are located on the 3.0 kb EcoRI genomic fragment present in the B0342 cosmid. These exons account for approximately 63% of the total protein. Since a portion of the 0.3 kb EcoRI fragment may be fused to the cosmid vector, as many as four of the six exons may be present in B0342. These four exons would account for approximately 75% of the total protein.  2.10. Identification of Upstream Coding Region  Analysis of DNA in the dpy-5 genomic region revealed the presence of another gene. This gene is located approximately 850 bp upstream of the dpy-5 translation start site (see Appendix 1). Genomic and cDNA sequence data for the upstream gene have been obtained from C. elegans (see Figure 26). The analysis of genomic and  cDNA sequences suggests that the upstream gene contains two introns and encodes a 249 amino acid protein (see Figure 27). Additional sequence data has also been obtained for the homologous gene in C. briggsae.  97  Results  Figure 25. Southern blot of C24H1 and B0342 Cosmids Cosmid DNA was digested with EcoRI and probed with a full length cDNA. Lane 1=C24H1 and Lane 2=B0342. In the C24H1 cosmid, three EcoRI fragments are detected measuring 3.0 kb, 1.1 kb, and 0.3 kb in size. In the B0342 cosmid, only the 3.0 kb fragment is detected. This result indicates that the B0342 cosmid does not contain the entire coding region for the dpy-5 gene.  98  Results  Figure 26. Alignment of dpy-5 upstream genomic and cDNA  sequences Genomic and cDNA sequences are aligned. Genomic DNA=C.eleg and cDNA=cDNA. Dashes indicate cDNA gaps corresponding to introns.  99  Results  ^GAATTCATGGATTTTGGATTTTGGATTACAAAATCAAAAAAACGTATCAAACTTTAGGGA ^60  1 1 1 1 1^III^III  ^GAATTCCC,GTGATATCATTGATTATGGA^28 ACCATCCGTCGTGAATACAACTACATGATAATCAGTATTCTTGGAAAAGATCTCTACCGT 120 ACCITIATIL ITIIIIIIIIIT111111111101 1111111CULITITIWg1 88 CTTCGTGCCGAACAACCGACTCGTTCATTCACTCTCAATACGACTACAAAGATTGCTCTT 180  A111111111111111111111111111WW11111111111111111W 1 148 GAAACTATTGAAGCTATTGAAGAGCTTCACAATATTGGATACCTGAGCCGTGATGTCAAG 240  11111111111111,11,111111111141111111111111111111111111111111  gillaigulguawwwwwwwwwffillaw4  208  CCAAGCAACTTTGCTCCAGGACAACGCGACAATGGACAGCATAAGACAATTTTCATGTTT 300 268  GACTTTGGACTTGCGAAGAAGTTTATTGATCGTGATAA.CAAGAAGCTGAAGTCTCGTGGA 360 alligIGWIRIIITUWWWillilligIUTCTATLI 328 GAGGTTGGATGGAGAGGTACTGTAAGATATGGAAGTCTCCAAGCTCTCAAACGTATGGAT 420 WITTLITIGIGIGITICWAIRTIWIWCWIOTWIWITITCLW 388 CTTGGAAGACGTGATGATGTTGAATGCTGGTTCTACATGCTCATTGAGATGTTGGTTGGA 480  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I I I I I I I III  CTTC,GAAGACGTGATGATGTTGAATGCTGGTTCTACATGCTCATTGAGATGTTGGTTGGA 448 GAGTTGCCATC,GCGTCATATGTCTGATCGTACACTTGTTGGACAATCGAAGCTCTCAATT 540 WITWIWWWITIWWWICIIIWILIWWWWIC1111 508 CGAAATGAATCTCGTCGTTTGTTCTTCAATCGAACTCCAAGACAGTTTGAGACGATCATG 600 Ca111111.11WITTIGWITAWGIUITICLUIWITIGAGIC11112111 568  auwwwwwwwwwwwwwwwwww  GATATGATCGATGGATACTCATTCGAAATCAGACCAGAGTACAGACATTTGAAGGCATTG 660 628  ATTAATGAGGTAACTAAAATTAGTCTAGATGGATATTTTTAGTTAGATCCC-GAP HERE 714  114111111^ 11111,1ffilia111111W1111114111,11W1111,11111111,1111111.  637  ATCCGTATGGAAAATATGATCCCTGATCGTTGCAAGTGGGACTGGCAAGTGGAAGAATCT 774  111111411w11.ralgiuglaluaglialllatali.11111,  697  CAACATTCGGAGCTCACCGAAACAGCTTCTGTCATGTCTGATATGGCAATTATGGCTGAA 834  100  757  Results CAAGGAGCTACTAACTACACTGATCGTGCCTGTGAGA t a tgttttattgtttttatttt 894  WiCLWITICULIWWWWITWIG:  794  ^Tritilliiiiniiiiiiiiii^ iiiiiiiii ctatatcaaaatatacatttttca  954 ACCAATAGGCATATACTCGTCTCACAACTGTTCTT 829  CATCACTACTCTTCTCATCAAATCTCGAATCGAATCGAGCACTTTCCAGTTCAATTTCCT 1014  11111111111.1.111111111111111111,11111111111111111111111114111  gwatagglwwwwwwilwwwwwwwall iwwwwwwwwagauwwwwwwwil  889  CTTTCTCTCACTCTTTTCCAATCCAAAGTAAAGAACCGATTGCCTCTTTTTGTATTCATT 1074 949  ATCATCATCGTCGTCGTTTGGTTTCAACTATACTACTAGATTTTTGTGCTGTGTAAATAA 1134  MMITITTIMI  1009  GTTTTTT11TATCTTI ATTGTAAGTTCCAACTATCACGGCGTTAGT 1194  ATGTATTTTTTTAAAAAAAAAAAACGGAATTC 1041 TTCTTTCCCAAGTTTGGCAAATTGAATATTAACTTGGATTAGAAGTGGTTAAACCTGAGC 1254 CAAACTTTGGCAAAACATAGTCTCCTGGAATCTTCTAGAAGTGATATGTTGACGGAAAGT 1314  101  Results  Figure 27. C. elegans genomic sequence data from the dpy-5 upstream gene Genomic sequence from the dpy-5 upstream gene is shown. Sequence data was derived from cDNA and genomic sequences. Upper case letters indicate coding sequence, while lower case letters indicate non-coding sequence. The inferred amino acids are shown below each codon.  102  ^  Results GAATTCATGGATTTTGGATTTTGGA  -60  TTACAAAATCAAAAAAACGTATCAAACTTTAGGGAACCATCCGTCGTGAATACAACTAC  -1  ATG ATA ATC AGT ATT CTT GGA AAA GAT CTC TAC CGT CTT CGT GCC  45  Met Ile Ile Ser Ile Leu Gly Lys Asp Leu Lys Arg Leu Arg Ala  15  GAA CAA CCG ACT CGT TCA TTC ACT CTC AAT ACG ACT ACA AAG ATT  90  Glu Gin Pro Thr Arg Ser Phe Thr Leu Asn Thr Thr Thr Lys Ile  30  GCT CTT GAA ACT ATT GAA GCT ATT GAA GAG CTT CAC AAT ATT GGA ^135  ^Ala Leu Glu Thr Ile Glu Ala Ile Glu Glu Leu His Asn Ile Gly ^45 ^TAC CTG AGC CGT GAT GTC AAG CCA AGC AAC TTT GCT CCA GGA CAA ^180  ^Lys Leu Ser Arg Asp Val Lys Pro Ser Asn Phe Ala Pro Gly Gin^60 ^CGC GAC AAT GGA CAG CAT AAG ACA ATT TTC ATG TTT GAC TTT GGA ^225  ^Arg Asp Asn Gly Gin His Lys Thr Ile Phe Met Phe Asp Phe Gly ^75 ^CTT GCG AAG AAG TTT ATT GAT CGT GAT AAC AAG AAG CTG AAG TCT^270  ^Leu Ala Lys Lys Phe Ile Asp Arg Asp Asn Lys Lys Leu Lys Ser ^90 ^CGT GGA GAG GTT GGA TGG AGA GGT ACT GTA AGA TAT GGA AGT CTC^315  ^Arg Gly Glu Val Gly Trp Arg Gly Thr Val Arg Lys Gly Ser Leu^105  ^CAA GCT CTC AAA CGT ATG GAT CTT GGA AGA CGT GAT GAT GTT GAA ^360  ^Gin Ala Leu Lys Arg Met Asp Leu Gly Arg Arg Asp Asp Val Glu^120 ^TGC TGG TTC TAC ATG CTC ATT GAG ATG TTG GTT GGA GAG TTG CCA ^405  ^Cys  Trp Phe Lys Met Leu Ile Glu Met Leu Val Gly Glu Leu Pro ^135  ^TGG CGT CAT ATG TCT GAT CGT ACA CTT GTT GGA CAA TCG AAG CTC ^450  ^Trp Arg His Met Ser Asp Arg Thr Leu Val Gly Gin Ser Lys Leu ^150 ^TCA ATT CGA AAT GAA TCT CGT CGT TTG TTC TTC AAT CGA ACT CCA^495  ^Ser Ile Arg Asn Glu Ser Arg Arg Leu Phe Phe Asn Arg Thr Pro ^165 ^AGA CAG TTT GAG ACG ATC ATG GAT ATG ATC GAT GGA TAC TCA TTC ^540  ^Arg Gin Phe Glu Thr Ile Met Asp Met Ile Asp Gly Lys Ser Phe^180 ^GAA ATC AGA CCA GAG TAC AGA CAT TTG AAG GCA TTG ATT AAT GAG ^585  ^Glu Ile Arg Pro Glu Lys Arg His Leu Lys Ala Leu Ile Asn Glu ^195 gtaactaaaattagtctagatggatatttttagttagatccc ^ATC CGT^636 ^Ile Arg^197 ^ATG GAA AAT ATG ATC CCT GAT CGT TGC AAG TGG GAC TGG CAA GTG^681  ^Met Glu Asn Met Ile Pro Asp Arg Cys Lys Trp Asp Trp Gin Val^212  ^GAA GAA TCT CAA CAT TCG GAG CTC ACC GAA ACA GCT TCT GTC ATG ^726  ^Glu Glu Ser Gin His Ser Glu Leu Thr Glu Thr Ala Ser Val Met ^227 ^TCT GAT ATG GCA ATT ATG GCT GAA CAA GGA GCT ACT AAC TAC ACT^771  ^Ser Asp Met Ala Ile Met Ala Glu Gin Gly Ala Thr Asn Lys Thr^242  103  Results GAT CGT GCC TGT GAG A gtatgttttattgtttttattttctatatcaaaatata 825 Asp Arg Ala Cys Glu A^ 247 catttttcag AC CAA TAG GCATATACTCGTCTCACAACTGTTCTTCATCACTACTC 881  sn Gin *^  249  TTCTCATCAAATCTCGAATCGAATCGAGCACTTTCCAGTTCAATTTCCTCTTTCTCTCAC 941 TCTTTTCCAATCCAAAGTAAAGAACCGATTGCCTCTTTTTGTATTCATTATCATCATCGT 1001 CGTCGTTTGGTTTCAACTATACTACTAGATTTTTGTGCTGTGTAAATAAATGTATTTTTT 1061 TAAAAGTATTTTACATCTTATTGTAAGTTCCAACTATCACGGCGTTAGTTTCTTTCCCAA 1121 GTTTGGCAAATTG 1134  104  Results  Figure 28. Comparison of C. elegans and C. briggsae DNA and amino acid sequences The figure contains genomic sequence data from C. elegans (C.eleg) and C. briggsae (C. brig), cDNA sequence from C. elegans (cDNA), and inferred amino acid sequences from C. elegans (CePROT) and C. briggsae (CePROT).  105  Results  C.brig C.eleg^GAATTCATGGATTTTGGATTTTGGATTACAAAATCAAAAAAACGTATCAAACTTTAGGGA 60 11111^111^111 cDNA^ GAATTCCGGTGATATCATTGATTATGGA 28 CePROT CbPROT C.brig  GGATCTCTACCGT 13 111111111111 C.eleg^ACCATCCGTCGTGAATACAACTACATGATAATCAGTATTCTTGGAAAAGATCTCTACCGT 120 111111111111111111111111111111111111111111111111111111111111 cDNA^ACCATCCGTCGTGAATACAACTACATGATAATCAGTATTCTTGGAAAAGATCTCTACCGT 88 CePROT^ MIISILGKDLYR 12 1^1^1^1 CbPROT^ DLYR4 C. brig^CTTCGTGCTGAGCAACCGAATCGTTCGTTCTCTCTCAACACCACTACCAAAATTGGATTG 73 11111111^11^1111111^111111^111^1111111^11^11111^11^1111^1 C.eleg^CTTCGTGCCGAACAACCGACTCGTTCATTCACTCTCAATACGACTACAAAGATTGCTCTT 180 111111111111111111111111111111111111111111111111111111111111 cDNA^CTTCGTGCCGAACAACCGACTCGTTCATTCACTCTCAATACGACTACAAAGATTGCTCTT 148 CePROT 32 CbPROT^LRAEQPNRSFSLNTTTKIGL 24  iiiTY P^ ITTITTiiiiiifAi  C. brig^GAGACTCTCGAGGCAATCGAAGAACTTCATGCCATTGGATACTTGAGTCGTGATGTCAAA 133 C.eleg^ILICTATTRAGITITTIIIIIGITICICAATITTIGIWCTIIICCITIIIITCAL 240 111111111111111111111111111111111111111111111111111111111111 cDNA^GAAACTATTGAAGCTATTGAAGAGCTTCACAATATTGGATACCTGAGCCGTGATGTCAAG 208 CePROT^ETIEAIEELHNIGYLSRDVK 52 II^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 CbPROT^ETLEAIEELHAIGYLSRDVK 44 C. brig^CCGAGTAATTTTGCTCCTGGAATGCGCGAATGTGGAGAACACAAAACGATTTACATGTTC 193 11^11^11^11111111^111^11111^1111^1^11^11^11^1111^111111 C.eleg^CCAAGCAACTTTGCTCCAGGACAACGCGACAATGGACAGCATAAGACAATTTTCATGTTT 300 111111111111111111111111111111111111111111111111111111111111 cDNA^CCAAGCAACTTTGCTCCAGGACAACGCGACAATGGACAGCATAAGACAATTTTCATGTTT 268 CePROT^PSNFAPGQRDNGQHKTIFMF 72 1^1^1^1^1^1^1^1^1^1^1^1^1^II CbPROT^PSNFAPGMRECGEHKTIYMF 64 C. brig^GATTTCGGGCTCGCCAAGAAGTATCTCGACCGTGAAGGGAAGAAAATGAAGAGTCGCGGA 253 11^11^11^11^11^1111111^1^1^11^11111^11111^11111^111^111 C.eleg^GACTTTGGACTTGCGAAGAAGTTTATTGATCGTGATAACAAGAAGCTGAAGTCTCGTGGA 360 111111111111111111111111111111111111111111111111111111111111 cDNA^GACTTTGGACTTGCGAAGAAGTTTATTGATCGTGATAACAAGAAGCTGAAGTCTCGTGGA 328 CePROT^DFGLAKKFIDRDNKKLKSRG 92 1^1^1^1^1^1^1^II^II^1111 CbPROT^DFGLAKKYLDREGKKMKSRG 84  106  Results  C. brig^GAGGTTGGATGGAGAGGAACCGTTCGTTACGGTAGTCTTCAAGCCCATAAACGATTGGAT 313  11111111111111111^11^11^1^11^11^11111^11111^1^11111^11111 111111111111111111111111111111111111111111111111111111111111 cDNA^GAGGTTGGATGGAGAGGTACTGTAAGATATGGAAGTCTCCAAGCTCTCAAACGTATGGAT 388  C.eleg^GAGGTTGGATGGAGAGGTACTGTAAGATATGGAAGTCTCCAAGCTCTCAAACGTATGGAT 420  i T 7 T i i Ti T i T 7 7 If ? i ' i 7 m ii CbPROT^EVGWRGTVRYGSLQAHKRLD104  CePROT  112  C. brig^CTTGGAAGACGTGATGATGTGGAATGCTGGTTCTACATGCTCATTGAGATGTATGCTGGG 373  C.eleg^11111iillA111111111/111111111111111111C1111111111111A 480  111111111111111111111111111111111111111111111111111111111111  cDNA^CTTGGAAGACGTGATGATGTTGAATGCTGGTTCTACATGCTCATTGAGATGTTGGTTGGA 448 CePROT^LGRRDDVECWFYMLIEMLVG132  1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1  CbPROT^LGRRDDVECWFYMLIEMYAG124  C. brig^GAGTTGCCATGGCGTCATATGACTGATCGCGCTCTTGTGGGACAGGCCAAACTTGCGATT 433 C.eleg^Ii11111111=1111TWIIIITAWIIIITIIIIIATLIIGACTIAW 540  111111111111111111111111111111111111111111111111111111111111  cDNA^GAGTTGCCATGGCGTCATATGTCTGATCGTACACTTGTTGGACAATCGAAGCTCTCAATT 468 CePROT TTTTSDITTICITIISTI(Sf152  i  II  P1  CbPROT^ELPWRHMTDRALVGQAKLAI144  C. brig^CGAAATGAGCAACGTCAAATCTTCTTCAATCGCATTCCAAGACAATTTGAAAAGATTATT 493 C.eleg^aiiilliATCTIIIIGTTLIWIIIIIIICIWIWIGIIIIIGICIIICIL 600  111111111111111111111111111111111111111111111111111111111111  cDNA^CGAAATGAATCTCGTCGTTTGTTCTTCAATCGAACTCCAAGACAGTTTGAGACGATCATG 470 CePROT STRL FFNRTPRQTTTfM172  ^1^1 11 7i^ ^1^ 1 ^ 1^1 ^ 1 CbPROT^RNEQRQIFFNRIPRQFEKII164  C. brig^GATTTGATTGATGGATACAGCTTTGAGATTAGACCGGATTATAGACAACTCAAGGGTTTG 553  111^1111^111111111^11^11^11^11111^11^11^11111^1^1111^111 111111111111111111111111111111111111111111111111111111111111 cDNA^GATATGATCGATGGATACTCATTCGAAATCAGACCAGAGTACAGACATTTGAAGGCATTG 526  C.eleg^GATATGATCGATGGATACTCATTCGAAATCAGACCAGAGTACAGACATTTGAAGGCATTG 660 CePROT^DMIDGYSFEIRPEYRHLKAL192  1^1^1^1^1^1^1^1^1^1^1^II^II^1  CbPROT^DLIDGYSFEIRPDYRQLKGL184 C.brig^TTITTrintron absent in C. briggsae ^  575  C.eleg^ATTAATGAGGTAACTAAAATTAGTCTAGATGGATATTTTTAGTTAGATCCC-gap here 714  111111111  cDNA^ATTAATGAG-intron in C. elegans ^ CePROT^  544 195  CbPROT^ I^1  187  107  Resul ts  C. brig^ATCCGCTTTGAAAACAACATTCCGGATCGATGCAAATGGGATTGGCAAATCGAGGAATCT 635 C.eleg^11111 tat iiilltit lalclictliTIGTIIIIIGIIIIICIIICIAGIGAAGIATIT 774 1111TIIITT111110111111111111111111111111111111111111111111 cDNA^ATCCGTATGGAAAATATGATCCCTGATCGTTGCAAGTGGGACTGGCAAGTGGAAGAATCT 604 CePROT  V fT m i I' N f Pi iiiTiii 1'^iii 2" CbPROT^IRFENNIPDRCKWDWQIEES207 C. brig^CAACATTCCGAGCTTACTGAAACCACTTCTGTCATGTCTGATGTTGCCATTCTGGCTGAA 695 11111111^11111^11^11111^11111111111111111^1^11^111^11111111 C.eleg^CAACATTCGGAGCTCACCGAAACAGCTTCTGTCATGTCTGATATGGCAATTATGGCTGAA 834 111111111111111111111111111111111111111111111111111111111111 cDNA^CAACATTCGGAGCTCACCGAAACAGCTTCTGTCATGTCTGATATGGCAATTATGGCTGAA 664 CePROT C II Aliii 235 CbPROT^QHSELTETTSVMSDVAILAE227 C. brig^CAAGGTGTCACCAACTACACTGATCGCGCTTGTGAGAGTGAGTTATATTCCTTTTTGAAC 755 11111^1^11^11111111111111^11^1111111 C.eleg^CAAGGAGCTACTAACTACACTGATCGTGCCTGTGAGAGTATGTTTTATTGTTTTTATTTT 894 1111111111111111111111111111111111111 ^ cDNA^CAAGGAGCTACTAACTACACTGATCGTGCCTGTGAGA^ 701 CePROT^QGATNYTDRACE^ 247 II^1^1^1^1^1^1^1^1^1 CbPROT^QGVTNYTDRACE^ 239  i7i  ITT'  li N 11' f m 7^ I' i  C. brig^TTTCAAAATTCATATTTCTTTTCAGATCAATAGAGGATCTCCCATCTCGTCATCTTCCCT 1 111111end of gene C.eleg^CTATATCAAAATATACATTTTTCAGACCAATAGGCATATACTCGTCTCACAACTGTTCTT ^ 11111111111111111111111111111111111 cDNA^ ACCAATAGGCATATACTCGTCTCACAACTGTTCTT CePROT  815  CbPROT^  241  7^1 T N Q *^  954 736 249  C. brig^TCTTTTTCTTTCACTCTCTGCTCATCAATCTCGAATCGAATCGAACACTTTCCAGTACAA 875 C.eleg^CATCACTACTCTTCTCATCAAATCTCGAATCGAATCGAGCACTTTCCAGTTCAATTTCCT 1014 cDNA^ CePROT  CITCICACTITICTIIIIIIIWWWWWWICITICIIITICIITTICIT  796  CbPROT C. brig^TTTCCTCTTTCTTTTTCCGCACTAAAGTAAAATACCAACCCTTCCAAATCCCTTCTTTCT 935 C.eleg^CTTTCTCTCACTCTTTTCCAATCCAAAGTAAAGAACCGATTGCCTCTTTTTGTATTCATT 1074 cDNA^glIggagglITUTIIIIIIIIIIIIIIAITIGIIIC1111111141111 856 CePROT CbPROT  108  Results C.brig  CTATTCACTATCATCATCGTCGTCGTTCCATATCTACTATGCTACTAGACCTTTGTATAT 995  C.eleg  iiiiiiiIiiiiiiiiiiiiiTiiiiiiiffiiiiiiiiiiiiiiiiiiiiiiiiiiiii  cDNA  CePROT  1"4 ATCATCATCGTCGTCGTTTGGTTTCAACTATACTACTAGATTTTTGTGCTGTGTAAATAA 916  CbPROT C.brig  AATAAATGTATTACTCCCACAATCACAATATCACTTTTTTTTATCTATAAAAACAGACAA 1055  C.eleg  ATGTATTTTTTTAAAAGTATTTTACATCTTATTGTAAGTTCCAACTATCACGGCGTTAGT 1194  cDNA  ITGIIIIIIIITIIII 1  CePROT  IIGGAATTC  948  CbPROT C.brig  GACTTTTATAATCCGTGTGTGATTTTCGTTATGTTCTAACAACTTTTCGAAATTGGTCTA 1115  C.eleg  TTCTTTCCCAAGTTTGGCAAATTGAATATTAACTTGGATTAGAAGTGGTTAAACCTGAGC 1254  cDNA  CePROT CbPROT  109  Analysis of the two homologues reveals an 84% identity at the amino acid level (see Figure 28). The C. briggsae homologue has only one intron compared to two introns in the C. elegans gene. The derived protein sequence was compared to other sequences in several databases (see Materials and Methods). Searches against these databases reveal a strong similarity to casein kinase I homologues in yeast in addition to other serine-threonine kinases (see Appendix 3).  110  IV. DISCUSSION  In this thesis Tcl transposition was studied in the Bristol strain of C. elegans and Tcl transposition was used to clone the dpy-5 gene and a casein kinase I homologue in the dpy-5 region of chromosome I. Southern blot analysis of CB51 and CB51-derived strains showed an altered pattern of Tcl hybridization in comparison to the pattern normally observed in Bristol strains. The short period of time in which strains with altered patterns of Tcl hybridization were isolated suggested that low level Tcl transposition had occurred. One of the additional Tcl-hybridizing bands was cloned and shown to be the result of a transposition event. Subsequent analysis revealed that one CB51-derived strain had become a mutator strain exhibiting high levels of Tcl transposition. Tcl transposition was also examined in several Tclinduced alleles of the dpy-5 gene in C. elegans. Tcl transposition was used as a tool in cloning the dpy-5 gene. Analysis of the Tc1-induced mutations shows that the mutant phenotype is the result of a Tcl insertion into a 1.1 kb EcoRI genomic fragment. Sequence analysis reveals that the gene encodes a 254 amino acid protein  111  Discussion containing a putative eukaryotic signal sequence. Partial sequence derived from the dpy-5 homologue in  C. briggsae shows that the gene is highly conserved between the two species. Searches against protein and DNA databases do not reveal significant similarity to any other gene. Northern analysis indicates that the dpy-5 message is present at low levels in the embryo and Li larval stages and increases in abundance from the L2 larval stage through to the adult stage. Analysis of DNA sequence data from the dpy-5 region revealed the presence of another gene located approximately 850 bp upstream of the dpy-5 gene. Additional sequence data was obtained from a cDNA clone and genomic clones from C. briggsae. Searches against protein and DNA databases indicate that the gene is a casein kinase I homologue.  1.1. Analysis of Tcl Transposition in CB51 Two types of Tcl activity have been observed in the CB51-derived Bristol strains: low level Tcl transposition and high level Tcl transposition associated with mutator activity. Our lab stock of CB51, KR579, was found to contain additional Tcl elements in comparison to other Bristol strains. In addition, the pattern of Tcl-  112  Discussion hybridizing bands which is representative of Tcl copy number, was observed to change over a brief period of time. The accumulation of Tcl-hybridizing bands was not the result of new EcoRI restriction sites and was not shown to be the result of unequal crossover events between Tcl elements. Investigations showed that additional Tcl-hybridizing bands are not present in other EMS-induced mutants that were generated at the same time as CB51 and the presence of additional Tcl-hybridizing bands is not correlated with the Unc-13 phenotype or the high recombination phenotype associated with certain CB51 derivatives.  1.2. Analysis of a Tcl Insertion Site in CB51 One of the additional Tcl elements was cloned and the insertion site was sequenced. Sequence analysis of the insertion site reveals a match at 7 out of 9 positions for the published insertion site consensus sequence (Mori et al. 1988; Eide and Anderson 1988). The similarity of the insertion site sequence to that of the consensus insertion site sequence is consistent with the idea that the additional cloned Tcl element in KR579 is the result of Tcl transposition into a new genomic site. The similarity of the insertion site suggests that the  113  Discussion mechanism of transposition in KR579 is similar to that described in Bergerac mutator strains.  1.3. The Observed Changes in Tc1 Transposition Over the course of three years, changes in Tcl number and behaviour were observed in CB51-derived strains. Initially, germline Tcl transposition occurred at a very low level with little or no change in the pattern of Tcl hybridization. However, at the end of a three year period, an isolate of KR579, KR1787, was found to contain at least 20 additional Tcl-hybridizing bands in comparison to the N2 strain. This strain was also found to exhibit mutator behaviour, with spontaneous mutations occurring at the unc-22 locus at a high frequency. The low level Tcl transposition observed in CB51 and CB51-derived strains could be the result of a mutation in either a Tcl element or a host regulatory gene. In Bergerac strains, mutator activity is associated with discrete genetic units that are capable of transposition (Mori. Moerman, and Waterston 1988). This evidence suggests that germline Tcl activity is the result of a mutation in a Tcl element that alters the regulation of Tcl transposition. Alternatively, Collins, Saari, and  114  Discussion Anderson (1987) have described an EMS-induced mutation that increases germline transposition for several transposons in C. elegans including the Tcl transposon. Since this mutation affects the germline transposition of more than one transposon, a cellular host factor associated with the regulation of transposition may be involved. Therefore, a mutation in either a Tcl element or a cellular host factor could have resulted in the germline Tc1 transposition observed in CB51-derived strains. A possible mechanism for the regulation of Tcl transposition is differential mRNA splicing between somatic and germline tissues. Tissue specific transposition is also observed for the P element in Drosophila where transposition activity occurs only in the germline (Engels and Preston 1979). In this example, tissue specificity comes about by differential splicing of the transposase message (Laski, Rio, and Rubin 1986). Comparison of the Tcl element in C. elegans and a related element in C. briggsae suggests the presence of an intron in the TclA transposase (Schukkink and Plasterk 1990; Prasad et al. 1991). This intron may be spliced out in somatic tissue resulting in a functional TclA protein. A mutation in either a Tcl element or a component of the  115  Discussion splicing machinery could allow germline Tcl transposition.^However, a single mutational event in a component of the splicing machinery does not explain the change from low level to high level Tcl transposition observed in the KR1787 strain. One explanation that could account for the change in Tc1 behaviour involves the transposition of a mutated Tcl element from one genomic location to another. In this case, a single mutation in a Bristol Tcl element could result in germline Tcl activity similar to that observed in Bergerac strains and the strain KR1787. However, if this Tc1 element was located in a part of the C. elegans genome that does not normally allow gene expression, mutator activity associated with high level Tcl activity might not occur. Under conditions such as heat-shock or starvation, gene expression in this region of the genome may occur allowing Tcl transposition. A subsequent increase in Tcl activity as observed in the KR1787 strain could be explained by transposition of the mutated Tcl element to a new position in the genome that allows increased gene expression. This hypothesis requires only one mutation to explain both the low level and high level Tcl activity observed in CB51-derived strains. Initially, low level  116  Discussion Tcl activity could be caused by poor expression of a mutated Tcl element. The change in Tcl activity could be the result of a subsequent transposition event involving the mutated Tcl element. A transposition event of this nature is a possible result of the initial low level Tcl activity and would explain the change in Tcl behaviour. This explanation suggests that the mutation occurred within a Tcl element normally found in the Bristol genome. It also suggests that genomic position can influence the level of Tcl transposition. Mori, Moerman, and Waterston (1988) have also suggested that the site of Tcl integration may affect Tcl transposition activity. In the strain KR579, Tcl activity may be influenced by environmental stimuli in a manner similar to that observed in maize (reviewed by Fedoroff 1989). Possible environmental pressures that may have occurred during stock handling include overcrowding and starvation. Either stimulus could have triggered expression of the mutated Tcl element resulting in a change in the pattern of Tcl hybridization in CB51-derived strains. The events leading up to the first demonstration of mutator activity in the KR1787 strain have been documented and progenitor strains frozen in liquid nitrogen. It may be possible by examination of these  117  Discussion  strains to characterize the nature of the changes that occurred in the generation of a Tcl mutator strain. An understanding of these changes may provide an insight into the constraints placed on Tcl transposition in Bristol strains.  1.4. Analysis of Tcl-induced Mutations of dpy-5  Six Tcl-induced alleles of the dpy-5 gene in C. elegans were examined. These mutations include one  allele isolated in a Bristol/Bergerac hybrid strain and five alleles isolated in Bergerac strains. Two spontaneous dpy-5 revertants isolated from the Tclinduced alleles were also examined. The genomic insertion site from the dpy-5(s1300) strain was cloned and sequenced. Unique flanking sequence was used to examine the insertion site for the other Tcl-induced alleles of dpy-5. In five out of six alleles the presence of a Tcl insertion was correlated with the Dpy-5 phenotype. In the strain carrying the dpy-5(s1300) allele, a novel Tcl element located in the dpy-5 region of chromosome I was cloned and unique flanking DNA was isolated. These cloned sequences in turn were used to examine other putative Tcl-induced dpy-5 alleles. In strains carrying  118  Discussion  the dpy-5 alleles s1300, h14, mn303, bx9, and bx10, the additional Tcl element was present. However, in strains carrying dpy-5 revertants of the alleles h14 and mn303, the additional Tcl element was absent. This evidence indicates that the Tcl insertion in this genomic fragment results in the Dpy-5 phenotype. In five of the six Tcl-induced alleles, the site of Tcl insertion can be positioned in a 1.1 kb EcoRI genomic fragment. Although the gene is spread over three EcoRI fragments, the 1.1 kb fragment appears to be the favoured site for Tcl insertion. This can be verified by examination of the restriction fragment length difference (RFLD) seen between the mutants carrying the Tcl insertion and the wildtype or wildtype revertants. In these examples, a 1.6 kb RFLD can be seen which is consistent with the insertion of a Tcl element. However, in one case, the Tcl-induced mutation dpy-5(m476) did not exhibit a 1.6 kb EcoRI RFLD. In this example, Tcl transposition may have been followed by an imprecise excision event. An imprecise excision would still result in a mutant phenotype, although no RFLD would be detected.  119  Discussion 1.5. Identification of the Phage Clone KR#85 and LG I Cosmid Contig Sequences flanking the Tc1-induced dpy-5(s1300) mutation were used to identify the genomic phage KR#85. In collaboration with John Sulston and Alan Coulson, the genomic phage was used to anchor a 350 kb cosmid contig to the genetic map. The placement of this contig on the genetic map was necessary in constructing the physical map of chromosome I in C. elegans. The placement of this DNA on the genetic map is a prerequisite for further alignment of the physical and genetic maps in C. elegans.  1.6. Sequence Analysis of the dpy-5 Gene Analysis of the dpy-5 gene reveals that the gene encodes a protein 254 amino acids in length. The gene does not contain any known protein motifs that might provide an insight into dpy-5 gene function. Searches of both nucleotide and protein databanks with the dpy-5 sequence do not reveal significant homology to any other genes. This indicates that at present no other gene similar to the dpy-5 gene has been identified and sequenced. Therefore the dpy-5 gene product represents a novel protein. The genes dpy-2, dpy-10 (Levy and Kramer, unpublished results), dpy-7 (Johnstone and Barry,  120  Discussion  unpublished results), and dpy-13 (von Mende et al. 1988) are known to encode collagen protein. The dpy-20 gene product is not a collagen and does not share homology with dpy-5 or any other known genes (Suleman, Clark and Baillie, unpublished results). From the knowledge obtained from the study of other dumpy mutations it is known that at least some of these genes are associated with cuticle formation. Since the  cuticle is composed primarily of covalently cross-linked collagen, it is not unexpected that collagen genes affect the overall shape of the worm. However, the dumpy collagen genes also appear to have a unique function within the collagen gene family. The function of dumpy collagens is not replaced by other collagen genes, since loss-of-function mutations are the predominant mutant class. One model for the defect associated with dumpy mutants suggests that the cuticle may have a greater extensibility than the wildtype cuticle (Kramer et al. 1988). Under high internal hydrostatic pressure the cuticle would tend to become more spherical, producing a short, fat animal. This model is compatible with the observation made for the dpy-5 cuticle (Ouazana, Garrone, and Godet 1985). The abnormal cuticle in dpy-5 animals  121  Discussion  may not have the same integrity as the wildtype cuticle. Since several protein components appear to be affected in the dpy-5 cuticle (Ouazana, Garrone, and Godet 1985), it is possible that the dpy-5 gene product is associated with modifications in cuticle proteins. Analysis of the protein sequence of the dpy-5 gene shows that the gene contains a putative secretory signal sequence (von Heijne 1990). This result is consistent with a suggested involvement with cuticle. The presence of the secretory signal suggests that the protein may be exported in a manner similar to pro A collagen -  polypeptide chains. Since the mutations in the dpy-5 gene appear to alter the cuticle, the most likely location for export would be the cuticle. In this location, the dpy-5 gene product could be involved in cuticle assembly. A possible role for the dpy-5 gene product is one that is associated with covalent crosslinking of cuticle proteins. Data from the protein composition of the dpy-5 cuticle shows that the pattern of protein banding is different in dpy-5 than in the wildtype cuticle (Ouazana, Garrone, and Godet 1985). This suggests that the dpy-5 gene product affects more than one protein in the cuticle. One way in which this  122  Discussion  may occur is through involvement in covalent crosslinking of cuticle proteins. It is known that the majority of the cuticle proteins are collagen type molecules (Cox, Kusch, and Edgar 1981; Cox, Staprans, and Edgar 1981). Molecular analysis of collagen genes shows that there are between 50 and 150 collagen type genes in C. elegans. The majority of these genes encode proteins that range in size from 30-40 kilodaltons (kd). However, the collagens found in C. elegans cuticles range in size from 50-200 kd (Cox, Kusch, and Edgar 1981). The most likely cause of this discrepancy is due to covalent cross-linking of cysteine and lysine residues between individual collagen genes (Siegel 1979). The dpy-5 gene product may be involved in the cross-linking of collagen genes. Examination of the dpy-5 cuticle shows that additional high molecular weight proteins are present that are not found in the wildtype worm (Ouazana, Garrone, and Godet 1985). These additional proteins may be the result of  abnormal cross-linking of smaller molecular weight proteins.  123  Discussion 1.7. Northern Analysis of the dpy-5 Gene  RNA expression was examined in the wildtype and several dpy-5 mutant strains. Northern analysis shows that the dpy-5 gene codes for a message approximately 950 bp in size. A message of this size is consistent with the 254 amino acid protein deduced from genomic and cDNA sequence analysis. The dpy-5 message is abundant in mixed population RNA samples. The dpy-5 message was examined in strains carrying the dpy-5(s1300), dpy-5(m476), and dpy-5(e61) mutations.  In the mutant strains examined, no visible alterations in message size were observed. This result is not unusual for strains carrying the mutations dpy-5(e61) and dpy-5(m476). The dpy-5(e61) mutation was  isolated in a screen using ethyl methane sulfonate (EMS) as a mutagen, and therefore is likely the result of a point mutation. Such an alteration in the gene may not affect the size of the dpy-5 message. For the Tclinduced mutation dpy-5(m476), a normal message size is expected based on molecular analysis of the mutation. In this case the strain carrying the dpy-5(m476) mutation does contain a Tcl insertion. If the mutation is the result of an imperfect excision event, a noticeable  124  Discussion  difference in the size of the message may not be observed. For the Tcl-induced mutation dpy-5(s1300) the wildtype message size may be explained by altered splicing of the mutant message. It is possible that a cryptic 3' splice acceptor or 5' slice donor may be utilized such that part or all of Tcl is spliced from the pre-mRNA, leaving a small insertion or deletion in the mature message. Several examples of an alternate splicing pattern have been observed for Tcl-induced mutants (A. Rushforth and P. Anderson). In these examples cryptic 3' splice acceptors and 5' splice donors were utilized to produce a near wildtype length message. Evidence for splicing of Tcl from message supports the idea that splicing of the Tcl element may be a general property of Tcl-induced alleles. In the case of the EMSinduced mutation dpy-5(e61), no alteration in the message size was observed in strains carrying the dpy-5(e61) mutation or the dpy-5(e61) smg-4(ma116) double mutation. Therefore, smg-suppression of the dpy-5(e61) allele does not appear to affect message size.  125  Discussion 1.8. Developmental Northern Blot Analysis The developmental expression of the dpy-5 gene was examined. Gene expression is lower in the embryo and Li stages of development and is then seen to increase from the L2 stage through to the adult. This pattern of gene expression is consistent with the phenotype exhibited for  dpy-5 worms. Observations of the dpy-5 worms indicate that the worm appears wildtype in length at the Li stage and begins to appear dumpy in the L2 stage. The difference in size between dumpy and wildtype worms becomes more dramatic with the increased age of the worm. The pattern of gene expression for dpy-5 is similar to other genes associated with cuticle formation. Examination of the cuticle reveals that both the ultrastructure and the protein composition of the cuticle vary between different cuticle stages (Cox, Kusch, and Edgar 1981; Cox, Staprans, and Edgar 1981). On the basis of the pattern of gene expression seen in Northern blots, it would appear that the dpy-5 gene is required primarily from the L2 stage through to the adult stage.  1.9. Injection Rescue of dpy-5 The cosmid line of B0342 has been used to rescue the  dpy-5(e61) mutation (H. Browning, unpublished results).  126  Discussion The cosmid B0342 was injected into homozygous dpy-5 animals and wildtype length animals were observed in the Fl generation. This information suggests that the B0342 cosmid carries sufficient genetic information to rescue the dumpy phenotype in dpy-5(e61) homozygotes. However, molecular analysis of the B0342 cosmid shows that it does not contain the entire gene cloned via transposon tagging. Several explanations may explain these data. One explanation is that the gene cloned via transposon tagging may not be the dpy-5 gene. In this instance, the presence of a Tcl element would interfere with gene expression some distance away from the insertion site. However, several lines of evidence argue against this possibility. Perhaps the strongest evidence is that the gene cloned via transposon tagging is an excellent candidate for the dpy-5 gene. This gene contains a putative secretory signal sequence that is consistent with a role in cuticle development. In addition, the pattern of gene expression for the transposon tagged gene is correlated with the onset of the Dpy-5 phenotype. Dpy-5 worms have a wildtype appearance until approximately the L2 stage when gene expression is first observed for the cloned gene.  127  Discussion  The suggestion that the transposon tagged gene is the dpy-5 gene is the simplest explanation of the results. Sequence data from the Tcl insertion site in worms carrying the dpy-5(s1300) mutation shows that Tcl inserts into coding DNA. The most likely result of this event is abnormal gene expression. One expectation of Tcl insertion into a coding region is an altered message. Northern analysis of gene expression in dpy-5(s1300) worms does not reveal a gross alteration in message size. However, results from the study of Tcl-induced mutations of the unc-54 gene suggest that Tcl often undergoes abnormal splicing resulting in near normal message size (A. Rushforth, unpublished results). Results from the unc-54 study also show that these abnormal messages do  not possess wildtype function. Therefore, normal message size for the transposon tagged gene is not an indication of an unaltered mRNA transcript. The presence of an upstream casein kinase I homologue also argues against a position effect due to Tc1 insertion. Next to the transposon tagged gene, the most likely candidate for disruption by Tcl insertion is the casein kinase I homologue. This gene is located approximately 2.5 kb upstream of the Tcl insertion site. Although the effect of disrupting casein kinase I gene  128  Discussion  expression is not known, the result is not likely a Dpy-5 phenotype. The dpy-5 gene exhibits a very weak dominant phenotype, such that heterozygote animals are slightly dumpy, but the effect is variable and hard to score reliably. This semi-dominant phenotype suggests that the dpy-5 gene is more likely a structural protein than a  serine-threonine kinase. Since the casein kinase I homologue is not a good candidate for the dpy-5 gene, the presence of a Tcl insertion would have to affect a more upstream gene. It seems unlikely that a Tcl insertion would affect a distant gene, but not the gene into which it had transposed or its nearest neighbour. Several other explanations may explain the B0342 result. For example, the entire dpy-5 gene may not be required for rescue. The B0342 cosmid contains three to four of the six exons that comprise the entire coding region. This coding material may be sufficient to produce a functional protein. Another possibility is that worms carrying the dpy-5(e61) mutation may produce a missense protein. The multimeric association of a missense protein from the worm and truncated protein from the cosmid may produce a wildtype phenotype. Alternatively, the B0342 cosmid may contain a suppressor of the Dpy-5 phenotype. Overexpression of the cosmid  129  Discussion could result in a wildtype phenotype. Gene conversion of the dpy-5(e61) mutant by an incoming cosmid carrying part of the dpy-5 gene may also result in cosmid rescue.  1.10. Identification of a Casein Kinase I Homologue Sequence analysis of DNA upstream of the dpy-5 gene reveals the presence of another coding region. This coding region is located approximately 850 bp upstream of the dpy-5 gene. Comparison of C. elegans genomic DNA, cDNA, and C. briggsae DNA sequences reveals the presence of a gene that encodes a 249 amino acid protein. Searches of the derived amino acid sequence against protein and nucleotide databases reveals a similarity to casein kinase I and other serine-threonine kinases. The mutant phenotype of this gene is not known; however, it represents a candidate for one of the lethal mutations that is located near the dpy-5 gene (J. McDowall, unpublished results).  1.11. Summary I. The presence of additional Tcl-hybridizing bands was investigated. Transposition was shown not to be associated with additional EcoRI sites, unequal crossover  130  Discussion  between adjacent Tcl elements, the Unc-13 or high recombination phenotypes.  2.  Germline transposition in the Bristol strain of  C. elegans was investigated and a Bristol mutator strain  was isolated. The spontaneous unc-22 forward mutation rate was measured in CB51 and the CB51-derived mutator strain.  3.  A Tcl insertion site from the Bristol strain was  cloned and sequenced. The presence of a Tcl consensus sequence suggests a similar mechanism of Tcl transposition as that observed in Bergerac mutator strains.  4.  Tcl-induced dpy-5 alleles and wildtype revertants  were examined. Tcl mutations were shown to be the result of Tcl insertion into a 1.1 kb EcoRI genomic fragment.  5.  Tcl was used as a tool in cloning the dpy-5 gene.  The Tcl insertion site was cloned and sequenced.  6. The dpy-5 gene was used in collaboration to anchor a 350 kb cosmid contig onto the genetic map.  131  Discussion 7.  Both genomic and cDNA clones of the dpy-5 gene were  isolated and sequenced. The dpy-5 gene codes for a novel protein associated with normal cuticle formation.  8.  dpy-5 gene expression was investigated using Northern  analysis. The developmental expression of the dpy-5 gene was also examined. The gene codes for a 950 bp developmentally expressed RNA message.  9.  A putative secretory signal sequence was identified  suggesting that the dpy-5 gene product is a secreted protein.  10. A casein kinase I homologue was identified immediately upstream of the dpy-5 gene and is a candidate for one of the lethal mutations mapped to this region of chromosome I.  1.12. Proposals for Future Research 1.  Attempt to isolate the putative Tcl element  responsible for Bristol germline transposition.  2.  Characterize the difference in genomic sites possibly  associated with altered Tcl regulation.  132  Discussion 3.  Determine if additional transposons are mobile in the  Bristol mutator strain.  4.  Map the mutator locus in the Bristol mutator strain  in order to determine its location for further study.  5.  Verify the tissue associated with dpy-5 gene  expression by antibody staining of the cuticle.  6.  Sequence dpy-5 mutations in order to determine the  sites important to gene function.  7.  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Biol. 77: 271-302.  141  APPENDIX 1 TABLE 1: dpy 5 and dpy 5 revertant alleles -  -  The following table contains a list of the dpy 5 alleles and dpy 5 -  revertant alleles used in this thesis work.  Strain  Genotype  Mutagen  Origin  BC1906  dpy 5(s1300)  Tcl  R. Rosenbluth  CB61  dpy 5(e61)  EMS  S. Brenner  DR1042  dpy 5(m476)  Tcl?  N. von Mende  EM9  dpy 5(bx9)  Tcl  S. Emmons  EM1012  dpy 5(bx10)  Tcl  S. Emmons  KR408  dpy 5(h14)unc 29(h2)  Tcl  L. Harris  KR1164*  unc 29  Tcl revertant  A. M. Howell  SP1091  dpy 5(mn303)  Tcl  R. Herman  wildtype  Tcl revertant  R. Herman  SP1092  *  -  -  -  -  -  -  -  -  -  *  Revertant of Tcl-induced dpy 5 allele -  142  -  Appendix 1 TABLE 2: Additional Strains Used in This Thesis Strain^Genotype^  Origin  BC82^unc 13(e51)^  A. Rose  -  BC187^dpy 5(e61) unc 15(e73) rec 1(s180)^A. Rose -  -  -  BC193^unc 13(e51)^  A. Rose  -  BC196^dpy 5(e61) dpy 14(e188) rec 1(8180)^A. Rose -  -  -  BC313^rec 1(s180)^  A. Rose  5C1906^dpy 5(s1300)^  R. Rosenbluth  CB30^sma 1(e30)^  S. Brenner  CB51^unc-13(e51)^  S. Brenner  CB1833^dpy-5(e61) unc-13(e51) ^  S. Brenner  KR408^dpy-5(h14) unc-29(h2)^  A. M. Howell  KR579^unc 13(e51)^  S. Brenner  -  -  -  -  KR1163^unc 38(e264) dpy 5(s1300) unc 87(e1459)^J. Babity -  -  -  KR1082^unc 13(e51)^  J. Babity  KR1787^unc 13(e51)^  J. Babity  -  -  143  APPENDIX 2 DNA and Protein Sequence From the dpy-5 Region  C. BRIG C.ELEG^GAATTCATGGATTTTGGATTTTGGATTACAAAATCAAAAAAACGTATCAAACTTTAGGGA 60  11111^III^III  cDNA^ CEPROT  GAATTCCGGTGATATCATTGATTATGGA 28  CBPROT C.BRIG^  GGATCTCTACCGT 13 111111111111 C.ELEG^ACCATCCGTCGTGAATACAACTACATGATAATCAGTATTCTTGGAAAAGATCTCTACCGT 120 111111111111111111111111111111111111111111111111111111111111 cDNA^ACCATCCGTCGTGAATACAACTACATGATAATCAGTATTCTTGGAAAAGATCTCTACCGT 88 CEPROT^ MIISILGKDLYR 12 1^1^1^1 CBPROT^ DLYR4 C. BRIG^CTTCGTGCTGAGCAACCGAATCGTTCGTTCTCTCTCAACACCACTACCAAAATTGGATTG 73 11111111^11^1111111^111111^111^1111111^11^11111^11^1111^1 C.ELEG^CTTCGTGCCGAACAACCGACTCGTTCATTCACTCTCAATACGACTACAAAGATTGCTCTT 180 111111111111111111111111111111111111111111111111111111111111 cDNA^CTTCGTGCCGAACAACCGACTCGTTCATTCACTCTCAATACGACTACAAAGATTGCTCTT 148 CEPROT T^ T1 T1^ A i I 32 CBPROT^LRAEQPNRSFSLNTTTKIGL 24  TiTY PIT^ T I C. BRIG^GAGACTCTCGAGGCAATCGAAGAACTTCATGCCATTGGATACTTGAGTCGTGATGTCAAA 133 11^111^1.^11^11^Ur^11111^11111^111111111^1111^11111111111 C.ELEG rilliiliiiiTTITITTiiiiiiiTITTiriTTITTIIIITTITTTYTTITTiTilia cDNA^GAAACTATTGAAGCTATTGAAGAGCTTCACAATATTGGATACCTGAGCCGTGATGTCAAG 208  240  CEPROT^ETIEAIEELHNIGYLSRDVK 52 II^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 CBPROT^ETLEAIEELHAIGYLSRDVK 44  C.BRIG^CCGAGTAATTTTGCTCCTGGAATGCGCGAATGTGGAGAACACAAAACGATTTACATGTTC 193 11^11^11^11111111^111^11111^1111^1^11^11^11^1111^111111 C.ELEG^CCAAGCAACTTTGCTCCAGGACAACGCGACAATGGACAGCATAAGACAATTTTCATGTTT 300 111111111111111111111111111111111111111111111111111111111111 cDNA^CCAAGCAACTTTGCTCCAGGACAACGCGACAATGGACAGCATAAGACAATTTTCATGTTT 268 CEPROT^PSNFAPGQRDNGQHKTIFMF 72 1^1^1^1^1^1^1^1^1^1^1^1^1^II CBPROT^PSNFAPGMRECGEHKTIYMF 64 C. BRIG^GATTTCGGGCTCGCCAAGAAGTATCTCGACCGTGAAGGGAAGAAAATGAAGAGTCGCGGA 253 11^11^11^11^11^1111111^1^1^11^11111^11111^11111^111^111 C.ELEG^GACTTTGGACTTGCGAAGAAGTTTATTGATCGTGATAACAAGAAGCTGAAGTCTCGTGGA 360 111111111111111111111111111111111111111111111111111111111111 cDNA^GACTTTGGACTTGCGAAGAAGTTTATTGATCGTGATAACAAGAAGCTGAAGTCTCGTGGA 328 CEPROT^DFGLAKKFIDRDNKKLKSRG 92 1^1^1^1^1^1^1^II^II^1^1^1^1 CBPROT^DFGLAKKYLDREGKKMKSRG 84  144  Appendix 2  C. BRIG^GAGGTTGGATGGAGAGGAACCGTTCGTTACGGTAGTCTTCAAGCCCATAAACGATTGGAT 313 C.ELEG^11111TaigallaTICTITAAGATITIGAIIIIICIIIIITCTCUITATIIIT 420  111111111111111111111111111111111111111111111111111111111111  cDNA^GAGGTTGGATGGAGAGGTACTGTAAGATATGGAAGTCTCCAAGCTCTCAAACGTATGGAT 388 CEPROT I^ IITLTTM? 112  ^Ti ITT^ CBPROT^EVGWRGTVRYGSLQAHKRLD104 C. BRIG^CTTGGAAGACGTGATGATGTGGAATGCTGGTTCTACATGCTCATTGAGATGTATGCTGGG 373  11111111111111111111^1111111111111111111111111111111^1^111 111111111111111111111111111111111111111111111111111111111111 cDNA^CTTGGAAGACGTGATGATGTTGAATGCTGGTTCTACATGCTCATTGAGATGTTGGTTGGA 448  C.ELEG^CTTGGAAGACGTGATGATGTTGAATGCTGGTTCTACATGCTCATTGAGATGTTGGTTGGA 480 CEPROT^LGRRDDVECWFYMLIEMLVG132  1 ^ 1 ^ 1 ^ 1^ 1 ^ 1^ 1^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1 ^ 1  CBPROT^LGRRDDVECWFYMLIEMYAG124 C. BRIG^GAGTTGCCATGGCGTCATATGACTGATCGCGCTCTTGTGGGACAGGCCAAACTTGCGATT 433  111111111111111111111^1111111^1^11111^11111^1^11^11^1111 11111111111111 cDNA^GAGTTGCCATGGCG-gap in cDNA ^  C.ELEG^GAGTTGCCATGGCGTCATATGTCTGATCGTACACTTGTTGGACAATCGAAGCTCTCAATT 540 468 CEPROT^ELPWRHMSDRTLVGQSTI(Sf152  1^1^1^1^1^1^1^II^1111  CBPROT^ELPWRHMTDRALVGQAKLAI144 C.BRIG^CGAAATGAGCAACGTCAAATCTTCTTCAATCGCATTCCAAGACAATTTGAAAAGATTATT 493  11111111^1111^1^11111111111^1^111111111^11111^1^111^11  C.ELEG^CGAAATGAATCTCGTCGTTTGTTCTTCAATCGAACTCCAAGACAGTTTGAGACGATCATG 600 cDNA ^ gap 470 CEPROT^TIITSTRLFIFITTTTTyFITTIM172 CBPROT^RNEQRQIFFNRIPRQFEKII164 C. BRIG^GATTTGATTGATGGATACAGCTTTGAGATTAGACCGGATTATAGACAACTCAAGGGTTTG 553  111^1111^111111111^11^11^11^11111^11^11^11111^1^1111^111 1111111111111111111111111111111111111111111111111111 cDNA^in cDNA-CGATGGATACTCATTCGAAATCAGACCAGAGTACAGACATTTGAAGGCATTG 526  C.ELEG^GATATGATCGATGGATACTCATTCGAAATCAGACCAGAGTACAGACATTTGAAGGCATTG 660  CEPROT^DMIDGYSFEIRPEYRHLKAL192  1^1^1^1^1^1^1^1^1^1^1^II^It^1  CBPROT^DLIDGYSFEIRPDYRQLKGL184 C.BRIG^ATCAATGAG-intron absent in C. briggsae ^  575  11^111111 C.ELEG^ATTAATGAGGTAACTAAAATTAGTCTAGATGGATATTTTTAGTTAGATCCC -gap here 714 111111111 cDNA^ATTAATGAG-intron in C. elegans ^ 544 CEPROT^I N E^ CBPROT^  195  I 1 I^  145  187  Appendix 2 C.BRIG^ATCCGCTTTGAAAACAACATTCCGGATCGATGCAAATGGGATTGGCAAATCGAGGAATCT 635 11111^1^11111^1^11^11^11111^11111^11111^111111^1^11^111111 tat C.ELEGatcc4  YY fi M iT'fiiiiiiiirriii 215 CBPROT^IRFENNIPDRCKWDWQIEES207  cDNA^ATCCGTATGGAAAATATGATCCCTGATCGTTGCAAGTGGGACTGGCAAGTGGAAGAATCT 604 CEPROT  C. BRIG^CAACATTCCGAGCTTACTGAAACCACTTCTGTCATGTCTGATGTTGCCATTCTGGCTGAA 695 C.ELEG^IIIIITICGUICICCIIIIIAGITICTITIATIIIIIITAIGICAITTAIllgal 834 111111111111111111111111111111111111111111111111111111111111 cDNA^CAACATTCGGAGCTCACCGAAACAGCTTCTGTCATGTCTGATATGGCAATTATGGCTGAA 664 CEPROT MI^235 IHMTT CBPROT^QHSELTETTSVMSDVAILAE227  YiliiiTT A iTifi  C. BRIG^CAAGGTGTCACCAACTACACTGATCGCGCTTGTGAGAGTGAGTTATATTCCTTTTTGAAC 755 C.ELEG^11111ALTAITIIIIWAIWITICCIIIIIIIGTATGTTTTATTGTTTTTATTTT 1111111111111111111111111111111111111 ^ cDNA^CAAGGAGCTACTAACTACACTGATCGTGCCTGTGAGA^ CEPROT A T1 Y1 T1 CBPROT^QGVTNYTDRACE^  894  C. BRIG^TTTCAAAATTCATATTTCTTTTCAGATCAATAGAGGATCTCCCATCTCGTCATCTTCCCT 1 111111end of gene C.ELEG^CTATATCAAAATATACATTTTTCAGACCAATAGGCATATACTCGTCTCACAACTGTTCTT ^ 11111111111111111111111111111111111 cDNA^ ACCAATAGGCATATACTCGTCTCACAACTGTTCTT CEPROT  815  TTA  7  CBPROT^  701 CE^ 247 7T i 239 954 736 249  T^ T I^241  N Q *^  C. BRIG^TCTTTTTCTTTCACTCTCTGCTCATCAATCTCGAATCGAATCGAACACTTTCCAGTACAA 875 C.ELEG^CATCACTACTCTTCTCATCAAATCTCGAATCGAATCGAGCACTTTCCAGTTCAATTTCCT 1014 cDNA^ CEPROT  11111WITWITCATC11111111111CIIITIMCITTICAGIWITITAT 796  CBPROT C.BRIG^TTTCCTCTTTCTTTTTCCGCACTAAAGTAAAATACCAACCCTTCCAAATCCCTTCTTTCT 935 C.ELEG^CTTTCTCTCACTCTTTTCCAATCCAAAGTAAAGAACCGATTGCCTCTTTTTGTATTCATT 1074 cDNA^ CEPROT  CITTIICIWWWWWWWWWWITIGAICTITITITAIWIT 856  CBPROT  146  Appendix 2  C. BRIG  CTATTCACTATCATCATCGTCGTCGTTCCATATCTACTATGCTACTAGACCTTTGTATAT 995  C.ELEG  ATCATCATCGTCGTCGTTTGGTTTCAACTATACTACTAGATTTTTGTGCTGTGTAAATAA 1134  cDNA CEPROT  1111111111/11111141141111111/11111111111411111111111111111 916  CBPROT C.BRIG  AATAAATGTATTACTCCCACAATCACAATATCACTTTTTTTTATCTATAAAAACAGACAA 1055  C.ELEG  ATGTATTTTTTTAAAAGTATTTTACATCTTATTGTAAGTTCCAACTATCACGGCGTTAGT 1194  cDNA CEPROT  1111111141141111  1  IIGGAATTC  948  CBPROT C.BRIG  GACTTTTATAATCCGTGTGTGATTTTCGTTATGTTCTAACAACTTTTCGAAATTGGTCTA 1115  C.ELEG  TTCTTTCCCAAGTTTGGCAAATTGAATATTAACTTGGATTAGAAGTGGTTAAACCTGAGC 1254  cDNA CEPROT CBPROT C.BRIG  ACACGATGTGTTGCCTAGGTCATTTTAACATGGTGGCCTAACTTTTTCAAGTTGAGTTTT 1175  C.ELEG  CAAACTTTGGCAAAACATAGTCTCCTGGAATCTTCTAGAAGTGATATGTTGACGGAAAGT 1314  cDNA CEPROT CBPROT C.BRIG  CTAGGCCACCAACTTCTATGACCTAAAAATGACATGATGTTGTACTGGTTTGTTTAATAG 1235  C.ELEG  TGGATCAAACTTTGATTACACTTGAGTGAAGCGTGAACCAAGTTTTCCAACTTTCAATCT 1374  cDNA CEPROT CBPROT C.BRIG  GCCATTTCGACATGGTGGTCTAACTTTTGCAAGTTGAGTTTTCTAGGCCAGCATGGTCAA 1295  C.ELEG  GAATTAATATTCTTTAAAATTGTGGTAGAGGAATCCGGCCTTGAAGGCAAGAATATAGCA 1434  cDNA CEPROT CBPROT  147  Appendix 2  C.BRIG  TGACCTAGAAAACGCATCAGGTGCATGTTAGGCGGTTTCTGGACCATCAACTACGGTGGC 1355  C.ELEG  TGTCACTTTTCCAACTTTTTTCTTATTTTCTCAATTATTGAAATTTTAAAATGAAATTAT 1494  cDNA CEPROT CBPROT C.BRIG  CTAGAAAAATAATTTCGAGAAAGTTAGGCCACACTATCCTCGCATGAAAACGGAGCGTCT 1415  C.ELEG  AGTTATAGATGTAATTTGATATGCTTAATCCAATTATTATACTGATTTTTTGGTTAACAA 1554  cDNA CEPROT CBPROT C.BRIG  GATTTTCGACAGCATCGAGAAAAATTTCGAAATCTCGTTCGAAAAGTCGTTCGAACGTAA 1475  C.ELEG  GTTTGAAAATGTCTGGAAATCCCAGAATGAATGGATATCTCATAGCAGATTGGCACTGTA 1614  cDNA CEPROT CBPROT C.BRIG  CGAAAAGCACACATTGATCACAGGACTCAAAAAATGAAAACGCTGATCCATAATGTTTCT 1535  C.ELEG  CTCTTCTGAACAAATTGAATTTGTACCGAAGTTTCTTTTCTGATATTTTTTTTCGAGTAA 1674  cDNA CEPROT CBPROT C.BRIG  TCTTCTAGAAGACATATTTTTCCGGCAAGTGTTCTTCAATCTCTATCACATCAAGATCAG 1595  C.ELEG  AACAGGGGCAAAAATTAAGTTTTTCCAAACTTTTCAAGGATTTTAAAGCACTGCTGGATA 1734  cDNA CEPROT CBPROT C. BRIG  TGTAATACTCCGAAGAGTCAAATGACCTCCAACTCCCCATCTTCTTCTTCTCTACCTTCT 1655  C.ELEG  TTTCGATTGCGTGGATCTTAATAATTTTACAACATGTCGTACTCCACCTTTCAGCATTGG 1794  cDNA CEPROT CBPROT  148  Appendix 2  C.BRIG  TTTTCTTCTTCCAACAACAAAATGCGTCTACTTCTGTTTTCTTTTGTTCTATTGCTTTCA 1715  C.ELEG  AAACCTATTTATTCAAGGAAATTAATAAACAAATTATTGTAATTTATGTTTACCGCATCA 1854  cDNA CEPROT CBPROT C.BRIG  TCTACGTCTTCTTTTGAGCATCAGAAGAAGCAACACATCGTCAACTTTTCTGTAAAATAC 1775  C.ELEG  ATCTTTACCATCGATTCAACGTCATCATTCTAGCCAGTTGTCAAAAAAAACATATCCGCA 1914  cDNA CEPROT CBPROT C.BRIG  TTTAACAAATCCCAAAGCGTACGTTCGATTGATATTTTGAACAAAAACTGGGATTCTATT 1835  C.ELEG  GATGTTATCTTTGATTTATTTATTGGGAGAACATCTGGTTGTGTAACAGAGTCAACTGAC 1974  cDNA CEPROT CBPROT C.BRIG  ACGAAAGTTGTGAATTTCAATAAAAATGGATCAAAGATAACGGGAAGAATTGGTTTCGAC 1895  C.ELEG cDNA CEPROT CBPROT C. BRIG  GAATCAATAGACAGAGTTATCCTGAAATTGGGTAACAGAACATCGATCAAGGTAAGTGAT 1955  C.ELEG cDNA CEPROT CBPROT C.BRIG  TTGACATTTCCAGAGGGTTCTATAATTTTTATTTATTCTACAGGTGGAGGTCTCTTCTCA 2015  C.ELEG cDNA CEPROT CBPROT  149  Appendix 2  C.BRIG^TGGGGCACNGTGGTAGGA^  2033  C.ELEG cDNA CEPROT CBPROT C. BRIG C.ELEG CTTCTCCCTTTTCATTCGTCCAAATCTTCATTTCTTTTCCAAAATGCACCTCTTTCTTTC 2034 III^111^11111111111111111111111111 cDNA^CGCGGAATCCGTTTTTTTTTTTTTTTTTTCCAAAATGCACCTCTTTCTTTC 999 CEPROT^ MHLFLS255 CBPROT C.BRIG C.ELEG TGTTTTTCTTCTGCTTATCCTACCGTTAATTTCCACCTCAGCAGTTGAGAATAATCAAAA 2094 111111111111111111111111111111111111111111111111111111111111 cDNA^TGTTTTTCTTCTGCTTATCCTACCGTTAATTTCCACCTCAGCAGTTGAGAATAATCAAAA 1059 CEPROT^VFLLLILPLISTSAVENNQN275 CBPROT C.BRIG C.ELEG TATTGTGGATTTTTCTGTGAAAGATTTCAACAAAAATATTAATTCAATCGATATTCTCAA 2154 111111111111111111111111111111111111111111111111111111111111 cDNA^TATTGTGGATTTTTCTGTGAAAGATTTCAACAAAAATATTAATTCAATCGATATTCTCAA 1119 CEPROT^IVDFSVKDFNKNINSIDILN295 CBPROT C.BRIG C.ELEG CAAAAAATGGGATATTGTTTCGAAATATGTGAAATTTAATGGAAACTCATCGAAATTGAA 2214 111111111111111111111111111111111111111111111111111111111111 cDNA^CAAAAAATGGGATATTGTTTCGAAATATGTGAAATTTAATGGAAACTCATCGAAATTGAA 1179 CEPROT^KKWDIVSKYVKFNGNSSKLN315 CBPROT C.BRIG C.ELEG TGGAAGACTCCGATTATCGGAAAAAATTGACAAAATCATTTTTAAATTGGGAGACGATGG 2274 111111111111111111111111111111111111111111111111111111111111 cDNA^TGGAAGACTCCGATTATCGGAAAAAATTGACAAAATCATTTTTAAATTGGGAGACGATGG 1239 CEPROT^GRLRLSEKIDKIIFKLGDDG335 CBPROT  150  Appendix 2  C.BRIG C.ELEG^AGCTAATCAGAGTAAGTTTTTTGAAATTTAAAAGTGGATTAGCGCCCTATGGTATGACTC 2334 11111111111 ^ 1250 cDNA^AGCTAATCAGA^ CEPROT^A N Q^ 338 CBPROT C.BRIG C.ELEG^CTATGATTCTAAAATGATAAATTTTCACAGTAAAACTTTGCGAAACTGGTTTGATATTTT 2394 cDNA CEPROT CBPROT C.BRIG C.ELEG^TAATAACATTTAAAAATGGTATTGATTCAGTTCTCAACTGTTATAATTTTGGAATATTCG 2454 cDNA CEPROT CBPROT C. BRIG C.ELEG^AATGTTCCAAAAAATTTATCTGAAAAAGTTCTGAAATCAAAAAAAAATTCTTCTACCAAA 2514 cDNA CEPROT CBPROT C.BRIG C.ELEG^TAGGCAAAATGTTTTCTACGACTTTTATATTTTAATCAAGTTAAGAAATATTTTTTGTTA 2574 cDNA CEPROT CBPROT C. BRIG C.ELEG^AGAAAAATAGTGCAAGAACGTTCAAAATTTCCGAAAAAAAAACCGAGTTTATCGAAAATT 2634 cDNA CEPROT CBPROT  151  Appendix 2  C. BRIG C.ELEG^TGGCAATTTGCCAAACTCTTACTGACTAGCAGCAATCCTCTTGTTTTCTAAAAATTTTAA 2694 cDNA CEPROT CBPROT C.BRIG C.ELEG TTTTCAGATGAAATAATAGTTTCCTTGGGTAATAGAAATACAACTCATATGACTGTGCTC 2754 ^ 11111111111111111111111111111111111111111111111111111 cDNA^ATGAAATAATAGTTTCCTTGGGTAATAGAAATACAACTCATATGACTGTGCTC 1303 CEPROT^NEIIVSLGNRNTTHMTVL356 CBPROT C.BRIG C.ELEG TCGGATTCATACAAAATGAAAGTATCCACTTCGAAGCAAGTTGTTCGGAAAGGTCTGTTG 2814 1111111111111111111111111111111111111111111111111111 ^ cDNA^TCGGATTCATACAAAATGAAAGTATCCACTTCGAAGCAAGTTGTTCGGAAAG ^1355 CEPROT^SDSYKMKVSTSKQVVRK^373 CBPROT C.BRIG C.ELEG^AAAAGGTTTTGAAAATTGTATTCTGTTTTACGTTGTTAACAAAGACAATTGCTCAAGCAG 2874 cDNA CEPROT CBPROT C.BRIG C.ELEG^CCTCAAGCCAGCAAGCCAAAACAAAACTTTAAAAAACTGAAATTCGGAATGAGAAAAAAA 2934 cDNA CEPROT CBPROT C.BRIG C.ELEG AAATACATAAAGGCTAATTAAAATTGTAATAAACCTTTCCAGAACTGTCTCTGAGAACTG 2994 ^ 111111111111111111 cDNA^ AACTGTCTCTGAGAACTG 1373 CEPROT^ ELSLRT 379 CBPROT  152  Appendix 2  CTTGGTTGT 2042  C.BRIG^  C.ELEG^GTTCATCAAACGAGAACGGATGTGCTTGTGTACACGGAAATTGTGCTTGCTGCCTAGAAA 3054 111111111111111111111111111111111111111111111111111111111111 cDNA^GTTCATCAAACGAGAACGGATGTGCTTGTGTACACGGAAATTGTGCTTGCTGCCTAGAAA 1433 CEPROT^GSSNENGCACVHGNCACCLE 399 CBPROT C. BRIG^CTTGGAAGCTCTGTCCCAGAATTTAGACATTCAGGNTTNTTGAAAAATTGTTTTGTAGAG 2102 C.ELEG^TTTCTGTTCCAGAATTCAGACATTCAGGTAATAGTTTTCCTTAAATTTTGTTTTCTTAAT 3114 111111111111111111111111111 ^ cDNA^TTTCTGTTCCAGAATTCAGACATTCAG^ 1460 CEPROT^ISVPEFRHS^ 408 CBPROT C. BRIG^TCATACAAAAACGTATTTCAGTTTGTGTCAATGCAACTTACAACCCAGTTTCCATTGGAT 2162 1111111111111111^11^11111111111111111^1 C.ELEG^TTTCCAATACTTTTAATTCAGTTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCT 3174 ^ 111111111111111111111111111111111111111 cDNA^ TTTGTGTCAATGCAACCTATAACCCAGTTTCCATTGGCT 1499 CEPROT^ VCVNATYNPVSIG 421 1^1^1^1^1^1^1^1^1^1^1^1 CBPROT^ CVNATYNPVSIG 253 C. BRIG^TGGATTTATCAGTCGGAGTGGATGGACACTACTTTACGGAAGAAGTTTCTCGTAAGGTTT 2222 1111111^11^1^11111^11111111^11^11^1^111111^1^111 C.ELEG^TGGATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTTGTATGTAAA 3234 111111111111111111111111111111111111111111111111111 ^ cDNA^TGGATTTGTCGATTGGAGTTGATGGACATTATTTCAGTGAAGAAATATCTT ^1550 CEPROT^LDLSIGVDGHYFSEEIS^438 1^1^1^1^1^1^1^1^1^1^1^II^1 CBPROT^LDLSVGVDGHYFTEEVS^270 C. BRIG^CTTTTTTTTGCCGAAACGTAGTGTG^  2247  C.ELEG^GAACTTTAAGATGGAATACGCGGTCAAATGGGTTCCATATGAATATGTTTAAATAATTCT 3294 cDNA CEPROT CBPROT C.BRIG C.ELEG^CAAATTCAGATATCCCACAAAAGGAATTCAGAAAAAGCGAGTTGAAACTACTAATCCAAC 3354 cDNA CEPROT CBPROT  153  ^  Appendix 2  C.BRIG C.ELEG^TTTCAAACTCGCGAATTTCTAACTCTGCTAATTTTTTGCTATTTAAATATAAATTTCACA 3414 cDNA CEPROT CBPROT C.BRIG  ^  CTTTATTTGCAGTCCGTAATCCACCTCC 2275 1^1^11^11^11^11 C.ELEG^GAACTTTTGTTTCACATAAAAAAACAATTGTTAATAATTTTCAGTGAGAAACCCCCCACC 3474 ^ 1111111111111111 cDNA^ TGAGAAACCCCCCACC 1566 CEPROT^ LRNPPP444 1^1^1^1^1^1 CBPROT^ LRNPPP276 C. BRIG^AATCTGTTTCTCTCTTCCAATTCCTGGCGCTGAACATATTGCAGGTGTTTGTGTTGCTTT 2335 1^1111111^11111111^11111^11111^11^11^11111111^11^11111^11^11 C.ELEG^AGTCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATT 3534 cDNA^AGTCTGTTTTTCTCTTCCGATTCCCGGCGCAGAGCACATTGCAGGAGTATGTGTGGCATT 1626 CEPROT^VCFSLPIPGAEHIAGVCVAF464 1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 CBPROT^ICFSLPIPGAEHIAGVCVAF296 C. BRIG^CACGAAAGTGAGTGACTAGTTCCATGAATTAAATCTTATTCCGATTTAAATGTTTTCAGT 111^11^ 1 C.ELEG^CACAAAGGTAAAATTGTTTCTGGTAACACGAAAA ^TTCCAATTTTATTTATTCAGT 1111111 ^ 1 cDNA^CACAAAG^ T CEPROT^T K^ 1^1 CBPROT^T K^  2395 3589 1634 466 298  C. BRIG^TAGATTTGGACAAGAAAGCCAAGATTCTATCAGGATGCATGGATTTCGAAGTTGAATTGA 2455 C.ELEG^IGGICIIIIIIIIIIIGGAGIIAITTITTTLIGITICITIIITTICIIIITGIIIITAI 3649 111111111111111111111111111111111111111111111111111111111111 cDNA^TGGACTTGGACAAGAAGGAGAAAATTCTTTCCGGATGCATGGATTTCGAAGTGGAATTAA 1694 CEPROT^LDLDKKEKILSGCMDFEVEL 486 1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 CBPROT^LDLDKKAKILSGCMDFEVEL 318 C. BRIG^TTCATTTGAGAGCTCTCAGTTTCCACTTGGGATGCTTCAGAATGCCAATTTGAGTGTTTG 2515 1111111^1111^111^1^1111^1^1^11111^111111111111!^111 end of C.ELEG^TTCATTTAAGAGTTCTTACTTTCAAGCTTGGATGTTTTAGAATGCCAATCTGAAATTAAA 3709 111111111111111111111111111111111111111111111111111111111111 cDNA^TTCATTTAAGAGTTCTTACTTTCAAGCTTGGATGTTTTAGAATGCCAATCTGAAATTAAA 1754 CEPROT^IHLRVLTFKLGCFRMPI *^503 1^1^1^1^1^1^1^1^1^1^1^1^1^1^1 CBPROT^IHLRALSFHLGCFRMPI *^335  154  Appendix 2  C. BRIG^AATAGCTGTTTTGCAAAAAATATTTTGATAAAGTTTTAATCTTTTGACTTCATTGTTTAC 2575 gene C.ELEG^CAAATATGTTATGTTGTAACAGTTTGGTGCAAACTGTTATGTCAAATCAAACAGAAAAAG 3769 cDNA^ CEPROT  CIIIIIIIIIITITTITIWIT/111=11111111111111111C11111111  1814  CBPROT C. BRIG^ACTTCAATGCATCCATGTGCATCCATGAGTGTACATGGAAACCAATACAGCGTTTAACCG 2635 C.ELEG^TTATGTTTTTTGGTTTTGCTCTTTGCTTGATTTTCAGAGACGAAGCTTCTCGTTCGTCTG 3829 111111111111 cDNA^TTATGTTTTTTGAAAAAAAAAACG^ 1838 CEPROT CBPROT C. BRIG^CAACTATTATGCTGGAATACAAAACGTGATGTGGAGGAGAAAATCCGGTGGCCGTGGAGG 2695 C.ELEG^CATTCGCTTATTTCATCGCCCATCTACACTCCCAATAGTGGTTTTTCATTTCTTCTACTG 3889 cDNA CEPROT CBPROT C.BRIG^AAGGTACGACAACTACAGTAACCCCCCTACGACAACTACAGTAACCTAAATGTTTTTATT 2755 C.ELEG^GAGATGATTCTGATAGACTTTATTTTGAGATGATCAAGTTTTGGGTGACAATGTACTCAA 3949 cDNA CEPROT CBPROT C. BRIG^TTCTGAGTTTTTTCTCGTCAATTTTTTGTTAGAAAAACTTCAATTGCCTGACTTTTTTTT 2815 C.ELEG^CGGGAACAATATATCCAGAGTGACACAGGATTCAGTAGATAAACTGGGGTGATATTATTT 4009 cDNA CEPROT CBPROT C. BRIG^GCAAAAAACAAAAGAAAAATCAGCAAAAAATTCGGGGGTACAATGATTCGGGGGTACAAG 2875 C.ELEG^TATTTGCCACGTTTGCGACGGATATTGACAAGATGAAAAATGTGTGAAATTTTTTTAAAA 4069 cDNA CEPROT CBPROT  155  Appendix 2  C.BRIG^GGGGTACAACTGATTTAGGGGTACAAAGTTGGTGATTCGGGGGNACCCAGCTTTTGTNCC 2935 C.ELEG^CTTTTATTTTAAAAAAAGACTGTAGCAAAAGGGATATTGAGGAAATTGAGACCTTGAAGA 4129 cDNA CEPROT CBPROT C.BRIG^NTTANTGNGGGTTCA^  2950  C.ELEG^GGATGTATGATCACATGTCGGTACTTGTAAAGCATGGTGACGACTGTCCAGAATCTGATC 4189 cDNA CEPROT CBPROT C.BRIG C.ELEG GGAAGGTTTGTGTGAATTGGATTTTCATAGTTAAAAACAACATTTCATATCTTTTCCGAT 4249 cDNA CEPROT CBPROT C.BRIG C.ELEG GCTTTCAAAGCAGTTCTATCGGCAACAAAGATTGACGATCTCTATCCAAACTGCATGTAT 4309 cDNA CEPROT CBPROT C. BRIG C.ELEG GACTCCATCCTCTGGAAATTATTCGCTGGGAAGTTAGCTTGTTCGCTGTTGGGTTGTGCT 4369 cDNA CEPROT CBPROT C.BRIG C.ELEG GAGGATTTGCGTGTCATCATTGGTCATCAGGATTTATGAAAACTATTGTATTTTATTGTA 4429 cDNA CEPROT CBPROT  156  Appendix 2 C.BRIG C.ELEG^AAATTATTTTCTTTTTCTTCGATAAATATTAATTTTCAATCATTGTTTGTTTTCATATTA 4489 cDNA CEPROT CBPROT C. BRIG C.ELEG CTTAGACGCGGAATTC 4505 cDNA CEPROT CBPROT  157  APPENDIX 3 Result of FASTA Run of the dpy-5 Upstream Gene vs. SWISS_PROT The best s cores are:^ initn^initl^opt YCKl_YEAST CASEIN KINASE I HOMOLOGUE 1 (EC 2.7.1. -).^202^99^283 YCK2_YEAST CASEIN KINASE I HOMOLOGUE 2 (EC 2.7.1. -). ^202^98^274 KDC2_DROME PROTEIN KINASE DC2 (EC 2.7.1.37).^91^55^75 KRBl_VACCV 30 KD PROTEIN KINASE HOMOLOGUE (EC 2.7.1. -) 81 81 204 KRB1 VACCC 30 KD PROTEIN KINASE HOMOLOGUE (EC 2.7.1. -) 81 81 203 IBMP CAMVD INCLUSION BODY MATRIX PROTEIN (VIROPLASMIN) 79 51 54 KS62=MOUSE RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. 78 49 111 POLG_STEVM GENOME POLYPROTEIN (CAPSID PROTEIN C; ENVEL 77 40 40 KS6B_XENLA RIBOSOMAL PROTEIN S6 KINASE II BETA (EC 2.7 76 48 114 KS6A_CHICK RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. 76 48 113 KS6A_XENLA RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. 76 48 113 KPKl_PHAVU PROTEIN KINASE PVPK -1 (EC 2.7.1. -). 74 74 85 DBF2 YEAST CELL CYCLE PROTEIN DBF2 (EC 2.7.1. -). 72 62 75 ITI2_HUMAN INTER -ALPHA -TRYPSIN INHIBITOR COMPLEX COMPO 72 55 55 TRPG_PHYBL ANTHRANILATE SYNTHASE COMPONENT II (EC 4.1. 71 71 71 KRAF_XENLA RAF PROTO-ONCOGENE SERINE/THREONINE KINASE 70 58 83 POLG_JAEVJ GENOME POLYPROTEIN (CAPSID PROTEIN C; ENVEL 69 40 47 ILVD_ECOLI DIHYDROXY -ACID DEHYDRATASE (EC 4.2.1.9). 69 49 51 CPTl_CHICK CYTOCHROME P450 XVIIA1 (P450-C17) ^(EC 1.14. 69 46 49 COX1 CAEEL CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9. 69 48 48 YCKl_YEAST CASEIN KINASE I HOMOLOGUE 1 (EC 2.7.1.-). initn= 202^initl= 99 opt= 283^26.2% identity in 225 aa overlap dur. P^  10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKI •  ^^ • •  YCK1 Y RDEYKTYKILNGTPNIPYAYYFGQEGLHNILVIDLLGPSLEDL-FDWCGRKFSVKTVVQV 110^120^130^140^150^160 40^50^60^70^80 dur. P ALETIEAIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK-LK ..^•  YCK1 Y AVQMITLIEDLHAHDLIYRDIKPDNFLIGRPGQPDANNIHLIDFGMAKQYRDPKTKQHIP - 170^180^190^200^210^220 90^100^110^120^130^140 dur. P SRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQ-S :..^•^.^.^••••^•^....^: .^......^.^. YCK1 Y YREKKSLSGTARYMSINTHLGREQSRRDDMEALGHVFFYFLRGHLPWQGLKAPNNKQKYE - 230^240^250^260^270^280 150^160^170^180^190^200 dur. P KLSIRNESRRLF-FNRT-PRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRC YCK1 Y KIGEKKRSTNVYDLAQGLPVQFGRYLEIVRSLSFEECPDYEGYRKLLLSVLDDLGETADG - 290^300^310^320^330^340  158  ^  Appendix 3  210^220^230^240 dur. P KWDW-QVEESQHSELTETASVMSDMAIMAEQGATNYTDRACENQ ...... YCK1 Y QYDWMKLNDGRGWDLNINKKPNLHGYGHPNPPNEKSRKHRNKQLQMQQLQMQQLQQQQQQ 350^360^370^380^390^400 YCK2_YEAST CASEIN KINASE I HOMOLOGUE 2 (EC 2.7.1.-). initn= 202^initl= 98 opt= 274 ^26.2% identity in 214 aa overlap dur. P^  10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKI  YCK2 Y KDEYRTYKILAGTPGIPQEYYFGQEGLHNILVIDLLGPSLEDL-FDWCGRRFSVKTVVQV — 120^130^140^150^160^170 40^50^60^70^80 dur. P ALETIEAIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK-LK ^• YCK2Y AVQMITLIEDLHAHDLIYRDIKPDNFLIGRPGQPDANKVHLIDFGMAKQYRDPKTKQHIP _ 180^190^200^210^220^230 90^100^110^120^130^140 dur. P SRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQ-S .^.^.^••••^•^....^: .^......^.^. YCK2 Y YREKKSLSGTARYMSINTHLGREQSRRDDMEAMGHVFFYFLRGQLPWQGLKAPNNKQKYE 240^250^260^270^280^290 150^160^170^180^190^200 dur. P KLSIRNESRRLF-FNR-TPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRC YCK2 Y KIGEKKRLTNVYDLAQGLPIQFGRYLEIVRNLSFEETPDYEGYRMLLLSVLDDLGETADG 300^310^320^330^340^350 210^220^230^240 dur. P KWDWQVEESQHSELTETASVMSDMAIMAEQGATNYTDRACENQ YCK2 Y QYDWMKLNGGRGWDLSINKKPNLHGYGHPNPPNEKSKRHRSKNHQYSSPDHHHHYNQQQQ — 360^370^380^390^400^410 KDC2_DROME PROTEIN KINASE DC2 (EC 2.7.1.37). initn= 91 initl= 55 opt= 75 ^38.7% identity in 62 aa overlap 10^20^30^40^50 dur. P^MIISILGKDLYRLRAEQPTRSFTLNTTTKIALETIEAIEELHNIGYLSRDV X: .:..^: • ... : . :^. KDC2 D WSTKDDSNLYMIFDYVCGGELFTYLRNAGKFTSQTSNFYAAEIVSALEYLHSLQIVYRDL - 260^270^280^290^300^310 60^70^80^90^100^110 dur. P KPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKKLKSRGEVGWRGTVRYGSLQALKRM KDC2_D KPENLLINR--DGHLKIT---DFGFAKKLRDRTWTLCGTPEYIAPEIIQSKGHNKAVDWW 320^330^340^350^360^370  159  ^  Appendix 3 ^120^130^140^150^160^170 dur. P DLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQSKLSIRNESRRLFFNRTPRQFETI KDC2 D ALGVLIYEMLVGYPPFYDEQPFGIYEKILSGKIEWERHMDPIAKDLIKKLLVNDRTKRLG 380^390^400^410^420^430 KRBl_VACCV 30 KD PROTEIN KINASE HOMOLOGUE (EC 2.7.1. -) initn= 81 initl= 81 opt= 204 ^23.9% identity in 201 aa overlap 10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKI .^ : KRB1 V WKKSHNIKHVGLITCKAFGLYKSINVEYRFLVINRLGADLDAVIRANNNR-LPKRSVMLI 70^80^90^100^110^120 dur. P^  40^50^60^70^80 dur. P ALETIEAIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK--L ^•^..^.. ^. .. : .... : .^.:.^::^•^•^••^•^• KRB1_V GIEILNTIQFMHEQGYSHGDIKASNIVLDQIDK---NKLYLVDYGLVSKFMSNGEHVPFI 130^140^150^160^170^180 90^100^110^120^130^140 dur. P KSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDR - - -TLV • •^:^: : ::X ^X.^•• KRBlV RNPNKMD-NGTLEFTPIDSHKGYVVSRRGDLETLGYCMIRWLGGILPWTKISETKNCALV _ 190^200^210^220^230^240 150^160^170^180^190^200 dur. P GQSKLSIRNESRRLFFNR---TPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI KRBl_V SATKQKYVNNTATLLMTSLQYAPRELLQYITMVNSLTYFEEPNYDEFRHILMQGVYY 250^260^270^280^290^300 210^220^230^240 dur. P PDRCKWDWQVEESQHSELTETASVMSDMAIMAEQGATNYTDRACENQ KRBl_VACCC 30 KD PROTEIN KINASE HOMOLOGUE (EC 2.7.1.-) initn= 81 initl= 81 opt= 203^23.9% identity in 201 aa overlap 10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKI .^ : KRB1 V WKKSHNIKHVGLITCKAFGLYKSINVEYRFLVINRLGVDLDAVIRANNNR-LPKRSVMLI 70^80^90^100^110^120 dur. P^  40^50^60^70^80 dur. P ALETIEAIEELHNIGYLSRDWPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK--L .. : .... : .^::^•^•^••^•^• ^•^..^• KRB1 V GIEILNTIQFMHEQGYSHGDIKASNIVLDQIDK---NKLYLVDYGLVSKFMSNGEHVPFI 130^140^150^160^170^180 90^100^110^120^130^140 dur. P KSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDR - - -TLV ^X•^•^•• • •^:^: : ::X KRB1 V RNPNKMD-NGTLEFTPIDSHKGYVVSRRGDLETLGYCMIRWLGGILPWTKISETKNCALV 190^200^210^220^230^240  160  ^  Appendix 3  ^150^160^170^180^190^200 dur. P GQSKLSIRNESRRLFFNR---TPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI • •^• ^— KRB1_V SATKQKYVNNTATLLMTSLQYAPRELLQYITMVNSLTYFEEPNYDEFRHILMQGVYY 250^260^270^280^290^300 210^220^230^240 dur. P PDRCKWDWQVEESQHSELTETASVMSDMAIMAEQGATNYTDRACENQ IBMP_CAMVD INCLUSION BODY MATRIX PROTEIN (VIROPLASMIN) initn= 79 initl= 51 opt= 54^31.0% identity in 29 aa overlap 40^50^60^70^80^90 dur. P EAIEELHNIGYLSRDWPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKKLKSRGEVG X: ^•^.. •^•^'X IBMP C FRTNCIKNTEKDIFLKIRSTIPVWTIQGLLHKPRQVIEIGVSKKVIPTESKAMESRIQIE - 330^340^350^360^370^380 100^110^120^130^140^150 dur. P WRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQSKLSIRNE IBMP C DLTELAVKTGEQFIQSLLRLNDKKKIFVNMVEHDTLVYSKNIKETDSEDQRAIETFQQRV 390^400^410^420^430^440 KS62 MOUSE RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. initii= 78 initl= 49 opt= 111^29.0% identity in 162 aa overlap dur. P^  10^20 MIISIL-GKDLY-RLRAEQPTRSFTLNTTT  KS62 M TKMERDILVEVNHPFIVKLHYAFQTEGKLYLILDFLRGGDLFTRLSKE---VMFT-EEDV 10^20^30^40^50^60 30^40^50^60^70^80 dur. P KIALETIE-AIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK ...^.. :^.^::.:X.:.^.^:^....^•^.. KS62_M KFYLAELALALDHLHSLGIIYRDLKPENILLD--EEGHIK---LTDFGLSKESIDHEKK70^80^90^100^110 90^100^110^120^130^140 dur. P LKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFY--MLIEMLVGELPWR---HMSDR •• •^•^•^•^ ...^•^.. KS 62_ M ^AYSFCGTVEYMAPEVVNRR--GHTQSADWWSFGVLMFEMLTGTLPFQGKDRKETM 120^130^140^150^160^170 150^160^170^180^190^200 dur. P TLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI KS 62 M TMILKAKLGMPQFLSPEAQSLLRMLFKRNPANRLGAGPDGVEEIKRHSFFSTIDWNKLYR 180^190^200^210^220^230 POLG_STEVM GENOME POLYPROTEIN (CAPSID PROTEIN C; ENVEL initn= 77 initl= 40 opt= 40^46.7% identity in 15 aa overlap  161  ^  Appendix 3  ^70^80^90^100^110^120 dur. P KTIFMFDFGLAKKFIDRDNKKLKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYML X:. :^.:::X POLG S^MSKKPGKPGRNRVVNMLKRGVSRVNPLTGLKRILGSLLDGRGPVRFML 10^20^30^40 130^140^150^160^170^180 dur. P IEMLVGELPWRHMSDRTLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEY POLGS _ AILTFFRFTALQPTEALKRRWRAVDKRTALKHLNGFKRDLGSMLDTINRRPSKKRGGTRS 50^60^70^80^90^100 KS6B_XENLA RIBOSOMAL PROTEIN S6 KINASE II BETA (EC 2.7 initn= 76 initl= 48 opt= 114^29.6% identity in 162 aa overlap dur. P^  10^20 MIISIL-GKDLY-RLRAEQPTRSFTLNTTT  KS 6B X TKMERDILADVHHPFVVRLHYAFQTEGKLYLILDFLRGGDLFTRLSKE---VMFT-EEDV 110^120^130^140^150^160 30^40^50^60^70^80 dur. P KIALETIE-AIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK :...Av ....^.. : . ::.:X.:.^.^:^.... •^.. KS6B__X KFYLAELALGLDHLHSLGIIYRDLKPENILLD--EEGHIK---LTDFGLSKEAIDHEKK170^180^190^200^210 90^100^110^120^130^140 dur. P LKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFY--MLIEMLVGELPWR---HMSDR •^• ^...^•^.. .^..^:::^:^.^.... :^•^ SSKS6B_X ^AYSFCGTVEYMAPEVVNRQ--GHSHGADWWSYGVLMFEMLTGSLPFQGKDRKETM 220^230^240^250^260^270 150^160^170^180^190^200 dur. P TLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI •^• KS6B X TLILKAKLGMPQFLSNEAQSLLRALFKRNATNRLGSGVEGAEELKRHPFFSTIDWNKLYR 280^290^300^310^320^330 KS6A_CHICK RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. initn= 76 initl= 48 opt= 113^29.6% identity in 162 aa overlap dur. P^  10^20 MIISIL-GKDLY-RLRAEQPTRSFTLNTTT  KS6A C TKIERDILADVNHPFVVKLHYAFQTEGKLYLILDFLRGGDLFTRLSKE---VMFT-EEDV 130^140^150^160^170^180 30^40^50^60^70^80 dur. P KIALETIE-AIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK :...Av ....^.. : . ::.:X.:.^.^:^.... •^..^• KS6A_C KFYLAELALGLDHLHSLGIIYRDLKPENILLD--EEGHIK---LTDFGLSKEAIDHEKK190^200^210^220^230  162  ^  Appendix 3  90^100^110^120^130^140 dur. P LKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFY--MLIEMLVGELPWR---HMSDR .^..^:::^:^.^.... :^•^•^•^• • •^•^• KS6AC _ ^AYSFCGTVEYMAPEVVNRQ--GHSHSADWWSYGVLMFEMLTGSLPFQGKDRKETM 240^250^260^270^280 150^160^170^180^190^200 dur. P TLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI KS 6A C TLILKAKLGMPQFLSAEAQSLLRALFKRNPANRLGSGPDGAEEIKRHPFYSTIDWNKLYR -'290^300^310^320^330^340 KS6A_XENLA RIBOSOMAL PROTEIN S6 KINASE II ALPHA (EC 2. initn= 76 initl= 48 opt= 113^29.6% identity in 162 aa overlap dur. P^  10^20 MIISIL-GKDLY-RLRAEQPTRSFTLNTTT  KS6A X TKMERDILADVHHPFVVRLHYAFQTEGKLYLILDFLRGGDLFTRLSKE---VMFT-EEDV - 110^120^130^140^150^160 30^40^50^60^70^80 dur. P KIALETIE-AIEELHNIGYLSRDWPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKK :...^v ....^.. : . ::.:X.:.^.^:^.... •^..^• KS6A__X KFYLAELALGLDHLHSLGIIYRDLKPENILLD--EEGHIK---LTDFGLSKEAIDHEKK170^180^190^200^210 90^100^110^120^130^140 dur. P LKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFY--MLIEMLVGELPWR---HMSDR .^..^:::^:^.^.... :^•^•^•^...^•^.. KS 6A_ X ^AYSFCGTVEYMAPEVVNRQ--GHSHSADWWSYGVLMFEMLTGSLPFQGKDRKETM 220^230^240^250^260^270 150^160^170^180^190^200 dur. P TLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMI •^• KS 6A X TLILKAKLGMPQFLSNEAQSLLRALFKRNPTNRLGSAMEGAEEIKRQPFFSTIDWNKLFR 280^290^300^310^320^330 KPKl_PHAVU PROTEIN KINASE PVPK-1 (EC 2.7.1.-). initn= 74 initl= 74 opt= 85^33.8% identity in 71 aa overlap 10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKIALETIE : X:. ::.^ • KPK1 P LQSLDHPFLPTLYTHFETEIFSCLVMEFCPGGDLHALRQRQPGKYFSEHAVRFYVAEVLL 290^300^310^320^330^340 dur. P^  40^50^60^70^80^90 dur. P AIEELHNIGYLSRDVKPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKKLKSRGEVGW . ::.:X.:^•^•^. : . KPKl_P SLEYLHMLGIIYRDLKPENVLV--REDGH---IMLSDFDLSLRCSVSPTLVKSSNNLQTK 350^360^370^380^390  163  ^  Appendix 3  100^110^120^130^140^150 dur. P RGTVRYGSLQALKRMDLGRADDVECWFYMLIEMLVGELPWRHMSDRTLVGQSKLSIRNES KPK1 P SSGYCVQPSCIEPTCVMQPDCIKPSCFTPRFLSGKSKKDKKSKPKNDMHNQVTPLPELIA 400^410^420^430^440^450 DBF2_YEAST CELL CYCLE PROTEIN DBF2 (EC 2.7.1.-). initn= 72 initl= 62 opt= 75 ^35.7% identity in 56 aa overlap 10^20^30^40^50^60 dur. P GKDLYRLRAEQPTRSFTLNTTTKIALETIEAIEELHNIGYLSRDWPSNFAPGQRDNGQH • X:^••^*X ^ DBF2_Y VPGGDFRTLLINTRCLKSGHARFYISEMFCAVNALHDLGYTHRDLKPENFLIDAKGH--250 260 270 280 290 300 80 70 90 100 110 120 dur. P KTIFMFDFGLAKKFIDRDN-KKLKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYM : ....^: • DBF2 Y --IKLTDFGLAAGTISNERIESMKIRLEKIKDLEFPAFTEKSIEDRRKMYNQLREKEINY 310^320^330^340^350^360 ITI2_HUMAN INTER-ALPHA-TRYPSIN INHIBITOR COMPLEX COMPO initn= 72 initl= 55 opt= 55 ^50.0% identity in 20 aa overlap 80^90^100^110^120^130 dur. P FGLAKKFIDRDNKKLKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGE X.::^: • ^ _ ITI2H IHIFNERPGKDPEKPEASMEVKGQKLIITRGLQKDYRTDLVFGTDVTCWFVHNSGKGFID 870^880^890^900^910^920 140^150^160^170^180^190 dur. P LPWRHMSDRTLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALI ITI2 H GHYKDYFVPQLYSFLKRP 930^940 TRPG PHYBL ANTHRANILATE SYNTHASE COMPONENT II (EC 4.1. init71-= 71^initl= 71 opt= 71^37.0% identity in 27 aa overlap 100^110^120^130^140^150 dur. P GEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQSKLS X: . .::.^••• ^•^*X TRPG_P VAKAREIVDALHKLPTRSSQLPVKSQKSIDWFDVQTEMVEQRVPWRPLVVGVFVNQSIEY 550^560^570^580^590^600 160^170^180^190^200^210 dur. P IRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRCKWDWQ TRPG P MSQVAVEAGLDLIQLHGTESAEIARFLPVPVIKAFHMDASSFHAGQIPYVTQPGNNQLLL 610^620^630^640^650^660 KRAF_XENLA RAF PROTO-ONCOGENE SERINE/THREONINE KINASE initn= 70 initl= 58 opt= 83 ^17.7% identity in 215 aa overlap  164  ^  Appendix 3  dur. P^  10^20^30 MIISILGKDLYRLRAEQPTRSFTLNTTTKIALETIE  KRAF X VLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLY-YHLHVLDTKPQMFQLIDIARQTAQ 390 400 410 420 430 440 40 60 50 70 80 90 dur. P AIEELHNIGYLSRDWPSNFAPGQRDNGQHKTIFMFDFGLAKKFIDRDNKKLKSRGEVGW KRAF X GMDYLHAKNIIHRDMKSNNIF---LHEGLTVKIGDFGLATVKTRWSGSQQVEQLTGSILW 450^460^470^480^490^500 100^110^120^130^140^150 dur. P RGTVRYGSLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVG--QSKLSIRN •• _ KRAFX MAP---EVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHIRDRDQIIFLVGRGGVVP 510^520^530^540^550 160^170^180^190^200^210 dur. P ESRRLFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRCKWDWQVEE .^: .... KRAF X DLSKL-YKNCPKAMKRLVADSIKKLRDERPLFPQILSSIELLQHSLPKINRSALEPSLHR 560^570^580^590^600^610 220^230^240 dur. P SQHSELTETASVMSDMAIMAEQGATNYTDRACENQ • KRAF X AAHTEDISSCALTSTRLPVF 620^630 POLG_JAEVJ GENOME POLYPROTEIN (CAPSID PROTEIN C; ENVEL initn= 69 initl= 40 opt= 47 ^34.5% identity in 29 aa overlap 110^120^130^140^150^160 dur. P SLQALKRMDLGRRDDVECWFYMLIEMLVGELPWRHMSDRTLVGQSKLSIRNESRRLFFNR : . . ..X::^:^::^:X. POLG J ATGLCHVMRGSYLAGGSIAWTLIKNADKPSLKRGRPGGRTLGEQWXEKLNAMSREEFFKY 2500^2510^2520^2530^2540^2550 170^180^190^200^210^220 dur. P TPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRCKWDWQVEESQHSELTET POLG J RREAIIEVDRTEARRARRENNIVGGHPVSRGSAKLRWLVEKGFVSPIGKVIDLGCGRGGW 2560^2570^2580^2590^2600^2610 ILVD_ECOLI DIHYDROXY-ACID DEHYDRATASE (EC 4.2.1.9). initn= 69 initl= 49 opt= 51^17.2% identity in 58 aa overlap 160^170^180^190^200^210 dur. P LFFNRTPRQFETIMDMIDGYSFEIRPEYRHLKALINEIRMENMIPDRCKWDWQVEESQHS ..X:^:.:X ILVD E DVKNVLGLTLPQTLEQYDVMLTQDDAVKNMFRAGPAGIRTTQAFSQDCRWDTLDDDRANG 350^360^370^380^390^400  165  ^  Appendix 3  220^230^240 dur. P ELTETASVMSDMAIMAEQGATNYTDRACENQ •^• _ ILVDE CIRSLEHAYSKDGGLAVL-YGNFAENGCIVKTAGVDDSILKFTGPAKVYESQDDAVEAIL 410^420^430^440^450^460 CPTl_CHICK CYTOCHROME P450 XVIIA1 (P450-C17) (EC 1.14. initn= 69 initl= 46 opt= 49 ^61.5% identity in 13 aa overlap 70^80^90^100^110^120 dur. P HKTIFMFDFGLAKKFIDRDNKKLKSRGEVGWRGTVRYGSLQALKRMDLGRRDDVECWFYM X:^::::X •: CPT1 C GTGRPRSLPALPLVGSLLQLAGHPQLHLRLWRLQGRYGSLYGLWMGSHYVVVVNSYQHAR — 30^40^50^60^70^80 130^140^150^160^170^180 dur. P LIEMLVGELPWRHMSDRTLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFEIRPE CPT1 C EVLLKKGKAFAGRPRTVTTDLLSRGGKDIAFASYGPLWKFQRKLVHAALSMFGEGSVALE 90^100^110^120^130^140 COX1 CAEEL CYTOCHROME C OXIDASE POLYPEPTIDE I (EC 1.9. initil= 69 initl= 48 opt= 48^31.4% identity in 35 aa overlap 130^140^150^160^170^180 dur. P WFYMLIEMLVGELPWRHMSDRTLVGQSKLSIRNESRRLFFNRTPRQFETIMDMIDGYSFE X:...^: . .^. COX1 C FAGTHGFPRKYLDYPDVYSVWNIIASYGSIIRTAGTFLFIYVLLESFFSYRLVIRDYYSN 440^450^460^470^480^490 190^200^210^220^230^240 dur. P IRPEYRHLKALINEIRMENMIPDRCKWDWQVEESQHSELTETASVMSDMAIMAEQGATNY :::X COX1 C RRPEYCMSNYVFGHSYQSEIYFRTTRLKN 500^510^520  166  

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