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Functional dissection of the gene, bli-4, in Caenorhabditis elegans Chiu, Readman Yue-Bok 1997

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FUNCTIONAL DISSECTION OF THE GENE, bli-4, IN CAENORHABDITIS ELEGANS by READMAN YUE-BOK CHIU B.Sc. Simon Fraser University 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics) we accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1997 © Readman Yue-Bok Chiu, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract The bli-4 gene of Caenorhabditis elegans encodes at least four gene products by the mechanism of alternative splicing. Termed blisterases A - D , these isoforms contain unique carboxyl termini and exhibit distinct structural homology to different members of the kexin family of proprotein convertases. Two different kinds of phenotypes have been identified in our collection of bli-4 mutants: blistering at the adult stage of the allele e937, and embryonic (or early larval) lethality of thirteen alleles, suggesting bli-4 plays a functional role not only in the assembly or maintenance of the adult cuticle but also in the early development of the animal. The goal of this thesis is to investigate the functions of the individual blisterases using isoform-specific minigenes in a bli-4 mutant background. Three minigenes were constructed. Together wi th pCeh226 containing the carboxyl terminus for blisterase A , these constructs provide minigenes specific for three of the isoforms, pCeh299 for blisterase B, pCeh308 and pCeh309 for blisterase C. The blistered mutant lacks the 3' exon of blisterase A . As expected, a high copy number of the minigene providing the blisterase A isoform rescued this phenotype. In addition, the blisterase A minigene also rescued the lethal mutants, suggesting either that blisterase A is required early in development or inappropriately provides the function of the other isoforms. A t high copy number, the blisterase B minigene also rescued both the blistering and the lethal phenotypes whereas a modified blisterase C minigene (pCeh309) only rescued the blistering phenotype. A t low copy number, three out of five transgenic lines containing the blisterase B minigene and one putative integrated line containing blisterase C (pCeh308) also rescued blistering. None of the arrays carrying a low copy number of minigenes rescued the lethal mutants tested. The results support the hypothesis that high copy number lines can inappropriately provide the function of other isoforms. This is compatible wi th the suggestion that there is functional redundancy between the isoforms, wi th blisterases A and B probably playing a more important role than blisterase C as indicated by the present results. Biological specificity may result from the developmental, or tissue-specific distribution of the processing enzyme with its substrate. iv Table of Contents Abstract i i List of Tables v i i List of Figures v i i i Acknowledgments x Introduction The genetics of bli-4 1 Molecular structure of bli-4 3 Mutational analysis 9 The kexins 9 Functional specificity of bli-4 isoforms 13 Materials and Methods Nematode growth conditions and strain designations 16 Agarose gel electrophoresis 18 Preparation of D N A for subcloning 18 Restriction digests 19 Ligation and bacterial transformation 20 Plasmid subcloning 21 Polymerase chain reaction (PCR) 22 Cloning of PCR products 23 Preparation of D N A for germline transformation 23 Germline transformation 24 Estimation of minigene copy number 26 Rescue of bli-4 lethal alleles with transgenic arrays 32 Results Section I. Construction of bli-4 minigene constructs 35 Section II. Generation of transgenic strains with high and low copy 39 copy number of minigenes V Section III. Estimation of copy number of minigene 49 Section IV Rescue of blistering using bli-4 minigenes Measure of rescue 56 A . pCeh226, the blisterase A minigene/ rescued blistering at 59 high, not low, copy number B. pCeh299, the blisterase B minigene, rescued blistering at 60 high copy number; a low copy number of pCeh299 also rescued blistering in particular lines C. pCeh308, the blisterase C short minigene, did not rescue 62 blistering at high copy number D. A putative integrated line containing a low copy number 62 of the C short minigene demonstrated complete rescue of blistering E. pCeh309, the blisterase C long minigene, rescued 63 blistering at high copy number Section V Rescue of lethal alleles using bli-4 minigenes Measure of rescue 63 A . pCeh226, the blisterase A minigene, rescued bli-4 lethal 65 alleles at high copy number B. pCeh299, the blisterase B minigene rescued bli-4 lethal 68 alleles at high copy number C. pCeh308 and pCeh309, the blisterase C minigenes, failed 68 to rescue bli-4 lethal alleles Discussion H i g h copy number of exogenous blisterases A and B rescued 69 blistering pCeh299 and pCeh238 demonstrated different rescue results 70 The unc-54 3' U T R improved rescuing ability of pCeh308 71 Extremely low copy number of blisterase A did not rescue 73 blistering L o w copy number of blisterase B rescued blistering 74 Integration improved rescuing ability of pCeh308 75 rol-6, the transformation marker, is a potential bli-4 substrate 76 Blisterases A , B rescued the lethal alleles h791 and s90 77 vi Low copy number of blisterases A and B did not rescue the 79 lethal phenotype Blisterase D is likely to play a functional role in early 79 development BLI-4 is a candidate cuticle-collagen processing enzyme 80 Functional redundancy of bli-4 isoforms 83 Other information or experimental approaches that may 84 contribute to our understanding of the individual function of bli-4 isoforms Conclusion 86 References 88 Appendix I 95 Appendix II 98 Appendix I 100 Appendix II 101 vu List of Tables Table 1. Abbreviations used in this thesis 17 Table 2 Transgenic strains constructed in this study 50 Table 3. Estimation of minigene copy number 55 Table 4. Rescue of blistering by bli-4 minigenes at high copy 57 number Table 5. Rescue of blistering by pCeh226 (A minigene) at low 58 copy number Table 6. Rescue of blistering by pCeh299 (B minigene) at low 58 copy number Table 7. Rescue of blistering by pCeh308 (C short minigene) at 58 low copy number Table 8. Rescue of lethal alleles by bli-4 minigenes 66 List o f Figures Figure 1. Mutant phenotypes of bli-4 2 Figure 2. Intracomplementation of bli-4 alleles 4 Figure 3. The molecular structure of bli-4 6 Figure 4. Structural comparison of BLI-4 with kexins 7 Figure 5. Location and molecular identities of bli-4 mutations 10 Figure 6. Microinjection of D N A for germline transformation 25 Figure 7. Location and sequence of primers used to verify the 27 presence of minigene constructs in transgenic strains Figure 8. Use of P C R to verify the presence of minigene constructs 29 in transgenic strains Figure 9. Location and sequence of primers used in the estimation 31 of copy number of minigenes in transgenic strains Figure 10. Strategy to determine transgenic rescue of bli-4 lethal 33 alleles Figure 11. Parent plasmids used to construct bli-4 minigenes 36 Figure 12. Structure of pCeh226 37 Figure 13. The putative carboxyl terminus of the variant blisterase C 38 transcript Figure 14. Construction of pCeh299, the B minigene 40 Figure 15. Construction of pCeh301 42 Figure 16. Construction of pCeh308, the C short minigene 44 Figure 17. Construction of pCeh.309, the C long minigene 46 Figure 18. Comparison of the genomic sequences of the 3' end of 48 blisterase C with pCeh308 (C short), and pCeh309 (C long) Figure 19. Estimation of minigene copy number by PCR/Southern 53 analysis Figure 20. Structural comparison of pCeh299 with pCeh238 61 Figure 21. Location and molecular lesion of bli-4(h791) 64 Figure 22. Comparison of minigene copy number in different 72 transgenic lines Figure 23. Summary of transgenic rescue results by bli-4 minigenes 81 Acknowledgments I would like to thank my research supervisor, Dr. Ann Rose, for giving me the opportunity to work on the bli-4 project, and for her support and guidance throughout the course of this work. I would like to especially thank Dr. Colin Thacker for his invaluable help, care, and support in both good and bad times. I thank my supervisory committee, Dr. Fred Dill and Dr. Carolyn Brown, for their constructive advice and support. Thanks to all the past and present members of the Rose lab for making it a nice environment to work in. Finally, thanks to my parents, God, and Ada for her love and support for my pursuit of this degree. 1 Introduction The genetics of bli-4 The bli-4 gene of C. elegans was originally identified as a recessive mutation, e937, that results in fluid-filled separations, or blisters, of the adult cuticle layers of the nematode (Brenner 1974; Figure 1). The phenotype is incompletely penetrant, as 10-20% of a population of isogenic e937 homozygotes do not develop blisters. This feature of reduced penetrance is heritable, as a "wild-type-looking" and a blistered e937 sibling both continue to produce progeny of similar blistering population frequency in the next generation. In addition, e937 homozygotes display variable expressivity of the blistering phenotype. Blisters may be restricted to one location or cover the entire length of the animal. Subsequent to the identification of e937, thirteen additional recessive mutations were mapped to the bli-4 locus on chromosome I by complementation testing (Howell et al, 1987; Peters et al., 1991., Thacker, Srayko and Rose, unpublished data). These alleles, however, result in either embryonic or early larval lethality, suggesting a functional role for bli-4 in early development in addition to adult morphology. Based on a complex heteroallelic complementation pattern, the current collection of bli-4 alleles are grouped into three classes (Peters et al., 1991; Figure 2). Class I is Figure 1. Mutant phenotypes of bli-4 Nomarski photographs (courtesy of C. Thacker) of Class I e937 homozygote showing the blistered phenotype at adulthood Class II q508 homozygote arresting development in late embryogenesis Class III s90 homozygote arresting development in early L l larval stage. 70% of s90 homozygotes appear as Class II homozygotes. 3 represented by the only viable allele, e937, as described above. Class II includes a group of twelve embryonic lethal alleles that fail to complement e937. Heteroallelic combination of most lethal class II alleles with e937 result in viable, blistered animals with, interestingly enough, close to complete penetrance of blistering. The implication of this observation is not further discussed in this thesis. Class III contains a single allele, s90, which complements e937 but fails to complement all the class II alleles. By genetic criteria, s90 is therefore classified as a member of bli-4 mutants. The majority of s90 homozygotes are embryonic lethals similar to the class II alleles yet about 30% delay arrest to the early Ll larval stage. The complicated complementation pattern indicates that bli-4 is a complex locus, while the two main kinds of phenotype (adult blistering and embryonic lethality) displayed by bli-4 alleles implies a multi-functional role of bli-4 gene product(s) at different development stages. Molecular structure of bli-4 The molecular structure of bli-4 was characterized by the efforts of Peters et al. (1992), Srayko (1995), and Thacker et al. (1995). bli-4 is composed of 21 exons stretched over a region of over 20 kb of genomic sequence (Figure 3). Transpliced to the leader sequence SL1, four gene products arise by alternative splicing of the first 12 exons to specific exons on the 3' end. 4 Class I Class II Class III Class I Blistered (-80%) Class II Blistered (-100%) ® Embryonic Arrest Class III Wild-type Embryonic Arrest ® / ^ Embryonic Arrest (-70%)/ L I Arrest (-30%) Figure 2. Intracomplementation of bli-4 alleles. The percentage within parenthesis represents the approximate population frequency of the phenotype. Class I: e937 Class II: h42, hl99, h254, h384, h.427, h520, h670 h791, hl010,hl403, q508, hl549 Class III: s90 5 Database searches revealed that BLI-4 shows homology to a group of serine endoproteases, the kex-2/subtilisin family of proprotein convertases (or kexins, to be discussed later). A protease domain based on sequence similarity is recognizable in bli-4 within exons 5-9, with the conservation of the important catalytic amino acids - Asp-202, His-241, Asn-342, and Ser-415 (Figure 4). In addition, the common region of the four isoforms, termed blisterases A-D, shares the same structural organization common to all members of this particular family of enzymes (Figure 4). The 3' exons of individual bli-4 isoforms encode unique carboxyl termini (C-termini) featured by the presence/absence of special structural domain(s) that are recognizable in their kexin homologues (Figure 4). Both blisterases C and D contain a cysteine-rich region (CRR) also found in, for example, furin, PACE4, and PC5/PC6. In addition, blisterase D contains a transmembrane domain (TMD) that is shared by both Kex2p and furin. Neither of these domains is present in blisterase A or B. The production of multiple gene products from bli-4 by alternative splicing is consistent with the association of multiple classes of mutant alleles and phenotypes with this locus. *3 ro Cn i-t-o ' o t> fD N <^ 3 CO c y a 3 cn a ffl tn O 3 5 ta a 3 r-t- h-i-t O 1-U ^ tr cn ft) O CL cr o X ft) co 3 I t ft) cn ft) 3 fD X o t> cn cn ft) 3 tr; s» n ti P) r-f-ft) CL ft) o tr cn _ . ro cn ,_Q O £ fD ts n fD cn O i t 3 ro o tr?TJ fD 3 cr < o x ft- fD tr cn CL i t t i : fD tS^ d fD t t fD fD cn •f t* Q) fD fD y t 3 5^ cn cn n " O 3 3 o tl 09 i t fD CO tr fD 3 o n ST t fD O cd )—' cd i—» Cd 2 cn cn cn terase terase terase terase a n cd > - f f l 5 9 7 Figure 4. A schematic comparison of the structures of bli-4 gene products and other kexin convertases. The vertical bar that cuts through the bli-4 isoforms represents the site of alternative splicing. O n the amino side, BLI-4 possesses all the structural features shared by the kexins: secretion signal peptides are shown stippled; potential autocatalytic cleavage sites are shown as vertical bars on the amino side of the protease domain; protease domains are shown as shaded with the positions of the catalytically important amino acids Asp , His , Asn , and Ser indicated by D, H , N , and S. The numbers within the protease domains represent the percentages of identical residues when compared wi th BLI-4. Structural divergence on the carboxyl termini of bli-4 isoforms displays homology to different kexin homologues: the cysteine-rich regions (CRR) are depicted as hatched boxes; transmembrane domains (TMD) are shown as dark boxes. Ovals indicate potential N-l inked glycosylations sites. (Figure modified from Thacker et al., 1995) 8 C. elegans Blisterase A £5 Blisterase B [ Blisterase C £ Blisterase D [~ C E L F U R I N CELPC2 D H N S I i D H I I T N S i y i n H \) s T i i T , i 670 730 1ZK 821 639 652 Vertebrates PC4 [;'| | | ^ V ' 62%-- j ^ | 628 PCI/3 [;| H I | - T ,57% - , \ " | 753 PC2 || | | 54% I I 638 Furin \4 III :- ••: .72% ' T j ? Vy^l ® ~ l 794 PACE4 | j | | 62% ' | Vy///Z&3&Zl 969 PC5/PC6 Ki I I J 60% T j Y/////7//J//71 915 PC7 | - | | T | T , | T 1 | 783 Drosophila Dfurinl j r T T | ' 1 j | | | T | , 62% — | T T T T f l I 1 1 0 1 Dfurin2 I II T | | : V 60% I " Bl680 Yeast 9 Mutational Analysis Molecular lesions corresponding to the majority of bli-4 mutants have been mapped and characterized using restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-based heteroduplex analysis (Srayko, 1995; Thacker et al., 1995, 1996). The original blistering allele, e937, results from a 3.5 kb deletion that removes the 3' exon unique to blisterase A (Figure 5). Northern and reverse-transcription-PCR (RT-PCR) analysis confirmed that the blistering phenotype of e937 results from the specific elimination of the expression of blisterase A (Thacker et al., 1995). Eleven of the class II lethal mutations were found to reside in the common region of the gene and presumably abolish the expression of all isoforms (Figure 5; Thacker and Rose, in preparation). Thus, they represent potential null alleles of bli-4. The molecular lesion associated with the class III mutation, s90, has yet to be identified. The kexins A large number of biologically important molecules are synthesized as inactive precursors and subsequently activated by proteolytic processing. In humans, this includes a host of hormones, neuropeptides, and receptor molecules (Steiner et al., 1993; Dubois et al., 1995; Nachtigal et al., 1996; 10 A / / / / rcf-/ / / / / / / / / § T •8- 1 o \ J: J; \ \ t-H pi uS uS \ \ O I X o CM N t S I D O * -c J: ' jH \ \ a; M u ra X H U X -d cu V '> o a c o n B O cn c o CCS 3 CU cu V u cj cu T3 £ rd O • r H H-» rcS CJ O - J LO CU r H bp -a cu CCS X CCS S o cu CC cu H-» O i-H O , cu X H-H cn cu r H cu cn cu CJ £ cu cr cu cn cu H-» r^cS I n £ ccS J-H H-» £ £ cu cn cu i-t a cu cn cu X o CU CJ •4—1 rci £ cu cn cu r H a, cu M cn cu "aJ VH cu r £ -4—' o £ o • r H H - J rci cu cn cu cn ccS CT) rS d o 'X S-H cu cn •S S H g ra o cn ^ O cn r-H rcS O CU cn £ a .2 -5 (U _ g "3 £ cu o « res CO P ^ cn S C rci cu LO O Cr- £ s « CQ CO o fxT r-O ctS CO rCl X cn cu N £ cu £ O • r H H-» CJ ' r H cn cu 11 Willnow et al., 1996). Viral coat proteins and bacterial toxins are also physiologically relevant molecules requiring this mode of post-translational modification (Decroly et al., 1996; Gu et al, 1996). The enzymes enforcing this significant process had been long sought after and their identities were recently unveiled. The kex2/subtilisin family of proprotein convertases, or kexins, are serine endoproteases featured by their proteolytic action at dibasic amino acid sites, usually Arg-Arg-i or Lys-Argi (for reviews, see Barr, 1991; Seidah and Chretien, 1992). The original prototype of kexins is the gene product of the yeast gene kex-2, a Ca++-dependent subtilisin-like protease which is responsible for the maturation of the pro-a-mating factor and the pro-killer toxin in the organism (Fuller et al., 1989a). Based on sequence homology to kex-2, seven members of mammalian kexins have been identified to date. They include PC1/PC3 (Seidah et al,. 1991; Smeekens et al, 1991), PC2 (Seidah et al., 1990; Smeekens and Steiner, 1990), PC4 (Nakayama et al., 1992; Seidah et al., 1992), PC5/PC6 (Lusson et al., 1993; Nakagawa et al., 1993) , PACE4 (Keifer et al., 1991), furin (Roebroek et al., 1986; Fuller et al, 1989b; van den Ouweland et al, 1990; Wise et al, 1990), and PC7 (Seidah et al, 1996). In addition, homologues of mammalian kexins are found in Drosophila (Roebroek et al., 1991,1992), Xenopus (Braks et al, 1992) and Hydra (Chan et al., 1992). In C. elegans, homologues of PC2 (Gomez-Saladin et al, 1994) , and furin (Thacker and Rose, unpublished finding) have been reported. bli-4, as mentioned in the previous section, encodes products homologous to various members of the kexin family (Figure 4). 12 The different members of kexins have been shown to function in either the constitutive or regulated secretory pathway of the cell, where different modes of post-translational modifications take place before molecules assume their mature structures. On the level of tissue distribution, different expression patterns are exhibited by the seven mammalian kexins, which accordingly have been generalized into two categories. One category exhibits expression restricted to the endocrine and neuroendocrine tissues and therefore assumes functional roles within the regulated secretory pathway. Members of this category include PC1/PC3 and PC2 (Seidah et al,. 1990, 1991; Smeekens and Steiner, 1990; Smeekens et al, 1991.) The other category, including furin and PACE4, displays ubiquitous expression patterns and participates within the constitutive secretory pathway (Roebroek et al., 1986; van den Ouweland et al., 1990; Kiefer et al, 1991). Members of this category also demonstrate a more elaborate cleavage specificity than the usual dibasic motif. They process substrates at a tetra-basic site: Arg-X-Arg/Lys-Argi (where X stands for any amino acid). PC5/6 and the recently identified PC7 also display widespread tissue distribution although the level of expression is predominant in intestinal tissue (PC5/6; Lusson et al, 1993), or in colon and lymphoid-associated tissues (PC7; Seidah et al, 1996). Therefore PC5/6 and PC7 may function in both types of secretory pathway. The functional roles of individual kexins appear to be a function of intracellular compartmentalization, which in turn depends on the structural 13 properties conferred, in particular, by the divergent C-termini. For example, the TMD on the carboxyl tail of furin is responsible for anchorage of the enzyme within the rrans-Golgi network (TGN). Further studies identified additional trafficking signal sequences adjacent to the TMD that direct cycling of furin between the cell surface and the TGN, thus establishing a dynamic multi-compartment distribution equilibrium for the enzyme (Molloy et al., 1994; Schafer et al., 1995; Jones et al., 1995). This characterization fits with the wide spectrum of substrates found to be processed by furin (Pei and Weiss, 1995). On the other hand, PCI/3 and PC2 were found sequestered to and functioned within secretory vesicles. Studies showed that the C-termini are determinants of the specific routing and storage pattern exhibited by these enzymes (Zhou et al., 1995; Creemers et al., 1996). Putative amphipathic domains were identified on the carboxyl tails of PCI/3 and PC2 which may facilitate such localization (Smeekens et al., 1991). Functional specificity of bli-4 isoforms The four gene products of bli-4 display structural homology to members of kexin family belonging to both the constitutive and regulated secretory pathways. Blisterases A and B are similar to PCI/3 and PC2 while blisterase C and D are similar to PC5/6, PACE4, and furin. Whether corresponding functional difference exists among the bli-4 isoforms is an intriguing question, bli-4 is not the only kexin gene that encodes multiple 14 isoforms through alternative splicing. Three mammalian kexins, PACE4, PC4, and PC5, also produce isoforms with divergent C-termini from a single gene (Tsuji et al., 1994; Seidah et al, 1992; Nakagawa et al., 1993b). Recent work by De Bie et al. (1997) demonstrated for the first time that the PC5-A and PC5-B isoforms, which differ in the length of the CRR and the presence of TMD (in PC5-B), exhibit different sub-cellular localization patterns. The dfur-1 gene of Drosophila also encodes multiple furin homologues with unique C-termini. The individual dfurin isoforms demonstrate different spatial and temporal expression patterns, suggesting they may play different physiological roles in the organism (De Bie et al., 1995). bli-4 is the only kexin member identified thus far in a multicellular organism for which mutants have been generated that facilitate functional characterization through genetic analysis. The different kinds of mutant phenotypes directly indicate that bli-4 plays distinct functional roles at different stages during development. The multitude of gene products further raises the possibility that functional specificity exists among individual bli-4 isoforms. The blistering phenotype of the viable e937 allele as a result of the specific elimination of blisterase A activity is suggestive of this possibility and provokes further investigation. To explore the hypothesis of functional specificity, one strategy we have adopted is to construct individual blisterase subclones to rescue different bli-4 mutants with the aim to correlate individual isoform function with specific mutant phenotypes. Preliminary data from the study of Srayko (1995) 15 using this approach indicated potential functional redundancy among the different isoforms. The present work extended this line of study by using individual minigenes specific for single blisterase isoform to rescue mutants representing the three classes of bli-4 alleles. Various studies reported the loss of substrate specificity when kexin convertases were overexpressed in vitro (Walker et al, 1994). With this complication in mind, two different concentrations of the minigenes were injected in order to generate transgenic strains containing different copy numbers of the minigenes. A comparison between the rescuing abilities and patterns exhibited by individual minigenes may then provide us with information on the functional specificity of individual bli-4 isoforms. 16 Materials and Methods Nematode growth conditions and strain designations All the C. elegans strains used in this study were maintained on petri dishes containing nematode growth medium (NGM) streaked with the Escherichia coli (E. coli) strain, OP50, at 20°C. The wild type strain, N2, is originated from Bristol, England (Brenner, 1974). Nomenclature of strains follows the formulations by Horvitz et al. (1979). Briefly, gene names consist of a three-letter code that usually describes the mutant phenotype or the function of the gene product in combination with a number for identification among other genes that display similar mutant phenotypes or encode similar products. Specific alleles are placed in parenthesis following the gene name. For example, bli-4(e937) identifies the original blistering allele of the bli-4 gene generated by Brenner in 1974. Transgenic animals are designated by the genotype of the parent strain followed by a hEx number which specifies the transgenic extrachromosomal array. For example, bli-4(e937); hExl04 represents an bli-4(e937) homozygote containing an extrachromosomal array which, in this example, is made up of the bli-4 A minigene and the transformation marker pRF4. The generation of transgenic animals is further explained below. Abbreviations used in this report are explained in Table 1. 17 Table 1. Abbreviations used in the thesis Abbreviat ion Description bli mutations in these genes give rise to a blistered phenotype Bli the blistered phenotype, fluid-filled separations of cuticular tissue BLI the gene product of bli dpy mutations in these genes result in a dumpy phenotype Dpy the Dumpy phenotype: short, fat body morphology unc mutations in these genes result in uncoordinated movement Unc the Uncoordinated phenotype: an impairment or abolishment of locomotion or sensation rol mutations in these genes result in a rolling phenotype Rol the Roller phenotype: a helical twisting of the body around the longitudinal axis, resulting in rolling motion as the worm moves forward h the Rose laboratory allele designation. A l l alleles, extra-chromosomal arrays, chromosomal rearrangements and D N A constructs (i.e. genotypic alternations) designed in this laboratory are issued an h number KR the Rose laboratory strain designation. A l l C. elegans strains isolated in the Rose laboratory are issued a KR number KRp all oligonucleotides, or primers, designed in the Rose laboratory for the purpose of Polymerase Chain Reaction are issued a KRp number pCeh D N A constructs are identified as plasmids subcloned from Caenorhabditis elegans in the Rose (h) laboratory hEx The exogenous D N A construct present within a nematode strain transformed via microinjection in the Rose (h) laboratory 18 Agarose gel electrophoresis Agarose gel electrophoresis was used for size-separation of DNA fragments. Agarose gel was prepared by dissolving agarose (Gibco BRL) in lx TBE (Tris-Borate, EDTA) buffer at the desired percentage (g/ml, usually between 0.6% and 1.5% in this study) for optimal resolution. For DNA extraction, low-melting point agarose was used with lx TAE (Tris-Acetate, EDTA) buffer. Electrophoresis was performed under 70-120 V. Ethidium bromide was added to the dissolved agarose at 0.5 ug/ml for visualization of DNA fragments under UV at 300 nm. Preparation of DNA for subcloning Plasmid DNA was prepared following the standard alkali-lysis method (Sambrook et al., 1989). Digested DNA fragment was isolated by excising the desired band from agarose gel and purified using the Qiaex Gel Extraction Kit from Qiagen. When electrophoresis was not necessary, DNA in restriction digests was directly purified by phenol /chloroform extraction. The volume of the reaction mix was increased to 200 ul and extracted with an equal volume 19 of phenol, followed by another extraction of the aqueous layer with an equal volume of a chloroform/isoamly alcohol (24:1) mix. The final DNA solution was usually concentrated by adding 1/10 volume of 3M sodium acetate pH 5.2, 3 ul of 200 ug/ml dextran, and 2 volumes of 95% ethanol and then centrifugation at 13200 rpm. The resulting DNA pellet was washed with 70% ethanol and dissolved in TE (Tris-EDTA pH 8.0). Restriction digests A typical restriction digest contained 5 units of restriction enzyme, lx reaction buffer (supplied), 1 |ig/ml of bovine serum albumin (BSA) and -100 Ug of plasmid DNA. The suppliers of restriction enzymes include New England Biolabs (NEB), Pharmacia, and Gibco BRL. Restriction digests were performed at 37°C for 1.5 hours unless specified otherwise by the manufacturer. Partial digestion was achieved using 0.5 unit of restriction enzyme for a brief incubation of 5 minutes. The enzyme was quickly inactivated by heating at temperature suggested by suppliers followed by phenol/chloroform extraction. For blunt-end ligation, 5' overhangs created by restriction digests were filled in by Klenow fragment (1 unit, Pharmacia, 30 minutes at room temperature) whereas 3' overhangs were removed by T4 DNA polymerase 20 (1 unit, NEB, same conditions) in the presence of 0.5 mM deoxyribonucleotides (dNTPs). Ligation and bacterial transformation DNA ligation was performed by incubating a mix of vector and insert DNA, lx ligation buffer, and 200 units of T4 DNA ligase supplied by NEB or Gibco BRL at 16°C overnight. When the vector DNA contained cohesive termini, it was first phosphatased to prevent self-ligation by 1 unit of calf intestinal alkaline phosphatase (CIAP, Gibco BRL) at 37°C for 1 hour. The enzyme was denatured by heat inactivation at 65°C for 10 minutes and completely removed by phenol-chloroform extraction. Plasmids were transformed into competent bacterial cells by the following procedure: 3 (out of 10) |il of the ligation mix was added to 50 ul of competent cells in a 15 ml falcon tube and the mix was incubated on ice for 30 minutes. The mix was then heat-shocked at 37°C for 45 seconds. After a 2-minute incubation on ice, 0.5 ml of SOC medium was added and the culture was inoculated at 37°C under shaking at 250 rpm for 1 hour. 50-200 ml of the inoculate was plated on an LB plate coated with ampicillin for selection of transformants. Most transformations in this study utilized the competent E. coli strain DH5oc from Gibco BRL. Certain restriction enzymes (e.g. Bel I) 21 process non-methylated DNA. In this case, the E. coli strain GM48 was used because it was deficient for the dam methylase. Plasmid subcloning All the constructs manufactured in this study were based on the vector plasmid pBlueScript SK (pBSSK) unless otherwise stated. This vector contains an ampicillin-resistance (ampR) gene that allows for positive selection of successful transformants. Furthermore, a blue/white color selection is supported. Insertion of a DNA fragment in the multiple cloning site of the vector disrupts the formation of (3-galactosidase and therefore produces a blue color in the presence of isopropyl-fj-D-thio-galactopyranoside (IPTG) and 5-bromo-4-chloro-3-indoyl-(3-D-galactopyranoside (X-gal). This enables easy identification of bacterial colonies containing recombinant plasmids. In most cases, however, appropriate restriction digests were required to discern the identities of engineered constructs because multiple cloning steps were involved. 22 Polymerase chain reaction (PCR) Template DNA for PCR in this study was either bacterial plasmid DNA or C. elegans genomic DNA from a single animal. 1 ng of plasmid DNA was normally used. To prepare template DNA from an animal, a single young adult nematode was transferred to a 5 ul mix of lx Taq buffer (Promega), 1.5 mM MgCl2, and 60 ug/ml of Proteinase K in a 0.65 ml microfuge tube and frozen at -70°C for 15 minutes. 40 ul of mineral oil was layered on top of the reaction mix before the lysis reaction was carried out at 57 °C for 60 minutes, followed by inactivation at 95°C for 15 minutes in a Perkin-Elmer/Cetus PCR thermocycler. PCR was then carried out in the same tube by adding appropriate amounts of required components. The final reaction mix contained 1 unit of Taq polymerase or Pfu polymerase (Stratagene), lx Taq buffer (Promega), MgCl2 (1.5 - 2.5 mM), primers (200 ng), and dNTPs (200 uM). Amplification was achieved by 30 cycles of denaturation (94°C, 45 seconds), annealing (54°C, depending on primers, 30 seconds), and extension (72°C, 1 minute, or 1.5 minutes when two sets of primers were used), followed by extension at 72 °C for 7 minutes. 23 Cloning of PCR products Blunt-end ligation was necessary to insert gel-purified PCR products into the Smal site of pBSSK. The vector was not phosphatased because PCR products did not possess 5'-phosphates. When Taq polymerase was used for PCR, the purified product was further polished by T4 DNA polymerase as described before ligation was performed. Pfu polymerase did not require the treatment. The blue/white selection scheme described above allowed easy identification of bacterial colonies containing ligation products. Preparation of DNA for germline transformation The various constructs used for injection were prepared using the Qiagen Miniprep Kit. 1 ul of purified DNA was loaded on an agarose gel alongside with known concentrations of standards to determine its approximate concentration. The minigenes in this study (except pCeh309) were injected at two concentrations to determine the effect of copy numbers on their rescuing abilities. A high-concentration injection mix contained about 50 ug/ml of the minigene construct and the same concentration of the transformation marker, pRF4. A low-concentration mix contained about 1 Ug/ml of the minigene construct, 50 ug/ml of pRF4, and the same 24 concentration of pBSSK to maintain the final concentration at approximately 100 Ug/ml. Germline transformation DNA was introduced into the nematode by injection into the distal gonadal arm in adult hermaphrodites based on the method of Mello et al.. (1991; Figure 6). The components of the injection mix were described above. The transformation marker pRF4, which contained the rol-6 (sul006) gene, was used to confer a dominant rolling phenotype (Rol) to transgenic animals. Once injected, the minigenes and the transformation marker molecules undergo recombination at homologous sequences (e.g. the ampR gene on pBSSK) to generate concatemeric hybrid molecules in the form of an extrachromosomal array. Arrays are assembled independently and incorporated into the oocytes. Resulting Fl transgenics may therefore contain arrays with different structures. The extrachromosomal array behaves similar to a free duplication such that it is transmitted in a non-Mendelian fashion during cell divisions. Transgenic animals therefore exhibit mosacism of the array. Transgenic Fls are singled and usually 10% of them lead to stable lines. Very rarely, the extrachromosomal array may be randomly integrated into the genome by this technique. The underlying mechanism of such integration event is not well understood. Figure 6. Microjection of plasmid D N A for germline transformation. D N A prepared for germline transformation was injected into the distal gonadal arm of young adult hermaphrodites (Mello, et a l , 1991) 26 Putative transgenic lines were confirmed by PCR to detect the presence of the minigene. For pCeh299, pCeh308 and pCeh309, specific bli-4 3' primers in combination with vector primers established the presence of the minigene (Figure 7, 8). For pCeh226, the A minigene, a product produced by primers specific to the blisterase A exon, which is deleted in e937 mutants (the parent strain used for injection), indicated the presence of the minigene. Estimation of minigene copy number An individual transgenic animal was lysed by the Proteinase K treatment as described. The resulting DNA was subjected to PCR amplification using the primers KRp44 and KRp51 to amplify a 886 bp fragment of the common region of bli-4, and the primers KRpl41 and KRpl42 to amplify a 380 bp fragment on the putative rec-1 gene as a control (Figure 9). The amount of the various components for the PCR are the same as above. 12 cycles of amplification were performed to maintain a linear relationship between the quantity of input template and product. Steps for a single cycle included: denaturation (94°C, 45 seconds), annealing (56°C, 30 seconds), and extension (72°C, 1.5 minutes), followed by a final extension at 72°C for 7 minutes. The PCR products were run on a 1.6% agarose gel and transferred by capillary action to a Zeta-Probe membrane (Bio Rad) while being denatured by O >4H 13 a; H-< CO cu -4-» cu cu 4^  cu bo cn o a> u C cu cn cu » H cu cu X -4-» g ) H JC! o cj o -4-» U 13 cu cn cn >H cu P H CU i H §> P4 U P H CU 13 • t - H > O )H CU •3 < cn cu C cu bO cn rt f H rt .a -a bO rt •4-< O ca cS u 'S cu bo cn C rt O cn cu O c cu CU o cu cj cn cu X cj CO T - H d o x cu cu <J cu cn cu >H CU cu ES cu > o 4 1 rt 13 cu +J cn cn JH cu CU cn "rt CU cj cj cu CU cn cu X H-> >> cu ^ Cu ^ g r C l CCS CO -+-» <j -a o IH cu cu -*-» CM CU U CU cu u cu CO cu *H CU cu X cn cu rt cj 1 3 CO »H cu VH CU cu cn i—i O >H c o cj cn O bO O g o CO CO f O Cu P4 1 3 rt CM CO Cu P H 1 3 cu H-> cj cu cu 1 3 CO rt < cu CO rt >H cu O cj cu CU CO cu IH rt O *-4-» CJ cu J H . O <-lH 13 cu cn 3 cu >H cu CO I H < U u < < u < £ u N ( J H i CM rH cu c< 13 C rt CO u O < u u o 11 c rt cu X •4-» CU cn rt cj cu 4 3 CU c cu bo '2 g U H U U H u < H i LO t - H r - H CU P< CU > • T—< -4-» cd CU cu bO .S bO JO ra3 Cu 4> o CO +-> o 41 rt cu g bO rt VH ra cu -*-» rt IH CU C cu bO g O p / <J U cn I—I CU > ^ CU u •s g 13 ^ U 1 3 •-—-3 cj cu C <u bO 28 to CJ O f-i P-. PH rO L O ON I O P H rO P H rQ V O C O P H x f CO cu u C <u 3 cr <u to < u CJ <: u u CJ u < u I LO C O u CJ CJ i—1 < u u U < i L O 2 cu g •c 9 P tx O N P H C O fx O N P H CN C O P H C O C O P H 01 cl cu (50 C H O N O N w is u P H C P PH C O H OO 1H O O C O X X cn cu IS S>1 o X —H 0> P H C N C O C H w-1 CO CO D H X. o> u p cu cr CD CJ 01 > P H 29 1 2 3 4 5 6 7 8 9 10 Figure 8. Use of P C R to verify the presence of minigene constructs in transgenic strains Lanes 1-9 show amplification products obtained using template D N A from KR3228 (1), KR3231 (2), KR3235 (3), KR3239 (4), KR3233 (5), KR3245 (6), KR3246 (7), KR3241 (8), KR3252 (9). Products obtained using primers specific for amplification of pCeh226 (A minigene) are shown in lanes 1 and 2, pCeh299 (B minigene) in lanes 3-5, pCeh308 (C short minigene) in lanes 6-8, and pCeh309 (C long minigene) in lane 9 (see Figure 7 for description of primers used). The 380 bp product seen in all lanes is the result of amplification using primers of the putative rec-1 gene, included as a positive control. Lane 10 contains a 100 bp D N A ladder (Gibco-BRL). 30 0.4 M NaOH overnight. The DNA was fixed on the membrane by baking in an oven at 65°C for 1 hour (Sambrook et al., 1989). PCR products from 30 cycles of amplification were purified using the Qiaex Gel Extraction Kit and used as probes in hybridization. Probes were prepared using the Random Primed Labeling Kit from Boehringer Mannheim. Approximately 30 ng of each PCR product were labeled with -30 on uCi [or P] dCTP and used for hybridization. The prepared membrane was incubated with a pre-hybridization solution, which contained 2x SSPE, 1% SDS, 0.5% dry milk, 100 ug single sperm salmon (SSS) DNA, at 65 °C for 1 hour to minimize unspecific binding. The probes were denatured at 95 °C for 5 minutes before hybridization was carried out at 65°C in a plastic bag containing 5 ml of the pre-hybridization solution, the probes, and the membrane with gentle shaking overnight. At the completion of hybridization, the membrane was washed successively in 2 x 15 minute -intervals by the following solutions: 2x SSC, 0.1% SDS -> 0.5x SSC, 0.1% SDS -» O.lx SSC, 0.1% SDS at 65 °C. Autoradiography was performed after washing. The autoradiograph was scanned and the intensities of individual bands were quantified using the ImageQuant 3.3 software from Molecular Dynamics. The average intensity of the bli-4 bands of three transgenic animals for each line was compared to that of three N2 animals in the same 31 KRp44 Xhol PstI 1 KRp51 KRp44 5 ' - T G T T G A A C G A T T G G A T T C A C - 3 ' KRp51 5 - T G G A C C A T C A A A A G T C T T T C - 3 ' • 886 bp Figure 9. Location and sequences of primers used for P C R amplification in the estimation of minigene copy number. The diagram shows the first 12 exons common to all bli-4 isoforms. The 5' untranslated region (5' UTR) is hatched whereas the protease domain is shaded. The sequences amplified by PCR are present in both the endogenous bli-4 gene and all the minigene constructs. Therefore the relative amount of P C R products in transgenic animals to the wild-type strain wi l l indicate the copy number of the bli-4 common region, which estimates the copy number of the actual transgenes. 32 experiment to provide an estimation of the copy number. Detailed calculations are outlined in Appendix I. Rescue of bli-4 lethal alleles with transgenic arrays Extrachromosomal arrays were crossed into bli-4 lethal-bearing strains based on the scheme depicted in Figure 10. Hermaphrodites of the balanced lethal strains were crossed with N2 males that resulted in Fl progeny (males and hermaphrodites) heterozygous for the lethal mutation and the Dpy and Unc markers. Fl males were then crossed with transgenic rollers generated from DNA transformation as described above. Rol hermaphrodites were isolated from the resulting F2 population and allowed to self-fertilize on individual plates. Plates of F2s who produced arrested embryos indicated the presence of the lethal allele in the F2 parent (even when the extrachromosomal array was integrated, see Appendix II). These plates were kept and scored for the following F3 phenotypes: non-Rol, Rol, dead eggs, and Dpy Unc. The extent of rescue was determined by comparing the transmission frequency of the extrachromosomal array and the percentage of rescue (see Table 8 and Results). 33 Figure 10. Strategy to determine transgenic rescue of bli-4 lethal alleles Lethal (let) alleles (e.g. h791, s90) are balanced by sDp2. Heterozygous Fl males were crossed into Rol strains carrying the extrachromosomal array. Rol F2 hermaphrodites were picked and set up individually for scoring. Rol F2 which did not produce any dead progeny were presumed not to carry the lethal allele and were not scored. (Modified from Srayko, 1995) 34 Fl dpy-5 Unc bli-4(let) unc-13 dPy~5 bli-4(let) J I unc-13 -i sDp2 <f X I dpy-5 bli-4(let) J I WT N2 unc-13 + pick wild-type -L (may also carry sDp2) F2 WT dpy-5 bli-4(let) 1_ 1 Bli unc-13 c f X | — L - j hEx (includes rol-6) Rol bli-4(e937) bli-4(e937) _ l 1 |—L-\ hEx (includes rol-6) WT dpy-5 1 bli-4(let) 1 unc-13 I + | + I + I + I bli-4(e937) 1 + I + | bli-4(e937) I + I dpy-5 1 Rol bli-4(let) 1 unc-13 I + | Rol + I + | + 1 bli-4(e937) 1 + I + I bli-4(e937) I + I |—L-\ hEx (includes rol-6) pick Rol ^ f o r scoring F3 progeny 35 Results Section I . Construction of bli-4 minigene constructs The strategy to construct minigenes specific for individual bli-4 isoforms was based on the previous study by Srayko (1995). Two "parent" plasmids, pCeh220 and 221, contained a 9 kb DNA fragment corresponding to the common genomic region of bli-4 (Figure 11). The 5 kb genomic sequences 5' of the transcription start site in exon 1 contain the putative promoter elements for the transcription of bli-4. Expression of a fusion of this region with a reporter lacZ gene was detected in selective tissues (hypodermal cells, the vulva, and the ventral nerve cord) at both the embryonic and adult stages (Thacker et al, 1995). Further analysis of the promoter region was provided by Jones (1997). 3' exons with neighboring intronic sequences specific to individual blisterases were subcloned and joined to the common region in the parent plasmids to complete the construction of bli-4 minigenes. In this study, minigenes specific for blisterase B (pCeh299), and blisterase C (pCeh308 and pCeh309) were constructed. pCeh226, constructed and tested for rescue by Srayko (1995), served as the blisterase A minigene (Figure 12). Previously, RT-PCR analysis detected a variant blisterase C transcript putatively resulting from incomplete processing of the last intron specific to blisterase C (Figure 13; Thacker et al., 1995). Therefore, to eliminate 36 A . Figure 11. The "parent plasmids" (constructed by C. Thacker) used in the construction of bli-4 minigenes in this study. The number within each box represents the exon number. The 5'-untranslated region (5' UTR) is hatched whereas the protease domain is shaded. Underlined restriction sites show the difference in cloning site available for subcloning downstream exons. A . pCeh220 was used for the construction of pCeh299, the B minigene B. pCeh221 was used for the construction of pCeh226 (A minigene, by Srayko), and pCeh301, which was then utilized for the construction of pCeh308 (C short minigene) and pCeh309(C long minigene) Restriction enzymes: (Xb) Xbal; (P) PstI; (E) EcoRI; (K) Kpril; (N) Notl. 37 ikb Xb Xh Sac II I Sal Sal E Sal Xh PEXbEEK Sac a1 1 E E P Sal I 1 I I pCeh226 E = EcoRI K = Kpnl P = Pstl Sac = Sacl Sal = Sail Xb = Xbal Xh = Xhol Figure 12. Structure pCeh226, the A minigene in this study (Srayko, 1995) 38 e x o n 1 7 in t ronl7 exon 18 G C 1' C A T T g t aag ttt tat aag agt ttc aga ata aaa agt taa tctccag[/VC G C T G A \ A D C K F Y K S F R I K S * Y A E Figure 13. The putative carboxyl terminus of the variant blisterase C transcript. (Modified from Thacker et a l , 1995) Amino acids potentially encoded within intron 17 are shown. The splice donor and acceptor are underlined. 39 possible expression of this variant transcript, intron 17 was exluded in both the blisterase C minigene constructs (Figure 16, 17). Moreover, in light of the extremely short 3'-untranslated region (3' UTR) of blisterase C (31 nucleotides) and the technical difficulty of including the small quantity of intronic sequences immediately downstream of the last blisterase C exon (exon 18), the unc-54 3' UTR, a commonly utilized 3' element in C. elegans gene expression vectors, was appended to the end of exon 18 in pCeh309 to ensure proper processing of transcript (Fire et al., 1990; Figure 18.). A detailed construction scheme for each minigene construct is outlined in Figures 14 to 17. Section II. Generation of transgenic strains with high and low copy numbers of bli-4 minigenes Transformation in C. elegans is usually performed using a DNA concentration of about 100 ug/ml. This range of concentration enables the generation of extrachromosomal arrays of optimal size for efficient transmission (Mello et al., 1991). In this study, minigenes were injected at two concentrations, 50 ug/ml and 1 ug/ml, to examine the effect of copy number on the rescuing ability of each minigene. To ensure the generation of transmissible extrachromosomal arrays, a constant total DNA concentration of 100 ug/ml was maintained. Therefore, 50 Ug/ml of pBlueScript, the backbone of each minigene construct, was added in addition Figure 14. Construction of pCeh299, the B minigene A. The substrate clones used B. A schematic representation of the cloning steps involved 41 A lkb Xb B pCeh221 1. digest with Sp 2. fill in Sp (blunt) 3. digest with B B D K Sp pCeh232 pCeh232 B / D E E P Sal ' * I I B = BamVO. E = EcoRI D = Dral K = Kpnl N = NofI P = Pstl Sac = Sad Sal = Sail Sp = Spel Xb = Xbal Xh = X/wI pCeh 221 \P/N N pCeh298 Xh Sac E E E P B D MP 520 bp Xh Sac E E E P B N pCeh 299 (B minigene) Figure 15. Construction of pCeh301 pCeh.301 contains the common region of bli-4 and the first exon of blisterases C and D A . The substrate clones used B. A schematic representation of the cloning steps involved 43 A lkb E Sal Sal E Sal Xh PE>jbEEK pCeh220 pCeh232 B Sph K H p 4+' PGeh232 isolate 858 bp ^ Sph-H fragment Sph H K Sau Sau Sau isolate 633 bp Sau-H fragment from partial digestion, clone into BSSK (B, H) pCeh 220 1. digest with P 2. fill in P (blunt) 3. digest with K E E P Sal ' * I I B = BamHl E = EcoRI H = HindlU K = Kpnl P = Psfl Sac = Sad Sal = Sail Sau = Sau3Al Sp = Spel Sph = Sphl Xb = Xbal Xh = XtoI Xb B K H pCeh300 1. digest with Xb 2. fill in Xb (blunt) 3. digest with K B K ±_jp (524 bp) Xh Sac E E E B K PCeh301 Figure 16. Construction of pCeh308, the C short minigene A . The substrate clones used B. A schematic representation of the cloning steps involved 45 A Xh Sac E _L E S i S i E Sal Xh PE >p EEK _igi i i >f w • • pSac K Ikb E E P S i L_Ii I pCeh220 pCeh232 pCehl80 taXh B = BamHl Bel = Bell E = EcoRl H = Hindlll K = Kpnl N = AMI P = Pstl Sac = Sacl Sal = Sail I Sma = Smal Sp = Spel Xb = X M Xh = X M B PCehl80 1. isolate 953 bp • Kfrag 2. fill in K (blunt) ^ *?cl Sal(pBSSK) : >• \~~wY 3. clone in N ^ y / pBSSK (Sma) pCeh303 replace Bel-Sal fragment with same portion from blisterase C cDNA (pCehl99) H Bel pCeh304 1.1 kb H fragment pCeh232 insert 990 bp B-H fragment into pBSSK B K H r%-^)-K(pBSSK) J_J pCeh305 pCeh308 (C short minigene) Figure 17. Construction of pCeh309, the C long minigene A. The substrate clones used The fragment containing the unc-54 3' UTR was cloned out from pPD49.26 (provided by A. Fire) B. A schematic representation of the cloning steps involved 47 A pCeh220 lkb E Sal Sal "J L E Sal Xh PE)jbEEK| Sac K E E pCeh232 pCehl80 p Sal i I toXh B B K pCeh306 pCeh301 insert unc-54 3' UTR (750 bp RV / Sp (blunted) fragment) at RV B K RV K B = BamHl E= EcoRI RV = EcoRV H = Hindm K = Kpnl P = Pstl Sac = Sad Sal = Sail Sp = Spel Xb = Xbal Xh = X7wI pCeh307 isolate 1.9 kb K fragment Y pCeh309 (C long minigene) 48 Xb Xb Genomic sequences of blisterase C pCeh308 (C short) pCeh309 (C long) P K intron 17 K L L _ K II < 3WR intron 17 Figure 18. Comparison of the genomic sequences of the 3' end of blisterase C with pCeh308 (C short), and pCeh309 (C long). Both pCeh308 and pCeh309 excluded intron 18 to eliminate the potential production of the C variant transcript (Figure 13). Hatched boxes represent exons specific to blisterase C. Restriction enzymes: Xb(Xfcal); P (Pstl); K (KpnT) 49 to the transformation marker pRF4 (also at 50 ug/ml) when injecting a low concentration of minigene. The injection procedures were kindly performed on e937 homozygotes by D. Janke (Simon Fraser University). Resulting stable transgenic lines were tested by PCR using specific primers to confirm the presence of minigene constructs (Figure 7, 8). Most of the lines demonstrated similar transmission frequencies (~ 40-50%) of the transgenic arrays and were maintained by selectively transferring rollers. A summary of lines generated by injections of different concentrations of the minigenes is recorded in Table 2. Section III. Estimation of copy number of minigene A semi-quantitative PCR-Southern analysis was used to determine the copy numbers of minigenes in the transgenic strains (Han and Sternberg, 1991). The use of PCR for quantitation analysis relies on the fact that the amount of PCR product is a linear function of input templates when other components (such as primers, Mg++, etc) remain in excess in the initial stage of amplification (Robinson and Simon, 1991). In this study, genomic DNA from single transgenic animals was isolated, and used as the template for PCR. Copy number was measured by the relative abundance of a DNA fragment of the common region of bli-4 in the transgenic animals to the same fragment in 50 o u C cu cr cu o cu u o co t o a <*> e rt H C rt bO at bD oi CD o s ~ O rt o u rt 01 C 0) bD rt x W OI a Ol bO rt CD ^ L O <0 o o o T-H t-H T-H X X X w w w A A -K K OD Ol O O O T-H T-H T-H w w w * « « O t-H CM T-H T-H T-H T-H rH T-H X X X W W W A A A r \ rv. co co co CT> O) 0~> cu cu cu ^ A A A CN T J T3 r\ rv co co co CTi Oi cu cu ta i ^ ^ A A A N K K co co co C~> Oi Oi Cu CU ^ I 1 I A A A a-) ^ LO T-H T-H r-H T-H T-H T-H X X X w w w ^ ^ ^ r\ t-H co CO co t-H Oi Ol Ol cu cu cu r-H T-H • "SU -sHA A A W W W A A A CN CN CN £ £ £ O r-H CN C N T-H r-H X X w w A A CTl ^ ^ rH K. 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CD CU IX IX CO co O i ON <u tu -sfrl "xr< A A A A A IX IX IX CO co co o> o> o> w x5i v i^ •xH "=* 1 1 ' r~-4 r-H* H r C ) r Q r Q VO cu O r-H X w rSi LO ^ CO CN 00 LX ON CO CN r-H CN VO T—I CO T-JH ro (N O — L o LO r-f! r X CO - « r-H T3 cu K S ON IX rC rC> r-H VO CU ON O rn X w o LO § > N ^ i CO "XS r-H T3 £ > > > > o o h J r J f» f> r> r* 'WJ O O O O . r T _L H-J _ J J X X U U U U U U U U U ^ CQ vo lx OO ON o T~H CN CO tx CO ^ LO LO LO LO LO CO CO CN CN CN CN CN CN CN CM CO CO CO CO CO CO CO CO CO CO CO CO CO CO PH PH PH PH 04 PH PH PH & r*H X, r ^ r ^ r ^ ON o CO X cu U a , II r J u o CO X CU u a , II CO U .ON ON CN X cu U T3 cu .S fi rH H-> cu H-» o fi ^ *-* r-H \ O bO rvj II * rS II r - H -CQ fi bO rt cu bO ra H-» c cu u r H CU DH vo CN 2 CU U II OH X 3 II - r H 0 ^ < E .fi crj JTI CJ >N r Q O r r H 52 N2 animals. A control set of primers that amplify part of the 5' portion of the putative rec-1 gene was included in every reaction to compare individual reaction efficiencies (C. Wicky, pers. com.). Judging from the intensities of the control bands of the different reactions, the efficiency of each single reaction appears reasonably uniform to allow cross comparison (Figure 19). Amplification was only allowed for 12 cycles to ensure all reaction components remained in excess and the linear relationship between quantities of templates and products still maintained (Appendix I). The resulting PCR product was subject to Southern analysis and the signals were quantified by densitometry. In general, the data indicated that the transgenic lines generated by injection of high concentration (50 (ig/ml) of minigene contained about 10-20 copies of transgenes on average, which is about five-fold higher than those lines generated by injection of low concentration (1 ug/ml) of minigene (Table 3). In particular, the signal from KR3231, obtained from injection of low copy number of pCeh226, the A minigene, could not readily be distinguishable from that of the wild-type control, indicating that the copy number of pCeh226 in this strain was very low (<1 according to our measurement). 53 Figure 19. Estimation of minigene copy number by PCR/Southern analysis Panels A-D display Southern blots of PCR products from DNA of a single animal used for the estimation of minigene copy number in the transgenic lines tested for rescue of bli-4 phenoytpes: Panel Lane Strain A 1, 2,3 KR3231 (A Low) A 4,5,6 KR3228 (A High) A 7,8,9 N2 B 1,2 KR3235 (B Low) B 3,4,5 KR3239 (B Low) B 6,7,8 KR3233 (B High) B 9,10,11 KR2868 ("B") C 1, 2,3 KR3245 (C Short Low) C 4, 5,6 KR3246 (C Short Low) C 7,8,9 KR3357 (C Short High) C 10,11,12 KR3252 (C Long High) D 1,2,3 N2 D 4,5, 6* KR3252 The top band in each panel represents the PCR product of bli-4 seqeunces, whereas the bottom band represents the PCR product of the putative rec-1 gene, as a positive control (see Figure 9 for explanation). The intensity of each band was scanned and used in the estimation of copy number of minigene (See Appendix I for calculation). * These three lanes serve to demonstrate the linear relationship between input template and resulting products under the reaction conditions used in this experiment. Lane 5 contains double the amount of template DNA in lane 4, whereas lane 6 contains double the amount in lane 5. See Appendix I for the evaluation of the linear relationship. 54 mm m 1 2 3 4 5 6 7 8 9 B 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 55 Table 3. Estimation of minigene copy number Transgenic Line Transgenic array Construct/ Minigene Injection concentration of minigene (High = 50 ng/ul Low = 1 ng/ul) Copy Number (+ 1 SD) KR3231 hExl07 pCeh226/A Low 0.7 ±0.1 KR3235 hExlll pCeh299/B Low 2.2 ± 0.9 KR3239 hExl!5 pCeh299/B Low 1.9 ±0.1 KR3245 hExlll pCeh308/CS Low 2.9 ± 0.7 KR3246 hExlll pCeh308/CS Low 2.8 ±1.1 KR3228 hExl04 pCeh226/A High 16.3 ±3.6 KR2868* hEx46 pCeh238/'B' N/A 3.5 ± 1.0 KR3233 hExl09 PCeh299/B High 9.7 ±1.9 KR3357 hExll7 pCeh308/CS High 20.5 ± 0.7 KR3252 hExl!8 pCeh309/CL High 21.5 ± 4.9 *KR2868 was constructed by Srayko (1995). The injection concentration of pCeh238 was not available. 56 Section IV. Rescue of blistering using bli-4 minigenes Measure of rescue The population frequency of blistered animals among transgenic rollers of stable lines was measured to determine the rescuing abilities of individual minigenes. Blistering frequency among rollers was compared to blistering frequency among non-rollers from the same line because both populations possessed identical genetic backgrounds except the presence of extrachromosomal arrays in the former. It is observed that the percentage of blistered animals among rollers was always lower than that among non-rollers within the same line (Tables 4-7). This observation was also true to a control transgenic line, KR2872, which contained only the transformation marker pRF4 (Tables 4), suggesting blistering was partially suppressed by the rolling phenotype (Appendix III). In order to control for the effect of the rolling phenotype on blistering, rescue was assessed by comparing the percentage of blistered animals among rollers of each line to the same measurement in KR2872. The rescue result is arbitrarily regarded as positive when <10% blistered roller was observed. When the frequency of blistering Table 4. Rescue of blistering by bli-4 minigenes at high copy number* Construct/ minigene Transgenic Line % Blistering among non-Rollers % Blistering among Rollers pCeh226/A KR3228 84.3 (507/601) 0 (0/640) pCeh299/B KR3233 60.0 (424/707) 0 (0/236) pCeh238/'B'a KR2868 73.1 (657/899) 31.6 (136/430) pCeh308/CS KR3357 86.1 (827/960) 61.8 (162/262) pCeh309/CL KR3252 86.2 (580/673) 0 (0/478) pCeh309/CL KR3253 81.6 (231/283) 0 (0/128) pRF4 KR2872 86.8 (548/631) 70.8 (235/332) - CB937 91.1 (246/270) -* KR2868 contained an intermediate copy number in comparison to other transgenic lines generated in this study (Table 3). The copy number of KR2872, the control line, was not determined. apCeh238 is a blisterase B subclone that also includes the first exon specific blisterases C and D (Figure 20). 58 Table 5. Rescue of blistering by pCeh226 (A minigene) at low copy number T r a n s g e n i c L i n e % B l i s t e r i n g a m o n g n o n - R o l l e r s % B l i s t e r i n g a m o n g R o l l e r s KR3231 92.0 (1058/1150) 67.1 (194/289) Table 6. Rescue of blistering by pCeh299 (B minigene) at low copy number T r a n s g e n i c L i n e % B l i s t e r i n g a m o n g n o n - R o l l e r s % B l i s t e r i n g a m o n g R o l l e r s KR3235 88.9 (335/377) 0 (0/431) KR3236 87.2 (156/179) 8.9 (9/101) KR3237 64.6 (62/96) 5 (3/57) KR3238 75.7(115/152) 59.6 (28/47) KR3239 77.3 (586/758) 66.2 (88/133) Table 7. Rescue of blistering by pCeh308 (C short minigene) at low copy number T r a n s g e n i c L i n e % B l i s t e r i n g a m o n g n o n - R o l l e r s % B l i s t e r i n g a m o n g R o l l e r s KR3245* n a 0 (0/1176) KR3246 89.1 (825/925) 67.2 (334/497) KR3247 83.6 (286/342) 72.6 (77/106) KR3348 77.2 (44/57) 41.5 (51/123) KR3249 86.8 (244/281) 89.7 (52/58) KR3250 69.4 (193/278) 63.5(115/181) KR3251 90.5 (86/95) 87.5 (63/72) * putative integrated line (100% Rollers) 59 among rollers is comparable (± 10%) with the control line KR2872, the rescue is considered negative. A. pCeh226, the blisterase A minigene, rescued blistering at high, not low, copy number The plasmid construct pCeh226 was chosen as the blisterase A minigene in this study because it demonstrated the "best" ability among other blisterase A subclones to rescue the lethal phenotype in the previous study (Srayko, 1995). In the present study, KR3228, containing at least 16 copies of pCeh226, demonstrated complete rescue of blistering as expected (Table 4). Only one stable line, KR3231, was obtained from injection of pCeh226 at low concentration. The copy number of pCeh226 in KR3231 was very low and blistering was not rescued in this strain (Table 5). 60 B. pCeh299, the blisterase B minigene, rescued blistering at high copy number; a low copy number of pCeh299 also rescued blistering in particular lines pCeh299 was designed to contain only exon 14, the exon specific to blisterase B, at the 3' end. It differs from the subclone constructed by Srayko (1995), pCeh238, which additionally contained the first blisterase C/D exon, exon 15 (Figure 20). KR2868, containing pCeh238, demonstrated only a partial rescue of blistering, in agreement with previous result (Table 4). On the other hand, complete rescue of blistering was shown by KR3233 with pCeh299 (Table 4). We found that KR2868 only contained about four copies of pCeh238 whereas KR3233 contained about ten copies of pCeh299 (Table 3). The difference in copy number may therefore account for the difference in rescue results demonstrated by the two strains. Five lines were generated from injection of pCeh299 at low concentration. KR3235 showed complete rescue of blistering while KR3236 and KR3237 showed -90% rescue. The other two lines were considered negative for rescue (Table 6). The copy numbers in KR3235 and KR3239 were determined. The insignificant difference in copy number is unlikely to contribute to the difference in rescuing performance (Table 3). 61 lkb Xb Xh Sac E E P Xh B E E K Sac K E E P 1 LL . Xb E Xh Sac E E E P E E K pCeh238 Xb E Xh Sac E E E P BEEN pCeh299 Figure 20 . A comparison of pCeh238 (Srayko, 1995) and pCeh299 Restriction enzymes: (Xb) Xbal; (Xh) Xhol; (P) Pstl; (Sal) Sail; (E) EcoRI; (K) Kpnl; (B) BamBl; (N) Notl. 62 C pCeh308, the blisterase C short minigene, did not rescue blistering at high copy number pCeh308, the C short minigene, was injected at high concentration into N2 animals and introduced into the e937 background by genetic crosses. The resulting transgenic line KR3357 contained about 20 copies of the minigene but displayed negative rescue of blistering (Table 4). D. A putative integrated line containing a low copy number of the C short minigene demonstrated complete rescue of blistering Six of the eight stable lines generated by injection of pCeh308 at low concentration, including KR3246 which contained about three copies of the minigene (Table 3), displayed negative rescue of blistering (Table 7). One line, KR3245, stably produced 100% rollers and completely rescued blistering (Table 7). A rare integration event may have occurred during transformation. KR3245 contained a copy number of the C short comparable to KR3246 (Table 3). The difference in rescue results between these strains indicates pCeh308 is able to rescue blistering when integrated. 63 E. pCeh309, the blisterase C long minigene, rescued blistering at high copy number pCeh309 at a high copy number was found able to completely rescue blistering in KR3252 and KR3253 (Table 4). A comparison of KR3252 with the negative rescue shown by KR3357, which contains a similar copy number of pCeh308, indicates that the unc-54 3' UTR may have enhanced the rescuing ability of the C minigene. Section V. Rescue of lethal alleles using bli-4 minigenes Measure of rescue Two lethal bli-4 alleles were tested for rescue by the minigenes: the class II K791 allele, and the single class III allele, s90. h791 is a nonsense mutation on Cys-245 (TGT—>TGA) in exon 6 (Figure 21). In this mutant, premature termination of translation within the protease domain is predicted, leading to truncated BLI-4. The molecular lesion in s90 is not identified yet. Extrachromosomal arrays carrying various minigenes were crossed into lethal strains based on the scheme depicted in Figure 10. To determine the extent of rescue, the percentage of rescued animals was compared to the 64 bli-4(h791) II C245 Nonsense S H M 11 -02r Truncated protein o T G A (stop) Figure 21. Location and molecular lesion of h791. A n EMS-induced transition changed Cys-245 to a premature stop codon (TGT->TGA), leading to the production of truncated protein. K791 is therefore a potential nul l allele of bli-4. (hatched boxes represent the 5' U T R and shaded boxes represent the protease domain) 65 transmission frequency of the extrachromosomal array (Table 8). A complete rescue was evident when the results from both measurements were similar. The genotype of rescued animals were scrutinized for the production of dead eggs. In addition, the rolling phenotype is epistatic to both the Dpy and Unc phenotype. Therefore, rescued animals were outcrossed with N2 males and the resulting production of Rol progeny verified the presence of extrachromosomal arrays in the putative rescued lines. In particular cases putatively rescued animals were found to perish after being transferred before adulthood. This indicated an incomplete rescue of the lethal allele by the minigene. A. pCeh226, the blisterase A minigene, rescued bli-4 lethal alleles at high copy number The array hExl04, containing a high copy number of pCeh226, rescued both K791 and s90, in agreement with previous experimental results (Srayko, 1995; Table 8A). However, hExl07, containing a very low copy number of pCeh226, did not manage to rescue either lethal mutant (Table 8B). CJ CU Resc T—H LO LO CO o CM T J T—1 "o PH V O LO CN CO LO CM CN Dead Egg O N CN CN LO CN o o CN ON IX CN LX CM CJ DpyU LO CM CN V O O CO 00 TP T~H CN t x CO O N r-H DpyU Rol O N co Q O 1353 CO LO ON O co LX t x o o Z * 1290 1067 1155 1224 1067 X « +* ^ ^ g ° fi d £ bo t! >< W o ON co X bo ffi < vo CN CN rC cu U CM r-H ON I X X bo ffi V O CN CN X CD X bo • < ffi PQ O N O N CN X cu U r-H ON I X X bO ffi PQ O N O N CN X cu U X bO * i-H ffi u O N o 2 cu u OH CU CO r * rf\ CCS C rH CU «H bO O _ £H (TJ «i o * § •2 g xs S - £ *s co CJ cy cS 3 (-1 H U crj g •i-H C crj . X cu CU o CH CO X o ^ OH « c o 3 r-H CU CJ CCS In CU X O H ^ r£> W c u ^ . 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CD X CO c O cj CD r 3 CJ CO 01 >H CD rC CD X H >N CD 60 O rl OH 60 c 'cj X O I-H OH -t-» o X X a> r C CO ' in 01 OH CO CO o fi 0 rH M-H rX 01 60 O |H OH U rX OH P X CD L , cu CU CU In > Tj & 3 cu I-I cu d u co cu I-I cu > d O H-» O TJ O Oi r\ TJ d rt O CO CX, -co cu cu cu ^ d T J rt cu rt 45 bo * cu rt Tj .d d cu bO O Sn CM o d CM Q o d ^ 1 1 TJ X, d 3 cu > o cn 4> bO rt S?* T j rt cu O TJ a) >*'rt d a O TJ T J CU d a rt X cu cu *-< cu £ cu CV >^  o c cu rt bO bO cu d o 4 3 cu d CJ co cu In CU cuT J CU o a O -d d d a a £ < cu jy bo £ o o T J cu CO cu -t-» CO rt I-I I-I < CO „ >N rt rt I-I cu f-< > rt 1+3 bO M-I d ° 3 a W rj cu d n I-I PH d ^ o <u d 4 3 cy T J 45 cu ' i H rt a a d IH O CO bO IH cu '1 d d a, o U CJ )H CO d o IO fr1 a rt X w I1 I1 o '•SI o o cu C cu bOl , <N CN CU U CU CQ ON ON CN CU CQ o\ o \ C N 45 CU U eg CO l u | 00 o C O 45 cu CO U | 00 o C O 45 cu co U oo o C O 45 cu U CM o r-H X r-H r-H r-H X w r-H r-H t-H TH X w CN r H w CN CN t-H X tq o, bO cn cu 68 B. pCeh.299, the blisterase B minigene rescued bli-4 lethal alleles at high copy number The array hExl09, containing a high copy number of pCeh299, was found able to rescue both h791 and s90 (Table 8A). Two arrays, which contained similar numbers of low copies of pCeh299 but demonstrated different rescue results of blistering, hExlll from KR3235 and hExll5 from KR3239 (Tables 3 and 6), both failed to rescue either of the lethal alleles (Table 8B). C pCeh308 and pCeh309, the blisterase C minigenes, failed to rescue bli-4 lethal alleles None of the arrays tested carrying either pCeh308 or pCeh309 at various copy numbers, managed to completely rescue either h.791 or s90 (Table 8B). Although Dpy Unc animals were observed from the introduction of hExl28, which contained a high copy number of pCeh309 (the C long minigene), into s90, none of the animals survived and therefore a partial rescue was registered (Table 8A). 69 Discussion The bli-4 gene of C. elegans encodes four structurally distinct products by alternative splicing that are homologous to the kexin family of proprotein convertases. In addition to the blistering phenotype of the original allele e937, the embryonic or early larval phenotype of potential null alleles suggests bli-4 plays a role not only in adult morphology but also in the early development of the animal. Based on the strategy adopted by Srayko (1995), this study further dissected the function of individual blisterases by using isoform-specific bli-4 minigenes to rescue different classes of bli-4 mutants. Four minigenes individually encoding blisterase A, blisterase B, and blisterase C were tested for rescue at both high and low copy number. The results indicated functional redundancy between blisterases A and B. The relatively inferior rescuing ability of the C minigenes implied a restricted functional role of blisterase C in vivo. High copy number of exogenous blisterases A and B rescued blistering The present data indicated that both blisterases A and B were able to rescue the blistering phenotype (Table 4). pCeh226, the A minigene, has been shown previously to completely rescue blistering (Srayko, 1995). The .70 experiment was repeated with the injection concentration monitored at 50 jig/ml in this study, and the positive rescue result was confirmed. As the blistering phenotype is the result of a deletion that causes the removal of the exon specific to blisterase A, the positive rescue result by pCeh226 is expected. pCeh299, the B minigene, also demonstrated complete rescue of blistering at high copy number in e937 animals. This is not surprising as blisterases A and B bear a high degree of structural similarity. The two isoforms differ in the length of the carboxyl tail in which no special structural features can be identified from sequence analysis. Previously, an RT-PCR experiment, although not fully quantitative, detected an increased level of the transcript of blisterase B in e937 animals. This suggests that the expression of blisterase B may be elevated to partially compensate for the loss of blisterase A in e937, and thus lead to the observed reduced penetrance of the blistering phenotype (Thacker et al., 1995). The rescue of blistering by both pCeh226 and pCeh299 support the notion of potential functional redundancy between blisterases A and B. pCeh299 and pCeh238 demonstrated different rescue results Previously, it was reported that a blisterase B subclone, pCeh238, only partially rescued blistering (Srayko, 1995). This contrasts the present positive rescue result demonstrated by pCeh299. pCeh238 differs from pCeh299 by its 71 incorporation of exon 15, the first blisterase C/D exon, and adjacent intronic sequences (Figure 20). As speculated before, the common region in pCeh238 could possibly be spliced onto exon 15 and hence produced an aberrant transcript, while simultaneously decreasing the production of functional blisterase B transcripts (Srayko, 1995). In the present study, a transgenic line containing pCeh268, KR2868, was picked and the test of blistering was repeated. A partial rescue was recorded, in agreement with the previous result (Table 4). KR2868 was included in the experiment to determine the copy number of transgenes. It was revealed that KR2868 contained a lower copy number of pCeh238 than pCeh299 in KR3233. This, in addition to the proposed undesired splicing event in pCeh238 as described, may explain why only a partial rescue of blistering was observed in KR2868. The unc-54 3' UTR improved rescuing ability of pCeh308 pCeh308, the C short minigene, did not rescue blistering at high copy number. However, pCeh309, which is identical to pCeh308 except with the addition of the unc-54 3' UTR, completely rescued blistering at a comparable copy number (Figure 22). The unc-54 3' UTR is commonly employed to serve as the 3' element for ectopic gene expression in C. elegans (Fire et al., 1990). Although unc-54 encodes the major myosin heavy-chain isoform in the animal, its 3' UTR was shown not to confer any tissue specificity for gene 72 TRANSGENIC LINE f/M% A minigene (PCeh226 - KR3231, KR3228) •gSgjgg B minigenes (pCeh299 - KR3235, KR3238, KR3233; pCeh268 - KR2868) C minigenes (pCeh308 - KR3245, KR3246, KR3357; pCeh309 - KR3252) Figure 22. A comparison of minigene copy number in different transgenic lines 73 expression (Okkema et al., 1993). Instead, this element permits expression in a wide range of different tissues and cell types specified by the upstream sequences (Fire et al., 1990). It has been known that many 3' UTRs function to stabilize messenger RNAs (Sachs, 1993). This may be the mechanism by which the unc-54 3' UTR contributes to improving the rescuing ability of pCeh309 over pCeh308. The natural 3' UTR of blisterase C is only 31 nucleotides long and is unusually short when compared with other C. elegans genes. The poor rescuing ability of pCeh308 may be due to the short length of the 3' UTR of blisterase C. In agreement with this reasoning, the endogenous amount of blisterase C transcripts was undetectable by Northern analysis (Thacker et al., 1995). Restricted expression of blisterase C indicates that this isoform may have a restricted functional role in the animal. Extremely low copy number of blisterase A did not rescue blistering KR3231 was the only stable line generated containing a low copy number of pCeh226, the A minigene. It was expected that an exogenous provision of the genetic material of blisterase A, which has been specifically deleted in the mutant animal, at even a low copy number may be sufficient for rescuing of the blistering phenotype. However, KR3231 did not demonstrate complete rescue of blistering. Nonetheless, the negative result 74 of KR3231 could be accounted for by the extremely low copy number of pCeh226 as compared to the copy numbers in other transgenic lines. The copy number was so low that the corresponding signal could not be differentiated from that from the non-trans genie control (Figure 22). Therefore, the comparison of the rescuing ability of pCeh226 as from KR3231 with the other minigenes at low copy numbers was inconclusive. Low copy number of blisterase B rescued blistering A group of five stable lines were generated by injecting a low concentration of pCeh299. Three of the five lines demonstrated complete or almost complete rescue blistering while the remaining two were considered negative for rescue (Table 6). The difference in results could probably not be caused by a difference in minigene copy number because KR3235, which demonstrated complete rescue, and KR3239, which displayed substantial amount of blistered rollers, were found to contain similar copy numbers of pCeh299 (Figure 22). The difference should also not be the result of a spontaneous second-site suppressor of blistering in the three rescued lines as the blistering frequency among non-rollers within these lines remains normally high (Table 6). We propose that the lines that showed rescue may contain more functional copies of the minigene (hence more transcripts) on their extrachromosomal arrays than the lines that were tested negative. This 75 is possible because extrachromosomal arrays were independently assembled during transformation (Mello et al, 1991). This hypothesis could not be detected by our use of PCR to determine the copy number of transgenes. However, Southern analysis can be applied to detect if structural difference indeed exists between the arrays (Mello et al., 1991). The demonstration of rescue of blistering by a low copy number of pCeh299 in three out of five low copy transgenic lines indicates that blisterase B is likely to function in the assembly or maintenance of the adult cuticle in vivo. Integration improved rescuing ability of pCeh308 A putative integrated line, KR3245, was generated from injecting pCeh308 at low concentration, as evidenced from 100% transmission of the Rol phenotype. Blistering was completely rescued in this line (Table 7). On the other hand, the other six non-integrated lines generated from the same injection event failed to completely rescue blistering (Table 7). Integration of the extrachromosomal array into the genome ensures faithful transmission of the transgenic material during mitotic cell divisions and therefore permits its expression in every tissue of the organism. As a result, expression of the transgene in the correct tissue is presumably secured. As discussed, one possible explanation for the poor rescuing ability of pCeh308 may be that the short 3' UTR failed to stabilize the blisterase C transcript. Integration of the 76 minigene may provide correct tissue localization so that even transient expression from pCeh308 may be sufficient for correcting the blistering defect. Alternatively, integration yer se may stabilize the expression of transgenic array. Furthermore, the site of integration may mediate the expression of the transgene. For example, pCeh308 may be juxtaposed near a collagen gene which is expressed at the adult stage so that co-expression occurs and promotes the function of blisterase C. Genetic mapping of the integration site in KR3245 can be performed to test this hypothesis. rol-6, the transformation marker, is a potential bli-4: substrate A positive rescue result for the blistering phenotype was defined in this report as a complete absence of blistered animals among the transgenic population. However, it is observed from that among the majority of transgenic lines that were considered negative for rescue of blistering, there is a slight decrease of blistering frequency (4-9%) when they are compared to the control line KR2872 (Table 4). This suggests that the different isoforms have at least partial overlapping capability of rescuing blistering. A more noticeable drop in blistering frequency is observed when the comparison is made against non-transgenic, or non-rollers, within the same line. This suggests that the Rol-6 phenotype is a partial suppressor of the Bli-4 phenotype. The rol-6 gene encodes a cuticle collagen that contains the 77 hallmark cleavage motif (Arg-X-Arg-Argi) for substrates of kexins (Yang et al., 1994). Given rol-6 as a candidate substrate of bli-4, the injection marker pRF4 may contain both temporal and spatial expression signals that interfere with the expression of the adjacent blisterase gene present on the same extrachromosomal array. In future, markers other than cuticular collagens, such as an antisense unc-4 plasmid, which displays a twitching phenotype, or unc-22, which displays an Unc phenotype, could be used for transformation with bli-4 subclones for precaution against undesired interaction between gene products of co-injected molecules (Mello and Fire, 1995). Blisterases A, B rescued the lethal alleles h791 and s90 Both pCeh226 and pCeh299, the A and B minigenes, managed to rescue the two lethal alleles h791 and s90 completely at high copy number while both the short and long versions of the C minigenes, pCeh308 and pCeh309, and all of the minigenes at low copy numbers failed to do so (Table 8). The A minigene notably demonstrated the best rescuing ability among the four constructs tested in this study - the majority of the rescued animals survived and produced a healthy amount of progeny. On the other hand, only a portion of the animals rescued by the B minigene survived, and they produced less progeny than the animals rescued by the A minigene. This difference may be a result of the lower transmission frequency of the 78 extrachromosomal array of pCeh299, hExl09 (Table 8). On the other hand, it may indicate that blisterase A is more specific than blisterase B for the function of bli-4 at the embryonic stage. Viable e937 mutants imply that blisterase A is not specifically essential for development. However, our rescue results indicate that blisterase A is capable of fulfilling the essential role of bli-4 in development. Preliminary result from RT-PCR on embryos detected expression of blisterase A (Thacker, pers. comm.). This suggests that blisterase A has a functional role at the embryonic stage which can be amplified by high copy number to maintain normal development in the absence of other isoforms. The rescue by the B minigene also indicates that blisterase B is capable of rescuing bli-4 lethals when present at high copy number. It is again noted that the positive rescue by pCeh226 contrasts the negative rescue result of pCeh238 reported previously (Srayko, 1995). The difference is probably due to the different structures of the constructs and the different transgene copy numbers between the two strains as discussed above. Blisterase B is not an abundantly expressed bli-4 isoform as indicated from Northern and RT-PCR analysis using mixed staged worm RNA (Thacker et al, 1995). However, structural similarity between blisterase B and blisterase A may explain the positive rescuing ability of pCeh299. pCeh309, the C long minigene, partially rescued the class III allele, s90. The molecular lesion of s90 is unknown but its phenotype (30% survive to the LI larval stage) suggests that s90 is a weak bli-4 lethal allele. The 79 incomplete rescue of a weak embryonic lethal allele demonstrated by the C long minigene indicates that blisterase C is probably not essential for the early development of the animal. Low copy numbers of blisterases A and B did not rescue the lethal phenotype That low copy numbers of pCeh226 and pCeh299 did not rescue the lethal alleles may indicate that blisterases A and B are not specific for early development. However, the present data is not convincing enough to support this conclusion. More efforts are required to generate transgenic lines containing higher copy numbers of minigenes. The present DNA concentration (1 ug/ml) used for generating low copy number of transgenes may need to be raised slightly to, for example, 10 |ig/ml. This may perhaps provide us with transgenic animals that contain enough copies of, in paritcular, the A minigene to rescue blistering but questionable for the rescue of embryonic lethality. Blisterase D is likely to play a functional role in early development Despite the positive rescue results from the A and B minigenes, blisterase D appears the most likely bli-4 isoform that ensures proper 80 development inside the embryo. Another construct, pCeh236, which encodes the blisterases B, C, and D also rescued the embryonic lethal alleles of bli-4 (Srayko, 1995; Figure 23). Comparable to blisterase A, blisterase D is also a highly expressed isoform as shown by Northern analysis (Thacker et al, 1995). It is, among the four isoforms, most structurally similar to human furin because of the presence of a TMD. Therefore, we speculate that blisterase D functions in the constitutive secretory pathway and processes molecules important to the development of the animal. Although construction of a minigene for blisterase D was unsuccessful (Appendix IV), we predict that blisterase D would also rescue the bli-4 lethal phenotype. BLI-4 is a candidate cuticle-collagen processing enzyme Various lines of evidence suggest that BLI-4 functions as a cuticle collagen processing endoprotease in the nematode. The C. elegans cuticle is secreted during the molting process that marks the transition between different developmental stages of the nematode. Cuticles are made up of different composition of collagens specific to each developmental stage (Johnstone, 1994; Kramer, 1994). One of the main functions of the cuticle is to act as an exoskeleton to model the shape of the animal (Priess et al., 1986). This functional role is evident in all developmental stages as cuticular defects lead to a variety of morphological phenotypes including early larval lethality 81 01 01 cj co o> C* CQ w VL JOT »H 2 O O O t-J ffi o + cu CO m C O w w-X CN CN OJ U DH o + c o , i + + PH " W -w X . X cE O N C T \ C N X o i U DH < N O PH" w-X. X SI X " cn U 00 0 C O X, 01 U DH + C N , + x 43 j x — r J u o CO X U DH + + X x -D U CO \ D C O C N X Oi u DH o 2 ccS cj OI r- -d OI o 3 cu CO CJ CU CO VH CU r3 t! DH 55 H_J CO 3 2 CU X > .s .s O \ rH + cu C cu cj cy co ^ X (OCT) CU | \ CO C rSt OJ 2 -H - > ( 1 ) co cu CU bJO cC cj X J . g CO >N CO -*-» r—H CO cu tH CU CJ CO CU rH M-l o & crj fi fi CO CO CN cu rH bO cu " .—i O (U rH r^ H CU crj r D r - , El ^ •fi -5 fi fi cu ro a; rH H-J M-( DH CO crj rH rc; 3 H fi cu o rC CU rH CJ CU co > cu o >-~ cu > CCJ bO ro cj X i co cu CU rH 3 „ r d cu o \ OJ m o C O CU u DH >N rO b b o V CJ c o cu _ .y ^ "1 -CJ QJ J U CH C O CU PH + JO ra o co c _o *H-» CJ cu CU fi ro co cu rC o rH X ! CU 3 co cu rH CO cu 3 l i b0 3 CU rH *H "3 bO fi & «3 CO s< bo cu fi >N fi ^ fi CO cu cu rH SH CU cu rc; 3 -rJ O - c o X5 CU rH CU CU rH o CJ -rH co +;) co 'co crj O S OH cu fi fi xi ro cu 1 0 3 £ o x r ^ X J 3 vu O rC £ bO co 3 fi O O •B fi rtj CU < -a 82 (van der Keyl et al., 1994). Similar to their vertebrate counterparts, the nematode collagens are synthesized as pro-collagen molecules and require limited proteolysis to acquire their mature forms. Many collagen molecules in C. elegans possess the hallmark substrate cleavage motif (Arg-X-Arg-Argi) recognized by kexin convertases, strongly suggesting bli-4 may encode the candidate collagen endoproteases (Yang et al., 1994). Expression studies on bli-4 using a fusion construct of the putative promoter region and a lac Z reporter gene detected predominant signals in, among other tissues, the seam cells of the hypodermis, which is involved with cuticle secretion, at both the embryonic and the adult stage (Thacker et al, 1995). Anatomical studies indicated that during lethargus, the period when new cuticle is deposited, densely packed Golgi bodies and vesicles predominate in the seam cells (Singh et al., 1978). Members of kexin convertases have been shown to localize to the TGN or the secretory vesicles in mammalian cells. The structurally divergent C-termini of kexins provide sorting signals for compartmentalization of the enzymes (Molloy et al, 1994; Jones et al., 1995; Schafer et al, 1995; Zhou et al., 1995; Creemers et al., 1996). It is very likely that the different blisterase isoforms are localized to Golgi bodies and secretory vesicles within the hypodermal seam cells, where processing of collagens occur en route to being secreted. The different bli-4 isoforms may possess different substrate specificities of collagen molecules, thus giving rise to stage-specific phenotypes, such as blistering in e937, when activity of specific bli-4 isoform is eliminated. 83 Functional redundancy of bli-4 isoforms Our results suggested functional redundancy exists among the different bli-4 isoforms. Rescue of specific mutant phenotype is not exclusive for any single isoform. Functional redundancy among kexin convertases which are differentially localized on the intracellular level is not a well-understood phenomenon. Despite reports of substrates processed by specific kexin convertases, there are also cases where a single substrate molecule may be processed by more than one kexin that are differently compartmentalized (Keller et al., 1995). C-terminally divergent dfurin isoforms of Drosophila exhibited similar catalytic activity or cleavage specificity (De Bie et al., 1995). The question of functional significance of the divergent C-terminus to the overall substrate specificity of kexins is even complicated by the consistent observation of C-terminal truncation during the biosynthesis of many kexin enzymes, leading to constitutive secretion of shortened yet functional molecules (Hatsuzawa et al., 1992). Whether truncated forms of kexins have any functional significance in vivo remains to be investigated. In the case of bli-4, pCeh221, a subclone which contained only the N-terminal genomic sequences common to all isoforms, reportedly rescued blistering (Srayko, 1995). This showed that aberrant expression of only the protease domain and the N-terminal features could also rescue a specific mutant phenotype 84 without any localization signals provided by the C-terminal sequences. Finally, the overall structurally similarity of collagen molecules, which are potential bli-4 substrates as mentioned, may indicate inconspicuous substrate specificity of the different blisterases that may not be easily unveiled by the current methodology (Kramer, 1994). Other information or experimental approaches that may contribute to our understanding of the individual function of bli-4 isoforms Functional specificities of the different blisterase isoforms should be influenced by individual temporal and tissue expression patterns. Individual cuticular collagen genes have been shown to demonstrate distinct temporal expression patterns (Johnstone and Barry, 1996). A temporal expression profile of the different blisterases will contribute to our identification of the specific functional role of individual isoform. Because of the technical difficulty of detecting the scanty amount of blisterases using Northern analysis, a semi-quantitative RT-PCR may be attempted to achieve the goal (Johnstone et al., 1996). Immunostaining using isoform-specific antibody or epitope-tagged minigene available in future will allow us to investigate the tissue distribution of the different isoforms. Another approach to dissect the functions of bli-4 gene products is to knock out sequences specific to individual isoforms. This can be achieved by 85 performing genetic screen using, for example, trimethylpsoralen as the mutagen which tends to produce small deletion mutations (Yandell et al., 1994). Alternatively, antisense RNA against a specific isoform can be injected to achieve a similar goal (Fire et al., 1991). It is expected that knock-outs of blisterases B and C may not produce observable phenotypes due to their possible minor roles and therefore high probability to be functionally compensated in the presence of the other isoforms. However, such experiments can be performed on e937 animals. For example, a specific deletion of blisterase D in wild-type or e937 background may provide us with additional information to verify our belief in the important functional role of this isoform. 86 Conclusion The present rescue data suggested that both blisterases A and B are more specifically involved in the role played by bli-4 in the adult cuticle than blisterase C. Blisterase C may have a restricted functional role unspecific to the adult cuticle. Alternatively, the overall expression of blisterase C may be limited by unfavorable sequence features such as the very short 3' UTR. On the other hand, the positive rescue results from pCeh299 at both high and low copy number despite the fact that blisterase B also contains a very short 3' UTR (23 nucleotides) demonstrates that blisterase B is potentially specific for the role of bli-4 within the adult cuticle in vivo. Although e937 mutant animals are viable, blisterase A is expressed at the embryonic stage and pCeh226, the A minigene, managed to rescue bli-4 lethal alleles at high copy number. These conflicting observations suggest strongly that functional redundancy exists between blisterase A and other bli-4 isoforms at the embryonic stage. The positive rescue of lethality by high copy number of blisterase B may be caused by its functional redundancy with blisterase A in light of the structural similarity between the two isoforms. Our rescue results suggest that blisterase C is not involved in embryogenesis. 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Chem. 270: 21509-21516 95 Appendix I A n estimation of the copy number of minigene is obtained by calculating the ratio of the intensity of the bli-4 PCR product in the transgenic animal to that of the wild-type animal (see Figure 9 for explanation). To adjust for the difference in the amount of template D N A and the efficiency between reactions, the intensity of the bli-4 band from the wild-type animal was first normalized by the difference in intensity of the control bands from the two animals. For example, to calculate the copy number of pCeh309 (the C long minigene), the intensity of band 4 in Figure A l was first normalized by dividing into the ratio of the intensity of band 10 to band 7 of the transgenic animal. The copy number is then computed as the ratio of the intensity of band 1 to the normalized intensity of band 4. 1 2 3 7 8 9 • bli-4 •control KR3252 (C Long) 4 5 6 10 11 12 • bli-4 • control wild-type Sample calculation Normalized intensity of band 4 = intensity of band4 X intensity of band 7 intensity of band 10 = 4.937 X = 4.494 6.955 7.396 Copy number intensity of band 1 normalized intensity of band 4 84.49 4.494 18.8 Figure A l 96 Normalization was repeated against the three wild-type animals for each transgenic animal and the average copy number was taken. The same procedure was repeated for 2-3 transgenic animals and the average was reported as the copy number for the transgenic strain (Table 3). The use of P C R to estimate the copy number of minigene relies on the linear relationship between template molecules and products in the initial stage of amplification. We performed a control in an attempt to demonstrate the linear relationship within the PCR conditions used in this experiment (Figure A2). A linear relationship can be observed in the first two lanes as the intensity of the signal doubled when the amount of D N A loaded was increased in a similar proportion. However, the reaction (top band) appeared saturated in the third lane. Most of the high-copy signals of the bli-4 bands appeared to be within the linear range except KR3231 which contained a high copy number of pCeh226, the A minigene. Therefore our estimation should reflect a minimum value of the actual copy number in this strain. bli-4 control Figure A 2 26.34 77.63 98.8 0.80 2.39 6.54 intensities of the corresponding y bands in the picture above 97 Table Al. Densitometry values corresponding to Figure 19. Panel.Lane Strain Intensity Top band Bottom band A.l KR3231 (A Low) 6.0 19.1 A.2 KR3231 (A Low) 3.2 10.9 A.3 KR3231 (A Low) 2.6 6.5 A.4 KR3228 (A High) 99.8 9.7 A.5 KR3228 (A High) 109.1 14.2 A.6 KR3228 (A High) 107.0 15.8 A.7 N2 4.9 14.3 A.8 N2 3.5 6.1 A.9 N2 3.6 4.6 B.l KR3235 (B Low) 12.2 8.6 B.2 KR3235 (B Low) 9.2 12.0 B.3 KR3239 (B Low) 9.0 9.1 B.4 KR3239 (B Low) 9.4 9.6 B.5 KR3239 (B Low) 11.2 12.5 B.6 KR3233 (B High) 72.3 18.7 B.7 KR3233 (B High) 87.7 15.1 B.8 KR3233 (B High) 72.0 14.3 B.9 KR2868 ("B") 22.9 10.8 B.10 KR2868 ("B") 20.5 17.3 B.ll KR2868 ("B") 19.1 9.8 C l KR3245 (C Short Low) 10.9 5.0 C.2 KR3245 (C Short Low) 17.0 8.0 C.3 KR3245 (C Short Low) 12.0 9.1 C.4 KR3246 (C Short Low) 9.6 7.0 C5 KR3246 (C Short Low) 12.1 8.5 C.6 KR3246 (C Short Low) 13.7 5.1 C.7 KR3357 (C Short High) 67.2 5.0 C.8 KR3357 (C Short High) 71.2 5.5 C9 KR3357 (C Short High) - -CIO KR3252 (C Long High) 84.5 7.0 C l l KR3252 (C Long High) 70.6 . 6.0 C.12 KR3252 (C Long High) 76.2 4.4 D.l N2 4.9 7.4 D.2 N2 3.8 6.5 D.3 N2 5.2 7.6 D.4 KR3252 (C Long High) 26.3 0.8 D.5 KR3252 (C Long High) 77.6 2.4 D.6 KR3252 (C Long High) 98.8 6.6 98 cu r H bp co PM T J CU > r H cu CO O CU J D CO bo bO cu TJ cC i—i <y t—( TJ X ^ •rr* L O f l CM 5 £ o« 2 CC cu bo rj cu CO C r H • r H CO £ • r H ?» « Si r H r H cd CC o co O fi O r H rC CJ cC r H x „ CU rj-J bO cu C r H O CU > Xj "co O DH CU rC r H CC bO CU TJ OI In OI rC ft fi o cn O fi O M rC (J r H OJ o en 0) fi o o l H rC u T J 13 l H bO 0) H-t .s tq rC > N (3 r H >H CU 5 X r C tX co. tq fx ON + I + co I rSN r X 13 PH X tx cn O N >> C QJ bO 0 r H OH TJ (0 01 TJ 01 > o c tq rx <r> O N cu cu CN UH cn rM ca 3 + + + c t x c o ON Mc + + + + f x f x 1= I \ c n rrj cn CO ON cr> ON cu cu I Is + + + 3 r o & CTi — cu m cS c n CO CO T-H c c-H i—1 CJ c j CJ CJ C C >, C a. 3 3 a cu o •a ^ c « D CO CO CO CO ±> c a . • a - t 3 - a 13 3 3 -Si x -Si •Si CO & 13 CO 13 3 *2 ±N T3 99 CD T J CD l-i CD X, 01 s o </l o 6 o »H X u TJ CD bO 01 UJ cu UJ fx X UJ CO 01 + CO 3 .52 co i PH c cu 6 0 O u a, (0 0) T J CD > "5b o CN OH t N c o ON cu t 2 x UJ t N CO CTi co i 3 CO PH O PH T J ca CD Q 6 0 6 0 0) T J a o> T J CD X CO PH CB cu H ^ cu CU ^ U CO Appendix III 100 The Rol-6 phenotype partially suppresses the Bli-4 phenotype The difference in the frequency of blistering in non-Rol and Rol of KR2872, the transgenic line which contained pRF-4, the injection marker was tested for statistical significance using the two-sample z-test (Freedman et al., 1991). The null hypothesis in this test is that the two frequencies are the same and the difference only represents chance variation. The alternative hypothesis states that the difference is real. The z-score is computed as the ratio of the difference between the frequencies to the root-mean square of the standard errors (SE) of the frequencies. SE is computed using the formula: {[p(l-p)]/n}l/2, where p = the percentage of blistering and n = the total number of animals scored (Freedman et al., 1991). The P-value represents the probability the recorded difference is observed if the null hypothesis is true i.e. by chance variation. A P-value of 0.01 (1%) is normally used as the criteria for rejecting the null hypothesis, i.e. the difference is statistically significant. Using the data from Table 4, P<0.0001 for the difference in blistering frequency between the non-Rol and the Rol populations of KR2872. For comparison, P<0.025 for the difference in blistering frequency between the non-Rol population of KR2872 and CB937, the original bli-4(e937) strain. Therefore, the Rol-6 phenotype partially suppresses the Bli-4 phenotype. 101 Appendix IV The construction of a minigene specific to blisterase D was attempted. One of the cloning schemes is shown in Figure A3. A l l of the cloning steps were completed except the final ligation of the D-specific exons with the common region (pCeh301). This step was repeatedly tried but the final construct was not able to be obtained. Figure A3 . Construction of blisterase D minigene A . Substrate clones used for cloning B. A schematic representation of the cloning steps 102 A Xb Xh Sac E E Sal Sal E Sal Xh PE)|bEEK| Sac K lkb E E P Sal 1 Li I pCehl80 toXh B B = BamHl Bel =Bc/I E = EcoRI RV = EcoRV H = HindW K = Kpnl N = Notl P = Pstl Sac = Sad Sal = Safl Sma = Smal Sp = Svel Xb = Xbal Xh = XtoI H Sp pCeh300 1. digest with Sp 2. fill in Sp (blunt) H K 3. digest with H 4. clone into BSSK (Sal (blunted), H) Sma-^y PCeh310 1. H 2. Sma pCeh 196 (blisterase D cDNA) • K H Bel replace Bcl-Bgl fragment ofpCeh311 with same portion from pCeh 196 K HBcl Bgl . pCehl80 1. digest with P 2. fillin P (blunt) 3. digest with H 4. isolate 4 Kb fragment pCeh311 pCeh312 replace Bcl-RV of pCeh306 fragment with same portion from pCeh312 Bel pCeh306 K RV pCeh301 RV Xh Sac E E B K D minigene 


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