Goodwin EB et al. (1994) Worm Breeder's Gazette "The laf-1 Gene Functions in the 3'UTR-Mediated Translational Repression of the Sex Determination Gene, tra-2"

Kimble JE, Goodwin EB

[Worm Breeder's Gazette, 1994]

The laf-l Gene Functions in the 3'UTR-mediated Translational Repression of the Sex Determination Gene, tra-2. Elizabeth Goodwin# and Judith Kimble University of Wisconsin, Madison, WI. # Dept. of CMS Biology, Northwestern Univ. Medical School, Chicago Ill.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755

van Luenen HGAM et al. (1999) Worm Breeder's Gazette "World wide worm SNPs (II)"

van Luenen HGAM, Thijssen KL, Plasterk RHA, Koch R, van der Horst M

[Worm Breeder's Gazette, 1999]

In the last WBG we presented single nucleotide polymorphisms (SNPs) in natural isolates from around the world (AB1 from Australia, CB4857 from California, RC301 from Germany and TR403 from Madison) (Koch et al., WBG 15#5, p17). We now sequenced over 700 kb and found 366 polymorphisms. The majority of polymorphisms (90%) are single base point mutations, but we also found small insertions and deletions of two or more base pairs. The lowest SNP frequency was found in TR403 (1:8750 base pairs), the highest frequency was found in CB4857 (1:1445 base pairs). On average we found 1 SNP per 2000 base pairs. After checking 63 potential SNPs, we found 3 to be a sequence error in N2. Based on this we estimate the error rate for the C. elegans genome to be 1 in 42,000. SNPs are enriched on the arms of the five autosomes, which fits with the idea of more rapid evolution on the chromosome arms (C. elegans sequencing consortium, Science. 1998 Dec 11;282(5396):2012-8) .

Bauer CC et al. (1995) Worm Breeder's Gazette "Myotonic Dystrophy Homologs in C. elegans?"

Fleming S, Epstein HF, Bauer CC

[Worm Breeder's Gazette, 1995]

We are interested in determining the possible use of C. elegans as an animal model to study the protein(s) indicated in the human disease Myotonic Dystrophy, (DM). We therefore performed a computer search of the dbEST database to locate cDNAs that were similar in sequence to the DM protein kinase gene, (DMPK). We found four different sequences with protein sequence similarities ranging from 50 to 95% over the short regions that had been sequenced. One cDNA (Accession # T01329) in the databases was originally found by Anthony Kerlavage at the Institute for Genomic Research in Gaithersburg, MD. The clone, CEESP52, came from a C. elegans embryonic cDNA library. One open-reading frame of the clone contained a protein sequence that matches at a score of 71% identity and 95% similarity to part of the catalytic domain of the human DM kinase gene. We have hybridized a probe made from CEESP52 to the Yac contig library and determined that the gene containing the coding sequences is found on Yac Y38B5. Yac Y38B5 is covered by known cosmids except for an approximately 20kb gap in the cosmid contigs. A Southern indicated that the region sequenced did not hybridize to any of the cosmids, therefore we believe that at least part, if not all, of the gene is located in the gap region. We have run pulse field gels and isolated Yac Y38B5 DNA and will be screening subclones of the Yac for the corresponding gene. We will then perform rescue experiments of known mutations or construct our own mutant. As another approach to locating DMPK homologs we have used is employing a monoclonal and polyclonal antibodies made to the human DMPK protein on Western blots containing C. elegans homogenates. The monoclonal antibody identified an approximately 80kDa band on the Western, whereas the polyclonal detected 5 different bands. We are in the process of isolating these genes from a lgt11 embryonic library. Below is a translated protein sequence match of the 4 closest homologs to DMPK found in the dbEST database over a overlapping 100 amino acid region. Two other cDNA sequences in the dbEST database have good similarity scores to DMPK equivalent to the C. elegans Ndr peptide score, but are not in the same region. Both have been mapped and are found on chromosome I (1,2). The partial sequence of the Ndr protein has been recently published and has homologs in both Drosophila and humans (3). .................................................................. Upper Sequence Human DMPK (all matches to the Human DMPK) . 2nd Sequence Accession # T01329 peptide Match Percentage 71% 3rd Sequence C. elegans Ndr peptide Match Percentage 45% 4th Sequence Accession # D37290 peptide Match Percentage 35% . . . : . : . : . : 1 DFEILKVIGRGAFSEVAVVKMKQTGQVYAMKIMNKWDMLKRGEVSCFREE |||:|||||:|||:|||||:|: |::|||||:|||:|:||:|||||| 1 DFEVLKVIGKGAFGEVAVVRMRGVGEIYAMKILNKWEMVKRAETACFREE :| |||||||||:|||: :||::|||||: | :|: : : : | | 1 HFKSLKVIGRGAFGEVRLVQKHDTGHIYAMKILRKSEMVEKEQTAHVRAE :||:|:|:||: :| :|: | :||||:: | :| : : | 1 NFALLRVLGKGAYGKVFLVRKDHNTIYAMKVLRKTRVLTKQKXLEHTMAE . . . : . : . : . 51 RDVLVNGDRRWITQLHFAFQDENYLYLVMEYYVGGDLLTLLSKFGERIP ||||| |||||||:||:|||||: ||:||:||:|||:|||||||:|| 51 RDVLVYGDRRWITNLHYAFQDEKNLYFVMDYYIGGDMLTLLSKFVDHIP ||:|:| |: :: ::||| : ||||||: |||::||| | : 51 RDILSEADCDWVVKMYYSFQDYSNLYLVMEFLPGGDMMTLLIKKATLTE | ||: | :||| : |::|||| ||:| | | | 51 RQVLERLRTPFLVNLXYAFQTDXKLHIVMEYVRGGELFTHLCSRGH .................................................................. 1) Gilchrist, E. and Baillie, D., (1995) A C. elegans Myotonic Dystrophy gene homologue. Abstracts of the 10th International C. Elegans meeting. University of Wisconsin, Madison. P233. 2) Wissmann, A., McGhee, J., Mains, P., (1995) Let-502 is a homolog of human Myotonic Dystrophy kinase. Abstractions of the 10th International C. elegans meeting. University of Wisconsin, Madison. P548. 3) Millward, T., Cron, P., Hemmings, B., (1995) Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase. Proc. Natl .Acad. Sci. USA 92:5022-6.

Rioux SD et al. (1986) Worm Breeder's Gazette "full length abundant protein and message from unc-15 Tc1 alleles."

Waterston RH, Rioux SD

[Worm Breeder's Gazette, 1986]

We have been analyzing spontaneous unc-15 alleles (r404, r405, r406, r407, r408) isolated from a mixed Bristol/Bergerac strain containing unc-105(n490) by Phil Anderson and Amy Bejsovec (Madison, WI). These unc-15 alleles revert spontaneously to wild type and have better muscle organization and better movement than the null e-1214. We used a lambda clone containing unc-15 specific sequence, kindly provided by Hiroaki Kagawa (Okayama U, Japan), to probe Southerns. Using several different enzymes we observed RFLDs that were 1.6kb larger in r408 than in an isogenic revertant. In order to determine if Tc1 was the cause of the mutation in r408 we cloned and sequenced this region of unc-15. The mutation was indeed due to Tc1 and the insertion site in r408 has homology to the consensus sequence derived by Ikue Mori et al (see this newsletter). [See Figure 1] The 600bp of DNA flanking the insertion site is not AT rich nor does it contain any long 0RFs with the predicted alpha-helix protein structure. unc-15 specific message is present in total and poly-A selected RNA from mixed worm populations in all of these mutants. The unc-15 message is the same size and in roughly the same amount in mutants, isogenic revertants, and N2, and there is no Tc1 hybridizing sequence in the unc-15 message. At the protein level paramyosin is easily detected on SD6 gels of whole worm extracts from these mutants. However, the amount of full length paramyosin detected by polyclonal and monoclonal antibodies in these alleles is reduced several fold from wild type levels. Just how the insertion of Tc1 into the unc-15 region produces these weak alleles is not known. From this preliminary data Tc1 appears to be reducing the amount of paramyosin produced. The DNA sequence flanking the insertion site in r408 is not intron or exon but may be in 5' or 3' regions of the gene. Further analysis of all of the alleles will be necessary to explain these observations.

Jeyaprakash A et al. (1988) Worm Breeder's Gazette "cloning of the unc-104 gene by transposon (Tc1) tagging."

Otsuka AJ, Hartshorne TA, Hedgecock EM, Franco R, Jeyaprakash A

[Worm Breeder's Gazette, 1988]

Mutations in the unc-104 gene result in lack of electron dense material at presynaptic nerve endings and absence of neuromuscular junctions. We have cloned unc-104 by transposon (Tc1) tagging as a first step towards understanding the molecular mechanism of this defect. Three alleles of the unc-104 gene (e2184, rh1016, rh1017) isolated from transposon high copy strains have been tested. Southern blots of DNA from wild type N2, mutant (e2184*7). and several spontaneous revertant strains were probed with Tc1. A unique 3kb EcoRI fragment always cosegregated with the mutant phenotype, but was absent in revertant strains. The 3kb fragment was cloned separately into bacteriophage lambda Charon 21A and pUC8 plasmid vectors. The relationship between the transposon (Tc1) containing clone and unc-104 was established by linkage analysis, excision of transposon Tc1 in several spontaneous revertants, and localization of Tc1 insertions in the alleles rh1016 and rh1017 to a nearby restriction fragment. The unc-104 gene has been isolated on 6 genomic clones containing 20 kb of contiguous DNA from a bacteriophage lambda N2 library (kindly provided by N. Nishiwaki and J. Miwa, NEC, Japan ). The genomic clones were mapped to a contig containing unc-104 and tra-2 and 4 cosmid clones containing 80 kb of contiguous DNA have been isolated by Alan Coulson and John Sulston ( MRC, England ). Three cDNA fragments have been isolated from an N2 cDNA library on bacteriophage lambda gt10 ( kindly provided by J. Arhinger and J. Kimble, Univ. of Wisconsin, Madison ) . A 0.75 kb cDNA is the 5' most fragment thus far isolated and corresponds to the region where Tc1 insertion occurs in the e2184 allele. A 1.1 kb cDNA fragment is derived from the downstream 1.4 kb EcoRI genomic fragment containing the sites of Tc1 insertions in the rh1016 and rh1017 alleles. The third cloned cDNA fragment hopefully contains sequences corresponding to the remaining 3' portion of the approximately 5.6 kb message detected by Northern blotting. Only weak homologies have been found to genes and proteins in the Genbank and PIR data banks. [See Figure 1]

Edgley ML (1985) Worm Breeder's Gazette "CGC news."

Edgley ML

[Worm Breeder's Gazette, 1985]

Strain requests: Please remember to request strains by letter, even if you've made a request by phone. Letters should list what strains you want (strain name or gene name is fine), briefly state what you want them for, and state your funding source (no numbers, just the institution or agency). Bibliography: The complete CGC bibliography (on paper) will shortly be mailed to each lab with a CGC laboratory designation. In addition, the bibliography is now available on computer diskette in a variety of formats, including dBase III, IBM-compatible ASCII, Apple II- and Macintosh-compatible ASCII. The ASCII data can be read with line editors and many word processors. To obtain a copy, send me a blank diskette (or diskettes) formatted with PC-DOS, MS-DOS or Apple DOS and tell me which format you want. The bibliography is about 300 kilobytes in size, so send enough diskettes to contain it all. For IBM-type drives, we can handle 1.2 Mb or 360 Kb diskettes. Complete file structure information will accompany each disk sent out. Films: The CGC now has a copy of the Encyclopaedia Britannica film 'Nematode' and a copy of Einhard Schierenberg's embryonic development film. We will loan these films for a period of two weeks to any laboratory with a CGC lab designation. Requests for such loans should be made by letter from the laboratory head to me or Don Riddle at the CGC. Electronic Mail: The University of Missouri is a node of the BITNET computer network, with connections (gateways) to the CSNET, CCNET, UUCP and MAILNET networks. People with local access to BITNET can send electronic mail to the CGC by directing it to 'BIOSCGC at UMCVMB'. Communicating over gateways to BITNET requires a little more complicated addressing. Anyone who wants to try electronic mail should contact me by regular mail first for detailed instructions, so I'll know if the message gets lost. According to our BITNET node list, the following worm-breeder institutions have direct access to BITNET: Cornell University, Columbia University, University of California at Berkeley, MIT, Harvard, University of Illinois at Chicago, North Carolina State University, Washington University, University of Houston, University of Wisconsin-Madison, University of Massachusetts, SUNY at Buffalo, University of California at Santa Cruz, Duke University, University of Texas at El Paso. There are certainly other BITNET nodes that are not on our list. In Europe, EARNET can be used to connect to BITNET.

Link CD (1993) Worm Breeder's Gazette "Senile Worms?"

Link CD

[Worm Breeder's Gazette, 1993]

As I described in my poster at the Madison meeting, I am interested in using transgenic C. elegans animals to model amyloid diseases, such as Alzheimer's disease. The basic idea is to engineer worms to express amyloid-forming human proteins, hope these proteins do something to worms, then use classic suppressor genetics to identify biological factors that are involved in amyloid formation or toxicity. (Whether or not this is a dumb idea is unclear.) On the assumption that constitutive expression of these proteins could possibly have a dominant lethal effect (suboptimal for recovering transformants!), my initial constructs (primarily using Andy Fire's expression vectors) used heat shock and her-1 promoters. These promoters were used to drive an artificial signal peptide/ 1-42 minigene (derived from the human amyloid precursor protein) or, as a control, wild-type human transthyretin. I was unable to detect any phenotypes in animals transgenic for these constructs. I have now done the experiments I should have done in the first place: express these proteins with a high-level promoter and see if any phenotypes result. I have recovered two independent transmitting lines with transthyretin driven by the unc-54 promoter/enhancer in Fire vector pDP30 .38.These animals stain strongly and fairly uniformly with anti-transthyretin antisera; some animals show intense coelomocyte staining. My interpretation of these results is that since this construct contains the natural signal peptide, it is being produced and secreted by the body wall muscle cells and scavenged by the coelomocytes. These transgenic animals have no obvious phenotypes. I have recovered 4 transmitting lines transgenic for the corresponding unc-54 /signalpeptide/ 1-42 minigene. At least two of these lines have a discernible phenotype: at 25 , V4 -V3of Rol progeny show a progressive paralysis as adults. All of these lines show muscle-specific deposits of anti- amyloid immunoreactivity; I do not have good enough data yet to determine if the sicker lines show more immunoreactivity. I do not have enough experience with unc-54 constructs to know if the unc-54 / 1-42results are exciting, or just what one expects when you express high levels of any generic heterologous protein. (Is a bogus phenotype better than no phenotype?) At present I have two candidate integrated lines generated by gamma irradiation; both are significantly sicker than the parental extrachromosomal array line, but neither have been out-crossed.

Lobel L et al. (1994) Worm Breeder's Gazette "The egl-19 Locus Might Encode a Homolog of the Alpha 1 Subunit of the Voltage-Gated Calcium Channel"

Horvitz HR, Avery L, Lobel L, Lee RYN

[Worm Breeder's Gazette, 1994]

We reported in the last issue of the Worm Breeder's Gazette (13(1): 46) the identification of multiple loci in C. elegans encoding homologs of the alpha 1 and beta subunits of the mammalian voltage-gated calcium channel. Since then, we have rescued egl-19 (n582 )IVmutant animals with cosmid clones that contain a homolog of the alpha 1 subunit of the voltage-gated calcium channel. egl-19 is an egg-laying defective mutant that is also long, and uncoordinated. Microinjection of the three cosmids C11E10 ,B0496 ,and C48A7 (localized to the contig on LGIV between lin-45 and col-4 )into egl-19 mutant animals yielded F1 progeny that displayed coordinated movement, normal length, and near wild-type egg-laying behavior (a few transformants were slightly egg-laying defective). Each of these cosmids hybridized to our PCR clone of the alpha 1 subunit of the voltage-gated calcium channel. We are currently narrowing the region of rescue with cosmid subclones. We also reported previously (C. elegans Meeting abstracts, Madison, WI, 1993, pg. 177) that unc-36 mutants, which are defective in the alpha 2 subunit of the voltage-gated calcium channel, are hypersensitive to the calcium channel antagonist verapamil. We have now found that egl-19 animals are also hypersensitive to verapamil. Greater than 95% of egl-19 animals assayed failed to pump in 3 mM verapamil, while wild-type animals displayed only a modest decrease in their pumping rates (about 10-15%). We have been studying the genetics of egl-19 .We suggested in the last WBG (13(1): 45) that the dominant eat-12 mutations n2368 and ad695 and the recessive egl-19 mutation n582 might be gain-of-function and loss-of-function mutations, respectively, in the same gene. New data support this hypothesis. First, we failed to recover wild-type recombinants among 12,800 progeny of egl-19 (n582)/eat-12 (n2368)unc-24 hermaphrodites, showing tight linkage (<0.05 map units) between n582 and n2368 .Second, two apparent intragenic revertants of n2368 partially failed to complement n582 . We also wondered if there might be genetic interactions between egl-19 and unc-36 .Preliminary results with single alleles of each locus are as follows: 1) unc-36 (e251);egl-19 (n582)double mutants are nearly completely paralyzed, while unc-36 and egl-19 single mutants, although slightly uncoordinated, can move fairly well; 2) unc-36 ;egl-19 /+worms are egg-laying defective, unlike either unc-36 or egl-19 /+animals. These results suggest that unc-36 and egl-19 might interact. We speculate that the protein products of these genes physically interact in muscles involved in egg-laying, as would be expected for the alpha 1 and alpha 2 subunits of the voltage-gated calcium channel.

Thacker CM et al. (1997) Worm Breeder's Gazette "The Growing Family of Kex2/subtilisin-like Proprotein Convertases in C. elegans"

Thacker CM, Rose AM

[Worm Breeder's Gazette, 1997]

Many physiologically important proteins are expressed as large inactive polypeptides, examples of which include growth factors, growth factor receptors, hormones, peptide neurotransmitters, viral glycoproteins and several metalloproteinases. One way in which the majority of these proproteins can be activated is by their limited proteolytic cleavage at dibasic residues, a reaction catalyzed by members of the kex2/subtilisin-like proprotein convertase family (kexins or PC) of serine endoproteinases. In vertebrates at least seven different members of the family have been identified and include furin, PC1/3, PC2, PC4, PC6, PC7 and PACE4. Our laboratory identified the first C. elegans member of the family as bli-4 which encodes at least four gene products that arise by alternative slicing. The combined efforts of ourselves, the genome sequencing consortium and the laboratory of Ian Dickerson at the University of Miami School of Medicine, have identified a further three nematode members of the family. These three members include genes with sequence similarity to furin and PC7, both of which reside on the right arm of chromosome I, and a PC2 homologue on chromosome V. We are interested in determining the role of each of the kexins during C. elegans development and towards this end we are attempting to isolate mutants for these three genes using the targeted approach described by Gary Moulder and Bob Barstead at the recent Worm meeting at Madison, WI (and personal communication). Continued studies on the bli-4 gene products suggests that the major role of these convertases is the processing of cuticle collagens during development, although we have not ruled out the possibility that other substrates are cleaved. We expect the furin homologue like its vertebrate counterpart, to recognize a large number of proproteins since this member of the PC family appears to be the major role player. Curiously the C. elegans gene differs markedly at the carboxy-terminus when compared with that from Drosophila and vertebrate proteins in that the worm protein does not contain a transmembrane domain even though the protease domain is highly conserved. Vertebrate PC2 is active in the regulated secretory pathway and has been shown to process a number of prohormones and peptide neural transmitters. PC7, like furin, is expressed ubiquitously in mammalian cells and is thought to be largely responsible, amongst other roles, for the processing of viral glycoproproteins including infection by HIV. Intriguingly, the nematode gene F32A7.6 is expressed as part of an operon with unc-54. The kexin gene is trans-spliced to SL2 which is consistent with it being the downstream gene in the operon. Obviously, expression of the kexin gene under the control of the unc-54 promoter raises the possibility that the role of the convertase is highly restricted to muscle tissue and we have recently isolated candidate mutants which may help us determine the function of this serine endoproteinase.