Rogalski TM et al. (1992) Worm Breeder's Gazette "The Sequence of unc-52"

Moerman DG, Rogalski TM

[Worm Breeder's Gazette, 1992]

The products of the unc-52 gene have an essential role in embryonic development, and are required for maintaining the normal structure of the body wall muscle cells and the somatic gonad in adult worms. We have completed the genomic sequence of this gene which is approximately 14.2 Kb in length and consists of 26 exons. Analysis of cDNAs indicates that there are at least three different transcripts with three different 3' ends. No full length cDNAs have been obtained so we do not known if there is more than one 5' end. The sequence of unc-52 suggests that its products are proteoglycans and are probably components of the extracellular matrix. The protein structure of three putative unc-52 products are shown in the accompanying diagram. All three proteins are shown with the same amino terminal sequences which consist of a signal peptide and an acidic region which may contain two heparin-sulfate binding sites. These are followed by a single C2 Immunoglobulin superfamily (Ig) repeat, three LDL-Receptor like repeats, another Ig repeat, two Laminin like repeats and 14 Ig repeats. One of the cDNAs obtained ends after the Laminin like repeats. The other two cDNAs differ in the number of Ig repeats, and all three cDNAs encode different carboxy terminal amino acids. The sizes of the three proteins are approximately 270,000, 230,000 and 125,000 Daltons. [See Figure 1]

Chan S et al. (1994) Worm Breeder's Gazette "The unc-40 Axon Guidance Gene Encodes a Transmembrane Receptor With Extracellular Fibronectin and Immunoglobulin Domains"

Culotti J, Chan S, Zheng H, Su M-W

[Worm Breeder's Gazette, 1994]

The unc-40 axon guidance gene encodes a transmembrane receptor with extracellular fibronectin and immunoglobulin domains. Shirley Chan, Ming-Wan Su, Hong Zhang, Joe Culotti Samuel Lunenfeld Research Institute of Mt. Sinai Hospital Toronto, Canada Ed Hedgecock, Johns Hopkins University, Baltimore unc40 primarily affects ventrally oriented pioneer growth cone and cell migrations that occur on the epideImis. unc40 also has rninor effects on dorsally oriented pioneer migrations (Hedgecock et al., Neuron 4:61-85,1990). We mapped unc40 relative to strain-specific DNA polymorphisms, then rescued the Unc and DTC defects by germline transformation with a cosmid and with a 14 kb phage from the region. Based on germline transformation experiments using overlapping cosmid and phage clones, we were able to identify a region required for rescue that encodes a single transcript by Northern analysis of 5.5 kb. We cloned and sequenced a 4.2 kb cDNA (S. Kim library) corresponding to this transcript. This cDNA contains a 3.3kb open reading frame. The predicted unc- 40 protein (so far) has a single hydrophobic transmembrane domain, six fibronectin type III (FNIII) domains and two immunoglobulin (IG) domains. FNIII and IG domains are found in a variety of transmembrane proteins, but these most commonly have three or five FNIII domains. Several transmembrane proteins that have extracellular IG and FNIII domains also have intracellular tyrosine kinase or phosphotyrosine phophatase domains, but the predicted 309 residue intracellular region of unc-40 has no obvious similarity to these domains. We are currently sequencing genomic DNA to identify the 5' end of unc40. Northern and Southern analyses have failed to reveal detectable deletions or rearrangements in a dozen unc40 mutants analyzed. In addition to affecting migrations on the dorsoventral coordinates of the epidermis, unc-40 mutations also affect body shape, normally an epidermal function. To determine where unc-40 is expressed and where it is required for guidance, we are constructing strains to do mosaic analysis and to analyze patterns of expression from the unc-40 promoter.

Felder H et al. (1990) Worm Breeder's Gazette "The Complete Nucleotide Sequence of the Retrovirus-Related Element TAS Present in the Genome of Ascarises"

Herczeg A, Mueller F, Aeby P, Tobler H, Felder H

[Worm Breeder's Gazette, 1990]

The proretrovirus like element TAS is found in about 50 copies in the haploid genome of the nematode A. Lumbricoides. One complete copy was sequenced. The putative gag region near the 5' LTR contains a 1962 bp long open reading frame which codes for a RNA binding domain. Near the center of the element we found on the +1 frame (relative to the gag frame) a region which shares some characteristics with known pol genes. However, this part of the sequence is interrupted by four stop codons. One of the putative polypeptides contains.the amino acid sequence YVDN, a slightly mutated version of the box YXDD which is present in almost all known reverse transcriptases. We conclude that stop codons as well as the change in the sequence of the YXDD box are the result of occasional point mutations and that the sequenced element may represent a nonfunctional copy of TAS. The YVDN box is carboxyterminally flanked by a conserved RNase H and a zinc finger motif. The latter characterizes a possible endonuclease domain. A further 2061 base pair spanning ORF near the 3' LTR of TAS corresponds to an env region. TAS represents obviously a retrovirus related sequence with a similar gene arrangement as, e.g., that of the mobile element 17.6 in the Drosophila genome and that found in some genomes of retroviruses.

Jefferson RA et al. (1984) Worm Breeder's Gazette "expression of a chimeric gene transformed into the worm."

Jefferson RA, Hirsh DI

[Worm Breeder's Gazette, 1984]

We have constructed fusions of the col-1 gene from the worm with the E. coli -glucuronidase gene, introduced them into the germline of the worm, and studied their expression. The fusions consist of the promoter region and the first several nucleotides of the col-1 coding sequence, the entire coding sequence of the bacterial -glucuronidase gene, col-1 sequences encoding its 3' intron and the terminator region. The fusions were either in frame or out of frame with the col-1 initiator codon. These constructions were injected into the gonad of gus-1(DH408) worms and detected in the progeny by dot blot hybridization. DH408 is a strain with no detectable -glucuronidase activity, and is used to increase the sensitivity with which we can measure the chimeric activity (Bazzicallupo, Jefferson and Hirsh, unpubl.). -glucuronidase activity was measured in extracts of the transformed worms. DH408 transformed with the in-frame fusion showed glucuronidase activity, while two independent transformed lines carrying the out-of-frame fusion showed no detectable activity. As the DNA was in the long extrachromosomal tandem array, we could use its characteristic instability to perform a cosegregation analysis. There was always a strict correlation between the presence of the transforming DNA and the glucuronidase activity. We verified that the glucuronidase activity in extracts from the transformed line was due to the bacterial enzyme by using affinity purified antibodies directed against the purified bacterial glucuronidase. col-1 has been shown to be transcribed primarily in the embryo ( presumably during the synthesis of the L1 cuticle) but also at lower levels in later stages (Kramer, Cox and Hirsh, in press). To see if the col-1: glucuronidase fusion was being regulated temporally, we prepared extracts from staged populations of transformed worms and measured glucuronidase specific activity. Specific activity is highest in eggs, and decreases markedly in later stages. These experiments are being repeated, but although they are consistent with the wild-type col-1 transcription profiles, they are not sufficient to indicate temporal regulation. To analyze spatial distribution of the glucuronidase activity, we have used enzyme histochemistry and immuno- cytochemical techniques. Although we can visualize activity in eggs using the histochemical stains, we have not yet achieved the resolution to say anything about localization. These experiments are in progress.

Rushforth AM et al. (1992) Worm Breeder's Gazette "Structure of Tc1-containing mRNAs"

Rushforth AM, Anderson P

[Worm Breeder's Gazette, 1992]

We previously characterized three Tc1 -inducedmutants of the regulatory myosin light chain gene, mlc-2 (WBG 12(2):59). Each of these mutants contains less than wild-type levels of mlc-2 mRNA. In each case all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. For mlc-2 (r948::Tc1),the 3' splice acceptor used within Tc1 is unusual; the last dinucleotide of the "intron" is GG rather than the canonical AG typical of eukaryotic introns. To determine if splicing to remove Tc1 from pre-mRNAs is unique to mlc-2 ,or if it is a general property of Tc1 -inducedalleles, we characterized the structures of mRNAs from Tc1 -inducedalleles of unc-54 ,the myosin heavy chain gene and of hlh-1 ,the C. elegans homolog of the vertebrate MyoD gene family (CeMyoD). We used the PCR in combination with sib-selection (WBG 11(5):65) to isolate Tc1 -inducedmutations of hlh-1 .hlh-1 (r1010::Tc1)is a Tc1 insertion within the hlh-1 second exon. r1010 homozygotes are viable and have no obvious muscle defects. In this mutant, the normal 5' splice donor of the first intron is spliced to a 3' splice acceptor within the right inverted repeat of Tc1 (at position 11). Use of these sites removes all of Tc1 ,except 12 nt, plus 18 nt of hlh-1 from the mature message. The resulting mRNA can be translated in-frame to yield a CeMyoD protein containing an insertion of 4 amino acids from Tc1 ,plus a deletion of 6 amino acids from CeMyoD. The 3' splice acceptor used within Tc1 is unusual; the terminal dinucleotide of this "intron" is TG rather than the canonical AG. We have also analyzed two Tc1 -inducedalleles of unc-54 that were previously isolated in a genetic screen for muscle defective mutants. These two insertions, r323::Tc1 and r328::Tc1 ,are at the same position, but in opposite orientations within the unc-54 third exon. We estimate that these mutants contain 20-50% of wild-type levels of an approximately normally sized unc-54 mRNA. In addition, r328 contains a small amount of an unc-54 mRNA that is approximately 1.6 kb larger than wild-type. In these mutants, a cryptic 5' splice donor, 33 nt upstream of the normal splice donor, is spliced to the normal 3' acceptor of the third intron. Use of these sites removes all of Tc1 plus 33 nt of unc-54 from the mature message. The resulting mRNAs can be translated in-frame to yield myosin heavy chain (MHC) proteins deleted for 11 amino acids. The 5' splice donor within unc-54 is unusual; the first two nucleotides of the "intron" are TT rather that the canonical GT typical of eukaryotic introns. In spite of the small in-frame deletions in the MHC proteins, r323 and r328 are indistinguishable from unc-54 null mutants. The 11 amino acids are deleted from the MHC globular head region; presumably these amino acids are important for MHC protein stability, function or thick filament assembly. For all six Tc1 -inducedmutations we characterized, a fraction of the mutant pre-mRNA is spliced in a manner that leaves the mature message in-frame. Perhaps a variety of different splice sites are used to splice Tc1 from the mutant pre-mRNAs. We expect mRNAs that are spliced in a manner that leaves nonsense or frameshift mutations in the message would be degraded by the C. elegans smg gene system (WBG 11(5):90). The small amount of in-frame mRNAs present in these mutants is probably stable because these mRNAs are translated throughout their entire length and therefore are not substrates for smg-dependent degradation.

Pretot RF et al. (1996) Worm Breeder's Gazette "ceh-26, a putative prospero (pros) homologue"

Pretot RF, Burglin TR

[Worm Breeder's Gazette, 1996]

In Drosophila the homeodomain encoding pros gene has been shown to be required for activation of the homeobox genes even-skipped (eve) and fushi-tarazu (ftz ) in ganglion mother cells (GMCs). During mitosis of the neuroblasts, the protein is segregated in a cortical crescent that becomes part of the resulting GMC. Only in established interphase, pros protein migrates into the nucleus, where it presumably acts as a transcription factor. The genome project sequenced an open reading frame (K12H4.1, which we call now ceh-26 ) with sequence similarity to pros*. The homeodomain of ceh-26 is atypical, with 3 extra amino acids between helix 2 and helix 3. In addition, there is a conserved domain following the homeodomain (see Fig. 1). Using the open-reading-frame (ORF) predicted by Genefinder, we succeeded in generating cDNAs using PCR. However, sequencing showed various PCR induced point mutations. With these PCR cDNAs, we isolated cDNAs from the Chris Martin and the Pete Okkema libraries. One consistent difference to the sequence from the genome project, an extra base, was found in all cDNAs looked at. Overall, the ORF prediction was confirmed, except for one splice donor. This difference in the splice pattern is necessary to compensate for the extra base to produce the correct ORF. The cDNAs are now being used to generate fusion proteins. Upon obtaining antibodies, we will be able to determine, whether there is a similar phenomenon of asymmetric protein localization in C. elegans.

Rogalski TM et al. (1990) Worm Breeder's Gazette "The unc-52 gene encodes a cell adhesion molecule."

Rogalski TM, Moerman DG

[Worm Breeder's Gazette, 1990]

Animals homozygous for certain unc-52 mutations exhibit a progressive paralysis, the onset of which can occur as early as the L3 stage or as late as the mature adult depending on the allele. Examination of body wall muscle in these mutants reveals that paralysis is due to the detachment of the muscle filaments from the outer membrane. Recently, recessive lethal alleles of unc-52 have been identified ( Williams and Waterston, WBG 11(1):45). Hermaphrodites heteroallelic for a non-lethal allele over a lethal allele exhibit the paralyzed phenotype and, in addition, are sterile. The gonad in adult animals is abnormal when viewed by Nomarski optics. Curiously, the same non- lethal allele over a deficiency for the region, jDf4, results in a fertile, paralyzed worm. The unc-52 gene has been cloned by Tc1-tagging, and two non- overlapping, partial cDNAs have been isolated and sequenced. One cDNA contains the Tc1 insertion site, and its deduced 700 a.a. open reading frame shows similarity to the cell adhesion members of the immunoglobulin superfamily. The most striking similarity is with a mouse basement membrane proteoglycan (Noonan et al., J. Biol. Chem. 263:16379-16387, 1988). The second cDNA is approximately 3 Kb upstream from the first and is transcribed in the same direction. This 350 a.a. open reading frame has homology to the laminin B chains, as well as a laminin-like domain of the mouse basement membrane proteoglycan mentioned above. The genomic DNA between these two regions is currently being sequenced to determine if they are both part of unc-52.

Costa M et al. (1988) Worm Breeder's Gazette "a putative mab-5 deletion disrupts a gene containing a homeobox."

Kenyon CJ, Costa M

[Worm Breeder's Gazette, 1988]

We are cloning the mab-5 gene to study the molecular mechanism by which patterns of cell types arise along the anteroposterior body axis of C. elegans after hatching. As a brief review, mab-5 is required to give cells located in the posterior body region their normal identities. This gene influences the fates of epidermal, neural, and mesodermal cells in a cell-autonomous fashion. It affects sequences of cell division, cell death, fates of specific neurons, production of mating structures and sensilla, cell fusion, and also directions of cell migration. The simplest way to explain most of the differences between mab-5 mutants and the wild type is to postulate that this gene allows posterior cells to develop differently than they would if located further anteriorly. Differences between mab-5 and wild type males and hermaphrodites also suggest that mab-5 interacts with elements of the sex determination pathway to generate much sexual dimorphism in the posterior body region. We identified and cloned a closely linked Tc1 element in the strain N62. We obtained cosmids in this region from John Sulston and Alan Coulson (thanks!) and used them to probe Southern blots of mab-5 mutants. In the mab-5 (e2088) mutant, we detected a deletion of about 150 bp located approximately one cosmid length, or 35 Kb, from the Tc1 element. To test whether this deletion could be in the mab-5 gene, we carried out high resolution mapping experiments. We found that the Mab-5 phenotype and the deletion always cosegregated in 102 recombinants within the 0.5 mu sma-3 to unc-36 interval. Since we examined 38 crossovers in the approximately 315 Kb interval between mab-5 and unc-36 (A. Coulson, pers. comm.), we expect about one crossover per 8 Kb. Thus it seems quite likely that this deletion is the mab-5 mutation. We have sequenced wild type genomic DNA in this region and determined that part of a large open reading frame is deleted in e2088. By Northern analysis, we found that this open reading frame is part of a polyadenylated RNA present in larvae (we have yet to look at other developmental stages). Currently, we are doing Northern analysis of the mab-5 mutants and transformation experiments to provide further evidence that we have cloned mab-5. We have sequenced partial cDNAs containing this open reading frame isolated from a library kindly given to us by C. Thacker and M. Capecchi. By comparing the sequence to known protein sequences, we have found that the open reading frame contains a homeobox highly homologous to those of Drosophila pattern formation genes. This putative mab-5 homeodomain is most similar to that of the Antennapedia gene. Of the 60 amino acids in the mab-5 homeodomain, 44 are identical to those of Antennapedia, and only 4 of the substitutions are not conservative. The figure below shows these homeodomains using dashes to represent amino acids identical to those of mab-5 with conservative substitutions underlined. Assuming that this sequence corresponds to mab-5, this similarity indicates that homeodomains are used to generate global aspects of body pattern in animals other than Drosophila. At a more mechanistic level, since homeodomain proteins are thought to be transcriptional regulators, it also suggests that mab-5 exerts its many influences on cell division, differentiation, and migration by regulating gene expression. [See Figure 1]

Hedgecock EM et al. (1990) Worm Breeder's Gazette "An SH3 Motif in the Cytoplasmic Domain of the unc-5 Protein?"

Hedgecock EM, Culotti JG, Leung-Hagesteijn CJ, Spence AM, Stern BD

[Worm Breeder's Gazette, 1990]

The unc-5 gene encodes a hypothetical trans-membrane receptor with an extracellular N-terminus, comprising one Ig and two tandem WSX domains, that could bind to unc-6 laminin B2, plus a large cytoplasmic C-terminus of unknown function (CSH Abstracts pp. 275, 276 (1989)). The cytoplasmic region contains a 50 aa sequence that is distantly related to the SH3 motif found in a variety of intracellular proteins associated with the membrane cytoskeleton including non-receptor tyrosine kinases (c-src, c-abl), phospholipase C (PLC), spectrin, type I myosins, and a yeast actin-binding protein (ABP1). All of these proteins interact, directly or indirectly, with actin microfilaments near the cell membrane. The function of the SH3 motif itself, however, is unknown for these proteins. unc-5 matches the SH3 consensus more poorly than bona fide SH3 proteins and may not be functionally related. However, unc-5 is required for guiding cell and axon migrations and local modulation of membrane cytoskeleton at the leading edge of migrating cells or growth cones has been proposed as a steering mechanism. In Drosophila, an SH3 protein, c-abl, affects axon growth in some mutant backgrounds. [See Figure 1]

Jin YS et al. (1999) Worm Breeder's Gazette "A C. elegans NeuroD gene functions in motor neuron fate specification"

Hallam SJ, Singer E, Jin YS, Waring DA

[Worm Breeder's Gazette, 1999]

The basic Helix-Loop-Helix (bHLH) transcription factor NeuroD has been implicated in neuronal fate specification, differentiation and survival . Here we report the expression and function of cnd-1, a C. elegans NeuroD gene. We isolated cnd-1(ju29) in a screen for mutations affecting the localization of Punc-25 SNB-GFP, a presynaptic marker expressed in GABAergic DD and VD motor neurons. ju29 animals kink when moving backward. ju29 was mapped to the dpy-17lon-1 interval on chromosome III by standard linkage analysis. A 10 kb subclone of cosmid ZC129 was capable of rescuing the uncoordinated phenotype. This subclone contains one predicted open reading frame that is most similar to NeuroD. The bHLH domain of cnd-1 shares approximately 55% identity and 72% similarity to vertebrate NeuroD proteins . cnd-1(ju29) contains a G to A transition in the splice acceptor site of exon 3, changing TTTCAG to TTTCAA.