Miller DM et al. (1992) Worm Breeder's Gazette "unc-4 LacZ Expression in A-Type Motor Neurons"

Miller DM, Niemeyer CJ

[Worm Breeder's Gazette, 1992]

unc-4 LacZ expression in A-type motor neurons David M. Miller and Charles J. Niemeyer, Dept. of Cell Biology, Duke Univ. Medical Ctr, Durham, NC 27710

Waddle JA et al. (1990) Worm Breeder's Gazette "Candidate CapZ genes from C."

Waterston RH, Waddle JA, Cooper JA

[Worm Breeder's Gazette, 1990]

CapZ is one of a family of actin binding proteins that may regulate actin dynamics in eukaryotic cells. This heterodimeric protein is located at the Z-line of vertebrate skeletal muscle in situ (1). Since the ends of actin filaments are also located at the Z-line, CapZ could function in the assembly or tethering of thin filaments at this location. The Z-lines of vertebrate skeletal muscle also contain the proteins alpha-actinin and vinculin. These two proteins have been localized to the dense bodies of nematode body wall muscles (2,3). Collectively, the above observations suggest that a CapZ homolog might also be present at the dense bodies or attachment plaques of nematode muscle. On this premise, we have initiated a search for CapZ homologs in C. elegans.Sequence data from cDNA clones encoding the CapZ subunits of chicken, (4,5), D. discoideum (6) and S. cerevisiae (7,8) were used to design degenerate oligonucleotide primers for the polymerase chain reaction. Direct amplification of nematode CDNA libraries (kindly provided by R. Barstead) generated candidate fragments for both subunits. A comparison between the deduced amino acid sequence from the nematode PCR products and the homologous regions in other organisms yielded the following table of

Sarkis GJ et al. (1986) Worm Breeder's Gazette "cathepsins Ce1, Ce2, and Ce3 in C elegans."

Sarkis GJ, Jacobson LA, Ashcom JD

[Worm Breeder's Gazette, 1986]

Two acid thiol proteases previously designated as cathepsins L 1 and L2 [Kurpiewski et al., Gazette Vol. 8, No. 3, 1984] have now been renamed cathepsins Ce1 and Ce2, since further studies have shown that they are not precisely analogous to the mammalian cathepsin L. Both proteases prefer Z-phe-arg-aminomethylcoumarin (AMC) to Z-arg-arg-AMC, and in this respect resemble mammalian cathepsin L rather than cathepsins B or H. On the other hand, both Ce1 and Ce2 show carboxydipeptidase activity toward the model substrate leu-trp-met-arg- phe-ala, and in this respect resemble cathepsin B rather than cathepsin L. Like the mammalian thiol proteases, both Ce1 and Ce2 are inhibited by EP-64, leupeptin and antipain. Both are insensitive to the trypsin inhibitors from soybean and pancreas, to alpha- 1 - antitrypsin and to pepstatin. Three lines of evidence indicate that Ce1 and Ce2 are distinct gene products. First, Ce1 has much more endoprotease activity (using FITC-casein as substrate) than Ce2, relative to the activity of each against Z-phe-arg-AMC. Second, isoelectric focusing in nondenaturing slab gels shows that Ce1 has a pI of 6.8, whereas Ce2 has a pI of 4.7. This seems too large a difference to attribute to variant post-translational processing. Finally, the age-dependences of Ce1 and Ce2 are different (Sarkis et al., this issue). We are still working on the development of differential screens for mutants deficient in each of these enzymes. A third protease has also been separated during purification of Ce1 and Ce2 on DEAE-cellulose. We call this enzyme cathepsin Ce3. It has strong endoproteolytic activity toward FITC-casein at pH 5.5, but no activity toward Z-phe-arg-AMC, Z-arg-arg-AMC, arg-AMC, N-succinyl-(ala) 3-AMC, glutaryl-phe-AMC, or any other model substrate we have tried. It does not hydrolyze the model hexapeptide leu-trp-met-arg-phe-ala. Unlike Ce1 and Ce2, it does not require a thiol for activity, but is inhibited by millimolar concentrations of DTT. It is insensitive to leupeptin, EP-64, pepstatin, antipain, STI and PTI. Its pI is about 7. 4. The major protein band in our Ce3 preparations reacts covalently with alpha-2-macroglobulin, a general protease-trapping inhibitor. This suggests that the major band is cathepsin Ce3. We have looked at crude extracts -- which have been depleted of cathepsin D by affinity chromatography -- by IEF and solid-phase casein zymography. The predominant activities are in the positions of cathepsins Ce 1 and Ce3, with a minor band at the position of cathepsin Ce2. Thus, most of the endoprotease activity at acid pH is attributable to cathepsins D, Ce1 and Ce3. For those of you whose interest in proteases is limited to protecting your favorite proteins or enzymes during isolation or assay, we recommend a mixture of pepstatin (an inhibitor of cathepsin D) and EP-64. The former is a queer pentapeptide; the latter is an active- site directed inhibitor based upon an active epoxide group. Neither is likely to interfere with your pet enzyme or protein. Both are available from Sigma. We suggest concentrations of about 10 M.

Roubin R et al. (1996) Worm Breeder's Gazette "AN FGF GENE IN THE WORM?"

Thierry-Mieg D, Birnbaum D, Roubin R

[Worm Breeder's Gazette, 1996]

A putative gene coding for a heparin-binding growth factor protein and presenting 30 % similarity with the Fibroblast Growth Factor 5 (FGF5) has been identified on chromosome III of C.elegans (Wilson, R et al, Nature, 1994, 368, 32-38, and Du, Z, unpublished data). The genomic clone CO5D11 carries a complete copy of the gene but no cDNA has yet been identified. In mammals, FGFs are important for embryonic development and have been implicated in some pathologies. Recently, Stern's group has shown that egl-15 encodes a member of the FGF receptor family in C.elegans (DeVore, D. l. et al, Cell, 1995, 83, 611-620). In both C.elegans and D.melanogaster, FGF receptors are involved in cell migration.

Gengyo-Ando K et al. (1986) Worm Breeder's Gazette "progress in sequencing of the unc-15 paramyosin gene."

Gengyo-Ando K, Kagawa H

[Worm Breeder's Gazette, 1986]

DNAfragments from -HK2 clone which contained paramyosin gene were processed and get results as follows. Nucleotide sequences of the HindIII 5kb were and HindIII 1.4kb fragments were nearly completed. There many ORFs with predicted-helix protein structure. Polarity of ORF was determined from the amino acid sequence predicted with the nucleotide sequence. Many fusion proteins(Miller et al, Pro. N. A. S.(1986) 83, 2305-2309) were isolated and were characterized by immunoassay. One of them which was corresponded to the 3'end part of the HindIII 5kb fragment crossreacted with a monoclonal antibody which only crossreacted with full sized paramyosin. Our results and the information from Stephan D. Rioux(personal communication and the worm gazette, Vol. 9, No.2, p39), Tc1 insertion site of TR605 munant located at the 3' region of the gene. There were three polyA site in the HindIII 1.4 kb fragment. Although we are not sure which site is a real polyA site the result suggests that Tc1 insertion into 3' noncording region might decrease the termination of the message. C. a fashionable organism to study in Japan. I do hope you could have a time to attend a workshop in Japan near future.

Hsu DR et al. (1987) Worm Breeder's Gazette "some X-linked deficiencies masculinize her-1(n695) XX animals."

Hsu DR, Meyer BJ

[Worm Breeder's Gazette, 1987]

n695 is a semi-dominant allele of her-1 which partially masculinizes XX animals. In an n695 homozygous strain, 100% of the XX hermaphrodites are egg-laying defective (Egl). A smaller subset of these animals are further masculinized and exhibit a partial transformation of the tail towards a more male fate (Tra) (Carol Trent, personal communication). These weak masculinizing effects of n695 are sensitive to small perturbations in the rest of the sex determination pathway. For example, n695 in combination with weak alleles of tra-2 masculinizes XX animals much more than either single mutation alone. In addition, duplications of X which have no phenotype in a her-1(+) background can suppress the masculinization caused by n695 (Anne Villeneuve, personal communication). Using the phenotype of n695 as an assay, we are attempting to identify regions of the X chromosome which interact with the sex determination pathway in a dose-dependent fashion by looking for X- linked deficiencies which increase the masculinization caused by n695. These deficiencies could interact with the rest of the sex determination pathway by removing a region of X that is important in the assessment of the X/A ratio. As a consequence, the apparent X/A ratio would be reduced and the animal would be directed toward a more male developmental fate. Alternatively, they might uncover a haplo insufficient gene that is required for hermaphrodite development in n695 XX animals, thus revealing a function that is likely to be important for proper sex determination in wild-type animals. Thus far, the n695-deficiency assay has localized several regions of the X chromosome, which when present in one copy, cause n695 XX animals to be highly masculinized. One masculinizing region, defined by the deficiency uDf1, results in the production of some n695 XX males which are able to mate. This deficiency, uDf1, has been shown by L. Miller to uncover the gene xol-1. This result is in agreement with the previous finding that some n695; xol-1(y9)/+ XX animals are transformed into males capable of mating (L. Miller, personal communication). Other masculinizing regions have been identified on the right arm of X. We have looked at the nested set of deficiencies on the right arm of X and have found that a subset of these also have a dominant masculinizing effect when placed in combination with n695 (Table 1, Figure 1). In one copy, these deficiencies increase the number of transformed animals. These deficiencies define two small regions which when present in one copy appear to result in further masculinization of n695 XX animals. [See Figure 1] [See Figure 2]

Holdeman R et al. (1995) Worm Breeder's Gazette "MES-2 Contains a Highly Conserved Motif Characteristic of Proteins Involved in Regulating Gene Expression via Chromatin Structure"

Srome S, Nehrt S, Holdeman R

[Worm Breeder's Gazette, 1995]

We have been studying maternal-effect sterile (mes) mutants in an effort to understand how development of the germ line of C. elegans is maternally controlled. Four genes, mes-2, mes-3, mes-4, and mes-6, all give a similar mutant phenotype. Specifically, progeny of mes/mes mothers undergo apparently normal embryogenesis and develop into heathy appearing adults but fail to produce a functional germ line. Mes-2 mutant larvae can produce as many as 80 germ nuclei by the L4 larval stage. There is then an obvious and extensive die-off of germ nuclei, which results in adult worms having only ~ nine germ nuclei and no mature gametes (Capowski et al., 1991; Paulsen et al., 1995). mes-3 and mes-6 have previously been cloned (mes-3: Paulsen et al., 1995); they encode proteins that lack significant similarity to any described proteins. Transformation rescue of Mes-2 mutants was accomplished by injecting overlapping genomic fragments of 7 and 13 kb in length from cosmid R06A4 on LGIIR. These fragments, which both recognize a 2.5 kb Northern transcript, were used to screen a cDNA library. A candidate cDNA clone of 2.5 kb, which also recognizes the 2.5 kb transcript, was identified in the screen. Injection of both sense and antisense RNA produced from this cDNA phenocopied the Mes-2 mutant phenotype in wild-type worms. Worms injected with control RNA did not show this phenotype. The mes-2 cDNA encodes a protein that contains a 150 amino acid motif known as the SET domain. This motif gets its name from three Drosophila proteins in which it is found: Trithorax (Trx), Suppressor of variegation 3-9 [Su(var)3-9], and Enhancer of zeste [E(z)] (see WBG for figure). SET domains have also been found in proteins from plants, yeast, and mammals. The E(z) protein, to which MES-2 is most similar, is a member of the Polycomb group (PC-G) genes in Drosophila. These genes are believed to be needed for the long-term maintenance of transcriptional inactivation of specific genes, such as the homeotic genes. PC-G proteins are thought to work by affecting chromatin structure, although they do not seem to bind DNA directly. Current thinking is that these proteins form large complexes with other gene products and control chromatin structure at specific sites (reviewed in Paro, 1995). Genetic evidence suggests that the Mes mutant phenotype may be due to defects in X-chromosome gene expression, specifically in the germ line. The similarity of MES-2 to E(z) and other SET domain-containing proteins, which have a long-term role in regulation of expression, is intriguing in light of our suspicion that mes genes may affect gene expression on the X chromosome. We are in the process of raising antibodies to MES-2 in order to further analyze this possibility. References: E. Capowski, P. Martin, C. Garvin, and S. Strome (1991) Genetics 129, 1061-1072. J. Paulsen, E. Capowski, and S. Strome (1995) Genetics, in press. R. Paro, TIG (1995) 11 (8), 295-297.

Seydoux GC et al. (1993) Worm Breeder's Gazette "A lac-Z Fusion Expressed in the C Lineage"

Fire A, Seydoux GC

[Worm Breeder's Gazette, 1993]

During the course of our screen for embryonic expression patterns (WBG 12#4, p20 ),we have isolated a lac-Z fusion that appears to be expressed specifically in the C lineage. This fusion (pGS 15.02) consists of a 3.2 kb fragment of C. elegans genomic DNA fused to the lacZ gene in the pPD21 .28vector. We found that 1 kb of the 3.2 kb is sufficient to drive C specific lac-Z expression (pGS 15.24) and have used three lines carrying an integrated array of pGS15 .24to characterize its expression pattern. To facilitate the identificadon of the lac-Z expressing cells, we have developed a modified method to stain lineaged embryos (thanks to Lois Edgar and Deborah Cowing for their helpful suggestions). Briefly, 2 to 4 cells embryos are transfered in a drop of modified lacZ staining mix (see below) onto a 1.5% agarose pad (also containing modified staining mix) and covered by a coverslip for Nomarski observadon. Embryos are allowed to develop and their lineage is recorded until a desired stage is reached (see WBG 12#2, p 19 for a descripdon of our recording system). At that time, the egg shell and vitelline membrane are disrupted using a laser microbeam to allow the staining mix to enter the embryo. 12 hours later, cells staining for lacZ can be idendfied based on their lineage idendty. In this manner, we found that in the 28 cell-stage embryo the four C-derived blastomeres (Caa, Cap, Cpa, Cpp) all express the fusion in their cytoplasm. (In nonlineaged embryos, we sometimes saw two lacZ-expressing cells in 24 to 26 cellembryos, suggesting that Cp and Ca may occasionally start expressing the fusion towards the end of their cell cycle). The four C-derived blastomeres, as well as their 8 progeny continue to express the fusion during their endre cell-cycle. By the 16 C-cellstage (172 cells), the expression of the fusion weakens but still remains confined to the C lineage. During morphogenesis, no expression of the fusion has been observed in lines carrying the integrated array. Curiously, lines carrying pGS15 .24on an extrachromosomal array continue to express the fusion up to the comma stage. At this dme, expression of the fusion is seen in the nuclei of C-derived hypodermal cells. (We note, however, that expression of the fusion appears somewhat deregulated in non-integrated lines: in the 28 cell-stage embryo, many more cells besides the C-derived blastomeres often express the fusion.) Sequencing pGS15 .24,we have found that it encodes a translational fusion between a novel C. elegans gene and lacZ. We have obtained and sequenced cDNAs corresponding to this gene and found that it has the potential to encode for a 38 kD protein containing an acidic region rich in glutamic acid. No homologies or other features have been noticed as of yet. By Northern analysis, this gene appears to be expressed predominantly in embryos, with no detectable expression in larvae and a low level of expression in adults. Modified Staining Mix: O.O5M NaPi pH 7.5 2mM MgCl2 5mM KFe/5mM KFo 1 ug/ml DAPI 0.004% SDS 5% Sucrose 0.125% X-gal 1% glutaraldehyde

Kurpiewski MR et al. (1984) Worm Breeder's Gazette "two distinct thiol proteases in C elegans."

Jacobson LA, Kurpiewski MR, Jen-Jacobson L

[Worm Breeder's Gazette, 1984]

We had previously observed that thiol-dependent, leupeptin-sensitive proteases make a significant contribution to proteolysis by crude extracts of C. elegans at pH 4.5-5.0 (approximating the intralysosomal pH). We have now resolved two distinct thiol proteases, which we designate cathepsin L1 and cathepsin L2, by chromatography on DEAE-Sephadex. The properties of cathepsin L2 from C. elegans correspond quite closely to those of vertebrate cathepsin L with respect to substrate specificity, pH optimum and molecular weight. Both proteases are optimally active toward Z-phe-arg-7-amino-4-methyl coumarin amide (MCA) at pH 5. Both are strictly thiol-dependent and leupeptin-sensitive, and neither is inhibited by pepstatin, bestatin or PMSF. Neither shows any activity toward Z-arg-arg-MCA, a substrate for vertebrate lysosomal cathepsin B, nor towards succinyl-(ala)(ala)( ala)-MCA, a substrate for the elastase-like protease of C. elegans. Cathepsins L1 and L2 are clearly distinct enzymes, since L1 is inhibited by soybean trypsin inhibitor (STI), whereas L2 is not. L2 digests denatured FITC-casein, whereas L1 does not. Cathepsin L2 preparations show a single major band on SDS gels with M=25,000, as detected after labeling the enzyme with [125I]. L2 acitivity emerges from a gel filtration column at a position corresponding to 24 kDa, so it appears likely that cathepsin L2 is active as a monomer. We are presently developing screening procedures for isolating mutants deficient in cathepsin L2. to contain several additional MSP genes. Thus in a 50Kb region there are at least five MSP genes. Two of them correspond to one of our cDNA sequences and two others correspond to one of Michael Klass's cDNA sequences so at least four of these genes must be expressed. If we can learn where these genes are in the genome it might be possible to eliminate them with a small deletion and so discover what happens to sperm with insufficient MSP. We are currently sequencing the genomic MSP genes to see if there are conserved regions outside the coding sequence. These might be either regulatory sites or the remnants of residual translocatable elements which could have participated in dispersing these genes throughout the genome.

Gilleard JS et al. (1996) Worm Breeder's Gazette "Regulation of cell and stage specific expression of the cuticle collagen gene dpy-7."

Barry JD, Johnstone IL, Gilleard JS

[Worm Breeder's Gazette, 1996]

We are interested in understanding how the spatial and temporal patterns of the cuticle collagen genes are regulated. To this end we have been studying the regulation of the cuticle collagen gene dpy-7. dpy-7/lac-Z translational reporter gene fusions with as little as 310 bp of 5! flanking sequence produce exactly the same pattern of expression as larger constructs (up to 1265bp). However translational reporter gene fusions containing just 165 bp of 5! flank produce no b-galactosidase expression showing sequences between -310 and -165 are necessary for expression. b-galactosidase expression is first seen in hypodermal cells at the late comma stage and is seen in all postembryonic developmental stages including the adult. The precise pattern of expression has been resolved by an IFA double labelling experiment using anti-lac Z monoclonal antibodies to detect the nuclear localised b-galactosidase and monoclonal antibody MH27 to delineate hypodermal cell membranes. The dpy-7 reporter constructs are expressed in hyp-7 , P cells and most of the hypodermal cells of the head and tail (hyp-5, hyp-6 and hyp-10 stain particularly strongly). However there is no expression in the V (or other) seam cells. Throughout development there is an increasing number of hyp-7 nuclei staining, with expression presumably beginning some time after each nuclei joins the syncitium. Since dpy-7 is trans-spliced with SL1 we have mapped the transcription initiation site by using a 5! RACE strategy designed to be specific for pre-mRNA by using an oligo immediately 5! to the SL1 splice acceptor site in conjunction with the anchor primer in the final round of PCR. Transcription appears to initiate from numerous sites between -210 and -159 relative to ATG (-206 and -155 relative to SL1 splice acceptor). We have also cloned the C.briggsae dpy-7 homolog and can repair the C.elegans dpy-7 mutant phenotype by microinjection of the C.briggsae gene showing that the regulation (and also the function) of dpy-7 is conserved between the two species. Similarly C.elegans reporter gene constructs produce the same pattern of expression when transformed into C.briggsae and sequence between -310 and -165 is also necessary for expression in C.briggsae. Comparison of the 5! flanking sequences of dpy-7 between the two species shows a block of homology (H1) in which 66 out of 72 residues are conserved. This lies within the region necessary for reporter gene expression and immediately upstream of the transcription initiation sites ( - 202 to -273 C.elegans and -185 to -256 C.briggsae). This is the only significantly conserved region of sequence between the dpy-7 ATG and the next predicted upstream gene at -346 (by genefinder analysis). Using RT-PCR we cannot detect any dpy-7 transcripts which are SL2 trans-spliced which, together with our analysis of the dpy-7 functional promoter and transcription initiation sites, show that dpy-7 is not a downstream gene of a polycistronic transcription unit. Transcriptional lac/Z reporter gene fusions with as little as 145bp encompassing the H1 homology and the transcription initiation sites are sufficient to produce the same pattern of expression as the translational fusions although expression in the later stages (post L1) is poor. A number of constructs have been examined and the low level of later stage expression does not appear to be due to the loss of a particular region of sequence but seems to be a general feature of our transcriptional fusion constructs. The transcriptional fusions are all orientation dependant and so the element appears to function as a true tissue specific promoter element which drives expression in all hypodermal cells except the seam cells. A series of fine 5! deletions through the H1 region shows that a single 15bp deletion completely ablates the b-galactosidase expression pattern. This deletion disrupts an AGATAA motif (which is conserved in C.briggsae) suggesting that a member of the GATA transcription factor family may play a role in the regulation of dpy-7 expression.