Han H-P et al. (1992) Worm Breeder's Gazette "Expression and Localization of the cha-1 and unc-17 Gene Products"

Rand JB, Duerr JS, Han H-P

[Worm Breeder's Gazette, 1992]

EXPRESSION AND LOCALIZATION OF THE cha-l AND unc-17 GENE PRODUCTS He-ping Han, Janet Duerr, and Jim Rand, Oklahoma Medlcal Research Foundation, Oklahoma Clty, OK 73104

Armand I et al. (1996) Worm Breeder's Gazette "The Control of Body Size"

Leroi AM, Armand I, Emmons SW

[Worm Breeder's Gazette, 1996]

Exactly 100 years ago (Jan 10, 1896) Edwin Conklin published a paper entitled "Cell Size and Body Size." In it he posed the following question: do species that vary in adult body size differ in cell number or in cell size? His answer, for a genus of intertidal snails, was that species that vary in body size vary in cell number, but that cell size is more or less constant over evolutionary time.

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

Livingstone D (1989) Worm Breeder's Gazette "some more unc-31 sequence."

Livingstone D

[Worm Breeder's Gazette, 1989]

I am continuing the molecular work on the unc-31 gene started by Roger Hoskins. Roger isolated a 17.5kb genomic clone in lambda2001 which he showed was capable of transformation rescue of unc-31 mutants. He also partially sequenced a 4.5kb fragment contained within this clone which covers some of the polymorphisms associated with mutant alleles of the gene. [See Figure 1] I have made subclones of this phage in lambda2001 and have used them in an attempt to narrow down the position of the gene by transformation rescue. A phage containing the 13.5kb XhoI fragment seems to rescue although this result has still to be confirmed. No rescue has been obtained with phage containing the 13.5kb SacI fragment or the 10.5kb SacI-XhoI fragment. I am currently sequencing the 13.5kb rescuing fragment. Several open reading frames have been found which could be spliced together inframe. Some of these have been confirmed by sequencing partial cDNA clones isolated from Julie Ahringers mixed stage cDNA library. The gene covers more than 8kb of genomic DNA. The 5' end is near or beyond the SacI site and the 3' end is beyond the end of the 4.5kb HindIII fragment. No homology has been found between predicted amino acid sequences and protein sequences present in the PIR and Swissprot databases.

Ward S (1978) Worm Breeder's Gazette

Ward S

[Worm Breeder's Gazette, 1978]

The brownish-yellow bacterium that commonly contaminates worm stocks is Pseudomonas aeruginosa. It is sensitive to gentamicin and colistin, we use gentamicin (10 g/ml) in some plates to control it. When present on plates, it reduces progeny production substantially and also causes rapid destruction of unfertilized oocytes. The bulk E. coli commercially available from Grain Processing Corp. can be grossly contaminated with Pseudomonas and other species. (When asked if they could prepare E. coli aseptically, the reply was 'No, the man that harvests the pellets scoops them up with his bare hands'. On special request he will wash his hands). After receiving a large batch of bacteria which would not grow worms well, we switched to growing our own E. coli in a Lab-Line High density fermentor.

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.

(1990) Worm Breeder's Gazette "CGC Bibliography in FileMaker and HyperCard (Mac) and Memory Mate ( IBM)"

[Worm Breeder's Gazette, 1990]

The CGC Bibliography has been translated into a couple of programs other than dBase by various worm people. David Barker and Andy Fire both have sent HyperCard versions to the CGC and Mark Blaxter has sent us a version in FileMaker 2.0. None of these is perfectly up-to-date, so you'll have to be somewhat familiar with the programs to add new references. The data files are available free from the CGC; to get yours, just send a blank 3.5' diskette to Mark Edgley at the CGC with a request letter. In addition, Lew Jacobson has translated the bibliography into a DOS program called Memory Mate and he is willing to distribute the data file to anyone who sends him a blank 5.25' 360 Kb diskette. Memory Mate can be operated as a TSR and called up with a hotkey from the middle of a word processor or other program. Addresses for Mark and Lew can be found in the Subscriber Directory Update in this issue.

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.

Riddle DL et al. (1980) Worm Breeder's Gazette "announcements"

Swanson MM, Riddle DL

[Worm Breeder's Gazette, 1980]

The Caehorhabditis Genetics Center (CGC), sponsored by the National Institute on Aging, was funded on September 30, 1979. Dr. Don Murray is the NIA Project Officer. As you know, the CGC is to be responsible for strain acquisition, banking and distribution. Strains will be available without cost to all qualified investigators pursuing genetics-related studies with C. elegans. Other CGC functions will include maintenance of the genetic map, coordination of genetic nomenclature and maintenance of a current data bank on all strains, including bibliographic information. During the first year, we will be developing a computerized system for storage and retrieval of this information. The program will be designed to allow other labs with CRT terminals remote access to the computer data files. We have recently mailed a questionnaire regarding your anticipated use of the Center's services. Since your responses may help us to modify the design of the services to be offered, we would appreciate your returning those questionnaires as soon as possible if you have not already done so. If you did not receive our mailing and anticipate using the CGC, please contact us. We plan to maintain a complete library of articles on Caenorhabditis, so we would appreciate it if you also would send us one copy of each of your Caenorhabditis publications. We have recently done a retrospective search of the literature and will soon be distributing a complete C. elegans bibliography as a supplement to this Newsletter. The new bibliography contains about 350 references, and we plan to update it with each future issue of the Newsletter. We would like to thank Bob Herman for the excellent job he has done as a volunteer stock distribution center. The immensely valuable work that Bob Herman and Bob Horvitz have done on the genetic map surely is apparent to everyone. Bob Herman will be supplying us with many strains he has received from other laboratories and we hope to have a relatively up-to-date C. elegans collection in a few months. The CGC is scheduled to be fully operational by the Fall of 1980, but some services are available now.

Yasuda H et al. (1990) Worm Breeder's Gazette "Trans-splicing in Tubulin?"

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1990]

At the last worm meeting (CSH, 1989), we reported the nucleotide sequence of the alpha-1 tubulin gene AT3 that was cloned by direct screening of a C. elegans cDNA expression library in a lambda gt11 vector (Bob Barstead), using a tubulin specific monoclonal anti-body 3A5 (provided by M. Fuller). This gene was previously identified by Linda Gremke using a chicken tubulin probe (Don Cleveland). Our clone SQ#TbA77 was cytologically mapped on chromosome I of C. elegans (Alan Coulson and John Sulston, MRC, LMB, UK). As Dr. Tom Blumenthal walked by our poster at the meeting, he pointed out the presence of an eight nucleotide non-coding sequence TTCAGGTT, which corresponds to the acceptor sequence of C. elegans in the trans-splicing reaction of mRNA. This is a rather novel feature for the tubulin gene, as the other alpha or beta tubulin genes, from C. elegans or other metazoans have not been reported to be trans-spliced at the mRNA level. We are currently trying to sequence the mRNA to test if this mRNA is indeed spliced. Figure below shows the physical map of the alpha-1 tubulin gene, and the nucleotide sequence showing the acceptor sequence ( marked by arrowed box), in the non-coding sequence of the tubulin gene. [See Figure 1]