Bowerman BA et al. (1994) Worm Breeder's Gazette "The Maternal Gene skn-4 and the Specification of Ventral Blastomere Fates in the Early C. elegans Embryo"

Bowerman BA, Shelton CA, Thorpe CJ, Martin PR

[Worm Breeder's Gazette, 1994]

THE MATERNAL GENE SKN-4 AND THE SPECIFICATION OF VENTRAL BLASTOMERE FATES IN THE EARLY C. ELEGANS EMBRYO Bruce Bowerman, Paula R. Martin, Christopher J. Thorpe, and Christopher A. Shelton. The Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

Hall DH et al. (1994) Worm Breeder's Gazette "Cytology of Degenerin-Induced Cell Death in the PVM Neuron"

Driscoll MA, Chalfie M, Gu GQ, Hall DH, Gong L

[Worm Breeder's Gazette, 1994]

Cytology of degenerin-induced cell death in the PVM neuron David H. Hall, Guoqiang Gu+, Lei Gong#, Monica Driscoll#, and Martin Chalfie+, * Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461 + Dept. Biological Sciences, Columbia University, New York, N.Y. 10027 # Dept. Molecular Biology and Biochemistry, Rutgers University, Piscataway, N.J. 08855

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Johnson TE (1980) Worm Breeder's Gazette "Generation of X-linked Mosaics"

Johnson TE

[Worm Breeder's Gazette, 1980]

We are investigating somatic loss of X-chromosomes as a means of producing mosaic worms at relatively high frequencies. We produce worms heterozygous for the X-linked muscle morphology mutant, unc-78( e1217), which are homozygous for various him's. Only one such mutant, him-2(e1065), generates spontaneous mosaics at a frequency greater than 0.1%. We are still examining two other lines and are exploring other means of inducing high-frequencies of X-linked mosaics . We have observed loss of X-chromosomes from worms irradiated by X- rays from 137Cs. The loss is seen in the F2 of irradiated worms (or the F1 or irradiated embryos). The number of males in this generation is in the range of 4-10%. The frequency of males is not distributed according to a Poisson but rather shows 'jack-pots' as might be expected from events that hit individual X chromosomes. The frequency of males in a jackpot can be as high as 40%. Most hermaphrodite siblings of these males are normal and show no increase in the frequency of male progeny. We asked whether only the irradiated chromosome was lost in an animal heterozygous for a marked irradiated X-chromosome and an unmarked unirradiated X-chromosome. Hermaphrodites marked with the X- linked marker, unc-78, were irradiated and then mated to N2 males. Wild type progeny were picked, cloned and scored for male offspring. Both wild type and unc-78 males were found at approximately equal frequency; apparently either chromosome can be lost.

Gu GQ et al. (1994) Worm Breeder's Gazette "mec-5 Encodes a Collagen Needed For Touch Sensitivity."

William CM, Chalfie M, Gu GQ

[Worm Breeder's Gazette, 1994]

mec-5 Encodes a Collagen Needed for Touch Sensitivity Guoqiang Gu, Chris William, and Marty Chalfie, Department of Biological Sciences, Columbia University in the City of New York, NY 10027 mec-5 is one of the genes required for mechanosensitivity in C. elegans. Although the touch receptor neurons do not function, they appear morphologically normal in mec-5 mutants. Peanut-lectin binding to the touch cell mantle, however, is absent (M. Chalfie and E. Hedgecock, unpublished data). By transformation rescue, we identified a 6.5 kb BamHI-Kpnl fragment in cosmid E03G2 which completely rescues the mec-5 phenotype. We isolated a 1.2 kb full length cDNA (Northern blots show a 1.2 kb mRNA) from the Chris Martin cDNA library. The cDNA sequence encodes a collagen. The protein (329 aa) has a 20 amino acid putative signal sequence at the N- terminus and 69 Gly-X-Y repeats starting at aa 63. These collagen repeats are followed by 60 additional amino acids. Because collagens are often glycosylated on hydroxyproline residues, the loss of MEC-5 may explain the lack of peanut- lectin binding. To verify that this collagen gene is mec-S, we probed genomic Southern blots with the 6.5 kb fragment and found that the 6.5 kb fragment was deleted in two mec-5 strains, the mut-2 derived strain TU828 and the Y-ray strain TU1031. We are now sequencing other mec-5 mutations and characterizing the expression pattern.

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.

Ainscough R et al. (1991) Worm Breeder's Gazette "Sequencing and mapping C. elegans cDNA's"

Craxton M, Durbin RM, Wilson RK, Martin C, Hillier L, Sulston JE, Ainscough R, Waterston RH, Halloran N, Coulson AR, Thomas K, Huynh C, Lee C, Green P, Metzstein MM

[Worm Breeder's Gazette, 1991]

As an adjunct to our genomic sequencing project we are taking advantage of a sorted cDNA library (generated by Chris Martin) consisting of 1743 cDNA clones, estimated to contain 1400 different cDNAs.This would represent 10% or so of the mRNAs currently thought to exist in C. elegans. Sorting was achieved by probing the library with pools of previously isolated clones, to avoid repeatedly re- isolating clones already represented in the selected subset. ABI 373A sequencers have been used to obtain sequence from the 5' end of the cDNAs. Both double-stranded plasmid DNA and PCR amplified inserts have been used as templates; the latter is faster but the former provides better quality sequence data. To date we have sequenced 550 clones. Each sequence has been analyzed by BLAST against the NCBI nonredundant database GENINFO. About one in three shows significant ( BLAST score of >100) similarity to known genes (see listing). Taking advantage of the almost complete physical map we are able to rapidly map the cDNA's by hybridization to the genomically ordered array of YAC clones ('polytene' filters). On average,this locates mapped cDNAs to a resolution of about 100kb. The sequence and mapping data generated by this project are being rapidly made available to the C. elegans community through the database acedb and the sequences are being submitted to the EMBL and GENBANK databases. The tagged and mapped cDNA's should prove useful and they should also provide a resource for checking predicted genes obtained in the genomic sequencing effort. The clones are available on request from the St. Louis lab. [See Figure 1]

Galas S et al. (1992) Worm Breeder's Gazette "A Quest for cdc-2-like Function in C. elegans"

Peuch CL, Doree M, Ferraz C, Thierry-Mieg D, Devault A, Galas S

[Worm Breeder's Gazette, 1992]

We are interested in the control of cell cycle in C. elegans and wish to use genetics to describe the various genes and their parts in the regulation of cell division. We started with cm4 g2 ,a cdc2 cDNA homolog from C. Martin, B. Waterston, J. Sulston et al.. We determined the complete cDNA sequence of the gene from this clone and a homologous clone selected from B. Barstead cDNA library: the protein is 72% identical (89% homologous) to human cdc2 .We mapped it on center II (Y24H1 ),then narrowed its position to about 20 kb on the physical map (thanks to Alan Coulson et al.). Connection to the genetic map was achieved by testing the presence of our gene in four deficiencies of the area, generated by B. Herman and provided by M. Edgley. We used PCR on single embryos, a marvellous technique developed by Barstead, Schrankc and Waterston, which in our hands works routinely on 80-cell embryos. Our cdc2 gene appears to be present in these four deficiencies, so it lies in a region that includes three known genes: a maternal effect lethal zyg-9 which affects the first embryonic mitosis, a zygotic lethal let 252 and a haplo-insufficient locus. zyg-9 looked like a good candidate from its phenotype. In addition four putative polymorphic alleles have been recovered, three mutator-induced by Phil Carter and Bob Edgar, one psoralen induced by Julie Ahringer. Using PCR we showed however that none of the four is structurally rearranged in the transcribed cdc2 region. We are now analyzing the alleles by genomic Southern, and trying to rescue the three known functions in the area by transformation with the cosmids. We are also interested in other cdc2 -1ikegenes -another atypical PSTAVRE (like cm4 g2 )containing gene has been studied in Paul Sternberg's- but is there a "true" PSTAIRE containing gene? What are the functions, inter-relations, specificities or redundancies of this set of genes ?

Levitan DJ et al. (1990) Worm Breeder's Gazette "Analysis of a par-2 cDNA"

Levitan DJ, Stinchcomb DT

[Worm Breeder's Gazette, 1990]

We have been cloning the maternal-effect lethal gene par-2. Embryos from a homozygous par-2 mother undergo symmetric and synchronous cleavages, a failure to localize P granules properly, a low expression (20%) of gut granules, and no morphogenesis (Kemphues et al. Cell 52:1988). The role of the wild type par-2 gene, as well as the other par genes, is therefore thought to be in the organization of the cytoplasm and the proper partitioning of cytoplasmic components in early embryogenesis. We have previously described the identification of a par-2 allele specific polymorphism, which consists of a 3 kb deletion in the allele it46. We have also shown that the DNA contained within this deletion identifies a 2.3 kb oocyte specific message when used to probe a Northern blot containing RNA isolated from fem-2 (sperm minus) and glp-1 (germline minus) animals (1989 CSH C. elegans meeting). Because this message seemed likely to be the par-2 message, we used the same probe to isolate cDNA's from the Schauer, Kim, Barstead, and Martin cDNA libraries. Although none of these cDNA's are full length, we are within a few hundred base pairs of the complete message. Hybridization experiments with 5' and 3' specific cDNA probes indicate that the it46 polymorphism deletes coding sequences found in this cDNA. These data strongly support the notion that this cDNA clone represents the par-2 message. We have sequenced 2.1 kb of this cDNA clone and searched several databases with the predicted protein product--however, no homologous proteins were found. The one notable feature of this sequence is the presence of an ATP-binding site of the myosin class. The figure below shows the ATP-binding site consensus sequence, the par-2 putative ATP- binding site, as well as other members of this class of proteins. While we don't yet understand the role this protein plays in early development, we are intrigued by the presence of the ATP-binding site which is consistent with a role for par-2 in cytoplasmic sorting during early cleavage divisions. [See Figure 1]

Klass MR (1977) Worm Breeder's Gazette "a method for isolating biochemical quantities of purified sperm."

Klass MR

[Worm Breeder's Gazette, 1977]

This procedure starts with a predominantly male culture isolated by the Nitex filter method described previously. I usually start with a culture 60-70% males (a total of 1 - 5 x 10+E5 males). These males are washed extensively with 4 liters of M-9 salts over a 20 m Nitex filter to remove the larvae (especially the L1 which can be a problem later!) and to remove the E. coli. The washed male culture is concentrated by settling into 2-3 ml of M-9 salts. The worms are then spread evenly in very small disperse droplets over the surface of an 11'x7'x3/8' Plexiglass plate. Another Plexiglass plate of equal size is placed on top. The worms are then squashed between these plates in a Carver Laboratory Press with a manual hydraulic jack. The worms are subjected to 15,000 psi. This pressure causes the males to eject their sperm as well as their intestinal cells. However the sperm are the only free floating cells. The intestinal cells of both the males and hermaphrodites adhere to the Plexiglass. The plates are then pried apart and rinsed off. The rinse is collected in a glass trough and placed in 125 ml conical centrifuge tubes on ice. Everything is kept on ice from this point on. (If some males don't get squashed in the first run, they can be squashed again, although resquashing usually only gives 10% additional sperm.) The rinse fluid contains the sperm (and worm carcasses) and is filtered through two 10 m Nitex filters fitted to a 25 mm millipore filter holder of the type used on syringes. This removes the worm carcasses and larger debris. The filtrate is centrifuged in a clinical centrifuge on setting 6 for five minutes. The supernatant is discarded and the pelleted sperm are washed in M-9 salts and filtered through an 8 m polycarbonate Nucleopore filter fitted to a 25 mm millipore filter holder. I routinely obtain 10+E7-10+E8 sperm, an average of about 100 sperm per male. One dimensional SDS polyacrylamide gel patterns of these sperm closely match the patterns seen on autoradiographs of hermaphrodites mated with radioactively labeled males. Some additional information: Sperm can be stored overnight in M-9 salts with a minimum of lysis (1%) but they seem more fragile and after 48 hours show 50% lysis. I have found that sonication works best to lyse sperm rather than freeze/thawing. I have used M-9 salts throughout these procedures and have not tried Ascaris Ringer's solution. These purified sperm are currently being used to investigate cell surface proteins, DNA binding proteins, sperm actin and other sperm- specific proteins and their modifications in various spermatogenesis mutants as well as being used as a source of germ line DNA.