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5 (FGF5) has been identified on chromosome III of C.elegans (Wilson, R et al, Nature, 1994, 368, 32-38, and Du, Z, unpublished data).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.

Consortium TCeGS (1995) Worm Breeder's Gazette "The C. elegans Genome Sequencing Project: A Progress Report."

Consortium TCeGS

[Worm Breeder's Gazette, 1995]

The C. elegans genome sequencing project: A progress report. The C. elegans Genome Consortium Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri, USA and Sanger Centre, Hinxton Hall, Cambridge, UK. We present here another progress report for the C. elegans genome sequencing project. The majority of chromosome I*II is now essentially complete, although some gaps remain where cosmids were not available (Figure 1). While we are rescuing these regions from YACs and lambda clones, we will continue to sequence the cosmid-rich regions of other chromosomes. In the previous issue of the WBG, we reported significant protein similarities (blastx score >100) for several cosmids in the central region of chromosome II. Since then, we have made considerable progress on much of the remainder of this region. Protein similarities for several additional cosmids from chromosome II are presented in Table 1 (all cosmids for which shotgun sequencing has been completed). We also have begun sequencing cosmids near the center of the X chromosome. The St. Louis group started with cosmid C23F12 (near kin-11) and are moving right to left along the physical map. The Sanger Centre group are moving left to right from the same starting point. Similarity data for X chromosome cosmids which have been processed through the initial shotgun phase are also included in Table 1. For each putative genomic locus, the highest blastx score and a brief identifier are listed. Although the cosmids which contain database hits may not be completely sequenced, the Consortium will make preliminary sequence data available to the community with the caveat that it is preliminary and may still contain errors. Finished cosmid sequences are now available by anonymous ftp at: ftp.sanger.ac.uk (directory: pub/C.elegans_sequences). For further information on blastx similarities, please contact LaDeana Hillier (lhillier@watson.wustl.edu) or Steve Jones (sjj@sanger.ac.uk). For information on sequencing plans or estimated completion times, please contact Richard Wilson (rwilson@watson.wustl.edu) or Alan Coulson (alan@sanger.ac.uk). All requests for cosmid clones should be addressed to Alan Coulson. For further information about the C. elegans genome project including our policy statement about sharing both data and sequencing expertise, please contact Richard Wilson.

Consortium TCeGS (1994) Worm Breeder's Gazette "The C. elegans Genome Sequencing Project: A Progress Report"

Consortium TCeGS

[Worm Breeder's Gazette, 1994]

We present here a progress report for the C. elegans genome sequencing project. A detailed description of 2.181 Mb of finished, contiguous sequence near the center of chromosome III has recently been accepted (1). This sequence, bracketed by cosmid clones ZK112 and ZK757 ,includes the mapped genes egl-45 ,lin-36 ,unc-36 ,unc-86 ,mig-10 ,unc-116 ,ceh-16 ,dpy-l9 ,sup-5 ,unc-32 ,lin-9 ,gst-l lin-12 ,glp-1 ,emb-9 ,tbg-1 and ncc-1 .All of the finished sequence data for this region has been placed in acedb and the GenBank and EMBL databases. Since the completion of the 2.181 Mb region, we have made considerable progress on other large stretches of chromosome III (Figure 1). Several small regions not shown between F45H7 and T21C12 are contained only on YAC clones and must be rescued and subcloned before the entire sequence can be completed. Protein similarity data for a part of the region is presented in Table 1 (database hits from the 2.181 Mb sequence are not included in this list). The two groups have used different criteria for determining which cosmids were included in the list. Cambridge has included only finished cosmids and those cosmids which are contiguous but still have one or more problem areas. St. Louis has also included cosmids which have one or two gaps but which are otherwise in good shape. Also, Cambridge has indicated the position and type of similarity within each cosmid, while St. Louis has listed the name and blastx score for the strongest hits. For future submissions we hope to be more consistent; our experience here should help us decide where to set boundaries for future Gazette releases. Although the cosmids which contain database hits may not be complete, the Consortium will make preliminary sequence data available to the community with the caveat that it is preliminary and may still contain errors. Furthermore, we are willing to help locate genes for persons having a bit of sequence data (or to provide an estimated completion time for a particular cosmid). In addition to chromosome III, sequencing has begun on chromosome II. Here, we have started near the lin-5 gene, with the St. Louis group proceeding left from cosmid C06A8 and the Cambridge group proceeding right from cosmid T05A6 .For information on homologies, please contact LaDeana Hillier (lhillier@watson.wustl.edu) or Richard Durbin (rd@sanger.ac.uk). For information on sequencing plans or estimated completion times, please contact Richard Wilson (rwilson@watson.wustl.edu) or Alan Coulson (alan@sanger.ac.uk). All requests for cosmid clones should be addressed to Alan Coulson. (1)R. Wilson et al. Nature, in press

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]

Nathoo A et al. (1997) Worm Breeder's Gazette "Putative buccalin-like and myomodulin-like neuropeptides in C. elegans"

Nathoo A, Hart AC

[Worm Breeder's Gazette, 1997]

Neuropeptides form the largest class of neuroactive substances, yet as a group their roles in nervous system function and development are still poorly understood. Only the FMRFamides and related neuropeptides (FaRPs) have been intensely studied in C. elegans. However, in Aplysia californica, over 40 neuropeptides have been identified and characterized. This suggests that many C. elegans neuropeptides have not yet been discovered. With the near completion of the C. elegans genome sequencing project, reverse genetic techniques can be applied to identify C. elegans neuropeptide genes. We are using FASTA and BLAST to search with previously identified neuropeptide sequences (stored in GENBANK) against the C. elegans genome sequence. We expect the products of neuropeptide genes to fit a prepropeptide model: they should contain a putative signal peptide region, followed by interrupted multiple repeats of the same or similar neuropeptide. Additionally, the predicted neuropeptides should be flanked by pairs of basic amino acids (KR, RR, KK, RK) or less frequently by monobasic residues (R or K) which are preferred sites of endoproteolytic cleavage. Potential C. elegans neuropeptide gene compatible with the prepropeptide model are then selected for molecular and genetic analysis. We are currently analyzing C. elegans genes encoding neuropeptides similar to Aplysia buccalin (C. elegans C01C4.1) and myomodulin (C. elegans T24D8.3, T24D8.4, T24D8.5 and T01B6.4). (See below.) Even though the predicted C. elegans neuropeptide sequences are not strikingly similar to the Aplysia neuropeptides, there is strong internal homology between neuropeptides within the same C. elegans genes. We are creating reporter constructs by fusing the promoter region of potential neuropeptide genes to the GFP gene using Fire laboratory vectors. We are injecting these reporter constructs to assess cellular expression patterns of these myomodulin-like and buccalin-like genes. We think that it is likely that the GENEFINDER predicted genes of T24D8.3, .4 and .5 are exons of a single gene. We will use RT-PCR to determine mRNA splicing patterns. Our long range goals include creating mutations in these genes and determining the function of these and other putative C. elegans neuropeptides. Identity is indicated with a colon, gaps with underlining. SLSMLRLG____ Aplysia Myomodulin A :MA:G:::LRPG T24D8.5 :MAYG:Q:FRPG # :IALG:S:FRPG :MAIG:A:MRPG T24D8.4 :IAIG:A:FRPG T24D8.3 N:LVG:Y:FRIG T01B6.4 DPNVDPYSYLPSVG Aplysia Buccalin _VNL::N:FRM:F: C01C4.1 ___M:ANAFRM:F: # ___M::NAFRM:F:

Sulston JE et al. (1991) Worm Breeder's Gazette "Genome Sequencing"

Waterston RH, Sulston JE, Metzstein MM, Ainscough R, Shownkeen R, Hawkins TL, Craxton M, Lutterbach B, Green P, Thierry-Mieg J, Wilson RK, Coulson AR, Kozono Y, Staden R, Qui QQ, Du Z, Weigelt LD, Durbin RM

[Worm Breeder's Gazette, 1991]

The Great Adventure has begun! The joint St. Louis Washington University/Cambridge Laboratory of Molecular Biology project to sequence three megabases of contiguous genomic DNA has been funded under the M.R.C. and N.I.H. Human Genome initiatives. The intention is to develop methods and strategies that will allow us to sequence 1Mb of DNA at each site in the final year of the three year project. It will thus act as a pilot project for the eventual sequencing of the entire genome. There are two elements to our general approach. The first is to reduce the redundancy associated with 'shotgun' sequencing schemes by maximizing the use of directed sequencing with synthetic oligonucleotides. The second is to make data retrieval and analysis more efficient by the use of direct read-out fluorescence-detection sequencing machines (both sites have ABI and Pharmacia devices) and sophisticated computer control of day to day data generation and analysis. We are currently addressing a number of problems: which vectors and insert sizes are most efficient for subcloning cosmids? What degree of shotgun redundancy should be obtained before oligo walking? How do the sequencing machines compare? Can double stranded sequencing yield data of good enough quality? (Molly Craxton has developed some excellent double-stranded sequencing protocols, available on request, which work well on cosmid and lambda DNA, in addition to sub-clones). We have begun to sequence in the cluster on chromosome III. The Cambridge group have started on the lin-9/unc-32 cosmid ZK637 and are proceeding rightwards to the edge of the cluster while the St. Louis group have started sequencing around sup-5 on the cosmid ZK370. Obviously, the genome map is essential in allowing us to take such a logical ordered approach. A 3Mb sequence should extend from the right edge of the cluster leftwards beyond mec-14. It is our intention to release sequence data to the community as rapidly as possible, probably within two months following completion ( of cosmid sized pieces). This will be done by incorporation into the databases currently being developed (see articles by R.D. and J. T-M in this issue and Bruce Schatz et al WBG 11/4, and submission to EMBL/Genbank libraries). Initial sequence analysis (predicted coding regions, splice sites, library comparisons etc.) will also be included.

Du Z et al. (1992) Worm Breeder's Gazette "Sequencing the C. elegans Genome: Progress Towards the First 1 MB."

Shownkeen R, Kershaw JK, Wohldmann P, Wilson RK, Du Z, Dear S, Smaldon N, Johnston L, Thierry-Mieg J, Vaudin M, Laister N, Smith A, Connell M, Kozono Y, Hawkins TL, Green P, Craxton M, Berks M, Halloran N, Staden R, Sulston JE, Waterston RH, Durbin RM, Thomas K, Favello T, Chau H, Jier CHM, Coulson AR, Copsey T, Hillier L

[Worm Breeder's Gazette, 1992]

The accompanying figure [See figure 2] illustrates the progress made in genomic sequencing to date. Cosmids (not drawn to scale) are shown at the top. Completed regions are ready for submission or have been submitted to the EMBL and GenBank databases. These completed regions have been analyzed by BLAST against the public databases, by Genefinder, and by comparison against the nematode cDNAs. Contiguous regions are contiguous sequences that have single stranded regions or problems such as compressions that have to be resolved. Shotgun regions consist of discontinuous fragments that can be searched by BLAST and cDNA comparison, but not by Genefinder. cDNA matches are shown by closed triangles for consortium clones and by open triangles for McCombie et al. clones. Significant similarities for BLAST are indicated. The total number of genes estimated by Genefinder analysis is shown for each completed segment.

Sakai T et al. (1994) Worm Breeder's Gazette "Analysis of the 5' Transcriptional Regulatory Regions of the let-23 Gene in C. elegans."

Ohshima YM, Sakai T, Koga M

[Worm Breeder's Gazette, 1994]

Thelet-23 gene encodes a receptor type tyrosine kinase(1) and is necessary for vulval induction, survival past theL1 stage, hermaphrodite fertility and for male spicule development. For the vulval induction, it was proposed thatLet-23 acts in vulval precursor cells(VPCs) to receive and transduce an inductive signal from the anchor cell(1). Genetic mosaic analysis of thelet-23 gene function supports this proposal(2). We have analyzed] the 5' regulatory regions of thelet-23 gene to understand the mechanism of the expression. We cloned alet-23 homologue from Caenorhabditis vulgaris (formerly called C. vulgarensis ) to find evolutionary conservations in the upstream sequences. The gene in C. vulgaris probably encodes a protein of 1332 amino acids, 76% of which are shared withLet-23 of 1323 amino acids in C. elegans. The exon 15 for the tyrosine kinase domain in C. elegans is split into two exons in C. vulgaris, and a total of 19 exons is found in the C. vulgaris gene. The 12kb EcoRI-SalI fragment carrying the C. vulgaris gene rescued the lethal phenotype in the germline transformation oflet-23 (mn23)mutant. Therefore, the gene cloned from C. vulgaris is homologous to the C. eleganslet-23 gene structurally and functionally. We have found sixteen 5' upstream sequences that are conserved between C. elegans and C. vulgaris (Fig. 1, a~p). We have also examined rescue activities of variouslet-23 5' deletion constructs. A construct which has 2984bp upstream of the initiation codon ATG rescuedlet-23 (mn23)lethal mutation. However, constructs carrying 2656bp, 2279bp or 2055bp did not rescue the mutation. The region from -2984bp to -2656bp includes two conserved sequences(Fig. 1, c and d). One of them (d) has a sequence AATCTTCA/CT which is similar to a consensus AATATNCAT for Pit-l/GHF-l binding sites(3). Constructs which has 2055bp, 1926bp,1672bp or 1487bp upstream of the ATG rescuedlet-23 (sy97)vulvaless mutation, but one with 1074bp did not. The region from -1487bp to -1074bp includes a conserved sequence(l in Fig. l) . These results indicate that the regions between -2984 and -2656 and that between -1487 and -1074 are required for the rescue of a lethal or vulvaless mutation, respectively. Two conserved sequences in the former (c and d) may be activating elements for the transcription of thelet-23 gene in the cells important for the larval survival. The conserved sequence in the latter (l) may be a cis-acting element required for the expression in VPCs. (1) Aroian, Koga, Mendel, Ohshima & Sternberg, Nature 348, 693-699(1990) (2) Koga & Ohshima, Abstracts of C. elegans Meeting, 248(1993) (3) Ingraham, Flynn, Voss, Albert, Kapiloff, Wilson & Rosenfeld, Cell 61,1021-1033(1990)

Setterquist RA et al. (1992) Worm Breeder's Gazette "Characterization of the msp-113 Gene Cluster in Strainsof Caenorhabditis elegans"

Setterquist RA, Fox GE, Smith GK, Ammons D

[Worm Breeder's Gazette, 1992]

We are using the major sperm protein (MSP) gene family found in nematodes as a model system to gain insight to genomic evolution. It would be of special interest to examine msp genes from strains or species which are sufficiently similar that a one to one correspondence of genes with those of the Bristol strain could be established in order to gain insight to how frequently new genes are created. To attempt a study of this type we selected the msp-113 gene cluster of C. elegans strain Bristol, which is known to have at least six msp genes of which four have been sequenced to date. This cluster is of special interest because its members are most alike in terms of gene sequence and therefore may be the most recently evolved cluster in the Bristol strain. Two C. elegans strains, the French strain BO and the Australian strain AB1 were examined. Earlier work (Thomas & Wilson Genetics 128: 269-279,1991) with the mitochondrial cytochrome oxidase gene showed that whereas strain BO 1 was virtually identical to the Bristol strain, the AB1 strain did exhibit some variation. In order to rapidly characterize genes in AB1 a msp-113 cluster specific PCR primer was constructed for a sequence region approximately 100 nucleotides upstream from the msp start codon and a conserved region surrounding the stop codon. Resulting amplification products were cloned into TA vector (InVitrogen Inc.) and sequenced using msp specific primers. A total of 11 clones yielded 9 unique msp gene sequences, eight of which belonged to the msp-113 cluster based on sequence comparison. Two of these genes were effectively identical to known Bristol sequences. Another two were unusual in that they contained a three amino acid insertion shortly after the start codon which is not found in any known msp gene (regardless of cluster) in Bristol. Two msp genes belonging to the msp-113 cluster were also sequenced from lambda clones of the BO strain. One of these was unique while the other was identical to one of the AB1 msp genes with the amino acid insertion. Are there genes in the AB1 analog of the msp-113 gene cluster that are not in the Bristol strain? We really can't tell at this stage. In order to accommodate the results there would have to be at least 10 genes in the msp-113 cluster of the Bristol strain. We are currently using the obvious PCR amplification approach to search for the missing genes in Bristol and plan a more formal examination of cosmids from the msp-113 region as well in order to localize any missing genes that are found.

Hoch RV et al. (1996) Worm Breeder's Gazette "mRNA expression patterns of a cell cycle regulator WEE1 in embryos"

Golden A, Hoch RV, Wilson MA

[Worm Breeder's Gazette, 1996]

We have initiated a project to examine the regulation of the cell cycle during C. elegans development (please see accompanying abstract by Wilson et al.). Biochemical and genetic experiments from other systems have revealed that the tyrosine kinase WEE1 is a negative regulator of cell cycle progression. WEE1 phosphorylates the serine/threonine kinase, CDC2, on a single tyrosine residue to maintain it as an inactive kinase during much of the cell cycle. A search of the available sequences from the Genome Consortium revealed a candidate wee1 gene (cosmid F35H8). The WEE1 candidate, as predicted by GENEFINDER, has all of the distinguishing features that define it as a member of the WEE1 tyrosine kinase family. Using SL1, SL2, and 3 UTR specific primers and RT-PCR, we identified a SL1-trans-spliced cDNA. Interestingly, the SL1 is trans-spliced to the second exon predicted by GENEFINDER. We have yet to detect a message that incorporates the predicted exon 1. We have used this cDNA for RNA in situ studies in embryos. The wee1 message appears not to be maternal. The first stage at which we observe mRNA expression is the 12-cell stage, in a single prometaphase nucleus. By co-staining with an anti-P-granule antibody (for orientation), we have determined that this single nucleus is that of the E blastomere. wee1 expression is then seen in 5-8 distinct prometaphase nuclei in the 16-cell embryo. We believe these 5-8 nuclei are those of the 8 AB-derived cells. Interestingly, the message is only observed in cells with condensed chromatin (as seen with DAPI staining). This expression is observed at a time in the cell cycle when nuclear envelope breakdown is occurring. The wee1 message does not appear to be cytoplasmic at all. We interpret this distribution as a burst of embryonic wee1 transcription (in these 5-8 prometaphase cells), followed by rapid mRNA turnover. Anaphase, telophase, and interphase cells appear devoid of wee1 message. We are currently attempting to determine whether this burst of embryonic transcription is truly cell cycle stage-specific and lineage-restricted. We have yet to observe any mRNA expression in the MS, C, D, or P3 (or P4) blastomeres. Also, no wee1 message has been observed beyond the 16-cell stage of embryonic development. We are currently testing WEE1 antibodies to determine the protein expression patterns of this cell cycle regulator during embryogenesis. We are also performing whole animal RNA and protein in situs to determine the expression patterns of this gene during the proliferation and development of the germline in the gonad. We would like to acknowledge and thank the C. elegans Genome Consortium for sharing all of their sequence information with the scientific community and for making this project possible. This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL.