Coulson AR et al. (1989) Worm Breeder's Gazette "genome map."

Ameer H, Albertson DG, Sulston JE, Coulson AR, Waterston RH, Fishpool RM

[Worm Breeder's Gazette, 1989]

The map is now widely distributed electronically (see WBG 10(3), 67), but we are once again providing a summary for the gazette in the form of an output from the routine CHPLT. Do note that this is a provisional best guess, and that some linkages may later go away: please enquire if you need to know about the status of particular areas. When you receive cosmid clones, as stabs, please IMMEDIATELY streak them out on selective medium, pick small colonies, and grow 4ml minipreps (protocol from Alan Coulson if needed). For some cosmids, larger preps are liable to yield deleted DNA. Check that cosmid DNA appears full size (runs slower than lambda on agarose gels), then freeze a sample of good cells in 20% glycerol at -70 C. MRC computer account 'ARC' does not exist; Alan and John share account JES. A database node is now open at Seattle: modem number 206-467-2957; operator Phil Meneely. The summary of clone types given on the next page may be helpful when you are deciding which clones to request for your research. To reveal the most suitable clones for microinjection, the buried clones need to be displayed by the routine CONTASS; we will help you to do this if you ask. [See Figures 1- 3]

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.

Greenwald IS (1984) Worm Breeder's Gazette "molecular cloning of lin-12."

Greenwald IS

[Worm Breeder's Gazette, 1984]

I have examined a total of eight independent lin-12 null mutations that arose spontaneously in a predominantly Bergerac genetic background. I probed genomic Southern blots with a 1.2 kb wild-type HindIII fragment that was defined by an insertion mutation (see last Newsletter) and/or with a cosmid 'contig' obtained from Alan Coulson and John Sulston that extends approximately 20 kb in each direction from the 1.2 kb HindIII fragment. The results thus far indicate that ( 1) seven of the eight mutations are associated with Tc1 insertions (2) the insertions are at different sites, and (3) all the insertion sites may lie within a single Pst fragment that is 2.8 kb in wild-type.

Sugimoto K et al. (1990) Worm Breeder's Gazette "Cloning, mapping and splicing of the tropomyosin gene, tmy-1 of Caenorhabditis"

Kagawa H, Sugimoto K

[Worm Breeder's Gazette, 1990]

Genomic restriction fragments expanding 6.3kb were cloned by using exon expression fragment as a probe and were sequenced. EcoRI-5kb fragment hybridized to the YAC clones;Y68B7 and Y38D6 LGI (Alan Coulson) and not hybridized to any fragment from cosmid clones K05C4 and F39B2. Hair pin structure of the intron part of the genomic fragment might interfere packaging of DNA fragment into phage particle. Following figure presents the genome structure and three different cDNA clones which was obtained from cDNA library (the last issue of WBG #11-2). Comparison with tropomyosin genes of Drosophila suggests that two were muscle type and one was cytoplasmic type. Missing 'head and tail ' of the genome will help to know how the gene expresses in the worm. [See Figure 1]

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

Hudson SJ et al. (1990) Worm Breeder's Gazette "unc-80 IS A STUPID GENE"

Hudson SJ, Morgan PG, Sedensky MM

[Worm Breeder's Gazette, 1990]

We have been trying to clone the gene unc-80 by the method of transposon tagging, using advice from David Miller, Tim Schedl, Susan Bektesh and Alan Coulson. We searched for spontaneously arising alleles in the mutator strain, RW7097. We obtained 11 such strains, eight of which had spontaneous revertants. We isolated a 3.4 kb BglII fragment which contains a Tc1 and correlates with the unc-80 phenotype 1.)after 14 outcrosses, 2.)after crosses with the linked marker dpy-21, and 3.)after a late occurring (after eight outcrosses) revertant. This fragment was ligated into the plasmid pBS (BamHI site) and labeled pCW1 (we're a real big lab) and we were happy. Restriction mapping showed that the fragment contained a 300 bp region, the Tc1, and a 1500 bp region. We subcloned the 300 bp fragment, the 1500 bp fragment and the 1800 bp fragment resulting from clipping out the Tc1 with EcoRV (pCW2, pCW3 and pCW4, respectively--this is really embarrassing, maybe we should have started with pCW500). [See Figure 1] Alan Coulson sent us his neat YAC library which we screened with pCW4 and (roll of drums) it lit up one clone. We were very happy. Then we identified the YAC as Y24H1 which contains part of chromosome II (unc-80 is on V). We were very, very unhappy. Then it turned out that Y24H1 contained more than just one YAC. We were slightly less unhappy. So we sent pCW4 to Bob Waterston, while we were probing our genomics with this plasmid. Both results showed that pCW4 contained repetitive DNA. About 10 YACs lit up, and the genomics showed about 15-20 bands. Very, very unhappy again. However, using pCW2 and pCW3 separately showed that pCW2 (300bp fragment) contains the repetitive stuff and pCW3 is a unique sequence. We don't know at present if the repetitive sequences are local or widely distributed. We are now sequencing both pieces and extending the cloned DNA by walking. The initial sequence (only a few hundred bp) shows no homology to anything thus far, but is about 50% GC. At present we are no longer suicidal.

Harfe BD et al. (1994) Worm Breeder's Gazette "ceh-24, a New NK Class Homeodomain Gene."

Okkema PG, Fire A, Harfe BD

[Worm Breeder's Gazette, 1994]

ceh-24, a new NK class homeodomain gene Brian Harfe 1, 2, Pete Okkema 1, and Andy Fire 1 1 Carnegie Institution of Washington, Department of Embryology, 2 Biology Graduate Program, The Johns Hopkins University The NK class of homeodomain proteins are a set of regulatory factors with a broad evolutionary distribution and a variety of expression patterns. Analysis of pharyngeal myosin regulation led to the identification of a member of this family, ceh-22, which appears to mediate aspects of pharyngeal gene expression (Okkema and Fire, Development 120, 2175). To determine the extent of the NK homeodomain family in C. elegans we have used a degenerate primer based PCR strategy. Among several candidates from the initial PCR, we have so far characterized one gene, denoted ceh-24. The CEH-24 homeodomain falls into the NK class, but is closer to diverse members of the NK family seen in other species than to CEH-22. In addition to the homeodomain, CEH-24 contains an acid activation domain C-terminal to the homeodomain. ceh-24 maps (thanks to Alan Coulson) to chromosome V just left of him-5.

Kagawa H et al. (1988) Worm Breeder's Gazette "structure of the paramyosin gene, unc-15."

Gengyo-Ando K, Kagawa H

[Worm Breeder's Gazette, 1988]

Complete genome structure of the paramyosin gene, unc15 was determined (see below). Twelve kilobases of DNA fragment from hybrid lambda HK2 were sequenced by MRC protocol of Sanger method and processed by ANALYSEQ. There were ten exons and nine introns. Consensus sequence of nine introns was GTAAGT---TTTCAG (Fig. 2). Tc1 was inserted in consensus sequence (Mori, I. et al PNAS, 85 (1988) 861-864) and was located at 196bp upstream of the initiation codon. Eight hundreds and forty amino acid residues with molecular mass of 97, 240 daltons were deduced from ten exons and the predicted amino acid sequence was completely matched to that of CNBr-fragment of the paramyosin at three different sites. The sequence was also confirmed by comparison to that of fusion protein with -galactosidase which were produced in bacterial cell and were crossreacted with anti- paramyosin serum and three monoclonal antibodies (Fig. 3). N- terminal 114 amino acid residues had 92 % homology to that of Dirofilia immitis paramyosin (Larry McReynolds, New England Biolabs). Alan Coulson sent me a letter that myo-1 contig joined to unc-15-col-7- unc-13 contig. We hope some unc-15 mutants will recover a motility with a gene therapy. [See Figures 1-3]

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]

Ishii N et al. (1988) Worm Breeder's Gazette "partial sequence of the unc-6 gene."

Ishii N, Stern BD, Culotti JG, Hedgecock EM

[Worm Breeder's Gazette, 1988]

In the previous issue (WBG 10(1) p.35), we reported cloning a small DNA fragment containing the Tc1 insertion which caused the unc-6 ( rh1035) mutation. Since then, we have isolated an 11.5 kb EcoRI fragment including this region from N2 DNA (NJ#14, EMBL4 vector) Alan Coulson has kindly positioned this fragment on the MRC genome map. We have now sequenced both the original 1.25 kb XbaI fragment of unc-6( rh1035) DNA (plasmid NJ#6) and a 2.7 kb HindIII fragment of N2 DNA subcloned from phage NJ#14. The 2.7 kb HindIII fragment has five exons which encode a hypothetical protein fragment of 360 residues. This fragment is not closely related to any protein in the current PIR database The last exon in the 2.7 kb HindIII fragment is followed by an intron which continues beyond the fragment. The first exon is preceded by a non-coding region which may be part of an intron or the unc-6 promoter region. In fact, a possible 21 residue signal sequence with an initiator methionine occurs in the first exon shortly upstream of the Tc1::rh1035 insertion site. TATA and CCAAT motifs occur about 90 and 120 bp upstream of the proposed initiator codon. The decanucleotide sequence TTTGCCTCTC is tandemly repeated about 200 bp upstream of this codon. [See Figure 1]