Simske JS et al. (1994) Worm Breeder's Gazette "Specification of Vulval Cell Fates By Sequential Signalling Pathways."

Simske JS, Kim SK

[Worm Breeder's Gazette, 1994]

Specification of vulval cell fates by sequential signaling pathways Jeffrey S. Simske and Stuart K. Kim, Dept. of Developmental Biology, Stanford University Medical Center, Stanford CA 94305

Otsuka AJ (1990) Worm Breeder's Gazette "ISOLATION AND CHARACTERIZATION OF UNC-104 CDNA CLONES"

Otsuka AJ

[Worm Breeder's Gazette, 1990]

Although the unc-104 fragments isolated from an Arhinger-Kimble library were instrumental in obtaining approximately 4 kb of the 6 kb mRNA sequence, it was desirable to obtain full length cDNA clones to both complete the cDNA sequence and to produce the recombinant protein product in bacteria. The discrepancy between the abundance of unc-104 cDNA clones in the Arhinger-Kimble library (approximately 1 per 10,000) and in the Kim-Horvitz (none in 200,000) and Thacker-Capecci libraries (none in 100,000), led us to screen the Barstead-Waterston and Schauer-Wood libraries. The abundance of cDNA clones in these additional libraries suggests that an insufficient number of clones was screened in the cases of the Kim and Thacker libraries. cDNA clones were isolated from 2.6 million phage (1.4 million phage screened from the Barstead-Waterston bank, 0.6 million the Schauer- Wood early embryonic and him-8 embryonic libraries). The probe was the 5'-most cDNA fragment obtained from the library. The results are shown below: [See Figure 1] The two cDNA clones from the Barstead library overlap and cover a region of 4.7 kb. DNA sequencing of the cDNA clones is currently in progress.

Rocco V et al. (1996) Worm Breeder's Gazette "Cloning of the Rad51 gene in C. elegans"

Ederle S, La Volpe A, Valenzi M, Rocco V, De Martino A

[Worm Breeder's Gazette, 1996]

We are interested in the mechanisms of homologous recombination in the nematode C. elegans. In prokaryotes, the RecA protein has a pivotal role in homologous recombination, and in vitro it has been demonstrated to catalyse the transfer of a DNA strand on a homologous molecule. In different eukaryotes (as yeast, mouse, man, and others) structural homologues of the bacterial RecA protein have been found, suggesting a certain level of conservation in some recombination pathways among living organisms (Shinohara et al. 1993 Cell 4: 239). In yeast an ATP- dependent DNA binding activity and, more recently, a strand transfer activity of the Rad51 gene product have been shown, whereas for the other Rad51 homologues the strand transfer activity has not yet been demonstrated (Sung and Robberson 1995 Cell 82). Experimental evidences in yeast (Milne and Weaver 1993 Genes Dev. 7:1755) suggest that the Rad51 product acts in a multiprotein complex in eukaryotes.

Rosenbluth RE et al. (1985) Worm Breeder's Gazette "update on linkage group V (left)."

Johnsen RC, McKim KS, Rosenbluth RE, Baillie DL, Turner LM

[Worm Breeder's Gazette, 1985]

Our analysis of LGV(left) is continuing. At present 10 deletions, providing 13 different breakpoints, divide the region into 14 zones ( figure). The table shows recessive lethal mutations that have been mapped to the zones. [See Figure 1] At Cold Spring Harbor (1985) we noted that deletion mapping placed unc-34 to the left of unc-60. This has been additionally confirmed by 3-factor mapping. The two genes are 0.8 - 3.4 m.u. apart. Another gene, let-336 has now been deletion-mapped to the left of unc-34 and therefore becomes the current left-end gene of LGV. One of us (Kim McKim) has begun an intragenic mapping study of unc- 60. We are in possession of the alleles, e677, e723, e890 and m35 and would greatly appreciate receiving others. We have noticed that at least e677 and m-35 differ greatly regarding their viability as heterozygotes over sDf28: e677/sDf28 barely survives while m35/sDf28 survives well. This relationship correlates with their respective homozygous phenotypes, - m35 being more mobile than e677.

Seydoux GC et al. (1995) Worm Breeder's Gazette "Evidence for a maternally-provided dowry of SL1 precursor RNA."

Seydoux GC, Fire A

[Worm Breeder's Gazette, 1995]

Thanks to Kim Ferguson and Joel Rothman for gifts of SL1 plasmid and zen-1 strains. We have previously described the embryonic distribution of RNAs containing the splice leader SL1 (Seydoux and Fire, 1994, Development 120, p. 2823). Using an oligo complementary to the 22 nucleotides of SL1 that are transpliced, we had detected SL1-containing RNAs in the cytoplasm of all cells, and in P granules, from the one-cell stage to the pretzel stage. We have now examined the distribution of the full-length SL1 precursor RNA. To do this, we used a probe complementary to the SL1 precursor RNA, under conditions where hybridization to the 22bp trans-spliced leader would not occur. We detected SL1 precursor RNA in the germline syncytium, in oocyte nuclei, and in interphase and prophase nuclei of early embryos (1-28/50 cell stage). No SL1 precursor RNA was detected past the 50-cell stage, except for occasional staining in 1 or 2 nuclei, which we tentatively identified as the nuclei of P4, and Z2/Z3. To test whether the SL1 precursor RNA detected in embryos was maternal or zygotic, we repeated our in situ hybridization using embryos derived from unc-76(e911) zen-1(e2482); wEx127 (pRF4, SL1/5SrRNA) hermaphrodites. These hermaphrodites segregate 65% viable embryos (which carry the SL1 gene on an extrachromosomal array) and 35% dead embryos (which presumably have lost the array and have no copy of SL1). If the embryonic distribution of SL1 was due to zygotic transcription, we would have observed 35% of embryos with no staining. Instead, we found that 95% of 1-28-cell stage embryos were positive for the SL1 precursor, indicating that this early RNA is independent of zygotic transcription. This result suggests that SL1 precursor RNA synthesized in the germline is inherited by the embryo and repeatedly localized to the nucleus following each mitosis. What about zygotic transcription of SL1? Kim Ferguson and Joel Rothman have demonstrated a zygotic requirement for SL1 as early as the 28-cell stage (wm95, p4). It appears, therefore, that SL1 is transcribed in the early embryo, in addition to being maternally inherited. We have not detected SL1 precursor RNA in somatic nuclei past the 50-cell stage: one possibility is that, in these later stages, SL1 precursor RNA no longer accumulates to detectable levels as increased numbers of zygotic transcripts are available for trans-splicing.

Pilgrim DB et al. (1992) Worm Breeder's Gazette "Preliminary Sequence Analysis of the daf-7 Gene"

Johnsen RC, Riddle DL, Pilgrim DB

[Worm Breeder's Gazette, 1992]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tc1 tagging. On a chromosomal walk toward fem-2 ,one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9 was used to probe a cDNA library constructed by Stuart Kim. Twenty cDNA clones of various sizes up to 2.3kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tc1 -insertionmutant DR1039 ,daf-7 (m434),revealed differences consistent with a 1.6kb Tc1 insertion in DR1039 DNA when compared with non-mutant DNA. For example, when probed with cDNA a 11kb BamHI fragment found in wild-type genomic DNA corresponds to a 12.6kb fragment in mutant DNA. We sequenced both ends of the largest (2.3kb) cDNA clone, and have orfs totaling 460 amino acids. Unfortunately, no appropriate initiator codon or poly A addition site are evident in the cDNA. Therefore, we are screening Bob Barstad's cDNA library to identify a more complete clone. The sequence so far shows no strong homology to anything in the NBRF-PIR data base, although it does have weak homology to some calcium dependent ATPases.

Huang LS et al. (1990) Worm Breeder's Gazette "Molecular cloning of lin-15 by the candidate gene approach"

Sternberg PW, Huang LS, Tzou P, Mendel JE

[Worm Breeder's Gazette, 1990]

We have rescued the lin-15 multivulva.(Muv; visible or AB) and synthetic (syn; class A and class B) Muv phenotypes with cosmid PS#74B3. This cosmid maps to the far left (formerly, far right) end of the clb-1 contig on the X chromosome and was originally isolated because it weakly hybridizes to the G-protein PCR clone 1, as reported in WBG11(2):32. As we had been trying to clone lin-15 (see WBG11(3) :60 for last update), we knew from the map data that lin-15 should lie either at the right edge of the clb-1 contig or in the gap off the end. Since PS#74B3 appeared to map closely to lin-15 on the physical map and because the putative G-protein was a candidate gene for a genetic locus presumably involved in signal transduction, we decided to microinject the cosmid to test whether it rescued lin-15. As we had hoped, PS#74B3 rescued lin-15, although the G-protein hybridizing region did not rescue. A 'reverse northern' analysis identified a 6.8 kb BamHI fragment which lay within the region defined as necessary for lin-15 function by injection. When the 6.8 kb fragment was used to screen Stuart Kim and Chris Martin's cDNA libraries, we identified two classes of non-cross hybridizing cDNAs which mapped to opposite sides of our 6.8 kb fragment, with cDNA2 apparently more abundant than cDNA1.

Finney M et al. (1990) Worm Breeder's Gazette "POU Family Genes in Worms and Mammals"

Burglin TR, Ruvkun GB, Hird SN, Finney M

[Worm Breeder's Gazette, 1990]

As part of the grand homeobox search (1), we have found nine loci that hybridize with oligonucleotides designed to pick out POU domain genes. The two of these that we have sequenced, ceh-6 and ceh-18, are real POU genes. We guess that there are at least 5-10 POU genes in C. elegans. ceh-6 is located on IC, near lin-28. Its POU domain sequence is most similar to a class of mammalian genes including Brn-1( 2) (the Brn genes were cloned by PCR, so sequence is available only for a small fragment). Another mammalian gene, Brn-3(2), has a POU domain more closely related to unc-86 than to other mammalian genes. Thus at least two subfamilies of POU domain genes have members in both worms and mammals. This strongly suggests that the unc-86 and ceh-6 subfamilies existed before worms split from mammals. ceh-18 was first cloned as cDNAs from the Kim library, and later placed somewhere on XL. Based on cDNA frequencies, it seems to be the most highly expressed POU gene. It's a big gene, 30-50 kb genomic, although all of the cDNAs are less than 3 kb. ceh-18 is the most divergent POU gene found so far the POU domain sequence differs from the previous consensus at seven locations, and it has the longest linker region between the POU- specific region and the homeodomain. Given the relationships mentioned above, we expect that genes related to ceh-18 will also be found in mammals. [See Figure 1]

Savage C et al. (1994) Worm Breeder's Gazette "More tolloid Homologs in the Worm."

Padgett RW, Finelli AL, Savage C, Cho SH

[Worm Breeder's Gazette, 1994]

More tolloid homologs in the worm Cathy Savage, Seo Hee Cho, Alyce L. Finelli, and Richard W. Padgett. Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855. Members of the tolloid/BMP-1 family of metalloproteases are thought to interact with extracellular growth factors to regulate a variety of developmental events. In particular, the Drosophila gene tolloid interacts with decapentaplegic (dpp), a member of the TGF-,B superfamily. Similarly, vertebrate BMP- 1 co-purifies with other BMPs (bone morphogenetic proteins) that are related TGF-,B-like ligands. Proteins in the tolloid/BMP-l family share a similar structure: they contain a predicted metalloprotease domain; EGF repeats; and CUB repeats, which are also found in blood complement proteins. These proteases are thought to interact with other proteins through the EGF and CUB repeats, and to regulate their activity through proteolysis. We have previously reported sequence analysis of the first C. elegans member of this metalloprotease family (WBG 12(4), p.30), which we have called toh-l (tollisk). Since then, the genome sequencing consortium has identified four more predicted ORFs with sequence similarity to tolloid. We have therefore isolated and sequenced cDNAs for these genes, to determine whether these new genes encode true members of the tolloid/BMP-1 family. One of the putative homologs displayed no protease homology. The predicted structures of the other three homologs are summarized below. Two of these, on the cosmids COSDl 1 and F42A10, contain related metalloprotease domains, but none of the characteristic protein interaction motifs of the tolloid/BMP-l family. Only one homolog, on the cosmid R151, was found to contain an EGF repeat and a CUB repeat. We therefore propose the name toh-2 for this gene. toh-2 represents a novel member of this family, because it contains repeats (TSP repeats) found in thrombospondin, an extracellular matrix protein. The presence of these two tolloid homologs in the worm raises several interesting questions: (1) Do these homologs act together in a TGF-,B-like signal transduction pathway, in separate pathways, or combinatorially with other, as yet unidentified, tolloid-like proteins? (2) What roles do these genes have in nematode development? (3) What is the function of the toh-2 thrombospondin repeats? Are there homologs in other organisms that also contain these repeats?

Miller LM et al. (1992) Worm Breeder's Gazette "Using Recombination in Yeast to Narrow the lin-31 Locus from 190 kb to 8.5 kb"

Gallegos ME, Kim SK, Miller LM

[Worm Breeder's Gazette, 1992]

We are initiating molecular studies to show how lin-31 acts to alternatively specify three different vulval cell fates, and to determine how upstream vulval determination genes regulate lin-31 activity (see also Morisseau and Kim, WBG, this issue). In our efforts to clone lin-31 ,we used a yeast recombination strategy that allowed us to rapidly narrow lin-31 rescuing activity within a large YAC clone. The cloning of lin-31 was initiated by identifying a candidate lin-31 Tc1 insertion by transposon tagging (S. Kim and H. R. Horvitz). Although this insertion site appeared to lie within the lin-31 locus (tight genetic linkage, insertions in multiple lin-31 alleles), we now know that this site is about 20 kb away from lin-31 (see below). Cosmid rescue experiments showed that lin-31 rescuing activity is not located to the right of the Tc1 insertion. We could not use similar experiments for the region to the left of the Tc1 insertion, due to the presence of a 10-30 kb cosmid gap located nearby. Instead, we showed that injection of Y14H12 (a 190 kb YAC that spans the cosmid gap) can rescue the lin-31 mutant phenotype. We then used recombination in yeast to generate a nested series of Y14H12 deletions. To do this, we cloned random fragments from F39D1 (the first cosmid to the left of the cosmid gap) into the yeast recombination vector pRB328 ,which contains the HIS selectable marker and vector sequences homologous to the end of the YAC (amp, ori). We then transformed the linearized library of random clones into a yeast strain carrying Y14H12 and selected for His+ colonies. These colonies represent cases in which the linear ends of the recombination vector have copied sequences from Y14H12 by gap repair (see Figure). The resulting recombination products contain inserts of worm DNA whose endpoints are defined by the random fragment from the cosmid F39D1 and the right end of the YAC Y14H12 .The sizes of the YAC derivatives were determined by Southern blot analysis using the starting cosmid (F39D1 )as a probe. This recombination technique is useful because a nested series of YAC deletion derivatives can be rapidly created. In our case, it allowed us to engineer a yeast clone that contained a cosmid gap. Also, circular YAC derivatives can be efficiently isolated from yeast by alkaline lysis. Microinjection experiments showed that a 100 kb circular YAC derivative, but not a 94 kb circular YAC derivative, contains lin-31 rescuing activity. This result suggested that a region contained within F39D1 is required for lin-31 rescuing activity, so we next injected F39D1 itself. Surprisingly, we found that F39D1 also has lin-31 rescuing activity, indicating that the Tc1 insertion sites are not in the lin-31 gene because they are not contained in the rescuing cosmid. Subsequent experiments further delimited the rescuing activity to 8.5 kb. Four lin-31 alleles (3 mutator, 1 EMS) are polymorphic in this 8.5 kb region. In addition, at least three related classes of cDNAs have been isolated and Northern blot analysis using one of the cDNAs as a probe has identified four transcripts in the region. Sequencing of the region is in progress. [See Figure 1]