Matthews LR et al. (1994) Worm Breeder's Gazette "A Potential C. elegans E2F Homolog: Is it zyg-9?"

Galas S, Matthews LR

[Worm Breeder's Gazette, 1994]

A potential C. elegans E2F homolog: Is it zyg-9? Lisa Matthews and Simon Galas, CRBM, CNRS Montpellier, France

Thacker CM et al. (1994) Worm Breeder's Gazette "Identifying Mutations in the bli-4 Gene, a Member of the Kex2-like Gene Family"

Srayko MA, Thacker CM, Rose AM

[Worm Breeder's Gazette, 1994]

We are studying the role of two genes that encode serine endoproteases that belong to the Kex2 -likefamily of prohormone convertases. These two genes include bli-4 and one identified as a sequenced cDNA designated cm1 Ob9 (Waterston et al., 1992, Nature Genet 1:114-123). Genetic analysis of bli-4 has enabled us to group mutant alleles into three classes (Peters et al, 1991, Genetics 129:95-102). The first mutant class includes the only viable mutation e937 which results in the blistering of the adult cuticle. Class II mutants include 11 lethal alleles which arrest development in late embryogenesis prior to hatching. These 11 alleles fail to complement e937 .Class III mutants comprise 2 lethal alleles that complement e937 yet fail to complement the class II lethal alleles. Class III animals predominantly arrest as early L1 larvae, although some lethality in late embryogenesis is also observed. The bli-4 gene was originally cloned in our laboratory by Ken Peters. The gene encodes at least three different proteins with extensive sequence similarity to the Kex2 -likefamily of serine endopeptidases. The three isoforms are produced by alternative splicing at the 3'-end of the gene as depicted in the figure of the gene structure below: So far we have been able to identify molecular lesions in three bli-4 alleles, e937 and two class II lethals. The e937 mutation is a 3.2kb deletion that removes the exon unique to blisterin A (Peters and Rose, W.B G. 11:28, 1991) resulting in the complete abrogation of expression of this product as demonstrated by northern analysis. Presumably, blisterin A is required for the production or maintenance of the adult cuticle. The class II mutant, h1 O10is a spontaneous allele isolated in a precomplementation screen for bli-4 alleles in a mut-6 background. The Tc1 insertion has been sequenced and found to reside in exon 9, 3' of the protease domain. A second class II lethal allele, q508 was found to contain a 366bp deletion that removes the splice acceptor and 33 nucleotides from exon 12. q508 was isolated by Lisa Kadyk in Judith Kimble's laboratory. Both of the lesions identified in the class II lethal alleles occur within the first 12 exons that are common to all of the bli-4 transcripts These results suggest that all of the class II alleles contain mutations either within this common region or in regulatory elements that control the expression of the gene, resulting in the loss of activity of all isoforms. We believe that the class III mutants are the result of mutations within the exons unique to either or both blisterin B and C, suggesting that these alternate isoforms have an essential function for early nematode development. We are currently attempting to identify the molecular lesions in the remaining class II and III lethal alleles using PCR-heteroduplexing and direct sequencing of PCR products. We have also initiated studies on a second gene which was originally identified by the random cDNA sequencing project (Waterston et al., 1992, Nature Genet. 1:114-123). The cDNA cm1 Ob9 has been mapped to the YAC clone Y51C6 in a region of chromosome III that is actively being sequenced by the genome sequencing group. Although the sequence of the gene that encodes cm1 Ob9 is not on the current ACEDB, we anticipate that it will be available in the next update. We have sequenced most of the cDNA and have determined that it represents a partial clone which does not include the protease domain. However, the sequence generated suggests that the cDNA encodes a product that most closely resembles Drosophila and vertebrate furin, a member of the Kex2 -likefamily. We are in the process of characterizing this gene in more detail by isolating cDNA clones that are more complete at the 5'-end. We would like to take this opportunity to thank Patty Wohldmann for forwarding cm1 Ob9 to us, and especially Lisa Kadyk in Judith Kimble's lab for the q508 allele. This work was supported by grants from the MRC (Canada) and the B.C. Health Foundation.

Darrell J Killian et al. (2002) Worm Breeder's Gazette "RNAi feeding to produce males"

E Jane Albert Hubbard, Darrell J Killian

[Worm Breeder's Gazette, 2002]

Heat-shock has traditionally been used as a means to obtain males, but particular worm strains will not tolerate this process. This problem may be overcome through genetic means by introducing a him mutation but this involves additional steps and may complicate later experiments. In an effort to obtain males from strains that are not amenable to heat-shock, we turned to RNA mediated interference as an approach. We made and tested a him-14 feeding construct (many thanks to Yuji Kohara for the him-14 cDNA and Lisa Timmons and Andy Fire for the L4440 vector) to see if males could be generated. Hermaphrodites that consume HT115(DE3) bacteria producing him-14 dsRNA consistently produce a low but significant number of males. Interestingly, the males appear among the progeny at the end of the brood. We typically see 5-7% males in the last 100 progeny from three pooled hermaphrodites at 20degC. Our best results are obtained by feeding L4 hermaphrodites for about 50-55 hours and then transferring these worms to a fresh RNAi plate and inspecting the progeny from the end of the brood for males. We did not observe an increase in the percentage of males if the RNAi feeding was carried over additional generations. To test if the effect persists once worms are removed from the dsRNA-producing bacteria, hermaphrodite siblings of RNAi-induced males were transferred to OP50 bacterial lawns. These animals threw fewer than 1% males (from a pooled total of 12 bulk-inspected hermaphrodite broods) in the first generation and none in the subsequent generation. We have also confirmed that males produced by him-14 RNAi can sire cross progeny. Finally, we did not see evidence of embryonic or larval lethality. We stress that feeding him-14 dsRNA to produce males is effective and reversible, but not efficient. We have not successfully obtained sufficient numbers of males from strains that are somewhat unhealthy or that have a reduced brood size. Since males appear at the end of a normal brood, low brood size may hinder the production of males by this approach. Furthermore, we found that the bacteria used to produce males must be freshly grown from a healthy plate colony and used immediately. Ultimately, other genes may be more useful for this purpose. Indeed, large scale RNAi screens have identified many genes that give Him phenotypes when targeted by RNAi (e.g., Fraser et al ., 2000). Theresa Stiernagle kindly agreed to distribute GC363, the HT115 (DE3) bacterial strain carrying pGC8 [ him-14 partial cDNA in the Fire L4440 vector]. Many thanks to David Greenstein for discussions that led to this experiment. These results were also described in a March, 2001 note to the C. elegans newsgroup.

Karashima T et al. (1997) Worm Breeder's Gazette "A C. elegans homologue of DAZ/boule is involved in progression through meiosis during oogenesis"

Karashima T, Sugimoto A, Yamamoto M

[Worm Breeder's Gazette, 1997]

Human DAZ (Deleted in Azoospermia), mouse Dazla, and Drosophila boule encode a family of proteins with a single RNP-type RNA-binding motif and are thought to be essential for meiosis during spermatogenesis (Nature, 381: 783-785, 1996). The C.elegans cDNA project identified a putative homologue of DAZ/boule. This cDNA (yk62b4, kindly supplied by Yuji Kohara) appears to contain the complete open reading frame for the gene and its predicted amino acid sequence contains a putative RNA-binding domain most similar to Boule, as well as a motif found in all DAZ family members (see figure). We tentatively call this gene Ce-boule . Ce-boule maps close to clr-1 on Chromosome II. To examine whether Ce-boule is involved in meiosis in C. elegans, antisense Ce-boule RNA was injected into gonads of wildtype L4 hermaphrodites. F1 hermaphrodite progeny derived from the injected P0s were sterile at high penetrance. The sterile F1s had normal somatic gonads and normal-looking sperm (by both Nomarski and DAPI staining), but oogenesis was severely affected: with Nomarski microscopy, gonad arms were filled with mostly small nuclei, and none of the cells displayed the characteristic morphology of developing or mature oocytes. By DAPI staining, majority of the nuclei looked similar to mitotic cells. Possible pachytene nuclei were occasionally observed in the proximal arms, but no diplotene-diakinesis nuclei were found. Although the morphology of the nuclei appears mitotic, the phenotype is not tumorous (unlike gld-1(null) or glp-1(gf)), judging from the density of the nuclei in the gonad. The injected P0s themselves showed a similar phenotype after they laid approximately 30 fertilized eggs. Thus, antisense Ce-boule injection seems to affect either the transition from mitosis to meiosis or the early stage of meiosis during oogenesis. The expression pattern of Ce-boule in the adult hermaphrodite germ line was studied by in situ hybridization and it was found that Ce-boule mRNA shows a graded pattern in the gonad. The signal was detected starting at the mitosis/meiosis transition zone and increased in intensity toward the pachytene region.The strong signal was maintained through the proximal arm except in mature oocytes, where the signal was distinctly weaker. No signal was seen in mature sperm. This expression pattern is consistent with the hypothesis that Ce-boule is required during oogenesis, for the meiotic entry and/or the prophase of meiosis I. It is surprising that DAZ/boule's possible counterpart in C.elegans appears to be essential for oogenesis and dispensable for spermatogenesis, as both DAZ and boule seem to be mainly associated with male sterility. For further characterization, we are currently screening for mutants of Ce-boule by a PCR-based method and by looking for sterile mutants in the corresponding chromosomal region. We are grateful to Tim Schedl and Lisa Kadyk for helpful suggestions and guidance.

Yingqi Teng et al. (2001) Worm Breeder's Gazette "Promoter - Dissection of C. elegans Hox Gene egl-5"

Scott W Emmons, Yingqi Teng

[Worm Breeder's Gazette, 2001]

C. elegans Hox gene egl-5 is involved in many cell-fate specification events in several tissues in the posterior body region. Jointly with Lisa Girard and Paul Sternberg, we have undertaken an egl-5 promoter dissection in order to understand how the complex egl-5 expression pattern is established. During male tail development, egl-5 is activated in seam cell V6.ppp, which defines the fate of V6.ppp as the precursor of three sensory rays, rays 4, 5, and 6. In an egl-5 null mutant, V6.ppp adopts the fate of its anterior counterpart V6.pap, which produces rays 2 and 3. Another Hox gene, mab-5 , activates egl-5 directly or indirectly. However, MAB-5 is present all through the V6 cell lineage in both hermaphrodites and males. Therefore, the temporally and spatially regulated, sex-specific expression pattern of egl-5 in V6.ppp remains unaccounted for. To study the mechanism of regulation underlying such a precise expression pattern, we decided to identify the regulatory sequence governing egl-5 expression in the seam. We've compared the DNA sequences of the 30kb intergenic region upstream of egl-5 gene in C. elegans and C. briggasae by blasting the two sequences against each other, and found that there are short stretches of noncoding sequences within the egl-5 promoter region that are well conserved between the two species. We have somewhat arbitrarily numbered the longest of these conserved sequences 1-14 from distal to proximal. To assign the function of these conserved sequences, we've made deletion constructs using a PCR-based method with one of our egl-5::gfp reporters, EM#285, as template. Based upon the criterion that deletion of a lineage-specific enhancer will inactivate our egl-5 reporter expression in that lineage, we've roughly mapped the enhancer of rectal epithelial cells to a region containing conserved sequences 7 and 8, and the enhancer of male tail hypodermal cells to a region containing conserved sequences 9 and 10. We've also found that conserved sequences 11 and 12, which lie 3kb upstream of the translation initiation site, are not only necessary for the expression of our reporter in the V6 lineage, but also sufficient to replicate the expression pattern of egl-5 in the V6 lineage when linked to a D pes-10::gfp reporter. Sequences 11 and 12 lie right next to each other within a highly conserved 300bp region, which implies that they are likely to share the function of the V6 lineage enhancer. The same region from C. briggasae gives exactly the same expression pattern driving D pes-10::gfp , further suggesting that the conserved sequences within this region represent the enhancer of the V6 lineage. We've found putative binding sites within the region for several transcription factors including TRA-1 (thanks to M. Sohrmann of the Sanger Center), POP-1 (TCF/LEF1 homolog), MAB-5/CEH-20 (Extradenticle homolog). Now we are making deletions and point mutations to eliminate the binding sites and looking for changes in the expression pattern of the reporter. Surprisingly, when we deleted the putative TRA-1 site, we got ectopic reporter expression in anterior seam cells, ventral cord neurons and head neurons in both male and hermaphrodite. We hypothesize that we have deleted another repressor binding site within or overlapping with this TRA-1 site, and now we are trying to identify this repressor.