Cinzia Rinaldo et al. (2001) International C. elegans Meeting "Roles and modes of action of rad-51"

Massimo Hilliard, Sara Ederle, Paolo Bazzicalupo, Adriana La Volpe, Cinzia Rinaldo

[International C. elegans Meeting, 2001]

Our understanding of genetic control of meiotic recombination comes from studies in the unicellular eukaryote Saccharomyces cerevisiae . Most gene involved in control of recombination and meiosis are conserved throughout evolution. However, metazoa have developed a germ cell line distinct from the somatic cell line requiring a different kind of regulation compared to unicellular eukaryotes. Furthermore, in distant species, in spite of evolutionary conservation, similar genes may be subject to different regulation and interactions. Accordingly, elimination of a conserved molecule or interference with a conserved pathway may lead to quite different results in different organisms. For this reason, genetic and biochemical studies in different organisms are likely to contribute to an understanding of the crucial features and fundamental components of recombination pathways and their evolution. Among eukaryotes, C. elegans is the only organism known so far provided with a single rec A like gene, the homolog of RAD51 . Surprisingly, the meiosis specific DMC1 gene present in fungi, plants and mammals, is absent in C. elegans. RNA interference of the rad-51 gene in C. elegans leads to a number of visible phenotypes such as i) high levels of embryonic lethality, ii) increase in the frequency of males, iii) reduced fertility, and iv) hypersensitivity to ionizing radiation in soma . We have analysed in details the mechanisms leading to the above described phenotypes, in order to understand the different functions and modes of action of rad-51 in somatic and germ line cells. We demonstrated that this gene is required at several steps of gametogenesis: gene inactivation, in fact, affects pre-meiotic repair, sister chromatid exchange and homologous recombination and triggers a meiotic checkpoint. Unlike what has been described in mammals, rad-51 expression is not required during embryogenesis, but is required and enhanced in soma in response to DNA damage induced by ionizing radiation.

Adriana La Volpe et al. (2002) European Worm Meeting "rad-51 gene in meiosis and in soma"

Cinzia Rinaldo, Sara Ederle, Adriana La Volpe, Massimo Hilliard, Paolo Bazzicalupo

[European Worm Meeting, 2002]

We have analyzed in detail the effects of rad-51 RNAi in several genetic backgrounds in order to dissect the pathways in which rad-51 is involved in C. elegans in the germline and to establish the role of this gene in the somatic cells. We have verified that the high levels of embryonic lethality are due to maternal effect. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery and postulated roles for such protein and for RAD-51 during meiosis. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced- 3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.

C Rinaldo et al. (1999) International C. elegans Meeting "Ce-hcr-51 gene, the RAD51/DMC1 homolog in Caenorhabditis elegans: a functional characterization"

P Bazzicalupo, S Ederle, M Mastrogiacomo, M Hilliard, A La Volpe, C Rinaldo

[International C. elegans Meeting, 1999]

We have cloned a RAD51/DMC1 homolog in Caenorhabditis elegans , we named Ce-hcr-51 for h omolog of Saccharomyces c erevisiae R AD 51 . This locus, we mapped on chromosome IV on the left of the mec-3 gene on fosmid H36F17, is transcribed into two alternative mRNAs potentially coding for proteins of 395 (Hcr51L) and 357 (Hcr51S) aminoacids in length (Rinaldo et al. 1998). Experiments of RNA interference show that the inactivation of the locus results in adult hermaphrodite sterility of the F1. The F2 embryos show gross abnormality, develop to different stages, but they do not hatch, suggesting a meiotic defect leading to aneuploidy. We are also analysing the gene expression pattern by injecting the promoter sequences upstream the GFP/LacZ coding sequences, and by RNA analysis. We are using a two hybrid system assay to study the ability of the putative products of hrc-51 to form homodymers and heterodymers in vivo . We identified two regions of the Hrc51S protein involved in the homodymerization process, one at the aminoterminal corresponding at the homodymerization domain already mapped in the yeast Rad51 protein (Donovan et al. 1994), and another located in the carboxiterminal half of Hrc-51 that seems to play a role either in the self-association activity or in stabilizing the protein. We are also studying proteins interacting with Ce-hcr-51 products. Donovan J. W., Milne G. T., and Weaver D. T. (1994) Genes Dev 8 : 2552-62. Rinaldo C., Ederle S., Rocco V., and Volpe A. L. (1998) Mol. Gen. Genet. 260 : 289-294.

Sara Vassalli et al. (2006) European Worm Meeting "Identification of Genes required for Anchor Cell Attachment and Invasion during Vulval Development"

ALEX HAJNAL, Sara Vassalli

[European Worm Meeting, 2006]

Sara Vassalli and Alex Hajnal. During vulval development in the hermaphrodite, three out of six equivalent vulval precursor cells (the VPCs P3.p to P8.p) are induced by a signal from the gonadal anchor cell (AC). The LIN-3 EGF growth factor produced by the AC activates the EGFR/RAS/MAPK pathway in the VPCs to specify the primary cell fate. Prior to vulval induction, the gonad is separated from the VPCs by two basal laminas covering each tissue such that the position of the AC relative to the VPCs is variable when the animal moves. At the time of vulval induction in the early L3 stage, however, the AC attaches to the basal side of the future primary cell P6.p. After the first round of vulval cell divisions (Pn.px stage), the basal laminas separating the gonad from the vulval cells start to dissolve precisely under the AC. After the second round of divisions (Pn.pxx stage), the basal laminas between the AC and the four primary vulval cells are interrupted, and the AC invades the vulval tissue.. While the mechanism of vulval induction is well studied, less is known about genes regulating the attachment of the AC to P6.p and the following invasion step. To study these two processes, we performed a forward genetic screen for mutants with defects in AC positioning and/or invasion. Since worms with an abnormal or missing connection between the gonad and the vulva usually display a protruding vulva (Pvl) phenotype, we first screened for animals with a Pvl phenotype and then examined them for defects in the positioning or invasion of the AC. Among 5300 F1 clones, we identified 8 sterile mutants with a misplaced AC and 2 mutants, in which the AC is normally positioned but fails to dissolve the basal laminas and invade the vulval tissue. Details about the cloning and analysis of the genes identified in this screen will be presented at the meeting.

La Volpe A et al. (1996) European Worm Meeting "DNA elements and proteins involved in regulation of recombination."

La Volpe A, Rocco V, Valenzi M, Rinaldo C

[European Worm Meeting, 1996]

The search for factors/elements triggering the regulation of homologous recombination in C. elegans is an intriguing issue. A few years ago we showed a close correlation between the distribution of some repetitive DNA elements and the spatial regulation of recombination along the chromosomes (Cangiano et al. 1993). It has been recently suggested (Barnes et al. 1995) that, among the repetitive DNA families we mapped, the the Cerep3 family is the one most likely to comprise recombination promoting elements. We will discuss what evidence, taken from the analysis of the available C. elegans genomic sequence, and classical genetic data in literature lead us to believe that actually best candidates for recombination promoting elements are the ITRs (Interstitial Telomeric Repeats). In order to confirm the validity of this indication we are trying to clone and characterise genes coding for proteins binding to the ITRs elements (see Ederle et al. this meeting). In a further approach to the study of factors involved in the regulation of homologous recombination we have cloned the C. elegans RAD51 homologue. In the extensive C.elegans ETS database we found a 447bp long ETS clone, named CEMSE47, identified by McCombie et al. that shares significant sequence homology with Rad51 cDNAs from humans, drosophila and yeast. To screen our cDNA library for clones of C. elegans RAD51 we performed a PCR with one primer from the library vector and a specific RAD51 primer derived from CEMSE47. In order to confirm the bona fide of the several products obtained, the PCR products were hybridized with an independent RAD51 oligonucleotide also derived from the original ETS. We subcloned the PCR products and obtained two small clones both at the 3' end of the gene. We used these clones to screen the entire library (about 1.000.000 plaques were plated) and we identified 91 positive clones. Each positive clone was subsequently screened for the insert length using the couple of primers used in the first PCR reaction (one at the 3' end of the gene and the other within the vector sequence). About a third of the clones were longer that 1kb. We have sequenced the longest cDNA (1371bp). We also identified, by primer extension, the 5' end of the mature mRNA. The Rad51 messenger RNA is 1486bp in length up to the polyA tail and the deduced aminoacid sequence shows a high level of identity with other eukaryotic Rad51 homologues. The Rad51 gene has been located on the C. elegans physical map (on the clones Y54E6 and Y42G11) and it maps on chromosome IV to the left of the mec-3 gene. The Rad51 gene will be used as bait in a yeast based two-hybrid system to select for other regulatory proteins in the recombination pathway. e-mail: lavolpe@iigbna.iigb.na.cnr.it