Worm Breeder's Gazette 14(2): 88 (February 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Cloning of the Rad51 gene in C. elegans

Vincenzo ROCCO, Arianna DE MARTINO, Sara EDERLE, Marco VALENZI, Adriana LA VOLPE

International Institute of Genetics and Biophysics - CNR- Naples - Italy

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.

We decided to clone the C. elegans Rad51 homologue to have a tool
to study interaction between different genes involved in recombination and
in recombinatorial repair in metazoan.

To identify Rad51 cDNA we designed two primers from a C.elegans
ETS named CEMSE47 (McCombie et al. Nature Genet., 1992, 1: 124), this
447bp long ETS shows homology to the yeast Rad51 gene. With these two
primers we performed a PCR on a C. elegans oriented cDNA library. The
PCR product, used as a probe on the same library, identified 91 positive
clones. Each positive clone was subsequently screened for the insert
length using one primer at the 3' end of the cDNA (chosen again within the
CEMSE47 sequence in between the first two oligos we used) and a second
primer present at the 5' end of the insert within the vector sequence of
the library.

We performed the DNA sequence of the longest cDNA. 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 (see fig.1 the comparison between the Rad51 proteins
f r o m Homo sapiens, Schizosaccharomyces pombe, Saccharomyces
cerevisiae, Drosophila melanogaster and Caenorhabditis elegans).

The Rad51 gene has been located on the C. elegans physical map (on
the clones Y54E6 and Y42G11) and it maps on chromosome IV on the left of
the mec-3 gene.

Our next goal is to use Rad51 cDNA to fish other recombination
proteins interacting with it by mean of the two hybrid system in yeast.

(A figure accompanies the original article.)