Worm Breeder's Gazette 10(3): 62
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.
In an attempt to identify genes involved in sex determination which may be evolutionarily conserved, I have tried looking for a worm homologue to mammalian Testis Determining Factor (TDF). TDF is thought to be the 'master' sex-determining locus in mammals. The putative human gene has been cloned (Page et al., Cell 51:1091 ), and the predicted protein sequence revealed the presence of repeated Cys/His 'zinc finger' domains, indicating a DNA binding function. There are homologues found on the sex chromosomes of most mammals tested. (The human X and Y homologues are apparently now referred to as zfx and zfy, respectively.) A full length TDF cDNA, isolated from a human testis library, was kindly provided by Mark Palmer (ICRF, London) . This was used at low stringency to probe a Southern Blot of N2 genomic DNA. 6-7 bands were seen, depending upon the restriction enzyme. The same probe was used to isolate 9 clones from a genomic lambda library, kindly provided by Alan Coulson. Four of the clones were chosen for further study. Two overlap, the other two appear to be discrete. The clones were fingerprinted by Alan Coulson, and compared with the physical map. The two overlapping inserts (CB#DP004, 005) lie on an unmarked contig which has since been localized by Rita Fishpool to LG V, using in situ hybridization. The third clone (DP002) lies on the rsn-8/myo-2/myb-1/col-9 contig on the right half of X ( although none of these genes has been genetically mapped). The last clone (DP003) has a small insert, and didn't match anything in the data base. The cross-hybridizing regions of each clone are being subcloned for further analysis. So far I have begun characterization of the DP004/005 clone. This insert accounts for three of the bands seen on the southern blot. A fragment of 3.5 kbp, which cross- hybridizes with the human cDNA, was subcloned, and used to probe a Northern Blot. A band of 3.9 kb was detected in pA+ RNA prepared from him-8 worms in L3, L4 and a mixed stage prep, but no band was detected in L2 pA+ or in total RNA. I have not yet looked to see if the message is sex-specific. When this subclone is used as a probe against the human cDNA, it cross reacts most strongly to the portion of the cDNA coding for 6 of the 'zinc-finger' repeats, as well as some nonfinger-coding sequence. I have begun to sequence this region of the DP004/005 clone to see if it also codes for a zinc-finger protein, and if so, if it has a role in sex in worms.