Nawrocki LW et al. (1988) Worm Breeder's Gazette "characterisationa and cloning of unc-24."

Thomson JN, Nawrocki LW, Southgate E

[Worm Breeder's Gazette, 1988]

The behavioral phenotype of unc-24 worms is characterized by severely kinked L1 worms that do not wriggle more than one quarter body length in either forward or backward directions, whereas worms from the L2 stage onward toward adults are only slightly kinked in motion and make progress similar to N2 worms. These observations appear to indicate that the unc-24 gene may be affecting the embryonic motoneurones (DAs, DBs and DDs). Reconstruction of three different alleles of unc-24 have not been consistent. The canonical allele (e138) reconstruction shows DA motoneurones to be lacking commissures to the dorsal musculature. A Tc1 allele (e2386) reconstruction contains one DA motoneurone with the same aberration but another with normal morphology. The P32 induced allele (e927) reconstruction presents DA motoneurones with normal morphologies, but the commissures of all D motoneurones (DAs, DBs and DDs) proceed toward the dorsal cord on the opposite side to the wild type condition. To clear up this ambiguity, a reconstruction of an L1 from, the canonical strain is being completed. The unc-24 gene has been isolated. A 5.6kb BamHI Tc1 containing fragment found in an allele (e2386) isolated from the TR679 strain that also shifts to a 4.0kb BamHI fragment in revertants and Bristol strains was cloned into pUC 8. A 1.0kb non-Tc1 containing fragment was isolated from this plasmid by BamHI and EcoRV digestion and was used as a probe to screen the Coulson and Sulston cosmid library. Two true positives were identified that mapped to a contig containing the vit-6 gene on linkage group IV. Fortunately, a larger cosmid in this contig which covered both newly identified cosmids had been used as an in situ hybridization probe by Rita Fishpool and Donna Albertson and showed binding to chromosome IV. The same 1.0kb non-Tc1 fragment was used to isolate 16 lambda clones from the 2a cDNA library of Julie Ahringer. The lambda inserts range in size from 0.6kb to 2.6kb. The largest of these was used as a probe and showed polymorphic band shifts on the genomic Southerns composed of different alleles of unc- 24. This insert is being sequenced.

Pilgrim DB (1988) Worm Breeder's Gazette "zinc finger proteins in worms ?"

Pilgrim DB

[Worm Breeder's Gazette, 1988]

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 [1987]), 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.