Worm Breeder's Gazette 11(4): 27b
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.
The gene tra-3 IV is required in hermaphrodites to permit the correct sexual differentiation of the soma and germ line. Its function is to positively regulate another gene tra-2 perhaps post- transcriptionally. Homozygous mutant animals from heterozygous mothers are completely maternally rescued and appear wild type; 100% of their progeny in turn are Tra and sterile. During the course of trying to clone tra-3 it became clear that much of the region where it had to lie was not represented by cosmids. However the YAC Y72F8U judging from its size (310kb) and position with respect to nearby cosmids was predicted to protrude from the end of the contig I was dealing with and contain the gene. It was important to establish a firm location for the gene and the simplest way was by YAC rescue. Rather than engineering of the YAC in yeast and subsequent isolation by sucrose gradients (Levitan, Mello and Stinchcomb WBG 11#1), I tried preparative PFG. With the assistance of John Sulston a 100ml culture of the appropriate strain of yeast was grown and processed into 20 plugs. These were loaded onto a 20-slot LGT gel which was run in our CHEF apparatus (130V 60s switch 24h run). The YAC band was cut out of the gel under long-wave UV. The total amount in the gel was about 1 g. The volume of the gel slice was about 5ml and this was melted, phenolized, precipitated, and processed as for cosmid injections. The final concentration of the YAC DNA was about 3 g/ml and this was injected syncitially into maternally rescued tra-3 animals without any viscosity problems. Of five animals injected one produced three partially rescued progeny; two had completely rescued tails but no recognizable gonad or vulva and one had an incompletely rescued tail but a wild type gonad and laid a complete brood of Tra animals. I interpret these three animals to be product rescuants; i.e. if any YAC DNA had been present in the gonad there should have been profound rescue of the F2 animals. To obtain clones covering the 120kb region of the YAC which protruded from the contig and was predicted to contain tra-3, I isolated a set of 54 lambda clones from a primary genomic lambda 2001 library (kindly provided by Alan Coulson) using a novel technique, the combinatorial YAC screen or CYS. The library was simultaneously screened with two partially overlapping YACs (Y37H7 270kb and Y1C12 210kb overlap 130kb) which meant I could immediately characterize clones as being Y37+Y1- which I picked. These correspond to the protruding part of the YAC. 54 of these survived rescreening. One clone was used to screen the YAC grids and all positives were grown up and isolated by John Sulston. All of these YACs were then further mapped by CYS onto the grid of 54 clones. 10 groups of clones were thus defined. To confirm that these 54 clones were comprehensive I pooled them all and injected six tra-3(m+z-) worms as before. One animal produced three completely rescued F1s; all three have established transmitting lines. Subpools defined by the CYS were injected and the rescuing activity has been localized to a single pool of 9 clones (9L) spanning 20-30kb. These injections were done with pRol6 which provides me with an easy marker for germ line loss in the arrays of transmitting lines: eEx24 animals are RolTra+ those which have lost the array are Tra+ (i.e. appear wild type). and then their progeny are Tra.As a postscript, it turns out that after fingerprinting of the 54 clones not only do the assignments by CYS exactly concord but they matched a small, bunched cluster of cosmids which includes the 9L pool. Only one was a lorist cosmid (i.e. able to participate in the YAC to cosmid screens), but for some reason it had never been included. [See Figure 1]