Worm Breeder's Gazette 11(4): 27b

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Cut Out the YAC and Get On With It

Tom Barnes

Figure 1

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]

Figure 1