Worm Breeder's Gazette 11(5): 18

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.

lin-22: Trying to Clone a Gene from HELL

Lisa Wrischnik and Cynthia Kenyon

Once upon a time, a young graduate student (me) optimistically set 
out to clone the lin-22 gene.  I thought this gene was especially 
interesting because gene dosage studies of lin-22 and mab-5 suggested 
that the two genes functionally antagonize one another.  The N62 
strain was used to identify a Tc1-element located 0.5 mu to the left 
of lin-22 (on the left arm of LGIV).  Based on other markers in this 
region, 0.5 mu might be only 1-2 cosmids.  I cloned a 3.3 kb fragment 
containing this Tc1-element and the two flanking sequences of 0.8 and 
0.9 kb.  When these fragments were used to probe worm genomic DNA 
Southern blots, a single, strongly hybridizing band was observed, with 
some high MW material hybridizing very weakly.  I then set out to 
obtain lambda clones so that I could map this DNA to a contig that 
would contain lin-22.After screening many, many genomes and isolating 
only 'positives' that mapped to at least two different chromosomes (
none of them even LGIV), I began to suspect that the 0.8 and 0.9 kb 
fragments contained repetitive DNA and that I had never found a true 
positive in our library.  I was able to isolate a smaller 450 bp 
fragment that appeared not to contain any repetitive DNA.  This probe 
was found to hybridize to absolutely nothing on the polytene YAC blot, 
and I grew despondent because the gene was probably in a gap in the 
physical map.  (There is a gap in the left arm of LGIV that is approx. 
4% the size of the chromosome based on in situ data.  If one divides 
the size of the genome by the no. of chromosomes, 10+E8 bp/6, one gets 
1.6x10+E7 bp per chromosome.  4% of this number, the size of this gap, 
is 666 kb-just a little piece of hell on LGIV).
Thinking that the presence of repetitive DNA in this region might 
have made clones unstable in our library, I then used the 450 bp 
fragment to probe an unamplified N2 library (thank you, Alan) grown on 
a recB-recC-sbcB- bacterial host.  I and my ever hopeful P.I.  learned 
what real positives looked like.  I began to make DNA from these 
unhappy phage in anticipation of the glorious contig awaiting my 
In the meantime, the 450 bp fragment had been sent to Bob 
Waterston's lab where Bart (my hero) used this fragment to probe their 
YAC library.  Some strong positives were identified; and I, older and 
battle-hardened, was finally united with a potential contig.
The putative lin-22 
K08D11/Y54G2cyB, Y55C10cyB, 
I am currently awaiting confirmation that this is indeed the region 
containing my probe before I start microinjecting.  This K08D11 contig 
might contain lin-22; but if it does not, at least the lin-22 gene is 
now flanked by two contigs: K08D11 to the left and the unc-33 contig 
to the right (unc-33 is the nearest cloned gene to lin-22 and is about 
2.5 mu to the right of lin-22).  In the meantime, I am drinking 
heavily and am preparing to walk into the gap from the left end of the 
unc-33 contig.  I will also use pulse-field gel electrophoresis of 
very large fragments of genomic DNA to ascertain the distance between 
the 450 bp lin-22 probe and the ZK226 cosmid at the end of the unc-33 
contig.  Insightful comments and encouraging remarks are welcome.