Worm Breeder's Gazette 11(5): 14

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.

YAC Nightmares

G. Cangiano and A. La Volpe

In the course of the experiments we carried out in collaboration 
with Bob Waterston to produce a method for linking genomic sequences 
cloned in yeast artificial chromosomes (YACs) making use of repetitive 
probes (Cangiano et al.  1990, Nucl.  Acids Res., 18, 5077-5081.), we 
came across several minor problems due to the very nature of YACs.
A good example of that is what we observed on three YACs (Y39C11, 
Y52D1, Y43E11), we identified in a first screening as overlapping 
because they shared common bands with two independent repetitive 
probes.  When we tried to reproduce these patterns with a new YAC DNA 
preparation, one of these YACs (Y39C11) seemed to have lost the 
ability to hybridize to one probe (pRcS5) and showed a different 
pattern with the other (pRcD1) .  What had happened?  We analyzed a 
few independent colonies from the same YAC strain (Y39C11) on a pulse 
field gel.  The original YAC strain turned out to contain not one but 
two YACs, one of which in fact overlapped the other YACs analyzed.  
The two YACs were present in some colonies of the strain together but 
more often one or the other was lost, furthermore when they were 
present together often the longer one was also partially deleted and 
additional fainter shorter bands were present when the pulse field gel 
was hybridized with a suitable cosmid probe (ZK384).
We may explain these events considering that the yeast host is under 
selective pressure to contain a copy of URA3 yeast gene carried by the 
YAC, but a single copy per cell is sufficient to restore prototrophy, 
so if two YACs are present together one or the other can be easily 
lost and/or undergo various rearrangements.  The occurrence of these 
rearrangements can be explained in several ways: i) high frequency of 
recombination in yeast, ii) the occurrence of segregator sequences (
SEG sequences described by Stinchcomb et al.  1985 Proc.  Natl.  Acad. 
Sci.  USA vol.  82, 4167-4171) might very well lead to dicentric YACs 
and eventually to their destruction, iii) many eukaryotic regulatory 
sequences are common to different species and we know that C.  elegans 
DNA may actively interact with nuclear machineries in yeast (La Volpe 
et al.  (1988) Nucl.  Acids Res.  16, 8213-8231); some of these 
sequences may be selected against leading to rearranged chromosomes.
We should all be as cautious is possible handling YACs and in 
general suspicious of the organization of cloned regions of YAC origin.