Worm Breeder's Gazette 11(1): 24

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.

A Case of Transformation by Homologous Integration: Alteration of Endogenous vit-2 Expression

Sherryl Broverman, Peg MacMorris, John Spieth and Tom Blumenthal

Figure 1

In C.  elegans integrative transformation generally occurs at random 
locations.  Although it would be extremely valuable to have a way to 
selectively alter the in vivo expression of genes that have been 
cloned, we still lack a way of increasing the frequency of homologous 
integration events above the level of a rare event.  (Note: Andy Fire 
has recently observed two cases of homologous interactions.) We report 
here a single example of a simple homologous integration event in 
which expression of an endogenous gene has been altered.
A single copy of the injected plasmid integrated in vit-2, 
apparently as the result of a single reciprocal crossover within the 4 
kb of DNA in which vit-2 and the plasmid are identical.  The injected 
plasmid, the endogenous vit-2 gene and the structure of the vit-2 
region in the transformant are diagrammed 
[See Figure 1]
We indirectly recognized this serendipitous occurrence.  We measured 
vit-2, ls by RNAse protection 
assays on a large number of integrative transformants containing a 
variety of deletions and point mutants in the vit-2 promoter.  We 
discovered one strain, homozygous for an insertion containing a 5' 
promoter deletion of 73 bp, that gave two surprising results: 1) 
elevated expression of the fusion gene mRNA, compared with full length 
promoter controls; and 2) reduced levels of endogenous vit-2 mRNA 
levels.  As shown in the diagram above, integrative transformation 
places the fusion gene under the control of the normal vit-2 promoter (
Pvit2), while the vit-2 gene comes under the control of the deleted 
fusion gene promoter (P174).  This particular fusion gene promoter has 
only the most 5' VPE1 element deleted, leaving 174 bp of upstream 
region intact and resulting in only a small reduction of promoter 
activity.  Had we been using one of our more severe mutations, such as 
a larger deletion or a point mutation in the most promoter proximal 
VPE1 element, the result would have been a completely inactive vit-2 
We have confirmed that the unexpected expression levels are in fact 
due to the promoter switch hypothesized: First, genomic Southern 
analysis demonstrates that the transformant has lost a vit-2-specific 
restriction fragment (a 22.1 kb KpnI fragment) and gained KpnI 
fragments predicted by the homologous event (6.5 and 23.8 kb).  Second,
genetic mapping data shows that both sup-7 activity and the synthesis 
of the vit-2/6 fusion protein are X-linked, and that the vit-2/6 
plasmid maps 2.5 map units from lon-2, consistent with the physical 
map position of vit-2 close to TCMEC2 on the X chromosome.  This 
experiment also places vit-2 on the genetic map.  We are now trying to 
obtain conditions that will increase the frequency of homologous 

Figure 1