Worm Breeder's Gazette 10(3): 25

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.

Analysis of vit-2 Promoter Mutations in Stable Transformants

Susan Greenspoon, Sherryl Broverman, John Spieth, Jerry Cane, Peg MacMorris and Tom Blumenthal

Figure 1

In the last gazette we reported that stable transgenic lines 
containing the vit-2/vit-6 fusion gene and only 247 base pairs of 
upstream sequence, expressed abundant quantities of the fusion protein 
(fp155) with correct developmental regulation.  We are now performing 
experiments to determine whether specific sequence elements within 
this 247 bp are responsible for the sex-, stage- and tissue-specific 
expression.  Recall that this region contains 5 sequences with a 6/7 
match to Box 1 (TGTCAAT) and 2 with a 6/7 match to Box 2 (CTGATAA), as 
shown in the Figure.  Perfect matches to the consensus are represented 
by filled symbols.
[See Figure 1]
The Figure shows the mutations we have made, the number of 
transgenic strains containing at least one complete copy of the gene, 
and estimates of expression in adult hermaphrodite animals.  The 
accumulation of fp155 was assessed by Coomassie-stained gels and 
Western blots, and copy number was determined by genomic Southerns.  
All of the strains contain either one or two copies of the stably 
integrated plasmid.  About half are homozygous viable.
While a very similar pattern of Box 1 and Box 2 elements has been 
considered in the eleven C.  elegans and C.  briggsae vit upstream 
regions, some of these elements can be removed or altered without 
total loss of promoter activity.  The expression of fp155 in 
transformants obtained with the pJ(delta)NN deletion demonstrates that 
the spacing between elements can be changed without completely 
destroying the promoter.  The deletion of the Box 1 at -180, which is 
present in all eleven promoters, also has no obvious effect on fp155 
accumulation.  Measurement of mRNA synthesis rates will be required 
before we can conclude whether these Box 1's play any role in promoter 
function.  On the other hand, the Box 1 closest to the TATA box, which 
is by far the most highly conserved element in all eleven promoters, 
appears to be absolutely required for activity.  Even though the point 
mutation we tested (pJ2478) leaves four other possible Box 1 elements 
intact, no fp155 has been detected in the two transgenic lines 
containing this plasmid.  While this Box 1 is necessary for expression 
it is not sufficient given the lack of expression from the pJ49 
promoter.  Thus sequences farther upstream also appear to play a role 
in regulating expression.

Figure 1