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.
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.