Worm Breeder's Gazette 10(1): 64

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.

Effect of a vit-2/vit-6 Fusion Gene on Expression of Endogenous vit Genes in Stable Transgenic Lines

J. Spieth, P. MacMorris and T. Blumenthal

Figure 1

The fusion gene shown below was incorporated onto a plasmid 
containing sup-7 and injected into tra-3 oocyte nuclei, ala Andy Fire. 
We obtained three stable transgenic lines containing multiple copies 
of the plasmid integrated as a tandem array.  One strain (temporarily 
designated 893) has two to four copies of the plasmid DNA and is not 
viable as a homozygote, while the other two strains (899 and 910) each 
have about 15 copies per haploid genome.  Surprisingly, both of the 
higher copy strains are viable as homozygotes.
The fusion genes appear to be expressed with the same temporal, 
tissue and sex specificity as the endogenous vitellogenin genes, 
indicating that sufficient DNA is present to specify correct 
developmental regulation.  The 155 kDa polypeptide product of the 
fusion gene, which is made in abundance, is not taken up into oocytes, 
but instead accumulates in the body cavity, and perhaps also in the 
intestine.  It appears to be secreted, but not as well as normal 
[See Figure 1]
On Western blots the fusion gene product is bound by antibodies 
specific for the endogenous yolk proteins, yp170 and yp115, but not by 
those for yp88, demonstrating for the first time that yp115 is at the 
C terminus of the vit-6 gene product.
In each transformed strain, the level of expression from the fusion 
genes is roughly proportional to the number of copies.  That is, 
homozygous 910 and 899 animals make more 155 kDa fusion protein than 
heterozygous individuals, which in turn make considerably more than 
893 heterozygotes.  Interestingly, in both strains containing about 15 
copies/haploid genome the level of mRNAs for all of the endogenous 
vitellogenins drops dramatically (as much as 80% reduction).  This has 
been demonstrated by Northern blots, by in vitro translation and by in 
vivo synthesis.  Furthermore, the level of endogenous vitellogenin 
synthesis is more severely competed in the homozygotes than in the 
heterozygotes.  The total vitellogenin mRNA accumulation in these 
transgenic strains remains approximately the same as wild-type except 
fusion gene mRNA is present in place of the normal mRNA.  It appears 
that 2-4 copies is not enough to compete since strain 893 makes normal 
levels of vitellogenin mRNAs in addition to the mRNA for the fusion 
Since there are six endogenous vitellogenin genes, it is remarkable 
that the presence of only 15 additional genes results in such a severe 
reduction in production of endogenous vitellogenin mRNA.  There are 
several possible explanations of these results: 1.  The extra 
promoters may be competing for a limiting activator protein.  2.  The 
transcriptional capacity of the intestine may be saturated.  3.  The 
fusion protein accumulating in the intestine may result in 
destablization of endogenous vitellogenin mRNA.

Figure 1