Worm Breeder's Gazette 13(5): 87 (February 1, 1995)
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.
|1||Lab. of Molecular Biology, Toyohashi University of Technology, Toyohashi 441, Japan.|
|2||Present address: Dept of Medical Biochemistry. University of Calgary Medical School, Calgary, Alberta, Canada|
DNA transformation is a widely used technique in C. elegans, but little is known about the effect of cotransformation markers on the pattern of gene expression in transgenic animals. We have examined the effect of cotransformation markers dpy-20 and rol-6 on the expression of tba-l::lacZ alpha-1 tubulin fusion gene in germline transformants. Cellular specificity of the fusion gene expression was found to be very different, depending on the cotransformation marker used. In case of the rol-6, the tubulin fusion gene expressed in neurons in the head and tail ganglia and a set of 38-39 excitatory motor neurons in the ventral cord along the body length of the animal, which we have identified as the set of DA, DB, VA, and VB neurons. In contrast, for the dpy-20 marker system, not only fewer neurons were stained in the head and tail ganglia, but the staining of motor neurons in the ventral cord was dramatically reduced both in their number and intensity. This down regulation of the fusion gene expression in motor neurons was observed, irrespective of the orientation of the dpy-20 chromosomal arrays whether in cis or trans. The dpy-20 arrays in trans configuration to the rol-6 and and tba-l::lacZ tubulin fusion gene arrays in the dpy-20(e2017) background was obtained as follows. Males from a dpy-20(e2017) transgenic culture, carrying the dpy-20 marker in extra-chromosomal arrays (phenotypically Wild Type), were mated with hermaphrodites carrying the tba-l::lacZ/rol-6 arrays (phenotypically Rollers). In the crossed progeny, many roller lines were selected and individually cloned to produce progeny. Among these, transgenic lines were identified that segregated rollers and dumpy animals (in a non-Mendelian ratio), suggesting that these lines carried the d*Y-20 and the rol-6 extrachromosomal arrays in the dpy-20(e2017) mutant background. Since these lines segregated roller and dumpy progeny independent of each other, clearly the two arrays (dpy-20 and rol-6) were not integrated in one extrachromosomal array in these animals. Histochemical staining of these animals revealed that dpy-20 marker suppressed the alpha tubulin fusion gene expresion in both the cis and trans orientations. In controls, where no cotransformation marker was used, the fusion gene expression closely resembled that of the rol-6 marker system. Similar suppression results were observed when the tba-2::lacZ alpha-2 tubulin fusion gene was used in the two systems. Whether the observed suppression is caused by an overexpression of the dpy-20 gene product, or due to the presence of a DNA sequence (e.g. in the promoter region of the dpy-20 gene) that inhibits the alpha tubulin expression in motor neurons (such as by titrating a specific transcription factor), remains to be seen. We thank D. Suleman, D. Baillie, J. Kramer. H. Yasuda, and A. Fire for gene markers and suggestions.