Worm Breeder's Gazette 12(1): 30 (September 1, 1991)
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As previously reported (WBG 11(2) p.63,11(4) p.52),we have been studying the gene structure and function of alpha-tubulin gene in C. elegans.
Now we have tried to study in situ hybridization and lacZ expression pattern of tubulir a lpha2 gene in C. elegans.
First,we tried the in situ hybridization with modified S. Mitani and M. Chalfie protocol [We changed the proteinase K (300-400µg/ml),and hybridization solution (Carrier is only salmon sperm DNA 200µg/ml)],and prepared SacI-EcoRI about 500bp fragment of tubulin a lpha2 allele in 3'-flanking region as a probe. Hybridization results show most of the worms were stained in the nerve ring,and some worms were stained in the pharynx and the snout (tip of the head region) in L3 -adultstages.
On the other hand,in lacZ expression,we constructed HindIII-PstI 822bp fragment of tubulin a lpha2 gene (5' flanking region was 400bp.) into the lacZ expression vector ( pPD16 .51)from A. Fire. LacZ expression pattern show staining of the nerve ring, pharynx and a few cells near the anus in adult,and the neural cells in L2 and L3 stages.
Apparently, both expression pattern are very similar (nerve ring and pharynx were strongly stained with both experiments.). But lacZ expression is more variable, we think the reason may be that the 400bp 5'-flanking region is not enough to exactly regulate this gene. We plan to construct BamHI about 5kb fragment of tubulin a lpha2 gene (5'-flanking region is about 4.5kb.) into the expression vector, and test the expression of tubulin a lpha2 gene in C. elegans.
Acknowledgement: We wish to thank Drs. S. Mitani and M. Chalfie for in situ protocol, Dr. Fire for expression vectors and Dr. J. Miwa for Discussions.
[See Figure 1]