CONSTRUCTION OF A TISSUE-SPECIFIC PUNCH PROMOTER:: LAC Z EXPRESSION VECTOR THAT ENCODES GTP CYCLOHYDROLASE IN THE FRUIT FLY DROSPHILA MELANOGASTER

摘要

The Punch locus of Drosophila encodes GTP cyclohydrolase I, a multimeric enzyme that catalyzes the firs step in pterin biosynthesis. One pterin, tetrahydrobiopterin (BH4), is a necessary cofactor for typrosine hydroxylase (TH) which is involved in neuro- transmitter biosynthesis. One Punch clone, pO.380, overlaps the 5' end of a 1.75 kb transcript. Punch mutations which alter the levels of this transcript also affect TH activity. A restriction map of pO.380 was generated; two restriction sites, EcoRI and NheI, were found to flank the promoter sequence. A 2.3 kb EcoRI-NheI DNA fragment was gel purified and ligated into the polycloning site of the expression vector, pJY505, which is upstream and in frame of the lacZ gene that encodes β galactosidase. This Punch pO.380 promoter::lacZ vector can be injected into Drosophial embryos, and tissue specific expression of this construct can be detected by staining tissues with the β galactosidase chromogenic substrate, X Gal.