Dihydrofolate reductase of Drosophila. Cloning and expression of a gene with a rare transcript.
Traditionally, dihydrofolate reductase (DHFR) has been isolated and the corresponding gene cloned from drug-resistant cell lines which have amplified DHFR genes after selection. A Dhfr sequence has now been obtained by nested polymerase chain reaction (PCR) from Drosophila bearing a single gene copy. Using the PCR-amplified partial cDNA as a probe, Dhfr was cloned by screening a Drosophila genomic library. It consists of regulatory regions as well as a 599-nucleotide coding region with a single 50-base pair (bp) intron and encodes a protein of 182 amino acids. Previously we have shown that the enzyme has kinetic properties characteristic of both "prokaryotic" and "eukaryotic" DHFRs. Here we show that the organization of Drosophila Dhfr is strikingly different from its mammalian counterparts and most similar to that of mosquito. A 790-bp transcript was detected by Northern blot analysis, with a single transcription start site located 27 bp upstream of ATG codon. The Drosophila genome contains a single Dhfr copy at 89E and a selected cell line has not amplified the gene. Confirmation of the identity of this gene has been obtained by kinetic studies of recombinant DHFR over-expressed in Escherichia coli cells.
Hao
H
H
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Tyshenko
M G
MG
Walker
V K
VK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004330:Drosophila / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase