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.

[1]  R. Haynes,et al.  Molecular characterization of the Saccharomyces cerevisiae dihydrofolate reductase gene (DFR1). , 1987, Nucleic acids research.

[2]  J. Hamlin,et al.  The mammalian dihydrofolate reductase locus. , 1990, Biochimica et biophysica acta.

[3]  V. Walker,et al.  The purification of dihydrofolate reductase from Drosophila melanogaster. , 1990, Biochimica et biophysica acta.

[4]  Z. Chen,et al.  Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene , 1989, Molecular and cellular biology.

[5]  J. Han,et al.  Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene , 1986, Molecular and cellular biology.

[6]  S. Beverley,et al.  Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Kaufman,et al.  Primary structure of chicken liver dihydrofolate reductase. , 1980, Biochemistry.

[8]  J. Bertino,et al.  Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. , 1978, The Journal of biological chemistry.

[9]  G. Attardi,et al.  Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes. , 1984, Journal of molecular biology.

[10]  C Benoist,et al.  Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[11]  V. Walker,et al.  Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster. , 1990, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[12]  M. Ashburner,et al.  Molecular analysis of the Adh region of the genome of Drosophila melanogaster. , 1985, Journal of molecular biology.

[13]  Douglas R. Smith,et al.  Nucleotide sequence of the E. coli gene coding for dihydrofolate reductase , 2022 .

[14]  A. Fallon,et al.  Transcripts from a mosquito dihydrofolate reductase gene: evidence for heterogeneity at the 5' end. , 1993, Insect biochemistry and molecular biology.

[15]  R. Schimke,et al.  Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. , 1982, The Journal of biological chemistry.

[16]  J. Maizel,et al.  The functional human dihydrofolate reductase gene. , 1984, The Journal of biological chemistry.

[17]  Stephen M. Mount,et al.  Splicing signals in Drosophila: intron size, information content, and consensus sequences. , 1992, Nucleic acids research.

[18]  L. Chasin,et al.  Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells. , 1983, Nucleic acids research.

[19]  D. Mead,et al.  Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning. , 1990, Gene.

[20]  A. Fallon,et al.  An amplified insect dihydrofolate reductase gene contains a single intron. , 1991, European journal of biochemistry.

[21]  G. Wahl,et al.  Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae. , 1988, Gene.

[22]  G. Clore,et al.  Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei. , 1985, Gene.

[23]  Stanley N Cohen,et al.  Structure and genomic organization of the mouse dihydrofolate reductase gene , 1980, Cell.

[24]  P. Farnham,et al.  Heterogeneity at the 5' termini of mouse dihydrofolate reductase mRNAs. Evidence for multiple promoter regions. , 1985, Journal of Biological Chemistry.

[25]  A. W. Phillips,et al.  The amino acid sequence of dihydrofolate reductase from the mouse lymphoma L1210. , 1979, The Journal of biological chemistry.

[26]  L. Hood,et al.  Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. , 1987, Proceedings of the National Academy of Sciences of the United States of America.