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. 1980 Oct;77(10):5794–5798. doi: 10.1073/pnas.77.10.5794

Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

D A Goldberg
PMCID: PMC350157  PMID: 6777776

Abstract

The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  3. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Buell G. N., Wickens M. P., Payvar F., Schimke R. T. Synthesis of full length cDNAs from four partially purified oviduct mRNAs. J Biol Chem. 1978 Apr 10;253(7):2471–2482. [PubMed] [Google Scholar]
  7. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  8. Fyrberg E. A., Kindle K. L., Davidson N., Kindle K. L. The actin genes of Drosophila: a dispersed multigene family. Cell. 1980 Feb;19(2):365–378. doi: 10.1016/0092-8674(80)90511-5. [DOI] [PubMed] [Google Scholar]
  9. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  10. Lepesant J. A., Kejzlarova-Lepesant J., Garen A. Ecdysone-inducible functions of larval fat bodies in Drosophila. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5570–5574. doi: 10.1073/pnas.75.11.5570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  12. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. March S. C., Parikh I., Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal Biochem. 1974 Jul;60(1):149–152. doi: 10.1016/0003-2697(74)90139-0. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McDonald J. F., Ayala F. J. Gene regulation in adaptive evolution. Can J Genet Cytol. 1978 Jun;20(2):159–175. doi: 10.1139/g78-018. [DOI] [PubMed] [Google Scholar]
  16. O'Donnell J., Gerace L., Leister F., Sofer W. Chemical selection of mutants that affect alcohol dehydrogenase in Drosophila. II. Use of 1-pentyne-3-ol. Genetics. 1975 Jan;79(1):73–83. doi: 10.1093/genetics/79.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Donnell J., Mandel H. C., Krauss M., Sofer W. Genetic and cytogenetic analysis of the Adh region in Drosophila melanogaster. Genetics. 1977 Jul;86(3):553–566. doi: 10.1093/genetics/86.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  19. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sofer W., Ursprung H. Drosophila alcohol dehydrogenase. Purification and partial characterization. J Biol Chem. 1968 Jun 10;243(11):3110–3115. [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. St John T. P., Davis R. W. Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell. 1979 Feb;16(2):443–452. doi: 10.1016/0092-8674(79)90020-5. [DOI] [PubMed] [Google Scholar]
  23. Thatcher D. R., Sawyer L. Secondary-structure prediction from the sequence of Drosophila melanogaster (fruitfly) alcohol dehydrogenase. Biochem J. 1980 Jun 1;187(3):884–886. doi: 10.1042/bj1870884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson J. N., Jr, Ashburner M., Woodruff R. C. Presumptive control mutation for alcohol dehydrogenase in Drosophila melanogaster. Nature. 1977 Nov 24;270(5635):363–363. doi: 10.1038/270363a0. [DOI] [PubMed] [Google Scholar]
  25. Vigue C., Sofer W. Chemical selection of mutants that affect ADH activity in Drosophila. III. Effects of ethanol. Biochem Genet. 1976 Feb;14(1-2):127–135. doi: 10.1007/BF00484879. [DOI] [PubMed] [Google Scholar]
  26. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wensink P. C., Finnegan D. J., Donelson J. E., Hogness D. S. A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster. Cell. 1974 Dec;3(4):315–325. doi: 10.1016/0092-8674(74)90045-2. [DOI] [PubMed] [Google Scholar]
  28. Woodruff R. C., Ashburner M. The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. I. Characterization of deficiencies and mapping of ADH and visible mutations. Genetics. 1979 May;92(1):117–132. doi: 10.1093/genetics/92.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Woodruff R. C., Ashburner M. The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. II. Lethal mutations in the region. Genetics. 1979 May;92(1):133–149. doi: 10.1093/genetics/92.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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