Cloned Drosophila Alcohol Dehydrogenase Genes are Correctly Expressed after Transfection into Drosophila Cells in Culture

We have obtained correct transcription of the cloned alcohol dehydrogenase (Adh) gene of Drosophila melanogaster after DNA-mediated gene transfer into Drosophila cells in culture. Supercoiled plasmids, each containing various regions of the Adh gene cloned in pBR327, were introduced into Schneider l...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-03, Vol.81 (6), p.1701-1705
Main Authors: Benyajati, Cheeptip, Dray, James F.
Format: Article
Language:English
Subjects:
Alcohol Dehydrogenase
Alcohol Oxidoreductases - biosynthesis
Alcohol Oxidoreductases - genetics
Base Sequence
Cell culture techniques
Cell lines
Cells, Cultured
Cloning, Molecular
Cultured cells
DNA
Drosophila
Drosophila melanogaster
Drosophila melanogaster - genetics
Drosophilidae
gene expression
Gene Expression Regulation
Genes
Larvae
Plasmids
RNA
RNA Processing, Post-Transcriptional
RNA Splicing
splicing
Transcription, Genetic
Transfection
transformation
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Summary:We have obtained correct transcription of the cloned alcohol dehydrogenase (Adh) gene of Drosophila melanogaster after DNA-mediated gene transfer into Drosophila cells in culture. Supercoiled plasmids, each containing various regions of the Adh gene cloned in pBR327, were introduced into Schneider line 2 (SL2) cells by the calcium phosphate--DNA transfection technique. Although these cells do not normally express their endogenous Adh genes, they do express the exogenous genes as shown by primer extension and nuclease S1 analyses of RNA isolated 48 hr after transfection. The resulting alcohol dehydrogenase (ADH) transcripts, both the larval and adult types, have the correct 5′ends and are properly spliced. The transfected cells have also acquired ADH enzyme activity. The levels of enzyme activity and of ADH protein crossreacting material in cells transfected with different Adh plasmids correlate directly with the level of ADH transcripts. When a mutant Adh gene cloned from an ADH-negative mutant fly with a defect in the splicing of ADH RNA is transfected into the Schneider line 2 cells, the resulting ADH RNA is not spliced properly and there is no synthesis of ADH; thus, the mutant gene transfection into cell culture mimics the mutant phenotypes observed in the mutant fly.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.6.1701