Introduction and expression of the bacterial genes cysE and cysK in eukaryotic cells.
The coding sequences of the cysE and cysK genes from Escherichia coli, which encode the enzymes of the cysteine biosynthetic pathway, namely, serine acetyltransferase (EC 2.3.1.30) and O-acetylserine sulfhydrylase (or cysteine synthase [EC 4.2.99.8]), were modified for expression in eukaryotic cells and introduced into murine L cells. A number of fusion genes comprising the cysE or cysK coding sequences joined to the promoter of the ovine metallothionein-Ia (MT-Ia) gene and various portions of the ovine growth hormone (GH) gene were prepared. Significant differences in the level of transcription were observed, depending on the amount and arrangement of the GH gene sequences used, the highest levels being obtained with the constructs MTCE10 and MTCK7, which contained only the GH 3' untranslated gene sequences. These two constructs were fused to produce the gene MTCEK1. In this single DNA sequence, each bacterial gene is under independent MT-Ia promoter control. Expression of the cysK sequence in this construct (MT-Ia promoter-cysE-3' GH sequence-MT-Ia promoter-cysK-3' GH sequence) was elevated compared with expression of the cysK gene in MTCK7. However, expression of the cysE sequence in MTCEK1 was only 40% of that of the cysE gene cloned into MTCE10. The double-promoter configuration, which enhances the expression of the second gene in MTCEK1, is proposed as a model for the modification of bacterial genes in general.
Leish
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Division of Animal Production, Commonwealth Scientific and Industrial Research Organisation, Blacktown, New South Wales, Australia.
Byrne
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Hunt
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Ward
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D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000096:biosynthesis / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012329:RNA, Bacterial / Q000032:analysis; D051050:Serine O-Acetyltransferase; D012756:Sheep / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D015032:Zinc