Light emission in marine bacteria is under the control of growth dependent induction. Associated with the induction is the increase in synthesis of the components of the two enzymes which catalyze the light emitting reaction, luciferase and fatty acid reductase. Large DNA fragments from three marine bacteria Vibrio fischeri, Vibrio harveyi and Photobacterium phosphoreum, were isolated from genomic libraries of these strains. Escherichia coli, harboring a recombinant plasmid containing a 16 kbp fragment of V. fischeri DNA, were found to express growth dependent luminescence. E. coli clones harboring analogous sequences of V. harveyi or P. phosphoreum lux DNA only expressed a low constitutive level of light which was dependent on the presence of aldehyde in the growth media. Cell extracts of E. coli expressing the lux genes from V. fischeri contained a fatty acid reductase activity analogous to the activity first identified in P. phosphoreum. This result showed that the genes for luciferase and fatty acid reductase were linked inV. fischeri. By differential acylation of the fatty acid reductase subunits in extracts of V. fischeri and the E. coli clone, the products of the luxC, luxD and luxE genes of V. fischeri were identified as the reductase, transferase and synthetase components, respectively. In addition, the lux genes from a fatty acid stimulatible dark mutant of V. harveyi were cloned and it was demonstrated that the luxD gene from this mutant synthesized a non-functional transferase polypeptide. / The lux structural genes are coordinately expressed to different levels during induction of luminescence. The lux polypeptides of V. fischeri were shown to be differentially synthesized when expressed under the control of a T7 promoter in E. coli. This result indicated that postranscriptional events were regulating the differential expression of the lux genes. A set of overlapping transcripts encoding the lux genes were shown to be induced during luminescence induction, suggesting that segmental differences in mRNA stability may be related to differential expression of the lux genes. / The luxA and luxB genes of V. harveyi encoding the alpha and beta subunits of luciferase, respectively, were fused by site-directed mutagenesis. This fused gene synthesized a monocistronic luxAB mRNA capable of programming the synthesis of a functional single subunit luciferase enzyme in E. coli, in a rabbit reticulocyte lysate and in Saccharomyces cerevisiae. This fused gene has potential applicability for studying gene expression in both prokaryotic and eukaryotic systems.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.75905 |
| Date | January 1988 |
| Creators | Boylan, Michael Owen |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000910250, proquestno: AAINL52264, Theses scanned by UMI/ProQuest. |
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