Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.Xenorhabdus luminescens is a luminous bacterium in the family Enterobacteriaceae (3,17,38 (1,6,7,18). The primary form is generally isolated from infective nematodes, but upon prolonged culture in various media secondary variants appear. In contrast to the primary form, the secondary variants are dim and lack detectable protease, lipase, antibiotic activity, protein crystals, and red pigment (6). The two forms also exhibit differences in colony morphology and staining properties (18). The secondary forms are deficient in providing optimum conditions for nematode reproduction (1, 7, 16).The physical and biochemical properties of the luminous system of X. luminescens are similar to those found in other bioluminescent bacteria (9,31,36 hyde to oxidized flavin and the corresponding long-chain fatty acid. A fatty acid reductase complex is required for the generation and recycling of fatty acid to aldehyde (34), and an NAD(P)H:flavin oxidoreductase supplies the reduced flavin (20). In rich media, the luminescence of X. luminescens increases dramatically during the late logarithmic or stationary phase of growth (36). The generation of secondary variants which are dim is another level of control of the luminous system of X. luminescens.The bioluminescence (lux) genes from Vibrio harveyi (8), Vibrio fischeri (12), Photobacterium leiognathi (11), and Photobacterium phosphoreum (22) have been cloned and expressed in Escherichia coli. Five structural genes are required for light emission: luxC, luxD, and luxE encode the fatty acid reductase complex, and luxA and luxB encode the alpha and beta subunits of bacterial luciferase (12,13,25). In all luminous bacteria that have been studied, these five genes are closely linked and luxA and luxB are flanked by the genes for the enzymes of the fatty acid reductase complex. In both P. phosphoreum and P. leiognathi there is another gene between luxB and luxE (19,22). The function of the product of this gene is not known.In this paper we report the cloning of the lux genes of X. luminescens and their expression when introduced into E. coli. We also present data showing the organization of the X. luminescens lux genes, the structure of the lux genes from the secondary form, and the homology of the luxA and luxB genes of X. luminescens Hm to DN...

Previous studies have demonstrated that the SP013 gene is required for chromosome separation during meiosis I in Saccharomyces cerevisiae. In the presence of the spol3-1 nonsense mutation, MATa/MATa diploid cells complete a number of events typical of meiosis I including premeiotic DNA synthesis, genetic recombination, and spindle formation. Disjunction of homologous chromosomes, however, fails to occur. Instead, cells proceed through a single meiosis I-like division and form two diploid spores. In this paper, we report the cloning of this essential meiotic gene and an analysis of its transcription during vegetative growth and sporulation. Disruptions of SP013 in haploid and diploid cells show that it is dispensible for mitotic cell division. Diploids homozygous for the disruptions behave similarly to spol3-1 mutants; they sporulate at wild-type levels and produce two-spored asci. The DNA region complementing spol3-1 encodes two overlapping transcripts, which have the same 3' end but different 5' ends. The major transcript is 400 bases shorter than the larger, less abundant one. The shorter RNA is sufficient to complement the spoi3-1 mutation. While both transcripts are undetectable or just barely detectable in vegetative cultures, they each undergo a >70-fold induction early during sporulation, reaching a maximum level about the time of the first meiotic division. In synchronously sporulating populations, the transcripts nearly disappear before the completion of ascus formation. Nonsporulating cells homozygous for the mating-type locus show a small increase in abundance (<5% of the increase in sporulating cells) of both transcripts in sporulation medium. These results indicate that (i) expression of the SP013 gene is developmentally regulated and (ii) starvation alone, independent of the genotype at MAT, can trigger initial induction.

The SPOII gene, required for meiotic recombination in Saccharomyces cerevisiae, has been cloned by direct selection for complementation ofthe spolIl-phenotype: lack of meiotic recombination and low spore viability. DNA sequencing indicates that the gene encodes a 398-amino acid protein having a predicted molecular mass of 45.3 kDa. There is no significant similarity between the SPOil protein and other protein sequences, including those from genes known to be involved in DNA recombination or repair. Strains bearing a disruption allele are viable, indicating that SPOil is dispensable for mitotic growth. RNA analyses demonstrate that SPOII produces a 1.5-kilobase transcript that is developmentally regulated and expressed early in the sporulation process.

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