摘要:

ABSTRACTIn this update we provide a list of the 71 P450 genes and the four P450 pseudogenes that have been characterized as of September 30, 1988. The chromosomal locations of many of these genes are also summarized. A modest revision of the initially proposed nomenclature of the P450 superfamily (Nebertet al., DNA 6, 1–11, 1987) is described specifically for the human and mouse chromosomal loci. The motivation for this revision is to conform to the rules of nomenclature for human and mouse genes. Recommendations for the naming of chromosomal loci include the root symbol "CYP" for human ("Cyp" for mouse), denoting "cytochrome P450." We recommend that this root also be used for other organisms. For a chromosomal locus, the root symbol is followed by an Arabic numeral designating the P450 family, a letter indicating the sub-family, and an Arabic numeral representing the individual gene within the family or subfamily. Numbers of the individual genes usually will be assigned in the order the genes are identified. This system is consistent with our earlier proposed nomenclature for P450 families and gene products from all eukaryotes and prokaryote

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.1

年代:1989

数据来源: MAL

摘要:

ABSTRACTBovine serum was used to identify a recombinant phage clone carrying thePasteurella haemolyticaleukotoxin gene. This fragment produced the 102-kD leukotoxin and several smallerP. haemolytica-specific protein antigens inEscherichia coli. An additional contiguous fragment, containing sequences upstream from the leukotoxin gene, was isolated from a plasmid library with a probe specific for the region near the 5′ end of the leukotoxin gene. Using these clones, we determined the nucleotide sequence of a 7745-bp region that included four open reading frames: an upstream gene,lktC; the leukotoxin gene,lktA; and two down-stream genes,lktB, andlktD. The predicted molecular weights of the proteins encoded by these genes were 19.9, 102, 79.6, and 54.7 kD, respectively. These genes and their predicted proteins were similar in organization and in sequence to the corresponding elements of the gene cluster that encodes anE. coliα-hemolysin and its activation and secretion functions. Expression of the leukotoxin was enhanced inE. coli, by fusing the gene to thelacpromoter. Under these conditions the leukotoxin was not secreted into the medium, as it is inP. haemolytica. However, in the presence of the α-hemolysin genes, the leukotoxin was secreted into the medium, demonstrating functional complementation by the hemolysin secretory sys

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.15

年代:1989

数据来源: MAL

摘要:

ABSTRACTCell-specific chemical toxicities may be influenced by P450-catalyzed biotransformation reactions. We have undertaken an analysis of P450 expression in isolated rat liver sections to assess better the cellular distribution of P450 gene products. Discriminatory 18-mer oligodeoxynucleotides directed to the phenobarbital (PB) inducible P450s, P450IIB1 (P450b) and P450IIB2 (P450e), were employed as probes forin situhybridization experiments. With these techniques we demonstrate that P450b and P450e mRNAs are each expressed in the hepatic lobule with similar spatial distribution. In animals pretreated with PB, only cells within the immediate periportal region were refractory to induction. Based on autoradiographic grain densities, responsive hepatocytes accumulated P450b mRNA at levels exceeding that for P450e. We employedin situhybridization methodology in combination with Northern blot analyses to compare the expression of these mRNAs in two rat strains, Sprague–Dawley and Marshall 520/N (the latter being deficient in the synthesis of P450e isozyme; Rampersaud and Walz, 1987). These strains provided valuable comparative models, demonstrating the specificity and sensitivity of the oligomer probes. The continued development ofin situhybridization methodologies, especially when used in conjunction with synthetic oligomer probes, will permit a detailed analysis of P450 expression in different tissues, under a variety of chemical exposures, and may be a valuable adjuvant to the prediction of target organ toxicitie

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.29

年代:1989

数据来源: MAL

摘要:

ABSTRACTThe developmental expression of an embryonic chicken myosin heavy-chain (MHC) gene homologous to the genomic clone pCM4.1 was examined by S1analysis. Transcripts homologous to pCM4.1 are first detected at day 12in ovo, and are maximally expressed between days 15–17in ovo. No pCM4.1 transcripts are detected at earlier stages of embryogenesis or at high levels in posthatch stages. This unique pattern of expression has led to the proposal that pCM4.1 represents a previously uncharacterized MHC gene, which is confined in its expression to late embryogenesis. Genomic hybridization data, in addition to a comparison between the DNA and amino acid sequences of pCM4.1 and other characterized chicken MHC 3′ end clones, provide further evidence for this proposal. We also present observations made during the sequence analysis of pCM4.1 that may be relevant to our understanding of the 3′-end processing of homologous primary transcripts, and of the mechanism controlling developmental MHC isoform transi

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.39

年代:1989

数据来源: MAL

摘要:

ABSTRACTA tRNACACValgene variant, pHtV4, was cloned from human placenta genomic DNA. This gene differs from a closely related, functional tRNACACValgene by four base exchanges: T residues in place of C25, C62, and C66create G:U pairs, and an A instead of G65creates an A:C mismatch in the corresponding RNA transcript. The tRNAValgene variant in pHtV4 is efficiently transcribed in HeLa cell nuclear extracts; however, the resulting pre-tRNA is processing-deficient,i.e., neither its 5′- nor its 3′-flanking sequences are removed to generate mature tRNA. Reversion of all four point mutations in pHtV4 by oligonucleotide-directed mutagenesis yielded a functional tRNACACValgene within the flanks of pHtV4, the pre-tRNA of which was processed to mature tRNA. Construction of a chimeric tRNAValgene and site-directed mutagenesis of the tRNAValgene in pHtV4, respectively, followed by transcription and processing studies showed that each of the four mutations contributes to the processing defect of the pHtV4-derived pre-tRNA. Moreover, this revealed that G:U pairs, which are common in all tRNAs, can impair pre-tRNA processing and therefore do not occur in certain positions in eukaryotic tR

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.51

年代:1989

数据来源: MAL

摘要:

ABSTRACTPrevious reports on retroviral vectors have shown them to be useful for transferring genes into animal cells. Genes placed under the retroviral long terminal repeat (LTR) act as dominant loci in recipient cells and can permanently alter their genotype and phenotype. Previous reports have shown that recombinant retroviruses containing genomic sequences with both introns and exons display a high frequency of deletion and abnormal kinetics of splicing of intron sequences. We report here our findings when a 2.9-kb fragment containing the entire human insulin gene was inserted into a Moloney-derived retroviral vector in the same transcriptional orientation as the LTRs. RNA transcripts synthesized in cells containing such constructs remain unspliced, as assessed by both RNA blot analysis and S1mapping. Ten subclones derived following viral passage showed no splicing, and failure to splice was observed regardless of cell type or species of origin, or number of viral passages. Thus, genomic sequences containing introns when situated within the context of a retroviral transcript do not in all instances exhibit expected kinetics of splicing.

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.59

年代:1989

数据来源: MAL

摘要:

ABSTRACTA strategy is presented for the efficient construction of λ ZAP genomic expression libraries. Procedures are described for the evaluation of the status of vector DNA at each stage of library construction to facilitate troubleshooting. Ligation of λ ZAP cohesive ends and preparation of the multiple cloning site were verified by restriction enzyme digestion of vector DNA. Sonication was a rapid way of producing random chromosomal fragments of a size range ideal for expression library construction. The advantages of cloning into theNotI site of the λ ZAP polylinker are discussed. The choice of this site eliminated the need to perform the methylation of chromosomal DNA, which is required when the conventionalEcoRI site is used. This method also facilitates restriction mapping of cloned inserts. Genomic expression libraries were constructed using this approach forSynechococcussp. PCC7942,Synechocystissp. PCC6803, andProchlorothrix hollandica. The utility of expression libraries andin vivoexcision was demonstrated by verifying the identity of clones coding forSynechococcussp. PCC7942 cytochrome f, since the correct reading frames of these cloned inserts were determined unambiguous

ISSN:1044-5498    

DOI:10.1089/dna.1.1989.8.69

年代:1989

数据来源: MAL