摘要:

ABSTRACTA nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1–2 years. Assignment of orthologous genes is presently uncertain in some cases—between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowle

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.1

年代:1987

数据来源: MAL

摘要:

ABSTRACTExamination of all mammalian, nonmammalian vertebrate, and invertebrate genomic sequences present in the GenBank data shows a striking distribution of the G + C (A + T) content. It has been known for a few decades that the G + C content in higher organisms averages 42%. The average of the sequenced genomic regions is significantly higher, with a distinct peak near the transcription initiation site. This distribution, as well as the overall A + T genomic incidence may be directly related to DNA geometry. Specific A-T sequences may facilitate DNA curving, allowing more tight packaging of chromatin in eukaryotic genomes. To examine further which particular oligomers may potentially contribute to recognition of transcription initiation sites, an extensive search was instituted for detecting recurrences of particular oligonucleotides in the regions surrounding these sites. Accordingly, the analyzed mammalian genomic sequences have been scanned for the occurrences of each of the 256 quartets. The CAAT box components yield moderate peaks. As expected, the TATA box is much more pronounced, though the ATAA sequence yields a more striking peak than the TATA. Tetranucleotides containing solely A + T are relatively rare downstream from coding regions. Upstream, their level is low except for the distinct peaks; the level of A4is very high upstream. All significant signals are enumerated. Of the G + C-rich quartets, CCCC and AGGG exhibit large peaks near transcription initiation sites. These may produce straight DNA segments.

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.13

年代:1987

数据来源: MAL

摘要:

ABSTRACTThe precursor to a seminal plasma protein reported to have inhibin-like activity was characterized through cDNA cloning and sequencing. It is a 114-amino-acid polypeptide which differs from its seminal plasma derivative mainly by the presence of a 20-residue amino-terminal extension, a putative signal sequence, carrying a possible N-glycosylation site. The protein is specified by a single gene per haploid genome. Its mRNA is detectable in the prostate but not in the testis, which suggests that it is primarily a prostatic secretory protein.

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.23

年代:1987

数据来源: MAL

摘要:

ABSTRACTThree chimeric cytochrome P450 cDNAs were constructed by replacing the central region, carboxy-terminal region, or both central and carboxy-terminal regions of cytochrome P450c cDNA with the corresponding regions of cytochrome P450d cDNA. These were inserted between the alcohol dehydrogenase I promoter and terminator of yeast expression vector pAAH5 to form expression plasmids pACDC2, pACCDl, and pACDD2. On introduction of each of these plasmids intoSaccharomyces cerevisiaeAH22 cells, chimeric cytochrome P450 proteins were expressed in AH22/pACDC2, AH22/pACCDl, and AH22/pACDD2 cells at the level of at least 105, 4 × 105, and 105molecules per cell, respectively. The reduced CO-difference spectra showed that AH22/pACCDl and AH22/pACDD2 cells contained 4 × 105and 10smolecules per cell of the corresponding chimeric cytochrome P450 hemoproteins, designated as cytochrome P450ccd and cytochrome P450cdd, respectively. Cytochrome P450ccd exhibited higher monooxygenase activities toward 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene than cytochrome P450c, although the substrate specificity of cytochrome P450ccd seemed to be the same as that of cytochrome P450c. Cytochrome P450cdd exhibited lower activities toward 7-ethoxycoumarin and benzo[a]pyrene, and a higher activity toward acetanilide as compared with those of cytochrome P450c and cytochrome P450ccd. Therefore, the substrate specificity of cytochrome P450cdd seemed to be the same as that of cytochrome P450d. These results suggest that the central one-third region of cytochrome P450c and cytochrome P450d is responsible for substrate-binding, and that the carboxy-terminal third of both cytochromes P450 plays an important role in electron transpor

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.31

年代:1987

数据来源: MAL

摘要:

ABSTRACTA human interferon-α2gene variant was expressed inEscherichia coliunder the control of the tryptophan operon promoter and of a series of synthetic partially overlapping ribosomal binding sites, which control initiation of translation. Variation in the distance between the start codon and such a set of ribosomal binding sites affected the level of interferon expression much less than previously described for single ribosomal binding sites

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.41

年代:1987

数据来源: MAL

摘要:

ABSTRACTA bovine P1 protamine cDNA from a bull testis cDNA library was isolated utilizing a series of oligonucleotide probes. Sequence analysis showed that the cloned cDNA insert extended 317 bp to the poly(A) tail. The 51-residue 6750-dalton protamine primary translated protein is encoded within a 156-bp segment. The protamine sequence predicted from the cDNA sequence differs from that previously reported for the amino acid sequence of bovine protamine P1 by the insertion of the tripeptide Cys-Arg-Arg from residues 39–41 in the carboxy-terminal region of the mature protein. Consistent with previous hybridization analysis, nucleotide sequence comparisons showed that trout protamine cDNA was more closely related to that of bovine than to that of mouse. However, bovine P1 protamine cDNA shared greater sequence homology with mouse P1. A common nucleotide sequence of 30 bp is conserved among all three of these species. Primer extension analysis revealed that, as with trout protamine mRNAs, the majority of the untranslated portion of the mRNA lies 3′ to the coding segment. Comparisons of their mRNA secondary structures by computer modeling indicate that the mRNAs fold back onto themselves, producing similar, extensively hydrogen-bonded, convoluted forms. These models support the view that translational regulation of protamine mRNA may be partially dependent on secondary structure. Southern analysis suggests that the bovine protamine P1 gene is not sex-linked and is present as one (or relatively few) copy within the bovine gen

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.47

年代:1987

数据来源: MAL

摘要:

ABSTRACTGenomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage λ and cosinid libraries. The entire GH/CS chromosomal locus was reconstructed utilizing overlapping restriction fragments characterized from the isolated clones. The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA in the order: 5′-(hGH-l/hCS-5/hCS-l/hGH-2/hCS-2)-3′, confirming analysis of cosmid clones obtained from a different human library (Barshet al., 1983). To complete the characterization of the hCS genes, the nucleotide sequence of the hCS-5 gene was determined. Sequence analysis revealed a mutation of the 5′ splice site at the exon II-intron B boundary, suggesting that the hCS-5 gene is a pseudogene. The nucleotide sequence of an allelic variant of the hCS-2 gene was determined and found to contain a single amino acid substitution and the deletion of a single codon. The hGH/hCS gene locus was further characterized by the localization of at least 27Alu-type repetitive sequences and identification of three unique sequences in the vicinity of several hGH and hCS genes which define the probable breakpoints of the evolutionary duplication units. These data, combined with the nucleotide sequences of all five GH and CS genes, indicate that the hGH/hCS gene locus has evolved by duplication mechanisms. Evidence for the occurrence of at least one gene conversion event involving the hCS-1 gene precursor and the hCS-2 gene was found, indicating that the hGH/hCS gene locus has evolved by concerted mechanisms. The structure of the hCS genes is discussed in light of recent studies of CS genes from other mammalian s

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.59

年代:1987

数据来源: MAL

摘要:

ABSTRACTcDNAs containing the entire coding sequence of endozepine, a putative ligand of the benzodiazepine receptor, were isolated from bovine and human cDNA libraries. These libraries were constructed using a novel oligonucleotide adapter molecule that allowed us to combine the use of G/C tailing with the preservation of the uniqueEcoRI site in the vector, λgtlO. The amino acid sequences derived from these cDNA clones are identical to those previously determined for the purified proteins and are homologous to a related rat protein termed diazepam-binding inhibitor. The endozepine proteins are highly conserved, as illustrated by the finding that the nucleotide sequences of the coding regions are 93% conserved between the bovine and human forms. Analysis of these sequences indicates that endozepine is not, as expected, derived from a precursor molecule containing a transient signal peptide. Moreover, Northern analyses using the cloned cDNAs as hybridization probes indicate that the 650-nucleotide endozepine mRNA is expressed in a number of peripheral tissues in addition to brain. These observations may be consistent with a recent report describing the presence in peripheral tissues of benzodiazepine receptors on the outer mitochondria! membrane (Anholtet al., 1986). In addition to the endozepine cDNAs, we also isolated a bovine cDNA clone which encodes a larger protein, a portion of which is homologous to endozepine. This related protein may be synthesized in a precursor form containing putative signal peptide and membrane-spanning domains

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.71

年代:1987

数据来源: MAL

摘要:

ABSTRACTTransfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves thein vitroreconstitution of foreign DNA–of viral or other origins–with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. Thein vitroformation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with<10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenical acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells. Ad2 DNA or plasmid preparations introduced into cells by the DNA-protamine complex method are as efficiently expressed as when transfected by the Ca2+-phosphate precipitation technique. Comparable levels of expression have been found both in transient and permanent express

ISSN:1044-5498    

DOI:10.1089/dna.1987.6.81

年代:1987

数据来源: MAL