摘要:

ABSTRACTA full-length cDNA for rat NADPH-cytochrome P-450 reductase was cloned by the procedure of Okayama and Berg (1982) from hepatic poly(A)RNA prepared from phenobarbital-induced rats. Both cDNA and amino acid sequences agreed with the sequences reported by Porter and Kasper (1985) except for four single base differences. Three expression plasmids were constructed by insertion of the reductase cDNA between yeast alcohol dehydrogenase I (ADH) promoter and terminator regions. Plasmids pARF1 and pTRF2 were constructed with slightly different lengths between the ADH promoter and the initiation codon; on introduction intoSaccharomyces cerevisiaeAH22 cells, they synthesized about 1 × l03and 5 × 103reductase protein molecules per cell, respectively. A third plasmid, pARM1, containing a cytochrome P-450MC cDNA expression unit located between two reductase cDNA expression units synthesized 4 × 105cytochrome P-450MC hemoprotein and 1 × 104reductase protein molecules per cell. The cellular extracts of the AH22/pARM1 strain, which synthesized both rat enzymes, showed higher cytochromecreductase and cytochrome P-450MC-dependent 7-ethoxycoumarinO-deethylation activities as compared to extracts of the AH22/pAMC1 strain, which synthesized only rat cytochrome P-450MC. 7-EthoxycoumarinO-deethylation activity in the cellular extract of the AH22/pARM1 strain was partly inhibited by the addition of anti-rat reductase IgG. In addition, whole AH22/pARM1 cells exhibited higher monooxygenase activity toward acetanilide and 7-ethoxycoumarin than control AH22/pAMC1 cells. These results indicated that a functional electron-transport chain consisting of rat NADPH-cytochrome P-450 reductase and rat cytochrome P-450MC was constructed inS. cerevisiaece

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.1

年代:1986

数据来源: MAL

摘要:

ABSTRACTEscherichia coliexpression vectors encoding an acid-labile aspartyl-proline (Asp-Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site forBamHI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptorin vitro.

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.11

年代:1986

数据来源: MAL

摘要:

ABSTRACTTransformation ofEscherichia colicells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin. The cDNA was modified by removal of a 5′ nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin. To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen from the consensus sequence around the initiation codon ofE. coligenes and by the amino acid sequence of the protein. Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformedE. colicells, and by reaction with specific antibody. Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of ispropyl-β-D-thiogalactopyranoside (IPT

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.21

年代:1986

数据来源: MAL

摘要:

ABSTRACTWe have characterized a genomic clone of the mouse equivalent of α1-antitrypsin (α1-antiprotease) a gene that is expressed in liver. There are at least three other genomic sequences similar to the gene expressed in mouse liver and all appear to be located on chromosome 12. The duplicated regions might include genes that are also expressed in the liver and/or in other tissues. Comparison of the sequences of the mouse α1-antitrypsin sequence expressed in liver with sequences of α1-antitrypsins from other mammals reveals great homology in the coding regions, particularly in the amino acids at the active site, including the neighboring methionine and serine thought to play a key role in the protease inhibit

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.29

年代:1986

数据来源: MAL

摘要:

ABSTRACTA series of mutant tRNA genes has been constructed by site-directed mutagenesis in pOP203, a colE1 derivative carrying a transcription unit under control of thelacUV5promoter. These mutant genes include all possible amber suppressing variants of tRNATrpwith single nucleotide substitutions at anticodon loop positions 32, 37, and 38 (numbered from the 5′end), and all possible paired base substitutions in the three base pairs nearest the anticodon loop. G at position 38 was not recovered as a single mutation, but rather in conjunction with an undirected mutation to T at position 32. The singly mutated G38 tRNA may not be active, though all the other tRNA derivatives are functional in the translation of amber codons.To construct the mutants, we ligated a synthetic deoxyoligonucleotide into a precisely formed single-stranded gap covering the anticodon arm region DNA, in an otherwise double-stranded fragment containing the tRNATrpgene. The resulting heteroduplex was then ligated into the plasmid and introduced intoEscherichia coli. This method of mutagenesis is simple, reproducible, and highly tolerant of varying degrees of heteroduplex in the gap, variations in temperature of ligation, and changes in the oligonucleotide concentration. Mutagenesis does not require a 5′-phosphorylated oligonucleotide. These qualities suit the gap method for intensive study of a region by site-directed mutagene

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.37

年代:1986

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.54

年代:1986

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.57

年代:1986

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1986.5.71

年代:1986

数据来源: MAL