Analysis of gene control signals by DNA fusion and cloning in Escherichia coli
TL;DR: Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription.
About: This article is published in Journal of Molecular Biology.The article was published on 1980-04-05. It has received 2442 citations till now. The article focuses on the topics: Cloning vector & Lac repressor.
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TL;DR: The recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability as predicted by the N-end rule.
Abstract: When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.
1,902 citations
TL;DR: A new family of lambda replacement vectors and derivatives containing amber mutations and derivatives of the EMBL3 vector allow the application of genetic screening procedures based on selection for the products of homologous recombination events, and for the selective cloning of DNA sequences linked to supF genes.
1,578 citations
TL;DR: Its sequence shows that the c-erb-B-2 gene encodes a possible receptor protein and allows an analysis of the similarity of the protein to the EGF receptor and the neu product.
Abstract: A novel v-erb-B-related gene, c-erb-B-2, which has been identified in the human genome, maps to human chromosome 17 at q21 (ref. 40), and seems to encode a polypeptide with a kinase domain that is highly homologous with, but distinct from, that of the epidermal growth factor (EGF) receptor. The c-erb-B-2 gene is conserved in vertebrates and it has been suggested that the neu gene, detected in a series of rat neuro/glioblastomas, is, in fact, the rat c-erb-B-2 gene. Amplification of the c-erb-B-2 gene in a salivary adenocarcinoma and a gastric cancer cell line MKN-7 suggests that its over-expression is sometimes involved in the neoplastic process. To determine the nature of the c-erb-B-2 protein, we have now molecularly cloned complementary DNA for c-erb-B-2 messenger RNA prepared from MKN-7 cells. Its sequence shows that the c-erb-B-2 gene encodes a possible receptor protein and allows an analysis of the similarity of the protein to the EGF receptor and the neu product. As a consequence of chromosomal aberration in MKN-7 cells, a 4.6-kilobase (kb) normal transcript and a truncated 2.3-kb transcript of c-erb-B-2 are synthesized at elevated levels. The latter transcript presumably encodes only the extracellular domain of the putative receptor.
1,259 citations
TL;DR: A series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes yielded hybrid proteins useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.
Abstract: We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.
971 citations
TL;DR: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes.
Abstract: The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
866 citations
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TL;DR: P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.
Abstract: Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.
4,372 citations
TL;DR: Fusions of lac genes to selected locations on the Escherichia coli chromosome are useful in discovering new types of regulation of gene expression, as was found in the case of the araC gene.
1,884 citations
1,415 citations
TL;DR: In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313, which has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which cleave the plasid at unique restriction sites.
984 citations