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Biomedical subjects

F Bolivar

Publications and source records attributed to F Bolivar.

At least 55 records · Page 3Linked to original sources
PubMed1982

Recombinant plasmids carrying the glutamate dehydrogenase structural gene from Escherichia coli K-12.

Plasmid pRSP1, isolated from the Escherichia coli gene bank of Clarke and Carbon (1975), has been shown to complement the gdh-1 mutation that affects the synthesis of glutamate dehydrogenase (GDH) in the E. coli strain PA340. The GDH structural gene of E. coli is present on a 2.3-Md DNA fragment from the hybrid plasmid pRSP1 and certain of its derivatives. Polypeptides synthesized by minicells carrying some of these plasmids, enabled us to make a preliminary determination of the direction of transcription of the gdh gene on the cloned fragments.

Chromosome Mapping↗
PubMed1982

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication.

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

Base Sequence↗
PubMed1980

Cloning and physical mapping of the glnA gene of Escherichia coli K-12.

We have isolated part of the glnA region of Escherichia coli K-12 as a 6.4 Md DNA fragment on the ColE1 hybrid plasmid pACR1. DNA fragments from pACR1 obtained by cleavage with certain restriction endonucleases were subcloned into the pBR322 cloning vehicle. Recognition sites for the endonucleases BamHI, SmaI, BglII, and EcoRI were localized inside the glnA gene sequence. De novo synthesized polypeptides, employing minicells that carried some of these plasmids, allowed us to determine the direction of transcription of the glnA gene relative to an adjoining gene that codes for a 65 000 dalton protein.

Bacteriocin Plasmids↗
PubMed1980

Characterization of a site-specific restriction endonuclease SphI from Streptomyces phaeochromogenes.

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5'-GCATGC and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times lambda DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.

Chromatography, DEAE-Cellulose↗
PubMed1980

Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325.

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.

Ampicillin↗
PubMed1980

Tight linkage of genes that encode the two glutamate synthase subunits of Escherichia coli K-12.

A hybrid deoxyribonucleic acid molecule, plasmid pRSP20, which was isolated from the Clarke and Carbon Escherichia coli gene bank, was shown to complement the gltB31 mutation, which affects the synthesis of glutamate synthase in E. coli strain PA340. We present evidence which demonstrates that plasmid pRSP20 carries an 8-megadalton E. coli chromosomal fragment, including the genes encoding the two unequal glutamate synthase subunits. Polypeptides with molecular weights of about 135,000 and 53,000, which comigrated with purified E. coli glutamate synthase subunit polypeptides and immunoprecipitated with antibodies to E. coli glutamate synthase, were synthesized by minicells carrying the pRSP20 plasmid.

Chromosomes, Bacterial↗
PubMed1979

Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313.

By employing a system of RNA polymerase binding and restriction endonuclease digestion, we demonstrate that the region in and around the Hind III site of pBR313 and pBR322 is the promoter for the tetracycline (Tc) resistance gene(s). Furthermore, it is shown that this region was transferred intact from pSC101 during the construction of the latter plasmids. The in vitro insertion of a few base pairs at the Hind III site produces a series of "down" promoter mutations in which the level of in vivo Tc resistance is reduced. Sequence analysis of the various promoter mutations revealed significant base pair rearrangements in the region between -40 and -12 of the promoter. While these base alterations do not appear to affect the firm binding of RNA polymerase, they do affect the ability of mutant promoters to initiate transcription. These observations suggest that the region from -40 to -12, previously designated as the "recognition region", is actually involved in the process of initiation of transcription.

Base Sequence↗
PubMed1979

Expression in Escherichia coli of chemically synthesized genes for human insulin.

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.

Amino Acids↗
PubMed1978

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.

Chromosome Mapping↗
PubMed1978

A general method for the purification of restriction enzymes.

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.

Bacteria↗
PubMed1977

Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin.

A gene for somatostatin, a mammalian peptide (14 amino acid residues) hormone, was synthesized by chemical methods. This gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pBR322. Transformation of E. coli with the chimeric plasmid DNA led to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. In vitro, active somatostatin was specifically cleaved from the large chimeric protein by treatment with cyanogen bromide. This represents the first synthesis of a functional polypeptide product from a gene of chemically synthesized origin.

Base Sequence↗
PubMed1977

Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9.

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. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.

Ampicillin↗
PubMed1977

Origin of replication of pBR345 plasmid DNA.

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.

Base Sequence↗
PubMed1977

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

Ampicillin↗