Robert C. Tait

Protein expression in E. coli minicells by recombinant plasmids

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.

In vitro maturation of germinal vesicle oocytes in stimulated intracytoplasmic sperm injection cycles.

In vitro enzymatic alteration of plasmid phenotype and in vitro construction of recombinant plasmids containing genetic information derived from the plasmid pSC101 have been used to investigate the mechanism of function of tetracycline resistance determined by the plasmid pSC101. The resistance has been shown to be inducible and involves the increased synthesis of membrane-associated polypeptides of 34,000, 26,000 and 14,000 daltons that are encoded for by the plasmid. The 34,000 dalton polypeptide along with another plasmid-encoded polypeptide of 18,000 daltons function in an ATP-independent manner to prevent the accumulation of tetracycline by the cell. These polypeptides are sufficient for resistance. A second component of plasmid-determined resistance involves the 14,000 dalton polypeptide and reduces the initial adsorption of tetracycline by sensitive cells, but is not alone sufficient for the generation of resistance. The role of the 26,000 dalton polypeptide in tetracycline resistance has not been identified.

The aminoglycoside-resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene

The nucleotide sequence of the probable C terminus of the kanamycin-resistance gene (KmR) and the probable complete sequence of the streptomycin-spectinomycin-resistance gene (SpR) of the IncW plasmid pSa have been determined. The two genes appear to be oriented in the same direction and separated by a spacer region of 53 bp, with transcription proceeding from the KmR gene into the SpR gene. An RNA transcript encompassing the C terminus of the KmR gene, the 53-base spacer, and the N terminus of the SpR gene has the potential to form a stem-loop structure with a free energy value of -68 kcal/mol. The SpR gene of pSa has extensive sequence homology with the aadA gene of the plasmid R538-1. Comparison of the proposed amino acid sequence of the KmR protein of pSa with those of two aminoglycoside phosphotransferases revealed a region of potential homology with those proteins.

Isolation of the origin of replication of the IncW-group plasmid pSa

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.

Altered tetracycline resistance in pSC101 recombinant plasmids

SummaryInvestigation of tetracycline resistance genetically determined by the plasmid pSC101 and several recombinants of pSC101 containing of EcoRI generated DNA fragments inserted at the EcoRI site has revealed significant differences in the phenotypic expression of that resistance. The altered phenotypes of the recombinant plasmids may be the result of the location of the EcoRI site of pSC101, which has been determined to be near the genetic elements involved with tetracycline resistance.

CHARACTERIZATION OF TETRACYCLINE AND AMPICILLIN RESISTANT PLASMID CLONING VEHICLES

Abstract: Our laboratory has been constructing plasmids with suitable genetic properties for the cloning of DNA fragments. We have combined the essential replication properties of the plasmid ColEl with two antibiotic resistance markers (tetracycline and ampicillin) to generate a series of related plasmids. During the course of the construction of these plasmids we have determined the positions of restriction sites used for cloning DNA fragments relative to the antibiotic resistance genes and the origin of DNA replication. We have mapped a number of restriction sites on a series of related plasmids. We will summarize the development of two plasmids, pBR313 and pBR322, which are useful cloning vehicles for in vitro recombinant DNA research. The characterization of these plasmids has prompted an investigation of the nature of tetracycline resistance determined by the plasmid pSC101 and the pMB9 series of plasmids.

Restriction endonuclease mapping of pSC101 and pMB9

SummaryA restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.

THE PRODUCTION OF PROTEINS BY BACTERIAL PLASMIDS CONTAINING EUKARYOTIC DNA FRAGMENTS

ABSTRACT The production of polypeptides by bacterial plasmids containing eukaryotic DNA fragments is a complex phenomenon that can be affected by the plasmid used as a molecular vehicle and by the nature of the inserted DNA fragment. This conclusion is based on the investigation of the polypeptide species produced in minicells by plasmids containing Drosophila melanogaster and cauliflower mosaic virus DNA fragments.