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At least 1,315 records · Page 73Linked to original sources
PubMed1988

[Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19].

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E. coli. The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19. Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E. coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119. The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A. Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted). In all these cases different levels of expression were observed (but only in the presence of IPTG). Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b. p. region in relation to the PT promoter, i. e. to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1985

[General approach to the engineering of synthetic DNA].

A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described.

Bacteriorhodopsins↗
PubMed1986

[Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12].

Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.

Bacteriophage lambda↗
PubMed1987

Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen.

A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.

Base Sequence↗
PubMed1987

Gene therapy for thioguanine-resistant human leukemia.

The feasibility of using retroviral gene therapy to overcome drug resistance was assessed by determining the efficiency by which a retrovirus containing the human HGPRT gene could sensitize hypoxanthine-guanine phosphoribosyltransferase (HGPRT) negative human promyelocytic leukemia cells to 6-thioguanine. A single three-hour exposure at a virus to cell ratio of 6 X 2:1 restored sensitivity to 70(+/- 18)% of the clonogenic cells. The efficacy varied as a function of virus concentration and duration of viral exposure; the time allowed for integration and expression between one and five days post-infection had little effect. Cells successfully sensitized contained a proviral insert and expressed HGPRT activity that ranged from 1 to 92% of that in the wild-type cells. The mutation rate of the inserted gene varied from the same as that of the endogenous HGPRT gene to 200-fold greater in different clones. Failure of sensitization following viral exposure was associated with absence of an integrated provirus, and clonogenic cells failing to be sensitized by one virus exposure were sensitized with approximately the same efficiency by a second viral exposure. These results demonstrate the feasibility of transferring a drug sensitivity gene to a human leukemia cell line.

Cell Line↗