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S Subramani

Publications and source records attributed to S Subramani.

123 records · Page 7Linked to original sources
PubMed1985

Rapid assay for extrachromosomal homologous recombination in monkey cells.

Most of the recombination assays based on the regeneration of selectable marker genes after transient infection or stable integration of DNA into mammalian cells are time consuming. We have used plasmids containing two truncated but overlapping segments of the neomycin resistance gene to rapidly quantitate and characterize the time course of extrachromosomal homologous recombination of DNA transfected into monkey COS cells. By transiently infecting cells with these recombination substrates, extracting Hirt DNA after 1 to 4 days, and transforming recombination-deficient Escherichia coli, we have shown that recombination between direct repeats occurs at frequencies of 1 to 4%. We have also used Southern blot analysis to directly characterize the recombination of this DNA in COS cells and to demonstrate that double-strand breaks in the region of homology increase recombination frequencies 10- to 50-fold.

Animals↗
PubMed1985

Recombination events after transient infection and stable integration of DNA into mouse cells.

To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.

Animals↗
PubMed1984

The minimum amount of homology required for homologous recombination in mammalian cells.

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.

Animals↗
PubMed1983

Nucleotide sequence of the Escherichia coli xanthine-guanine phosphoribosyl transferase gene.

The Escherichia coli gene coding for the enzyme xanthine-guanine phosphoribosyl transferase (gpt) has been widely used as a dominant selectable marker in a variety of mammalian cells. We have determined the complete nucleotide sequence of the 1057 base pair (bp) segment of DNA containing this gene. The coding sequence for the enzyme is 456 nucleotides long and can code for a 152 amino acid (16.9 Kd) polypeptide. A comparison of the amino acid sequence of the bacterial enzyme with that of the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) reveals no significant homology between the two polypeptides.

Amino Acid Sequence↗
PubMed1983

Elements in the long terminal repeat of murine retroviruses enhance stable transformation by thymidine kinase gene.

We have investigated the effects of long terminal repeats (LTRs) of murine retroviruses on the frequency of obtaining stable transfectants by the herpes virus thymidine kinase (TK) gene. The results indicate that addition of LTRs enhances the number of TK+ transformants by 10-20 fold. A 5-12 fold enhancement was also observed when chromosomal DNA from either human or hamster cells was mixed with a plasmid containing LTR sequences and transfected onto LTR- cells. The LTR sequences involved in the enhancement were localized in the region which contains tandem repeats. All other regions of the LTR did not show any enhancement of stable TK+ transfectants. The location or the orientation of the enhancer sequences with respect to the TK gene did not exert any influence on the frequency of transformation. The enhancement effect does not appear to be linked to either increased numbers of chromosomal integrations or elevated levels of transcription of the TK gene.

Animals↗
PubMed1983

Analysis of gene expression using simian virus 40 vectors.

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.

Animals↗
PubMed1983

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.

Animals↗
PubMed1981

Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.

Animals↗
PubMed1980

Introduction of liposome-encapsulated SV40 DNA into cells.

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.

Animals↗
PubMed1977

Ligand-promoted weakening of intersubunit bonding domains in aspartate transcarbamolylase.

THE COOPERATIVITY AND FEEDBACK INHIBITION EXHIBITED BY THE REGULATORY ENZYME, ASPARTATE TRANSCARBAMOYLASE (CARBAMOYLPHOSPHATE: L-aspartate carbamoyltransferase; EC 2.1.3.2), from Escherichia coli are generally attributed to ligand-promoted conformational changes involving alterations in the subunit interactions. However, no quantitative estimates have been made of the effect of ligands on the strength of the intersubunit bonding domains. The native enzyme, composed of two catalytic trimers "crosslinked" by three regulatory dimers, shows little tendency to dissociate in neutral buffers at room temperature. In addition, very little exchange was observed in 2 hr between subunits within the intact enzyme and free subunits. Although exchange was enhanced in solutions of low ionic strength containing the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, the rates of exchange were too small to permit reliable estimates of the weakening of the bonding domains caused by the ligand. Studies were conducted, therefore, on a less stable oligomeric complex which resembles the native enzyme in structure and allosteric behavior but lacks one regulatory subunit. These molecules, containing only four bonding domains between the catalytic and regulatory polypeptide chains (compared to six in the native enzyme), disproportionate to form the more stable native enzyme and free catalytic subunits. An electrophoretic technique is described for measuring the rate of disproportionation which is controlled by the rupture of the intersubunit bonding domains. This rate is enhanced about 300-fold upon the addition of the active-site ligand. Hence the ligand-promoted allosteric conversion of the enzyme-like complex from the constrained to the relaxed conformation involves a substantial weakening of the intersubunit interactions corresponding to about 1.7 kcal/mole (7.1 kJ/mole) per bonding domain between a catalytic and a regulatory chain.

Aspartate Carbamoyltransferase↗
PubMedDate not supplied

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors.

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.

Animals↗