Biomedical subjects

W R Guild

Publications and source records attributed to W R Guild.

6 recordsLinked to original sources

PubMed1979

Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases.

Bacteriophages↗

PubMed1979

A plasmid in Streptococcus pneumoniae.

Plasmid deoxyribonucleic acid has been detected in three related laboratory strains of Streptococcus pneumoniae. Strains D39S, R36, and R36NC each contain a minimum of two copies per cell of a 2.0-megadalton plasmid (pDP1). A plasmid twice as large as this smaller one is also present in much lower quantity in these strains, but neither plasmid is present in four strains related to these or in a drug-resistant clinical isolate from Paris. The plasmid yield was not amplified in the presence of chloramphenicol. No phenotype has been correlated with the presence of pDP1, which has existed in strains carried for many years in laboratory collections.

Anti-Bacterial Agents↗

PubMed1979

Organization and transfer of heterologous chloramphenicol and tetracycline resistance genes in pneumococcus.

The cat and tet genes of chloramphenicol- and tetracycline-resistant clinical isolates of Streptococcus pneumoniae from Paris and Japan were shown to be contained in adjacent heterologous insertions into the chromosome. The two insertions transformed laboratory strains at frequencies that were low, unequal, and, for tet, very sensitive to the length of the donor deoxyribonucleic acid strand. In contrast, the transforming activity of cat was relatively stable. There was an unusual asymmetric cotransfer, in that a majority of the tet transformants also acquired cat, whereas only a few of the cat transformants also acquired tet. The evidence for chromosomal insertion came from genetic data showing linkage of cat to a chromosomal gene and from cosedimentation of cat with chromosomal markers in both velocity and dye-buoyancy experiments. Genes on a known plasmid introduced into pneumococcus from Streptococcus faecalis showed very different physical behavior. Most of the transformation properties of these genes can be readily accounted for by analogy to transformation of deletions of normal genes. Whether transposition contributes any of the transfers remains to be determined. The presence of one of the genes in the recipient promoted the integration of the other, demonstrating enhanced accumulation of heterologous genes by a process that did not involve plasmids in the species of concern.

Chloramphenicol↗

PubMed1978

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

Bacteriophages↗

PubMed1976

Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency.

A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.

DNA Repair↗

PubMed1976

Characterization of some pneumococcal bacteriophages.

The growth of pneumococcal phages at high cell and phage densities is enhanced strongly by the substitution of potassium for sodium in the medium. Initial titers of 2 X 10(10) to 4 X 10(10) PFU/ml are readily obtained, and concentrated stocks are stable in a storage buffer described here. The mechanism of the cation effect is obscure. Phages omega3 and omega8 each have linear double-stranded DNA of 33 X 10(6) daltons per particle, with an apparent guanine plus cytosine content of 47 to 49 mol%, as determined by buoyancy and melting temperature, but with an unusual absorbance spectrum. Efficiency of plating is high if sufficient time is allowed for a relatively slow adsorption, which differs several-fold in rate between the two phages. Morphologically, these and other pneumococcal phages are similar to coliphage lambda but with a longer tail and tail fiber. Upon UV inactivation, omega3 and omega8 have D37 values of 33 and 55 J/m2, respectively, and each shows multiplicity reactivation. A total of 13 ts mutants have been isolated from the two phages, representing only two complementation groups; complementation and recombination occur between omega3 and omega8 mutants. Both phages provoke high-titer antisera with extensive cross-reactivity against a number of newly isolated pneumococcal phages.

Adsorption↗