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PubMed1968

Recombination of nonchromosomal mutations: a 3-point cross in the green alga Chlamydomonas reinhardi.

The heterothallic, unicellular green alga Chlamydomonas reinhardi possesses both chromosomal and nonchromosomal systems of inheritance. Mutations belonging to the latter system are capable of recombination. The experiments reported show that double and triple recombinants can be obtained from crosses employing three phenotypically distinct mutations and that certain pairs of mutants recombine more freely than others.

Crosses, Genetic↗
PubMed2000

A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Antiviral Agents↗
PubMed1971

Resistance of Pseudomonas to quaternary ammonium compounds. II. Cross-resistance characteristics of a mutant of Pseudomonas aeruginosa.

Tube dilution experiments showed that benzalkonium chloride (BC)-resistant mutants of Pseudomonas aeruginosa grown in the presence of 1,000 mug of BC per ml were at least 20 times more sensitive to polymyxin B and colistin sulfate than the BC-sensitive (BCS) parent strain. BCS cells selected for resistance to 500 mug of polymyxin B per ml remained sensitive to BC. There was little difference in the amount of carbenicillin, gentamicin sulfate, or rifampin needed to prevent growth of either the BCS or BC-resistant (BCR) strains. Growth of BCR cells was inhibited by ethylenediaminetetraacetate at a concentration of 400 mug/ml or less, whereas the BCS strain grew at ethylenediaminetetraacetate levels of 10,000 mug/ml. Phenylmercuric acetate and thimerosal inhibited growth of BCR and BCS cells at concentrations of 10 mug/ml or less. BCR cells were cross-resistant to >1,000 mug/ml concentrations of five other quaternary ammonium compounds, including three with C(16) alkyls and two with alkyl groups of shorter length. The BCS strain was also resistant to >1,000 mug/ml concentrations of the three quaternary ammonium compounds with C(16) alkyl groups but, in addition to BC, was inhibited by 200 mug/ml levels or less of the two quaternary ammonium compounds containing alkyl groups of less than 16 carbon atoms.

Anti-Bacterial Agents↗
PubMed1974

Clinical studies on a transformation test for identification of Acinetobacter (Mima and Herellea).

Deoxyribonucleic acid (DNA) from 250 strains of aerobic, nonfermentative, gram-negative coccobacilli and rods were tested for the ability to transform a stable competent auxotroph of Acinetobacter (strain trp E 27) to prototrophy by using the method established by Juni. Several modifications of Juni's original procedure were made to adapt it for use in a clinical diagnostic laboratory. These modifications were directed primarily towards shortening the procedure to allow completion in a time framework consistent with current procedures. The modifications included changes in sterilization temperature, incubation time and temperature of the competent auxotroph and DNA preparation, overnight incubation temperature, and variations in the age of the auxotroph culture when used. Under these conditions, the transformation can easily be performed in 24 h, the final 16 to 18 h being an overnight uninterrupted incubation period. When used in conjunction with the glucose oxidative fermentative basal metabolism test, it provided a rapid highly efficient means for grouping and identifying acinetobacters which is far superior to a biochemical schema. Without exception, the 141 strains of DNA from Acinetobacter species were able to transform the auxotroph to prototrophy. None of the 105 oxidase-positive nonfermenters possessed DNA which was able to transform the Acinetobacter auxotroph to prototrophy.

Acinetobacter↗
PubMed1973

Transmission of lac by the sex factor E in Erwinia strains from human clinical sources.

Lactose-utilizing (Lac(+)) strains of Erwinia spp. from human clinical material transfer lac by conjugation to plant strains of Erwinia herbicola and Erwinia amylovora, to other Erwinia strains from human clinical sources, and also to Escherichia coli, Paracolobactrum arizonae, Salmonella typhimurium, and Shigella dysenteriae. The frequency of this transfer varies with the donor and recipient strains employed. The lac genes appear stable in these exconjugants, and they are not cured by acridine orange. The Lac(+) exconjugants transfer lac to an Escherichia coli F(-) Lac(-) strain; the frequency of this transfer is high with E. herbicola and S. typhimurium exconjugants and relatively low with other exconjugants. The most studied Erwinia donor strain from human clinical material (EH133) and its Lac(+) exconjugants are insensitive to the F-specific phage, M13. P1-mediated transduction of lac, by using a Lac(+) exconjugant of E. coli as the donor and an E. coli F(-) Lac(-) strain as the recipient, revealed that all 50 Lac(+) transduced clones tested also inherited donor ability, suggesting a close linkage between the Erwinia sex factor (designated as E) and the lac genes. The E. coli culture harboring E-lac (E and the lac genes linked to it) does not restrict phages T1, T7, and lambdavir. E-lac is compatible with F'his, R100 drd-56 (F-like), and R64 drd-11 (I-like); cells harboring F'his or one of the R factors do not show super-infection immunity to the incoming E-lac, and E-lac plus one of the other plasmids can coexist stably in the same cell. The fertility of cells harboring F'his or R100 drd-56-as determined by the frequency of conjugal transfer of his or of the resistance determinant (Tet(r) in case of R100 drd-56) and also by sensitivity to F-specific phage (M13)-is not altered by the presence of E-lac, and this suggests that the sex factor E might belong to the fi(-) class.

Bacteriological Techniques↗
PubMed1970

Fertility regulation in F-like resistance transfer factors.

Mutants of the R factor R100 have been isolated that mediate high-frequency transfer of the R factor during conjugation. Complementation tests revealed two classes of mutants, operator-constitutive and repressor-negative. Some of the latter class were suppressible by amber and ochre suppressors. The results support a simple model of regulation for the control of R-factor-mediated piliation.

Conjugation, Genetic↗
PubMed1970

Intercellular effects on development of competence in Bacillus subtilis.

The intercellular transfer of competence during growth under the conditions specified by the transformation procedure of Spizizen was investigated with Bacillus subtilis 168. The rate of competence development as assayed uniformly in medium B was not affected by variations in the cell concentration, although the first appearance of transformants occurred earlier with high cell densities in medium A, approximately in proportion to the onset of the stationary phase in the culture. Growth in the presence of Pronase enhanced the frequency of transformation, but did not detectably alter the kinetics of competence development. The rate of competence increase in physiologically noncompetent cultures was not changed by mixing with competent cultures either in medium A or in medium B; however, an early appearance of transformants was noted in mixed cultures in which the proportion of competent to noncompetent cells prevented exponential growth of the noncompetent strain. These experiments indicate that the normal development of competence in B. subtilis is not mediated by a soluble or loosely bound protein factor capable of transmitting competence directly via cell contact. The onset of competence is thus a function of internal physiological changes which are induced by the overall metabolic state of the culture.

Bacillus subtilis↗
PubMed1970

Genetic mapping and dominance of the amber suppressor, Su1 (supD), in Escherichia coli K-12.

In this report Su1 (supD), which is known to suppress "amber" mutations by means of a specific transfer ribonucleic acid, has been mapped relative to his and uvrC and has been found to be located at about 37.5 min on the Taylor and Trotter genetic map of the Escherichia coli K-12 chromosome. In addition, Su1(+) has been shown to be dominant over Su1(-), which supports the idea that Su1 is the structural gene for the suppressing transfer ribonucleic acid.

Alkaline Phosphatase↗
PubMed1970

Genetic mapping in Escherichia coli K-12 by radiation-induced crossing-over.

Conventional methods for chromosomal mapping in Escherichia coli are (i) interruption of matings to obtain minimum marker entry times, (ii) linkage analysis of recombinants, and (iii) cotransduction. Method (i) has a resolution of about 0.5 min (5 x 10(4) nucleotides) and is not useful for distances less than about 1 min; methods (ii) and (iii) are capable of better resolution but are generally not very reproducible and no general theory is available for translating crossing-over and cotransduction frequencies into physical chromosomal distances. We found that when merozygotes are irradiated (X rays or ultraviolet light) soon after marker transfer, high linkage values (0.8 to 1.0) between nearby marker pairs decrease with radiation dose to 0.5. Our results are quantitatively consistent with the idea that radiations induce crossing-over lesions proportional to dose, and the number of such lesions between two markers is proportional to the physical separation of the markers in the range that can also be measured by interruption of mating (0.5 to 4.0 min). Additivity relations among markers are also satisfied. We used this technique to measure the distances (0.1 to 1.0 min) between several pairs of closely linked markers.

Chromosome Mapping↗
PubMed1970

Differential depression of staphylococcal plasmid and chromosomal penicillinase genes by a class of unlinked chromosomal mutations (R2-).

We described previously a class of mutations (phenotypic designation R2(-)) in Staphylococcus aureus that were unlinked to penicillinase genes but nevertheless imposed constitutive synthesis of this enzyme upon wild-type plasmids. The plasmids affected by these mutations have been found to possess the same maintenance-compatibility type (I) and to form penicillinase of serotype A. Three plasmids that had a different maintenance-compatibility type (II) and formed penicillinase of serotype C were only slightly derepressed in R2(-) mutants. Three chromosomal penicillinase genes were variably derepressed, the range extending from negligible to nearly complete derepression. The differences in degree of derepression among the three types of penicillinase linkage groups were tentatively ascribed to position effects.

Chromosomes, Bacterial↗
PubMed1970

Uptake of synthetic polynucleotides by competent cells of Bacillus subtilis.

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.

Amino Acids↗
PubMed1971

Valine accumulation by alpha-aminobutyric acid-resistant mutants of Serratia marcescens.

alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator.

Aminobutyrates↗
PubMed1971

Pleiotropic effect of the rec A gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid replication.

A defective recA gene, which is involved in recombination, is shown in this article to permit limited cell division, when deoxyribonucleic acid (DNA) synthesis is blocked. Thymidine starvation or nalidixic acid blocked DNA synthesis, and stopped cell division of a rec(+)thy(-) strain of Escherichia coli. However, with the same treatments, a recAthy(-) strain could continue to divide for at least 5 hr, and cell numbers increased 2.5- to 4-fold. After several hours of thymidine starvation, the culture contained very long cells (snakes) and small (normal-sized) cells. The short cells contained very little, if any, DNA. Cells of all ages divided in the absence of thymidine. Specific differences in membrane proteins were observed between thymidine-starved rec(+) and recA cells, as expected from previous experiments in which these proteins were associated with cell division and DNA synthesis. It is proposed that septum formation is controlled negatively by the recA(+) gene.

Arginine↗
PubMed1971

Relationship between competence for transformation of Bacillus subtilis with native and single-stranded deoxyribonucleic acid.

The response of populations of Bacillus subtilis to both native deoxyribonucleic acid (DNA) and denatured DNA was investigated at maximal competence and at various times during the development of compentency. The results indicate that competence for transformation with native and denatured DNA increases and decreases simultaneously. Competition occurs between native and single-stranded DNA during transformation, and the same cells in a population can be doubly transformed by DNA molecules of both configurations.

Bacillus subtilis↗