Search PubMed⌕ Search

Biomedical subjects

C Anagnostopoulos

Publications and source records attributed to C Anagnostopoulos.

At least 73 records · Page 4Linked to original sources
PubMed1976

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.

Adenosine Triphosphatases↗
PubMed1976

Differences in the genetic structure of Bacillus subitilis strains carrying the trpE26 mutation and strain 168.

It was previously shown that in strains of Bacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region. Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other. These data concern evidence of linkage of tre-12- to markers in the translocation (hisH2, tyrA1, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA. They explain the previously observed absence of linkage of markers in the translocated segment to cysB3. The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding.

Bacillus subtilis↗
PubMed1975

Studies on the size of the diploid region in Bacillus subtilis merozygotes from strains carrying the trpE26 mutation.

Simultaneous selection of transformants for trpE26 and a second unliked marker of B. subtilis in many cases yields double heterogenotic clones. Several chromosome areas analyzed in this way were found to be involved in the diploid condition. Diploids for areas on the left hand side of trpE26 on the map (and as near as the aroB locus) are in general unstable while stable merodiploids can be obtained for areas on the right hand side of this marker (as far as the ilvA locus). Merozygotes for regions other than the "aromatic segment" are also formed by transformation of already diploid (stable and unstable) clones. Stable diploids give rise to new heterogenotes only for markers on the right hand side of trpE26. Through reversion of untransformed markers in unstable and stable diploids it was found that these clones are homodiploid for loci situated at a long distance from (or between) the areas which were involved in the transformation. This indicates that the diploid state covers a continuous segment of the chromosome, the length of which can be determined. The segregation pattern of unstable multiple merodiploids suggests that exchange of genetic material must take place between the two homologous regions. The data presented are in agreement with the hypothesis that the merodiploids possess a very long duplication on their chromosome. In the case of the stable clones this duplication is shorter.

Bacillus subtilis↗
PubMed1975

Induction and transmission of a merodiploid condition near the terminal area of the chromosome of Bacillus subtilis.

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB-ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4+ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be cotransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.

Aneuploidy↗
PubMed1975

Evidence for the Translocation of a Chromosome Sement in Bacillus subtilis Strains Carrying the trpE26 Mutation.

The replication order of markers was studied in Bacillus subtilis strains bearing the trpE26 mutation by the use of the density transfer technique. An important difference in this order was observed in comparison with that of strain 168 T-. All markers tested of a chromosome segment extending from trpD to ilvA replicated early, after purB6 and before thr-5. Two markers flanking this region, trpE8 and citK7, replicated late as usual. The results suggested that this segment was shifted in trpE26 strains to a region closer to the origin of replication. PBS-1-mediated transduction crosses corroborated this hypothesis and revealed the position of the translocated segment. (i) Linkage was demonstrated for markers in the segment (hisH2, tryA1, met B3, ilvA2) to thr-5 and ald; (ii) aroB2 and citK7 were found to be linked; and (iii) linkage of cysB3 to thr-5 was lost in trpE26 strains. These findings made it possible to account for the characteristics of the trpE26 mutation and to propose a model explaining the fact that all trpE26+ transformants or transductants are merodiploid. The model calls for fusion of two genetic elements: two independent chromosomes, or two arms of a replicating structure. The resulting chromosome bears a long tandem duplication. Most of the features of this system of merodiploid formation can be interpreted by use of this model: the segregation pattern of the diploids, the stabilization of the unstable clones, and the length of the duplicated region. A relatively stable diploid strain was also studied by the density transfer technique. The data show that it remained diploid for the region corresponding to the translocated segment and are in agreement with the structure predicted by the model.

Bacillus subtilis↗
PubMed1973

Genetic studies relating to the production of transformed clones diploid in the tryptophan region of the Bacillus subtilis genome.

Transformation experiments with Bacillus subtilis strains carrying trpE26 (the marker responsible for the detection of merodiploid clones after transformation or transduction) have established the precise position of this marker on the "aromatic region" of the chromosome, at the distal end of the anthranilate synthetase locus. Integration efficiency of the mutant allele (trpE26) seems to be very low. Co-transfer of markers situated on either side of it is almost nil when both donor and recipient carry this mutation. The "exclusion" of trpE26 does not, however, affect recombination frequencies for nearby markers. To explain these facts we considered the hypothesis of a preferential breakage of the deoxyribonucleic acid (DNA) at the trpE26 site or that of an insertion mutation. These studies have also demonstrated the establishment of physical linkage of a marker from the exogenote (hisH2) to a resident marker (tyrA1) in stable and unstable merodiploid clones, thus confirming integration of the donor DNA segment into a genetic structure of the recipient. Furthermore, duplication was shown in merodiploid clones (through reversion and transformation) for a locus of the recipient (tyrA) which was not involved in the initial transformation. This suggests that the diploid condition in this region extends beyond the transformed area. Interpretation of the genetic constitution of these partial diploids calls for postulation of the existence of long duplications, a second (incomplete) chromosome, or an episome-like element.

Alleles↗
PubMed1971

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis.

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.

Adenosine Triphosphate↗
PubMed1970

Chromosomal location and properties of radiation sensitivity mutations in Bacillus subtilis.

Transformation and transduction crosses involving recA1, recB2, and urv-1 mutations have shown that these mutations belong to three distinct unlinked genetic loci. The precise position of these loci on the Bacillus subtilis chromosome map has been determined. The behavior of recB2 strains in transformation studies suggested a dominance of recB2(+) function over recB2 and an early expression of this phenotype during transformation. Strains bearing two ultraviolet sensitivity markers possess a phenotype characteristic of the marker with the most adverse effect on recombination. The possibility that the effects of the two mutations are additive was also considered. Results are also presented which show that a phage-induced enzyme is not responsible for the high transducibility of recA1 strains.

Bacillus subtilis↗
PubMed1967

Transformation and transduction in recombination-defective mutants of Bacillus subtilis.

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.

Bacillus subtilis↗