Biomedical subjects

M Dosmar

Publications and source records attributed to M Dosmar.

6 recordsLinked to original sources

PubMed1979

Deoxythymidine nucleotide metabolism in Bacillus subtilis W23 infected with bacteriophage SP1Oc: preliminary evidence that dTMP in SP10c DNA is synthesized by a novel, bacteriophage-specific mechanism.

Despite the fact that mature SP10c DNA contains dTMP, the acid-soluble fraction of infected cells contained no dTTP during the interval of phage replication. However, infected cells contained normal cellular levels of dATP, dGTP, and dCTP. Upon infection of deoxythymidine-starved Bacillus subtilis M160 (a deoxythymidine-requiring mutant of B. subtilis W23), mature phage DNA with a normal dTMP content was made. SP10c codes for an enzyme that seems to catalyze the tetrahydrofolate-dependent transfer of 1-carbon fragments to the 5 position of dUMP. The transfer of 1-carbon fragments is not accompanied by oxidation of tetrahydrofolage to dihydrofolate, implying that the enzyme in question is not a dTMP synthetase. It is proposed that dTMP in mature SP10c DNA is derived by the postreplicational modification of some other nucleotide and not by the direct incorporation of dTTP into DNA.

Bacillus subtilis↗

PubMed1978

Production and expression of dTMP-enriched DNA of bacteriophage SP15.

Normal DNA of Bacillus subtilis phage SP15 contains approximately equimolar quantities of dTMP and a hypermodified nucleotide, 5-dihydroxypentyl-dUMP (DHPdUMP). Deoxythymidine (dThd) rescue of phage DNA synthesis in 5-fluorodeoxyuridine (FUdR)-inhibited cultures resulted in the synthesis of SP15 DNA containing enhanced levels of dTMP and correspondingly reduced levels of DHPdUMP. This rescued system was used to probe possible roles of DHPdUMP in phage development. The results suggested that normal levels of DHPdUMP were not required for proper transcription of phage DNA, but normal amounts of DHPdUMP were indispensable for phage assembly and/or DNA maturation. The amount of exogenous dThd required to rescue phage DNA synthesis in FUdR-inhibited cultures was 20-fold higher than the concentration required to rescue cellular replication, whereas the same low concentrations of dThd sufficed to rescue viral and bacterial DNA syntheses in aminopterin-inhibited cultures. Normal SP15 DNA was made in rescued, aminopterin-inhibited cultures. We suggest that FUdR (but not aminopterin) partially suppresses biosynthesis of the hypermodified nucleotide and that there is a barrier to replacement of DHPdUMP by dTMP; therefore, exceptionally large amounts of dThd must be salvaged in FUdR-inhibited cultures to force replacement of the unusual nucleotide by dTMP.

Aminopterin↗

PubMed1977

SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.

Aminoglycosides↗

PubMed1977

Effect of antibiotics on certain aspects of bacteriophage SP-15 development in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with bacteriophage SP-15. Two waves of phage-specific RNA synthesis were observed. Wave I was prereplicative, and wave II was coincident with replication of the viral genome. To determine the temporal appearance of general classes of phage-coded messengers and proteins, we studied the dependence of lysozyme synthesis, phage production, and DNA synthesis on time of addition of transcriptional and translational inhibitors. Lysozyme synthesis started to become refractile to a variety of transcriptional inhibitors (rifampin, streptolydigin, and actinomycin D) between 20 and 22 min postinfection and was completely refractile by 30 min. Nevertheless, functional enzyme did not appear until 45 to 47 min postinfection; lysozyme was maximal by 65 min. Rna isolated from SP-15 phage-infected cells was used to program the cell-free synthesis of lysozyme. The messenger was synthesized exclusively between 20 and 30 min postinfection. Lysozyme messengers were stable. The data imply that lysozyme messengers were present 52 min prior to their translation. Progeny virus formation remained sensitive to transcriptional inhibitors until 40 to 50 min postinfection, and sensitivity to chloramphenicol lasted 65 min. The first progeny viruses appeared at 75 min. Again, an unusually long lag between completion of functional messengers and their translation was evident. The aforementioned data indicated that transcription of lysozyme messengers and, at least, some messengers, whose products are essential for phage production, are uniquely associated with waves I and II of RNA synthesis, respectively. However, messengers whose products are essential for normal amounts of DNA synthesis were apparently synthesized during both waves; transcription of these messengers was transiently repressed (using the term broadly) between 30 and 40 min postinfection. Judging from the dependence of DNA synthesis on time of chloramphenicol addition, proteins essential for normal amounts of DNA synthesis were also synthesized in two discrete waves, each yielding sufficient protein for half-maximal levels of DNA synthesis. An hiatus in the synthesis of the proteins in question was evident between 45 and 65 min postinfection; evidence cited in this paper indicates that this hiatus did not result from messenger depletion, which, in turn, implied some type of translational-level control. This latter conclusion is substantiated by the lysozyme synthesis that occurred during the same interval when synthesis of certain proteins for DNA replication was transiently repressed.

Aminoglycosides↗

PubMed1976

Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.

Chloramphenicol↗

PubMedDate not supplied

The Water Pressure Integrity Test--a new integrity test for hydrophobic membrane filters.

Sterilizing grade hydrophobic filters are used for the sterile filtration of gases in pharmaceutical and biological applications. Until now the integrity of these membrane filters and their ability to retain bacteria, has been correlated to a solvent based nondestructive integrity test. Current methods use solvents to wet the membranes in order to perform bubble point and diffusion integrity tests. Solvent based integrity tests make it difficult to test in situ following sterilization because of the risk of downstream solvent contamination. A newly developed method, the Water Pressure Integrity Test (WPIT), allows for the integrity testing of hydrophobic filters eliminating the problems associated with traditional test methods employing solvents. A prime advantage of WPIT is that it may be performed in situ post sterilization without any downstream manipulations. The test has been directly correlated to the retention of bacterial challenges. Data will be provided to show the reliability and sensitivity of this easy to perform test.

Evaluation Studies as Topic↗