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M D Mamet-Bratley

Publications and source records attributed to M D Mamet-Bratley.

At least 19 recordsLinked to original sources
PubMed1997

Toxicity and repair of psoralen adducts in bacteriophage T7.

Psoralens react photochemically with DNA to form interstrand crosslinks as well as two types of monoadduct (furan-side and pyrone-side adducts). To investigate the relative roles of these adducts in toxicity, we have studied the interaction of 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) with bacteriophage T7. These two derivatives differ in the fraction of pyrone-side monoadducts formed, TMP producing very small amounts of this type of adduct. The results show similar phage survival for the two psoralen analogs at equivalent numbers of crosslinks per DNA molecule. However, the survival fraction of treated phage is significantly lower than the fraction of noncrosslinked DNA molecules. Phage survival decreases after secondary irradiation which is used to transform monoadducts into crosslinks, but this decrease is not due solely to crosslinks; at doses beyond that required to transform all crosslinkable monoadducts into crosslinks, phage survival continues to decrease, pointing to the production of other genotoxic lesions during secondary irradiation. These results indicate that, although crosslinks can kill phage T7, as shown by the secondary irradiation results, they are not sufficient in number to explain the psoralen toxicity after primary irradiation. Therefore monoadducts, both furan-side and pyrone-side types, must in large part be responsible for phage inactivation.

Bacteriophage T7↗
PubMed1997

Taxonomic changes in tailed phages of enterobacteria.

Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented.

Coliphages↗
PubMed1994

Transcription by T7 RNA polymerase of DNA containing abasic sites.

The effects of abasic (AP) sites on RNA synthesis were studied in vitro, using T7 RNA polymerase and a plasmid template containing a T7 promoter. The presence of increasing numbers of AP sites caused a progressive decline in RNA synthesis. The average RNA chain length, calculated from the ratio of initiation to chain elongation, decreased with increasing numbers of AP sites, revealing that complete blocks must occur during synthesis. The probability that RNA polymerase would be blocked at an AP site in the DNA template strand was estimated to be 0.3 in our experimental conditions. These results demonstrate that RNA synthesis by T7 RNA polymerase is inhibited by AP sites and that readthrough of the lesion occurs more frequently than premature chain termination. Chemical reduction of AP sites in the template did not change the block/bypass pattern.

Apurinic Acid↗
PubMed1994

Effect of abasic sites on bacteriophage T7 protein synthesis.

We have examined protein synthesis directed by bacteriophage T7 which had been alkylated with methyl methanesulfonate so as to produce apurinic sites in its DNA in vivo. Both repair-proficient and repair-deficient (xth nfo mutant) strains of Escherichia coli served as host cells. In repair-proficient cells, all three classes of phage proteins were synthesized, although with significant delays. In mutant cells, only class I proteins were produced and their synthesis was delayed and reduced, demonstrating a perturbation of protein synthesis and providing the first in vivo indication that transcription is inhibited by abasic sites. However, the proposed effects of abasic sites on transcription appear to be weaker than those on replication.

Alkylation↗
PubMed1993

Mechanism of toxicity of 3-methyladenine for bacteriophage T7.

Treatment of bacteriophage T7 with methyl methanesulfonate perturbed phage-specific genetic expression in both repair-proficient and repair-deficient Escherichia coli cells. In wild-type cells (AB1157), the time course of protein synthesis was slowed down but an entire complement of phage proteins was synthesized. In cells (BK2114, tag-) unable to repair 3-methyladenine, the toxic lesion produced by methyl methanesulfonate, alkylated phage produced only early (class I) proteins. These results suggested that late transcription was inhibited in infected tag- cells. These cells were shown to contain a significant amount of active T7 RNA polymerase, a class I protein. Thus, the cause of inhibition appeared to be the inability of T7 RNA polymerase to use unrepaired DNA as template. In vitro transcription assays with alkylated T7 DNA as template supported this proposal. T7 RNA polymerase proved to be very sensitive to the presence of alkylation lesions. In addition, the phage enzyme was much more sensitive to these lesions than was its bacterial counterpart, E. coli RNA polymerase. These results suggest that 3-methyladenine exerts its toxic action, in the T7 system, at the level of transcription by T7 RNA polymerase. To further characterize the reduced activity of the T7 enzyme, an in vitro transcription assay using linearized plasmid DNA with one T7 promoter was devised. Gel electrophoresis revealed that only one transcript of well-defined length was synthesized by T7 RNA polymerase on this template. Alkylation of the template did not alter the size of the transcript produced. Simultaneous measurement of chain initiation and chain elongation confirmed this result by showing that both steps were reduced to the same extent by alkylation of template DNA. Thus T7 RNA polymerase does not appear to be blocked by 3-methyladenine. Rather the lesion must hinder translocation of T7 RNA polymerase along the DNA template during chain elongation.

Adenine↗
PubMed1992

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.

Adenine↗
PubMed1992

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli.

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.

Bacterial Proteins↗
PubMed1992

[protein synthesis in alkylated bacteriophage T7].

We have studied the toxic effects of alkylating agents with a well characterized model: phage T7. Treatment of bacteriophage T7 with methyl methanesulfonate led to perturbation of phage-specific protein synthesis. Synthesis of class I and II proteins was prolonged, while production of class II and III proteins was delayed. This delay increased for proteins coded by genes located further to the right on the T7 genetic map. In extracts prepared from cells infected by alkylated phage, the specific activity of T7 RNA polymerase was decreased. These results suggest that the toxic action of methyl methanesulfonate is directed towards viral transcription.

Alkylation↗
PubMed1987

Characterization of morphogenetic intermediates and progeny of normal and alkylated bacteriophage T7.

Analysis of thin sections of Escherichia coli B cells infected by normal (nonalkylated) or alkylated bacteriophage T7 showed that alkylation altered phage morphogenesis. To understand these morphogenetic alterations, we have isolated phage-related particles from infected-cell lysates by differential and sucrose gradient centrifugation. Cells infected by normal and by alkylated phage produced mature phage particles, empty heads, and proheads; however, production of proheads and mature phage particles was less in the case of alkylated phage. These lysates also contained sedimentable material which migrated more slowly than empty heads on sucrose gradients. In the case of alkylated phage, this peak contained radioactive material in amounts nearly equal to that in either proheads or empty heads; for normal phage, this peak represented a smaller fraction of the total radioactivity. Examination of the gradient fractions by electron microscopy revealed appreciable quantities of phage tails and tail-related particles. The same gradient fractions contained phage tail proteins: gene products (gps) 11, 12, and 17 as well as smaller amounts of gp 8, the head-tail connector. In addition, these fractions contained two other proteins which we believe to be of bacterial origin. These proteins may be related to tail formation or function as part of the phage receptor. On the basis of our data, we propose an alternative morphogenetic pathway for T7 tail formation, a pathway which would involve formation of a complex of tail proteins prior to association with the phage head.

Alkylation↗
PubMed1986

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7.

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.

DNA Repair↗
PubMed1982

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204-213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5-10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.

DNA Glycosylases↗
PubMed1982

Alkylation of T7 bacteriophage blocks superinfection exclusion.

Alkylation of T7 bacteriophage by methyl methane sulfonate blocked superinfection exclusion. This blockage could be correlated with a delay in the synthesis of phage-specific proteins. Therefore we conclude that protein synthesis directed by the primary infecting phage is required for efficient exclusion of superinfecting phage particles.

Alkylation↗
PubMed1982

Injection defect in alkylated and depurinated T7 bacteriophage: analysis by DNA ejection.

Using DNA ejection in vitro as a model, we have studied the DNA injection defect caused by alkylation and depurination of T7 bacteriophage. Phage was alkylated with 0.02 M methyl methanesulfonate for 2 h at 37 degrees C; alkylated phage was then incubated 24 h at 30 degrees C to induce depurination. These samples were treated with formamide to cause DNA ejection without dissociation of the phage capsid. After ejection, the phage preparations were analyzed by electron microscopy. DNA lengths in capsid-DNA complexes were measured; relative numbers of full, empty, and partially empty phage heads were determined. To establish the direction of DNA ejection, E. coli RNA polymerase was bound to capsid-DNA complexes. The results showed that DNA was partially ejected from both alkylated and depurinated phages. In the alkylated sample, RNA polymerase was bound to the DNA end distal to the capsid; this showed that ejection started from the genetic left end. We interpret these results to show, in confirmation of earlier results obtained by marker rescue, that alkylation causes T7 phage to partially inject its DNA, starting from the genetic left end. For depurinated phage, our results suggest that partial DNA injection is responsible, in this case as well, for the already documented injection defect.

Alkylation↗
PubMed1981

Biological consequences of infection of Escherichia coli B by alkylated T7 bacteriophage.

Alkylation of T7 bacteriophage considerably delayed phage development and reduced the phage's killing action on host cells. Only a small fraction of infected cells produced phage. For these phages, the latent period was markedly prolonged but the burst was equivalent to or only slightly lower than that of untreated phage. In the progeny of alkylated phage, there was an increase in the fraction of defective particles as well as a change in their morphology. These data show that infection with alkylated T7 bacteriophage is to a large degree abortive; hence, biological consequences of this infection are very different from those characteristic of a normal virus infection.

Alkylation↗
PubMed1979

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.

Alkylation↗