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C K Mathews

Publications and source records attributed to C K Mathews.

At least 91 records · Page 5Linked to original sources
PubMed1977

Enzyme associations in T4 phage DNA precursor synthesis.

A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.

Coliphages↗
PubMed1977

Further studies on bacteriophage T4 DNA synthesis in sucrose-plasmolyzed cells.

This paper describes several technical improvements in the sucrose-plasmolyzed cell system used in earlier experiments on DNA synthesis in situ with Escherichia coli infected by DNA-defective mutants of bacteriophage T4 (W. L. Collinsworth and C. K. Mathews, J. Virol. 13:908-915, 1974). Using this system, which is based primarily on that of M. G. Wovcha et al. (Proc. Natl. Acad. Sci. U.S.A. 70:2196-2200, 1973), we reinvestigated the properties of mutants bearing lesions in genes 1, 41, and 62, and we resolved some disagreements with data reported from that laboratory. We also asked whether the DNA-delay phenotype of T4 mutants is related to possible early leakage of DNA precursors from infected cells. Such cells display defective DNA synthesis in situ, even when ample DNA precursors are made available. Thus, the lesions associated with these mutations seem to manifest themselves at the level of macromolecular metabolism. Similarly, we examined an E. coli mutant defective in its ability to support T4 production, apparently because of a lesion affecting DNA synthesis (L. Simon et al., Nature [London] 252:451-455). In the plasmolyzed cell system, reduced nucleotide incorporation is seen, indicating also that the genetic defect does not involve DNA precursor synthesis. The plasmolyzed cell system incorporates deoxynucleotide 5'-monophosphates into DNA severalfold more rapidly than the corresponding 5'-triphosphates. This is consistent with the idea that DNA precursor-synthesizing enzymes are functionally organized to shuttle substrates to their sites of utilization.

Coliphages↗
PubMed1977

Bacteriophage T4 virion dihydrofolate reductase: approaches to quantitation and assessment of function.

This paper is concerned with the physiological role(s) of T4 phage-coded dihydrofolate reductase, which functions both in DNA precursor metabolism and as a virion protein. (i) We have detected enzyme activity in noninfectious particles produced under restrictive conditions by gene 11 mutants. This supports the conclusion of Kozloff et al. (J. Virol. 16:1401-1408, 1975) that the protein lies in the baseplate, covered by the gene 11 protein. (ii) We have obtained further evidence for virion dihydrofolate reductase as the target for neutralizing activity of T4 dihydrofolate reductase antiserum and as a determinant of the heat lability of the virion. This derives from our observation that the reductases specified by T4B and T4D differ in several properties. (iii) We have investigated several anomalous properties of T4 mutants bearing deletions that reportedly extend into or through the frd gene, which codes for dihydrofolate reductase. Evidence is presented that the deletions in fact do not extend through frd. These strains direct the synthesis of material that cross-reacts with antiserum to homogeneous dihydrofolate reductase. Moreover, they are all quite sensitive to the phage-neutralizing effects of this antiserum. In addition, they are restricted by several of the hospital strains, wild-type strains of Escherichia coli supplied by the California Institute of Technology group. (iv) We have attempted to detect dihydrofolate reductase among early-synthesized proteins present in T4 tails. Two such proteins are seen, one of which is evidently the gene 25 product and one that is a bacterial protein. Quantitation of our electrophoretic technique has allowed determination of the number of molecules of some T4 tail components present per virion. (v) Finally, we have compared the T4 dihydrofolate reductase with the corresponding enzyme specified by two plasmids conferring resistance to trimethoprim (Skold and Widh, J. Biol. Chem. 249:4324-4325, 1974). Although the enzymes are similar in some properties, they differ in several important respects, including immunological activity.

Coliphages↗
PubMed1976

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.

Cell-Free System↗
PubMed1975

Bacteriophage-host interaction and restriction of nonglucosylated T6.

Nonglucosylated T6 phage (T6gtam 16am30, hereafter called T6alpha gt-) were found to have two structural anomalies when compared with wild-type T6. The DNA of T6alpha gt- phage contains single-strand interruptions. These can be seen both during infection, in the pool of replicating DNA, and in DNA extracted from purified phage. In addition, the sodium dodecyl sulfate-polyacrylamide gel pattern of T6alpha gt- phage structural proteins reveals a protein band not found in T6. The altered protein has a mobility slightly faster than that of the major head protein, and it is not removed by osmotic shock. The restriction activity of Escherichia coli B directed against T6alpha gt- phage is abolished by preinfection of the cells for 4 min with T4 im m2. The shut-off of restriction is observed either by the rescue of superinfecting T6alpha gt- or by the failure to detect degradation of incoming T6alpha gt- DNA. This effect is resistant to rifampin and chloramphenicol.

Carbon Radioisotopes↗
PubMed1974

Continued synthesis of bacterial DNA after infection by bacteriophage T4.

Early in infection by bacteriophage T4, before replication has commenced, one can detect the presence of newly synthesized DNA which cosediments with parental phage DNA on sucrose gradients. As shown earlier (R. E. Murray and C. K. Mathews, 1969), some of this represents covalent attachment of new material to parental phage DNA molecules. However, as shown herein, most of it is bacterial DNA, which is synthesized after infection and presumably degraded to T4 DNA-sized pieces. The small amount of phage-specific DNA synthesis which occurs is apparently a repair process, for its extent is greatly increased if the phage are irradiated with ultraviolet light prior to infection. Analysis by means of pulse labeling with [(3)H]thymidine and DNA-DNA hybridization shows that host DNA synthesis continues at a significant rate (40 to 80% of the preinfection rate) as late as 10 min after infection at 37 C. Very early in infection this is primarily replicative synthesis, but later a repair process predominates. Presumably this represents attempted repair of damage being inflicted on host DNA by phage-coded nucleases.

Coliphages↗
PubMed1974

Biochemistry of DNA-defective amber mutants of bacteriophage T4. IV. DNA synthesis in plasmolyzed cells.

Requirements for bacteriophage T4 DNA synthesis have been investigated in situ by use of plasmolyzed infected cells. When such cells are incubated with dATP, dGTP, dTTP, hydroxymethyldeoxycytidine triphosphate, and rATP, significant semiconservative synthesis of DNA occurs. This DNA hybridizes preferentially to T4 DNA. T4 amber mutants defective in genes 44 and 45, which display a DNA-negative phenotype in vivo, are unable to synthesize DNA in situ. By contrast, T4 amber mutants bearing lesions in genes 41 and 62, which also display a DNA-negative phenotype in vivo, do allow DNA synthesis in situ, the extent of synthesis being 80 to 90% that of the wild-type synthesis under the same conditions. Cells infected with gene 42 mutants (dCMP hydroxymethylase) are unable to synthesize DNA in situ even though exogenous nucleotides are provided. Also one gene 1 mutant (deoxynucleotide kinase) was found to synthesize DNA in situ, but two other gene 1 mutants did not. These results point to possible roles of hydroxymethylase and kinase in DNA metabolism, in addition to provision of essential DNA precursors, as has recently been suggested by Wovcha et al. (1973).

Adenosine Triphosphate↗
PubMed1973

Inactivation of T4D bacteriophage by antiserum against bacteriophage dihydrofolate reductase.

Antiserum was prepared against highly purified T4D bacteriophage-induced dihydrofolate reductase (DFR). This serum not only inactivated the enzyme but also inactivated all strains of T4D examined. T6 was inactivated to a lesser extent, and T2L, T2H, and T5 were unaffected by the antiserum. The phage-killing power of the serum could be blocked by prior incubation with partially purified T4D dfr obtained from host cells unable to make phage structural proteins. These observations confirm earlier results that the phage dfr is a structural component of the phage particle, and they offer new evidence on the manner in which this enzyme in incorporated into the tail structure.

Animals↗