Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase

Dihydrofolate reductase plays a dual role in bacteriophage T4, first, as an enzyme of thymidylate metabolism, and second, as a protein component of the tail baseplate. Antibody to the purified enzyme has been used to study its synthesis and intracellular turnover. The antibody specifically precipitates one protein from T4D-infected cell extracts. This has been identified as dihydrofolate reductase, although the polypeptide molecular weight (22,000) is lower than that earlier determined for this enzyme. The protein comigrates on gels with pY, a genetically undefined protein component of the baseplate. However, it is not pY, for pY is synthesized late in infection, whereas virtually no dihydrofolate reductase synthesis occurs later than 10 min after infection at 37°C. Dihydrofolate reductase, once formed, is neither degraded nor converted to proteins of higher or lower molecular weight. Thus, it is probably incorporated into virions at the same molecular weight as that of the soluble enzyme. 125I-radiolabeled antibody binds to the wedge substructure of the baseplate, and this binding is blocked by preincubation with purified T4 dihydrofolate reductase. Thus, the enzyme protein seems to be a component of the wedge. tect the reductase protein as a component of the wedge subassembly of the baseplate. (These results were taken from a Ph.D. thesis presented by R.A.M. to the University of Arizona.) MATERIALS AND METHODS Cell and bacteriophage 8trains. The wild-type phage strain T4D has been maintained in this laboratory for some time, as have Escherichia coli strains 94

Section: Fig 3 Specificity Of the Double Antibody Precipi-supporting

confidence: 71%

mentioning

confidence: 70%

See 1 more Smart Citation

Bacteriophage T4-coded dihydrofolate reductase: synthesis, turnover, and location of the virion protein

Mosher¹,

Mathews²

1979

“…Note also that two point mutants in the frd gene also form this cross-reacting material. One of these, frdll, is an amber mutant, but, evidently, it synthesizes an incomplete polypeptide nearly as long as the wild-type protein (17). These observations involve us in the same apparent paradox as do our similar experiments with thymidylate synthetase antiserum: mapping data indicate the presence of long 4.…”

Section: 8mentioning

confidence: 51%

Bacteriophage T4 Virion Dihydrofolate Reductase: Approaches to Quantitation and Assessment of Function

Mosher

DiRenzo

Mathews

1977

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 ofthe 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 ofEscherichia 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.

“…This paper gives the detailed properties of these two mutants and presents additional evi-dence that the td gene product is a tail plate component adjacent to the dfr in the tail plate. A tentative scheme for the structural relationships of the tail plate folate (14), dfr (12), and td is proposed.…”

mentioning

confidence: 99%

Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase

Kozloff¹,

Crosby²,

Lute³

1975

Self Cite

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrlstdls and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.