Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

Capco, Gloria R.; Mathews, Christopher K.

doi:10.1016/0003-9861(73)90568-7

Cited by 30 publications

(20 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, in addition to its catalytic role the synthase molecule forms a structural component of the phage base plate (28,29). In this regard, the splicing event may provide a means for differentially regulating synthesis of the catalytically active and structural components of thymidylate synthase.…”

Section: Resultsmentioning

confidence: 99%

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

Chu¹,

Maley²,

Maley³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

178

View full text Add to dashboard Cite

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption two-thirds of the way through the nucleotide sequence of the structural gene. The 5'-and 3'-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the second coding region of the synthase is not incorporated into the final protein product.

show abstract

Section: Resultsmentioning

confidence: 99%

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

Chu¹,

Maley²,

Maley³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

178

View full text Add to dashboard Cite

show abstract

“…In this paper we describe several approaches to directly demonstrating and quantitating virion dihydrofolate reductase activity and to evaluating the role of this enzyme as a virion protein. The accompanying paper (18) from Kozloff's laboratory presents complementary data, both for dihydrofolate reductase and for thymidylate synthetase, another early enzyme that has been implicated as a virion protein (6,16).…”

mentioning

confidence: 99%

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

Mosher

DiRenzo

Mathews

1977

J Virol

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Immunological methods allow us to design experiments which focus upon the synthesis, turnover, and fate of specific protein entities. The antiserum used in our earlier work (2,24) had been used up. Accordingly, we prepared new antiserum against purified T4 DFR and used this for electrophoretic analysis of labeled proteins in immunoprecipitates.…”

Section: Resultsmentioning

confidence: 99%

“…In track 3 the anti-DFR serum was incubated with an extract of cells infected by T4 frdll, an amber mutant (6). The band corresponding to the enzyme is not seen, but there is a faint band, of lower molecular weight (marked with dots), which may represent a DFR amber peptide, since it seems clear that this mutant does make cross-reacting material (2,18,26). Tracks 4 and 5 represent comparable experiments carried out with T4 del(63-32)7 and del(63-32)9, respectively.…”

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

confidence: 99%

See 1 more Smart Citation

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

Mosher¹,

Mathews²

1979

J Virol

View full text Add to dashboard Cite

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

show abstract

Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

Cited by 30 publications

References 18 publications

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

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

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

Contact Info

Product

Resources

About