Bacteriophage-coded thymidylate synthetase: Characteristics of the T4 and T5 enzymes

Capco, Gloria R.; Krupp, Jan R.; Mathews, Christopher K.

doi:10.1016/0003-9861(73)90567-5

Cited by 25 publications

(8 citation statements)

References 20 publications

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“…Growth media and procedures for growth and infection of cells, preparation of cell-free extracts, purification of phage, assay for enzymes, and immunodiffusion have been described in previous publications from this laboratory (5,6,21,(24)(25)(26)(27).…”

Section: Methodsmentioning

confidence: 99%

“…itself migrates at an anomalously slow rate in ias a molecular our system [R. A. Mosher, unpublished reymidylate synthe-sults]). In an effort to relate the 40,000-dalton Llton subunits (5). band or the smaller polypeptide to precursor or cted in the corre-cleaved forms of the td or frd gene product, we , which is in the crossed the gene 23 mutation into dell, del7, molecular weights, del9, frdll, and amN54.…”

Section: 8mentioning

confidence: 99%

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

Mosher

DiRenzo

Mathews

1977

J Virol

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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.

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Section: Methodsmentioning

confidence: 99%

Section: 8mentioning

confidence: 99%

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

Mosher

DiRenzo

Mathews

1977

J Virol

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“…Standard procedures were used for plasmid preparation, restriction enzyme digestions, ligations, transformations, and agarose gel electrophoresis. The T4 thymidylate synthase gene (TD gene) was amplified from the T4 phage by PCR (1,878 bp), and then TD⌬I was prepared with deletion of the intron (550 bp to 1,566 bp region) by a gap deletion method for obtaining an active enzyme, since the T4 TD gene is transcribed as preform mRNA and then the intron is deleted by self-splicing (4,8,12). The T4 nrdC gene and the T4 nrdA-nrdB operon were amplified by PCR with the flanking primers including 6-nt restriction site extensions for easy cloning and ligated to each other with a ribosome binding site to construct an artificial operon (nrdC-nrdAnrdB).…”

Section: Vol 75 2009 Production Of Thymidine By Recombinant E Colimentioning

confidence: 99%

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Lee¹,

Kim²,

Kim³

et al. 2009

Appl Environ Microbiol

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Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter ؊1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA Nglycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ϳ1.2-fold (740.3 mg liter ؊1 ). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

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“…[E or QI d: lack of immunity to superinfection (42,197 (47) 18,000 13729,000 6846,000 9540,000 9610,000-o8,900 (196) 11,700-o10,000 (196) 21,200-o18,300 (196) 14733,000 14710,000 9629,000 (28) 10,500 (127) 53,700 (196) [psu+SB] The following general articles, not referred to in the table, are useful sources of additional information and references as follows: for the original assignment of essential gene functions (67); for early functions of phage-coded enzymes (128,129); for the original classification of gene functions in assembly of phage particles using in vitro complementation (63,219); for recent work on phage assembly (29,64,76,171,212,221); for gene functions in general (26, 34, 58, 136, 140, 155a, 178).…”

Section: Gene Classes and Gene Namesmentioning

confidence: 99%

The genome of bacteriophage T4.

Wood¹,

Revel²

1976

Bacteriological Reviews

123

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Bacteriophage-coded thymidylate synthetase: Characteristics of the T4 and T5 enzymes

Cited by 25 publications

References 20 publications

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

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

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

The genome of bacteriophage T4.

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