Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes.
J Gen Virol 1986 Nov
PMID:The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans. 302 36

The thymidine kinase (TK) gene from herpes simplex virus type 1 strain SC16 was cloned into bacteriophage M13 mp8 so that functional HSV-1 TK was expressed in bacteria infected with the recombinant bacteriophage, M13/TK. Oligonucleotide site-directed mutagenesis was then employed to introduce single nucleotide changes into the TK gene in M13/TK in order to alter the codon for cysteine 171 in the wild-type enzyme to a codon specifying either serine or glycine. Analysis of the mutant enzymes in bacterial extracts showed that these substitutions had little effect on the activity of the enzyme, indicating that the side chain of this residue is not involved in nucleoside binding and is not essential for the catalytic activity of the enzyme.
J Gen Virol 1987 Jan
PMID:Analysis of the role of the cysteine 171 residue in the activity of herpes simplex virus type 1 thymidine kinase by oligonucleotide-directed mutagenesis. 302 47

Herpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.
J Gen Virol 1987 Feb
PMID:Restricted replication of herpes simplex virus type 1 in murine embryonal carcinoma cells. 302 90

Using both selection enrichment and site-directed mutagenesis, a herpes simplex virus type 1 (HSV-1) strain 17 genome lacking all four XbaI sites has been generated. The site at 0.45 map units which lies within the gene coding for a polypeptide of 28,000 molecular weight was removed by selection enrichment, while the site at 0.29 map units which lies within the gene coding for glycoprotein H was removed by site-directed mutagenesis. The parental virus from which these two XbaI sites were deleted had previously had the sites at 0.07 and 0.63 map units removed through selection enrichment. The variant devoid of XbaI sites (X4) showed normal growth characteristics; its phenotype was normal apart from the absence of the thymidine kinase protein, which is believed to be unrelated to the loss of XbaI sites.
J Gen Virol 1987 Apr
PMID:Generation of a herpes simplex virus type 1 variant devoid of XbaI sites. 303 32

We report the isolation of a variant (X2D) of herpes simplex virus type 1 strain 17 which has a deletion of 5 X 10(6) mol. wt. in the long unique and long inverted repeat regions, such that one copy of the immediate early (IE) gene 1 and two unique open reading frames coding for polypeptides of 20K and 22K are deleted. The mutant X2D synthesizes reduced levels of VmwIE110, and also apparently fails to synthesize VmwIE63, at both the protein and RNA levels, despite there being no apparent deletion in the coding or controlling regions of the IE2 gene. X2D also fails to synthesize the thymidine kinase polypeptide but exhibits normal growth characteristics in tissue culture.
J Gen Virol 1987 May
PMID:A herpes simplex virus type 1 variant which fails to synthesize immediate early polypeptide VmwIE63. 303 39

To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a "supersuppressor" selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HATr) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT- mutants - selected by their resistance to 6-thioguanine (TGr) - and TK- mutants - selected by their resistance to 5-bromodeoxyuridine (BrdUrdr) - are HATs. Therefore, from HPRT- TK- double nonsense mutants, whose phenotype would be TGr BrdUrdr (HATs), simultaneous HPRT+ TK+ double phenotypic revertants could be obtained by selecting HATr (TGs BrdUrds) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TGr variants, 53 of which were HATs and the rest HATr. Among 36 TGr (HATs) variants tested, 23 did not revert to HATr, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT- nonsense mutants because they were very stringent (had less than 2% of wild-type [3H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrdr) in 7 of these strains allowed us to isolate 29 TGr BrdUrdr (HATs) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HATr (TGs BrdUrds) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.
Mol Gen Genet 1988 Jul
PMID:Isolation and characterization of mammalian cell lines carrying suppressible mutations. 322 37

A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9 X 10(-5). Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK+ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.
Mol Gen Genet 1985
PMID:Transfer of mink genes into mouse cells by means of isolated lipid-encapsulated nuclei. 386 8

The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism. The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1985
PMID:Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila. 392 18

Various micro-organisms (131 strains of 73 species) were studied for their ability to produce thymidine kinase (TK; EC 2.7.1.21). Taking the specific TK activity of Escherichia coli K12 [specific activity of sonicated cell extracts 95-194 pmol min-1 (mg protein)-1] as 100%, the test organisms had the following relative specific TK activities. In the Gram-positive cocci, Staphylococcus aureus (21-84%) showed higher activity than Staph. epidermidis (1-20%) and Streptococcus (1-7%) except for one strain of Strep. pyogenes (29%). Neisseria sicca, a Gram-negative coccus, lacked TK. Gram-positive endospore-forming rods showed significant activity (Bacillus, 13-51%; Clostridium perfringens, 9-18%) except for one strain of B. megaterium (2%) and C. difficile (1-3%). Among the Gram-positive asporogenous rods, Listeria monocytogenes and six species of Lactobacillus (especially L. brevis, L. buchneri and L. casei) had moderate to high activity (23-348%) but L. acidophilus, L. bulgaricus, L. lactis and L. cellobiosus had low activity (0-8%). Of the species of Pseudomonas studied, most lacked TK but Ps. fluorescens and Ps. maltophilia had significant TK activity (15-53%). Of the Gram-negative facultative anaerobes, Vibrio lacked TK, while Enterobacteriaceae, including Salmonella (148-1120%), Escherichia (59-141%), Klebsiella (78-299%) and Serratia (61-110%), had a high activity. Proteus had a somewhat lower activity (0-34%) except for 'Pr. rettgerella' (307%). Propionibacterium and Bifidobacterium and related organisms other than Streptomyces, Nocardia, Rhodococcus, Corynebacterium and Mycobacterium lacked TK. The seven species of Candida tested, and Cryptococcus neoformans, essentially lacked TK.(ABSTRACT TRUNCATED AT 250 WORDS)
J Gen Microbiol 1985 Nov
PMID:Further studies on thymidine kinase: distribution pattern of the enzyme in bacteria. 409 63

Purified herpes simplex virus thymidine kinase has been used to immunize mice for the production of monoclonal antibodies to the enzyme. Monoclonal antibodies were successfully produced against both herpes simplex virus type 1 and type 2 enzymes. These antibodies should prove useful for detecting the enzyme under a variety of experimental conditions. We also demonstrate that the antibodies can provide an alternative method for obtaining large amounts of purified thymidine kinase.
J Gen Virol 1984 Sep
PMID:Monoclonal antibodies to herpes simplex virus thymidine kinase. 608 85


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