Thymidine kinase mutant

Black, M. E., Newcomb, T. G., Wilson, H. M., and Loeb, L. A. (1996). Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc. Natl. Acad. Sci. USA, 93, 3525-3529. 

Munir KM, French DC, Loeb LA (1993) Thymidine kinase mutants obtained by random sequence selection. Proc Natl Acad Sci USA 90 4012-4016... 

Black ME, Kokoris MS, Sabo P. Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-medialed tumor cell killing. Cancer Res 2001 61(7) 3022-3026. 

BVdU is degraded by thymidine phosphorylase more rapidly than the natural substrate, thymidine. This rapid enzymic degradation may present a problem in its clinical use. Moreover, herpes vimses develop resistance to BVdU, apparendy because of mutant vimses that have lower thymidine kinase activity. G. D. Seade has dropped further development of BVdU because of increased animal tumor incidence induced by prolonged dosing (1). 

Cifone, M.A., Myhr, B., Eiche, A. and Bolisfodi, G. (1987). Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/— cells. Mutation Res. 189 39-46. 

In HSV and VZV, the most common mechanism of resistance to acyclovir involves mutations that result in decreased thymidine kinase activity. Therefore, these viral mutants exhibit cross-resistance to other antiviral agents that require thymidine kinase activation, such... 

Resistance to foscamet may result from mutation of viral DNA polymerase. Because this drug does not require phosphorylation for activation, thymidine kinase-deficient mutants should not be resistant to fos-carnet. 

Famciclovir is the diacetyl ester prodrug of 6-deoxypencidovir, an acyclic guanosine analog (Figure 49-2). After oral administration, famciclovir is rapidly deacetylated and oxidized by first-pass metabolism to penciclovir. It is active in vitro against HSV-1, HSV-2, VZV, EBV, and HBV. As with acyclovir, activation by phosphorylation is catalyzed by the virus-specified thymidine kinase in infected cells, followed by competitive inhibition of the viral DNA polymerase to block DNA synthesis. Unlike acyclovir, however, penciclovir does not cause chain termination. Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient these are cross-resistant to acyclovir and famciclovir. 

Puhlmann M, Brown CK, Gnant M, Huang J, et al. 2000. Vaccinia as a vector for tumor directed gene therapy Biodistribution of a thymidine kinase-deleted mutant. Cancer Gene Ther. 7 66-73. 

Penciclovir triphosphate has lower affinity for the viral DNA polymerase than acyclovir triphosphate, but it achieves higher intracellular concentrations and has a more prolonged intracellular effect in experimental systems. The most commonly encountered clinical mutants of HSV are thymidine kinase-deficient and are cross-resistant to acyclovir and famciclovir. 

Saada, A., Shaag, A., Mandel, H., Nevo, Y.,Eriksson, S., andElpeleg, O., Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat. Genet. 29, 342—344 (2001). 

THYMIDINE KINASE (TK) An enzyme involved in the utilization of the nucleoside thymidine (which ultimately becomes part of the structure of DNA) catalyzes the phosphorylation of thymidine to thymidine monophosphate mutants that lack TK are resistant to the toxic effects of several thymidine analogues, including bromodeoxy-uridine and trifluorothymidine selection of these drug-resistant mutants provides the basis of several... 

Mutants lacking, or with much reduced levels of the enzyme thymidine kinase survive as this enyzme is not essential. Thus the cell can make dTMP from dUMP using folic acid as the one carbon donor (Fig. 13.1). 

By growing cells in the presence of increasing concentrations of aminopterin a number of resistant cells lines have been isolated (Hakala and Ishihara, 1962 Littlefield, 1969). These have been characterised as having either an altered permeability to the drug or an altered folate reductase or an increased rate of synthesis and hence increased amounts of the enzyme (Alt et al., 1976), resulting, at least in part, from a selective amplification of the dihydrofolate reductase gene (Alt et al., 1978 Schimke et al., 1988). The problem is considered in more detail in 11.8.1. The importance of the antifolates lies in their role in the HAT selection technique ( 13.5) devised by Szybalski (1962) (see also Szybalski et al., 1962 and Littlefield, 1964) for the isolation of hybrids between mutant cells defective on the one hand in thymidine kinase and on the other hand... 

The MLA is used for the detection of point mutations, structural aberrations and aneugenicity. The principle of the assay is that cells deficient in thymidine kinase (TK) due to the tk+/" or tk/ mutation are resistant to the cytotoxic effects of the pyrimidine analogue trifluoro-thymidine (TFT). TK proficient cells are sensitive to TFT. This influences the cellular metabolism and leads finally to an inhibition of further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain TK, are not. The major advantage of the assay is its ability to detect a broad range of mutagenic events represented by optimal detection of both large and small colonies. 

In order to determine the relationship between protein structure and function and to create mutant enzymes with altered properties useful for biotechnology and cancer therapy, a directed evolution approach has been explored and novel proteins developed for Pol I DNA polymerase enzymes thymidylate synthase, thymidine kinase and 06-alkylguanine-DNA alkyltransferase. In every case the creation of a large variety of altered proteins has been achieved, and the emerging picture is that even highly conserved proteins can tolerate wide-spread amino acid changes at the active site with-... 

HAT medium. Mutant cells unable to synthesize nucleotides by salvage pathways are very useful tools in molecular and cell biology. Suppose that cell A lacks thymidine kinase, the enzyme catalyzing the phosphorylation of thymidine to thymidylate, and that cell B lacks hypoxanthine-guanine phosphoribosyl transferase. 

Aciclovir is active against Herpes simplex virus type 1 (HSV-1), HSV-2, Varicella zoster virus (VZV), Herpesvirus simiae, and to a lesser degree Epstein-Barr virus (EBV). Resistant strains of HSV can arise owing to the emergence of thymidine kinase-deficient mutants. Other forms of resistance patterns are less common (2,3). 

The two most popular myeloma cell lines used for this purpose are those lacking hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK). Other kinds of mutants can be used as well, such as those resistant to ouabain (in combination with HGPRT- Baker et al., 1974). Myeloma cells treated with biochemical inhibitors can also be rescued by fusion with untreated lymphocytes (Wright, 1978). 

Fig. 5.1. Two pathways in normal cells to synthesize DNA precursors. The mutant myeloma cell line, which has the capacity to grow indeflnitely in vitro and can confer this property on antibody-producing lymphocytes through cell fusion, lacks the salvage pathway (no thymidine kinase (TK) or hypoxanthine-guanine phosphori-bosyl transferase (HGPRT)). This cell line would, therefore, not grow in the presence of folic acid antagonists. Fused cells, however, may grow since the antibody-producing cells contribute the salvage pathway.

B. Moss, Vaccinia vims expression vector a new tool for immunologists, Immunology Today, 6, 1985, 243-5 J. A. McCart et al., Systemic cancer therapy with a tumor-selective vaccinia vims mutant lacking thymidine kinase and vaccinia growth factor, Cancer Research, 61, 2001, 8751-7. 

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