Thymidine mutants

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). 

Stock cultures are established from frozen ampoules of cells that have been treated with thymidine, hypoxanthine, methotrexate and glycine for 24 h, which purges the culture of pre-existing TK / mutants. This cell stock is used for a maximum of two months. 

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. 

Kandell and Bernstein published one of the earliest reports to suggest that bile acids also demonstrate DNA-damaging effects in eukaryotic cells. They showed that human foreskin fibroblasts underwent unscheduled DNA synthesis (indicating DNA repair), as measured by tritiated thymidine incorporation when cells were treated with increasing concentrations of sodium deoxycholate or chenodeoxycholate. Utilising mutant Chinese hamster ovary cells deficient in strand rejoining (EM9), the authors were able to demonstrate that the repair of deoxycholate-induced DNA damage was dependent on strand break repair capacity. 

A similar experiment but with thymidine and adenine exchanging roles was devised by Ozaki and co-workers to analyze mixtures of shorter oligonucleotides with high resolution, supporting the notion that hydrogen bonding can be considered the key factor for separation (45). The pivotal role of Mg+ ions supports that view. As an example, single mutants in poly-... 

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. 

A mutant strain of Escherichia coli Y-10, defective in L-rhamnose synthesis, accumulates109 a considerable amount of thymidine diphosphate sugars. Three of them were identified110 as the esters of thymidine 5 -pyrophosphate with 6-deoxy-a-D-xyZo-hexopyranos-4-ulose (28), 6-deoxy-D-glucose, and D-fucose. The fourth component was found to be a 4-acetamido-4,6-dideoxy-D-galactose derivative111,112 (29) it occurs also in Pasteurella pseudotuberculosis102 and its... 

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). 

Conversely, HPRT4 cells can be selected for in the presence of hypoxanthine-aminopterin-thymidine medium. It should be possible to select for HPRT cells in normal HPRT+ persons with one of the analogues, and this was one of the first systems used to detect spontaneously occurring mutants in freshly cultured cells from humans.88 388... 

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... 

Autoradiographic analysis is the only method which can determine the proportion of cells incorporating a radioactive precursor and the site of that incorporation. Thus, tritiated thymidine is incorporated into DNA in the nuclei of those cells in S-phase and tritiated hypoxanthine appears first in the nucleus and later in the cytoplasm of cells with HPRT but not in mutants lacking the enzyme (see 13.2). 

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... 

Black, M. E., and Loeb, L. A. (1993). Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection analysis of selected mutants in vitro. Biochemistry, 32, 11618—11626. 

Fig. 2. Mutagenesis strategy. A neo gene replaced one-third of the ORF and was used as a positive selectable marker. The HSV thymidine gene (HSV-ffc) was used for negative selection. The vector was linearized with Xbal digestion. The targeted events were screened by Southern blot. Two Xbal sites are in the outside of the targeting vector (the 3 -end one is just at the end of the vector, which cannot be digested by Xbal if the vector is randomly inserted). An outside probe is located at the 5 -end. The genotype of the mice can be easily identified based on the size of the hybridized bands, 6.7 kb (mutant) vs 6 kb (wild-type).

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. 

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

CD-I mice were exposed to purified air or benzene by inhalation at 0.04, 0.1, or 1.0 ppm for 22 hours per day, 7 days per week for 6 weeks (Ward et al. 1992). The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period, lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The increased frequencies of mutant spleen lymphocytes were significant at the low and mid, but not the high dose, and the method does not take into account possible clonal expansion. Further evaluation of the induction of gene mutation at these dose levels seems warranted. 

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-... 

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