Xanthine-guanine Phosphoribosyltransferase Research Articles

Novel Targets Against Helicobacter Pylori : A Bioinformatic Approach

Evaluation of: Duckworth M, Menard A, Megraud F, Mendz GL: Bioinformatic analysis of Helicobacter pylori XGPRTase: a potential therapeutic target.Helicobacter 11, 287–295 (2006). Helicobacter pylori is a globally spread pathogen, with roughly 50% of the human population being contaminated. It causes severe gastrointestinal diseases, such as chronic gastritis, peptic ulcers and gastric cancer, which lead to significant morbidity and mortality. Much effort is dedicated by scientists to design alternative strategies to treat this infection, due to the widespread emergence of resistance to the presently used pharmacological agents. The xanthine–guanine phosphoribosyltransferase (XGPRTase) enzyme of H. pylori may be one of these targets, as the enzyme was recently purified, characterized and shown to be essential to the life cycle of the parasite. Duckworth and colleagues used a bioinformatic approach to investigate this target. The parasite enzyme has been compared with those present in various bacteria, archaea and eukaryotes, and its main features have been deduced by using conserved domain analysis, multiple sequence alignment and phylognetic analysis, as well as protein 3D modeling. However, significant findings did not emerge after this work for the design of XGPRTase inhibitors.

Purification of human malaria parasite hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) using immobilized Reactive Red 120

Malaria is caused by Plasmodium parasite infection. The human malarial parasite does not have a de novo pathway for synthesis of nucleotides and the purine salvage pathway enzyme hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) is critical for survival. In our efforts to find inhibitors of the malarial parasite HGXPRT, we have developed a simple but effective purification protocol for this protein expressed in Escherichia coli without an affinity tag. The protocol consists of tandem columns of anion exchange and immobilized Reactive Red 120 resins. The enzyme is inactive as isolated but can be activated by incubation with substrate(s).

Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.

A Comparative QM/MM Simulation Study of the Reaction Mechanisms of Human and Plasmodium falciparum HG(X)PRTases

QM/MM hybrid potential free-energy simulations are performed to compare the reaction mechanisms of human hypoxanthine guanine phosphoribosyl transferase (HGPRTase) and the corresponding enyzme from Plasmodium falciparum (Pf), hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRTase). These enzymes share 44% of sequence identity but display very different affinities for xanthine. The calculations show that in both enzymes phosphoribosyl transfer proceeds via a dissociative mechanism from an anionic form of the substrate. Nevertheless, there are significant differences in the geometries of critical structures along the reaction paths which it may be possible to exploit for the design of specific inhibitors against the Pf enzyme.

Bioinformatic Analysis of Helicobacter pylori XGPRTase: A Potential Therapeutic Target

Xanthine-guanine phosphoribosyltransferase (XGPRTase) is an enzyme of purine nucleotide salvage synthesis. The gpt gene of Helicobacter pylori has been annotated as encoding an XGPRTase and proposed as essential for survival of the bacterium in vitro. The aims of this work were to investigate the structure of H. pylori XGPRTase and to compare the key features of the enzyme to other phosphoribosyltransferases employing computational, modelling, and bioinformatic tools. XGPRTase activity was measured in the cytosolic fraction of H. pylori by (31)P-nuclear magnetic resonance spectroscopy, and also in recombinant XGPRTase produced by a cell-free expression system. Bioinformatics was employed to analyze the phylogeny of XGPRTase, and a structural model of the XGPRTase was built using threading techniques. The observed interactions of purine phosphoribosyltransferases with immucillin-GP were used to study the theoretical interactions of H. pylori XGPRTase with this transition-state analog. It was demonstrated that the gpt gene of H. pylori encodes a functional XGPRTase enzyme. Analyses of the XGPRTase sequence showed that the enzyme is significantly divergent from equivalent mammalian enzymes. Modelling served to identify specific features of the enzyme and key residues involved in catalysis. The H. pylori XGPRTase is structurally similar to other phosphoribosyltransferase enzymes, but there were significant differences between the hood domain of H. pylori XGPRTase and other purine salvage phosphoribosyltransferases. Significant differences were found between the interactions of the H. pylori and human enzymes with a purine phosphoribosyltransferase inhibitor.

Construction and isolation of recombinant vaccinia virus using genetic markers.

The standard approach for the isolation of vaccinia virus recombinants involves homologous recombination between a transfected plasmid and the replicating viral DNA. In a typical infection/transfection experiment, recombinant viruses only account for a tiny proportion (10-4 to 10-3) of the progeny virus; thus, genetic markers are often included in the transfected plasmid to facilitate the selection of recombinant viruses. This chapter describes in detail two different selection procedures: one relies on plaque formation phenotype using the vaccinia virus gene F13L; the other relies on antibiotic resistance using the Escherichia coli xanthine-guanine phosphoribosyl transferase gene.

Engineered delta ribozymes can simultaneously knock down the expression of the genes encoding uracil phosphoribosyltransferase and hypoxanthine–xanthine–guanine phosphoribosyltransferase in Toxoplasma gondii

Engineered delta ribozymes or HDV ribozymes were used as gene expression modulators in Toxoplasma gondii. The substrate recognition sequence of the trans-acting delta ribozyme, which was derived from the self-cleaving motif located on the antigenomic strand of the hepatitis delta virus genome, was modified to target T. gondii transcripts. The mRNA encoding two well-documented genes, namely uracil phosphoribosyltransferase ( UPRT) and hypoxanthine–xanthine–guanine-phosphoribosyltransferase ( HXGPRT) of T. gondii were chosen as the targets for the ribozymes. UPRT and HXGPRT are the operative enzymes of the pyrimidine and purine salvage pathway, respectively. The knockdown of UPRT or HXGPRT expression by the engineered ribozymes resulted in parasites with lower levels of the corresponding transcripts and diminished their abilities to assimilate radioactive pyrimidine or purine analogs. Five out of six engineered ribozymes could cleave their substrates and gave rise to the products, which were detected by primer extension assays. Upon electroporation of individual active ribozymes against the UPRT gene, the uracil incorporation was decreased. Similarly, the ribozymes against HXGPRT caused decreased incorporation of hypoxanthine. When the most active ribozyme against UPRT was used in a combination with the best HXGPRT specific ribozyme, the incorporation of both uracil and hypoxanthine were decreased. Northern blot analysis revealed that the target transcripts were lowered to an undetectable level when specific ribozymes were used, and that the transcripts of the housekeeping gene remained intact. The ribozyme system should thus prove to be effective for the study of gene expression in T. gondii.

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes.

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

The purine salvage enzyme hypoxanthine guanine xanthine phosphoribosyl transferase is a major target antigen for cell-mediated immunity to malaria.

Although there is good evidence that immunity to the blood stages of malaria parasites can be mediated by different effector components of the adaptive immune system, target antigens for a principal component, effector CD4(+) T cells, have never been defined. We generated CD4(+) T cell lines to fractions of native antigens from the blood stages of the rodent parasite, Plasmodium yoelii, and identified fraction-specific T cells that had a Th1 phenotype (producing IL-2, IFN-gamma, and tumor necrosis factor-alpha, but not IL-4, after antigenic stimulation). These T cells could inhibit parasite growth in recipient severe combined immunodeficient mice. N-terminal sequencing of the fraction showed identity with hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT). Recombinant HGXPRT from the human malaria parasite, Plasmodium falciparum, activated the T cells in vitro, and immunization of normal mice with recombinant HGXPRT reduced parasite growth rates in all mice after challenge.

A point mutation at the subunit interface of hypoxanthine–guanine–xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis

Hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Plasmodium falciparum catalyzes the phosphoribosylation of hypoxanthine, guanine and xanthine. The functionally active form of HGXPRT is a tetramer but interface residues do not contribute to catalysis. Here we report the characterization of an interface mutant Y96C, which has a decreased k cat, an increase in the K m for phosphoribosyl pyrophosphate (PRPP) and no change in K m for the purine bases when compared to the wild type enzyme. The mutant enzyme does not tetramerize in the presence of PRPP, unlike the wild type in which the tetramer is stabilized by PRPP. This is the first report of a HGXPRT mutation, at a unique interface where non-adjacent subunits interact, that impairs catalysis.

Evidence for Multiple Active States of Plasmodium falciparum Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferase

The lack of de novo purine biosynthesis in the malaria parasite Plasmodium falciparum makes the purine salvage enzyme hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) a drug target. However, high activities for the purified recombinant enzyme have not been achieved, indicating that the P. falciparum enzyme requires very precise conditions for its maximal activity. In this report we have standardized the activation conditions necessary for high levels of activity, which is critically dependent on the ratios of enzyme, phosphoribosylpyrophosphate (PRPP), hypoxanthine, and buffer conditions. We demonstrate that excess substrates will push the enzyme to a less active state. We also present evidence for the existence of different kinetic states of the enzyme during activation and storage. Our kinetic data show that hypoxanthine is the substrate with highest affinity for the enzyme with a Km well below 1 μM. The activated enzyme has a maximum activity of 8.370 μmol/min/mg for hypoxanthine which is 10.8 times more than the previous reports. We discuss the biological relevance and implications of these results on drug design efforts.

Correlation between secreted and membrane-bound IgG in mouse myeloma cells transfected with chimeric immunoglobulin heavy and light chain genes

Mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt) and the neomycin (neo) selection marker genes. A broad distribution in the level of mouse-human chimeric IgG expression was observed with series of independently isolated transfectoma clones. The relative amounts of secreted to membrane-bound antibodies correlated closely, which suggested, that fluorescence-activated cell sorting could be a valuable tool for the selection of high-yielding production cell lines. However, a single cycle of cell sorting did not steer the cloning process significantly toward cells that produce enhanced amounts of recombinant IgG. Only in cases in which the polyclonal transfectoma population contained a large percentage of nonproducing cells, these were successfully separated from the IgG-producing cell population. (c) 1996 John Wiley & Sons, Inc.

Purification and characterization of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase and comparison with the human enzyme

The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229±2 Da is in excellent agreement with the calculated value of 26 232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. falciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KCl. Kinetic constants were determined for 5-phospho- α- d-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine–guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design.

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen.

Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.

Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 Å resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 Å resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5′-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 Å resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by ∼24° compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.

Preferential completion of human immunodeficiency virus type 1 proviruses initiated with tRNA3Lys rather than tRNA1,2Lys.

All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1Lys, tRNA2Lys, and tRNA3Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3Lys, tRNA1Lys and tRNA2Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3Lys, not tRNA1Lys or tRNA2Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3Lys from proviral DNA isolated from gpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2Lys. The results of these studies suggest that the selection of tRNA3Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.

Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule.

Development of a novel CHL/IU cell line with an incorporated gpt shuttle vector for concurrent analysis of gene mutations and chromosome aberrations

A cosmid shuttle vector containing the target gene of Escherichia coli gpt coding xanthine-guanine phosphoribosyl transferase was constructed. The shuttle vector was designed to be rescued into the gpt-deficient Escherichia coli from Chinese hamster CHL/IU cells through an in vitro packaging method. Mutations occurred at the target gene can be detected with a selective agent, 6-thioguanine (6-TG). The shuttle vector was stably transfected into CHL/IU cells to give several cell lines containing copies of the shuttle vector in the chromosomes. Each cell line exhibited a characteristic rescue efficiency (0 to 1.9×10 5 CFU/μg of genomic DNA) of the shuttle vector and spontaneous mutation frequency (3.9×10 −5 to over 10 −2) at the 6-TG selection. One transgenic cell line (KN63), which showed a higher rescue efficiency and a low spontaneous mutation frequency, was selected and tested for the ability to respond to a genotoxic agent, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). MNNG increased both the mutation frequency at the target gene and the number of the cells with chromosome aberrations. DNA sequence analysis of 6-TG mutants showed that predominant mutations (10/14) were identified as G:C to A:T transitions in MNNG-induced mutants, whereas tranversions were predominant (5/9) in spontaneous mutants. These results suggest that this transgenic CHL/IU cell line can be a useful tool for analyzing the relation between gene mutations and chromosome aberrations.

Expression of a bacterial endo (1-4)- β-glucanase gene in mammalian cells and post translational modification of the gene product

An endo (1-4)- β-glucanase gene C6.5 from Bacillus subtilis has been expressed in Chinese hamster ovary (CHO) cells and pancreatic 266-6 cells. The fusion gene, stably transfected into CHO cells consisted of the mouse Amy-2.2 signal peptide coding sequence and the endoglucanase gene C6.5 transcribed from the early SV40 promoter/enhancer, using the dihydrofolate reductase gene as a selective marker. The gene construct transfected into pancreatic 266-6 cells consisted of the mouse Amy-2.2 promoter/enhancer and signal peptide coding sequence and the same C6.5 sequences using the xanthine-guanine phosphoribosyl transferase gene (gpt) as the selective marker. The stably transfected CHO cells synthesized endoglucanase at 1.1 U/mg cell protein in a 72 h culture, with 89% of the activity secreted into the culture fluid in a glycosylated form of 66 kDa as compared with the unglycosylated 53 kDa form expressed in E. coli. Glycosylation did not change the specific activity, protease resistance, or cellulose binding of the endoglucanase as compared to the unglycosylated form of the enzyme from E. coli. The level of expression in the stably transfected pancreatic cells was substantially lower at 0.3 mU/mg cell protein with all detectable activity present in the culture fluid. The secreted enzyme from pancreatic cells was glycosylated with a mass similar to that secreted from CHO cells.

Polymerase chain reaction-based deletion analysis of spontaneous and arsenite-enhanced gpt mutants in CHO-AS 52 cells

In this study, we have examined the mutagenicity of sodium arsenite at the xanthine-guanine phosphoribosyltransferase locus ( ypt) in a pSV2 gpt-transformed CHO cell line, AS52. Our results provide very weak evidence for arsenite as a gene mutagen because the chemical at high doses and at high cytotoxicity enhances barely a doubling of mutant frequency (MF) and a doubling of the gpt gene deletion frequency compared to controls. We suggest that the increase in MF in arsenite-treated cells results from arsenic, as comutagen, enhancing the induction effect of any unknown endogenous or exogenous factors on the spontaneous mutagenesis of AS52 cells. Nested PCR analysis mutants has a total deletion of the gpt gene. For the spontaneous, 50 μM arsenite- and 100 μM arsenite-enhanced spontaneous mutants in AS52 cells, the percentages of total deletion of the gpt gene are 36.00%, 54.72% and 66.67%, respectively. We suggest that a high proportion of the gene deletion in arsenite-enhanced mutants may be due to the high cytotoxicity of the chemical.