The RHD gene is highly detectable in RhD-negative Japanese donors.

Recent molecular studies on the Rh blood group system have shown that the Rh locus of each haploid RhD-positive chromosome is composed of two structural genes: RHD and RHCE, whereas the locus is made of a single gene (RHCE) on each haploid RhD-negative chromosome. We analyzed the presence or absence of the RHD gene in 130 Japanese RhD-negative donors using the PCR method. The RhD-negative phenotypes consisted of 34 ccEe, 27 ccee, 17 ccEE, 26 Ccee, 19 CcEe, 1 CcEE, and 6 CCee. Among them, 36 (27.7%) donors demonstrated the presence of the RHD gene. Others showed gross or partial deletions of the RHD gene. These results were confirmed by Southern blot analysis. Additionally, the RHD gene detected in the RhD-negative donors seemed to be intact through sequencing of the RhD polypeptide cDNA and the promoter region of RHD gene. The phenotypes of these donors with the RHD gene were CC or Cc, but not cc. It suggested that there is some relationship between the RHD gene and the RhC phenotypes in RhD-negative individuals. In Caucasian RhD-negative individuals, the RHD gene has not been found outside of the report of Hyland et al. (Hyland, C.A., L.C. Wolter, and A. Saul. 1994. Blood. 84:321-324). The discrepant data on the RHD gene in RhD-negative donors between Japanese and Caucasians appear to be derived from the difference of the frequency of RhD-negative and RhC-positive phenotypes. Careful attention is necessary for clinicians in applying RhD genotyping to clinical medicine.

A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.

Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1).

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Species difference in liver microsomal and cytosolic enzymes involved in mutagenic activation of N-hydroxy-N-2-fluorenylacetamide.

The mutagenic activations of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by subcellular fractions of the livers of the Sprague-Dawley rat, C57BL mouse, Hartley guinea pig, Syrian golden hamster, and Macaca fuscata fuscata monkey were examined for sensitivity to paraoxon, which inhibits a deacetylase but not an arylhydroxamic acid acyltransferase. The mutagenic activation by liver microsomes was almost entirely mediated by a paraoxon-sensitive enzyme in all the animals tested. In contrast, the mutagenic activation by liver cytosol was mediated mostly by a paraoxon-sensitive enzyme in mice and guinea pigs, mostly by a paraoxon-resistant enzyme in rats and hamsters, and by both enzyme types in the monkey. In rats and guinea pigs, attempts were made to identify the enzymes causing mutagenic activation in the liver cytosol by a comparison of the elution positions of these enzymes in gel filtration and DEAE-cellulose column chromatography with those of known enzymes. In the rat liver cytosol, the mutagenic activation was mediated not only by acyltransferase but also by an unknown enzyme, which was resistant to paraoxon and differed from acyltransferase in chromatographic behavior. In the guinea pig liver cytosol, the activation was due to deacetylase activities, which could be separated into four fractions by gel filtration. These data indicate that species differ in the kinds of liver cytosolic enzymes involved in mutagenic activation of N-OH-2-FAA but not in the kind of liver microsomal enzyme involved.

Unusual sensitivity to bleomycin and joint resistance to 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine of mouse FM3A cell mutants with altered ribonucleotide reductase and thymidylate synthase.

This paper describes unusual sensitivity to bleomycin; resistance to aphidicolin, 9-beta-D-arabinofuranosyladenine, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea; and a mutator phenotype as common phenotypes of two classes of previously reported mouse FM3A cell mutants with distinct biochemical defects. One class of mutants, selected for aphidicolin resistance, contains altered ribonucleotide reductase desensitized to deoxyadenosine triphosphate, a negative allosteric effector, and the other class contains temperature-sensitive thymidylate synthase. Abnormal levels of deoxyribonucleoside triphosphate pools, especially those of deoxyadenosine triphosphate, were the common biochemical trait for both classes of mutants. The aphidicolin-resistant thymidylate synthase mutants, isolated independently of aphidicolin resistance, showed reduced levels of thymidylate synthase and increased levels of ribonucleotide reductase. Bleomycin was more lethal to both classes of mutants than to wild-type cells, as determined by colony-forming ability. It also caused more marked scissions of DNA in the ribonucleotide reductase mutant cells than in the wildtype cells as analyzed by sedimentation on an alkaline sucrose gradient. Thus, the activity or level of ribonucleotide reductase seems to be one factor determining the bleomycin sensitivity of cells by potentiating the capacity of bleomycin to cleave DNA strands.

Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells.

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied under a variety of conditions. In medium containing 10 microM thymidine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 microM thymidine and 50 microM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidylate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 microM thymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 microM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular tetrahydrofolate pool.

Chromosome breakage induced by thymidylate stress in thymidylate synthase-negative mutants of mouse FM3A cells.

In thymidylate synthase-negative mutants of mouse FM3A cells, thymidine starvation rapidly decreased mitotic activity and resulted in cell death (thymineless death). When the thymidine starvation was reversed by an addition of thymidine, mitotic activity was recovered, but the majority of mitotic cells exhibited extensive chromosome aberrations, including chromatid breaks, chromatid exchanges, and pulverizations. Autoradiographic examination revealed that chromosome instability was induced only in cells arrested in the S phase during thymidine starvation. Furthermore, the most sensitive sites to the chromosome-damaging effect appeared to be sites which had replicated just prior to thymidine starvation. During thymidine starvation, cells at other stages in the cell cycle were accumulated at the G1-S boundary, and they were insensitive to the chromosome-damaging effect. Thymidine starvation was also found to be recombinagenic. Complete removal from the medium of a thymidine analogue, 5-bromo-2'-deoxyuridine, resulted in a dramatic increase in the frequency of sister chromatid exchanges. These results support the view that thymidine starvation in mammalian cells results in thymineless death via induction of DNA double-strand breaks, leading to chromosome fragmentation as well as rearrangements in the cells synthesizing DNA.

Effect of tunicamycin on production by mouse fibroblast L929 cells of the factor-stimulating differentiation of mouse myeloid leukemic cells and the colony-stimulating factor.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as a single peak with an apparent molecular weight of 67,000. D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies.

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.

DNA synthesis in isolated chromatin. Nature of activities, and relationship to kinetics of DNA polymerase release from chromatin DNA.

Chromatin isolated from Ehrlich ascites tumor cells showed two DNA synthetic activities differing in sensitivity to N-ethylmaleimide. For studies on the nature of activities and relationship to kinetics of DNA polymerase, a new method was developed for detecting the activity of DNA polymerase released from chromatin DNA during DNA synthesis in vitro. The activity of DNA polymerase released was measured in a reaction mixture for DNA synthesis using exogenously added poly(dA-dT) as a template-primer in the presence of actinomycin D. Evidence that the DNA polymerase released was actually involved in DNA synthesis of chromatin was obtained in experiments using chromatin isolated from cells treated with various concentrations of 1-beta-D-arabinofuranosylcytosine and chromatin from adult mouse liver. The experiments showed that chromatin isolated from cells in which only small amount of DNA polymerase was engaged in DNA synthesis released a negligible amount of DNA polymerase, especially N-ethylmaleimide-sensitive polymerase. Kinetic analysis of DNA polymerase during chromatin DNA synthesis by the new method suggested that KCl at the optimal concentration (10-20 mM) for the N-ethylmaleimide-sensitive chromatin activity enhanced the binding of the N-ethylmaleimide-sensitive DNA polymerase to chromatin DNA. From the findings that addition of actinomycin D or omission of dNTPs from the preincubation mixture prevents this binding, it is suggested that the binding of DNA polymerase is followed by the DNA chain synthesis and that the DNA polymerase involved in this reaction is N-ethylmaleimide sensitive. Data on the effect of KCl on the rate of chromatin DNA synthesis and on the size of the DNA chain favor this assumption.

Size difference in catalytic polypeptides of two active forms of mouse DNA polymerase alpha and separation of the primase subunit from one form, DNA replicase.

There are two active forms of DNA polymerase alpha in mouse cells. One form (DNA replicase) is a DNA polymerase associated with primase activity and the other form (7.3 S polymerase) has no primase activity (Yaugar, T., Kozu, T. and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127). The primase activity was dissociated from partially purified DNA replicase by hydroxyapatite column chromatography in buffer containing dimethyl sulfoxide and ethylene glycol. Nearly homogeneous primase, consisting of a 58 kDa polypeptide was obtained by glycerol gradient sedimentation and DEAE-cellulose column chromatography. Experiments on the effect of proteinase treatment and measurement of the molecular weight of the catalytic polypeptide of DNA replicase after its dissociation from the primase polypeptide indicated that the primase is not part of the DNA polymerase molecule, but an independent protein associated with DNA polymerase alpha, and that the latter is a 115 kDa catalytic polypeptide. The other form of DNA polymerase alpha, 7.3 S polymerase, consists of a 72 kDa catalytic polypeptide. Thus, the two forms of mouse DNA polymerase alpha have partially, if not completely, different catalytic polypeptide structures, suggesting that the 7.3 S polymerase is not simply formed from DNA replicase by dissociation of the primase subunit.

Structural changes in the glutamine-chargeable Escherichia coli transfer RNA-Trp produced by chemical modification with sodium bisulfite.

Glutamine-mischargeable tRNA produced by sodium bisulfite-treated Escherichia coli tRNA-Trp was isolated by dihydroxyboryl-cellulose affinity column chromatography. This tRNA was shown to have dual specificity tryptophan and glutamine, and, when charged with either amino acid, bound to ribosomes in response to the non-sense codon UAG but not in response to the tryptophan codon UGG. The results were consistent with the reported properties of Su+7 tRNA. The bisulfite-treated tRNA-Trp migrated as two bands during polyacrylamide gel electrophoresis. The faster moving band (band I) coincided with that of untreated tRNA-Trp. The slower moving band (band II) coincided with the glutamine-chargeable tRNA-Trp. Su+7 tRNA behaved like band II tRNA upon gel electrophoresis. Nucleotide sequence analysis showed that a cytidine-uridine transition occurred at the 1st or the 2n position of the anitcodon of band II tRNA. Band I and band II tRNAs differed from each other in their thermal melting profiles. It is suggested that the single base change in the anticodon is responsible for the altered conformation of band II tRNA.

Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate.

Human diploid fibroblasts were synchronized in the resting phase by incubation in medium containing a low level of serum and then stimulated to proliferate by adding a high concentration of serum. DNA replication started 12 hours after addition of serum, and reached a maximum after 24 hours. Thymidylate synthase activity was very low in resting cells, but began to increase 12 hours after growth stimulation and thereafter continued to increase. Thymidylate synthase mRNA in the growing cells was compared with that in resting cells, using cloned human thymidylate synthase cDNA as a probe. Results showed that the mRNA content as a percentage of total RNA began to increase six hours after stimulation, reaching a level about 14 times that in unstimulated cells after 24 hours. However, the mRNA content relative to poly(A)+ RNA had increased two- to fourfold by 24 hours after growth stimulation. Transcription of the thymidylate synthase gene, determined by hybridizing labelled nascent transcripts obtained in isolated nuclei to immobilized human thymidylate synthase cDNA, was similar in the nuclei of resting and of growth-stimulated cells. These results show that the increase in thymidylate synthase mRNA in growth-stimulated cells is caused by an increase in post-transcriptional events.

Aphidicolin-resistant mutants of mouse lymphoma L5178Y cells with a high incidence of spontaneous sister chromatid exchanges.

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.

Expression of glutaredoxin in human coronary arteries: its potential role in antioxidant protection against atherosclerosis.

Oxidative stress is considered an important factor in atherogenesis. Mammalian cells have a complex network of antioxidants such as catalase, superoxide dismutase, and glutathione peroxidase. However, the mechanisms that regulate the cellular redox state in the vessel wall remain unclear. Recent study has shown that thioredoxin, a thiol-disulfide oxidoreductase, is expressed in atherosclerotic plaques of human carotid arteries. In this study, we investigated the localization and expressional change of glutaredoxin and thioredoxin, two important members of the thiol-disulfide oxidoreductases, in autopsy samples of human coronary arteries. In nonatherosclerotic coronary arteries, glutaredoxin was expressed in endothelial cells, in fibroblasts of the adventitia, and most intensely in medial smooth muscle cells. Interestingly, in atherosclerotic lesions such as hypercellular lesions, the infiltrating macrophages highly expressed glutaredoxin. The expressional pattern of thioredoxin was quite similar to that of glutaredoxin. Western blot analysis demonstrated that hydrogen peroxide stimulated the expression of glutaredoxin in a time- and dose-dependent manner in cultured human coronary artery smooth muscle cells. Fluorescence microtopography with dihydroethidium demonstrated that the generation of reactive oxygen species was associated with the expression of glutaredoxin. These results suggest the possible involvement of thiol-disulfide oxidoreductases in antioxidant protection in human coronary arteries.

Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1.

A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds.

Murine mammary carcinoma (FM3A TK-/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ng/ml, respectively; i.e., a concentration 5000 to 20 000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action of BVDU and related compounds.

Thymidylate synthetase-positive and -negative murine mammary FM3A carcinoma cells as a useful system for detecting thymidylate synthetase inhibitors.

The murine mammary FM3A/O and the thymidylate (dTMP) synthetase-deficient FM3A/TS- carcinoma cell lines can be considered as a novel and useful test system for the detection of nucleoside analogues which are directly aimed at the thymidylate synthetase. These compounds should be inhibitory for FM3A/O but not for FM3A/TS- cells, and their inhibitory effects on FM3A/O cell growth should be readily reversed by exogenous dThd within the concentration range of 5-20 microM.

Differential effect of a microsomal deacetylase inhibition on the mutagenicity in Salmonella typhimurium of 2-acetylaminofluorene by liver homogenates of guinea pigs, mice and rats.

The effect of paraoxon, a microsomal deacetylase inhibitor, on the mutant genicity of 2-acetylaminofluorene (AAF) by liver homogenates was compared between the AAF carcinogenesis-resistant guinea pigs and the susceptible mice and rats. The mutagenicity of AAF was mostly abolished by paraoxon, not only in the 3 kinds of untreated animals but also in guinea pigs treated with a combination of phenobarbital and 5,6-benzoflavone, whereas about 50% of the mutagenicity was resistant to paraoxon in treated mice and rats. We suggest that microsomal deacetylase activity is crucially involved in the mutagenic activation of AAF by guinea pig liver homogenates, while the enzyme activity other than the deacetylase activity is also important in the activation by liver homogenates from treated mice or rats.