Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.     

Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells

The M2 subunit of mammalian ribonucleotide reductase was purified to homogeneity from hydroxyurea-resistant, M2-overproducing mouse cells. The purification procedure involved affinity chromatography on an anti-tubulin antibody-Sepharose column and high performance gel permeation chromatography. The pure protein is a dimer of Mr = 88,000, containing stoichiometric amounts of a non-heme iron center and a tyrosyl free radical. The radical is destroyed by hydroxyurea but can readily be regenerated on incubation of the radical-free protein alone with iron-dithiothreitol in the presence of air. The ability to spontaneously regenerate the tyrosyl radical distinguishes protein M2 from the corresponding subunit of Escherichia coli ribonucleotide reductase, protein B2, but apart from that the two proteins are very similar.     

Localization of ribonucleotide reductase in mammalian cells.

The results of immunocytochemical studies using two different monoclonal antibodies against the M1 subunit of ribonucleotide reductase show an exclusively cytoplasmic localization of this subunit both in cultured MDBK and mouse 3T6 cells, and in cells from various rat tissues. By fluorescent light microscopy, there is a diffuse staining of the cytoplasm, while by electron microscopy the immunoreactive material appears to be associated with ribosomes. In the rat tissues, only actively dividing cells show M1-specific immunofluorescence revealing a strong correlation between the presence of protein M1 and DNA synthesis. Therefore M1 immunofluorescence could be used to study cell proliferation in normal, inflammatory or neoplastic tissue. A lesser variation in M1 staining is observed between individual cells in tissue culture, where most cells are positive, but neither here nor in the tissues examined are any cells with nuclear staining detected. We interpret our results to mean that in mammalian cells ribonucleotide reduction takes place in the cytoplasm and from there the deoxyribonucleotides are transported into the nucleus to serve in DNA synthesis.     

Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.     

Regulation of ribonucleotide reductase activity in mammalian cells

Mammalian ribonucleotide reductase catalyzes the rate-limiting for the de novo synthesis 2'-deoxyribonucleoside 5'-triphosphates. There is some suggestion that this step may also be the rate-limiting step of DNA synthesis. It is apparent that the level of the enzyme, ribonucleotide reductase, varies through the cell cycle and is highest in those tissues with the greatest proliferation rate. This increase in activity is associated with increased protein synthesis. The purified enzyme has been shown to be subject to strict allosteric regulation by the various nucleoside triphosphates and it has been proposed that allosteric regulation plays an important role in the level of ribonucleotide reductase activity which is expressed. All experimental data relating to this point, however, do not support the role of deoxyribonucleoside triphosphates as a major factor in determining cellular reductase activity during normal cell division. Several naturally occurring factors have been isolated from cells which lower ribonucleotide reductase activity in vitro. These factors have been found in tissues of low growth fraction and appear to be absent or low in tissues or high growth fraction such as tumor, regenerating liver and embryonic tissues. The expression of intracellular ribonucleotide reductase activity is therefore controlled at various levels and by various factors and the prevailing mode of regulation may vary throughout the cell cycle transverse and also in the various types of cells.     

Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells

Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation. However, results showing direct causal linkages between specific ROS (reactive oxygen species) and the ionizing radiation-induced mutator phenotype are lacking. The present study demonstrates that ionizing radiation-induced genomically unstable cells (characterized by chromosomal instability and an increase in mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and GPx (glutathione peroxidase) activity. Treatment of unstable cells with PEG-CAT (polyethylene glycol-conjugated catalase) reduced the mutation frequency and mutation rate in a dose-dependent fashion. In addition, inhibiting catalase activity in the stable clones using AT (3-aminotriazole) increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists for many generations following exposure of mammalian cells to ionizing radiation.     

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.     

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.     

Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase.

Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size.     

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.     

Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells

To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (G0) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A lambda-cDNA library was prepared using poly(A)+ RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the G0 to S transition, in contrast to the "immediate-early" rise of c-myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to beta-actin, lactate dehydrogenase, and alpha-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified "late response" genes in the pre-DNA synthesis pathway are in progress.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.     

Molecular cloning and expression of a mouse thiamin pyrophosphokinase cDNA

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with a Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression of mTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 microM versus 3.0 nM). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.     

Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. We have produced and characterized rat polyclonal and monoclonal antibodies directed against protein M2 of mouse ribonucleotide reductase. Using these antibodies for immunocytochemical studies, an exclusively cytoplasmic localization of protein M2 was demonstrated both in cultured parent and hydroxyurea-resistant, M2-over-producing mouse TA3 cells, and in cells from various mouse tissues. These data, together with the previously demonstrated cytoplasmic localization of the M1 subunit, clearly show that ribonucleotide reductase is a cytoplasmic enzyme. Combining the anti-M2 antibodies with a monoclonal anti-M1 antibody allowed for double-labelling immunofluorescence studies of the two subunits in individual cells. Only approximately 50% of the cells in a logarithmically growing culture contained immunodetectable protein M2, while the M1-specific staining was present in all cells. The M2 staining correlates well with the proportion of cells in the S-phase of the cell cycle. In tissues, only actively dividing cells stained with either antibody and there were always fewer cells stained with the M2-antibodies than with the M1-antibody. Our data therefore present independent evidence for the earlier proposed model of a differential regulation during the cell cycle of the M1 and M2 subunits of ribonucleotide reductase.     

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.