We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.
Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter regionJane Clifford Azizkhan, James P. Vaughn, Robert J. Christy, and Joyce L. HamlinCite this: Biochemistry 1986, 25, 20, 6228–6236Publication Date (Print):October 7, 1986Publication History Published online1 May 2002Published inissue 7 October 1986https://pubs.acs.org/doi/10.1021/bi00368a059https://doi.org/10.1021/bi00368a059research-articleACS PublicationsRequest reuse permissionsArticle Views35Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
We have utilized a dihydrofolate reductase (DHFR) probe in combination with selected probes from other positions along the 2q chromosome arm in a two-color fluorescence in situ hybridization analysis of early DHFR gene amplification events in CHO cells. These studies show clearly that the most frequent initiating event is the formation of a giant inverted duplication, resulting from chromosome breakage and terminal fusion or a reverse unequal sister chromatid exchange. The dicentric chromosomes thus formed initiate bridge/breakage/fusion cycles that appear to mediate subsequent amplification steps to higher copy number.
In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.
The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L.(1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of ∼2.5 h. We have shown previously that [3H]mimosine can be specifically photo-cross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly− cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly− cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer. The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L.(1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G1/S boundary, and blocks DNA replication when added to S phase cells after a lag of ∼2.5 h. We have shown previously that [3H]mimosine can be specifically photo-cross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [3H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly− cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly− cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer. INTRODUCTIONOur laboratory's interest is the regulation of DNA synthesis in mammalian cells and, in particular, the nature of origins of replication. Although it is known that mammalian DNA is replicated from bidirectional origins spaced ∼100 kilobase pairs apart(1.Huberman J.A. Riggs A.D. J. Mol. Biol. 1968; 32: 327-341Crossref PubMed Scopus (632) Google Scholar), the molecular mechanisms of this process remain elusive (see (2.Hamlin J.L. Mosca P.J. Levenson V.V. Biochim. Biophys. Acta. 1994; 1198: 85-111PubMed Google Scholar), for review).In the absence of a viable assay for identifying the genetic elements (replicators) that control initiation in mammalian cells, attention has been focussed on determining the positions at which replication initiates, which should lie close to replicators. This approach requires methods for obtaining cell populations in which initiation at a given origin is occurring at the same time. In a commonly used synchronization protocol, cells are first arrested in the G0 (non-proliferating) compartment by nutritional or serum starvation, followed by release into an inhibitor of DNA synthesis (e.g.(3.Hamlin J.L. Pardee A.B. Exp. Cell Res. 1976; 100: 265-275Crossref PubMed Scopus (59) Google Scholar, 4.Heintz N.H. Hamlin J.L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4083-4087Crossref PubMed Scopus (120) Google Scholar, 5.Tobey R.A. Crissman H.A. Exp. Cell Res. 1972; 75: 460-464Crossref PubMed Scopus (85) Google Scholar)). The drug treatment is enforced for a time long enough to allow all cells in the population to arrive at the beginning of the S period (a time when at least some origins are sure to be firing); the drug is then removed, allowing cells to enter S in a semi-synchronous wave. Unfortunately, this protocol is not entirely satisfactory for examining initiation events at the beginning of S, because even the most efficacious replication inhibitors do not inhibit initiation per se; rather, they slow the rate of replication fork movement by affecting DNA polymerases (e.g. aphidicolin (6.Huberman J.A. Cell. 1981; 23: 647-648Abstract Full Text PDF PubMed Scopus (315) Google Scholar)) or by lowering deoxyribonucleotide pools (e.g. hydroxyurea (7.Skoog L. Nordenskjold B. Eur. J. Biochem. 1971; 19: 81-89Crossref PubMed Scopus (230) Google Scholar) and 5′-fluorodeoxyuridine(8.Rueckert R.R. Mueller G.C. Cancer Res. 1960; 20: 1584-1591PubMed Google Scholar)).About five years ago it was reported that the plant amino acid, mimosine, arrests mammalian cells at a specific point in the late G1 phase of the cell cycle(9.Lalande M. Exp. Cell Res. 1990; 186: 332-339Crossref PubMed Scopus (178) Google Scholar, 10.Lalande M. Hanauske A.H. Exp. Cell Res. 1990; 188: 117-121Crossref PubMed Scopus (31) Google Scholar). Therefore, mimosine could be a superior agent for synchronizing cells prior to initiation at early-firing origins. However, when we examined the effects of mimosine on cell cycle progression, specific G1 arrest was not observed(11.Mosca P.J. Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 4375-4383Crossref PubMed Scopus (123) Google Scholar). Instead, we showed that the drug inhibits replication per se, but in a manner different from any known chain elongation inhibitor. For example, mimosine completely prevents the uptake of [3H]thymidine into DNA when added to CHO ( 1The abbreviations used are: CHOChinese hamster ovaryp5050-kDa polypeptidemSHMTmitochondrial serine hydroxymethyltransferasecSHMTcytosolic serine hydroxymethyltransferasePAGEpolyacrylamide gel electrophoresis.) cells that have already entered the S period, but only after ∼2.5 h, whereas aphidicolin and hydroxyurea inhibit replication almost immediately (e.g.(12.Levenson V. Hamlin J.L. Nucleic Acids Res. 1993; 21: 3997-4004Crossref PubMed Scopus (86) Google Scholar)). After the 2.5-h lag, mimosine effectively prevents S phase cells from progressing any further in the cell cycle for at least 48 h, as assessed by fluorescence-activated cell sorter analysis; this contrasts with aphidicolin and hydroxyurea, which are relatively leaky even at high concentrations and allow cells to slowly traverse the S period (11.Mosca P.J. Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 4375-4383Crossref PubMed Scopus (123) Google Scholar, 13.Walters R.A. Tobey R.A. Hildebrand C.E. Biochem. Biophys. Res. Commun. 1976; 69: 212-217Crossref PubMed Scopus (76) Google Scholar). When added to cells as they attempt to cross the G1/S boundary, mimosine completely prevents the formation of replication forks in the dihydrofolate reductase origin in CHO cells, while aphidicolin and hydroxyurea do not(12.Levenson V. Hamlin J.L. Nucleic Acids Res. 1993; 21: 3997-4004Crossref PubMed Scopus (86) Google Scholar, 14.Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 3715-3722Crossref PubMed Scopus (111) Google Scholar). Finally, the initial rate of DNA synthesis is zero regardless of how long cells are maintained in mimosine after release from a G0 block, suggesting again that mimosine prevents the formation of replication forks; in contrast, with aphidicolin or hydroxyurea, the initial rate of [3H]thymidine incorporation increases with the duration of the block, arguing that both initiation and a significant amount of chain elongation occur in their presence(14.Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1992; 12: 3715-3722Crossref PubMed Scopus (111) Google Scholar).Thus, the possibility arose that mimosine could inhibit initiation itself, either by interfering with an initiator protein or by somehow preventing the formation of replication forks. However, it is also known that mimosine chelates iron, which is required by ribonucleotide reductase(15.Reichard P. Science. 1993; 260: 1773-1777Crossref PubMed Scopus (498) Google Scholar). Indeed, at relatively high concentrations, mimosine has been reported to lower deoxynucleotide pools in mammalian cells(16.Gilbert D.M. Neilson A. Miyazawa H. DePamphilis M.L. Burhans W.C. J. Biol. Chem. 1995; 270: 9597-9606Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17.Dai Y. Gold B. Vishwanatha J.K. Rhode S.L. Virology. 1994; 205: 210-216Crossref PubMed Scopus (75) Google Scholar). In addition, it has no apparent effect on DNA synthesis either in frog embryos (18.Wang Y. Zhao J. Clapper J. Martin L.D. Du C. DeVore E.R. Harkins K. Dobbs D.L. Benbow R.M. Exp. Cell Res. 1995; 217: 84-91Crossref PubMed Scopus (29) Google Scholar) or in in vitro replication extracts prepared from mammalian cells(16.Gilbert D.M. Neilson A. Miyazawa H. DePamphilis M.L. Burhans W.C. J. Biol. Chem. 1995; 270: 9597-9606Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17.Dai Y. Gold B. Vishwanatha J.K. Rhode S.L. Virology. 1994; 205: 210-216Crossref PubMed Scopus (75) Google Scholar), ( 2R. F. Kalejta, unpublished data.) both of which contain large deoxynucleotide pools. Furthermore, the inhibitory effects of mimosine on DNA replication in CHO cells can be overcome by adding iron to the culture medium(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar). It has therefore been suggested that mimosine functions solely by inhibiting ribonucleotide reductase(16.Gilbert D.M. Neilson A. Miyazawa H. DePamphilis M.L. Burhans W.C. J. Biol. Chem. 1995; 270: 9597-9606Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17.Dai Y. Gold B. Vishwanatha J.K. Rhode S.L. Virology. 1994; 205: 210-216Crossref PubMed Scopus (75) Google Scholar).It is difficult to prove or disprove the latter assertion (see arguments in (19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar)). However, a simple lowering of deoxyribonucleotide pool levels by inhibiting ribonucleotide reductase does not explain why mimosine is such an efficacious inhibitor and why it appears to prevent initiation of nascent DNA chains. We recently obtained evidence for a different (or additional) intracellular target. We showed that [3H]mimosine can be specifically cross-linked to a 50-kDa polypeptide (p50) both in vivo and in vitro at concentrations equal to the minimum effective dose in vivo(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar, 20.Hamlin J.L. Mosca P.J. Dijkwel P.A. Lin H.-B. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 467-473Crossref PubMed Scopus (7) Google Scholar). Furthermore, we demonstrated that the p50 mimosine binding activity is almost absent in a CHO cell line selected for resistance to 1 mM mimosine (∼10 times the lethal dose; 19, 20). p50 partitions largely with the soluble cytoplasmic and nuclear fractions, and the ability to bind mimosine does not fluctuate demonstrably during the cell cycle(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar).In the present study, we have partially purified the major 50-kDa mimosine binding activity and have sequenced six of its tryptic peptides by tandem mass spectrometry(21.Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1043) Google Scholar). The sequences of all six peptides (58 residues total) are consistent with 96.5% similarity and at least 77.5% identity to sequences in rabbit mitochondrial serine hydroxymethyltransferases (mSHMT), and all are closely related to rabbit cytosolic SHMT (cSHMT). Both of these enzymes are involved in the biogenesis of thymidine (among other activities; (22.Schirch V. Shostak K. Zamora M. Guatam-Basak M. J. Biol. Chem. 1991; 266: 759-764Abstract Full Text PDF PubMed Google Scholar) and (23.Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (296) Google Scholar)). Evidence is presented that mSHMT and cSHMT are, indeed, the major mimosine-binding species in vivo, as well as bona fide targets for the drug.Given the role of SHMT in deoxyribonucleotide metabolism, mimosine would be expected to have an effect only on chain elongation, even though its overall effect on replication differs greatly from other drugs that inhibit replication by lowering nucleotide pools (e.g. hydroxyurea, methotrexate, and 5′-fluorodeoxyuridine). Possible reasons for this dichotomy are discussed.MATERIALS AND METHODSCell CultureA clonal derivative of CHO-K1 cells with a doubling time of ∼13 h was the source of purified p50 in this study. CHO-K1 and CHOC 400 (a methotrexate-resistant CHO variant; (24.Milbrandt J.D. Heintz N.H. White W.C. Rothman S.M. Hamlin J.L. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6043-6047Crossref PubMed Scopus (101) Google Scholar)) were maintained in minimal essential medium supplemented with non-essential amino acids (Life Technologies, Inc./BRL), 10% Fetal Clone II serum substitute (Hyclone), and 50 μg/ml Gentamicin (Life Technologies, Inc./BRL) in an atmosphere of 5% CO2. Human HeLa and 293 cells, monkey COS and CV-1 cells, murine 3T3 cells, and Chinese hamster CHEF 18 cells were maintained in the same manner.Purification of the 50-kDa Mimosine-binding ProteinCHO-K1 cells were plated into 20-40 15-cm tissue culture dishes (∼3 × 107 cells/plate) 36 h before use and were harvested and photo-cross-linked to [3H]mimosine (Amersham, custom labeling) while still in exponential growth. All operations were carried out at room temperature except where noted. Plates were washed once with PSG (135 mM NaCl, 5 mM KCl, 1.1 mM KH2PO4, 1.1 mM Na2HPO4, pH 7.2) and were drained almost to dryness. To each plate were added 100 μl of lysis buffer (20 mM Hepes, pH 7.4, 3 mM MgCl2, 1 mM EDTA, 2% Triton X-100, 1 mM dithiothreitol, 40 μg/ml trypsin inhibitor, 40 μg/ml leupeptin, 1 μM 1-chloro-3-tosylamido-7-amino-2-heptanone). Cells were scraped with a plastic policeman, triturated to a uniform suspension, pooled into 1.5-ml microfuge tubes, and centrifuged for 3 min at 5,000 rpm in an Eppendorf microcentrifuge (final volume of the cell pellet was 180-200 μl/plate). The supernatant was removed, 225-μl aliquots were distributed to the wells of a microtiter dish, and 25 μl of [3H]mimosine (100 μCi; 7.4 Ci/mmol; Amersham custom labeling) was added. Samples were then incubated at 37°C for 30 min and were cross-linked with a Xenon lamp (ILC model LX3000 UV) for 15 s at a distance of 10 cm while maintaining the plates on ice.Each sample was then transferred to a 1.5-ml tube and centrifuged at 8,000 rpm for 5 min in a microcentrifuge. The supernatants were brought to 40% (NH4)2SO4, incubated on ice for 10 min, and centrifuged at 10,000 rpm for 20 min at 4°C in an HB-4 rotor (DuPont/Sorvall). Pellets were resuspended in lysis buffer without Triton X-100 and were subjected to gel filtration on a 1.2 × 100-cm Sepharose CL-6B column (Sigma). Fractions (1.2 ml) were collected and 100-μl aliquots were analyzed on a 10% sodium dodecyl sulfate-polyacrylamide gel (PAGE)(25.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205982) Google Scholar). The gel was impregnated with ENTENSIFY (DuPont Fluorofor kit) following the manufacturer's instructions, and radioactive spots were identified fluorographically, using Kodak X-Omat AR film. The 3H-labeled p50 protein fractions thus identified were combined and subjected to DE52 anion exchange chromatography (1.5 × 10 cm, Whatman): after washing the column with 40 mM KCl in lysis buffer lacking Triton X-100, fractions were eluted sequentially with 100, 200, 500, and 1,000 mM KCl in lysis buffer without Triton X-100. The p50-containing fraction (the 100 mM KCl wash) was then separated by PAGE and blotted onto a nitrocellulose membrane. The membrane was stained with Ponceau S, and the p50 spot or band, as well as several surrounding ones, were individually excised. Approximately 5% of each was used for scintillation counting to confirm the identity of 3H-labeled p50, and the remainder was subjected to tryptic digestion (see below).Enrichment and recovery values at each purification step were determined by assessing protein content with the Bradford assay (26.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213254) Google Scholar) and radioactivity by trichloroacetic acid precipitation. Although a large percentage of the [3H]mimosine label in the initial cell lysate is cross-linked to proteins other than p50(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar, 20.Hamlin J.L. Mosca P.J. Dijkwel P.A. Lin H.-B. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 467-473Crossref PubMed Scopus (7) Google Scholar), the majority is associated exclusively with p50 after the 8,000 rpm centrifugation step (e.g. see Fig. 2).Peptide SequencingThe stained p50 band (1-2 μg of protein) was excised from the nitrocellulose sheet and digested in situ with trypsin, following the procedure of Aebersold et al.(27.Aebersold R.H. Leavitt J. Saavedra R.A. Hood L.E. Kent S.B.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6970-6974Crossref PubMed Scopus (630) Google Scholar). The stain was removed from the proteins by washing in 200 μM NaOH, followed by several washes with water. The nitrocellulose fragment was then incubated in 0.5% polyvinylpyrrolidone (Sigma) in 0.6% acetic acid for 30 min at 37°C; excess polyvinylpyrrolidone was removed by extensive rinsing with water, and the fragment was finally submerged in 100 mM ammonium acetate, pH 8.0, containing 0.1 μg of sequencing grade modified trypsin (Promega Biotech; just enough liquid to cover the sample). Digestion was carried out overnight at 37°C. The digestion solution was removed, the nitrocellulose fragment was rinsed twice with 100 μl of digestion buffer, and all solutions were pooled. The digest was then acidified with acetic acid (1% final concentration), and the volume was reduced to ∼40 μl in a Speed Vac. Aliquots of the digest were analyzed on a Finnigan-MAT TSQ-70 triple-quadrupole mass spectrometer equipped with an APCI source (San Jose, CA) interfaced to a polyimide-coated fused-silica microcapillary high performance liquid chromatography column (inner diameter 75 μm; outer diameter 200 μm; Polymicro Technologies, Phoenix, AZ) packed with Poros R2/H material (PerSeptive Biosystems, Cambridge, MA). The digest was eluted into the mass spectrometer with an Applied Biosystems 140B Solvent Delivery System (Foster City, CA)(21.Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1043) Google Scholar). Protein data base searches were submitted to the National Center for Biotechnology Information of the National Institutes of Health (Bethesda, MD), using the BlastP program(28.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69020) Google Scholar).Immunoprecipitation of p50 from Whole Cell LysatesTen microliters of a polyclonal antiserum raised against rabbit cSHMT (L. Schirch, Virginia Commonwealth University) were incubated with 10 μl (packed volume) of Protein A beads (Sigma) at 37°C for 1 h with periodic agitation. After washing the beads extensively with phosphate-buffered saline, 7 μl of [3H]mimosine-labeled cell lysate (∼20 μg of protein) were added to the beads and incubated at 37°C for 3 h. The beads were washed three times with phosphate-buffered saline, boiled in PAGE sample buffer, and pelleted in a microcentrifuge. The supernatants were subjected to one-dimensional SDS-PAGE.Schiff Base ReductionAliquots of cell lysate (80 μl; ∼100 μg of protein) were incubated with different concentrations of sodium borohydride for 10 min at 37°C (see figure legend). As a control, [3H]mimosine was pretreated with 1 mM sodium borohydride at 37°C for 30 min and was added to the lysate and cross-linked as described above. Separate aliquots of lysate were treated identically, substituting NaOH, dithiothreitol, or β-mercaptoethanol for sodium borohydride, to control for any possible effects of pH and reducing equivalents on the NaBH4-treated extract.SHMT Enzyme AssaysCHO cell lysates were prepared exactly as described for cross-linking studies, except that 60 mM potassium phosphate buffer (pH 7.5) was substituted for Hepes buffer in the cell washing and lysis steps. Triton X-100 was removed by extraction with isoamyl alcohol and protein concentrations were measured with the Bradford assay(26.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213254) Google Scholar). The assay for SHMT enzyme activity measures the transfer rate of a [14C]methylene group from serine to tetrahydrofolate, and was performed according to the method of Snell(29.Snell K. Biochem. J. 1980; 190: 451-455Crossref PubMed Scopus (31) Google Scholar), with the following modifications: 1) solid L-tetrahydrofolic acid (Sigma) was added just prior to assay to minimize oxidation; 2) [14C]serine was present at 20 mM and 20 μCi/ml final concentration; and 3) mercaptoethanol was replaced with dithiothreitol. Data were plotted using Sigmaplot (version 4.1), and the means of duplicate determinations is shown. Error bars indicate the range of values of two determinations in each case.RESULTSConservation of p50 in Mammalian CellsIn previous studies on CHO-K1 cells, we showed that [3H]mimosine can be photo-cross-linked to a 50-kDa polypeptide (p50)(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar, 20.Hamlin J.L. Mosca P.J. Dijkwel P.A. Lin H.-B. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 467-473Crossref PubMed Scopus (7) Google Scholar). However, the 50-kDa mimosine binding activity was virtually undetectable in CHO-K1 cells that had been selected for resistance to 1 mM mimosine (∼10 times the lethal dose; (19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar) and (20.Hamlin J.L. Mosca P.J. Dijkwel P.A. Lin H.-B. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 467-473Crossref PubMed Scopus (7) Google Scholar)). This result strongly suggested that p50 is a biologically relevant target for mimosine. Since mimosine also inhibits DNA synthesis in cell lines derived from human, monkey, and murine sources (9.Lalande M. Exp. Cell Res. 1990; 186: 332-339Crossref PubMed Scopus (178) Google Scholar, 10.Lalande M. Hanauske A.H. Exp. Cell Res. 1990; 188: 117-121Crossref PubMed Scopus (31) Google Scholar) ( 3R. F. Kalejta, P. J. Mosca, and H.-B. Lin, unpublished observations.) it would therefore be predicted that each of these cell lines should also exhibit [3H]mimosine binding activity.To address this question, extracts from eight different cell lines of human, monkey, mouse, and hamster origin were incubated with [3H]mimosine and illuminated with a xenon lamp. The extracts were separated on a polyacrylamide gel, and the corresponding radioactive products were detected by fluorography. The Coomassie Brilliant Blue-stained gel is shown in Fig. 1A and the fluorogram in Fig. 1B. Seven of the eight cell lines contain a prominent [3H]mimosine-binding species that migrates at ∼50 kDa. Although the CV-1 extract displays only a faint band in this experiment, p50 mimosine binding activity has been observed in other preparations. ( 4H.-B. Lin, unpublished observations.) Also note that monkey COS cells, which were derived from CV-1 cells, display a prominent radioactive band at ∼50 kDa. Thus, a mimosine binding activity appears to be conserved among four different mammalian species. Furthermore, although some minor differences in molecular weight are detected (e.g. compare human 293 and monkey COS cell extracts, Fig. 1B), the binding proteins all migrate in the size range 50-53 kDa.Figure 1:A 50-kDa [3H]mimosine-binding polypeptide is detected in a wide variety of mammalian cell lines. Cell extracts were prepared and irradiated in the presence of 100 μM [3H]mimosine, as described under "Materials and Methods." Samples (∼100 μg of protein) were run on a 10% denaturing polyacrylamide gel, which was stained and photographed, and then treated with ENTENSIFY and dried onto filter paper. Panel A, Coomassie Brilliant Blue-stained polyacrylamide gel. Panel B, fluorograph of the gel shown in panel A, showing tritium-labeled polypeptides (7 day exposure).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Additional, less prominent 3H-labeled bands are also detected, as well as significant amounts of labeled high molecular weight material at the top of the gel. Since the spectrum and intensity of the faint bands are not reproducible from experiment to experiment, and since additional experiments indicated that they are probably not relevant (see below), we have not studied any of these bands further. The high molecular weight material is not detected in non-irradiated extracts(19.Mosca P.J. Lin H.B. Hamlin J.L. Nucleic Acids Res. 1995; 23: 261-268Crossref PubMed Scopus (31) Google Scholar), ( 5P. J. Mosca, unpublished observations.) and we therefore assume that it represents aggregates of p50, either with itself or with other polypeptides. We have also examined extracts prepared from Saccharomyces cerevisiae and Escherichia coli, but did not reproducibly observe specific [3H]mimosine binding activity at any position in the gels.5 This is consistent with the observation that mimosine does not inhibit DNA replication in either of these species at concentrations as high as 1 mM. ( 6R. F. Kalejta and J. L. Hamlin, unpublished observations.) Purification of p50 from CHO-K1 CellsThe observation that p50 is present in all of the cell lines that are sensitive to mimosine inhibition further argues that p50 represents a biologically important target for drug action. We therefore developed a fractionation scheme for obtaining p50 of sufficient purity to sequence by tandem mass spectrometry(21.Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1043) Google Scholar).Exponentially growing CHO-K1 cells in tissue culture dishes were harvested, and a soluble extract was prepared by lysing with Triton X-100 and removing nuclei by centrifugation. The resulting extract was cross-linked with [3H]mimosine and aggregated material was removed by low speed centrifugation. The bulk of the p50 was then precipitated in a 40% saturated ammonium sulfate solution, and the resulting pellet was redissolved and fractionated by size on Sepharose CL-6B. The p50-enriched fractions were pooled and subjected to chromatography on DE52, and those fractions containing the [3H]mimosine binding activity were finally subjected to preparative polyacrylamide gel electrophoresis.The approximate degrees of purification and recovery at each step in a typical experiment are outlined in Table 1, and the progress of the purification is shown in Fig. 2. Note that equal amounts of protein were loaded into each well of the polyacrylamide gel in this experiment. As can be seen in the stained gel in Fig. 2A and the corresponding fluorogram in Fig. 2B, DE52 chromatography and polyacrylamide gel electrophoresis represented the most effective purification steps. The arrow in Fig. 2A indicates the stained band that co-migrates with the [3H]mimosine-binding band in Fig. 2B. This stained band must represent several 50-kDa polypeptide species in the early stages of the purification, since its intensity relative to total protein does not markedly increase in the first two steps, while the [3H]mimosine binding activity at the corresponding position does increase. This raises the question whether the 50-kDa band in the final polyacrylamide gel represents a single species. In fact, on a two-dimensional gel, the 50-kDa band separates into one major and one very minor radioactive spot that differ only slightly in isoelectric point, and each of these corresponds to a stained spot.4 These data suggest that the bulk of the material in the 50-kDa band on the one-dimensional polyacrylamide gel binds to [3H]mimosine.Tabled 1 Open table in a new tab In a typical purification protocol, ∼6 × 108 cells yielded 1-2 μg of p50 (estimated by Ponceau S staining intensities on nitrocellulose blots relative to standards). This represented an average purification of 6,000-fold and an average recovery of ∼57% from the starting extract (Table 1).Primary Sequence Analysis of p50 by Tandem Mass SpectrometryAfter transfer of the polypeptides in the gel to nitrocellulose, the immobilized p50 band (∼1 μg) was excised and incubated with trypsin as
In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.
Abstract We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate‐resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In one cell line (A3), replication of the HSR requires, at most, 3 hours of a 7‐hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique. The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.
