Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.     

Cytogenetic manifestations associated with the reversion, by gene amplification, at the HGPRT locus in V79 Chinese hamster cells

Some HGPRT spontaneous revertants were isolated from a mutant line (E2) of V79 Chinese hamster cells and phenotypically characterized. Dot-Blot hybridization with a 32P-labelled HGPRT probe revealed an increase in the number of HGPRT sequences in some of these revertants, suggesting the occurrence of gene amplification. Cytogenetic analysis performed in three of these revertants showed a characteristic abnormally banding region (ABR) on the elongated p arm of the X chromosome. In situ hybridization in one revertant (RHE2) showed that the amplified sequences reside on the p+ arm of the X chromosome in two different localizations. Because of the very probably clonal origin of the revertant, these features indicate that the amplified sequences might rearrange after their integration into the chromosome.     

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Evidence for linkage of 3-phosphoglycerate kinase,hypoxanthine-guanine-phosphoribosyl transferase,and glucose 6-phosphate dehydrogenase loci in Chinese hamster cells studied by using a relationship between gene multiplicity and enzyme activity

The PGK activity was assayed in diploid, hyperdiploid, tetraploid, and hybrid cells all originating from the same Chinese hamster cell line (DON line). A relationship between gene multiplicity and enzyme activity was observed. Selective pressure on the HGPRT locus by growth of hybrid cells in the presence of 8-azaguanine resulted in decreased levels of PGK activity. Growth of these hybrids in the presence of 5-BUdR did not influence the enzyme activity. It was concluded that the genes coding for HGPRT, PGK, and G6PD are linked in the Chinese hamster. The TK locus seems to be linked neither to the HGPRT, PGK, and G6PD loci nor to the 6PGD locus.     

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T. Van der Velde-Koerts, V. Ling, and P. Borst, Mol. Cell. Biol. 6:1671-1678, 1986). We report here that only a subset of these, encoding the 170-kilodalton P-glycoprotein, are consistently amplified in three different multidrug-resistant Chinese hamster lung cell lines, selected with vincristine, daunorubicin, or actinomycin D. Within each cell line, genomic sequences homologous to the P-glycoprotein cDNA probe were amplified to different levels. The pattern of differential amplification was consistent with the presence of at least two and possibly three P-glycoprotein genes. In the actinomycin D-selected cell line, these genes were disproportionately overexpressed relative to the associated levels of amplification. These results underline a central role for P-glycoprotein in multidrug resistance. In the daunorubicin-selected cell line, another, as yet uncharacterized, gene was amplified but disproportionately underexpressed. Its amplification was therefore fortuitous. We present a tentative map of the region in the hamster genome that is amplified in the multidrug-resistant cell lines which were analyzed.     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

Gene amplification as a mechanism of reversion at the HPRT locus in V79 Chinese hamster cells

Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl-transferase (HPRT) temperature-sensitive V79 Chinese hamster cells were selected by plating a temperature-sensitive mutant in HAT medium at 39 degrees C. The incidence of such revertants was approximately 2 X 10(-4) per cell. The majority of the revertants examined had increases of between three- and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39 degrees C. When the revertants were cultivated in the absence of HAT, they recovered their HAT-sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied: the Km for both PRPP and hypoxanthine was significantly different in the temperature-sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind III restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5-fold in one revertant and 1.4-fold in the other. Reintroduction into non-selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized. We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.     

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes.

Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb (COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5' end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human x Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.     

DNA-mediated transfer of a human gene required for low-density lipoprotein receptor expression and for multiple Golgi processing pathways.

Transfection of a hamster cell mutant with human DNA corrected both the low-density lipoprotein receptor-deficient phenotype and the multiple glycosylation defects of the cells. Independently transfected colonies contained a small set of common human DNA fragments. These fragments may correspond to the human analog of a single gene required for several different Golgi processing pathways.     

Cloning and expression of a mouse adenine phosphoribosyltransferase gene

A functional mouse adenine phosphoribosyltransferase (APRT) gene was identified and cloned by screening a mouse sperm genomic DNA library in lambda Charon 4A. The probe utilized for screening was a restriction fragment encoding much of the hamster APRT gene. Six recombinants that hybridized with the probe were identified, and after digestion with restriction enzymes EcoRI and PvuII revealed three different patterns of digestion for each enzyme. Of the six recombinants, five representing two of the restriction patterns possessed transforming activity. A sixth recombinant, which has a unique restriction pattern, lacks transforming activity but hybridizes well with hamster APRT coding sequences and is a possible candidate for a pseudogene. We used three criteria for conclusively identifying the mouse APRT genes. (1) DNA from the recombinant lambda phage hybridizes with DNA encoding hamster APRT. (2) The recombinant lambda phages and their DNAs transform mouse, hamster and human APRT- cells to the APRT+ phenotype. (3) The hamster and human transformants display APRT activity that migrates with a mobility characteristic of mouse APRT and not of hamster or human. A 3.1-kb EcoRI-SphI restriction fragment which retains transforming activity has been subcloned into the plasmid pBR328. Comparison of restriction enzyme sites with those contained in a mouse APRT cDNA, coupled with loss of transforming activity after enzyme digestion, indicates that the mouse APRT gene is larger than 1.8 kb and contains at least three introns.     

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.     

Assignment of the structural gene coding for albumin to human chromosome 4

Summary Albumin is a developmentally regulated serum protein synthesized in the liver mainly during adulthood. Family studies using variant forms of albumin established autosomal linkage between albumin and group-specific component protein (GS). Since GC has been assigned to human chromosome 4, albumin can be indirectly assigned to the same chromosome; however no direct assignment has been made. Recently, the human albumin cDNA probe has been isolated and characterized. It thus permits a direct chromosomal assignment of the albumin gene in the human genome. When the cDNA probe was hybridized to the HindIII digested total human DNA, an intense band at 6.8 kb was present. When the probe was hybridized to the HindIII digested Chinese hamster CHO-K1 DNA, a less intense band at 3.5 kb was found, plus three other faint bands. When the probe was hybridized to a series of human/CHO-K1 cell hybrids retaining a complete hamster genome and various combinations of human chromosomes, it was evident that hybrids containing human albumin gene sequences could be readily distinguished from hybrids containing no human albumin gene. Analysis of 22 primary cell hybrids for the presence or absence of human albumin sequences has assigned the albumin gene to human chromosome 4. Similar results were obtained using another restriction endonuclease EcoR1. Thus, by direct assay of the genomic albumin gene sequences in the cell hybrids, we provide evidence for a direct assignment of the structural gene for human albumin to chromosome 4.