Fine structure analysis of the Chinese hamster AS gene encoding asparagine synthetase.

Overlapping cDNAs for Chinese hamster ovary (CHO) asparagine synthetase (AS) were isolated from a library prepared from an AS-overproducing cell line. The sequence was determined and shown to contain an open reading frame encoding a protein of Mr 64,300. The predicted amino acid sequence for the CHO AS enzyme was compared to that of the human AS enzyme and found to be 95% homologous. A potential glutamine amide transfer domain, with sequence similarity to amidotransferases from bacteria and yeast, was identified in the N-terminal portion of the protein. The cDNAs were used to screen a library of phage containing wild type CHO DNA and the genomic AS sequences were detected on three overlapping phages. Determination of the fine structural organization showed that the CHO AS gene spanned 19 kilobases and was composed of 12 exons, three of which contained the glutamine amidotransferase domain. The 5' flanking sequences were highly G + C-rich and, like other housekeeping genes, lacked TATA and CAAT boxes.

Isolation, structure and expression of mammalian genes for histidyl-tRNA synthetase.

A full length cDNA clone that codes for human histidyl-tRNA synthetase (HRS) and cDNA clones that span the full length transcript of hamster HRS have been isolated. The full length human HRS cDNA was expressed after transfection into Cos 1 cells and a CHO ts mutant defective in the gene for HRS. The complete nucleotide sequence of the hamster and human gene were obtained and extensive homologies were observed in three regions on comparing these sequences between themselves and with the sequence of HRS derived from yeast. These results provide unequivocal evidence that we have indeed cloned the hamster and human gene for HRS. Three overlapping phage recombinants containing the complete hamster chromosomal gene for HRS have also been isolated. The genomic HRS is divided into 13 exons. The precise locations of each of the 5' and 3' exon-intron boundaries were defined by sequencing the appropriate regions of the cloned genomic DNA and aligning them with the sequence of HRS cDNAs. These studies provide the basis for future structural and functional analysis of the gene for HRS. In particular, it will be of interest to examine if different exons of HRS correlate to different domains of the HRS polypeptide.

Structural analysis of the 5' region of the chromosomal gene for hamster histidyl-tRNA synthetase.

The chromosomal gene (HRS) coding for hamster histidyl-tRNA synthetase, like many other housekeeping genes, lacks many of the features associated with promoters of RNA-polymerase-II-transcribed genes. HRS transcripts have multiple start points. Using RNase protection analysis, we also identified a 300-bp exon located only 36 bp away from the 5'-most start point of the HRS transcript. This exon hybridizes to a 3.5-kb transcript which transcribes from a different strand of DNA in the 5' region of the HRS gene. This divergent 3.5-kb transcript also has multiple transcription start points. The identity and function of this 3.5-kb transcript is not known.

Stability of retrovirally transduced markers in a rat cell line.

A MoMLV-based retroviral vector capable of transmitting and expressing both the human hypoxanthine phosphoribosyltransferase (hprt) coding sequence and the Herpes simplex type 1 thymidine kinase (tk) gene has been constructed. After infection of a rat cell line, cell clones were selected on the basis of expressing both markers. They were subsequently found to contain a single provirus of the expected topology. The ease with which loss of expression of the markers can be monitored has allowed us to make observations on the stability of proviral genes. In particular, we have found indirect evidence of strong position effects on proviral gene expression by comparing the characteristic frequency of marker loss in different clonal proviral lines. Effects of the selection protocol on the apparent frequency of variants have also been noted. Finally, a combination of molecular and genetic observations lead us to invoke chromosome loss as the major factor influencing marker stability in this system.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Characterization of single step albizziin-resistant Chinese hamster ovary cell lines with elevated levels of asparagine synthetase activity.

The amino acid analog, albizziin, which acts as a competitive inhibitor of asparagine synthetase with respect to glutamine was used to isolate mutants of Chinese hamster ovary cells with alterations in levels of the target enzyme. These mutant lines have been characterized biochemically and genetically. Mutants selected in a single step are up to 40-fold more resistant to the drug than the parental line, express levels of asparagine synthetase activity 6-17-fold greater than that of wild type cells, and act co-dominantly in hybrids. Several classes of mutations can be distinguished on the basis of cross-resistance to beta-aspartyl hydroxamate, another amino acid analog. Studies on asparagine synthetase indicate that resistance to albizziin may be due to altered regulation of asparagine synthetase, structural mutations of the enzyme, and gene amplification.

Amphotericin B enhances efficiency of DNA-mediated gene transfer in mammalian cells.

Using plasmids containing the genes for thymidine kinase (tk) and neomycin resistance (neo), we have shown that DNA-mediated genotypic transformation of L and Chinese hamster ovary (CHO) cells is increased several-fold by the presence of the sterol-binding polyene antibiotic, amphotericin. Transformation into the same host cells, using genomic DNA, was also enhanced by amphotericin. Phenotypic expression of beta-galactosidase activity of a plasmid containing the gene for the enzyme was also markedly elevated when the antibiotic was added at transfection. Other sterol-binding polyene antibiotics also showed activity in these DNA-mediated gene transfer assays.

Molecular cloning of a cDNA for Chinese hamster ovary asparagine synthetase.

In previous reports we have described the isolation and characterization of a number of Chinese hamster ovary (CHO) cell mutants resistant to the amino acid analogue albizziin (Alb). Multistep mutants were derived which showed a high degree of drug resistance and expressed increased levels of asparagine synthetase (AS) levels up to 300-fold over that of the parental cell line. Karyotypic analysis of these mutants revealed homogeneously staining regions (HSRs) usually indicative of gene amplification. In the present work, we provide further proof for gene amplification by showing that the mutants greatly overproduce functional AS mRNA, as evidenced by in vitro translation of purified mRNA and immunoprecipitation of AS. By using these overproducing mutants as sources of mRNA coupled with velocity centrifugation, we have been able to greatly enrich for AS sequences in our mRNA preparations to the point where they represent 1-5% of the total message. This facilitated cloning and selection of the cDNA sequences complementary to the gene. Utilizing these cloned cDNAs, we have demonstrated a correlation between gene copy number and enzyme expression in the parent and Alb-resistant mutants, thus providing direct evidence that drug resistance is due to gene amplification.

Metastatic properties of distinct phenotypic classes of lectin-resistant mutants isolated from murine MDAY-D2 cell line.

Single-step mutants were isolated from the murine metastatic MDAY-D2 cell line after selection in toxic concentrations of wheat-germ agglutinin. They were partially characterized by measuring their relative level of resistance to WGA, PHA, Con A, RIC, and LCA (Lec phenotype), and by comparing their karyotype and their ability to produce metastases upon transplantation into syngeneic DBA/2 mice. Based on their Lec phenotype, a total of 19 independent isolates were ranked into 10 distinct classes. Among them, two EMS-induced mutants were nontumorigenic (Lec II, Lec III), one nonmetastatic (Lec IV), and one spontaneous mutant (Lec I) failed to produce blood-borne metastases. Other spontaneous mutants belonging to Lec I, Lec II, and other classes were as metastatic as their parents. The Lec IV phenotype was found to segregate independently from metastatic potential in somatic hybrids. Metastatic ability was recovered in mutants expressing the Lec IV phenotype, after further selection for resistance to RIC. Our results strongly suggest that the loss or reduction of the invasive property of tumor cells is associated with only few Lecr1 phenotypes and, therefore, that a restricted number of cell surface glyconjugates are essential for this particular function.

Multidrug-resistance phenotype in Chinese hamster ovary cells.

Multidrug resistance is a complex pleiotropic phenotype of cross-resistance and collateral sensitivity to unrelated compounds observed in many mammalian cell mutants selected for resistance to single agents. In Chinese hamster ovary cells, colchicine-resistant mutants expressing this phenotype have been characterized extensively. Such mutants arise apparently from a single genetic event, and the basis of this phenotype appears to be localized at the membrane level, resulting in altered drug permeability. Expression of a 170,000-dalton surface glycoprotein (P-glycoprotein) has been identified to correlate with the multidrug-resistance phenotype. Selection of a second mutation in colchicine-resistant mutants, for resistance to phytohemagglutinin, results in an alteration of the carbohydrate moiety in P-glycoprotein and other surface components. This mutation does not noticeably affect the multi-drug-resistance phenotype. The altered permeability of mutant cells to drugs, however, can be modulated by nonionic detergents or metabolic inhibitors. These findings are consistent with a molecular mechanism of multidrug resistance whereby the pleiotropic response of the cell is mediated by an overexpression of a cell-surface protein, the P-glycoprotein.

Analysis of a break in chromosome 14 mapping to the region of the immunoglobulin heavy chain locus.

We have detected restriction fragment length polymorphisms associated with the immunoglobulin heavy chain C gamma genes. DNA from both parents of an individual having an unbalanced rearrangement of the long arm of chromosome 14, region q32 [Cox, D. W., Markovic, V. D. & Teshima, I. E. (1982) Nature (London) 297, 428-430], revealed distinctive patterns of BamHI fragments which hybridized with cloned probes from the C gamma 2-C gamma 4 gene cluster. The number of hybridizing fragments in both cases (five) equaled the number of known C gamma genes. Pedigree and densitometric analyses indicated that the proband did not have any maternal complement of C gamma gene-hybridizing fragments. Included on the deleted chromosomal segment was a C gamma gene having properties of the previously reported C gamma pseudogene. We also examined DNA from this family with a probe for the highly polymorphic locus D14S1, which recently was demonstrated to be tightly linked to the C gamma 1 gene locus [Balazs, I., Purrello, M., Rubinstein, P., Alhadeff, B. & Siniscalco, M. (1982) Proc. Natl. Acad. Sci. USA 79, 7395-7399]. EcoRI and EcoRI-BamHI fragments from both parents hybridized with a probe for this locus in DNA from the proband, indicating that, unlike the C gamma gene family, D14S1 was not deleted from the abnormal chromosome. Thus, the chromosomal breakpoint in the proband lies within region 14q32 between the two tightly linked markers, D14S1 and the C gamma 1 heavy chain gene locus. The D14S1 locus must lie proximal to the centromere relative to the C gamma gene family. The genetic variability detected with C gamma gene probes may prove useful for genetic analysis of structural rearrangements involving this region of chromosome 14.

Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

Isolation of mutants of CHO cells resistant to 6(p-hydroxyphenylazo)-uracil I. A novel BrdU cross-resistant phenotype.

Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPURA exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.

Isolation of mutants of CHO cells resistant to 6 (p-hydroxyphenylazo)-uracil II. Mutants auxotrophic for deoxypyrimidines.

Two classes of CHO mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been characterized. Both classes exhibited a nutritional requirement that could be satisfied by deoxypyrimidines and uridine but not other ribopyrimidines. A biochemical investigation of these mutants revealed a structural defect in ribonucleotide reductase resulting in a two- to fourfold increase in the Km for UDP and CDP. As a consequence of this lesion, the cells had imbalanced deoxypyrimidine pools and showed an increase in the rate of spontaneous mutation to 6-thioguanine resistance but not emetine resistance.

Expression of bacterial beta-galactosidase in animal cells.

A recombinant plasmid containing the gene for bacterial beta-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.

Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells.

We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells. In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.

DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression.

Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.