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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase.