Cytological evidence for a relationship between normal hemotopoietic colony-forming cells and cells of the lymphoid system.

The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.

Comparison of phenotypic expression with genotypic transformation by using cloned, selectable markers.

The frequency of phenotypic expression of the herpes simplex virus type 1 tk and Escherichia coli gpt genes was compared with the frequency of genotypic transformation after calcium phosphate-mediated DNA transfection of a number of tk- and hprt- cell lines. In three of the five lines tested, the frequency of phenotypic expression was at most 10-fold higher than that of genotypic transformation as indicated by frequency of HAT resistance. The remaining two lines showed phenotypic responses which were 50- to 100-fold greater than the genotypic responses. The data indicate that the efficiency of DNA-mediated transformation with some cell lines can be limited by events after the uptake and expression of transfected DNA.

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.

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.

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. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

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.

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.

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.

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.

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.

Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells: characteristics of some recently developed selective systems.

Selection conditions have been optimized in the Chinese hamster ovary (CHO) cell system for a number of genetic markers. The genetic systems studied include resistance to the protein-synthesis inhibitors emetine (Emtr) and diphtheria toxin (Dipr), resistance to methylglyoxalbisguanylhydrazone (Mbgr) which affects polyamine transport, resistance to the nucleoside analogs toyocamycin and tubercidin (Toyr), and resistance to thioguanine (Thgr) and ouabain (OuaR). The optimal expression time following mutagenesis for various markers was between 2 and 6 days. A linear dose--response relationship between the concentration of mutagen (ethyl methanesulfonate) and mutation frequency has been observed over the range of 10--700 micrograms/ml, for all of the above markers except Toyr. The response of these markers to other mutagens such as tritium (3H) decay and ICR-191 show some specificity. Since the response of a number of genetic markers can be studied simultaneously in the CHO system, it should prove very useful for studies of quantitative mutagenesis and in assay systems for mutagen detection.

Complementation between mutants of CHO cells resistant to a variety of plant lectins.

Chinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8--10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (RicRII) behaves dominantly. Tests for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complimentation groups can be delineated among the phenotypically recessive mutants.

Selection and characterization of Chinese hamster ovary cells resistant to the cytotoxicity of lectins.

Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Phar) phenotype is stable in the absence of selection; its frequency in a sensitive-population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminal alpha-mannose residues is dramatically reduced (less than or equal to 5% of the activity detected in wild-type CHO cells) in several independent PhaR clones. These clones also exhibit (a) a decreased ability to bind [125I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) and Lens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytoxocity of concanavalin A (Con A); (d) an increased ability to bind 125I-Con A; and (e) decreased surface galactose residues - all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistance closes from each of two complementary CHO auxitrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC, or LCA, and 4-5 "lectin-resistance" phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con AR mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A.

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.

The relationship between transformation and somatic mutation in human and Chinese hamster cells.

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.

The isolation and preliminary characterization of somatic cell mutants resistant to the protein synthesis inhibitor-emetine.

Emetine reversibly inhibits protein synthesis in Chinese hamster ovary (CHO) cells. Stable mutants which are 20-80 fold more resistant to the cytotoxic action of the drug can be isolated in a single step at a frequency of about 2-5 X 10(-7). The frequency of such mutants is increased 30-50 fold by ethyl methane sulphonate mutagenesis, and the spontaneous rate of mutation to emetine resistance as measured by Luria-Delbruck fluctuation analyses is 4.9 X 10(-7) mutations per locus per generation. Protein synthesis in extracts of the mutant cells is resistant to the inhibitory action of the emetine, indicating that the molecular lesion in these cells lies in the protein synthesis machinery.

Diphtheria toxin resistance in Chinese hamster cells: genetic and biochemical characteristics of the mutants affected in protein synthesis.

Diphtheria toxin (DT) resistant mutants (Dipr) have been isolated from a number of different Chinese hamster lines. Among mutants affected in protein synthesis (DiprII class), two distinct phenotypes have been identified. In one class, the entire elongation factor-2 (EF-2) activity becomes resistant to DT-catalyzed ADP-ribosylation (DiprIIa class); these mutants behave recessively upon hybridization with sensitive cells. The second kind of protein synthesis mutants contain nearly normal levels of the ADP-ribosylatable EF-2 activity (DiprIIb class). The hybrids the two types of protein synthesis mutants complement each other indicating that mutations in different genes are responsible for them. While the DiprIIa class of mutants are presumably affected in the EF-2, the lesion in DiprIIb mutants seems to have occurred in a yet unidentified protein synthesis factor. Interesting differences are also observed in the characteristics of mutants that presumably are defective in the entry of toxin into cells (DiprI class).

DRB resistance in Chinese hamster and human cells: genetic and biochemical characteristics of the selection system.

Stable mutants resistant to the nucleoside analog 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), which interferes with RNA synthesis, have been selected in Chinese hamster ovary (CHO) and human diploid fibroblasts. In CHO cells, upon treatment with the mutagen ethyl-methane sulfonate (EMS), a linear dose--response between the concentration of mutagen and the frequency of DrbR mutants was observed in the range of 20--300 micrograms/ml. The selection system did not show cell density or cross-feeding effects, and the optimal expression time following mutagenesis was found to be 2--3 days for CHO cells and 5--6 days for human fibroblasts. The DrbR mutation behaved codominantly in DrbR x DrbS hybrids. Addition of DRB affected nucleoside uptake to a similar extent in both wild-type and mutant cells, indicating that the drug was able to enter the mutant cells. The failure of DrbR mutants to show any cross-resistance to other toxic nucleoside analogs examined suggests that the action of DRB does not involve the initial phosphorylation step. DRB addition did not cause any marked inhibition of either RNA polymerase I or RNA polymerase II activity from both wild-type and mutant cells in vitro, indicating that its effect on RNA synthesis may be indirect.