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.     

Chinese hamster ovary cells resistant to beta-aspartylhydroxamate contain increased levels of asparagine synthetase

The growth of Chinese hamster ovary cells in a complete medium lacking asparagine is inhibited by beta-aspartylhydroxamate. The inhibition is overcome by the presence of asparagine in the growth medium. beta-Aspartylhydroxamate inhibits the activity of both asparagine synthetase and asparaginyl-tRNA synthetase in vitro. beta-Aspartylhydroxamate-resistant clones of Chinese hamster ovary cells have been isolated and three of these have been characterized. One clone, AH12, is 3-fold more resistant to beta-aspartylhydroxamate than the parental line and has 2 times higher levels of asparagine synthetase activity. Strains AH2 and AH5 are 6- to 7-fold more resistant to beta-aspartylhydroxamate and have 5 times higher levels of asparagine synthetase. The regulation of the expression of asparagine synthetase is altered in all three resistant cell lines. Whereas asparagine synthetase activity varies 2- to 3-fold in response to the asparagine content of the medium or to the extent of aminoacylation of tRNALeu in the parental cells, the activity of asparagine synthetase in the resistant cells is elevated under all growth conditions. No significant changes in the Km for substrates, Ki for beta-aspartylhydroxamate, or thermal stability were found for the asparagine synthetase of the resistant cells. These variants should prove useful in understanding the mechanisms involved in regulating the levels of asparagine synthetase in mammalian cells.     

Chinese hamster ovary cells resistant to borrelidin overproduce threonyl-tRNA synthetase

Chinese hamster ovary cell lines that are 1000-fold more resistant to the threonyl-tRNA synthetase inhibitor borrelidin than the sensitive parental cells were isolated after stepwise selection for growth in increasing concentrations of the drug. These cells show a 10-20-fold increase in threonyl-tRNA synthetase activity. Quantitation of the amount of threonyl-tRNA synthetase protein by immunological techniques indicated a 60-100-fold increase compared to sensitive cells. No significant changes in the Km for substrates, inhibition by borrelidin or thermal stability were found for the threonyl-tRNA synthetase of resistant cells. These data suggest that the resistant cell lines may have amplified the gene encoding threonyl-tRNA synthetase, but no evidence of homogeneously staining regions or double minute chromosomes was found. The resistant cell lines should prove useful for the study of the regulation of threonyl-tRNA synthetase.     

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.     

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.     

Interspecific somatic cell hybridization between asparagine-requiring and drug-resistant cell lines

Interspecific hybrids between Walker 256 carcinosarcoma rat cells which are asparagine requiring, and LMTK^t mouse cells which are drug resistant and asparagine independent have been isolated. The hybrids were selectively isolated by taking advantage of the asparagine requirement, or, in some cases, combining the asparagine requirement with an azaguanine resistance marker. The hybrids: (a) possessed a chromosome complement which was additive between the two parent lines; (b) showed two marker chromosomes; (c) possessed both rat and mouse forms of a number of different isozymes. The specific activities of asparagine synthetase was measured in the two parents and the hybrids. The enzyme level in the hybrids was found to be higher than the levels observed in the W 256 line, but only 10% of that observed in the LMTK⁻. The results are in agreement with, but do not prove, the hypothesis that asparagine requirement is due to a mutation in a structural cistron specifying the asparagine synthetase polypeptide.     

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.     

The human QARS locus: assignment of the human gene for glutaminyl-tRNA synthetase to chromosome 1q32–42

Summary We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.     

Asparagine-Requiring Tumor Cell Lines and Their Non-Requiring Variants: Cytogenetics, Biochemistry and Population Dynamics

Asparagine-requiring Jensen and Walker rat tumor cells and their asparagine-independent variants have been analyzed. The following results were obtained: (1) Both cell lines have very low levels of asparagine synthetase, and non-requiring revertants isolated from these lines have elevated levels of the enzyme. (2) No differences in chromosome number were detected between the parent Jensen line and five Jensen non-requiring revertants isolated from it. (3) Both Jensen and Walker cells undergo asparagineless death when deprived of this amino acid, although the Jensen cells do so at a more rapid rate. (4) Jensen requiring lines are at a selective advantage when grown in competition with non-requiring variants in complete medium, and their growth rate is more rapid when grown separately. The selective coefficients for the variant with respect to the asparagine-requiring parent ASN(-) line were 0.94 for the competition experiments and 0.83 for growth rate estimates. (5) A somatic cell hybrid between Chinese hamster cells (which require asparagine at low densities, and posses measurable synthetase activity) and the Walker line was found to be asparagine-independent, and it possessed enzyme levels equivalent to the hamster parent. The results of these investigations suggest a parallel with microbial auxotrophic mutants and can be understood in terms of alterations within nuclear structural genes.     

The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat.

Barley yellow dwarf virus (BYDV) resistance has been transferred to wheat from a group 7 chromosome of Thinopyrum (Agropyron) intermedium. The source of the resistance gene was the L1 disomic addition line, which carries the 7Ai-1 chromosome. The resistance locus is on the long arm of this chromosome. BYDV resistant recombinant lines were identified after three or more generations of selection against a group 7 Th. intermedium short arm marker (red coleoptile) and selection for the presence of BYDV resistance. One recombinant line produced by ph. mutant induced homoeologous pairing and 14 recombinant lines induced by cell culture have been identified. Resistance in seven of the cell culture induced recombinants has been inherited via pollen according to Mendelian segregation ratios for up to eight generations. Meiotic analysis of heterozygotes indicates that the alien chromatin in the cell culture induced recombinants is small enough to allow regular meiotic behaviour. The ph-induced recombinant was less regular in meiosis. A probe, pEleAcc2, originally isolated from Th. elongatum and that hybridizes to dispersed repeated DNA sequences, was utilised to detect Th. intermedium chromatin, which confers resistance to BYDV, in wheat backgrounds. Quantification of these hybridization signals indicated that the translocations involved a portion of alien chromatin that was smaller than the complete long arm of 7Ai-1. Restriction fragment length polymorphism analysis confirmed the loss of the short arm of 7Ai-1 and indicated the retention of segments of the long arm of 7Ai-1. Two 7Ai-1L DNA markers always assorted with the BYDV resistance. A third 7Ai-IL DNA marker was also present in seven of eight recombinants. In all recombinants except TC7, the 7Ai-1L markers replaced the 7DL markers. None of the wheat group 7 markers was missing from TC7. It is concluded that all the resistant lines are the result of recombination with wheat chromosome 7D, except line TC7, which is the result of recombination with an unidentified nongroup 7 chromosome.     

Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies

Thirteen stable hybridoma cell lines producing monoclonal antibodies specific for asparagine synthetase were established and one monoclonal antibody was chosen to produce an immunoaffinity resin for the purification of asparagine synthetase. Bovine pancreatic asparagine synthetase was purified to a specific activity of 395 nmol of Asn produced/min/mg. Electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 54,000 polypeptide. Prior cross-linking with dimethyl suberimidate resulted in a band at Mr = 52,500 (monomer) and two additional bands at Mr = 97,000 and 98,000 (dimers), suggesting the possibility of a heterogeneous enzyme population with slight differences in subunit composition. The ratio of Gln-dependent and NH3-dependent asparagine synthetase activities was constant for immunoaffinity-purified enzyme, but the ratios of glutaminase activity to synthetase activities varied, suggesting separate aspartate and glutamine binding sites. The monoclonal antibodies were tested as inhibitors of the Gln-dependent and NH3-dependent asparagine synthetase activities as well as for inhibition of the glutaminase activity of the enzyme. Two antibodies inhibited Gln- and NH3-dependent synthesis of asparagine, but did not affect the glutaminase activity of immunoaffinity-purified asparagine synthetase. A third monoclonal antibody inhibited Gln-dependent synthesis of asparagine and glutaminase activity, but activated NH3-dependent asparagine synthetase activity. These data are discussed in terms of multiple substrate binding domains within the asparagine synthetase molecule.     

Separation of Two Forms of Anthranilate Synthetase from 5-Methyltryptophar Susceptible and -Resistant Cultured Solarium tuberosumCells

Potato cell suspension cultures (Solanum tuberosumL. cv. Merrimack) have been selected which are resistant to growth inhibition by D,L-5-methyltryptophan. Anthranilate synthetase activity in crude extracts from resistant cells was less sensitive to feedback inhibition by L-tryptophan and D,L-5-methyltryptophan than the activity from the sensitive line. This altered feedback control apparently accounts for the cell's resistance to growth inhibition since there is a 48-fold increase in free tryptophan in one of the resistant cell lines. Preparative polyacrylamide gel electro-phoresis separated feedback-sensitive and -resistant forms of anthranilate synthetase in extracts from both 5-methyltryptophan-susceptible and -resistant cells, with a predominance of the corresponding form in the respective cell type. The anthranilate synthetase activity from the 5-methyltryptophan-resistant line was inactivated more slowly by incubation of crude extracts at 50°C than the activity from the sensitive line. These results suggest the presence of two isoenzymes of anthranilate synthetase in cultured potato cells.     

Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.     

Is there a functional equivalence between abnormalities of the long arm of chromosome 1 and the presence of Epstein-Barr virus in continuous lines of Burkitt's lymphoma?

Chromosome 1 long arm abnormalities (translocations, partial of complete trisomies) are non-randomly but inconstantly associated with specific translocations involving chromosomes 8, and 2, 14 or 22 in Burkitt's lymphomas and leukemias. All nine Burkitt's lymphoma cell lines not associated with Epstein-Barr virus (EBV) were shown to exhibit a chromosome 1 long arm abnormality and were present in only 3 out of 18 EBV positive cell lines. Bands 1q23 - 1q24 were involved in EBV-negative cell lines. It was thus hypothesised that genetic information resembling that included in viral genome exists on chromosome 1 long arm. This hypothesis implies new possible aspects of relationship between Burkitt's cell line proliferation and EBV.     

Development and characterization of wheat- Leymus racemosus translocation lines with resistance to Fusarium Head Blight

Wheat scab (Fusarium Head Blight, FHB) is a destructive disease in the warm and humid wheat-growing areas of the world. Finding diverse sources of FHB resistance is critical for genetic diversity of resistance for wheat breeding programs. Leymus racemosus is a wild perennial relative of wheat and is highly resistant to FHB. Three wheat- L. racemosus disomic addition (DA) lines DA5Lr#1, DA7Lr#1 and DALr.7 resistant to FHB were used to develop wheat- L.racemosus translocation lines through irradiation and gametocidal gene-induced chromosome breakage. A total of nine wheat-alien translocation lines with wheat scab resistance were identified by chromosome C-banding, GISH, telosomic pairing and RFLP analyses. In line NAU614, the long arm of 5Lr#1 was translocated to wheat chromosome 6B. Four lines, NAU601, NAU615, NAU617, and NAU635, had a part of the short arm of 7Lr#1 transferred to different wheat chromosomes. Four other lines, NAU611, NAU634, NAU633, and NAU618, contained translocations involving Leymus chromosome Lr.7 and different wheat chromosomes. The resistance level of the translocation lines with a single alien chromosome segment was higher than the susceptible wheat parent Chinese Spring but lower than the alien resistant parent L. racemosus. At least three resistance genes in L. racemosus were identified. One was located on chromosome Lr.7, and two could be assigned to the long arm of 5Lr#1 and the short arm of 7Lr#1.     

Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified approximately 50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.     

Dihydrofolate reductase activity in adriamycin and methotrexate sensitive and resistant P388 leukemia cells

P388 murine leukemia cells 18.4-fold more resistant to methotrexate (MTX) than the parent, drug susceptible line, were shown to possess a 1.5-fold higher dihydrofolate reductase (EC1.5.1.3) (DHFR) activity. This is in contrast to a MTX-resistant line, obtained from adriamycin-resistant cells, which is 27.9-fold more resistant to MTX and exhibits a 22.4-fold higher DHFR activity than that of the parent. The susceptibility of the enzyme to inhibition by MTX does not markedly change with the acquired drug resistance of the cell lines studied. Thus MTX-resistant cells obtained from an adriamycin-resistant line acquired resistance due to increased activity of the target enzyme, whereas other mechanisms are responsible for the resistance of cells derived from the adriamycin-sensitive parent.