Influence of targeted asparagine starvation on extra- and intracellular amino acid pools of cultivated Chinese hamster ovary cells

1995 ◽  
Vol 44 (3-4) ◽  
pp. 344-350
Author(s):  
T. Seewöster ◽  
J. Lehmann
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Intracellular Amino Acid

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

scholarly journals Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells

1998 ◽  
Vol 329 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Richard HEAL ◽  
John McGIVAN
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Asparagine Synthetase ◽  
Mrna Levels ◽  
Amino Acid Deprivation ◽  
Ovary Cells ◽  
Protein Levels

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and α-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.

N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 552-559 ◽  
Author(s):  
Lijun Xia ◽  
Vishwanath Ramachandran ◽  
J. Michael McDaniel ◽  
Kiem N. Nguyen ◽  
Richard D. Cummings ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Terminal Region ◽  
Wild Type ◽  
Ovary Cells ◽  
N Terminus

P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 andO-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components andO-glycosylation to bind to P-selectin than does human PSGL-1.

scholarly journals Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

1984 ◽  
Vol 4 (4) ◽  
pp. 799-808 ◽  
Author(s):  
J Moffett ◽  
E Englesberg
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Amino Acid Transport ◽  
Actinomycin D ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Acid Transport ◽  
Ovary Cells ◽  
Proline Transport

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.

Control of amino acid transport into Chinese hamster ovary cells

2018 ◽  
Vol 115 (12) ◽  
pp. 2908-2929 ◽  
Author(s):  
Darren Geoghegan ◽  
Claire Arnall ◽  
Diane Hatton ◽  
Joanne Noble-Longster ◽  
Christopher Sellick ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Amino Acid Transport ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Acid Transport ◽  
Ovary Cells
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