Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport.

Mutants of CHO-K1 cells with varied levels of A system activity, probably the result of increases in absolute amount of the A system transporter, have corresponding increases in levels of peptides banding at 62-66 and 29 kDa. Mutant alar4-H3.9, showing the highest increase of A system activity and of 62- to 66- and 29-kDa peptides, was selected for this study.

The competence progression model in CHO-K1 cells: the relationship between protein kinase C and immediate early gene expression in the insulin mitogenic signal.

CHO-K1 cells grow in a defined medium with insulin, at physiological concentrations, as the only hormone. IGF-I can substitute for insulin. Quiescent cells require a 9-10-h lag, subsequent to the addition of insulin, to synthesize DNA.

Analysis of the genes involved in the insulin transmembrane mitogenic signal in Chinese hamster ovary cells, CHO-K1, utilizing insulin-independent mutants.

CHO-K1 cells, wild type (WT), grow in a defined medium with insulin as the only essential hormone. When starved for insulin, these cells accumulate in G0/G1 stage. Insulin binding to its receptor stimulates DNA synthesis and cell division and induces an increase in abundance of mRNA for c-fos, c-jun, Krox-20, Krox-24 (zif/268), fra-1, jun-B, c-myc, and JE.

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Previous work suggested that the structural gene for the A system transporter and the mRNA for the alpha subunit of the Na+,K(+)-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance of the alpha 1 subunit of Na+,K(+)-ATPase mRNA over that found in repressed WT.

Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes.

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone.

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Insulin is the only hormone required for continued growth of Chinese hamster ovary CHO-K1 cells in the defined medium M-F12. When CHO-K1 cells are incubated in M-F12 without insulin for 48-72 hr, the cells accumulate in G1. In response to physiological concentrations of insulin an 18-fold increase in rate of DNA synthesis occurs due to cells entering S phase after an 8- to 10-hr lag; cell division begins after 24 hr.

alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable 86Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and K1/2 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type.

Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1.

Growth of CHO-K1, a proline auxotroph, is inhibited by amino acids that prevent proline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, alar4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, alar4-H2.

Control of A-system amino acid transport by a second regulatory gene R2 in Chinese hamster ovary cells CHO-K1 and the possible connection of this gene with insulin activity.

Evidence based on a study of alanine-resistant (Alar), constitutive mutants of CHO-K1 cells and the conditions that favor stimulation of the A system of amino acid activity supports the model that the A system of amino acid transport in these cells is repressible and under negative control of regulatory gene R1. In this study, we show that mutant Alar6, when grown under conditions of repression, has an A system of amino acid transport activity similar to that of the derepressed parental cell line, CHO-K1 (wild type) and of the fully constitutive mutant in gene R1, Alar4. However, the A system of Alar6 is further derepressible.

Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants.

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.

Regulation of the A system of amino acid transport in Chinese hamster ovary cells, CHO-K1: the difference in specificity between the apo-repressor inactivator (apo-ri) and the transporter and the characterization of the proposed apo-ri.

When amino acids that are generally transported through the A system are added to derepressed cultures of CHO-K1 cells or to cultures that are undergoing starvation-derepression, as in the co-repressor (co-r), co-inactivator (co-i), (co-ri) assay, the A system undergoes trans-inhibition, inactivation, and repression. The effect of inactivation and repression is not related to the ability of amino acids to bind to the A system transporter but supports a model in which these amino acids act as co-r's/co-i's, and by binding to a aporepressor/inactivator (apo-ri), the product of gene R1, convert it into a repressor/inactivator (ri). For example, beta-alanine acts as a strong co-r but does not inhibit proline transport through the A system.

A defined medium for and the effect of insulin on the growth, amino acid transport, and morphology of Chinese hamster ovary cells, CHO-K1 (CCL 61) and the isolation of insulin "independent" mutants.

Insulin, FeSO4, or transferrin are major requirements together with HEPES buffer and selenium for the growth of CHO-K1 (CCL 61) in a modified F12 medium (M-F12). Insulin stimulates growth at 1 ng/ml to 10 micrograms/ml. In the defined medium minus insulin, CHO-K1 grows slowly as elongated, elliptical cells in parallel arrays typical of normal diploid fibroblasts in contrast to round-to-cuboid cells in loosely overlapping arrays in the presence of serum or insulin.

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.

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.

Recessive 2-(methylamino)-isobutyrate (MeAIB)-resistant mutant of Chinese hamster ovary cells (CHO-K1) with increased transport through ASC system.

Mutants of Chinese hamster ovary cells (CHO-K1 Pro-), resistant to the proline transport antagonist 2-(methylamino)-isobutyrate (MeAIB) were isolated by a single-step procedure. Mutation rates to Pro+ and to Pro- MeAIB resistance (MeAIBr) are 1.7 X 10(-6) and 2.

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems.

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake.

Inhibition of growth of proline-requiring Chinese hamster ovary cells (CHO-k1) resulting from antagonism by a system amino acids.

CHO-K1 requires proline for growth. Two proline-independent revertants were isolated--K1-J and K1-6. CHO-K1 pro- is much more sensitive than the pro+ cell lines to inhibition of growth by addition to the medium of amino acids and amino acid analogues that are transported through the A system.

Inhibition of growth of cells in culture by L-phenylalanine as a model system for the analysis of phenylketonuria. I. aminoacid antagonism and the inhibition of protein synthesis.

Phenylalanine in high concentrations inhibits the growth of mouse A9 cells. Protein synthesis is inhibited earlier and more severely than RNA or DNA synthesis. Phenylalanine inhibits the uptake and decreases the intracellular pool of several amino acids.

Elucidation of an A and L system for amino acid transport in the human lymphoblast using a membrane filtration technique.

Optimum conditions have been established for the measurement of amino acid transport by human lymphoblastoid cell lines using a membrane-filtration technique. The parameters we found to be important for the reproducibility of the method are: the types and combination of filters, the strength of the vacuum applied to the filters and the density of the cultures at the time of harvesting and during uptake and filtration. We found that bovine serum albumin added to phosphate buffered saline (PBS) glucose in which the cells are washed, resuspended and assayed is essential for the maintenance of viability, the prevention of clumping and the retention of the accumulated amino acid.

Constitutive mutations in the controlling site region of the araBAD operon of Escherichia coli B/r that decrease sensitivity to catabolite repression.

Strains of Escherichia coli B/r containing a deletion of the regulatory gene araC are Ara-. Slow-growing revertants of these strains were isolated and designated aralc because they contain a second mutation in a controlling site, aral, that allows for a low level of constitutive expression of the araBAD operon (Englesbert et al., 1969).

The site for catabolite deactivation in the L-arabinose BAD operon in Escherichia coli B/r.

A series of deletions beginning in the leu operon and continuing into the araC gene and also into the ara controlling site region were analyzed in reciprocal merodiploids, e.g., F' A2Cc67/B24delta719, F' B24delta719/A2Cc67, for their effects on catabolite deactivation (CD).

Isolation and characterization of 5-fluorotryptophan-resistant mutants with altered L-tryptophan transport.

Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM.