Evidence for a regulatory element controlling amino aced transport system L in Chinese hamster ovary cells

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO–;tsO25C1 has a temperature-sinsitive mutationin leucyl-tRNA synthetase. At the nonpermissive temperature of 39^oC, CHO–tsO25C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell, CHO–C11B6, that has constitutively elevated system L transport activity. The CHO–C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads of increased intracellular leucine levels, which compensate for the defective. Hybrid cells formed by fusion of the temperature-sensitive CHO–;tsO25C1 cells the temperature-resistant CHO–C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO–C11B6 cell line.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.     

Association of human chromosome 14 with a ts defect in G1 of Chinese hamster K12 cells.

Somatic cell hybrids between human lymphoblastoid cells (Raji) and temperature-sensitive Chinese hamster cells (K12) were selected from monolayer cultures in MEM at 40 degrees C. A total of 21 hybrid clones were isolated and karyotyped. All clones contained a near complete set of Chinese hamster chromosomes and 1 to 5 human chromosomes. Human chromosome 14 present in the hybrid cells of all clones; and was the only human chromosome retained in 10 clones. The presence of human chromosome 14 in hybrids was further confirmed by the demonstration of human nucleoside phosphorylase activity in the hybrid cells. Only one hybrid clone was positive for EBNA, the Epstein-Barr virus antigen present in Raji cells. These findings indicate that human chromosome 14 contains the necessary information for the K12 cells to overcome their G1 defect in the cell cycle and grow at non-permissive temperature. The present study lends strong support to the possibility that different steps in the G1 phase of the cell cycle are controlled by genes located on different chromosomes.     

Characteristics of somatic cell hybrids (mouse x Chinese hamster) with a different ratio of chromosome sets from the parent species. II. Thymidine kinase activity]

The activity of thymidine kinase (TK) was studied in series of somatic cell hybrids between the mouse cell line 3T3-4E (TK-) and Chinese hamster cells M-15-1 (HGPRT-). Four groups of hybrid lines with different ratio of parental chromosome sets have been investigated: 1) three lines containing one hamster and one mouse chromosome set (1 hs+1 ms); 2) one line with 2 hs+1 ms; 3) one line containing 3 hs+1 ms and 4) one line containing 1 hs+2 ms. Mixtures of extracts from the parental cells were shown to possess the expected TK activity. The calculation of the activity per cell revealed that the 1 hs+1 ms and 2 hs+1 ms hybrid lines possessed about 50% of the initial hamster cell TK activity. The decreased TK activity in these hybrids might be due either to a loss of hamster chromosomes or to some inhibitory effect of mouse genome in cells with the studied ratio of parental sets. The enzyme activity in the 3 hs+1 ms hybrid was as expected, about three times greater than that of hamster cells.     

Chromosome replication in somatic hybrids of mouse and temperature sensitive Chinese hamster cells

Hybrid clones were obtained between a mouse cell line (3TP) and a temperature-sensitive Chinese hamster cell line (K12) unable to grow at 40° because of a ts defect apparently located at the G1/S transition. The great majority of hybrid clones grew at 40°, showing the ts defect to be “recessive.” Chromosome DNA replication was analyzed in some detail in three hybrid clones with balanced complements. Although the S period of these hybrids was longer than that of K12, DNA replication in mouse and hamster chromosomes started and ended synchronously. Upon prolonged culture, mouse chromosomes were lost as they are in hybrids involving a non ts Chinese hamster partner, in which case asynchronous chromosome replication appears to be the rule. It seems therefore that asynchronous replication is not the determining factor in chromosome loss from cell hybrids.     

Chromosomally depleted interspecific hybrid cell clones selected with cytotoxic antisera: Utilization in the study of control of murine leukemia virus host-range

A chromosomally stable mouse-Chinese hamster hybrid cell line was subjected to five rounds of selection with cytotoxic antisera raised in rabbits against either the parental mouse 3T3 cells or the parental Chinese hamster Wg-1 cells. Routine karyological analysis of clones isolated at each stage of serum selection revealed that treatment with either serum resulted in a limited loss of chromosomes (compared to the untreated hybrid cell cultured in parallel) and that the pattern of chromosome loss could not be correlated with the particular antiserum used for selection. However, more detailed analysis with the SSC-formamide C-banding technique, which identifies chromosomes containing a mouse centromere region, demonstrated that while large-scale chromosome loss was not achieved as a result of antiserum selection, the limited loss of chromosomes did, in fact, reflect a specific depletion of chromosomes in response to treatment with cytotoxic antiserum. Specific chromosomal elimination was shown to occur as early as the first round of antiserum treatment. Antigenic analysis of the serum-selected clones revealed a quantitative decrease in the expression of the species-specific surface antigens selected against, but no qualitative loss of antigens was detected. The results suggest that treatment with cytotoxic antiserum may select for clones that have lost specific chromosomes bearing genes regulating the expression of species-specific surface antigens, rather than for those demonstrating large-scale depletion of chromosomes bearing the corresponding structural genes. Some of these chromosomally depleted hybrid cell clones have been used (along with pseudotype viruses containing the genome of vesicular stomatitis virus within the envelope of murine leukemia virus, VSV [MuLV]), to study the mechanisms regulating MuLV replication in Chinese hamster cells. The results indicate that the restriction of MuLV replication in Chinese hamster cells operates at two levels: (a) an inability to adsorb to or penetrate Chinese hamster cells; and (b) an additional intracellular block which is dominant in the mouse-Chinese hamster hybrid cell clones examined. This latter block is presently under study.     

A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex

The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts025Cl with a defect in leucyl-tRNA synthetase (LeuRS) does not have an inherently more thermolabile LeuRS, but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same K _m for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS.This work was supported by NIH Grant GM 19506 to A.E.H. and GM 20737 to D.E.O.     

A temperature-sensitive auxotropic variant of Chinese hamster ovary cells

We used a conventional procedure involving treatment with 5-bromodeoxyuridine and visible light to isolate a stable, temperature-sensitive, auxotrophic variant-TsNd-6-from its parental Chinese hamster ovary cell clone K₁. At the nonpermissive temperature of 39.5 °C, TsNd-6 requires thymidine, hypoxanthine, and glycine for growth. Folinic acid can substitute for hypoxanthine and glycine at the elevated temperature.     

Simultaneous activity of nucleolus organizer regions of human and Chinese hamster chromosomes in somatic cell hybrids

In an interspecific human-Chinese hamster hybrid that retains 13 and 85.6% of the chromosomes of each parental complement, activity of nucleolus-organizing regions (NOR) of both type chromosomes is observed in 18.9% of the cells. Interspecific chromosomal associations are also noted. Unlike the parental lines of Chinese hamster cells, the hybrids show the associations of the NOR of Chinese hamster chromosomes. In hybrid cells, there occurs partial suppression of NOR activity in human and Chinese hamster chromosomes, while the NOR of the 3d chromosome of the Chinese hamster is completely suppressed.     

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.     

A mammalian temperature-sensitive mutation affecting G1 progression results from a single amino acid substitution in asparagine synthetase.

ts11 is a temperature-sensitive (ts) mutant isolated from the BHK-21 Syrian hamster cell line that is blocked in the G1 phase of the cell cycle at the non-permissive temperature (39.5 degrees C). We previously showed that the human gene encoding asparagine synthetase (AS) transformed ts11 cells to a ts+ phenotype and that ts11 cells were auxotrophic for asparagine at 39.5 degrees C. We show here that ts11 cells exhibit a ts phenotype for AS activity, and that the ts11 AS was much heat-labile than the wt enzyme. We have isolated AS cDNAs from wt BHK and ts11 cells and found that wt, but not ts11 AS cDNAs were capable of transformation. The deduced amino acid sequence of Syrian hamster AS showed 95% identity to the human protein as well as the same number of residues. The inability of the ts11 AS cDNAs to transform was due to a single base change, a C to T transition, that would result in the substitution of leucine with phenylalanine at a residue located in the C-terminal fourth of the enzyme. Thus the ts11 mutation identifies a mutated, thermolabile AS.     

Isolation of a line of immortal chicken embryo fibroblasts after transfection with the nuclei of Rous sarcoma virus-transformed Chinese hamster cells

Secondary cultures of chicken embryo fibroblasts were transfected with purified nuclei from lysed cells of a clonal line of temperature-sensitive Rous sarcoma virus (tsRSV)-transformed Chinese hamster fibroblasts. After propagation for 3 months an established cell line designated ChR32 was obtained in one chicken cell culture. The cells of this line have been propagated so far for 18 months, whereas normal chicken embryo fibroblasts died after 2 months. The established cells were heteroploid with a diploid modal number of macrochromosomes and two Z chromosomes. No Chinese hamster chromosomes could be identified. Southern blot analysis of DNA from the uncloned ChR32 cells and the clones provided evidence that these established cells were, in fact, clonal in origin and contained full-length RSV proviruses and no defective proviruses. Furthermore, they contained, at the 3' end proviral-cellular junction, Bg/II, HpaI, KpnI, SacI, and XbaI fragments of the same size as the Chinese hamster donor cells, suggesting that the cellular sequence adjacent to the provirus is of Chinese hamster origin. The cells after establishment were able to grow continuously at 37 degrees or 41 degrees C and produce a large amount of ts sarcoma virus particles. A corollary finding was that these virus particles were non-leaky for the transforming function at the non-permissive temperature.     

Hybridization of Chinese hamster cells sensitive to ultraviolet irradiation

Resistance to UV-light was studied in two UV-sensitive aneuploid Chinese hamster cell clones to different origin and degree of sensitivity, their respective polyploids and somatic cell hybrids. The karyotype of the parental clones, cell hybrids and polyploids was analyzed in parallel. A great variability of karyotypes was detected in hybrid cells. Serial cultivation of hybrids was accompanied by chromosome loss. Soon after fusion the hybrid clones proved to be more resistant to UV than the parental sensitive cells. However, their sensitivity increased with passages. The comparison of UV-sensitivity with data on karyotype analysis allowed to assume that the increase in sensitivity was correlated with the loss of particular chromosomes or chromosome regions. The results obtained indicated the existence of a polygenic control of UV-sensitivity, the multiple genes being assigned to different chromosomes. A reverse effect of ploidy was detected, i.e. a decrease in the resistance to the lethal action of UV-light in polyploids as compared to the parental clones.     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.     

Complementation between two temperature-sensitive mammalian cell mutants, each defective in the G1 phase of the cell cycle

Two mammalian temperature-sensitive (ts) G1 cell cycle mutants of different species origin (Syrian hamster and mouse) have been tested for complementation using somatic cell hybrid analysis. All hamster-mouse hybrid clones tested were found to exhibit normal growth properties at the restrictive temperature, while neither mutant alone was capable of normal growth at this temperature. The two mutant lines therefore complement for growth in a somatic cell hybrid and most likely represent ts lesions in different cellular functions specific to the G1 phase of the cell cycle.     

Novel cytogenetic expression of gene amplification in actinomycin D-resistant somatic cell hybrids: transfer of resistance by centric chromatin bodies

SEWATC13 mouse cells, resistant to 0.1 g/ml of actinomycin D (AMD), were fused to AMD-sensitive cells of the Chinese hamster ovary cell line (CHO). Twentytwo hybrid clones were isolated and put into serial culture in the selective medium. Unexpectedly, identifiable mouse chromosomes were found only in one of the hybrids. All the others had only hamster chromosomes and, in addition, numerous chromatin bodies (CBs), mostly small and irregularly shaped, but also larger, more chromosome-like ones. The CBs were distinctly C-band positive and a mouse satellite probe hybridized strongly to them. The AMD resistance of the murine parental cells had previously been attributed to gene amplification in two large homogeneously staining regions (HSR-AMD1 and 2). They were not observed in the hybrid cells but had supposedly reappeared in the guise of the CBs. It was established by Southern DNA blot analysis that amplified DNA sequences, localized to the HSRAMD1 and 2 of the SEWA parent were present in multiple copies in the hybrids. It was also established by in situ hybridization that they were located in the CBs. Unlike double minutes (DMs) the CBs were all centric.     

Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.     

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.     

Esterase gene expression in Chinese hamster and mouse lymphoma hybrids isolated under nonselective pressure

Hybrids between a fibroblastic Chinese hamster cell line (CH23) and a mouse lymphoma cell line (P388F36) were produced and isolated by a simple new method without using selective media and avoiding contact with the parental cells. The chromosomal situation in the two hybrid types (PCM and PCS) isolated suggested that growth on glass surface (PCM) or in suspension (PCS) depended on the number of hamster and mouse chromosomes which existed in the hybrids. Chromosomal stability in hybrids grown as monolayers (PCM) was reached at a stage in which two to four mouse chromosomes coexisted with no fewer than 19 hamster chromosomes. In a study of gene linkage utilizing clones of this hybrid population, five out of nine genes regulating the synthesis of different esterases in the mouse cells used were found to be unlinked.