Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells.

Using the S49 T-cell lymphoma system for the study of immunodeficiency diseases, we characterized several variants in purine salvage and transport pathways and studied their responses to the cytotoxic action of adenosine (5-20 micron) in the presence of adenosine deaminase (ADA) inhibitors. Both an adenosine transport deficient mutant and a mutant lacking adenosine (ado) kinase activity are resistant to the cytotoxic effects of adenosine up to 15 micron. Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase are sensitive to the killing action of adenosine. We monitored the intracellular concentrations of purine and pyrimidine nucleotides, orotate, and PPriboseP in mutant and wild-type cells following the addition of adenosine and an ADA inhibitor. We conclude that at low concentrations, adenosine must be phosphorylated to deplete the cell of pyrimidine nucleotides and PPriboseP and to promote the accumulation of orotate. These alterations account for one mechanism of adenosine toxicity.     

Further characterization of a fodrin-containing transmembrane complex from mouse T-lymphoma cells

A transmembrane complex containing fodrin (an actin-binding protein) and a major surface glycoprotein (GP 180) was previously isolated from mouse T-lymphoma cells by the complementary techniques of non-ionic detergent extraction and sucrose gradient centrifugation (Bourguignon et al. (1985) J. Cell Biol. 101, 477-487). The analysis of this complex has been extended to verify the structural association and further define the interaction between fodrin and GP 180. The association between fodrin and GP 180 has been confirmed by the following evidence: co-sedimentation of fodrin and GP 180 in a single peak on a sucrose gradient with a sedimentation coefficient of 20 S; a constant ratio of fodrin and GP 180 across the 20 S peak; the specific co-precipitation of GP 180 with fodrin from the 20 S peak using anti-fodrin antibody; and the colocalization of fodrin and GP 180 from the 20 S peak on actin filaments using an immuno-electron microscopic technique. Furthermore, this fodrin-GP 180 complex can be readily dissociated and reassembled in the presence and absence of 0.6 M NaCl, respectively. The fact that this fodrin-GP 180 complex displays actin-binding ability indicates that this transmembrane complex may play an important role in the linking event between receptors and the cytoskeleton during lymphocyte patching and capping.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.     

Isolation of Thy-1 caps and analysis of their phospholipid composition in mouse T-lymphoma cells

In this study we have used a density perturbation method to isolate anti-Thy-1 antibody-induced Thy-1 caps from mouse T-lymphoma cells in the absence of detergents, and then compared the phospholipid composit on of these capped membranes with that of uncapped membranes. Initial phospholipid analysis by two-dimensional thin layer chromatography (2-D TLC) reveals a significant increase in the amount of ³²P-labeled phosphatidylcholine in the Thy-1 capped membrane. In contrast, no significant changes are observed in the labeling of phosphatidylserine, phosphatidylethanolamine, or the sphingomyelins. Therefore, it is suggested that phosphatidylcholine may be involved in the organization and/or regulation of Thy-1 antigen redistribution. The composition of phosphoinositide in uncapped and capped membranes was analysed separately using one-dimensional thin layer chromatography (1-D TLC) to resolve phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4, 5-bisphosphate (PIP₂) from all other phospholipids. This analysis reveals a significant reduction in levels of PIP and PIP₂, but not PI, in Thy-1 caps. Through the use of ion exchange column chromatography, we have found an increased production of all three species of inositol phosphates during anti-Thy-1 antibody-induced capping. Inositol 1, 4, 5 -triphosphate (IP₃) shows the most significant increase, compared to the much smaller increases in inositol 4, 5-bisphosphate (IP₂) and inositol monophosphate (IP). These results suggest that the binding of anti-Thy-1 antibody to Thy-1 antigen activates phospholipase C which, in turn, initiates polyphosphoinositide turnover and IP₃ production. It is proposed that these observed effects are the result of early signal transducing events which are prerequisite steps in Thy-1 receptor cap formation.     

Multiple forms and inhibitors of uridine-cytidine kinase in neoplastic cells

1. Two forms (isozymes) of uridine (urd)-cytidine (cyd) kinase are present in the 30-50% ammonium sulfate fraction of the cytosols of L1210 ascites leukemia cells and a human malignant lymphoma. 2. These findings confirm those which described multiple forms of urd-cyd kinase in tumors with rapid growth rate. 3. Studied of inhibitors (nucleoside analogs) of urd-cyd kinase derived from L1210 and 6410 leukemia cells resulted in the finding of four possible inhibitors of this enzyme.     

UV sensitivity in thymidine kinase deficient mouse erythroleukaemia cells

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.     

Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity

Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map.     

Glial tumor cells deficient in thymidine kinase: isolation and characterization

Rat astrocytoma cells were treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine and grown in the presence of high concentrations of the thymidine analog 5-bromo-2′-deoxyuridine. A surviving clone, designated C₆TK⁻, lacked thymidine kinase but exhibited differentiated functions such as catecholamine-sensitive adenylate cyclase activity, cortisol-inducible glycerol-1-phosphate dehydrogenase activity, and S-100 protein biosynthesis.     

Clonal variation in cultured neuroblastoma cells. I. Isolation and characterization of variants

Two spontaneously arising variant clones were selected from the N18 neuroblastoma cell line solely on the basis of their flattened morphology and tight adherence to the culture flask. Two other clones having the round loosely adherent morphology typical of the parent line were also selected, and flat variants were shown to arise in them upon prolonged cultivation. The flat variant clones have slower growth rates in culture, lower cloning efficiencies in suspension, and reduced acetylcholinesterase inducibility when compared with either the parent N18 line or the round cell clones. Cells of both morphologic types have high levels of plasminogen activator and are tumorigenic, although the variants have a slower growth rate in vivo, consistent with their slower growth rate in culture. SDS-polyacrylamide gel electrophoresis of total protein from the two cell types shows that the flat variants have increased amounts of a 200,000 molecular weight polypeptide that has tentatively been identified as the heavy chain of myosin. Round morphological revertants from one of the flat variant clones exhibited growth characteristics typical of the parent N18 line, but their content of myosin heavy chain, although reduced, was not so low as that in the round cell clones originally isolated. The possibility of a causal relationship between flat morphology, reduced suspension cloning efficiency, and increased content of myosin heavy chain is discussed.     

Isolation and characterization of tropomyosin-containing microfilaments from cultured cells

We have developed a new method for the rapid isolation of tropomyosin-containing microfilaments from cultured cells using anti-tropomyosin monoclonal antibodies. Anti-tropomyosin monoclonal antibodies induce the bundle formation of microfilaments, which can be easily collected by low speed centrifugation. Electron microscopic studies of the isolated microfilaments show periodic localization of tropomyosin along the microfilaments of nonmuscle cells with a 33-34 nm repeat. Furthermore, the isolated microfilaments have the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin to almost the same extent as skeletal muscle F-actin (filamentous actin). This microfilament isolation method is applicable to a variety of cell types, including REF-52 cells (an established rat embryo line), L6 myoblasts, 3T3 fibroblasts, Chinese hamster ovary cells, baby hamster kidney (BHK-21) cells, mouse neuroblastoma cells, gerbil fibroma cells, and chicken embryo fibroblasts. Sodium dodecyl sulfate-polyacrylamide gel analysis shows that, in addition to actin, microfilaments isolated from REF-52 cells contain five species of tropomyosin with apparent Mr = 40,000, 36,500, 35,000, 32,400, and 32,000, alpha-actinin, and as yet unknown proteins with apparent Mr = 83,000 and 37,000. The molar ratio of total tropomyosin (dimer) to actin in the isolated microfilaments is 1:8. The patterns of these multiple forms of tropomyosin were found to change when REF-52 cells were transformed with SV40 or adenovirus type 5.     

Isolation and characterization of an 18 kDa hypusine-containing protein from cultured NB-15 mouse neuroblastoma cells

An 18 kDa protein can be metabolically labeled by [3H]putrescine or [3H]spermidine in various mammalian cells. The labeling is due to a post-translational modification of one lysine residue to hypusine using the aminobutyl moiety derived from spermidine. In view of the lack of knowledge of the function of this spermidine-modified protein, we decided to use the radioactivity associated with the [3H]spermidine-labeled 18 kDa protein as a tracer to develop a simple procedure for purifying this protein from cultured cells. We first screened more than 15 different affinity adsorbents for their ability to bind the labeled 18 kDa protein. This approach enabled us to develop a four-step procedure to purify the labeled 18 kDa protein from NB-15 mouse neuroblastoma cells. The procedure, including a Cibacron Blue column, an omega-aminooctyl-agarose, a Sepharose G-50, and a Mono Q column, resulted in an 800-fold purification of the labeled 18 kDa protein. Two-dimensional gel analysis of fractions enriched in the labeled 18 kDa protein revealed (i) the presence of isoforms of hypusine-containing 18 kDa protein, with pI values ranging from 4.7 to 5.2, and (ii) the presence of an additional labeled protein with an apparent molecular mass of 22 kDa and a pI value of 5.0. The labeling intensity of the 22 kDa protein, however, was less than 5% of that of the 18 kDa protein. Peptide map analysis, using the V-8 proteinase digestion method, indicated that the 18 kDa hypusine-containing protein obtained from NB-15 cells was similar to eukaryotic initiation factor 4D isolated from rabbit reticulocytes.     

Isolation and characterization of proteoglycans synthesized by cultured mesangial cells

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.     

Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells

This article describes the selection of auxotrophic mutants of Chinese Hamster Ovary (CHO) cells and the genetic and biochemical characterization of two mutant lines. AUXB1 is auxotrophic for glycine, adenosine, and thymidine (GAT-), whereas AUXB3 requires only glycine and adenosine (GA-). These mutants do not complement since hybrid cells formed between them are also auxotrophic. Experiments concerned with the reversion of AUXB1 to prototrophy suggest that a single genetic lesion is responsible for the multiple auxotrophy. Biochemical analysis indicates that the multiple auxotrophy of both AUXB1 and AUXB3 is a result of low levels of intracellular folates in mutant cells. Phenotypic reversion to complete or partial prototrophy can be accomplished by growing these cells in high concentrations of folic or folinic acids. However, neither the folate transport nor the dihydrofolate reductase are defective in mutant cells. Chromatographic analysis of intracellular folate derivatives indicates that while folates extracted from wild type cells exist almost exclusively as polyglutamyl derivates (primarily pentaglutamates), AUXB1 cells contain primarily folate derivates in monoglutamyl form and AUXB3 cells contain mono-, di-, and perhaps some triglutamates. This observation suggests that the enzyme responsible for linking glutamate residues onto intracellular folate derivates is the site of the biochemical lesion in the mutant cells. Our results also suggest that a possible function of polyglutamyl residues is to aid cellular retention of folates.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.