Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.     

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.     

Nucleotide excision repair is not required for the antiapoptotic function of insulin-like growth factor 1

Potential genetic determinants of dengue virulence were studied by sequencing the entire genomes of eight dengue 2 virus strains isolated from patients exhibiting different disease severities during an epidemic season in northeastern Thailand in 1993. The isolates came from one dengue shock syndrome (ThNH-7/93), three dengue hemorrhagic fever, and four dengue fever patients. Phylogenetic analysis showed that the isolates belonged to the Southeast Asian genotype. The 3' noncoding regions showed distinctive secondary structures, with one specific structure for the isolate ThNH-7/93. Analysis of the predicted polyprotein showed several amino acid (aa) changes scattered mostly in the nonstructural region. Of 30 positions with aa changes, 7 were unique to the isolate ThNH-7/93 and 3 of those led to radical alterations in aa character. Several aa changes coincided with previous studies relating genome sequence and virulence. Minimal changes in computer-predicted protein secondary structures were observed. Infective particles in the inoculum for all isolates were approximately equal as measured by focus formation on BHK-21 cells, but this did not correlate with the number of plaques formed on LLC-MK2 cells. Isolates from patients that experienced secondary infection were shown to have significantly larger plaques than the isolates from primary infection patients.     

Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells

To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.     

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.     

Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L-iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.     

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.     

Regeneration and luminescence of aequorin in Chinese hamster ovary cells transformed with cDNA for apoaequorin

Aequorin, a photoprotein which is regenerated from apoaequorin by incubation with coelenterazine, emits light when it binds Ca2+. The aim of this study was to determine if apoaequorin could be used in adherent mammalian cells for measuring cytosolic Ca2+, and imaging Ca2+, at the single cell level. Chinese hamster ovary (CHO-K1) cells were stably transformed with apoaequorin cDNA and expressed apoaequorin while attached to the culture dishes. Maximal luminescence intensity was obtained when 0.5 x 10(6) cells/ml were grown and incubated with 2.5 microM coelenterazine for 4 hr at 20 degrees C. Ca2+ mobilizing agents (ionomycin and maitotoxin) induced luminescence in CHO-K1 transformed cells. However, imaging of light emission from single cells proved to be unsuccessful. Ca2+ could be readily measured in the adherent CHO-K1 cells, but imaging was not possible at the single cell level.     

Cloning and characterization of cDNA for adenosine kinase from mammalian (Chinese hamster, mouse, human and rat) species. High frequency mutants of Chinese hamster ovary cells involve structural alterations in the gene

In a previous investigation of bifidobacteria isolated from human dental caries (V. Scardovi and F. Crociani, Int. J. Syst. Bacteriol. 24:6-20, 1974), 40 strains were assigned to the new species Bifidobacterium dentium. In this study we examined 70 new strains of bifidobacteria isolated from dental caries. The morphological characteristics, biochemical reactions, fermentation patterns, end products from glucose metabolism, protein electrophoretic patterns, levels of DNA hybridization, and DNA G+C contents of these organisms revealed that they belong to three different taxa. One of these taxa was identified as B. dentium. The other two are described as the following new Bifidobacterium species in this paper: Bifidobacterium inopinatum (type strain, DSM 10107) and Bifidobacterium denticolens (type strain, DSM 10105). The two new species differ from other Bifidobacterium species in their morphological characteristics (especially B. inopinatum, with its very small coccoid cells), in their carbohydrate fermentation patterns (most strains ferment dextran, and B. inopinatum does not ferment galactose), and in their DNA base compositions (especially B. inopinatum).     

Activation of aniline by extracts from plants and induction of chromosomal damages in Chinese hamster ovary cells

Activation of aniline by plant extracts was studied by a chromosomal damage induction assay in Chinese hamster ovary (CHO) cells in vitro. Extracts from roots of Vicia faba activated aniline and the activation caused increases in chromosomal aberrations (CAs) and endoreduplicated cells (ERCs), but did not cause sister-chromatid exchanges (SCEs). Extracts from Pisum sativum and Lactuca sativa, however, did not activate aniline. All C-hydroxylated metabolites of aniline, o-aminophenol, m-aminophenol and p-aminophenol, induced not only CAs but also SCEs in CHO cells. These results show that the pathway for aniline activation by Vicia extracts is by means other than the C-hydroxylation.     

Functions of the N-glycans of rat leukemia inhibitory factor expressed in Chinese hamster ovary cells

A non-covalently coimmobilized bienzymic reactor of horseradish peroxidase (HRP) and cholesterol oxidase (COD), operating in a continuous organic flowing stream of 1X10-3 M p-anisidine in buffer-saturated (pH 7.0) toluene, has been employed for cholesterol determination in animal greases, such as pig, beef, and chicken fat, and codfish liver oil. The method provides a good linear relationship up to 1.8 X 10-3 M cholesterol and average recoveries of 99.5%, a high sensitivity, with a detection limit of 1 X 10-6 M of cholesterol and a good precision (an interday RSD of 1.8% for the determination of total cholesterol in a codfish oil sample). The method permits the direct spectrophotometric determination of free cholesterol present in animal grease samples without any pre-treatment, and the total cholesterol determination after a microwave-assisted saponification. The bienzymic reactor exhibits a good stability in the water-restricted environment, being possible to perform more than 180 analyses during a period of 10d with 1mg of HRP, and 1 mg of COD, equivalent to 220 purpurogallin units and 23 U, respectively.     

Biosynthesis of the prohormone convertase PC2 in Chinese hamster ovary cells and in rat insulinoma cells

The biosynthesis of the prohormone convertase PC2 was studied in Chinese hamster ovary cells stably transfected with PC2 cDNA (CHO/PC2) and in rat insulinoma cells (Rin5f). The major form of PC2 synthesized by CHO/PC2 cells was a 75-kDa protein corresponding to proPC2; this protein was retained intracellularly for 2-4 h following synthesis, suggesting prolonged intracellular residence. In contrast, the major form of PC2 within Rin cells initially exhibited a molecular mass of 72 kDa and was then progressively converted to a 64-kDa species. This 64-kDa species, which required 1-2 h to be released, was the major PC2 form detectable in Rin cell medium. Calcium-dependent benzyloxycarbonyl-Arg-Ser-Lys-Arg-aminomethylcoumarin cleaving activity was found in spent Rin cell medium; this activity could be immunoprecipitated with a carboxyl-terminal PC2 antibody, but not with preimmune serum. In neither cell line did intracellular PC2 become endoglycosidase H-resistant over time. PC2 released from Rin cells was also endoglycosidase H-sensitive. Microsequencing and endoglycosidase H results indicate that 75-kDa CHO cell PC2 and 72-kDa Rin cell PC2 both represent proPC2. We speculate that (a) PC2 undergoes unusual glycosylation, which may be related to its slow release from cells, and (b) the 64-kDa molecule detectable in spent Rin cell medium represents the enzymatically active form of PC2.     

Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

Responses of individuals to the loss of a primary attachment object may be quite variable. In humans, it has been suggested that only about 25% of bereavements result in substantial psychological or medical morbidity (Hamburg et al. 1975). In nonhuman primates, which are used to model responses to separation and loss, a similar estimate of about 25% has also been obtained (McKinney 1985). In addition, there are wide-ranging species differences in vulnerability with regard to the nature and severity of the response to maternal separation and/or loss. All of these findings suggest that there are important processes, intrinsic and/or extrinsic to the individual, that contribute to the probability that a loss will produce a major behavioral or physiological response. We have been systematically examining some of the factors that may account for a portion of this variability in two species of macaques (bonnet monkeys Macaca radiata; and pigtail monkeys, M. nemestrina).     

Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells

Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]-AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. Scatchard analysis of saturation isotherms for the specific binding of [3H]-AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nM and a maximum receptor density (Bmax) of 1580 fmol mg(-1) protein, 5230 fmol mg(-1) protein and 7020 fmol mg(-1) protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC-21268, OPC-31260, SR 49059 and SR 121463A, to inhibit binding of [3H]-AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC-31260>SR 121463A> >OPC-21268 and at hV2 receptors, YM087=SR 121463A>OPC-31260>SR 49059> >OPC-21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059> >SR 121463A=YM087=OPC-31260=OPC-21268. In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nM and 0.90 nM, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nM. The potency order of antagonists in inhibiting AVP-induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.     

Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27

1. Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and alterations in muscle insulin-stimulated glucose uptake lead to derangements in whole body glucose disposal. 2. Furthermore, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes, which suggests that increasing GLUT4 in muscle by pharmacological intervention may be an effective therapy in insulin-resistant states. 3. This highlights the importance of understanding the pathways that upregulate GLUT4 glucose transporter expression in muscle. 4. We review studies describing the regulation of GLUT4 and the information currently available on the mechanisms that control GLUT4 expression in muscle.     

Activation of phospholipase A2 by the nociceptin receptor expressed in Chinese hamster ovary cells

To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A2 (PLA2) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca2+-dependent cytosolic PLA2 (cPLA2) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA2, suggesting that phosphorylation of cPLA2 is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA2 phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA2 through the action of PTX-sensitive G proteins and suggest that cPLA2 is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Thirty-six isolates, from man or swine, of Pasteurella multocida subsp. multocida producing (n = 13) or not producing (n = 23) the dermonecrotic toxin (DNT) were studied by numerical analysis, capsular typing and ribotyping. Toxigenic strains were also characterised by restriction fragment length polymorphism (RFLP) of the toxA gene and pulsed-field gel electrophoresis (PFGE). Numerical analysis differentiated the Pasteurella species and subspecies, but did not discriminate between toxigenic and nontoxigenic strains. RFLP demonstrated that toxA was located in a conserved part of the chromosome of all toxigenic strains. Ribotyping provided evidence of a close association between DNT production and one of the six EcoRI ribotypes designated as E2. In contrast, PFGE provided evidence for significant DNA polymorphism amongst the toxigenic strains. Results of phenotypic and genotypic studies suggested that toxigenic strains do not form a clone within the subspecies multocida. No difference was found between toxigenic strains of porcine or human origin by biochemical characterisation, capsular serotyping or genomic typing methods.