Identification of cellular proteins required for simian virus 40 DNA replication

Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.     

Expression of a naturally occurring angiotensin AT(1) receptor cleavage fragment elicits caspase-activation and apoptosis

Several transmembrane receptors are documented to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. Our prior studies indicate that a population of the 7-transmembrane angiotensin type-1 receptor (AT(1)R) is cleaved in a ligand-augmented manner after which the cytoplasmic, carboxy-terminal cleavage fragment (CF) traffics to the nucleus. In the present report, we determine the precise cleavage site within the AT(1)R by mass spectrometry and Edman sequencing. Cleavage occurs between Leu(305) and Gly(306) at the junction of the seventh transmembrane domain and the intracellular cytoplasmic carboxy-terminal domain. To evaluate the function of the CF distinct from the holoreceptor, we generated a construct encoding the CF as an in-frame yellow fluorescent protein fusion. The CF accumulates in nuclei and induces apoptosis in CHO-K1 cells, rat aortic smooth muscle cells (RASMCs), MCF-7 human breast adenocarcinoma cells, and H9c2 rat cardiomyoblasts. All cell types show nuclear fragmentation and disintegration, as well as evidence for phosphotidylserine displacement in the plasma membrane and activated caspases. RASMCs specifically showed a 5.2-fold increase (P < 0.001) in CF-induced active caspases compared with control and a 7.2-fold increase (P < 0.001) in cleaved caspase-3 (Asp174). Poly(ADP-ribose)polymerase was upregulated 4.8-fold (P < 0.001) in CF expressing cardiomyoblasts and colocalized with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). CF expression also induces DNA laddering, the gold-standard for apoptosis in all cell types studied. CF-induced apoptosis, therefore, appears to be a general phenomenon as it is observed in multiple cell types including smooth muscle cells and cardiomyoblasts.     

A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.     

Cytoplasmic fraction of Lactococcus lactis ssp. lactis induces apoptosis in SNU-1 stomach adenocarcinoma cells

Lactic acid bacteria are known to have antitumor activity, but the underlying mechanisms remain unclear. Recently we showed that a cytoplasmic fraction - but not peptidoglycan - of Lactococcus lactis ssp. lactis (L.lac CF) had strong antiproliferative activity on SNU-1 human stomach adenocarcinoma cells. The present study investigated whether the antiproliferative activity of L.lac CF on SNU-1 is linked to the induction of apoptosis. Treatment of L.lac CF inhibited the proliferation of SNU-1 cells in a dose- and time-dependent manner. Furthermore, treatment of the cells with 50 microg/ml and 100 microg/ml L.lac CF resulted in DNA fragmentation and chromatin condensation, respectively. The results indicate that the inhibitory effect of L.lac CF on SNU-1 cell growth is mainly attributable to the induction of apoptosis.     

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.     

Autolytic activity associated with competent group H streptococci

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.     

Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky).

Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Serological reactions associated with the clumping factor of Staphylococcus aureus

Rotter, Joan (University of Oklahoma Medical Center, Oklahoma City), and Florene C. Kelly. Serological reactions associated with the clumping factor of Staphylococcus aureus. J. Bacteriol. 91:588-594. 1966.-Evidence that the substance which causes staphylococci to clump in the presence of fibrinogen (clumping factor) is antigenically similar in strains which are serologically diverse according to agglutination reactions has been obtained from fibrinogen-cell clumping inhibition tests. Antisera for clumping factor (CF)-positive strains inhibited the clumping reaction of all strains tested. After adsorption with homologous cells or with cells of other CF-positive strains, the antisera no longer inhibited clumping. When antisera were adsorbed with trypsin-treated, CF-positive cells, or with cells of CF-negative mutants, the ability to inhibit the clumping reaction persisted. Antibody to CF activity was not associated with coagulase. Latex coated with extracts derived from the cells of five CF-positive and six CF-negative strains was, in each instance, agglutinated by sera from rabbits immunized with CF-positive cells. After adsorption with trypsinized, CF-positive cells, antisera still agglutinated latex which had been treated with the CF-positive extracts, but not with the CF-negative extracts. Similar results were obtained after antisera were adsorbed with the cells of CF-negative mutants. Cell agglutination titers of sera from rabbits immunized with CF-negative staphylococci were significantly lower than those produced in response to CF-positive cells, regardless of their coagulase activity. If the CF-inhibiting antibody also functions as an agglutinin, it apparently is not solely responsible for this difference.     

An IgG-Fc receptor induced in cytomegalovirus-infected human fibroblasts.

Cytomegalovirus- (CMV) infected cultured human fibroblasts incubated with normal human serum were shown to react with IgG by direct and indirect fluorescent antibody tests. The binding of IgG to infected cells was unrelated to the presence of anti-CMV complement fixing (CF) antibodies and was not observed with uninfected cells. The reaction was first observed 36 hours after infection as diffuse cytoplasmic staining which by 72 hr became localized into a dense perinuclear structure. The cytoplasmic fluorescence was not detected with anti-IgA or anti-IgM fluorescent conjugates. The reaction was seen with purified IgG and Fc fragments but was only minimally detectable with Fab fragments. It occurred in fibroblasts infected with three standard laboratory strains of CMV and seven recent CMV isolates from patients and was observed in six separate lots of human foreskin fibroblast cultures as well as in WI-38 cells. We conclude that CMV infection induces the formation of a IgG receptor in human fibroblasts.     

Ploidy Effects in Isogenic Populations of Alfalfa : III. Chloroplast Thylakoid-Bound Coupling Factor 1 in Protoplasts and Leaves

The influence of polyploidization on chloroplast coupling factor 1 (CF1) was examined in leaves and leaf protoplasts from isogenic diploid-tetraploid (DDC 2X-4X) and tetraploid-octoploid (IC 4X-8X) sets of alfalfa (Medicago sativa L.). Alfalfa CF1 was purified to homogeneity and was found to contain five subunits with molecular weights of 57,900, 54,300, 38,700, 23,100, and 15,200. Rocket immunoelectrophoresis with anti-spinach CF1 gamma immunoglobulins was used to quantify CF1 from protoplasts and leaves. In the DDC 2X-4X set, fresh weight per cell and cellular content of CF1, chlorophyll (Chl), and DNA doubled with ploidy. Ratios of CF1 to Chl of leaf protoplasts and leaves were similar between diploids and tetraploids. In the IC 4X-8X set, octoploid protoplasts were 90% higher in Chl than were comparable tetraploids, whereas octoploids were 50 to 60% higher than tetraploids in fresh weight per cell and cellular content of CF1 and DNA. Concentrations of CF1 and Chl in leaves and ratios of CF1 to DNA in protoplasts were similar across ploidy levels of both isogenic sets. Therefore, cellular content of CF1 increases proportionately with the amount of DNA per cell or gene dosage.     

Template-directed assembly of receptor signaling complexes

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.     

Inactivation of DNA-Dependent Protein Kinase by Protein Kinase Cδ: Implications for Apoptosis

Protein kinase Cδ (PKCδ) is proteolytically cleaved and activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. A role for PKCδ in apoptosis is supported by the finding that overexpression of the catalytic fragment of PKCδ (PKCδ CF) in cells is associated with the appearance of certain characteristics of apoptosis. However, the functional relationship between PKCδ cleavage and induction of apoptosis is unknown. The present studies demonstrate that PKCδ associates constitutively with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The results show that PKCδ CF phosphorylates DNA-PKcs in vitro. Interaction of DNA-PKcs with PKCδ CF inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate its downstream target, p53. The results also demonstrate that cells deficient in DNA-PK are resistant to apoptosis induced by overexpressing PKCδ CF. These findings support the hypothesis that functional interactions between PKCδ and DNA-PK contribute to DNA damage-induced apoptosis.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

Analysis of DNA polymorphism haplotypes linked to the cystic fibrosis locus in North American black and Caucasian families supports the existence of multiple mutations of the cystic fibrosis gene.

Strong linkage disequilibrium (LD) was found between DNA marker XV2c and the cystic fibrosis (CF) locus (delta = 0.46) and between DNA marker KM19 and CF (delta = 0.67) in 157 CF and 138 normal chromosomes from U.S. Caucasians. DNA haplotypes with nine polymorphic sites were created in 54 Caucasian families. There is a strong LD between the haplotypes and the presence of the mutant CF genes. This implies that the DNA polymorphisms examined are close to the CF gene and that one mutation of the CF gene predominates in the Caucasian population. Haplotype analysis can also be used to refine estimates of CF carrier risk in Caucasians. Data for XV2c and MET markers in 16 American black patients and their families revealed a different haplotype distribution and LD pattern with the CF locus. These data suggest that racial admixture alone does not explain the occurrence of CF in American blacks and that multiple alleles of the CF gene may exist in this population.     

Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.     

Small coelomic epithelial cells of the starfish Asterias rubens L. that are able to proliferate in vivo and in vitro

Echinoderms, due to their outstanding potential for regeneration, are widely used as experimental models for research in regenerative biology. One of the main problems in this field concerns identification and characterization of cells responsible for the restoration of lost body parts and organs in adult animals. In this study, we analyze the probable candidates for this role in the starfish Asterias rubens L., namely, small coelomic epithelial cells with a high nuclear–cytoplasmic ratio that have the ability to proliferate. These cells are one of several cell types common to the coelomic epithelium (CE) and coelomic fluid (CF). They are analyzed with respect to morphology, proportion in the total cell pool, dynamics after injury and distribution between CE and CF. The results of whole-mount and scanning electron microscopy provide evidence that these small cells occupy a boundary position between CE and CF. Moreover, a novel subpopulation of CE cells is identified that is enriched (up to 50 %) with small epitheliocytes capable of migrating from CE into the CF. As shown in experiments with BrdU incorporation and anti-phospho-histone H3 antibody staining, small epitheliocytes cultured on laminin retain proliferative activity for at least 1 month and can form colony-like aggregates. Two types of small proliferating cells are distinguished by their behavior in culture: some cells remain attached to the substrate and form aggregates, while others detach from the substrate during culturing. The morphology of small epitheliocytes, their proliferative activity in vivo and in vitro and the ability to migrate suggest that they possess certain properties characteristic of stem cells.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.