Deoxyribinucleic acid-binding proteins in virus-transformed cell lines.

The synthesis of proteins with affinity for DNA has been studied in clones of a Syrian hamster cell line (NIL) and subclones of this line transformed by polyoma virus (NIL-Py) or hamster sarcoma virus (NIL-HSV). The results show that the synthesis of DNA-binding proteins in NIL and in its virus-transformed derivatives NIL-Py and NIL-HSV is very similar in exponentially growing cells, but in dense culture there is a very significant difference in the level of a protein (P8), which is much higher in the transformed lines than in untransformed NIL. The high levels of P8 in dense transformed cells have been observed in all the clones of transformed cells examined, indicating that this behavior of P8 is related to transformation and not simply due to a fortuitous clonal selection from the NIL. Experiments with synchronized cells indicate that the time of maximal P8 synthesis relative to cellular DNA synthesis in NIL-HSV precedes that observed in NIL cells. P8 has a molecular weight of 30,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is present in large amounts in the transformed cells in dense culture, where it makes up 0.5 to 1% of the total soluble protein.     

DNA binding of a nonstructural reovirus protein

The specific early inhibition of DNA synthesis in reovirus-infected cells suggests that the cell nucleus is a target for virus-induced damage. We have now examined the affinity of reovirus proteins for DNA, postulating that such affinity could provide a mechanism for the inhibition. Cytoplasmic and nuclear extracts of cells labeled with [35S] methionine from 6 to 8.5 h after infection at high multiplicity was subjected to chromatography on denatured DNA - cellulose columns. Fractions from both cytoplasm and nucleus eluted with 0.6 N NaCl contained a protein with the same electrophoretic mobility of polyacrylamide slab gels as the nonstructural (NS) reovirus protein of the sigma size class. The protein also exhibited affinity for native DNA - cellulose and denatured DNA - agarose. Electrophoretic analysis is tube gels of cell extracts labeled for 48 h before infection with [14C] leucine and from 6 to 8.5 h after infection with [3H] leucine showed increased 3H label in this protein indicating it is reovirus specific. Small amounts of mu proteins also had DNA affinity. Purified virus did not bind strongly to DNA, suggesting that the binding protein is not a structural protein of the sigma size class on the outer surface of the virus. Our results provide evidence that the sigma NS protein binds to DNA. This affinity could interfere with chromosome function in the infected cell.     

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.     

Specific immune serum to the Epstein-Barr virus DNA polymerase.

Epstein-Barr virus (EBV) DNA polymerase was released from phorbol ester-treated tamarin (Saguinus oedipus) cells (B95-8) and prepared for use as an antigen by sequential column chromatography with DEAE-Sephadex A-25, DEAE-cellulose, phosphocellulose, and single-stranded DNA cellulose. Proteins from single-stranded DNA cellulose with DNA polymerase activity in 100 mM ammonium sulfate were mixed with complete Freund adjuvant and injected intradermally into rats and rabbits. Immune sera that were screened for specific antibody by indirect immunofluorescence procedures reacted with approximately 3% of the cells in EBV-producer cultures (B95-8 and P3HR-1) but not with EBV genome-negative cells (BJAB). In functional enzyme assays, immune sera or the immunoglobulin fraction inhibited the activity of purified EBV DNA polymerase 90%. Inhibition of enzyme activity was not affected by absorption of immune sera with insoluble matrices of proteins prepared with tamarin and human cells which lacked the EBV genome. Cellular DNA polymerase alpha was not inhibited by immune sera to the EBV enzyme.     

Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome.

DNA of bacteriophage PRD1 has protein P8 at its termini. Extracts of infected cells are able to derivatize P8 in vitro with labeled dGTP. Two early proteins, P1 and P8, products of genes I and VIII, respectively, are the only phage proteins necessary for the formation of the protein P8-dGMP complex. This was shown by complementation of extracts from cells infected with mutants and by use of extracts from cells carrying cloned genes I and VIII. With Escherichia coli mutants that are temperature sensitive for DNA synthesis, it was possible to show that the formation of the protein P8-dGMP complex was dependent upon the host replication apparatus. The analysis of the purified protein P8-dGMP complex by hydrolysis and enzymatic digestion showed that there is a covalent phosphodiester bond between tyrosine and 5'-dGMP.     

Early changes in the synthesis of proteins with affinity for single-stranded DNA during the onset of transformation in NRK cells.

Early alterations in the synthesis of proteins which bind to single-stranded DNA have been examined following the onset of transformation in NRK cells transformed by a heat-sensitive mutant (ts339) of Rous sarcoma virus. Transformation was initiated by shifting quiescent cultures from nonpermissive to permissive temperatures. Cultures were prelabelled with [3H]leucine for several generations at the non-permissive temperature, and with [35S]methionine at times after shift to the permissive temperature. Cytosol extracts were passed through sequential columns of double-stranded and single-stranded DNA bound to cellulose. Within the first hour of transformation there was an increase in the synthetic rate of proteins binding tightly to single-stranded DNA, but not to double-stranded DNA. More loosely bound protein fractions showed no such early synthetic increase. Electrophoresis of the fraction eluted from single stranded DNA-cellulose with 2 M NaCl demonstrated the presence of a major protein of 93 000 daltons, which comprised more than 0.1% of the cytosol protein. The synthesis of the 93 000 dalton protein increased continuously over the first 4 h interval after the onset of transformation. The synthetic rate of a 35 000 dalton protein, a major DNA-binding polypeptide found in mammalian cells, began to increase after a 1-h lag, following the onset of transformation. The protein fraction containing the 93 000 dalton protein had considerable unwinding activity, depressing the melting temperature of poly(dA-dT) by 39 degrees C. The protein fraction containing the bulk of the 35 000 dalton protein did not have unwinding activity. Transformation-induced DNA synthesis was measured in cells made permeable to deoxyribonucleoside triphosphates at times after shift to the permissive temperature. It was determined that synthesis of DNA began within the first 1--2 h after the onset of transformation. We conclude that the early transformation-associated synthesis of SS93 and perhaps other proteins binding to single-stranded DNA may be related to early transformation-associated changes preparatory to DNA replication and subsequent growth.     

Binding specificity of a HeLa DNA-binding protein to DNA and homopolymers

A protein which has affinity for single-stranded DNA but not for double-stranded DNA has been isolated from HeLa cells by DNA-cellulose chromatography. This protein having a molecular weight of 34,000 was accounted for approximately 3% of total soluble proteins. Its binding specificity to DNA and nucleotide homopolymers has been investigated by Sephadex G-200 column chromatography. Specific binding to single-stranded DNA has been confirmed also by this method and furthermore strong binding to poly U has been found.     

Impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses

The study aims on affinity matrix selection for a cell culture derived influenza virus capture step in downstream processing. Euonymus europaeus lectin (EEL) was used as an affinity ligand. Human influenza A/Puerto Rico/8/34 (H1N1) virus produced in MDCK cells was chosen as a model strain. The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine sulphate and heparinized agarose. The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.     

The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.     

Molecular basis of cellulose biosynthesis disappearance in submerged culture of Acetobacter xylinum

Acetobacter xylinum strains are known as very efficient producers of bacterial cellulose which, due to its unique properties, has great application potential. One of the most important problems faced during cellulose synthesis by these bacteria is generation of cellulose non-producing cells, which can appear under submerged culture conditions. The reasons of this remain unknown. These studies have been undertaken to compare at the molecular level wild-type, cellulose producing (Cel(+)) A. xylinum strains with Cel(-) forms of cellulose-negative phenotype. Comparison of protein profiles of both forms of A. xylinum by 2D electrophoresis allowed for the isolation of proteins which were produced exclusively by either Cel+ or Cel- cells. Sequences of peptides derived from these proteins were aligned with those of proteins deposited in databases. This analysis revealed that Cel(-) cells lacked two enzymes: phosphoglucomutase and glucose-1-phosphate uridylyltransferase, which generates UDP-glucose being the substrate for cellulose synthase. DNA was analyzed by ligation-mediated PCR carried out at low denaturation temperature (PCR-MP). Two DNA fragments of different thermal stability (218 and 217 bp) were obtained from the DNA of Cel(+) and Cel(-) forms, respectively. The only difference between these Cel(-) and Cel(+) DNA fragments is deletion of one T residue. Alignment of those two sequences with those deposited in the GenBank database revealed that similar fragments are present in the genomes of some bacterial cellulose producers and are located downstream from open reading frames (ORF) encoding phosphoglucomutase. The meaning of this observation is discussed.     

Membrane-associated DNase activity controlled by genes 46 and 47 of bacteriophage T4D and elevated DNase activity associated with the T4 das mutation.

Lethal, amber mutations in T4 genes 46 and 47 cause incomplete degradation of host DNA, premature arrest of phage DNA synthesis, accumulation of abnormal DNA replication intermediates, and defective recombination. These phenotypes can be explained by the hypothesis that genes 46 and 47 control a DNA exonuclease, but in vitro demonstration of such a nuclease has not yet been reported. Membrane and supernatant fractions from 46- and 47- mutant-infected and 46+ 47+ control-infected cells were assayed for the presence of the protein products of these genes (i.e., gp46 and gp47) and for the ability to degrade various DNA substrates to acid-soluble products in vitro. The two proteins were found only on membranes. The membrane fraction from 46- 47- mutant-infected cells digested native or heavily nicked Escherichia coli DNA to acid-soluble products three to four times slower that the membrane fraction from control-infected cells. No such effect was found in the cytoplasmic fractions. The effect on nuclease activity in membranes was the same whether 46- and 47- mutations were present singly or together. NaClO4, a chaotropic agent, released both gp46 and gp47 from 46+ 47+ membranes, as well as the DNase activity controlled by genes 46 and 47. DNA cellulose chromatography of proteins released from membranes by NaClO4 showed that gp46 and gp47 bound to the native DNAs of both E. coli and T4. Thus, the overall enrichment of gp46 and gp47 relative to total T4 protein was 600-fold (10-fold in membranes, 2-fold more upon release from membranes by NaClO4, and 30-fold more upon elution from DNA cellulose). T4 das mutations, which partially suppress the defective phenotype of 46- and 47- mutants, caused a considerable increase in vitro DNase activity in both membrane and cytoplasmic fractions, We obtained evidence that the das+ gene does not function to inhibit E. coli exonuclease I or V, endonuclease I, or the UV endonuclease of gene uvrA or to decrease the activity of T4 exonuclease A or the T4 gene 43 exonuclease.     

Two strains of Epstein-Barr virus (B95-8 and a P3HR-1 subclone) that lack defective genomes induce early antigen and cause abortive infection of Raji cells.

The heterogeneity of Epstein-Barr virus (EBV) obtained from P3HR-1 cells has permitted derivation of a distinct subclone of P3HR-1 (L. Heston, M. Rabson, N. Brown, and G. Miller, Nature (London) 295:160-163, 1982). We have analyzed the biologic properties and genomic structure of this subclonal virus (clone 13) compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV proteins in Raji cells superinfected with virus derived from P3HR-1, clone 13, and B95-8 was analyzed both by fluorography of radiolabeled proteins and by immunoblotting. Highly concentrated preparations of clone 13 and B95-8 virus induced most of the spectrum of EBV proteins in Raji cells with the exception of the 145,000-, 140,000-, and 110,000-molecular-weight proteins, which were either undetectable or reduced. Moreover, both clone 13 and B95-8 viruses also induced the same patterns of early antigen diffuse components as the parental P3HR-1 virus did. However, only P3HR-1 virus could induce EBV DNA synthesis in superinfected Raji cells, as determined both by buoyant density centrifugation and by in situ cytohybridization with biotinylated recombinant EBV DNA probes. Defective heterogeneous molecules present in P3HR-1 virus have been implicated in early antigen induction after superinfection of Raji cells. Therefore, Southern blots of clone 13, P3HR-1, and B95-8 viruses were hybridized to recombinant EBV fragments representing the sequences contained within the defective molecules in P3HR-1. The parental P3HR-1 contained the previously described defective molecules. No evidence for defective molecules was found in clone 13 or B95-8 viruses. These data indicate that concentrated preparations of both clone 13 and B95-8 viruses can induce abortive infection in Raji cells, but while the defective molecules are not needed for induction of early antigen diffuse components, they may be required for the induction of viral DNA synthesis.     

A DNA binding protein showing sequence specificity for a region containing the replication origin of Xenopus laevis mitochondrial DNA.

In Xenopus laevis mitochondria up to 14 different polypeptides with affinity for the DNA, have been identified by the protein blotting technique. Under stringent binding conditions only one polypeptide displayed specific affinity for a restriction fragment containing the H strand origin of replication of the Xenopus laevis mt chromosome. The proteins were fractionated by double stranded DNA cellulose chromatography. Under conditions which favor high affinity interactions between proteins and DNA, a protein of the 2M NaCl step shows specific binding to the DNA fragments containing the D-loop region. Some physical properties of the protein have been studied. It has a MW of 21.5 Kd and a globular shape as can be inferred from the relationship between MW and sedimentation coefficient (2.7 S). It binds non cooperatively to DNA and forms relatively stable complexes as demonstrated by DNA competition experiments.     

Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel electrophoresis of their AMP-binding domains in the trypsin-digested protein fragments. The results also suggested that these isozymes have been derived from the same primordial DNA sequence or from the same precursor protein. The purification scheme and the data obtained will be instrumental for the further elucidation of the biological roles of various DNA ligases from human cells.     

Two separable protein species which both restore uvrABC endonuclease activity in extracts from uvrC mutated cells

Two different protein species which both complement the detective repair endonuclease (uvrABC endonuclease) in uvrC mutated cells have been detected. These proteins have quite different chromatographic properties and were easily separated by ion exchange chromatography. One has affinity for DEAE cellulose and co-cromatographs with the uvrB protein. The other has strong affinity for phosphocellulose and appears to be the uvrC protein itself. The uvrB associated uvrC+ activity is absent from both uvrC and uvrB mutated cells, indicating that this species result from an interaction between uvrB+ and uvrC+ functions at the protein level.     

Isolation and partial characterization of EGF-like growth factor from rat glioma tissue

Using acid-ethanol extraction, two proteins with Mr=8 and 12 kD were extracted from rat glioma tissue induced with ethylnitrosourea. These proteins were shown to complete for the receptor with [125I]EGF (epidermal growth factor) on A431 cells. The 8 kD protein exhibited a marked mitogenic effect by stimulating DNA synthesis in resting NIH 3T3 cells. Stepwise chromatography of the acid-ethanol extract on Biogels P-60 and P-10 resulted in preparative amounts of the protein and allowed for its partial characterization. It was found that the half-maximum stimulation of DNA synthesis in NIH 3T3 cells was achieved at growth factor protein concentration of 5 micrograms/ml. The preparation obtained possessed the EGF-competing activity of 10 ng-equiv. EGF per 1 microgram of protein and stimulated protein phosphorylation of the 170 kD protein in NRK cell membranes. The data obtained suggest that this factor may be related to the family of the so-called EGF-like growth factors.     

Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.     

Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication.

The UL30 protein of herpes simplex virus type 1 (HSV-1) is a catalytically active DNA polymerase which is present in virus infected cells in a heterodimeric complex with an accessory subunit, the UL42 polypeptide. Both proteins are essential for viral DNA synthesis but because the UL42 protein is much more abundant it has been difficult to determine whether its role is related to, or independent of, its interaction with the UL30 protein in vivo. Since the C-terminal region of UL30 has been shown to be important for interaction with the UL42 protein but dispensable for DNA polymerase activity, a recombinant baculovirus which overexpresses a UL30 protein truncated by 27 amino acids at its C-terminus was constructed and used to assess the significance of the protein-protein interaction. The mutated protein was as active as wildtype (wt) UL30 in a DNA polymerase assay in which activated calf thymus DNA was used as template. However, in contrast to the wt protein, the activity of the truncated polymerase on this template was not stimulated by addition of purified UL42. A monoclonal antibody against the UL42 protein co-precipitated the full length but not truncated polymerase from extracts of cells which had been co-infected with a UL42-expressing recombinant baculovirus. Finally, the truncated protein was not active in a transient assay for HSV-1 origin-dependent DNA replication performed in insect cells in tissue culture. These results indicate that sequences at the C-terminus of the UL30 protein which are dispensable for DNA polymerase activity play essential roles both in viral DNA replication and interaction with the UL42 protein, and strongly suggest that the interaction between the proteins is important in vivo.