Effects of phenethyl alcohol on transport reactions, nucleotide pools and macromolecular synthesis in Novikoff rat hepatoma cells growing in suspension culture

At cytostatic concentrations, phenethyl alcohol has immediate and reversible effects on multiple metabolic processes of Novikoff rat hepatoma cells growing in suspension culture. These include an inhibition of the transport of various low molecular weight substances into the cell, an inhibition of DNA and protein synthesis and the processing of ribosomal RNA, and a degradation of ribosomal RNA. All effects might be explained as resulting from an interaction of the chemical with cellular membranes. Phenethyl alcohol does not have an immediate effect on RNA synthesis per se. The immediate failure of phenethyl alcohol-treated cells to incorporate uridine from the medium into RNA is due to an inhibition of the uridine transport reaction.     

Identification of mammalian DNA repair factors using a reconstituted subcellular system : Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12000–15000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase β-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.     

Triton X-100 activates nucleoside triphosphate-dependent, recBC-dependent DNA synthesis in toluene-treated Escherichia coli.

Escherichia coli cells whose chromosome replication has been terminated in vivo, either by growth into stationary phase or by incubation of a mutant carrying a temperature-sensitive initiation mutation under restrictive conditions, are inactive in in vitro DNA synthesis as measured in toluene-treated cells. Addition of the non-ionic detergent Triton X-100 to such inactive systems results in a marked stimulation of ATP-dependent in vitro DNA synthesis. This Triton-stimulated DNA synthesis appears to proceed by a semi-conservative mechanism, in that DNA synthesized in vitro in the presence of a density labeled precursor bands in CsCl equilibrium centrifugation at a hybrid density. Neutral sucrose gradient centrifugation demonstrates that most of this hybrid material exhibits a molecular weight in excess of 1 X 10(7). Triton-stimulated synthesis requires the presence of DNA polymerase III, as does normal in vivo replication. We show here, however, several anomalous properties of the DNA synthesis in the Triton/toluene system. In particular, Triton-stimulated synthesis is absent in cells harboring a recB mutation which lack the ATP-dependent exonuclease V, an enzyme implicated in recombinational repair synthesis in vivo. Furthermore, the ATP requirement for Triton-stimulated synthesis is relatively non-sepcific, and a variety of nucleoside triphosphates can effectively substitute for ATP. Finally, despite their high molecular weight in neutral sucrose gradient centrifugation, Triton-stimulated DNA synthesis generates DNA molecules of low molecular weight (less than 500 000) as determined by alkaline sucrose gradient centrifugation. In contrast, DNA synthesis in the normal toluene-treated cell system is not dependent on recB activity, shows a nearly absolute requirement for ATP which cannot be replaced by other nucleoside triphosphates, and produces molecules of far greater molecular weight as measured on alkaline sucrose gradients. Taken altogether the data strongly suggest that Triton activates an unusual form of DNA synthesis in toluene-treated cells which shows both repair and replicative aspects. These results caution against the use of Triton-activated toluene-treated cells system, for studying simple replicative DNA synthesis.     

Synthesis of DNA polymerase alpha analyzed by immunoprecipitation from synchronously proliferating cells

Synchronously proliferating TC7 monkey and 3T3 mouse cells were pulse labeled with [35S]methionine. Radioactively labeled DNA polymerase alpha was immunoprecipitated with polymerase-specific monoclonal antibodies. The precipitated polypeptides were identified by gel electrophoresis and fluorography. The increase in DNA polymerase alpha activity during S phase was accompanied by an increased synthesis of the enzyme. Some DNA polymerase alpha was synthesized in growth-arrested TC7 cells whereas the synthesis of the large polymerase subunit in 3T3 cells was strictly coupled to the replicative phase of the cell cycle. We also found that DNA polymerase alpha was more prone to proteolysis in TC7 cells than in 3T3 cells. In 3T3 cells, a polymerase subunit with an apparent molecular weight of 186 000 was observed; this subunit was most probably associated with two smaller subunits of Mr 74 000 and 52 000. Synthesis of these three polymerase-associated polypeptides appeared to be regulated differently.     

Modification of membrane lipids. Phenethyl alcohol-induced alteration of lipid composition in Tetrahymena membranes.

Tetrahymena pyriformis NT-I cells in the early-logarithmic phase were incubated with phenethyl alcohol (2-phenylethanol) and effects on the lipid composition were examined in various membranes. 1. There was a marked modification in phospholipid head, as well as fatty acyl group composition in pellicles, mitochondria and microsomes of the phenethyl alcohol-treated cells. Compared with membranes of the control cells, the membranes from phenethyl alcohol-treated cells were found to contain a higher level of phosphatidylcholine content with the compensating decrease in phosphatidylethanolamine, while 2-aminoethylphosphonolipid showed only a slight decrease in these membranes. The acyl group profile of membrane phospholipids in the presence of phenethyl alcohol was also modified so that a profound elevation of the content of polyunsaturated fatty acids, linoleic and gamma-linolenic acids. The major monounsaturate, palmitoleate decreased. Such lipid alteration is a reversible process, and therefore upon removal of phenethyl alcohol the modified lipid composition returned to normal. 2. By freeze-fracture electron microscopy in combination with temperature quenching, the outer alveolar membrane of the phenethyl alcohol-treated cell was observed to reveal less aggregation of intercalated-membrane particles, as compared with the control membrane. The quantitative analysis of the thermotropic lateral movement of membrane particles provided evidence that the membrane in the phenethyl alcohol-treated cell became more fluid. Such fluidizing effects may result from an increase in the acyl group unsaturation and also in the phosphatidylcholine content. 3. With regard to the mechanism responsible for the marked decrease in palmitoleate in membrane phospholipids, there was found a depressed conversion of the palmitate to palmitoleate in the phenethyl alcohol-treated cells. It was further suggested that the drug may have an inhibitory effect on the synthesis of palmitoyl-CoA desaturase involving the (16 : 0 leads to 16 : 1) conversion. Also, it was demonstrated that the increase in a precursor-product fashion of phosphatidylcholine with the corresponding decrease in phosphatidylethanolamine was not due to transformation of phosphatidylethanolamine to phosphatidylcholine through stepwise methylation.     

Leading and lagging strand synthesis at the replication fork of bacteriophage T7. Distinct properties of T7 gene 4 protein as a helicase and primase

Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork. A minimum of three proteins is required to catalyze leading and lagging strand synthesis. The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities. A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity. Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis. Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases. Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis. The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms. Removal of the large form results in the loss of primase activity but not of helicase activity. Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms. These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms. The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.     

Inhibition of protein and nucleic acid synthesis of animal cells in vitro mediated by high molecular weight inhibitors in human liver extract.

1. The addition of human liver extract to HeLa cells induces a reversible inhibition of the incorporation of [3H] thymidine into the DNA, [3H] uridine into the RNA, and 14C-labelled amino acids into the protein of HeLa cells. The inhibitory effects appear after treatment for 1 h and reach a maximum after 4-8 h. These effects do not depend on a defective precursor penetration, isotopic dilution or degradation of labelled precursor (thymidine-degrading enzymes were inactivated by the addition of unlabelled thymine), reduced activity of thymidine and uridine kinase, medium impairment, or an impairment of the cell-membrane function. 2. The nucleic acid synthesis-inhibiting activity of the extract seems to be dependent on cellular protein synthesis but independent of RNA synthesis which indicates that the inhibitors act in an indirect way. Furthermore, the inhibitors seem to lack the tissue-specific character of chalones. 3. The extract contains separate inhibitors of DNA, RNA and protein synthesis. These inhibitors were found to have different physical-chemical characteristics and to be macromolecules with a protein or conjugated protein character (mol. wt. approx. 90 000). 4. The possibility that the activity of the high molecular weight inhibitors resides in low molecular weight factors (bound to protein carriers) was tested: No true low molecular weight inhibitors could be liberated by extraction with trichloroacetic acid/organic solvents or by dialysis/enzymatic treatments. Nucleosides such as thymidine, uridine, and cytidine, however, were liberated and could be shown to interfere with the uptake of [3H] thymidine/[3H] uridine.     

DNA polymerase activities during erythropoiesis : Effects of erythropoietin, vinblastine, colcemid, and daunomycin

Erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring approx 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis, increased ⁵⁹Fe incorporation into heme, and an increase in the activity of the cytoplasmic high molecular weight DNA polymerase (DNA polymerase-α). In contrast, the activity of the low molecular weight DNA polymerase (DNA polymerase-β) does not significantly change after administration of erythropoietin. Vinblastine, colcemid, and daunomycin prevent the effects of erythropoietin on mouse spleen, so that increases in DNA synthesis, DNA polymerase-α, and ⁵⁹Fe incorporation do not occur. DNA polymerase-α may have a short half-life in cells since its activity is barely detectable 12 to 24 h after administration of inhibitors of cellular proliferation or nucleic acid synthesis. The half-life of DNA polymerase-β may be long since it is unaffected by these inhibitors. Cytoplasmic rather than nuclear DNA polymerases appear to play a major role in erythropoietin-stimulated DNA synthesis and replication of erythroid cells.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.     

Calcification inhibitors in rat and human serum and plasma

Rat and human serum and plasma were shown to contain considerable amounts of calcium phosphate precipitation inhibitors. Two general classes of inhibiting molecules were observed for both species: high molecular weight (approx. 30 000-200 000) and low molecular weight (less than 1000). The high molecular weight components eluted from a Bio-Gel P-200 column in two peaks, one at approx. 158 000 and a broader peak at approx. 43 000. The identity of these inhibitors is unknown at present. Low molecular weight inhibitors include magnesium, pyrophosphate, and citrate ions and at least one unidentified component that coelutes with pyrophosphate and citrate on a Bio-Gel P-4 column. Quantitatively, most of the inhibitor activity resides in the high molecular weight components and it is possible that it is this activity which is responsible for maintaining the metastability of the circulating fluids. The role of the low molecular weight components may be to regulate calcification at sites inaccessible to high molecular weight molecules.     

Interaction between membrane properties and protein synthesis: In vitro synthesis of membrane proteins during erythropoiesis

Membrane protein synthesis was investigated by incubating rabbit reticulocytes, in vitro, with radioactive amino acids. The kinetics of membrane protein synthesis showed linear incorporation for approx. 15 min, after which there was only a slight increase in incorporation. On the other hand, intracellular protein synthesis was linear for an incubation period of 60 min. Membranes isolated from such rabbit reticulocytes were analysed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Two major radioactive bands were found in the 50–60 000 D region, whilst another labelled band had a molecular weight of 43 000 D. This latter band had an electrophoretic mobility identical with rabbit muscle actin (and chick brain actin), when run on one-dimensional SDS polyacrylamide gels. Absolute identity between rabbit brain actin and a newly synthesized reticulocyte membrane protein was shown by comigration on a two-dimensional (first dimension isoelectric focusing and second dimension SDS gel) electrophoresis system. Another band that was radioactively labelled was found to have a molecular weight of approx. 32 000 D. Separation of reticulocytes into different age groups showed that young reticulocytes synthesized a membrane protein species that was not radioactively labelled in the old reticulocyte population.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Kinetics of the decamer - dimer dissociation of arginine decarboxylase.

Aurintricarboxylic acid inhibited replicative DNA synthesis in nucleotide-permeable mouse ascites sarcoma cells. DNA polymerase activity assayed with activated DNA template and DNA polymerase purified partially from sarcoma cells was also inhibited by aurintricarboxylic acid. The inhibition of DNA polymerase activity was probably due to the inhibitory interaction of aurintricarboxylic acid with DNA polymerase. The replicative DNA synthesis might be inhibited by aurintricarboxylic acid interacting with some essential protein component(s), such as DNA polymerase of the replication machinery.     

Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains.

With the use of an in vitro complementation assay to measure activity, the gene 4 protein of bacteriophage T7 has been purified 1000-fold to yield a nearly homogeneous protein. The purified gene 4 protein is a single polypeptide having a molecular weight of 58,000. In addition to being essential for T7 DNA replication in vivo and in vitro, the gene 4 protein is required for DNA synthesis by the purified T7 DNA polymerase on duplex T7 DNA templates. In the absence of ribonucleoside 5'-triphosphates, DNA synthesis by the gene 4 protein and the T7 DNA polymerase is dependent on phosphodiester bond interruptions containing 3'-hydroxyl groups (nicks) in the duplex DNA. The reaction is specific for the T7 DNA polymerase, but any duplex DNA containing nicks can serve as template. The Km for nicks in the reaction is 3 x 10(-10) M.     

Changes in the acid-soluble nucleotide composition of red cells of cattle at various ages.

DNA polymerase was extracted from HeLa cell mitochondria with high salt concentrations (1M) and Nonidet-P 40 (0.2%). Subsequently the enzyme was purified stepwise by DEAE-cellulose-, phosphocellulose-, hydroxyapatite-Ultrogel-, DNA-cellulose chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme exhibited a molecular weight between 100 000 – 110 000 and was devoid of endonuclease activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this enzyme preparation revealed two protein bands suggesting that the mitochondrial DNA polymerase might consist of two subunits with the molecular weights of 45 000 and 60 000.     

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Heat-shock response in Xenopus oocytes during meiotic maturation and activation

After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight.