Purification of host DNA synthesis-suppressing factor (DSF) produced by infection with measles virus.

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

The effect of the DNA-suppressing factor (DSF) on host DNA synthesis in synchronized cell cultures

Purified host DNA-suppressing factor (DSF) produced into culture fluid of HeLa C-9 cells infected with measles virus inhibited cellular DNA synthesis in HeLa cells. When purified DSF was added into cultures of synchronous HeLa cells at the early G1-phase, cellular DNA synthesis was irreversibly inhibited. However, DSF did not affect the stability of native double-stranded DNA nor the chain-elongation of single-stranded DNA in cells of the S-phase.     

Transformation in bacillus subtilis: fate of transforming DNA in transformation deficient mutants.

Summary Transformation deficient mutants were isolated by means of selection for sensitivity to methylmethane-sulfonate (MMS). The mutations were introduced into a multiple auxotrophic highly transformable recipient. The transformation deficient strains were characterized with respect to their sensitivity to UV-irradiation and treatment with MMS and mitomycin-C (MC) and with respect to both the physico-chemical and biological properties of reextracted donor DNA.As has been established previously (Davidoff-Abelson and Dubnau, 1973b) in the transformation proficient wild-type, double-stranded fragments (DSF), single-stranded fragments (SSF) and donorrecipient complex (DRC) are formed from donor DNA.The mutants we report on are of various types: Mutant 7G-73 (transformation frequency about 25 times lower than in the wild-type) is sensitive to UV-irradiation and treatment with MMS and MC, and is extremely deficient in the production of DRC.Mutant 7G-84 (transformation frequency about 12 times lower than in the wild-type) shows also sensitivity to UV-irradiation and treatment with MMS and MC. However, although it forms DSF, SSF, and DRC, the biological activity of DNA re-extracted from transforming cultures of 7G-84 is much reduced as compared to that of the wild-type.Mutant 7G-97 (transformation frequency about 500 times lower than in the wild-type) shows approximately wild-type resistance to UV-irradiation and treatment with MMS and MC, and forms DSF exclusively; the donor DNA is not processed further.Double mutants6G-103 and 6G-105, constructed by transformation of mutant 7G-97, with DNA from 7G-84 and 7G-73, are about 1250 and 5000 times less transformable than the wild-type respectively. They are sensitive to UV-irradiation and treatment with MMS and MC. Mutants 6G-103 and 6G-105 also produce DSF, which are not processed further.     

Release of nerve growth factor by human glial cells in culture

Non-transformed human glial cells obtained from brain biopsies (lines U-787 CG, U-1169 CG and U-1508 CG) release to their culture medium a factor which, in bioassay, induces neurite outgrowth in spinal and sympathetic embryonic chick ganglia. The neurite-stimulating activity, which was enhanced after pressure dialysis of glial-conditioned medium, is inhibited by specific antiserum prepared to mouse βnerve growth factor (NGF). The glial factor was partially purified by gel filtration on Sephadex G-200 of concentrated, serum-containing, conditioned medium. The activity eluted close to a molecular weight of 30000, as did mouse NGF run under identical conditions. Ion-exchange chromatography on DEAE-Sepharose CL-6B and flat-bed electrofocusing of conditioned medium showed the activity to be associated with a heat-labile entity having an isoelectric point of about 4.1. All purified preparations were blocked by anti(mouse)-βNGF. The results demonstrate the existence of a human glial NGF which in several respects resembles the mouse submandibular gland NGF.     

DNA-mediated cotransfer of unlinked mammalian cell markers into mouse L cells

Purified DNA from three different types of mammalian cells was precipitated with calcium phosphate and added to mouse L cells deficient in thymidine kinase (TK). Donor DNA was prepared from three cell lines: (a) mouse cells transfected with UV-inactivated herpes simplex virus (HSV) type 1, or a purified fragment of HSV carrying the TK gene (b) human HeLa cells, and (c( CHO, a cell line derived from Chinese hamster ovaries. Several hypoxanthine-aminopterin-thymidine resistant colonies were isolated from each experiment. The origin of the TK that is expressed in these cells was studied by polyacrylamide gel electrohporesis, isoelectric focusing, or heat stability. The TK in all instances was of the donor origin. To determine the extent of gene transfer we have assayed the CHO and HeLa DNA transfectants for galactokinase (GALK), a marker closely linked to TK, and 25 other isozymes representing a large number of different chromosomes. No cotransfer of GALK was observed, indicating that the size of the transferred DNA segment is limited. We observed that, in one instance, esterase-D, an unlinked marker of Chinese hamster origin, was transferred along with TK. These experiments indicate that nonselected markers can be transferred by this method, although at a low efficiency.     

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.     

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.     

Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus.     

Insulin-Like Growth Factor I/Somatomedin-C: A Rapid Isolation Procedure with FPLC

Insulin-like growth factor I (IGF l)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000 – 10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH₃ CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS poly-acrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pi of 8.3 0.1. SDS polyacryl-amide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measureing the (³H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.     

Partial purification and chemical characterization of macrophage cytotoxicity factor (MCF, MAF) and its separation from migration inhibitory factor (MIF)

Macrophage cytotoxicity factor (MCF) was purified in 3 consecutive steps including adsorption chromatography on Matrex Gel Red A, hydrophobic chromatography on phenylalanine-Sepharose, and isoelectric focusing. MCF was characterized as a protein with a m.w. of approximately 30,000 by gel filtration on Sephadex G-100 with 2 isoelectric points at 7.4 and 8.4 in the presence of urea. The unpurified supernatant was fairly stable provided that manipulations favoring adsorption to membrane materials used for dialysis or ultrafiltration were omitted. The partially purified preparation was highly unstable. Trypsin treatment did not affect MCF activity, whereas chymotrypsin destroyed it. Treatment with glycosidases and neuraminidase or cultivation of cells in the presence of 2-deoxy-D-glucose or tunicamycin did not impair the MCF activity. MCF was separated from migration inhibitory factor (MIF) by 2 methods: first, isoelectric focusing in the presence of urea, and second by gel filtration on Ultrogel. MCF could be separated from interferon by chromatography on poly(I)-Sepharose.     

Separation of a New Deoxyribonucleic Acid Polymerase from Vaccinia-infected HeLa Cells

HeLa cell deoxyribonucleic acid (DNA) polymerase was purified about 100-fold by sequential column chromatography on phosphocellulose, hydroxylapatite, and Bio Rex 70. A new form of DNA polymerase found in vaccinia virus-infected cells was separated from HeLa DNA polymerase by chromatography on diethylamino-ethyl cellulose. The new form was also purified approximately 100-fold in the same manner as the HeLa DNA polymerase. In addition to chromatographic differences, the two enzymes differed with regard to primer response, relative activity at high pH, inactivation by heat and p-chloromercuribenzoate, and inhibition by vaccinia antiserum.     

Novel form of DNA polymerase alpha associated with DNA primase activity of vertebrates. Detection with mouse stimulating factor

With a specific stimulating factor of mouse DNA replicase for its detection, a novel form of DNA polymerase alpha (DNA replicase) associated with DNA primase activity was partially purified from several vertebrates, i.e. the cherry salmon Oncorhyncus masou, the frog Xenopus laevis, the chick, and human (HeLa cells). Activity similar to DNA replicase was also partially purified from embryos of the sea urchin Anthocidaris crassispina. In all vertebrates examined, two forms of DNA polymerase alpha were separated by chromatography on ion-exchange columns; one form (DNA replicase) was associated with DNA primase activity and could utilize unprimed single-stranded DNAs as template, and the other could not utilize unprimed single-stranded DNAs. The sedimentation coefficient of the former, the novel form, obtained from each vertebrate in a glycerol gradient at high ionic strength was slightly larger than that of the other form which had no primase activity, except in the case of chick embryos where the sedimentation coefficients of the two forms were almost the same. The initiator RNA synthesized with the DNA primase activity associated with DNA replicase obtained from salmon, chick, HeLa cells, and sea urchin was 8 to 10 nucleotides long. The stimulating factor obtained from Ehrlich ascites cells has been found to stimulate both the activities of DNA primase and DNA polymerase in DNA replicase obtained from all the vertebrates examined, when unprimed single-stranded DNA was used as template, while the factor failed to stimulate both the activities of the enzyme of sea urchin embryos. This factor thus should be an effective tool in studies on the mechanism of vertebrate DNA replication.     

A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells

An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Purification and partial characterization of prolactin from the California ground squirrel (Spermophilus beecheyi)

Prolactin (Prl) secreted by cultured ground squirrel (Spermophilus beecheyi) pituitaries (SbPrl) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on Polybuffer Exchanger 94. Purification from culture medium from 190 pituitaries yielded 1.1 mg of purified SbPrl. The SbPrl has an apparent molecular weight of 27,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 6.3, and does not contain any asparagine-linked carbohydrate. Purified SbPrl displaces 125I-labeled ovine Prl from binding sites on lactating rabbit mammary gland membranes and stimulates secretion of alpha-lactalbumin by cultured mouse mammary gland epithelial cells.     

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.     

Two distinct human DNA diesterases that hydrolyze 3''-blocking deoxyribose fragments from oxidized DNA.

Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis. Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells. The two activities were purified to differing degrees from HeLa cells. One enzyme had the properties of the known HeLa AP endonuclease (Mr approximately 38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein. The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity. This second enzyme may have been detected in other studies but never characterized. In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities. It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected. The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused.     

Characterization and sequence-specific binding to mouse mammary tumor virus DNA of purified activated human glucocorticoid receptor

Activated glucocorticoid receptor (GR) from the human cell line HeLa S3 was purified by differential chromatography on DNA-cellulose followed by DEAE-Sepharose chromatography to 50-60% homogeneity according to sodium dodecyl sulfate gel electrophoresis and densitometric scanning of silver-stained gels. These gels routinely demonstrated a main band of Mr 94,000 (94K band) and two minor bands of Mr 79,000 (79K band) and 39,000 (39K band), respectively. Photoaffinity labeling indicated that the hormone was bound to the 94K and 79K components. In some preparations, a 72K band was observed. Further characterization of the purified receptor by gel permeation chromatography on Sephadex G-200 revealed a receptor complex with a Stokes radius of 5.8 nm. The sedimentation coefficient of the purified receptor was 4.4 Sw. In analogy to the rat hepatic GR, limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of 28K and 39K fragments, respectively. In addition, no difference in the protease digestion pattern using Staphylococcus aureus V8 protease was observed. Immunoblotting using a monoclonal antibody raised against the 94K GR from rat liver demonstrated cross-reactivity with the human 94K and 79K proteins from HeLa S3 cells, indicating similar antigenic characteristics between rat and human GR. In our study, five out of nine tested monoclonal antibodies against the rat liver GR cross-reacted with human GR. DNase I and exonuclease III protection experiments demonstrated binding of the purified human GR to specific GR binding regions in mouse mammary tumor virus DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and properties of cathepsin G-like proteinase of mouse myeloid leukemia M1 cells

A proteinase extracted with 1M NaCl from particulate fraction of the postnuclear fraction of mouse myeloid leukemia M1 cells was partially purified by Bio-Gel HTP treatment and Sephadex G-75 gel filtration. The apparent molecular mass of the proteinase was 26,000 Da and the isoelectric point was about pH 10. The enzyme activity was inhibited by phenylmethanesulfonylfluoride, chymostatin, and soy-bean trypsin inhibitor. It hydrolysed specifically Suc-Ala2-Pro-Phe-4-methylcoumaryl-4-amide (MCA). NaCl and KCl enhanced several times the activity for Suc-Ala2-Pro-Phe-MCA, but not that for fluorescein-labeled albumin and fibrinogen. These enzymic properties of the major proteinase are similar to those of chymotrypsin and cathepsin G. The role of a cathepsin G-like proteinase in relation to M1 cell differentiation is discussed.