Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis (original) (raw)
Related papers
Purification of DNA-dependent RNA polymerase fromStreptomyces granaticolor
Folia Microbiologica, 1983
R~qA nucleotidyltransferase (EC 2.7.7.6) of Streptomycea granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA-cellulose and gell 'filtration on Biogel A 1.5 m. SDS-polyacrylamide gel eleetrophoresis revealed 8 protein bands, of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be ~ and ~ subunits, respectively. The role of other subunits of the enzyme is discussed.
Folia Microbiologica, 1991
Partially purified DNA-dependent RNA polymerase of Streptomyces granaticolor was further separated on phosphocellulose in 50 % glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg,/mol. These four subunits represented 93 % proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage q~29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases from Eschericlu'a coil and Bacillus subtilis. *Present address: Institute of Entomology, Czechoslovak Academy of Sciences, 370 05 t~x.sk6 Bud/~jovice, Czechoslovakia. 1~"~ J. ~MARDOVA eta/.
Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels
European Journal of Biochemistry, 1985
A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a postition corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400 -900 kDa, in some cases, close to 200 kDA.
Biotechnology Techniques, 1998
A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 µg up to 3 mg per gram of tissue with an average purity measured as A 260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen™ reagent and of PCR products with the PicoGreen™ reagent.
European Journal of Biochemistry, 1991
The 3'-terminal two-thirds of the Streptococcus pneumoniae polA gene was cloned in an Escherichia coli genefusion vector with inducible expression. The resulting recombinant plasmid (pSM10) directs the hyperproduction of a polypeptide of 70.6 kDa corresponding to the C-terminal fragment of pneumococcal DNA polymerase I. Induced cells synthesized catalytically active protein to the extent of 7% of the total soluble protein in the cells. The polymerase fragment was purified to greater than 90% homogeneity with a yield of 1.5 mg pure protein/ culture. The protein has DNA polymerase activity, but no exonuclease activity. The enzyme requires a divalent cation (MgC1, or MnCl,) for polymerization of DNA. Comparison of the mutant and wild-type pneumococcal polymerases shows that the construction did not affect the enzymatic affinity for the various substrates. The mutant protein, like its parent DNA polymerase 1, exhibited an intermediate level of activity with primed single-stranded DNA. At high molar ratio of enzyme/DNA Substrate, the polymerase fragment catalyzes strand displacement and switching after completing the replication of a primed single-stranded M 13 DNA molecule.
The Journal of Antibiotics, 1982
Covalently closed circular (ccc) DNAs were isolated by a technique involving alkaline denaturation from the spiramycin producer Streptonryces annbofaciens KA-1028 and also from spiramycin non-producing strains AF-1 I and QN-25; plasmids could not be detected in these strains by a cleared lysate method. It was found that most of the ccc DNA in these strains was present in the chromosomal and membrane fractions. These ccc DNAs had identical mobilities in agarose gel electrophoresis. The size was calculated to be 53.1 x 106 daltons from the contour length measurements. The ccc DNA gave one fragment on digestion with Hind 111, three fragments with Eco Rl, and twenty-eight fragments with Bain HI. Various ccc DNAs have been isolated from streptomycetes1~13) Their involvement in various biological functions, such as antibiotic production14) and resistance4.11), fertility2,15) , differentiation16~18) and release of extracellular enzyme19,20), has been studied; the involvement of plasmids in antibiotic production in streptomycetes has been suggested by "curing experiments"16). Isolation of ccc DNAs from streptomycetes has generally been performed by cleared lysate methods although YAGISAWA et al.11 and OMURA et al.12) employed alkaline denaturation. THE JOURNAL OF ANTIBIOTICS APR. 1982 This paper deals with the isolation and distribution of plasmid DNA from spiramycin-producing and non-producing strains of S. ambofaciens and with the characterization of pSAI DNA. Materials and Methods Organisms Streptomyces ambofaciens KA-1028 (ISP-5053), a spiramycin-producer, and strains AF-11 and QN-25, spiramycin non-producing strains, were used. Strains AF-11 and QN-25 were non-producing strains obtained by treatment of S. ambofaciens KA-1028 with acriflavine and quinacrine, respectively. Chemicals CsCl was purchased from Nakarai Chemicals Ltd., Kyoto, Japan, lysozyme and ethidium bromide from Sigma Chemicals Co., RNase A from Boehringer Mannheim Gm13H, restriction endonucleases, Eco R1, Hlnd 111, and Bam HI, from Takara Shuzo Co., Ltd., Kyoto, Japan, and [methyl-1H]thymidine (41 Ci/mmole) and [2-14C]thymidine (42 mCi/mmole) from New England Nuclear. Media and Growth Strains were incubated in Tryptic Soy Broth (BBL) at 27°C for 48 hours, and 0.2 ml of the culture was transferred into 10 ml of a medium containing 0.25 % glucose, 0.5 low phosphate content Casamino Acids (when low phosphate content Casamino Acids was not used, mycelial pellets were often formed that interfered with lysozyme digestion), 0.3 % L-asparagine • H2O, 0.3 % NaCl, 0.05 % MgSO4 • 7H2O, 0.1% NaNO2, 0.01 % CaCl2.2H2O, 0.009 % KH2PO4, 0.4 % trace elements solution, 0.02 % 2'-deoxyadenosine, 0.1 M tris-(hydroxymethyl)aminomethane hydrochloride (tris-HCl), pH 7.2, and 1-10 t1Ci of [methyl-3H]thymidine (41 Ci/mmole) or 1 1LCi of [2-11C]thymidine (42 mCi/mmole) per ml and then cultivated at 27°C for 18 hours. Mycelia were harvested by centrifugation at 12,000 xg for 10 minutes. Low phosphate content Casamino Acids was prepared as follows. Twenty grams of Casamino Acids (Difco) was dissolved in 100 ml of distilled water and then 2.54 g of MgCl2.6H2O and 11 ml of concentrated NH4OH were added. The stirred mixture was chilled at 0°C for at least 2 hours, and centrifuged at 2,000 xg for 5 minutes at 0°C to remove the precipitate. The excess ammonia was evaporated from the supernatant fluid under reduced pressure. After the pH was adjusted to 7.0 with I N HCl, the solution was lyophilized to give a powder of low phosphate content Casamino Acids.
Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus
Molecular and General Genetics MGG, 1988
A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5' termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.
Two forms of DNA-dependent RNA polymerase α subunit in streptomycetes
FEMS Microbiology Letters, 2000
We demonstrated two different DNA-dependent RNA polymerase (RNAP) K subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa. The 43-kDa subunit was also found in vegetative cells. These two proteins are highly similar to each other as well as to other bacterial RNAP K subunits. The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators. The gene for RNAP K from S. granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli. In vitro experiments using purified RNAP K showed that the cell free extract from spores of S. granaticolor exhibits proteolytic activity responsible for the K subunit shortening, whereas that from vegetative cells does not. This modification of K subunit might point to a novel mechanism of transcriptional control in streptomycetes. ß