Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120–140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.     

Large-scale preparation of biologically active measles virus haemagglutinin expressed by attenuated vaccinia virus vectors

A procedure described here allows the efficient and rapid purification of histidine-tagged measles virus haemagglutinin that is synthesized under the control of powerful promoters (PSFJ1-10 and PSFJ2-16) of the highly attenuated vaccinia virus (VV) strain LC16mO. A single affinity chromatography step purifies recombinant haemagglutinin proteins from the lysates of cells infected with the recombinant VVs. The recovery and purity are both very high (a yield of 0.5-2.8 mg/10(8) cells and purity of >94-98%), indicating that this procedure is approximately 400 times more efficient than the conventional methods used to prepare haemagglutinin. The haemagglutinins are correctly transported to the cell surface and have haemadsorption activity. Moreover, the recombinant haemagglutinin proteins cooperate with the measles virus fusion protein to elicit cell fusion activity. In addition, the antibody titres against measles virus, as measured by enzyme-linked immunosorbent assay using the purified haemagglutinin as the capture antigen, correlated closely with neutralization test titres (R(2) = 0.84, p < 0.05), indicating the preservation of immunologically relevant antigenicity. Such recombinant haemagglutinin preparations will be useful in diagnostic tests that measure functional anti-measles immunity and investigate the biological functions and structure of the haemagglutinin.     

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.     

Purification of the T4 endonuclease V

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.     

抗甲肝病毒基因工程单抗分离纯化与鉴定

为克服血源免疫球蛋白制品的不足,开发了抗甲肝病毒基因工程单克隆抗体anti-HAV IgG。用无血清培养基培养rCHO工程细胞株,上清液经过rProtein A SFF亲和层析→脱盐→离子交换层析→超滤换液纯化后,所得anti-HAV IgG纯度达99%以上,比活性约100IU/mg,anti-HAV IgG活性回收率40%。所纯化的anti-HAV IgG分子量150kD,等电点8.4～9.3。免疫印迹实验证实anti-HAV IgG为人源全抗体分子。亲和层析介质rProtein A SFF确实存在亲和配基脱落问题,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti-HAV IgG各项纯度检测指标均达到我国对基因工程产品的质量要求。     

DNA repair in Saccharomyces cerevisiae: purification and characterization of apurinic endonucleases

Five chromatographically distinct apurinic endonucleases (D1, D2, D3, D4, and E) were purified from Saccharomyces cerevisiae 234, 122, 1,000, 4,550, and 5,490-fold, respectively. All appeared to be class II apurinic endonucleases and were not contaminated with exonuclease or nonspecific endonuclease activities under the reaction conditions used. All had similar pH optima, but endonucleases D4 and E showed higher salt requirements and endonuclease D4 had a lower Mg2+ requirement for optimal activity than the other endonucleases. Endonuclease D4 also nicked OsO4-treated DNA. The molecular weights of the apurinic endonucleases as determined by glycerol gradient sedimentation analysis were 37,000, 49,000, and 10,000, for endonucleases E, D4, and D2, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples of radioiodinated endonuclease E showed the presence of two proteins.     

High-capacity multimodal anion-exchange membranes for polishing of therapeutic proteins

This contribution reports on a study using Purexa™-MQ multimodal anion-exchange (AEX) membranes for protein polishing at elevated solution conductivities. Dynamic binding capacities (DBC₁₀) of bovine serum albumin (BSA), human immunoglobulins, and salmon sperm DNA (ss-DNA) are reported for various salt types, salt concentrations, flowrates, and pH. Using 1 mg/ml BSA, DBC₁₀ values for Purexa™-MQ were >90 mg/ml at conductivities up to 15 mS/cm. The membranes maintained a high, salt-tolerant BSA DBC₁₀ of 89.8 ± 2.7 (SD) over the course of 100 bind-elute cycles. Polishing studies with acidic and basic monoclonal antibodies at >2 kg/L loads showed that Purexa™-MQ had higher clearance of host cell proteins and aggregate species at high conductivity (13 mS/cm) and in the presence of phosphate than other commercial AEX media. Purexa™-MQ also had a high ss-DNA DBC₁₀ of 50 mg/ml at conductivities up to 15 mS/cm, markedly outperforming other commercial products. In addition to the effectiveness of Purexa™-MQ for protein polishing at elevated solution conductivities, its unusually high binding capacity for ss-DNA indicates potential applications for plasmid DNA purification.     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification

A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis.     

Two new site-specific endonucleases from Staphylococcus species strain D5

Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5;-GGTGA(8N/8N) downward arrow-3; on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3;-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5'-ATGCA T-3' and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 degrees C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol).     

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M_r 21 000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.     

Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.

Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized. Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M). Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate. Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.     

Action of recombinant human apoptotic endonuclease G on naked DNA and chromatin substrates: cooperation with exonuclease and DNase I

Endonuclease G (endoG) is released from mitochondria during apoptosis and is in part responsible for internucleosomal DNA cleavage. Here we report the action of the purified human recombinant form of this endonuclease on naked DNA and chromatin substrates. The addition of the protein to isolated nuclei from non-apoptotic cells first induces higher order chromatin cleavage into DNA fragments > or = 50 kb in length, followed by inter- and intranucleosomal DNA cleavages with products possessing significant internal single-stranded nicks spaced at nucleosomal ( approximately 190 bases) and subnucleosomal ( approximately 10 bases) periodicities. We demonstrate that both exonucleases and DNase I stimulate the ability of endoG to generate double-stranded DNA cleavage products at physiological ionic strengths, suggesting that these activities work in concert with endoG in apoptotic cells to ensure efficient DNA breakdown.     

Isolation of high-mobility-group proteins HMG1 and HMG2 in non denaturing conditions and comparison of their properties with those of acid-extracted proteins

We describe a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of protein tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.     

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.     

Restriction endonucleases from various bacterial strains exhibit an ice-nucleating activity

Six strains containing site-specific endonucleases II were selected from a collection of 45 ice-nucleating bacterial strains isolated from rhizosphere of plants growing in various geographical regions. Endonucleases Pfl211I, Psp8I, and Psp23I were isolated and purified from two Pseudomonas sp. strains and a Pseudomonas fluorescens strain. Restriction endonucleases Pfl21I and Psp23I were shown to recognize and cleave the DNA nucleotide sequence 5'-CTGCA decrease G-3'. Endonuclease Psp8I recognized and cleaved the DNA nucleotide sequence 5'-G decrease GATCC-3'. These endonucleases were found to be true isoschizomers of PstI and BamHI, respectively.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.     

Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications.

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.     

A general method for the purification of restriction enzymes.

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA.