A DNA-modification methylase from Bacillus stearothermophilus V.

A type II modification methylase (M BstVI) was partially purified from the thermophilic bacterium Bacillus stearothermophilus V. The methylase catalyses the transfer of methyl groups from S-adenosyl-L-methionine to unmodified double-stranded DNA. The product of methylation was identified by paper chromatography as N6-methyladenine. Since M BstVI protects DNA against cleavage by BstVI and XhoI restriction endonucleases, it follows that it methylates the adenine residue in the sequence 5'-C-T-C-G-A-G-3'.     

Crystals of a nucleosome core particle containing defined sequence DNA

Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.     

Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5.

High pressure liquid chromatography on the RPC-5 reversed-phase ion exchange system has been shown to have several potential applications as an initial high capacity step in the isolation of specific DNA restriction fragments. The fractionation of the Hinc II digest of lambda DNA, which contains 35 fragments with "flush ends" ranging in size from 3 x 10(6) to 7 x 10(4) daltons, has been used as a model system. Under certain conditions there are some restriction fragments whose elution relative to other fragments is different on RPC-5 chromatography than it is on gel electrophoresis. In some special circumstances it is possible to obtain satisfactory yields (60-70%) of a pure restriction fragment after a single passage through an RPC-5 column.     

Stage-specific DNA methylation in a fungal plant pathogen.

Significantly more 5-methylcytosine residues were found in the DNA from the dormant sclerotia of Phymatotrichum omnivorum than in the DNA from the metabolically active mycelia of the fungus, as shown by high-pressure liquid chromatography of acid-hydrolyzed DNA digests and by restriction of the DNA with the isoschizomers MspI and HpaII. N6-Methyladenine was not detected in GATC sequences in the DNA isolated from either stage.     

Genomic DNA extraction from<Emphasis Type="Italic">Victoria amazonica</Emphasis>

DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP) (Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A_260/230 ratio of 1.836, A_260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion.     

A new packing for separation of DNA restriction fragments by high performance liquid chromatography

TSK-GEL SW was found to be useful as a packing in high performance liquid chromatography for the separation of double-stranded DNA restriction fragments. DNA fragments smaller than 300 base pairs were separated as discrete peaks depending solely upon difference in chain length. The recovery of DNA fragments was higher than 90%.     

Characterisation of the novel restriction endonuclease <Emphasis Type="Italic">Sui</Emphasis>I from <Emphasis Type="Italic">Sulfolobus islandicus</Emphasis>

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70°C.     

Unique modification of adenine in genomic DNA of the marine cyanobacterium Trichodesmium sp. strain NIBB 1067.

The genomic DNA of the marine nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067 was found to be highly resistant to DNA restriction endonucleases. The DNA was digested extensively by the restriction enzyme DpnI, which requires adenine methylation for activity. The DNA composition, determined by high-performance liquid chromatography (HPLC), was found to be 69% AT. Surprisingly, it was found that a modified adenine which was not methylated at the usual N6 position was present and made up 4.7 mol% of the nucleosides in Trichodesmium DNA (15 mol% of deoxyadenosine). In order for adenine residues to be modified at this many positions, there must be many modifying enzymes or at least one of the modifying enzymes must have a degenerate recognition site. The reason(s) for this extensive methylation has not yet been determined but may have implications for the ecological success of this microorganism in nature.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

The replication origin of proplastid DNA in cultured cells of tobacco

Summary When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.     

Size fractionation of DNA fragments by liquid-liquid chromatography

A method for the fractionation of double-stranded DNA fragments from 150 to 22000 b.p. in size by liquid-liquid chromatography is described. The procedure makes use of the fact that the partitioning of DNA in a polyethylene glycol-dextran system is size dependent and can be altered by alkali metal cations. Cellulose or celite are used as supports for the stationary, dextran-rich phase. Examples show the fractionation of digests of T7 DNA produced by Dpn II and Hind II restriction endonulceases as well as lambda DNA digests produced by Hind III and Eco RI restriction endonucleases.     

DNA global hypomethylation in EBV-transformed interphase nuclei.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.     

Derivation of a restriction map of bacteriophage T3 DNA and comparison with the map of bacteriophage T7 DNA.

The DNA of bacteriophage T3 was characterized by cleavage with seven restriction endonucleases. AvaI, XbaI, BglII, and HindIII each cut T3 DNA at 1 site, KpnI cleaved it at 2 sites, MboI cleaved it at 9 sites, and HpaI cleaved it at 17 sites. The sizes of the fragments produced by digestion with these enzymes were determined by using restriction fragments of T7 DNA as molecular weight standards. As a result of this analysis, the size of T3 DNA was estimated to be 38.74 kilobases. The fragments were ordered with respect to each other and to the genetic map to produce a restriction map of T3 DNA. The location and occurrence of the restriction sites in T3 DNA are compared with those in the DNA of the closely related bacteriophage T7.     

O6-methylguanine in place of guanine causes asymmetric single-strand cleavage of DNA by some restriction enzymes

The interactions of restriction enzymes with their cognate DNA recognition sequences present a model for protein-DNA interactions. We have investigated the effect of O6-methylguanine on restriction enzyme cleavage of DNA; O6-methylguanine is a carcinogenic lesion and a structural analogue of the biological restriction inhibitor N6-methyladenine. O6-Methylguanine was synthesized into oligonucleotides at unique positions. The oligonucleotides were purified and analyzed by high-pressure liquid chromatography to assure that, within the limits of our detection, O6-methylguanine was the only modified base present. These oligonucleotides were annealed with their complement so that cytosine, and in one case thymine, opposed O6-methylguanine. DNA cleavage by restriction enzymes that recognize a unique DNA sequence, HpaII, HhaI, HinPI, NaeI, NarI, PvuII, and XhoI, was inhibited by a single O6-methylguanine in place of guanine (adenine for PvuII) within the appropriate recognition sequences. However, only the modified strand was nicked by HpaII, NaeI, and XhoI with O6-methylguanine at certain positions, indicating asymmetric strand cleavage. For all the restriction enzymes studied but AhaII, BanI, and NarI, lack of double- or single-strand cleavage correlated with inability of the O6-methylguanine-containing recognition sequence to measurably bind enzyme. None of the restriction enzymes studied were inhibited by O6-methylguanine outside their cognate recognition sequences.     

The influence of energy restriction and developmental state on DNA 5-methyldeoxycytidine in rat mammary and liver tissues

Female rats were fed either ad libitum or 30% energy-restricted diets from 5 weeks through 25 weeks of age. Genomic DNA was extracted from mammary tissue and liver at 7, 9, 14 (mid-pregnancy), 16.5 (mid-lactation), and 25 weeks of age. The 5-methyldeoxycytidine content of DNA was determined by high pressure liquid chromatography. As animals aged from 7–25 weeks, 5-methyldeoxycytidine increased in mammary tissue, whereas in liver it decreased. This suggests that 5-methyldeoxycytidine exhibits tissue specific patterns. No changes in 5-methyldeoxycytidine content due to 30% energy restriction were observed in either mammary tissue or liver.     

猪生长激素cDNA的分子克隆

用改进的氯化锂沉淀沉法和寡聚(dT)一纤维素亲和层析法由猪垂体制得总mRNA。以此总mRNA为模板,合成cDNA。钝端连接重组到质粒pUC19的SmaI位点,转化E.coli JM107,筛选出阳性克隆。用限制性内切酶酶切签定及5’端部分核苷酸序列分析证明:克隆了全长猪生长激素cDNA,其长度约为896bp。     

Characterization of the NgoAXP: phase-variable type III restriction–modification system in Neisseria gonorrhoeae

Methyltransferases associated with type III restriction–modification (RM) systems are phase-variably expressed in a variety of pathogenic bacteria. NgoAXP, the type III RM system encoded by Neisseria gonorrhoeae , was characterized in this study. The cloned resngoAXP and ngoAXPmod genes were expressed in Escherichia coli strains. The restriction and modification activities of NgoAXP were confirmed in vivo by the λ phage restriction and modification test and in vitro by the methylation of DNA substrates in the presence of [ methyl -³H]AdoMet. As in all known type III systems, the restriction activity needed the presence of both genes, while the presence of the ngoAXPmod gene was sufficient for DNA methylation. Following its overexpression, the DNA methyltransferase M.NgoAXP was purified to apparent homogeneity using metal affinity chromatography. The specific sequence recognized by this enzyme was determined as a nonpalindromic sequence: 5'-CCACC-3', in which the adenine residue is methylated. We observed that in E. coli cells, the expression of the restriction phenotype associated with NgoAXP switched randomly. This phase variation was associated with the change in the number of pentanucleotide repeats (5'-CCAAC/G-3') present at the 5'-end of the coding region of the ngoAXPmod gene.     

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.     

Preparation and characterization of a viral DNA molecule containing a site-specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens

The synthetic oligonucleotide heptamer 5'-ATCCGTC-3' was reacted in vitro with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single N-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of M13 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex M13 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the HincII recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the M13 DNA containing the site-specific AF adduct with HincII, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA molecule at this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion.