The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Caenorhabditis elegans DNA does not contain 5-methylcytosine at any time during development or aging.

DNA, isolated from age-synchronous senescent populations of Caenorhabditis elegans has been quantitatively and qualitatively analyzed for the presence of 5-methylcytosine. High performance liquid chromatography on two wild-type and several mutant strains of C. elegans failed to detect any 5-methylcytosine. The restriction endonuclease isoschizomers, HpaII and MspI, were used to digest genomic DNA after CsCl purification and failed to detect any 5' cytosine methylation at any age. We conclude that C. elegans does not contain detectable (0.01 mole percent) levels of 5-methylcytosine.     

The 5'-GGATCC-3' cleavage specificity of BamHI is increased to 5'-CCGGATCCGG-3' by sequential double methylation with M.HpaII and M.BamHI

Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at 'non-canonical' DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5'-GGATCm5C efficiently, whereas M.BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M.HpaII, M.BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5'-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.     

The restriction modification system of Bacillus licheniformis MS1 and generation of a readily transformable deletion mutant

Restriction modification systems (R-M systems), consisting of a restriction endonuclease and a cognate methyltransferase, constitute an effective means of a cell to protect itself from foreign DNA. Identification, characterization, and deletion of the restriction modification system BliMSI, a putative isoschizomer of ClaI from Caryophanon latum, were performed in the wild isolate Bacillus licheniformis MS1. BliMSI was produced as recombinant protein in Escherichia coli, purified, and in vitro analysis demonstrated identical restriction endonuclease activity as for ClaI. A recombinant E. coli strain, expressing the heterologous bliMSIM gene, was constructed and used as the host for in vivo methylation of plasmids prior to their introduction into B. licheniformis to improve transformation efficiencies. The establishment of suicide plasmids in the latter was rendered possible. The subsequent deletion of the restriction endonuclease encoding gene, bliMSIR, caused doubled transformation efficiencies in the respective mutant B. licheniformis MS2 (∆bliMSIR). Along with above in vivo methylation, the establishment of further gene deletions (∆upp, ∆yqfD) was performed. The constructed triple mutant (∆bliMSIR, ∆upp, ∆yqfD) enables rapid genome manipulation, a requirement for genetic engineering of industrially important strains.

     

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.     

Effects of 5-azacytidine on methylation and expression of specific DNA sequences in C3H 10T1/2 cells.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.     

A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.     

Microarray-based method to evaluate the accuracy of restriction endonucleases HpaII and MspI

A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.     

乙肝病毒核心蛋白钉突部位基因工程改造对其功能的影响

通过在乙肝病毒核心蛋白钉突部位插入标签蛋白EGFP及小片段多肽,研究各种改造对HBc功能的影响。采用RLIC方法,构建野生型HBc、HBc钉突部位带不同接头的EGFP融合重组体、缩短的EGFP融合重组体,并构建与HBc功能互补的质粒HBV1.1c－,将不同重组体与HBV1.1c－共转染HEK293细胞,通过观察荧光及Southern blotting检测病毒复制中间体,判断相应基因工程改造对重组蛋白中不同结构域功能的影响。RLIC方法可有效地用来进行片段缺失,且缺失片段大小及位置无明显限制。带柔性或刚性接头的重组HBc-EGFP均可产生绿色荧光,但荧光在细胞内分布形态不同,两种重组HBc-EGFP均不能支持正常的HBV复制,各种截短的插入片段以及aa79-80单独缺失体亦不能支持HBV复制。结果表明RLIC方法是一种基因工程改造的有力工具,不同类型接头对重组蛋白的结构和功能有不同影响,aa79-80对维持HBc的主要功能之一——支持HBV复制有重要作用。     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.     

The construction of bifunctional fusion proteins consisting of MutS and GFP

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.     

The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.     

Characterization of an<Emphasis Type="Italic">Eco</Emphasis>R 124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit,which results in a novel DNA specificity

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.     

In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants

The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.     

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.     

Tissue- and cell-specific casein gene expression. II. Relationship to site-specific DNA methylation

The relationship between DNA methylation and the expression of the gamma- and beta-casein genes was investigated in both expressing and nonexpressing tissues and in isolated tumor cell subpopulations displaying differential casein gene expression. MspI/HpaII digestions of DNA isolated from liver, a totally nonexpressing tissue, indicated that specific sites of hypermethylation existed in these genes as compared to the DNA isolated from casein-producing lactating mammary gland. The positions of these sites were mapped in the gamma-casein gene by comparing total genomic DNA Southern blots to the restriction digests of several overlapping phage clones constituting the gamma-casein gene. In contrast, the methylation status of the HhaI sites in the gamma-casein gene was found to be invariant regardless of the expression status of the gene. The inverse correlation between the hypermethylation of certain MspI/HpaII restriction sites in the casein genes and their potential expressibility was further substantiated by studies in 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary carcinomas, which have an attenuated casein gene expression, and in cell subpopulations isolated from the 7,12-dimethylbenz(a)-anthracene tumor which were either depleted or enriched in casein-producing cells. Analysis of total tumor DNAs indicated that the casein genes were hypermethylated at the same sites observed in liver. However, a very faint hybridization signal was observed in the HpaII digests, suggesting cell-specific methylation differences. We have confirmed the hypomethylation of at least two of these MspI/HpaII sites within the subpopulation containing the casein-producing cells at a level consistent with the relative enrichment in that fraction. These results demonstrate differential site-specific casein gene methylation not only between tissues but also between cell subpopulations within a single tissue.     

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.