Restriction Enzyme Isoschizomers Research Articles

Short telomere subtelomeric hypomethylation is associated with telomere attrition in elderly diabetic patients 1.

Telomere shortening is well known to be associated with the aging process and aging-associated diseases, including diabetes. The telomere length and subtelomeric methylation status in peripheral leucocytes (LTL) were compared in elderly type 2 diabetes (T2D) patients and diabetes-free controls (C). The methylation status was analyzed between MspI-TRF lengths and HpaII-TRF lengths by using methylation-sensitive and -insensitive restriction enzyme isoschizomers, MspI and HpaII, respectively. The mean telomere lengths, MspI-TRF or HpaII-TRF, were not significantly different between C and T2D patients. The percentage of fractionated densitometry showed that long and middle telomeres (>9.4 kb, 4.4-9.4 kb) were unaltered but short telomeres (<4.4 kb) in T2D patients were increased compared with C group. The methylation status revealed subtelomeric hypomethylation in short telomeres of T2D patients. When some patients with T2D were treated with 3-hydroxy-3-methylglutaril coenzyme A (HMG-CoA) reductase inhibitors (statin), results seen in short telomere of T2D patients were not observed and were not different from C. This suggested that this altered subtelomeric hypomethylation may be associated with the accelerated telomere shortening in elderly diabetic patients. These results also mean that the subtelomeric hypomethylation can also be influenced by statin treatment in T2D.

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells.

The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis

*In plants, epigenetic variations based on DNA methylation are often heritable and could influence the course of evolution. Before this hypothesis can be assessed, fundamental questions about epigenetic variation remain to be addressed in a real-world context, including its magnitude, structuring within and among natural populations, and autonomy in relation to the genetic context. *Extent and patterns of cytosine methylation, and the relationship to adaptive genetic divergence between populations, were investigated for wild populations of the southern Spanish violet Viola cazorlensis (Violaceae) using the methylation-sensitive amplified polymorphism (MSAP) technique, a modification of the amplified fragment length polymorphism method (AFLP) based on the differential sensitivity of isoschizomeric restriction enzymes to site-specific cytosine methylation. *The genome of V. cazorlensis plants exhibited extensive levels of methylation, and methylation-based epigenetic variation was structured into distinct between- and within- population components. Epigenetic differentiation of populations was correlated with adaptive genetic divergence revealed by a Bayesian population-genomic analysis of AFLP data. Significant associations existed at the individual genome level between adaptive AFLP loci and the methylation state of methylation-susceptible MSAP loci. *Population-specific, divergent patterns of correlated selection on epigenetic and genetic individual variation could account for the coordinated epigenetic-genetic adaptive population differentiation revealed by this study.

Cloning of NruI and Sbo13I restriction and modification sstems in E. coli and amino acid sequence comparison of M.NruI and M.Sbo13I with other amino-methyltransferases

BackgroundNruI and Sbo13I are restriction enzyme isoschizomers with the same recognition sequence 5' TCG↓CGA 3' (cleavage as indicated↓). Here we report the cloning of NruI and Sbo13I restriction-modification (R-M) systems in E. coli. The NruI restriction endonuclease gene (nruIR) was cloned by PCR and inverse PCR using primers designed from the N-terminal amino acid sequence. The NruI methylase gene (nruIM) was derived by inverse PCR walking.ResultsThe amino acid sequences of NruI endonuclease and methylase are very similar to the Sbo13I R-M system which has been cloned and expressed in E. coli by phage selection of a plasmid DNA library. Dot blot analysis using rabbit polyclonal antibodies to N6mA- or N4mC-modified DNA indicated that M.NruI is possibly a N6mA-type amino-methyltransferase that most likely modifies the external A in the 5' TCGCGA 3' sequence. M.Sbo13I, however, is implicated as a probable N4mC-type methylase since plasmid carrying sbo13IM gene is not restricted by Mrr endonuclease and Sbo13I digestion is not blocked by Dam methylation of the overlapping site. The amino acid sequence of M.NruI and M.Sbo13I did not show significant sequence similarity to many known amino-methyltransferases in the α, β, and γ groups, except to a few putative methylases in sequenced microbial genomes.ConclusionsThe order of the conserved amino acid motifs (blocks) in M.NruI/M.Sbo13I is similar to the γ. group amino-methyltranferases, but with two distinct features: In motif IV, the sequence is DPPY instead of NPPY; there are two additional conserved motifs, IVa and Xa as extension of motifs IV and X, in this family of enzymes. We propose that M.NruI and M.Sbo13I form a subgroup in the γ group of amino-methyltransferases.

Assessing the DNA methylation status of single cells with the comet assay

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated ‘reference plasmids’), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these ‘reference plasmids’ revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).

In the cerebral cortex of female and male mice, amyloid precursor protein (APP) promoter methylation is higher in females and differentially regulated by sex steroids

The over-expression of amyloid precursor protein (APP) gene in certain areas of the brain indicates abnormalities in gene regulation as an important factor for the development of Alzheimer's disease (AD). We have reported recently that APP mRNA expression is lower in female as compared to male and is regulated by sex steroids. As methylation of promoter is crucial for such regulation, we have used isoschizomeric restriction enzymes MspI and HpaII to analyze the pattern of APP promoter methylation in the cerebral cortex of intact, gonadectomized, testosterone- and estradiol-treated adult and old mice of both sexes. Southern blots of DNA digested with HindIII/MspI or HindIII/HpaII from different groups of mice were probed with a 1.27-kb DIG-11-dUTP labeled APP promoter fragment. The results revealed four distinct bands of 315 bp, 596 bp, 911 bp, and 2.6 kb by MspI, a single band of 2.6 kb by HpaII in all groups and an additional 2.9 kb in intact and estradiol treated old male and testosterone-treated adult female mice. The intensity of 2.6-kb band was relatively lower in intact female as compared to male and varied with different treatments in both ages, indicating the modulation of methylation. Thus, these findings showed that APP promoter methylation is higher in female and differentially regulated by sex steroids in the mice cerebral cortex, suggesting a strong correlation between promoter methylation and transcriptional silencing of APP.

A Unique Pair of GATC Specific DNA Methyltransferases in Mitsuokella multiacida

Two GATC specific methylases together with Sau3AI isoschizomeric restriction endonuclease were partially characterized in Mitsuokella multiacida 46/5. This is the first report on the presence of solitary Dam methyltransferase alongside GATC specific restriction-modification system resulting in the unusual two-fold methylation of the GATC motifs.

GATC-specific restriction--modification systems in ruminal bacteria.

The GATC-specific restriction and modification activities were analyzed in 11 major bacterial representatives of ruminal microflora. Modification phenotype was observed in 13 out of 40 ruminal strains. MboI isoschizomeric restriction endonucleases were detected in 10 bacterial strains tested; three strains lacked any detectable corresponding endonuclease activity. The only examined strain of Mitsuokella multi-acida was found to possess a different type of endonuclease activity. This is the first report on restriction activity in ruminal treponemes M. multiacida and Megasphaera elsdenii.

GATC-specific restriction and modification systems in treponemes.

To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. This is the first report on restriction activity in rumen treponemes.

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Transcriptional Regulation of the Human CYP1B1 Gene: EVIDENCE FOR INVOLVEMENT OF AN ARYL HYDROCARBON RECEPTOR RESPONSE ELEMENT IN CONSTITUTIVE EXPRESSION

The cytochrome P450 1B1 gene (CYP1B1) is expressed constitutively and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the human breast adenocarcinoma cell line MCF-7 but not in the human hepatoma cell line HepG2. Genomic DNA isolated from both cell lines was digested with the methylation-sensitive restriction enzyme isoschizomers MspI and HpaII, and subjected to Southern analysis with a probe for the CYP1B1 promoter/enhancer region. Although differences were observed in methylation patterns for the CYP1B1 gene from MCF-7 and HepG2 cells, treatment with the demethylating agent 5-azacytidine (10 microM for 6 days) did not activate CYP1B1 mRNA expression in HepG2 cells. Furthermore, treatment with the histone deacetylase inhibitor trichostatin A (100 nM for 24 h) did not activate CYP1B1 mRNA expression in HepG2 cells. Comparative analysis of the constitutive expression of luciferase/1B1 reporter constructs containing a series of deletions in the 5' enhancer region indicated that in MCF-7 cells the region from -987 to -732 (relative to the transcription start site) was necessary for maximal levels of activity. Mutation of the aryl hydrocarbon receptor response elements (dioxin response elements) in this region showed that the dioxin response elements located at -833 is essential for constitutive gene expression in MCF-7 cells. In HepG2 cells, reporter gene activity was at least equal or greater than the activity observed in MCF-7 cells, which is in marked contrast to the expression of the native CYP1B1 gene. Taken together these findings indicate that the observed cell-specific differences in CYP1B1 constitutive expression are not mediated by DNA promoter/enhancer methylation, but are likely due to either 1) inaccessibility of the 5'-enhancer region in HepG2 cells to transcriptional activators due to a higher order chromatin structure that does not involve histone acetylation, or 2) the action of a repressor protein at cis-elements located outside of the -2296 to +25 region examined with the CYP1B1 reporter constructs. Furthermore, at least one of the dioxin response elements in the enhancer region is required for constitutive expression of CYP1B1.

Prevalence of CTGCAG Recognizing Restriction and Modification Systemsin Ruminal Selenomonades

Analysis of restriction and modification activities in natural population of Selenomonas ruminantium have revealed the prevalence of CTGCAG (Pst I isoschizomers) recognizing restriction and/or modification systems in these bacteria. Pst I isoschizomeric restriction endonucleases were detected in 4 out of 15 strains tested. In one strain, the Pst I isoschizomeric restriction system was accompanied by another restriction and modification system recognizing GAATTC sequence (Eco RI isoschizomer). Four other strains contained CTGCAG specific methylases which lacked cognate endo-nuclease activities. Presence of identical restriction and modification systems in both of subspecies of S. ruminantium , as well as the occurrence of Pst I isoschizomers in various combinations, indicate the possibility of horizontal transfer of genes coding for these systems.

PMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size.

pMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size.

Methylation of mitochondrial DNA in carrot (Daucus carota L.).

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed.

Prochlorothrix hollandica PCC 9006: genomic properties of an axenic representative of the chlorophyll a/b-containing oxyphotobacteria

Prochlorothrix hollandica is an oxygenic photosynthetic prokaryote that differs from the cyanobacteria in having chlorophyll a/b-protein complexes instead of phycobilisomes as major light-harvesting antennae. We report the isolation and culturing of an axenic strain of P. hollandica, available from the Pasteur Culture Collection of Cyanobacteria as strain PCC 9006. The strain has a mean DNA base composition of 51.6 +/- 0.1 mol% G+C and a genomic complexity of 3.37 +/- 0.17 x 10(9) daltons (5,505 kb). A reiterated DNA sequence represents approximately 4.4% of the genome. Restriction enzyme isoschizomers with different sensitivities to base methylation were used to demonstrate that most A residues in the sequence GATC are methylated in P. hollandica DNA and that this methylation increases with culture age. Furthermore, some C residues are methylated, although the specificity of the C methylation system does not match that of well-characterized C methylases. Nucleotide analysis showed that up to approximately 3.5% of both dA and dC residues are methylated in P. hollandica DNA.

Methylation of somatic and sperm DNA in the homosporous fern Ceratopteris richardii.

Plants, in general, have a high proportion of their CpG and CpNpG nucleotide motifs modified with 5-methylcytosine (5mC). Developmental changes in the proportion of 5mC are evident in mammals, particularly during gametogenesis and embryogenesis, but little information is available from flowering plants due to the intimate association of gametes with sporophytic tissues. In ferns, sperm are uninucleate and free-swimming and thus are easily isolated. We have examined 5mC in DNA isolated from fern sperm and other tissues with methylation-sensitive and -insensitive restriction enzyme isoschizomers, Southern blots probed with chloroplast and nuclear ribosomal RNA genes and end-labeled restriction fragments. We conclude that fern sperm DNA is methylated to a similar or greater degree than DNA isolated from either sporophytes or gametophytes.

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10(-1), and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10(-1)-2 and 2-10(2), respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.

Cloning of a pair of genes encoding isoschizomeric restriction endonucleases from bacillus species: the BspEI and BspMII restriction and modification systems

The respective genes ( R− M) encoding restriction and modification systems from two Bacillus species which recognise the same nucleotide sequence, 5′-TCCGGA, have been cloned and expressed in Escherichia coli. The BspEI R-M genes were cloned on a 3.6-kb HindIII fragment, whereas the BspMII R-M genes were cloned on three contiguous HindIII fragments totalling 9.8 kb. Upon thermal induction, E. coli carrying the bspEIR clones under the control of the phage λ PL promoter, express high levels of R·BspEI (10 6 units/g wet cell paste). The bspMIIR gene, on the other hand, is only poorly expressed (about 4 × 10 3 units/g wet cell paste) following induction. Although the enzymes of both R-M systems recognize the same sequence and the restriction endonucleases (ENases) cleave DNA at the same position, the modification specified by the methyltransferases (MTases) differ. The internal cytosine is the site for M·BspMII modification (TC meCGGA), whereas the external cytosine is modified by M·BspEI (T meCCGGA). The two R-M systems probably evolved independently.

Methylation of cottontail rabbit papillomavirus DNA and tissue-specific expression in transgenic rabbits

Transgenic rabbits carrying multiple copies of CRPV genomic DNA were described previously (Peng et al. (1993) J. Virol. 67, 1698–1701). CRPV DNA was detectable in all tissues of the transgenic rabbits, howeverm the transcripts of CRPV genes only were found in skin and skin tumors. Tumor development was also restricted to skin. To study the mechanism involving tissue-specific expression of CRPV genes in rabbits, cellular DNAs, isolated from different normal tissues and skin tumors, were digested with the two isoschizomeric restriction endonucleases Msp I (methylation resistant) and Hpa II (methylation sensitive), respectively, and analyzed by Southern blot. CRPV DNA, especially its upstream regulatory region (URR), was extensively methylated in all normal tissues, but methylation was remarkably reduced in skin tumors. These data suggest that extensive methylation of CRPV genome, especially in the URR, might be a factor in controlling its tissue-specific expression.