Methylation-sensitive Enzyme Research Articles

Sensitive Measurement of Unmethylated Repeat DNA Sequences by End-Specific PCR

We describe a new method that is well-suited for the determination of the methylation level of repetitive sequences such as human Alu elements. We have applied the method to the analysis of cell and tissue DNAs and expect it to have wide utility in studies of DNA methylation in cancer and other disease states, in monitoring response to epigenetic cancer therapies and in epidemiological studies. Only 1 ng DNA is needed for a duplex, one-tube real-time PCR in which methylation level and the amount of input DNA are concurrently measured. The relative cutting by the methylation-sensitive enzyme BstUI is compared with that of the methylation-insensitive enzyme DraI to give a measure of DNA methylation. The method depends upon the use of 5'-tailed, 3'-blocked oligonucleotides called facilitator oligonucleotides (Foligos). Only cut DNAs with specific matching sequences at their 3' ends can copy the tails of the Foligos and thus become tagged and available for subsequent PCR. Both the tagging and PCR are carried out by the same enzyme, Taq DNA polymerase. Because amplification only occurs if suitable ends have been generated in the target DNA, we have called this method end-specific PCR (ESPCR). ESPCR avoids the bisulfite treatment step that is usually required to measure methylation.

Optimized design and data analysis of tag-based cytosine methylation assays

Genome-wide, tag-based cytosine methylation analysis is optimized.

Investigation of SNP haplotypes in the H19 imprinted gene

Differentially methylated SNPs upstream of the human H19 imprinted gene were investigated in DNA samples from Japanese, German and African (Nigerian and Ghananian) individuals. We examined a roughly 1.2 kb region of DNA and successfully detected a parental allele (haplotype of SNPs) after treatment of the genomic DNA by a methylation-sensitive enzyme. Our investigation revealed nine new SNPs that are largely specific to a single ethnic group, and the new haplotype system composed of over 20 SNPs, designated H19HP, revealed a high diversity of genotypes in each ethnic group. This haplotype system may therefore be a useful tool for ethnicity determination and personal identification.

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3-1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

No Link Between X Chromosome Inactivation Pattern and Simple Goiter in Females: Evidence from a Twin Study

Simple goiter (SG) comprises diffuse (DG) and nodular (NG) benign nonautoimmune nontoxic goiter. In nonendemic goiter areas, the ratio of females to males may exceed 5:1, indicating that gender and/or sex hormones may play a role in the etiology of SG in these areas. Theoretically, as shown for autoimmune thyroid disease, X chromosome inactivation (XCI) and resultant tissue chimerism could offer a novel explanation for the female preponderance of SG. To examine whether skewed XCI is associated with SG, we first compared XCI in 71 twin individuals with SG with that in 142 unrelated healthy control twin individuals, and then performed a within-pair comparison of XCI in 48 twin pairs discordant for SG. DNA was extracted from peripheral blood cells. XCI analysis was performed by predigestion of DNA using the methylation-sensitive enzyme Hpall, followed by polymerase chain reaction of the polymorphic CAG repeat of the androgen receptor gene. A polymerase chain reaction product is obtained from the inactive X chromosome only. The XCI pattern was classified as skewed when 80% or more of the cells preferentially inactivated the same X chromosome. Twin zygosity was established by DNA fingerprinting. The frequency of skewed XCI in female twins with SG, DG, and NG was 11% (8/71), 13% (6/46), and 8% (2/25), respectively, which was not significantly different from the prevalences in the corresponding control populations, 14% (20/142, p = 0.56), 14% (13/92, p = 1.00), and 14% (7/50, p = 0.71), respectively. Essentially, similar results were obtained when comparing the prevalence of skewed XCI in twin pairs discordant for SG (48 pairs), DG (30 pairs), and NG (18 pairs). In a sample of Danish female twins, we did not find evidence for involvement of skewed XCI in the etiology or the female preponderance of SG.

Analysis of skewed X-chromosome inactivation in females with rheumatoid arthritis and autoimmune thyroid diseases

IntroductionThe majority of autoimmune diseases such as rheumatoid arthritis (RA) and autoimmune thyroid diseases (AITDs) are characterized by a striking female predominance superimposed on a predisposing genetic background. The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of several autoimmune diseases.MethodsWe examined XCI profiles of females affected with RA (n = 106), AITDs (n = 145) and age-matched healthy women (n = 257). XCI analysis was performed by enzymatic digestion of DNA with a methylation sensitive enzyme (HpaII) followed by PCR of a polymorphic CAG repeat in the androgen receptor (AR) gene. The XCI pattern was classified as skewed when 80% or more of the cells preferentially inactivated the same X-chromosome.ResultsSkewed XCI was observed in 26 of the 76 informative RA patients (34.2%), 26 of the 100 informative AITDs patients (26%), and 19 of the 170 informative controls (11.2%) (P < 0.0001; P = 0.0015, respectively). More importantly, extremely skewed XCI, defined as > 90% inactivation of one allele, was present in 17 RA patients (22.4%), 14 AITDs patients (14.0%), and in only seven controls (4.1%, P < 0.0001; P = 0.0034, respectively). Stratifying RA patients according to laboratory profiles (rheumatoid factor and anti-citrullinated protein antibodies), clinical manifestations (erosive disease and nodules) and the presence of others autoimmune diseases did not reveal any statistical significance (P > 0.05).ConclusionsThese results suggest a possible role for XCI mosaicism in the pathogenesis of RA and AITDs and may in part explain the female preponderance of these diseases.

An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines

BackgroundDNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance.ResultsWe here describe a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.ConclusionThe MMSDK method will be a valuable tool to increase the current knowledge of genome wide DNA methylation profiles.

A Sensitive Molecular Method for Detection of Disseminating Hematopoietic Tumor Cells by Using Specific Epigenetic DNA Methylation Biomarkers

Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients' survival.Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens.Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10−3 in a single-step PCR and 0.00001% or 10−6 in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR.Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14% (5/36), or 55% (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

DNA methylation and demethylation in Arabidopsis.

DNA methylation and demethylation in Arabidopsis.

Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

BackgroundThe study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK), a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci.ResultsGenomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05) after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes.ConclusionThe genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models of complex human diseases in a mixed cell population and might be a valuable technology to determine whether environmental exposures can lead to epigenetic changes.

형질전환 토마토에서 Cytosine Methylation에 의한 유전자발현 억제

토마토 형질전환체에서 NPTII 및 TPSP 유전자의 발현양상을 조사하였다. 4개 line 토마토 형질전환체가 선발배지에서 kanamycin에 저항성을 보이며 생산되었다. 그러나, 1번 line과 11번 line의 후대에서 NPTII 유전자의 발현이 억제되었으며, Kanamycin 저항성을 보이지 못했다. Southern 분석 결과, 1번 11번 line은 여러 copy의 외래 유전자를 가지고 있었으며, 2번, 10번 line은 1-2 copy의 외래유전자를 가지고 있었다. 형질전환 line 11번은 methylation sensitive 제한효소처리 후 DNA methylation 분석 결과, TPSP coding부위 및 도입유전자의 발현을 조절하는 CaMV35S 프로모터 주위에서 CpG methylation이 축적되었음이 확인되었다. 따라서 여러 copy의 외래유전자를 가진 토마토 형질전환 line 11번에서 NPTII 유전자 및 TPSP유전자의 발현이 억제된 것은 transcriptional gene silencing 기작에 의한 것으로 추정되었다. Transgene expression was analyzed in tomato plants. Four lines of neomycin phosphotransferase II gene (NPTII) and the trehalose biosynthetic fusion gene (TPSP) transformed <TEX>$T_0$</TEX> plants showed kanamycin resistance on selection medium. However, the analysis of phenotype (kanamycin resistance) and mRNA expression in <TEX>$T_1$</TEX> plants indicated that the expression of the NPTII and TPSP transgenes was down-regulated to an undetectable level in two independent lines 1 and 11. Southern analysis demonstrated that the lines 1 and 11 had multicopies of the transgenes, whereas the typical transgenic lines 2 and 10 had 1 or 2 copies. DNA methylation analysis using methylation sensitive enzyme detected accumulated CpG DNA methylation on TPSP coding region and CaMV35S promoter region in the line 11, but not the typical transgenic line 2. These results suggest that multicopy transgene in plants is attributed to down-regulation of the transgene expression via transcriptional gene silencing.

Gene expression and clonality analysis of the androgen receptor and phosphoglycerate kinase genes in polygonal cells and cuboidal cells in so-called pulmonary sclerosing hemangioma

The histogenesis of polygonal cells and cuboidal cells in so-called pulmonary sclerosing hemangioma remains unclear. To understand their histogenesis, polygonal and cuboidal cells were obtained from pulmonary sclerosing hemangioma tissue using a laser capture microdissection technique. Genomic DNA and total RNA were extracted and mRNA levels of cytokeratin, epithelial membrane antigen, vimentin, surfactant protein B, thyroid transcription factor-1, synaptophysin, and chromogranin-A were analyzed by RT-PCR. DNA was digested with the methylation-sensitive enzymes HhaI or HpaII, followed by nested PCR of the androgen receptor and phosphoglycerate kinase genes. Samples with polymorphisms were identified and a clonality analysis was performed. The cytokeratin, epithelial membrane antigen, and surfactant protein B genes were clearly expressed in cuboidal cells, while the vimentin and synaptophysin genes were clearly expressed and the epithelial membrane antigen gene was weakly expressed in polygonal cells. Thyroid transcription factor-1 was expressed in both cell types, while neither cell type expressed chromogranin-A. Clonality analysis showed the same loss of allele in both cell types (clonality ratio=0) or an unbalanced methylation pattern (clonality ratio <0.25). Polygonal and cuboidal cells in pulmonary sclerosing hemangioma exhibited a uniform pattern of monoclonality, indicating that both cell types are highly likely to originate from a common precursor. The differences in their morphological phenotype might result from their different mature status.

Skewed X inactivation in an X linked nystagmus family resulted from a novel, p.R229G, missense mutation in the FRMD7 gene

Aims:This study aimed to identify the underlying genetic defect of a large Turkish X linked nystagmus (NYS) family.Methods:Both Xp11 and Xq26 loci were tested by linkage analysis. The 12 exons...

An unusual S-adenosylmethionine synthetase gene from dinoflagellate is methylated

BackgroundS-Adenosylmethionine synthetase (AdoMetS) catalyzes the formation of S-Adenosylmethionine (AdoMet), the major methyl group donor in cells. AdoMet-mediated methylation of DNA is known to have regulatory effects on DNA transcription and chromosome structure. Transcription of environmental-responsive genes was demonstrated to be mediated via DNA methylation in dinoflagellates.ResultsA full-length cDNA encoding AdoMetS was cloned from the dinoflagellate Crypthecodinium cohnii. Phylogenetic analysis suggests that the CcAdoMetS gene, is associated with the clade of higher plant orthrologues, and not to the clade of the animal orthrologues. Surprisingly, three extra stretches of residues (8 to 19 amino acids) were found on CcAdoMetS, when compared to other members of this usually conserved protein family. Modeled on the bacterial AdeMetS, two of the extra loops are located close to the methionine binding site. Despite this, the CcAdoMetS was able to rescue the corresponding mutant of budding yeast. Southern analysis, coupled with methylation-sensitive and insensitive enzyme digestion of C. cohnii genomic DNA, demonstrated that the AdoMetS gene is itself methylated. The increase in digestibility of methylation-sensitive enzymes on AdoMet synthetase gene observed following the addition of DNA methylation inhibitors L-ethionine and 5-azacytidine suggests the presence of cytosine methylation sites within CcAdoMetS gene. During the cell cycle, both the transcript and protein levels of CcAdoMetS peaked at the G1 phase. L-ethionine was able to delay the cell cycle at the entry of S phase. A cell cycle delay at the exit of G2/M phase was induced by 5-azacytidine.ConclusionThe present study demonstrates a major role of AdoMet-mediated DNA methylation in the regulation of cell proliferation and that the CcAdoMetS gene is itself methylated.

Restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment. It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome. RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. Compared with the conventional gene-detection technologies, such as Southern blot analysis and PCR, RLGS has the following advantages even though it needs specially designed instruments: high-efficiency scanning capacity, scanning extensibility by using alternate restriction enzyme combinations, applicability to any organism, a spot intensity that reflects the copy number of restriction landmarks, and the ability, by using a methylation-sensitive enzyme, to screen the methylated state of genomic DNA. The RLGS protocol can be accomplished in 5 days to 2 weeks.

MMASS: an optimized array-based method for assessing CpG island methylation

We describe an optimized microarray method for identifying genome-wide CpG island methylation called microarray-based methylation assessment of single samples (MMASS) which directly compares methylated to unmethylated sequences within a single sample. To improve previous methods we used bioinformatic analysis to predict an optimized combination of methylation-sensitive enzymes that had the highest utility for CpG-island probes and different methods to produce unmethylated representations of test DNA for more sensitive detection of differential methylation by hybridization. Subtraction or methylation-dependent digestion with McrBC was used with optimized (MMASS-v2) or previously described (MMASS-v1, MMASS-sub) methylation-sensitive enzyme combinations and compared with a published McrBC method. Comparison was performed using DNA from the cell line HCT116. We show that the distribution of methylation microarray data is inherently skewed and requires exogenous spiked controls for normalization and that analysis of digestion of methylated and unmethylated control sequences together with linear fit models of replicate data showed superior statistical power for the MMASS-v2 method. Comparison with previous methylation data for HCT116 and validation of CpG islands from PXMP4, SFRP2, DCC, RARB and TSEN2 confirmed the accuracy of MMASS-v2 results. The MMASS-v2 method offers improved sensitivity and statistical power for high-throughput microarray identification of differential methylation.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

Methylation-specific digital karyotyping

Epigenetic alterations, including DNA methylation and histone modifications, are known to regulate various physiological and pathological processes. In mammalian cells, DNA methylation occurs at cytosines of CpG dinucleotides. Several methods have been developed for the genome-wide analysis of methylation patterns. However, none of these are quantitative or sequence-based, and the identification of the exact location of the methylated CpG is difficult. In this protocol, we describe a recently developed method--methylation-specific digital karyotyping (MSDK)--that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. The number of tags in an MSDK library reflects the methylation status of the mapping enzyme sites. Generation of MSDK libraries can be completed in 7-10 days, whereas sequencing and data analysis requires an additional 3-4 weeks.