Variation In Methylation Levels Research Articles

Evaluation of DNA methylation status at differentially methylated regions in IVF-conceived newborn twins

To examine the effect of assisted reproductive technology on the stability of DNA methylation at differentially methylated regions (DMRs) in twins conceived by IVF. Prospective clinical observational study. IVF center, university-affiliated teaching hospital. Fifty-nine pairs of twins were recruited, including 29 pairs conceived through IVF and 30 pairs of naturally conceived twins. Collection of umbilical cord blood samples. DNA was extracted from umbilical cord blood. Two maternally methylated regions (KvDMR1 and PEG1) and one paternally methylated region (H19/IGF2 DMR) were analyzed using bisulfite-based technologies. Although H19/IGF2 DMR and KvDMR1 showed slightly more variable levels of methylation in IVF cases than in spontaneous cases, methylation indices did not reveal significant differences at three DMRs between IVF-conceived and naturally conceived twins. Our results suggest no significant increase in imprint variability at these DMRs, but the greater variance in the IVF twins has a biologically meaningful consequence and may be a topic for future investigation. Large cohorts are needed to systematically assess the potential epigenetic risk in twins conceived with IVF.

A comparative analysis of DNA methylation across human embryonic stem cell lines

BackgroundWe performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines.ResultsWe find that heavily methylated non-CG sites are strongly conserved, especially when found within the motif TACAG. They are enriched in splice sites and are more methylated than other non-CG sites in genes. We next studied the relationship between allele-specific expression and allele-specific methylation. By combining bisulfite sequencing and whole transcriptome shotgun sequencing (RNA-seq) data we identified 1,020 genes that show allele-specific expression, and 14% of CG sites genome-wide have allele-specific methylation. Finally, we asked whether the methylation state of transcription factor binding sites affects the binding of transcription factors. We identified variations in methylation levels at binding sites and found that for several transcription factors the correlation between the methylation at binding sites and gene expression is generally stronger than in the neighboring sequences.ConclusionsThese results suggest a possible but as yet unknown functional role for the highly methylated conserved non-CG sites in the regulation of HESCs. We also identified a novel set of genes that are likely transcriptionally regulated by methylation in an allele-specific manner. The analysis of transcription factor binding sites suggests that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.

Abstract P3-04-01: Integrated Analysis of Epigenetic Signatures Associated with Inflammatory Breast Cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary epithelial breast cancer and differs from non-IBC both from a clinical and a biological perspective. The high metastatic potential of IBC is highlighted by the low 5-year survival rate of 50%. The biological features that make IBC unique are its rapid local growth, its high angioinvasive capability with early metastasis and its aggressive behavior from inception. These differences could be the result of key changes in the epigenetic profile of IBC. To address this issue, we compared the levels of epigenetic regulation in IBC (N=19) and non-IBC (N=43) samples at the methylome (DNA methylation), miRome (miRNA) and transcriptome (mRNA) level using high-throughput molecular technologies. Differences in gene expression profiles allowed the classification of samples according to tumor subtype (IBC vs non-IBC). The analysis of 27,578 CpG loci demonstrated that the largest variation in methylation levels between samples was attributable to differences in metastatic status (P=0.026) and patients’ age (P=0.049). About 2% of all interrogated CpG loci was differentially methylated in IBC and non-IBC (FDR <0.1). Annotated gene functions for these CpG loci were enriched for biological processes related to inflammation and immune response. Using a whole genome approach, we observed significant, negative correlations between methylation and mRNA levels for 38% of genes. For 8% of genes, a significant, positive correlation was observed. Interrogated CpG loci for these genes were more frequently located in the gene body (>500 bp from TSS) (P=0.008). Our analysis of 322 miRNAs showed that the largest variation in miRNA levels between samples was attributable to differences in ER status (P=0.001) and histological grade (P=0.002). About 4% of all miRNAs was expressed at different levels in IBC and non-IBC. Using a whole genome approach, we identified 17,295 of highly correlated miRNA-mRNA pairs for these IBC-associated miRNAs. Based on this miRNA target gene information, we were able to detect a variety of biological processes that may indicate function of these miRNAs and to indicate potential associations of these miRNAs with prognosis in breast cancer. Furthermore, the comparison of mRNA levels of key miRNA processing genes revealed the upregulation of Ago2 (P=0.002) and the downregulation of Dicer (P=0.004) in IBC when compared to non-IBC, which may suggest higher RISC activities and, therefore, that miRNAs regulate target gene expression in IBC with higher efficiency. In conclusion, this study represents the first integrated analysis of epigenetic signatures in IBC. Using high-throughput technologies, we were able to identify a number of epigenetic markers specifically associated with IBC. A global initiative has been taken to set up an IBC tissue repository that will allow the confirmation of these kinds of studies on a larger IBC sample population. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-04-01.

Rapid and reliable detection of LINE-1 hypomethylation using high-resolution melting analysis

Objectives The aim of the present study was to evaluate the precision and reproducibility of the LINE-1 high-resolution melting (HRM) assay to detect LINE-1 hypomethylation. Design and methods We first evaluated a methylated DNA dilution matrix and a panel of human cancer cell lines. We then applied this LINE-1 HRM assay to a set of 37 archival prostate cancer tissue samples. Results Our LINE-1 HRM assay revealed small and reproducible run-to-run and bisulfite-to-bisulfite variations. As expected, we found a large variation in methylation levels between different cancer cell lines. All results were confirmed with MethyLight and pyrosequencing as indicated by the high correlation coefficient. Finally, we successfully applied the LINE-1 HRM assay to archival prostate cancer tissues. Conclusions The present LINE-1 HRM assay represents a novel, accurate, and cost-effective method to measure global hypomethylation, which makes it suitable for high- and low-throughput laboratories.

Patterns of DNA methylation in individual colonic crypts reveal aging and cancer-related field defects in the morphologically normal mucosa

Methylation of CpG islands (CGIs) in the promoter regions of tumour suppressor genes is common in colorectal cancer and occurs also in an age-dependent manner in the morphologically normal colorectal mucosa. In this study, we quantified the level of methylation of six genes associated with the Wnt signalling pathway (adenomatous polyposis coli, DKK1, WIF1, SFRP1, SFRP2 and SFRP5) together with long-interspersed nuclear element-1 as a surrogate for global methylation. DNA methylation was analysed in 260 individual colorectal crypts obtained from eight female patients with no evidence of colorectal disease and five with colorectal cancer. Significant variation in methylation levels for each of the six genes existed between crypts from the same biopsy. The variation in both global and gene-specific CGI methylation between crypts from the same individual was significantly less than that between individuals. Bisulphite sequencing provided insight into the mechanism of aberrant methylation showing that CGI methylation occurs in an 'all-or-none' manner by the directional spreading of methylation from further upstream. Univariate statistical analyses revealed that there were significant differences in crypt-specific methylation associated with both aging and disease status. A multivariate statistical modelling approach was able to distinguish both subject age and health status based on crypt-specific methylation profiles. Our results indicate that the differential methylation of genes associated with the Wnt signalling pathway affecting individual morphologically normal crypts may contribute to the age-dependent generation of the colonic field defect and, in combination with mutations, to the stepwise development of colorectal neoplasia.

Regulation of NF-κB responses by epigenetic suppression of IκBα expression in HCT116 intestinal epithelial cells

Intestinal epithelial cells play critical roles in regulating mucosal immunity. Since epigenetic factors such as DNA methylation and histone modifications are implicated in aging, carcinogenesis, and immunity, we set out to assess any role for epigenetic factors in the regulation of intestinal epithelial cell immune responses. Experiments were conducted using the HCT116 cell line, and a subclone was genetically engineered to lack DNA methyltransferases (DNMT). The induction of the chemokine interleukin-8 and the antiapoptotic protein cFLIP by tumor necrosis factor-alpha were markedly less in HCT116 cells lacking DNMT than in parental cells. These effects were accompanied by lower monocyte chemotaxis and higher caspase signaling in HCT116 cells lacking DNMT than parental cells. Tumor necrosis factor-alpha-induced NF-kappaB activation was blocked and IkappaBalpha expression was higher in HCT116 cells lacking DNMT than in parental cells. A CpG island in the IkappaBalpha gene promoter region was found to contain variable levels of methylation in parental HCT116 cells. Chromatin immunoprecipitation analysis of histone proteins bound to the IkappaBalpha gene promoter revealed that higher levels of IkappaBalpha expression in HCT116 cells lacking DNMT compared with parental cells were accompanied by more chromatin marks permissive to gene transcription. These findings show that epigenetic factors influence the NF-kappaB system in intestinal epithelial cells, resulting in a previously unrecognized mechanism of innate immune regulation.

FT-IR spectroscopy supported by PCA–LDA analysis for the study of embryonic stem cell differentiation

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterizedin situby FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm–1), protein (1700–1600 cm–1) and in the nucleic acid (1050–850 cm–1) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm–1and 899 cm–1) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm–1shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research.

Tailored Synthetic Polyamines for Controlled Biomimetic Silica Formation

Organic compounds isolated from diatoms contain long-chain polyamines with a propylamine backbone and variable methylation levels and chain lengths. These long-chain polyamines are thought to be one of the important classes of molecules that are responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe a synthetic route based on solid-phase peptide synthesis from which well-defined long-chain polyamines with different chain lengths, methylation patterns, and subunits can be obtained. Quantitative silica precipitation experiments together with structural information about the precipitated silica structures gained by scanning and transmission electron microscopy revealed a distinct dependence of the amount, size, and form of the silica precipitates on the molecular structure of the polyamine. Moreover, the influence of the phosphate concentration was elucidated, revealing the importance of divalent anions for the precipitation procedure. We were able to derive further insights into the precipitation properties of long-chain polyamines as functions of their hydrophobicity, protonation state, and phosphate concentration, which may pave the way for better control of the formation of nanostructured silica under ambient conditions.

Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability

Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.

Does promoter methylation of the SLC30A5 (ZnT5) zinc transporter gene contribute to the ageing-related decline in zinc status?

A decline in Zn status with ageing may contribute to the development of frailty, including impaired immune function, and increased incidence of age-related degenerative diseases. This decline may be a result of reduced dietary Zn intake and/or impaired Zn absorption in the gut. The Zn transporter ZnT5 may play a key role in the absorption of dietary Zn. The corresponding gene (SLC30A5) has a CpG island in its promoter region, so could be regulated by epigenetic mechanisms. It is hypothesised that methylation of the SLC30A5 promoter region is increased with age and that a resulting reduction in ZnT5 expression contributes to the decline in Zn status observed with ageing. This hypothesis has been addressed through (1) studies of effects of SLC30A5 promoter methylation on gene expression in vitro and (2) in vivo measurements of the DNA methylation status of this gene domain. It has been established in vitro that methylation of the human SLC30A5 promoter region results in reduced expression of an associated reporter gene. Second, this gene region shows variable levels of methylation in vivo. Correlation between the level of methylation at this locus and age would support the hypothesis that age-related hypermethylation of this region has the potential to modulate dietary Zn absorption. This premise is being investigated by analysis of additional samples from a human adult cohort to test the hypothesis that methylation of the SLC30A5 promoter region contributes to the age-related decline in Zn status.

From biosilicification to tailored materials: Optimizing hydrophobic domains and resistance to protonation of polyamines

Considerable research has been directed toward identifying the mechanisms involved in biosilicification to understand and possibly mimic the process for the production of superior silica-based materials while simultaneously minimizing pollution and energy costs. Molecules isolated from diatoms and, most recently sponges, thought to be key to this process contain polyamines with a propylamine backbone and variable levels of methylation. In a chemical approach to understanding the role of amine (especially propylamine) structures in silicification we have explored three key structural features: (i) the degree of polymerization, (ii) the level of amine methylation, and (iii) the size of the amine chain spacers. In this article, we show that there are two factors critical to their function: the ability of the amines to produce microemulsions and the presence of charged and uncharged amine groups within a molecule, with the latter feature helping to catalyze silicic acid condensation by a proton donor/acceptor mechanism. The understanding of amine-silicate interactions obtained from this study has enabled the controlled preparation of hollow and nonporous siliceous materials under mild conditions (circumneutral pH, room temperature, and in all aqueous systems) possibly compatible with the conditions used by biosystems. The "rules" identified from our study were further used predictively to modulate the activity of a given amine. We believe that the outcomes of the present contribution will form the basis for an approach to controlling the growth of inorganic materials by using tailor-made organic molecules.

Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA.

Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests.

Aberrant DNA methylation imprints in aborted bovine clones

Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.

Arginine Methylation of Yeast mRNA-binding Protein Npl3 Directly Affects Its Function, Nuclear Export, and Intranuclear Protein Interactions

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels of methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmt1. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmt1, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins.

Relationship among expression, amplification, and methylation of FE4 esterase genes in Italian populations of Myzus persicae (Sulzer) (Homoptera: Aphididae)

The wide use of insecticides containing an esteric group selected resistant Myzus persicae populations characterised by the overproduction of one of two closely related carboxylesterases (E4 and FE4). In this paper, we present data collected from Italian population indicating that all the 22 populations analysed possess amplified FE4 gene only. The estimation of FE4 copy number, carried out by densitometric scanning of dot and Southern blots, puts in evidence that the different populations possess a gene copy number ranging from 6 to 104. Statistical analysis shows the existence of a high positive correlation between gene copy number and total esterase activity. In aphid strain with low FE4 copy number, these genes are almost totally methylated. On the contrary, aphid strains with high FE4 gene number evidenced highly variable methylation levels and absence of correlation between the number of genes and their methylation state. The same result has been observed when comparing FE4 methylation levels and esterase activity.

Enzymatic Regional Methylation Assay for Determination of CpG Methylation Density

DNA methylation is a critical mechanism for epigenetic gene regulation. In mammalian cells, CpG methylation density often influences the transcription level of related genes. Here, we report a short and accurate method to determine the CpG methylation density of any DNA region by sequentially applying bisulfite PCR and SssI (CpG) methylase. We introduced simple and efficient binding and washing steps that greatly improve the previous methodology and considerably enhance the accuracy of the assay. The accuracy of this method allows the detection of differences at the level of a single CpG methylation site when homogeneous PCR product was used as substrate. We validated our method by bisulfite sequencing multiple clones of samples with variable levels of CpG methylation. We envision that for its accuracy, simplicity, low-cost, flexibility, minimum instrumentation requirements, and high-throughput potential our method could greatly benefit research on DNA methylation.

CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene.

Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.

Differentiation of metaxylem cell line in the root ofAllium cepa L.

The differentiation processes of the metaxylem cell line in the root ofAllium cepa are characterized by amplification phenomena of repetitive DNA sequences mainly localized in heterochromatic regions of metaphase chromosomes. Moreover, these sequences are heavily methylated. This paper presents additional results on variation in endogenous DNA methylation in different developing root segments. The results show that methylation is higher in apical meristematic cells than the differentiating segments; contrastingly, total RNA synthesis seems to be correlated with undermethylation. Addition of labelled methyl groups to DNA by eukaryotic methylase, DNA digestions with different restriction enzymes specific for methylated sites and HPLC analysis confirmed the above results. Moreover, variation in methylation levels during differentiation occur not only at the internal cytosine of the-CCG-sites, but also at external cytosine. Furthermore, methylation affects other sites containing the trinucleotides-CXG-. In conclusion, root differentiation inAllium cepa seems to be correlated with gene activation modulated by the methylation/demethylation of particular DNA sequences.

Mouse mammary tumor virus dna methylation: Tissue-specific variation

Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpaII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpaII site in Mtv-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.

Taxonomically significant 8-hydroxyflavone glucuronides from the marchantialean liverwort, bucegia romanica

The monotypic genus, Bucegia, was not investigated in an earlier comparative flavonoid study of the Marchantiaceae. In the present communication Bucegia romanica is shown to elaborate solely a range of 8-hydroxyapigenin and 8-hydroxyluteolin glucuronides with variable levels of methylation. Such flavonoid biochemistry is unique within this family and the taxonomic implications of this are discussed. One new flavonoid was identified as 5,7-dihydroxy-8,4'-dimethoxyflavone (bucegin) 7- O-β- D-glucuronide.