We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D-Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D-Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution. D uring recent years, there has been a growing interest in how patterns of DNA cytosine methylation in gene promoter regions, particularly for those genes containing CpG-rich promoters or CpG islands, play a role in the regulation of gene expression. Although these islands are normally unmethylated for most genes, hypermethylation is associated with loss of expression of one copy in the normal settings of inactivation of the female X chromosome and the silenced alleles for parentally imprinted genes (1, 2). Loss of function of key genes during human tumorigenesis can occur in association with aberrant hypermethylation (3). For example, loss of expression of the tumor suppresser genes VHL in sporadic renal cancer (4), p16 in multiple tumor types (5, 6), and hMLH1 in colorectal cancer (7-9) has been correlated with hypermethylation of the corresponding promoter, and this serves as an alternative mechanism for loss of tumor suppressor gene function.The development of sensitive PCR techniques and genomic sequencing to assess cytosine methylation status has facilitated studies of CpG island methylation (10-12). However, these procedures can be performed only with DNA extracted from total cell populations. These methods cannot easily address critical issues, such as the precise timing of DNA methylation changes in specific cell types during embryonic development and oncogenesis, or define the extent of heterogeneity for loss of expression during tumor evolution. Understanding suc...

Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.

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The goal of this project was to isolate representative Fe(III)-reducing bacteria from kaolin clays that may influence iron mineralogy in kaolin. Two novel dissimilatory Fe(III)-reducing bacteria, strains G12T and G13T, were isolated from sedimentary kaolin strata in Georgia (USA). Cells of strains G12T and G13T were motile, non-spore-forming regular rods, 1–2 μm long and 0.6 μm in diameter. Cells had one lateral flagellum. Phylogenetic analyses using the 16S rRNA gene sequence of the novel strains demonstrated their affiliation to the genus Geobacter. Strain G12T was most closely related to Geobacter pelophilus (94.7 %) and Geobacter chapellei (94.1 %). Strain G13T was most closely related to Geobacter grbiciae (95.3 %) and Geobacter metallireducens (95.1 %). Based on phylogenetic analyses and phenotypic differences between the novel isolates and other closely related species of the genus Geobacter, the isolates are proposed as representing two novel species, Geobacter argillaceus sp. nov. (type strain G12T=ATCC BAA-1139T=JCM 12999T) and Geobacter pickeringii sp. nov. (type strain G13T=ATCC BAA-1140T=DSM 17153T=JCM 13000T). Another isolate, strain R7T, was derived from a primary kaolin deposit in Russia. The cells of strain R7T were motile, spore-forming, slightly curved rods, 0.6×2.0–6.0 μm in size and with up to six peritrichous flagella. Strain R7T was capable of reducing Fe(III) only in the presence of a fermentable substrate. 16S rRNA gene sequence analysis demonstrated that this isolate is unique, showing less than 92 % similarity to bacteria of the Sporomusa–Pectinatus–Selenomomas phyletic group, including ‘Anaerospora hongkongensis’ (90.2 %), Acetonema longum (90.6 %), Dendrosporobacter quercicolus (90.9 %) and Anaerosinus glycerini (91.5 %). On the basis of phylogenetic analysis and physiological tests, strain R7T is proposed to represent a novel genus and species, Pelosinus fermentans gen. nov., sp. nov. (type strain R7T=DSM 17108T=ATCC BAA-1133T), in the Sporomusa–Pectinatus–Selenomonas group.

Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination. STEM CELLS 2006;24:531-546