Sanjoy K. Bhattacharya

A mammalian protein with specific demethylase activity for mCpG DNA.

DNA-methylation patterns are important for regulating genome functions, and are determined by the enzymatic processes of methylation and demethylation. The demethylating enzyme has now been identified: a mammalian complementary DNA encodes a methyl-CpG-binding domain, bears a demethylase activity that transforms methylated cytosine bases to cytosine, and demethylates a plasmid when the cDNA is translated or transiently transfected into human embryonal kidney cells in vitro. The discovery of this DNA demethylase should provide a basis for the molecular and developmental analysis of the role of DNA methylation and demethylation.

Antisense MBD2 gene therapy inhibits tumorigenesis.

Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes.

DNA Demethylase Is a Processive Enzyme

DNA methylation patterns are generated during development by a sequence of methylation and demethylation events. We have recently demonstrated that mammals bear a bona fidedemethylase enzyme that removes methyl groups from methylated cytosines. A general genome wide demethylation occurs early in development and in differentiating cell lines. This manuscript tests the hypothesis that the demethylase enzyme is a processive enzyme. Using bisulfite mapping, this report demonstrates that demethylase is a processive enzyme and that the rate-limiting step in demethylation is the initiation of demethylation. Initiation of demethylation is determined by the properties of the sequence. Once initiated, demethylation progresses processively. We suggest that these data provide a molecular explanation for global hypomethylation.

Toxicity and biodegradation of formaldehyde in anaerobic methanogenic culture.

Formaldehyde is present in several industrial wastewaters including petrochemical wastes. In this study, the toxicity and degradability of formaldehyde in anaerobic systems were investigated. Formaldehyde showed severe toxicity to an acetate enrichment methanogenic culture. As low as 10 mg/L (0.33 mM) of formaldehyde in the reactor completely inhibited acetate utilization. Formaldehyde, however, was degraded while acetate utilization was inhibited. Degradation of formaldehyde (Initial concentration < or =30 mg/L) followed Monod model with a rate constant, k, of 0.35-0.46 d(-1). At higher initial concentrations (> or =60 mg/L), formaldehyde degradation was inhibited and partial degradation was possible. The initial formaldehyde to biomass ratio, S(0)/X(0), was useful to predict the degradation potential of high formaldehyde concentrations in batch systems. When S(0)/X(0) < or = 0.1, formaldehyde was completely degraded with initial concentration of up to 95 mg/L; when S(0)/X(0) > or = 0.29, formaldehyde at higher than 60 mg/L was only partially degraded. The inhibition of formaldehyde degradation in batch systems could be avoided by repeated additions of low concentrations of formaldehyde (up to 30 mg/L). Chemostats (14-day retention time) showed degradation of 74 mg/L-d (1110 mg/L) of influent formaldehyde with a removal capacity of 164 mg/g VSS-day. A spike of 30 mg/L (final concentration in the chemostat) formaldehyde to the chemostat caused only a small increase in effluent acetate concentration for 3 days. But a spike of 60 mg/L (final concentration in the chemostat) formaldehyde to the chemostat resulted in a dramatic increase in acetate concentration in the effluent. The results also showed that the acetate enrichment culture was not acclimated to formaldehyde even after 226 days. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 727-736, 1997.

Modified Oligonucleotides as Bona Fide Antagonists of Proteins Interacting with DNA HAIRPIN ANTAGONISTS OF THE HUMAN DNA METHYLTRANSFERASE

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.

Construction of a multi RE module: Exploitation of mechanochemistry of restriction endonucleases

We describe the construction of a multi-immobilized restriction endonuclease module (Multi RE module). We demonstrate that the applied mechanical stress enables modulation of enzyme activity and modulation of recognition site selectivity (in oligonucleotides of approximately 200 bp) of immobilized restriction endonucleases. The central module which is consisted of different strips of immobilized restriction endonucleases allows limited digestion of a large DNA sample in a controlled manner as a function of applied mechanical stress on strips. The stress-activity relationship and the effect of repeated cycles of stress and relaxation on the immobilized strips are presented here.