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Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively.Analysis of substrate specificity with synthetic DNAs and restriction fragments of 4X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methylaccepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.DNA methylation in cells from vertebrates is a post-replication process involving the transfer of methyl groups from S-adenosyl L-methionine (AdoMet) to the 5 position of cytosine residues through the action of DNA methyltransferase [DNA MeTase; DNA (cytosine-5-)-methyltransferase, EC 2.1.1.37]. Methylated cytosine residues are located primarily within the dinucleotide 5' CpG 3' (1) and are found in tissue-specific amounts and positions (2). Sano and Sager (3) reported that, in bovine satellite I DNA, 5-methylcytosine (m5C) residues are located exclusively 5' to G residues, occur in clusters, and tend to be located within short self-complementary sequences; the distribution of m5C residues within this DNA showed striking tissue-specific variation.Changes in amounts and locations of m5C residues in DNA appear to be a part of the developmental program in higher cells (reviewed in ref. 4), and it has been demonstrated for many different genes that the loss of methyl groups from specific sites is correlated with the transcriptional activation of adjacent sequences (5-9). Also, in vitro enzymatic methylation of CpG dinucleotides by the bacterial restriction methyltransferase MHpa II inhibits expression of viral (10) and cellular (11) genes after introduction of the methylated DNA into cells. The pattern of methylation is maintained during subsequent cell divisions (12). Treatment of cultured cells with inhibitors of DNA methylation leads to the induction of certain genes with a concomitant reduction in the number of associated methylated CpG sites (13).Despite the apparent biological importance of DNA methylation it is not at all clear how patterns of methylation are established and maintained within the genome. DNA MeTases prepared from a number of rodent (14-17) and human (18) sources have been shown to require CpG-containing DNA, although additional sequence requirements have not been identified. A crude preparation of DNA MeTase from mouse ascites cells showed a strong pre...

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (VL)12.3, rescued toxicity in a neuronal model of HD. We also found that VL12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. VL12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets