No abstract

Epigenetic inheritance of heterochromatin structure is an important cellular process whose mechanism remains elusive. In this article, we describe the identification of nine enhancers of the silencing defect of a Saccharomyces cerevisiae-PCNA mutant by screening a library of Ϸ4,700 viable yeast deletion mutants. Of the nine mutants identified, six (hir1, hir3, sas2, sas4, sas5, and sir1) were previously known to reduce silencing synergistically with a mutation in Cac1p, the large subunit of chromatin assembly factor 1 (CAF-1). The predicted gene products that are affected in three other mutants (nam7, msh2, and rtt106) have not been implicated previously in silencing. Characterization of the rtt106⌬ allele revealed that it synergistically reduced heterochromatin silencing when combined with a mutation in Cac1p but not with a mutation in Asf1p (a histone H3 and H4 chaperone). Moreover, Rtt106p interacted with histones H3 and H4 both in vitro and in vivo, and it displayed a nucleosome assembly activity in vitro. Furthermore, Rtt106p interacts with CAF-1 physically through Cac1p. These biochemical and genetic data indicate that Rtt106p is a previously uncharacterized histone chaperone connecting S phase to epigenetic inheritance. epigenetic silencing ͉ nucleosome assembly ͉ chromosome assembly factor 1

We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 ϫ 10 9 plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t 1/2 of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME 359 ), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME 359 expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.

Background:The calcium-binding protein DREAM/KChIP3 binds DNA and other proteins to regulate neuronal function. Results: DREAM/KChIP3 binds the EF-hand protein, calmodulin, in the presence but not in the absence of calcium. Calciumbound DREAM/KChIP3 enhances calmodulin-dependent calcineurin activity. Conclusion: DREAM/KChIP3 binds and regulates calmodulin activity. Significance: DREAM/KChIP3 heterodimerizes with the EF-hand protein, calmodulin, and regulates calmodulin activation of calcineurin.