Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity

Expression of enzymatically active reverse transcriptase in Escherichia coli

Reverse transcriptase of murine retroviruses is a monomeric protein of approximately 80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the pol gene of Moloney murine leukemia virus. The inserted pol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein

Localization of BDNF mRNA with the Huntington's disease protein in rat brain

<p>Abstract</p> <p>Background</p> <p>Studies have implicated reduced levels of brain-derived neurotrophic factor (BDNF) in the pathogenesis of Huntington's disease. Mutant huntingtin (Htt) protein was previously reported to decrease BDNF gene transcription and axonal transport of BDNF. We recently showed that wild-type Htt is associated with the Argonaute 2 microRNA-processing enzyme involved in gene silencing. In dendrites, Htt co-localizes with components of neuronal granules and mRNAs, indicating that it might play a role in post-transcriptional processing/transport of dendritic mRNAs.</p> <p>Results</p> <p>We conducted imaging experiments in cultured cortical neurons to demonstrate the co-localization of endogenous Htt and BDNF mRNA in fixed cells, and co-trafficking of BDNF 3'UTR mRNA with endogenous and fluorescently tagged Htt in live neurons. We used an enhanced technique that combines FISH and immunofluorescent staining to co-localize BDNF mRNA with Htt, Ago2, CPEB and dynein in thick vibratome sections of the rat cortex.</p> <p>Conclusions</p> <p>In cultured neurons and sections of the rat cortex, we found BDNF mRNA associated with Htt and components of neuronal RNA granules, which are centers for regulating RNA transport and local translation. Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neurotrophic support and neuron survival.</p

Target genes of the largest human SWI/SNF complex subunit control cell growth

The largest subunit of the mammalian SWI/SNF-A orBAF (BRG1-associated factor) chromatin-remodelling complexis encoded by two related cDNAs hOsa1/BAF250a andhOsa2/BAF250b that are unique to the BAF complex and absentin the related PBAF (Polybromo BAF). hOsa/BAF250 has beenshown to interact with transcriptional activators and bind toDNA suggesting that it acts to target the remodelling complexto chromatin. To better understand the functions of hOsa2,we established inducible stable HeLa cell lines over-expressingFLAG&#8722;hOsa2 or a derivative lacking the ARID (AT-richinteractive domain) DNA-binding domain. Immunopurificationof complexes containing hOsa2 that was followed by massspectrometry and immunoblotting demonstrated the presenceof BRG1 and known BAFs, but not hOsa1 or hBRM.Deletion of the ARID did not compromise the integrity ofthe complex. Induction of hOsa2 expression caused impairedcell growth and accumulation of cells in the G0/G1 cell cyclephase. Elevated levels of the p53 and p21 proteins weredetected in these cells while c-Myc mRNA and protein levelswere found to decrease. Chromatin immunoprecipitation andreporter assays suggested that hOsa2 had a direct effect on cmycand p21 promoter activity. Thus hOsa2 plays an importantrole in controlling genes regulating the cell cycle