We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer. Comparison of all three microarray data sets indicates the majority of Dicer-regulated genes are not dependent on RDE-4 or RDE-1, including the X-linked genes. However, all three data sets are enriched in genes important for innate immunity and, specifically, show increased expression of innate immunity genes.
Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer's disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab, consistently identified the predicted amino-terminal fragment (∼18kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain.
There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to cis-acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5 end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3 end of the minus-strand DNA a poor template for initiation.Hepadnaviruses are a family of small, enveloped DNA viruses that display a narrow host range and a tropism for the liver and are capable of both acute and chronic infections in their hosts. The prototype member of the family, human hepatitis B virus (human HBV), is a major worldwide health problem, exposing ca. 350 million chronic carriers to an increased risk of developing hepatocellular carcinoma (10). As viral replication is necessary for maintenance of the chronic carrier state, elucidation of the underlying molecular mechanisms of replication may uncover therapeutic targets. The hepadnavirus family consists of a number of mammalian and avian viruses, all sharing similar genetic organization and a general replication strategy. The avian viruses duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV) are useful models for studying many aspects of hepadnavirus biology, including replication (reviewed in reference 4).Hepadnaviruses are related to RNA-containing retroviruses and retroelements as they use reverse transcription to replicate their genomes. Reverse transcription commonly involves a series of template switches, the process whereby the nascent strand of DNA switches from one template to another template. This can occur either intramolecularly or intermolecularly. During hepadnavirus replication, template switches are thought to occur intramolecularly as there is only believed to b...
In a recent issue of Science, Nishikura and colleagues provide the first evidence that editing of a microRNA (miRNA) precursor by ADARs can modulate the target specificity of the mature miRNA (Kawahara et al., 2007).
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