The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo–formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.     

Exon repetition: a major pathway for processing mRNA of some genes is allele-specific

Exon repetition describes the presence of tandemly repeated exons in mRNA in the absence of duplications in the genome. Its existence challenges our understanding of gene expression, because the linear organization of sequences in apparently normal genes must be subverted during RNA synthesis or processing. It is restricted to a small number of genes in some of which over half of the mRNA contains specific patterns of repetition. Although it is sometimes assumed to arise by trans-splicing, there is no evidence of this and the efficiency is very much higher than for examples of bona fide trans-splicing in mammals. Furthermore, a potentially ubiquitous reaction such as trans-splicing is not consistent with a phenomenon that involves such a high proportion of the products of so few genes. Instead, it seems more probable that exon repetition is caused by a specific trans-acting factor. We have tested this and demonstrate for the two best characterized examples that the property is restricted to specific alleles of the affected genes and is determined in cis. It is not determined by exonic splicing signals, as had been suggested previously. In heterozygotes, RNA transcribed from the two alleles of an affected gene can have fundamentally different fates.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Transposable Element Regulation in Rice and Arabidopsis: Diverse Patterns of Active Expression and siRNA-mediated Silencing

Transposable elements (TEs) are DNA segments that can mediate or cause movement within genomes. We performed a comprehensive, whole-genome analysis of annotated TEs in rice (Oryza sativa L.) and Arabidopsis thaliana, focusing on their expression (mRNA data) and silencing (small RNA data), and we compared these data with annotated genes that are not annotated as transposons. TEs demonstrated higher levels of antisense mRNA expression in comparison to non-TE genes. The majority of the TEs were silenced, as demonstrated by higher levels of small RNAs and a lack of mRNA MPSS data. When TEs were expressed, their activity was usually limited to just one or a few of the mRNA libraries. When we examined TE expression at the whole-genome level and across the complete mRNA dataset, we observed that most activity was contributed by a few highly expressed transposable elements. These TEs were characterized by their low copy number and few matching small RNAs. Our results help define the relationship between gene expression and gene silencing for TEs, and indicate that TE silencing can impact neighboring genes, perhaps via a mechanism of heterochromatin formation and spreading. These data may be used to define active TEs and families of transposable elements that continue to shape plant genomes.     

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w ^67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w ^67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. P. Georgiev     

Gene silencing：Double－stranded RNA mediated mRNA degradation and gene inactivation

INTRODUCTIONThe genome structure of plants can be alteredby genetic transformation. During the process ofgene transfer, Agrobacterium tumefaCJens integratepart of their genome into the genome of susceptiblespecies. Recently, genetic transfOrmation techniqueshave been used to modify significantly the organi-zation of the genome. Introducing transgenes intop1ants can both modify the number of copies of agiven sequence and affect gene expression. Becausethe expression of a transgene cannot…     

Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.