Isolation and characterization of regulatory mutations affecting the expression of the guaBA operon of Escherichia coli K-12

Summary We isolated strains of Escherichia coli K 12 in which the lac structural genes were fused to the structural genes of the guaBA operon. These strains were used to isolate regulatory mutations that increased the expression of the guaBA operon under normal repressing conditions as compared to the wild type parental fusion strain. Three classes of guaBA specific regulatory mutations were identified. Class I regulatory mutations were trans-acting and unlinked to the guaBA operon as shown by bacteriophage P1 transduction. Class II regulatory mutations were tightly linked to the guaBA operon, cis-dominant to the wild type allele in a cis-trans analysis and were regarded as control region mutations. Class III regulatory mutations were tightly linked to the guaBA operon and trans-recessive to the wild type allele in a cis-trans analysis. We have designated the locus responsible for the class III regulatory mutations as guaR. The guaR locus is tightly linked and was mapped to the counterclockwise side of the guaBA operon. The guaR locus is proposed to specify a trans acting regulatory element involved in the regulation of the guaBA operon.     

cis-Acting Signals in Bromovirus RNA Replication and Gene Expression: Networking with Viral Proteins and Host Factors

Bromoviruses are representative members of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses. Tractable biochemical and genetic features have made bromoviruses useful systems forin vivoandin vitrostudies ofcis-acting RNA sequences andtrans-acting factors in RNA replication, subgenomic mRNA synthesis, translation, encapsidation, and virus–host interactions. Among other findings, bromoviruscis-acting RNA replication signals are large, structurally complex, and conserve potential conformational switches that may coordinate RNA replication with other infection processes. The tRNA-like 3′ ends of bromovirus RNAs are required for negative-strand synthesis and recognized by multiple tRNA-specific host enzymes. The presence of additional host regulatory sequence motifs in other bromoviruscis-acting regions suggests that their function also involves interaction with host as well as viral factors.     

Definition of the minimal cis-acting sequences necessary for genome maturation of the herpesvirus murine cytomegalovirus

Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail-linked genomes. Genome maturation is carried out by the terminase, a protein complex that mediates both insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs called pac1 and pac2 lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. However, the potential importance of other sequences has not been fully investigated. We have undertaken to define all of the sequences necessary for efficient genome maturation for a herpesvirus by inserting ectopic cleavage sites into the murine cytomegalovirus genome and assessing their ability to mediate genome maturation. A combination of deletion and substitution mutations revealed that the minimal cleavage site is large (~180 bp) and complex. Sequences distal of pac1 (relative to the point of cleavage) were dispensable, suggesting that pac1 may be the sole cis-acting element on this side of the cleavage site. In contrast, a region distal to pac2 up to 150 bp from the point of cleavage was essential. Scanning substitutions revealed that the pac2 side of the cleavage site is complex and may contain multiple cis-acting sequence elements in addition to pac2. These results should facilitate the identification of trans-acting factors that bind to these elements and the elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.     

Validating Discovered Cis-Acting Regulatory Genetic Variants: Application of an Allele Specific Expression Approach to HapMap Populations

Background

Localising regulatory variants that control gene expression is a challenge for genome research. Several studies have recently identified non-coding polymorphisms associated with inter-individual differences in gene expression. These approaches rely on the identification of signals of association against a background of variation due to other genetic and environmental factors. A complementary approach is to use an Allele-Specific Expression (ASE) assay, which is more robust to the effects of environmental variation and trans-acting genetic factors.

Methodology/Principal Findings

Here we apply an ASE method which utilises heterozygosity within an individual to compare expression of the two alleles of a gene in a single cell. We used individuals from three HapMap population groups and analysed the allelic expression of genes with cis-regulatory regions previously identified using total gene expression studies. We were able to replicate the results in five of the six genes tested, and refined the cis- associated regions to a small number of variants. We also showed that by using multi-populations it is possible to refine the associated cis-effect DNA regions.

Conclusions/Significance

We discuss the efficacy and drawbacks of both total gene expression and ASE approaches in the discovery of cis-acting variants. We show that the ASE approach has significant advantages as it is a cleaner representation of cis-acting effects. We also discuss the implication of using different populations to map cis-acting regions and the importance of finding regulatory variants which contribute to human phenotypic variation.     

Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript

Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the cis-acting elements and trans-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (FGG) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on FGG pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other trans-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for FGG pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating FGG pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.     

Cis-acting RNA elements in human and animal plus-strand RNA viruses

The RNA genomes of plus-strand RNA viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. Those structures that are functionally important are called cis-acting RNA elements because their functions cannot be complemented in trans. They can be involved not only in RNA/RNA interactions but also in binding of viral and cellular proteins during the complex processes of translation, RNA replication and encapsidation. Most viral cis-acting RNA elements are located in the highly structured 5′- and 3′-nontranslated regions of the genomes but sometimes they also extend into the adjacent coding sequences. In addition, some cis-acting RNA elements are embedded within the coding sequences far away from the genomic ends. Although the functional importance of many of these structures has been confirmed by genetic and biochemical analyses, their precise roles are not yet fully understood. In this review we have summarized what is known about cis-acting RNA elements in nine families of human and animal plus-strand RNA viruses with an emphasis on the most thoroughly characterized virus families, the Picornaviridae and Flaviviridae.     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

Novel aspects of RNA regulation in Staphylococcus aureus

A plethora of RNAs with regulatory functions has been discovered in many non-pathogenic and pathogenic bacteria. In Staphylococcus aureus, recent findings show that a large variety of RNAs control target gene expression by diverse mechanisms and many of them are expressed in response to specific internal or external signals. These RNAs comprise trans-acting RNAs, which regulate gene expression through binding with mRNAs, and cis-acting regulatory regions of mRNAs. Some of them possess multiple functions and encode small but functional peptides. In this review, we will present several examples of RNAs regulating pathogenesis, antibiotic resistance, and host-pathogen interactions and will illustrate how regulatory proteins and RNAs form complex regulatory circuits to express the virulence factors in a dynamic manner.