Mesoderm-specific B104 expression in the Drosophila embryo is mediated by internal cis-acting elements of the transposon

The Drosophila melanogaster genome contains about 100 copies of the B104 transposable element, which is strongly expressed during embryogenesis. Here we show that B104 expression is restricted to the esophageal and amnioproctodeal regions of the embryo and to the developing mesoderm. Mesoderm-specific B104 expression requires the activity of the mesoderm-determining factors twist and snail. Virtually the same expression patterns were observed in Drosophila yakuba, a species that a separated from D. melanogaster by some 15 million years of evolution. We show that B104 expression is directed by internal sequences of the retrotransposon that are capable of acting as a cis-acting regulatory element in front of a heterologous Drosophila promoter. Our findings suggest that retrotransposon insertions can affect the expression patterns of endogenous genes by adding and distributing specific cis-acting control elements throughout the host genome. We therefore propose that transposable elements in addition to reducing the fitness of their hosts may also provide a rich pool of cis-acting sequences that contribute to the long-term evolutionary potential of the population in a beneficial manner.     

Serotype variation in Bordetella pertussis is governed by cis-acting elements

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.     

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation. To gain insight into such mechanisms, we addressed the involvement of cis and trans viral factors in HCV IRES activity by using a cell-based RNA reporter system. We found that the HCV 3' noncoding region (NCR) strongly stimulates IRES efficiency in cis, depending on the genotype and the cell line. Moreover, we confirmed the role of the core protein in viral gene expression as previously reported in vitro. Surprisingly, we observed a similar effect, i.e. a twofold increase under low amounts of NS5B RNA polymerase, followed by a decrease at higher concentrations. However, no contribution of NS5A to HCV IRES-mediated translation was noted and no cooperative effect could be detected between 3' NCR and viral proteins or between proteins. Collectively, these results suggest that HCV RNA translation is regulated, and that the switch from translation to replication might involve a sequential requirement for both cis and trans viral factors, because of their apparent lack of synergy, probably with the aid of host factors.     

Splicing of branchpoint-distant exons is promoted by Cactin,Tls1 and the ubiquitin-fold-activated Sde2

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5′-splice site (5′ss), branchpoint (BP) and 3′-splice site (3′ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3′ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP–3′ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3′ss towards the spliceosome''s catalytic centre by folding the RNA between the BP and 3′ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.     

The direct repeat sequence upstream of Bacillus chitinase genes is cis-acting elements that negatively regulate heterologous expression in E. coli

To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.     

Gelatinase A expression in endothelial cells is regulated by at least two cis-acting promoter elements.

Increased expression of gelatinase A is associated with both angiogenesis and alterations in blood vessel structure. Heart-derived endothelial cells derived from spontaneously hypertensive rats (SHR) were found to express significantly more gelatinase A in culture, both at the protein and mRNA level, than endothelial cells from normotensive Wistar-Kyoto (WKY) rats. Other matrix metalloproteinases, as well as their tissue inhibitors, were not differentially regulated. A 1683 bp gelatinase A promoter fragment linked to a luciferase reporter demonstrated up to 40-fold more activity when transfected into SHR-derived cells versus WKY-derived cells. The promoter region between -1324 and -1272, previously termed RE1, contributed up to a five-fold increase in basal promoter activity in both cells, but contributed only 12% of the promoter activity in SHR-derived cells compared to 85% in WKY-derived cells. In SHR-derived cells, but not in WKY-derived cells, a second region between -1435 and -1375, termed RE2, contributed 60% of the total activity of the 1683 bp promoter fragment. Both electrophoretic mobility shift assays and Southwestern blots demonstrated differences in RE2-specific binding factors in nuclear extracts derived from the two cell types. SHR-derived endothelial cells thus represent a new model system to study the regulation of gelatinase A expression, which itself may contribute to the abnormal vascular structure seen in the SHR.     

The role played by exons in genomic DNA sequence correlations

The codon structure inside exons imposes a strong modulation with period-3 for genomic composition correlations. A new formalism for calculating nucleotide correlations along DNA sequences in terms of an irreducible set of six correlation functions is presented. New procedures to extract the corresponding period-3 modulations are also developed. These modulations are seen to be stronger for the irreducible self-correlation C_zz(k), which accounts only for the binding strength of dinucleotides (z stands for adenine or thymine minus cytosine or guanine concentrations). We investigate and model the relationship between exon distribution and genomic period-3 correlations for the D. melanogaster genome.     

Interleukin-6 and leukemia inhibitory factor induction of JunB is regulated by distinct cell type-specific cis-acting elements.

Interleukin (IL)-6 plays an important role in a wide range of biological activities, including differentiation of murine M1 myeloid leukemic cells into mature macrophages. At the onset of M1 differentiation, a set of myeloid differentiation primary response (MyD) genes are induced, including the proto-oncogene for JunB. In order to examine the molecular nature of the mechanisms by which IL-6 activates the immediate early expression of MyD genes, JunB was used as a paradigm. A novel IL-6 response element, -65/-52 IL-6RE, to which a 100-kDa protein complex is bound, has been identified on the JunB promoter. Leukemia inhibitory factor (LIF)-induced activation of JunB in M1 cells was also mediated via the -65/-52 IL-6RE. The STAT3 and CRE-like binding sites of the JunB promoter, identified as IL-6-responsive elements in HepG2 liver cells were found, however, to play no role in JunB inducibility by IL-6 in M1 myeloid cells. Conversely, the -65/-52 IL-6RE is shown not to be necessary for JunB inducibility by IL-6 or LIF in liver cells. It appears, therefore, that immediate early activation of JunB is regulated differently in M1 myeloid cells than in HepG2 liver cells. This indicates that distinct cis-acting control elements participate in cell type-specific induction of JunB by members of the IL-6 cytokine superfamily.     

Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.