Transcription Of Foreign Genes Research Articles

Salmonella expresses foreign genes during infection by degrading their silencer

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogen Salmonella enterica serovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes-even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

Excessive Promoters as Silencers of Genes Horizontally Acquired by Escherichia coli.

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming “promoter islands.” We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the “promoter island” of a foreign gene appY and the promoter region of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a sequence with improved -35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.

Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region

The repressive activity of ancestral histone-like proteins helps integrate transcription of foreign genes with discrepant AT content into existing regulatory networks. Our investigations indicate that the AT-rich discriminator region located between the −10 promoter element and the transcription start site of the regulatory gene ssrA plays a distinct role in the balanced expression of the Salmonella pathogenicity island-2 (SPI2) type III secretion system. The RNA polymerase-binding protein DksA activates the ssrAB regulon post-transcriptionally, whereas the alarmone guanosine tetraphosphate (ppGpp) relieves the negative regulation imposed by the AT-rich ssrA discriminator region. An increase in the GC-content of the ssrA discriminator region enhances ssrAB transcription and SsrB translation, thus activating the expression of downstream SPI2 genes. A Salmonella strain expressing a GC-rich ssrA discriminator region is attenuated in mice and grows poorly intracellularly. The combined actions of ppGpp and DksA on SPI2 expression enable Salmonella to grow intracellularly, and cause disease in a murine model of infection. Collectively, these findings indicate that (p)ppGpp relieves the negative regulation associated with the AT-rich discriminator region in the promoter of the horizontally-acquired ssrA gene, whereas DksA activates ssrB gene expression post-transcriptionally. The combined effects of (p)ppGpp and DksA on the ssrAB locus facilitate a balanced SPI2 virulence gene transcription that is essential for Salmonella pathogenesis.

ChemInform Abstract: Development of Recombinant Adenovirus Carrying MicroRNA‐Regulated Gene Expression System

AbstractReview: 23 refs.

Development of Recombinant Adenovirus Carrying MicroRNA-regulated Gene Expression System

Target tissue-specific delivery and transcription of foreign genes are desirable for safe and effective gene therapy. Two approaches for this purpose, "Targeted Delivery" and "Targeted Expression", have been mainly reported. Among "Targeted Expression" approaches, microRNA (miRNA)-mediated "post-transcriptional de-targeting" has been recently demonstrated, and much attention has been focused on this approach. MiRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region (UTR) of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. First, in order to reduce the hepatic transduction by Ad vectors, complementary sequences of liver-specific miRNA, miR-122a, were inserted into the 3'-UTR of the transgene expression cassette. Intratumor injection of this Ad vector resulted in approximately 100-fold lower hepatic expression than that of the conventional Ad vector, without reducing gene expression in the tumor. Second, complementary sequences for miRNAs selectively down-regulated in tumor cells were inserted into the E1 gene expression cassette in oncolytic Ads, which exhibit tumor cell-specific replication and antitumor effects. Recent studies demonstrated that expression of several miRNAs is exclusively reduced in tumor cells. Oncolytic Ads containing the miRNA complementary sequences showed reduced replication in the normal cells, without altering the antitumor effects. MiRNA-regulated gene expression system mediates "post-transcriptional de-targeting", in which translation of transgene is suppressed in a tissue-specific manner; however, tissue-specific transgene expression can be achieved by taking tropism of gene delivery vehicles into consideration and reducing the transgene expression in untargeted organs via miRNA-regulated gene expression system.

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

The H-NS nucleoid-associated DNA-binding protein is an important global repressor of transcription in Gram-negative bacteria. Recently, H-NS has been implicated in the process of xenogeneic silencing, where it represses the transcription of foreign genes acquired by horizontal transfer. This raises interesting questions about the integration of the horizontally acquired genes into the existing gene regulatory networks of the microbe. In particular, how do bacteria derepress silenced genes in order to benefit from their expression without compromising competitive fitness through doing so inappropriately? This article reviews current knowledge about the derepression of genes that are transcriptionally silenced by H-NS. It describes a variety of anti-silencing mechanisms involving (i) protein-independent processes that operate at the level of local DNA structure, (ii) DNA-binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins related to AraC, and (iii) modulatory mechanisms in which H-NS forms heteromeric protein-protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST. The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists. There is also evidence that in a number of cases more sophisticated regulatory processes have been superimposed on these rather simple anti-silencing mechanisms, broadening the range of environmental signals to which H-NS-repressed genes respond.

An additional copy of the homologous region (hr1) sequence in the Autographa californica multinucleocapsid polyhedrosis virus genome promotes hyperexpression of foreign genes.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus genome contains nine homologous region (hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5) sequences that are thought to be involved in viral replication and activation of transcription. Our results show that the 750 bp hr1 sequence is capable of functioning as an enhancer of transcription of foreign genes from the homologous late polyhderin gene promoter and the heterologous Drosophila heat shock protein (hsp70) promoter in insect cells. Introduction of an additional copy of the complete hr1 element downstream to the polyhedrin locus in the viral genome, while not affecting the stability of the recombinant virus for at least 30 serial passages, led to hyperexpression of reporter genes. The enhancement in the expression levels of foreign genes varied from 40 to 90-fold depending on the promoter used.

T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei

The study of RNA editing and other molecular processes in the trypanosome mitochondrion would benefit greatly from the ability to insert and express exogenous DNA in the organelle. However, even with a method to introduce DNA, the current lack of knowledge about mitochondrial transcription would hinder efforts to obtain expression. To circumvent this problem, Leishmania tarentolae promastigotes and Trypanosoma brucei procyclic cells have been transfected with bacteriophage T7 RNA polymerase targeted to the mitochondrion. Mitochondria isolated from the transfectants contained active T7 RNA polymerase, as shown by a comigration in density gradients of mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electroporation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix either in a transient assay using electroporation of DNA into isolated mitochondria, or in a stable assay using cells transfected with DNA by the biolistic gun method.

Involvement of host factors in transcription from baculovirus very late promoters - a review

The baculovirus expression vector system has emerged as the system of choice for the expression of a number of heterologous genes of both prokaryotic and eukaryotic origin. This system utilizes the baculovirus very late, hyperactive polyhedrin and p10 promoters to drive the transcription of foreign genes. Regulation of transcription from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified. Fresh insight into host-virus interactions during baculovirus pathogenesis is now offered by the identification of insect host factors that interact with transcriptionally essential motifs of these promoters as well as cis-acting enhancer-like elements upstream from the promoter.

Chromosomal position effects and the modulation of transgene expression.

Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.

DNA methylation: eukaryotic defense against the transcription of foreign genes?

DNA methylation: eukaryotic defense against the transcription of foreign genes?

The construction and characterisation of plasmid vectors suitable for the expression of all DNA phases under the control of the E. coli tryptophan promoter.

A DNA fragment flanked in the E. coli genome by Hinf I sites and containing the E. coli tryptophan promoter-operator and nucleotides specifying the leader sequence and first seven amino acids of trp E, has been cloned in the Hind III site of pBR322 with the aid of Hind III linkers. The Hind III site upstream from the trp promoter in the transcriptional sense was deleted to generate a Hind III cloning vector, designated pWT111, suitable for the expression of cloned DNA sequences as fusion products. Derivatives of this plasmid were constructed, designated pWT121 and pWT131, whose Hind III cloning sites differ with respect to their translation phasing relative to the initiator ATG of the trp E gene. Both pWT121 and pWT131 have a higher copy number per cell than pWT111. The tetracycline genes of all three plasmids are under trp promoter control and could be used to monitor the transcription of foreign genes inserted at the Hind III site of the vectors.