Trans-acting Elements Research Articles

Amphipathic trans-acting phosphorothioate DNA elements mediate the delivery of uncharged nucleic acid sequences in mammalian cells

Amphipathic trans-acting phosphorothioate DNA elements deliver PNA and PMO oligomers in mammalian cells.

저산소와 NO 조건에서 Mycobacterium smegmatis의 adenylyl cyclase 유전자 발현

In Mycobacterium tuberculosis H37Rv 16 adenylyl cyclase (AC) genes have been identified, while 10 AC genes have been found in non-pathogenic Mycobacterium smegmatis. Expression of 6 AC genes (MSMEG_0218, MSMEG_3243, MSMEG_3780, MSMEG_4279, MSMEG_4477, and MSMEG_6154) among 10 AC genes in M. smegmatis was increased when M. smegmatis was subjected to hypoxic growth conditions. On the other hand, only MSMEG_3780 and MSMEG_4279 were slightly induced in the presence of NO. The cAMP levels in cells and culture media were 450and 9.8-fold increased, respectively, when M. smegmatis was grown under hypoxic conditions relative to those grown aerobically. Intracellular levels of cAMP were increased 5.8-fold on the exposure to NO. The DevSR two-component system is known to be involved in the induction of many genes whose expression is induced under hypoxic conditions and in the presence of NO. Expression of 6 hypoxically induced AC genes was observed to be induced in a devR deletion mutant grown under hypoxic conditions, indicating that the induction of the 6 AC genes under hypoxic conditions is independent of the DevSR two-component system. In order to identify a trans-acting regulatory element that is pertinent in the hypoxic induction of MSMEG_3780, ligand-fishing analysis was performed using the upstream DNA of MSMEG_3780 and MSMEG_5136 protein was identified.

The Type I toxin–antitoxin par locus from Enterococcus faecalis plasmid pAD1: RNA regulation by both cis- and trans-acting elements

The pAD1 par determinant was the first Type I toxin–antitoxin system identified in Gram-positive bacteria and has recently been shown to be the prototype of a family of loci that is widespread in these organisms. All family members have (i) convergently transcribed toxin message and regulatory RNAs, (ii) three non-contiguous complementary regions for potential interaction, and (iii) intramolecular structures within the toxin message that modulate translation and transcript stability. Therefore, the detailed information available on the par locus provides a paradigm for studying the function and mechanism of regulation of the related loci.

Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians.

Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species. Much research has focused on Ciona or Halocynthia spp. but development in other ascidians remains poorly characterized. In this study, we surveyed the multipotent myogenic B7.5 lineage in Molgula spp. Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval, cardiac, and pharyngeal muscle progenitors. Moreover, the expression patterns of key regulators are conserved, but cross-species transgenic assays uncovered incompatibility, or 'unintelligibility', of orthologous cis-regulatory sequences between Molgula and Ciona. These sequences drive identical expression patterns that are not recapitulated in cross-species assays. We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements, hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis.

Refined candidate region for F4ab/ac enterotoxigenic Escherichia coli susceptibility situated proximal to MUC13 in pigs.

F4 enterotoxigenic Escherichia coli (F4 ETEC) are an important cause of diarrhea in neonatal and newly-weaned pigs. Based on the predicted differential O-glycosylation patterns of the 2 MUC13 variants (MUC13A and MUC13B) in F4ac ETEC susceptible and F4ac ETEC resistant pigs, the MUC13 gene was recently proposed as the causal gene for F4ac ETEC susceptibility. Because the absence of MUC13 on Western blot from brush border membrane vesicles of F4ab/acR+ pigs and the absence of F4ac attachment to immunoprecipitated MUC13 could not support this hypothesis, a new GWAS study was performed using 52 non-adhesive and 68 strong adhesive pigs for F4ab/ac ETEC originating from 5 Belgian farms. A refined candidate region (chr13: 144,810,100–144,993,222) for F4ab/ac ETEC susceptibility was identified with MUC13 adjacent to the distal part of the region. This candidate region lacks annotated genes and contains a sequence gap based on the sequence of the porcine GenomeBuild 10.2. We hypothesize that a porcine orphan gene or trans-acting element present in the identified candidate region has an effect on the glycosylation of F4 binding proteins and therefore determines the F4ab/ac ETEC susceptibility in pigs.

The new insights into cadmium sensing.

Cadmium (Cd) is non-essential heavy metal, which in excess, exhibits deleterious effects to the most of the organisms. Mobilization of defense mechanisms against this toxic agent requires rapid activation of signaling pathways. The article presents recent advances in the research concerning cadmium signal transduction in plants. New insights into the involvement of reactive oxygen species (ROS), nitric oxide (NO), plant growth regulators, and Cd-induced protein modifications are reviewed. Moreover, the role of recently recognized Cd-associated signal elements, including micro RNAs and several cis- and trans-acting elements is discussed.

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon.

Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon.

DNA Methylation and Regulation of the CD8A after Duck Hepatitis Virus Type 1 Infection

BackgroundCluster of differentiation 8 (CD8) is expressed in cytotoxic T cells, where it functions as a co-receptor for the T-cell receptor by binding to major histocompatibility complex class I (MHCI) proteins, which present peptides on the cell surface. CD8A is critical for cell-mediated immune defense and T-cell development. CD8A transcription is controlled by several cis-acting elements and trans-acting elements and is also regulated by DNA methylation. However, the epigenetic regulation of CD8A in the duck and its relationship with virus infection are still unclear.ResultsWe investigated the epigenetic transcriptional regulatory mechanisms, such as DNA methylation, for the expression of the CD8A and further evaluated the contribution of such epigenetic regulatory mechanisms to DHV-I infection in the duck. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed the highest level of CD8A expression to be in the thymus, followed by the lungs, spleen, and liver, and the levels of CD8A expression were very low in the kidney, cerebrum, cerebellum, and muscle in the duck. RT-qPCR also demonstrated that the CD8A mRNA was down-regulated significantly in morbid ducklings treated with DHV-1 and up-regulated significantly in non-morbid ducklings in all the tissues tested. In addition, hypermethylation of CD8A was detected in the morbid ducklings, whereas relatively low methylation of CD8A was evident in the non-morbid ducklings. The CD8A mRNA level was negatively associated with the CpG methylation level of CD8A and global methylation status.ConclusionsWe concluded that the mRNA level of the CD8A was negatively associated with the CpG methylation level of CD8A and global methylation status in the duck, suggesting that the hypermethylation of CD8A may be associated with DHV-1 infection. The first two CpG sites of the CD8A promoter region could be considered as epigenetic biomarkers for resistance breeding against duckling hepatitis disease in the duck.

Transactional Distance and Student Ratings in Online College Courses

Student ratings were compared in courses offered either exclusively online (n = 13,416) or face-to-face (n = 5,272). Data from 105 institutions were accessed from archived files of the IDEA Student Ratings of Instruction system. If a course was taught online, the instructor was less likely to lecture and more likely to use discussion, especially in hard disciplines. A course was less likely to have been taught online if the instructor was rated high in establishing rapport and if the course was within hard and pure disciplines. A high rating on structuring classroom experiences and expecting students to share in responsibility for learning increased the odds the course was offered online. However, high ratings on stimulating student interest and student effort in the course made it less likely. Results are discussed with respect to transactional distance elements of dialogue, structure, and learner autonomy.

The work of commissioning: a multisite case study of healthcare commissioning in England's NHS

ObjectiveTo examine the work of commissioning care for people with long-term conditions and the factors inhibiting or facilitating commissioners making service change.DesignMultisite mixed methods case study research, combining qualitative analysis...

The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron–exon recognition

The essential cis- and trans-acting elements required for RNA splicing have been defined, however, the detailed molecular mechanisms underlying intron-exon recognition are still unclear. Here we demonstrate that the ratio between stability of mRNA/DNA and DNA/DNA duplexes near 3'-spice sites is a characteristic feature that can contribute to intron-exon differentiation. Remarkably, throughout all transcripts, the most unstable mRNA/DNA duplexes, compared with the corresponding DNA/DNA duplexes, are situated upstream of the 3'-splice sites and include the polypyrimidine tracts. This characteristic instability is less pronounced in weak alternative splice sites and disease-associated cryptic 3'-splice sites. Our results suggest that this thermodynamic pattern can prevent the re-annealing of mRNA to the DNA template behind the RNA polymerase to ensure access of the splicing machinery to the polypyrimidine tract and the branch point. In support of this mechanism, we demonstrate that RNA/DNA duplex formation at this region prevents pre-spliceosome A complex assembly.

Viral Genome Maintenance and Latent Replication of Human Gammaherpesviruses

During gammaherpesvirus latency, only a few genes are expressed and required for maintenance of viral latency over a long period. While the expressed latent viral proteins play functional roles in viral latent DNA replication, they do not have replication-associated enzymatic activity such as polymerase or helicase activity. Viral genomes are detected in a similar copy number per infected cell, suggesting that the viral genome is replicated and segregated using host replication machinery. Kaposi’s sarcoma-associated herpesvirus and EBV have trans-acting elements required for viral genome maintenance during latency; LANA1 and EBNA1, respectively. The proteins recruit host replication-associated proteins at their latent origins, leading to initiation of viral replication and segregation with host chromosomes once per cell cycle. In addition, viral latent origins (cis-elements) provide trans-element-binding sites as well as a sufficient space for recruitment of cellular factors. In this review, we describe the molecular mechanisms required for replication of the viral genome during latency, including interactions with cellular factors and the interplay between viral trans- and cis-elements.

Plant Nucleotide Binding Site–Leucine-Rich Repeat (NBS-LRR) Genes: Active Guardians in Host Defense Responses

The most represented group of resistance genes are those of the nucleotide binding site–leucine-rich repeat (NBS-LRR) class. These genes are very numerous in the plant genome, and they often occur in clusters at specific loci following gene duplication and amplification events. To date, hundreds of resistance genes and relatively few quantitative trait loci for plant resistance to pathogens have been mapped in different species, with some also cloned. When these NBS-LRR genes have been physically or genetically mapped, many cases have shown co-localization between resistance loci and NBS-LRR genes. This has allowed the identification of candidate genes for resistance, and the development of molecular markers linked to R genes. This review is focused on recent genomics studies that have described the abundance, distribution and evolution of NBS-LRR genes in plant genomes. Furthermore, in terms of their expression, NBS-LRR genes are under fine regulation by cis- and trans-acting elements. Recent findings have provided insights into the roles of alternative splicing, the ubiquitin/proteasome system, and miRNAs and secondary siRNAs in the regulation of NBS-LRR gene expression at the post-transcriptional, post-translational and epigenetic levels. The possibility to use this knowledge for genetic improvement of plant resistance to pathogens is discussed.

Hofstede’s Cultural Model, LMX and Pakistani Historicsocio-political Discourse: An Analytical Critique

This paper grounds its analytical historical discourse of Pakistani culture, society and leadership (political, social and economic) in Hofstede’s model of national and organizational culture. It borrows from LMX theory of leadership which entails both transactional and transformational elements and applies it in the context of Pakistani leadership style. Since the founding ideology of Pakistan is the religion of Islam, the paper also undertakes a detailed account of Divine Commands based socio-economic Islamic precepts. It also presentssome empirical evidence reflecting the present Pakistani cultural and societal values embedding both the culture and leadership style of the country. The paper challenges Hofstede’s labeling of Pakistani culture as collectivistic and calls for future research on the ‘Dark Side’ of LMX practices prevalent in Pakistani discourse.

Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence

RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence.

Transcriptional and post-transcriptional regulation of cGMP-dependent protein kinase (PKG-I): pathophysiological significance.

The ability of the endothelium to produce nitric oxide, which induces generation of cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG-I), in vascular smooth muscle cells (VSMCs), is essential for the maintenance of vascular homeostasis. Yet, disturbance of this nitric oxide/cGMP/PKG-I pathway has been shown to play an important role in many cardiovascular diseases. In the last two decades, in vitro and in vivo models of vascular injury have shown that PKG-I is suppressed following nitric oxide, cGMP, cytokine, and growth factor stimulation. The molecular basis for these changes in PKG-I expression is still poorly understood, and they are likely to be mediated by a number of processes, including changes in gene transcription, mRNA stability, protein synthesis, or protein degradation. Emerging studies have begun to define mechanisms responsible for changes in PKG-I expression and have identified cis- and trans-acting regulatory elements, with a plausible role being attributed to post-translational control of PKG-I protein levels. This review will focus mainly on recent advances in understanding of the regulation of PKG-I expression in VSMCs, with an emphasis on the physiological and pathological significance of PKG-I down-regulation in VSMCs in certain circumstances.

BK Polyoma Virus Induces Aberrant Methylation of Host Genome to Promote Epithelial Mesenchymal Transition and Fibrosis

Although most people are infected during early childhood, BK polyoma virus (BKV) reactivates and causes disease primarily in renal transplant patients. BKV is currently the leading cause of graft loss during the first two years after renal transplant. Activation of BKV is associated with inflammation, tissue destruction and progression to fibrosis through a process involving the transformation of epithelial cells in to mesenchymal phenotype. Understanding how BKV causes these changes will help us in better prediction and management of BKV nephropathy. As the primary site of BKV tropism is renal tubular epithelial cells, it needs to device ways to promote the transition and proliferation of these terminally differentiated cells. We hypothesized that BK may activate epigenetic mechanisms to promote these changes by inducing or inhibiting key genes involved in maintaining cellular integrity and stability. DNA CpG methylation is one such epigenetic mechanism which can control gene regulation by causing changes to the DNA which inhibit the accessibility of transcription factors to gene promoters. For our experiments, we have used primary human proximal renal tubular epithelial cells (HPTC) isolated from donor kidneys or procured from ATCC. Cells were infected with BKV for different time points. Real-time expression profiling was done to identify key genes modulated by BKV. Bisulfite conversion and Methylation specific PCR was used to identify specific CpG changes induced by BKV. Sequencing of the bisulfite converted DNA was done to identify the methylation status of entire CpGs islands in the promoters of these genes. Promoter region with specific CpGs was then cloned in pGL3 luciferase reporter vector to specify the role of particular CpGs in regulation and rule out the effect of trans-acting elements. For luciferase assays, CCD1105 KIDtr and HPTC cells were transfected using Lipofectamine- LTX and Plus reagent, infected with BK virus and assayed using Dual luciferase assay. Cell cycle analysis was done using propidium iodide staining and flow cytometry. Infection of renal epithelial cells by BKV caused induction of proinflammatory genes such as Cox-2 and interleukin 1 Beta along with Vimentin which is the marker for epithelial mesenchymal transition. More importantly, it inhibited retinoblastoma (RB1) and E-cadherin (CDH1) genes. We have for the first time identified that BK inhibits these genes by causing hypermethylation of specific CpG islands in their upstream regulatory promoters. These genes are important regulators of cell-cell adhesion (CDH1) and cell cycle (RB1), and their inhibition may lead to loss of adhesion and cell proliferation respectively. Luciferase Reporter assay using cloned RB1 promoter confirmed the involvement of these CpG islands and showed progressive decrease in RB1 promoter activity with increasing BK infection. Our studies also suggest that BKV infection may lead to increased aneuploidy in these cells further contributing to cellular instability and transformation. The changes induced by BKV may ultimately lead to inflammation, cellular proliferation and progression to renal fibrosis and graft loss. Specific CpG methylation patterns induced by BKV may also serve as biomarkers to predict the initiation of BKV nephropathy and help in devising relevant control strategies.

Clinical utility gene card for: pseudohypoparathyroidism.

1.5 Mutational spectrum In E70–80% of cases, PHP-Ia is caused by haploinsufficiency because of maternally-inherited heterozygous inactivating mutations of GNAS gene1,2 (personal data). In very few cases, large deletions including part or the whole gene have been reported,3,4 while in a further subset of patients cytosine methylation defects of the GNAS promoters have been identified, a pattern typically found in PHP-Ib.5–8 Patients affected with PHP-Ib share a loss of methylation on the maternal A/B exon of GNAS. Those with the autosomal dominant form of PHP-Ib (AD-PHP-Ib) display an isolated loss of methylation at exon A/B associated with a recurrent 3-kb deletion in the STX16 gene, although a 4.4-kb deletion has been described in one family with AD-PHP-Ib.9,10 In four families with AD-PHP-Ib, NESP55 and NESPAS deletions have also been described leading to the loss of all maternal GNAS imprints (epimutations).11–13 In most cases, PHP-Ib is sporadic, and is characterized by complete loss of methylation at the NESPas, XLas and A/B promoters, and no other changes in cisor trans-acting elements have been found to explain this loss of methylation. In some cases (2–20% of the patients with PHP-Ib) paternal 20q disomies have been described.14–17

Transcriptional and Epigenetic Regulation of KIAA1199 Gene Expression in Human Breast Cancer

Emerging evidence has demonstrated that upregulated expression of KIAA1199 in human cancer bodes for poor survival. The regulatory mechanism controlling KIAA1199 expression in cancer remains to be characterized. In the present study, we have isolated and characterized the human KIAA1199 promoter in terms of regulation of KIAA1199 gene expression. A 3.3 kb fragment of human genomic DNA containing the 5′-flanking sequence of the KIAA1199 gene possesses both suppressive and activating elements. Employing a deletion mutagenesis approach, a 1.4 kb proximal region was defined as the basic KIAA1199 promoter containing a TATA-box close to the transcription start site. A combination of 5′-primer extension study with 5′RACE DNA sequencing analysis revealed one major transcription start site that is utilized in the human KIAA1199 gene. Bioinformatics analysis suggested that the 1.4 kb KIAA1199 promoter contains putative activating regulatory elements, including activator protein-1(AP-1), Twist-1, and NF-κB sites. Sequential deletion and site-direct mutagenesis analysis demonstrated that the AP-1 and distal NF-κB sites are required for KIAA1199 gene expression. Further analyses using an electrophoretic mobility-shift assay and chromatin immunoprecipitation confirmed the requirement of these cis- and trans-acting elements in controlling KIAA1199 gene expression. Finally, we found that upregulated KIAA1199 expression in human breast cancer specimens correlated with hypomethylation of the regulatory region. Involvement of DNA methylation in regulation of KIAA1199 expression was recapitulated in human breast cancer cell lines. Taken together, our study unraveled the regulatory mechanisms controlling KIAA1199 gene expression in human cancer.

The mystery of mitochondrial RNases

The central dogma states that DNA is transcribed to generate RNA and that the mRNA components are then translated to generate proteins; a simple statement that completely belies the complexities of gene expression. Post-transcriptional regulation alone has many points of control, including changes in the stability, translatability or susceptibility to degradation of RNA species, where both cis- and trans-acting elements will play a role in the outcome. The present review concentrates on just one aspect of this complicated process, which ultimately regulates the protein production in cells, or more specifically what governs RNA catabolism in a particular subcompartment of human cells: the mitochondrion.