921 resultados para RNA GENE
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Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes.
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The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.
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DNA sequencing, RNA mapping, and protein expression experiments revealed the presence of a gene, tfoX+, encoding a 24.9-kDa polypeptide, that is transcribed divergently from a common promoter region with the Haemophilus influenzae rec-1+ gene. H. influenzae strains mutant for tfoX failed to bind transforming DNA and were transformation deficient. Primer extension experiments utilizing in vivo total RNA from precompetent and competent H. influenzae cells demonstrated that transcription of tfoX+ increased immediately upon competence induction, suggesting that tfoX+ is an early competence gene. Similar experiments showed that the expression of the late competence-specific gene, com101A+, was tfoX+ dependent. Moreover, expression of plasmid-borne tfoX+ in H. influenzae resulted in constitutive competence. The addition of cyclic adenosine monophosphate (cAMP) to strains carrying a tfoX::lacZ operon fusion resulted in an immediate increase in beta-galactosidase activity that correlated with an increase in genetic transformability. Collectively, our results suggest that TfoX may play a key role in the development of genetic competence by regulating the expression of late competence-specific genes.
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Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.
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Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.
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As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the nonprotein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.
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Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.
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Thesis (Ph.D.)--University of Washington, 2016-06
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Thesis (Master's)--University of Washington, 2016-06
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Thesis (Ph.D.)--University of Washington, 2016-06
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Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.
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Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.
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Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.
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Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and in a subset of tissue macrophages and dendritic cells. It is expressed at lower levels in the parenchymal cells of the liver, glomerular mesangial cells of the kidney and pancreatic acinar cells. We have identified novel TRAP mRNAs that differ in their 5-untranslated region (5'-UTR) sequence, but align with the known murine TRAP mRNA from the first base of Exon 2. The novel 5'-UTRs represent alternative first exons located upstream of the known 5'-UTR. A similar genomic structure exists for the human TRAP gene with partial conservation of the exon and promoter sequences. Expression of the most distal 5'-UTR (Exon 1A) is restricted to adult bone and spleen tissue. Exon 1B is expressed primarily in tissues containing TRAP-positive nonhaematopoietic cells. The known TRAP 5'-UTR (Exon 1) is expressed in tissues characteristic of myeloid cell expression. In addition the Exon 1C promoter sequence is shown to comprise distinct transcription start regions, with an osteoclast-specific transcription initiation site identified downstream of a TATA-like element. Macrophages are shown to initiate transcription of the Exon 1C transcript from a purine-rich region located upstream of the osteoclast-specific transcription start point. The distinct expression patterns for each of the TRAP 5'-UTRs suggest that TRAP mRNA expression is regulated by the use of four alternative tissue- and cell-restricted promoters. (C) 2003 Elsevier Science B.V. All rights reserved.
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Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and P-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that Classical reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells. (C) 2003 Elsevier Inc. All rights reserved.