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Untranslated Regions (untranslated + regions)
Selected AbstractsA simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesisFEBS JOURNAL, Issue 4 2006Macarena Morillo-Huesca We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3,-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3,-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes. [source] Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP)FEBS JOURNAL, Issue 17 2000Holger Thiele In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3, untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue-specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5,-flanking regions of two TPT1 pseudogenes. [source] Megalencephalic leukoencephalopathy with subcortical cysts: an update and extended mutation analysis of MLC1,HUMAN MUTATION, Issue 6 2006P. K. Ilja Boor Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive cerebral white matter disorder in children. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation. MLC is caused by mutations in the gene MLC1, which encodes a novel protein, MLC1. Since the first report, 50 mutations in this gene have been found. Mutations occur throughout the entire coding region and include all different types: 11 splice-site mutations; one nonsense mutation; 24 missense mutations; and 14 deletions and insertions. Until now, six polymorphisms within the coding sequence of MLC1 had been reported. In about 20% of the patients with a typical clinical and MRI picture, no mutations in the MLC1 gene are found. Several of the families, in which no mutations are found, also do not show linkage with the MLC1 locus, which suggests a second gene involved in MLC. The absence of mutations may also be the consequence of performing standard mutation analysis that can miss heterozygous deletions, mutations in the promoter, 3, and 5, untranslated regions (UTRs), and intron mutations, which may influence the amino acid composition of the end product. In this work we describe 13 novel mutations, including those found with extended mutation analysis on MLC patients. This study shows that extended mutation analysis is a valuable tool to identify at least some of the missing mutations. Therefore, we suggest extended mutation analysis for the MLC1 gene, if no mutations are found during standard analysis. Hum Mutat 27(6), 505,512, 2006. © 2006 Wiley-Liss, Inc. [source] Influenza virus assays based on virus-inducible reporter cell linesINFLUENZA AND OTHER RESPIRATORY VIRUSES, Issue 5 2009Yunsheng Li Background, Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5,- and 3,-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression. Findings, Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses. Conclusions, These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers. [source] Detection and analysis of alternative splicing in the silkworm by aligning expressed sequence tags with the genomic sequenceINSECT MOLECULAR BIOLOGY, Issue 2 2005X.-F. Zha Abstract We identified 277 alternative splice forms in silkworm genes based on aligning expressed sequence tags with genomic sequences, using a transcipt assembly program. A large fraction (74%) of these alternative splices are located in protein-coding regions and alter protein products, whereas only 26% are in untranslated regions. From the alternative splices located in protein-coding regions, some (43%) affect protein domains that bind various biological molecules. The vast majority of the detected alternative forms in this study appear to be novel, and potentially affect biologically meaningful control of function in silkworm genes. Our results indicate that alternative splicing in silkworm largely produces protein diversity and functional diversity, and is a widely used mechanism for regulating gene expression. [source] Cloning and characterization of a 70 kDa heat shock cognate gene (HSC70) from two species of ChironomusINSECT MOLECULAR BIOLOGY, Issue 1 2003N. K. Karouna-Renier Abstract In the present study we carried out the isolation and characterization of an HSC70 gene from two midges, Chironomus tentans and C. yoshimatsui. The HSC70 cDNAs are approximately 2424 (C. tentans) and 2464 bp (C. yoshimatsui) long, and contain 1950 and 1956 bp open reading frames, respectively. Analysis of genomic DNA revealed the presence of two introns in these genes. The 5, untranslated regions of the HSC70 genes are adenosine-rich, a feature found in inducible HSP70 genes. The nucleotide and amino acid sequences exhibit high identity with cytosolic HSC70s from other Dipterans. Northern hybridization indicated that HSC70 is expressed at all developmental stages, from embryo to adult, and Southern hybridization confirmed the presence of multiple HSP70 genes in Chironomus. [source] Isolation and molecular characterization of Musca domestica delta-9 desaturase sequencesINSECT MOLECULAR BIOLOGY, Issue 6 2002A. L. Eigenheer Abstract We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two ,1.66 kb cDNAs were recovered. They had identical coding regions and 3, untranslated regions (UTRs), but differed in their 5, UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a ,9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more ,9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family. [source] Chronic hepatitis C infection: Influence of the viral load, genotypes, and GBV-C/HGV coinfection on the severity of the disease in a Brazilian populationJOURNAL OF MEDICAL VIROLOGY, Issue 1 2002Leila M.M.B. Pereira Abstract The distributions of the different genotypes of the hepatitis C virus (HCV) and GBV-C virus (GBV-C/HGV) vary geographically and information worldwide is still incomplete. In particular, there are few data on the distribution of genotypes (and their relationship to the severity of liver disease) in South America. Findings are described in 114 consecutive patients from Northeast Brazil (median age 52 years, range 18,72 years) who had abnormal levels of serum aminotransferases and seropositivity for HCV RNA. The patients were recruited from an outpatient clinic between November 1997 and April 1998. Quantitative HCV RNA and GBV-C/HGV RNA estimations were carried out by double-nested polymerase chain reaction (PCR) using primers from the 5,-untranslated regions (UTRs) of the genomes. HCV genotypes were determined by restriction fragment length polymorphism (RFLP) analysis with 5,-UTR primers and by PCR with type-specific 5,-UTR primers. GBV-C/HGV-RNA genotypes were determined by RFLP with specific 5,-UTR primers and phylogenetic trees were constructed using the Neighbour-Joining and Drawtree programs. Histological features were graded and staged according to international criteria. Of the 114 patients, 35 (30.7%) patients had cirrhosis and 22 (27.8%) had mild, 51 (64.6%) had moderate, and 6 (7.6%) had severe chronic hepatitis. Median HCV viral load was 106 genome equivalents per millilitre (range 104,109/ml). Frequencies of genotypes were 5.3% type 1a, 44.7% type 1b, 3.5% type 2, 41.2% type 3, and 5.3% mixed types. GBV-C/HGV-RNA was detected in the sera of 12 (10.5%) patients and was distributed among three phylogenetic groups. There were no significant differences between patients with the predominant HCV genotypes (1b and 3) with respect to gender, age group, viral load, severity of liver disease, or coinfection with GBV-C/HGV. J. Med. Virol. 67:27,32, 2002. © 2002 Wiley-Liss, Inc. [source] Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expressionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001Maria K. Dahle Abstract By using 5′ RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIα subunit of cAMP‐dependent protein kinase that differed in their 5′ untranslated regions. Two of these 5′‐regions showed similarity with the human RIα exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIα 1b and RIα 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIα 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIα protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIα 1c haploid‐specific mRNA, therefore, may be important for the regulation of RIα expression in sperm. Mol. Reprod. Dev. 59:11–16, 2001. © 2001 Wiley‐Liss, Inc. [source] Research note: Characterization of a cDNA encoding glutamine synthetase II from Gelidium crinale (Rhodophyta)PHYCOLOGICAL RESEARCH, Issue 1 2002D. Wilson Freshwater SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5,- and 3,-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5, and 3, untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution. [source] Molecular characterization of the CP gene and 3,UTR of Chilli veinal mottle virus from South and Southeast AsiaPLANT PATHOLOGY, Issue 3 2008W. S. Tsai Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3, terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3, untranslated regions (3,UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3,UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV. [source] Postnatal transcription profile and polymorphism of the ADIPOR1 gene in five pig breedsANIMAL GENETICS, Issue 1 2010M. Stachowiak Summary As a result of its role in energy homeostasis regulation, the ADIPOR1 gene is a candidate for fat deposition, an important production trait, in the pig. The aim of the study was to conduct a comparative analysis of the ADIPOR1 postnatal transcript level, in order to establish its promoter and 5,UTR sequences and to search the gene for polymorphisms. The transcription level was examined in longissimus dorsi and semimembranosus muscles collected from 180 pigs at 60,210 days of age, representing five pig breeds: Duroc, Polish Large White, Polish Landrace, Pietrain and Pulawska. We calculated highly significant breed by age by muscle interaction (P < 0.0001) and breed by muscle interactions (P < 0.01). The 5,UTR and promoter region of the porcine ADIPOR1 gene were amplified for the first time and their sequences were deposited in the GenBank database. In total, 21 novel and two previously described polymorphisms were found in the ADIPOR1 promoter, coding, intronic, 5, and 3, untranslated regions. The only SNP detected in the coding region was a synonymous substitution. Two polymorphisms in 3,UTR (c.*129A>C and c.*536A>G) showed no significant effect on the transcript level. Our results showed a high polymorphism of the ADIPOR1 and a complexity in its transcription level in the studied muscles. This complexity indicates that conclusions based on such studies should be carefully gradated. [source] Ovine acyl CoA:diacylglycerol acyltransferase 1, molecular characterization, polymorphisms and association with milk traitsANIMAL GENETICS, Issue 5 2009M. C. Scatà Summary The objective of this work was to characterize the complete coding region of the ovine acylCoA:diacylglycerol acyltransferase 1 (DGAT1) gene of three Italian sheep breeds: Sarda, Altamurana and Gentile di Puglia. Characterization was accomplished by direct sequencing of 8676 bp of the relevant DNA, including introns and partial 5, and 3, untranslated regions (UTRs). We detected five novel SNPs; one SNP (g.5553C>T) is located in intron 2, has similar frequencies in the three breeds and showed a negative association with milk fat content. More interesting is an SNP in the 5, UTR (g.127C>A), the occurrence of which is rare in the higher milk-fat breeds (Altamurana and Gentile di Puglia); it is located in the core sequence of Sp1, a putative binding site of transcription factors. This SNP showed a significant negative association with milk fat content in the Sarda sheep. Because DGAT1 plays a fundamental role in triacylglycerol synthesis, the novel detected SNP in the 5, UTR of the DGAT1 gene might explain, at least partially, the variation of fat content in the milk of Sarda sheep. [source] Distribution of FMR1 and FMR2 alleles in Javanese individuals with developmental disability and confirmation of a specific AGG-interruption pattern in Asian populationsANNALS OF HUMAN GENETICS, Issue 2 2001SULTANA M. H. FARADZ The number of trinucleotide repeats in the 5, untranslated regions of the FMR1 and FMR2 genes was determined by PCR in 254 Fragile XA-negative Javanese male children with developmental disabilities. The distribution of FMR1 and FMR2 trinucleotide repeat alleles was found to be significantly different in the Indonesian population with developmental disability compared to that in developmentally disabled populations in North America and Europe (p < 0.021). Sequence analysis was performed on the trinucleotide repeat arrays of the 27 individuals with FMR1 alleles in the ,grey zone' (35,54 repeats). A repeat array structure of 9A9A6A9 was found in 16 unrelated individuals with 36 repeats, confirming earlier observations in intellectually normal Japanese. We propose that this FMR1 array pattern is specific for Asian populations and that Javanese and Japanese populations arose from a single progenitor population. [source] MicroRNA-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 5 2009Yuji Nakamachi Objective To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression. Methods Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription,polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay. Results We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G1 phase. We identified a putative consensus site for miR-124a binding in the 3,-untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3,-untranslated regions of CDK-2 and MCP-1 mRNA. Conclusion The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes. [source] Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel,Lindau diseaseCANCER SCIENCE, Issue 5 2006Keiko Hattori Germline mutations of the VHL gene are responsible for VHL. Approximately 70% of VHL families display small intragenic mutations detectable by sequencing, whereas partial- or whole-gene deletions have been described in the majority of the remaining families. For such large deletions, complex genetic techniques other than sequencing might have to be used. In this study, we describe an RQ-PCR assay with TaqMan fluorescent probes to detect germline VHL deletions. The RQ-PCR primer/probe sets were designed for the three VHL coding exons as well as for the 5, promoter and 3, untranslated regions. The RQ-PCR assay for 30 normal and 10 known VHL deletion control samples demonstrated high sensitivity and specificity. We then screened 29 individuals from 19 classical VHL families (16 type 1, 2 type 2A, and one type 2B) and one PHEO family, as well as four solitary suspected cases, none displaying any sequence changes, for VHL deletions by the RQ-PCR assay. We detected germline deletions in 17 (89%) classical families including 15 type 1, one type 2A, and one type 2B. We also identified two mutation carriers and two non-carriers in our family cohort. The one PHEO family and four solitary cases did not display any deletion patterns. These findings indicated that the TaqMan-based RQ-PCR assay is a simple and potent technique for the rapid, sensitive, and specific investigation of VHL genetic diagnoses that could be used profitably before more complex large-deletion detection techniques. (Cancer Sci 2006; 97: 400 ,405) [source] | |||||||||||||