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Upstream Regions (upstream + regions)
Selected AbstractsEctopic activation of the transcription promoter for the testis-specific mouse Pgk-2 gene on elimination of a cis -acting upstream DNA regionDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2000Hiroshi Ando Transgenic mice carrying the coding sequence of ,-galactosidase, for which expression was driven by various upstream regions including the transcription promoter of the testis-specific mouse Pgk-2 gene, were generated. Expression of ,-galactosidase mRNA driven by the region between nucleotide positions , 1404 and + 61, with respect to the transcription initiation site numbered + 1, was examined by reverse transcription-mediated polymerase chain reaction, blot hybridization and in situ hybridization, and compared with that of endogenous Pgk-2 mRNA. The results revealed that the 1.4 kb DNA region is sufficient for determining the organ-specific, developmental stage-specific and spermatogenic stage-specific transcription of the mouse Pgk-2 gene. When the region between , 684 and + 61 was used to generate transgenic mice, ,-galactosidase mRNA was detectable not only in the testis, but also in other organs such as brain and lung. However, the timing and cell-type specificity of testicular expression of ,-galactosidase mRNA were retained in these mice. Because the region between , 1404 and , 685 repressed the Pgk-2 promoter in somatic cell-derived cell lines, it is suggested that the organ specificity of Pgk-2 transcription is achieved at least partly by negative regulation. [source] Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum)FEBS JOURNAL, Issue 18 2003Susan E. Douglas Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (,) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides. [source] Deletions of SCN1A 5, genomic region with promoter activity in Dravet syndrome,HUMAN MUTATION, Issue 7 2010Tojo Nakayama Abstract Mutations involving the voltage-gated sodium channel ,I gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. Here we investigated the upstream regions of the SCN1A 5, noncoding exons and found two major regions with promoter activity. These two major promoters were simultaneously active in various brain regions and in most neurons. Using multiplex ligation-dependent probe amplification (MLPA) assays with probes for the 5, noncoding exons, their upstream regions, and all coding exons of SCN1A, we investigated 130 epileptic patients who did not show any SCN1A mutations by sequence analysis of all coding exons and exon,intron boundaries. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5, noncoding exons and regions with promoter activity but not affecting the coding exons. We also identified four patients with deletions/duplication in the coding region. One patient with symptomatic focal epilepsy also showed a deletion in the coding region. This study provides the first case of microdeletion limited to the SCN1A 5, promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome. Hum Mutat 31:,11, 2010. © 2010 Wiley-Liss, Inc. [source] Cloning and characterization of Dorsal homologues in the hemipteran Rhodnius prolixusINSECT MOLECULAR BIOLOGY, Issue 5 2009R. Ursic-Bedoya Abstract Rhodnius prolixus is an ancient haematophagous hemipteran insect capable of mounting a powerful immune response. This response is transcriptionally regulated in part by transcription factors of the Rel/Nuclear Factor kappa B (Rel/NF-,B) family. We have cloned and characterized three members of this transcription factor family in this insect. Dorsal 1A is primarily expressed in early developmental stages. In contrast, dorsal 1B and 1C, both differentially spliced products of dorsal 1A, are expressed primarily in the adult fat body in response to septic injury, suggesting their exclusive role in immunity. Additionally, we identified putative ,B binding sites in the 5, upstream regions of target genes known to be involved in the innate immune response of insects. [source] Calcium Channel TRPV6 Expression in Human Duodenum: Different Relationships to the Vitamin D System and Aging in Men and Women,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006FRCP, Julian RF Walters MA Abstract Intestinal absorption of calcium affects bone mineralization and varies greatly. In human duodenum, expression of the calcium channel TRPV6 was directly related to blood 1,25-dihydroxyvitamin D in men, but effects of age with lower median vitamin D receptor levels were more significant in women. Introduction: The TRPV6 calcium channel/transporter is implicated in animal studies of intestinal calcium absorption, but in humans, its role and relationship to differences in mineral metabolism is unclear. We aimed to characterize TRPV6 expression in human intestine including defining relationships to the vitamin D endocrine system. Materials and Methods: TRPV6 transcript expression was determined in endoscopic mucosal biopsies obtained from normal duodenum. Expression was compared with that in ileum and with in situ hybridization in archival tissues and related to sequence variants in genomic DNA. TRPV6 expression was related in 33 subjects to other transcripts involved in calcium absorption including the vitamin D receptor (VDR) and to blood vitamin D metabolites including 1,25-dihydroxyvitamin D [1,25(OH)2D]. Results: TRPV6 transcripts were readily detected in duodenum but not in ileum. Expression was highest in villous epithelial cells. Sequence variants in the coding and upstream regions of the gene did not affect TRPV6 expression. The relationship between duodenal TRPV6 expression and 1,25(OH)2D differed in men and women. In men, linear regression showed a strong association with 1,25(OH)2D (r = 0.87, p < 0.01), which was unaffected by age. In women, there was no significant overall relationship with 1,25(OH)2D, but there was a significant decrease with age (r = ,0.69, p < 0.001). Individual expression of TRPV6 and VDR was significantly correlated. The group of older women (>50) had lower median levels of both TRPV6 and VDR transcripts than younger women (p < 0.001 and 0.02, respectively). Conclusions: Duodenal TRPV6 expression is vitamin D dependent in men, but not in older women, where expression of TRPV6 and VDR are both reduced. These findings can explain, at least in part, the lower fractional calcium absorption seen in older postmenopausal women. [source] Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeaeMOLECULAR MICROBIOLOGY, Issue 1 2009Vincent M. Isabella Summary Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope-tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe,2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a Kd of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co-ordination of a NO-sensitive Fe,S centre required for DNA binding. [source] Microarray analysis of chitin elicitation in Arabidopsis thalianaMOLECULAR PLANT PATHOLOGY, Issue 5 2002Katrina M. Ramonell Summary Chitin oligomers, released from fungal cell walls by endochitinase, induce defence and related cellular responses in many plants. However, little is known about chitin responses in the model plant Arabidopsis. We describe here a large-scale characterization of gene expression patterns in Arabidopsis in response to chitin treatment using an Arabidopsis microarray consisting of 2375 EST clones representing putative defence-related and regulatory genes. Transcript levels for 71 ESTs, representing 61 genes, were altered three-fold or more in chitin-treated seedlings relative to control seedlings. A number of transcripts exhibited altered accumulation as early as 10 min after exposure to chitin, representing some of the earliest changes in gene expression observed in chitin-treated plants. Included among the 61 genes were those that have been reported to be elicited by various pathogen-related stimuli in other plants. Additional genes, including genes of unknown function, were also identified, broadening our understanding of chitin-elicited responses. Among transcripts with enhanced accumulation, one cluster was enriched in genes with both the W-box promoter element and a novel regulatory element. In addition, a number of transcripts had decreased abundance, encoding several proteins involved in cell wall strengthening and wall deposition. The chalcone synthase promoter element was identified in the upstream regions of these genes, suggesting that pathogen signals may suppress the expression of some genes. These data indicate that Arabidopsis should be an excellent model to elucidate the mechanisms of chitin elicitation in plant defence. [source] Genome-wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a ,phoP mutantPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Antonio Rodríguez-García Abstract Phosphate limitation in Streptomyces and in other bacteria triggers expression changes of a large number of genes. This response is mediated by the two-component PhoR,PhoP system. A Streptomyces coelicolor ,phoP mutant (lacking phoP) has been obtained by gene replacement. A genome-wide analysis of the primary response to phosphate limitation using transcriptomic and proteomic studies has been made in the parental S. coelicolor M145 and in the ,phoP mutant strains. Statistical analysis of the contrasts between the four sets of data generated (two strains under two phosphate conditions) allowed the classification of all genes into 12 types of profiles. The primary response to phosphate limitation involves upregulation of genes encoding scavenging enzymes needed to obtain phosphate from different phosphorylated organic compounds and overexpression of the high-affinity phosphate transport system pstSCAB. Clear interactions have been found between phosphate metabolism and expression of nitrogen-regulated genes and between phosphate and nitrate respiration genes. PhoP-dependent repressions of antibiotic biosynthesis and of the morphological differentiation genes correlated with the observed ,phoP mutant phenotype. Bioinformatic analysis of the presence of PHO boxes (PhoP-binding sequences) in the upstream regions of PhoP-controlled genes were validated by binding of PhoP, as shown by electrophoretic mobility shift assays. [source] | |||||||||||||