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Sequence Upstream (sequence + upstream)
Selected AbstractsRNA interference by expressing short hairpin RNA in the Ciona intestinalis embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008Aya Nishiyama We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos. [source] Diversity of soil mycobacterium isolates from three sites that degrade polycyclic aromatic hydrocarbonsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007C.D. Miller Abstract Aims:, This paper investigates the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading mycobacterium isolates from three different sites within United States: Montana, Texas and Indiana. Methods and Results:, All five mycobacterium isolates differed in chromosomal restriction enzyme-fragmentation patterns; three isolates possessed linear plasmids. The DNA sequence between the murA and rRNA genes were divergent but the sequence upstream of nidBA genes, encoding a dioxygenase involved in pyrene oxidation, was more highly conserved. Long-chain fatty acid analysis showed most similarity between three isolates from the same Montana site. All isolates were sensitive to rifampicin and isoniazid, used in tuberculosis treatment, and to syringopeptins, produced by plant-associated pseudomonads. Biofilm growth was least for isolate MCS that grew on plate medium as rough-edged colonies. The patterns of substrate utilization in Biolog plates showed clustering of the Montana isolates compared with Mycobacterium vanbaalenii and Mycobacterium gilvum. Conclusion:, The five PAH-degrading mycobacterium isolates studied differ in genetic and biochemical properties. Significance and Impact of the Study:, Different properties with respect to antibiotic susceptibility, substrate utilization and biofilm formation could influence the survival in soil of the microbe and their suitability for use in bioaugmentation. [source] Directed Evolution of Metabolically Engineered Escherichiacoli for Carotenoid ProductionBIOTECHNOLOGY PROGRESS, Issue 6 2000Chia-wei Wang We have previously introduced a reconstructed isoprenoid pathway into Escherichia coli that exhibits amplified biosynthetic flux to geranylgeranyl diphosphate (GGPP), a common isoprenoid precursor. It was shown that GGPP synthase is an important rate-controlling enzyme in this reconstructed isoprenoid pathway. In this investigation, we applied directed evolution to GGPP synthase from Archaeoglobusfulgidus to enable the enhanced production of carotenoids in metabolically engineered E. coli. Eight mutants were isolated, and the best one increased lycopene production by 100%. Among the mutants that were isolated, mutation points were clustered in four "hot regions". The "hottest" region is located in the sequence upstream of the coding region, which presumably improves the expression level of the enzyme. The other three are within the coding sequence and are believed to improve the enzyme-specific activity in E coli. These results demonstrate that modulating both enzymatic expression and specific activity are important for optimizing the metabolic flux distribution. [source] Structural and functional differences between the promoters of independently expressed killer cell Ig-like receptorsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2005Bergen, Jeroen van Abstract Killer Ig-like receptors (KIR) are important for the recognition and elimination of diseased cells by human NK cells. Myeloid leukemia patients given a hematopoietic stem cell transplantation, for example, benefit from KIR-mediated NK alloreactivity directed against the leukemia cells. To establish an effective NK cell repertoire, most KIR genes are expressed stochastically, independently of the others. However, the sequences upstream of the coding regions of these KIR genes are highly homologous to the recently identified KIR3DL1 promoter (91.1,99.6% sequence identity), suggesting that they are regulated by similar if not identical mechanisms of transcriptional activation. We investigated the effects of small sequence differences between the KIR3DL1 promoter and other KIR promoters on transcription factor binding and promoter activity. Surprisingly, electrophoretic mobility shift assays and promoter-reporter assays revealed significant structural and functional differences in the cis-acting elements of these highly homologous KIR promoters, suggesting a key role for transcription factors in independent control of expression of specific KIR loci. Thus, the KIR repertoire may be shaped by a combination of both gene-specific and stochastic mechanisms. [source] Sigma factor selectivity in Borrelia burgdorferi: RpoS recognition of the ospE/ospF/elp promoters is dependent on the sequence of the ,10 regionMOLECULAR MICROBIOLOGY, Issue 6 2006Christian H. Eggers Summary Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (PospF) and ospE (PospE) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by ,70. Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to PospF, PospE, or two hybrid promoters in which the ,10 regions of PospF and PospE were switched [PospF (E , 10) and PospE,(F , 10) respectively]. We found that the PospF,10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while ,70 specificity for PospE is dependent on elements outside of the ,10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to ,70. Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have ,10 regions virtually identical to that of PospF. Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the PospF,10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the ,10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding. [source] Structure of the restriction,modification controller protein C.Esp1396IACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009N. Ball The controller protein of the Esp1396I restriction,modification (R,M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43,46 in the loop region flanking the DNA-recognition helix are displaced by up to 10,Å. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4,Å displacement of the recognition helices when the dimer is bound to its DNA target. [source] | ||||||||||