Home About us Contact
|
Home About us Contact | ||
|
|
||
Vitro Transcription (vitro + transcription)
Terms modified by Vitro Transcription Selected AbstractsAmplification of low quantity bacterial RNA for microarray studies: time-course analysis of Leptospirillum ferrooxidans under nitrogen-fixing conditions,ENVIRONMENTAL MICROBIOLOGY, Issue 6 2006Mercedes Moreno-Paz Summary We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3, end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130,150 µg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram-negative bacterium Leptospirillum ferrooxidans. We further validated our amplification method in parallel experiments with non-amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time-course of growth. [source] Laser capture microdissection and microarray analysis of dividing neural progenitor cells from the adult rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007Ulf Gurok Abstract Neural progenitor cells reside in the hippocampus of adult rodents and humans and generate granule neurons throughout life. Knowledge about the molecular processes regulating these neurogenic cells is fragmentary. In order to identify genes with a role in the proliferation of adult neural progenitor cells, a protocol was elaborated to enable the staining and isolation of such cells under RNA-preserving conditions with a combination of immunohistochemistry and laser capture microdissection. We increased proliferation of neural progenitor cells by electroconvulsive treatment, one of the most effective antidepressant treatments, and isolated Ki-67-positive cells using this new protocol. RNA amplification via in vitro transcription and subsequent microarray analysis revealed over 100 genes that were differentially expressed in neural progenitor cells due to electroconvulsive treatment compared to untreated control animals. Some of these genes have already been implicated in the functioning of neural progenitor cells or have been induced by electroconvulsive treatment; these include brain-derived neurotrophic factor (Bdnf), PDZ-binding kinase (Pbk) and abnormal spindle-like microcephaly-associated (Aspm). In addition, genes were identified for which no role in the proliferation of neurogenic progenitors has been described so far, such as enhancer of zeste homolog 2 (Ezh2). [source] Identification of a novel staining pattern of bile duct epithelial cells in primary sclerosing cholangitisINFLAMMATORY BOWEL DISEASES, Issue 2 2010Brita Ardesjö PhD Abstract Background: Primary sclerosing cholangitis (PSC) is an inflammatory disease of the bile ducts with an unknown etiology. A number of autoantigens have been proposed, but an early diagnostic marker is still lacking. Our aim was to identify such an autoantigen. Methods: Immunostaining was performed on normal human bile duct with sera from patients with PSC and controls. To identify an autoantigen a cDNA library from normal human choledochus was constructed and immunoscreened with patient sera. Using in vitro transcription and translation and immunoprecipitation we examined the immunoreactivity against PDZ domain containing 1 (PDZK1) in 35 patients with PSC, 198 control patients, and 94 healthy controls. Results: We observed a previously unpublished staining pattern in which cytoplasmatic granules and apical cell membranes of biliary epithelial cells were stained by PSC sera. Strong immunoreactivity to these structures was obtained with 12 out of 35 PSC sera (34%) but not with sera from healthy controls. By screening the cDNA library we identified PDZK1 as a candidate antigen. Immunoreactivity against PDZK1 was detected in 9% of PSC patients, 2% of inflammatory bowel disease (IBD) patients, 8% of autoimmune pancreatitis patients, 18% of Grave's disease patients, and 1% of healthy controls. Conclusions: Previously unpublished, specific, and strong autoantibodies against epithelial cells of the bile duct in PSC sera were identified. Furthermore, PDZK1 is suggested as a potential new autoantigen. Inflamm Bowel Dis 2009 [source] Cloning and characterization of a trypsin-encoding cDNA of the human body louse Pediculus humanusINSECT MOLECULAR BIOLOGY, Issue 1 2004A. H. Kollien Abstract From a cDNA library of the whole insect, a trypsin gene of Pediculus humanus has been cloned and sequenced. The 908 bp clone has an open reading frame of 759 bp, which encodes a pre-proenzyme with 253 amino acid residues. A sixteen-residue N-terminal signal peptide is followed by a twelve-residue activation peptide with putative cleavage sites at Gly16 and Tyr28. The deduced amino acid sequence has several features typical of trypsin proteases and an overall identity of 35,43% with the trypsins of several haematophagous Diptera. The 1.0 kb genomic trypsin gene contains three introns of 102, 79 and 80 nucleotides following the codons for Gly16, Gln74 and Ala155, respectively. Only a single gene seems to be present. In Northern blot analysis, unfed first instar larvae have an identical or slightly lower level of trypsin mRNA than fed adult lice, and in adults 2,24 h after the bloodmeal this gene shows a constitutive expression. After in vitro transcription and translation, the activation peptide is cleaved by chymotrypsin, a so far unreported phenomenon in trypsin activation. [source] The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of ,54 -dependent transcriptionMOLECULAR MICROBIOLOGY, Issue 3 2006Lisandro M. D. Bernardo Summary The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from ,70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo,54 transcription even though they do not have any major direct effects on ,54 transcription in reconstituted in vitro transcription and ,-factor competition assays, (ii) that previously defined mutations rendering the housekeeping ,70 less effective at competing with ,54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from ,54 promoters in vivo reflects the innate affinity of the promoters for ,54 -RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of ,54 -dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated ,70 promoters. [source] In vitro transcription of PrfA-dependent and -independent genes of Listeria monocytogenesMOLECULAR MICROBIOLOGY, Issue 1 2001M. Lalic-Mülthaler In vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas transcription from Phly, PplcA and PactA is strictly PrfA-dependent, that from Piap, PprfA1/2 and, unexpectedly, also from Pmpl is independent. Initiation of in vitro transcription at all tested promoters except PprfA requires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript. RNA polymerase prepared from L. monocytogenes cultured either in rich culture medium (RNAPBHI), exposed to heat shock conditions (RNAP48) or conditioned in minimal essential medium (RNAPMEM) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters. Transcription starting from the PrfA-dependent promoters PactA and Phly is enhanced with RNAP48 and RNAPMEM (in relation to Piap,mediated transcription), while transcription from the other promoters is reduced when compared with RNAPBHI. These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor. [source] Novel human testis-specific cDNA: Molecular cloning, expression and immunobiological effects of the recombinant proteinMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001Ramasamy Santhanam Abstract A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-,gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122,124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S -transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the ,17 kDa recombinant TSA-1, and a ,24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility. Mol. Reprod. Dev. 60: 1,12, 2001. © 2001 Wiley-Liss, Inc. [source] Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitroTHE PLANT JOURNAL, Issue 1 2005Carla E. Hegeman Summary None of the macromolecular components of the chloroplast RNA editing apparatus has yet been identified. In order to facilitate biochemical purification and characterization of the chloroplast RNA editing apparatus, we have identified conditions suitable for production of chloroplast extracts from the model plant Arabidopsis that are capable of editing exogenous substrates produced by in vitro transcription. A simple poisoned primer extension assay readily quantified editing extent of mutated and wild-type substrates. Maximum editing efficiency typically varied from 10 to 40% with different chloroplast preparations. Substrates carrying as little as 47 nt surrounding the psbE editing site were as efficiently edited as longer substrates. Editing activity was stimulated when either ATP, CTP, or dCTP was provided to the extract, an unusual observation also recently seen with plant mitochondrial editing extracts. Editing was sensitive to a zinc chelator, also a characteristic of the mammalian APOBEC editing enzyme, which is a zinc-dependent cytidine deaminase. [source] The effect of ,-amanitin on RNA polymerase II ubiquitination,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2006Jozsef Szeberenyi Terms to be familiar with before you start to solve the test: RNA polymerase II, transcription, ,-amanitin, protein ubiquitination, synchronization of cell cultures, affinity adsorption, SDS-polyacrylamide gel electrophoresis, Western blotting, protein phosphorylation, protein kinases, in vitro transcription, plasmid, promoter, general and regulatory transcription factors, autoradiography. [source] De novo synthesis and assembly of multiplex riboswitches in vitroBIOTECHNOLOGY PROGRESS, Issue 5 2009Hao-Hua Sun Abstract Pools of short synthetic oligonucleotides (oligos) are required in the multiplex and parallel DNA construction. Microarray technology provides a fast and economical mean for massive parallel synthesis of oligos. The method of oligo synthesis with the programmable microfluidic PicoArray could simultaneously synthesize the designed oligos for multiple riboswitch genes. The synthetic oligos were recovered and purified as a pool of oligo mixture (OligoMix). Three temperature steps were employed to denature, anneal and extend the designed OligoMix until, after multiple rounds of thermocycling, the riboswitches with the desired length are obtained. The OligoMix was amplified using this PCR-based technique and the flanking adapter segments were cleaved for following assembly. Based on these oligos derived from 197 riboswitch sequences, the method of simultaneous assembling multiplex riboswitches (SAMRs) showed high fidelity by sequence identification. The resultant error rate was determined to be 2.78,. With the templates from SAMRs, in vitro transcription was applied to produce milligram amounts of biologically active riboswitches. With the verification of biological activity based on the high specificity of recognizing small-molecule metabolites as well as the DNA sequence redivivus by RT-PCR, the assembled riboswitches can be used for further gene operation and biological application. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] ,-Thalassaemia intermedia in a Turkish girl: homozygosity for G,A substitution at +22 relative to the ,-globin cap siteBRITISH JOURNAL OF HAEMATOLOGY, Issue 1 2001R. Öner We provide the first description of a homozygote patient for the G,A substitution in the 5, UTR of the ,-globin gene. The proband was a 17-year-old girl with ,-thalassaemia intermedia who had never received a blood transfusion. The physical examination revealed a well-developed women with no facial or bony abnormalities. There was mild paleness and mild splenomegaly which was 2 cm below the costal margin. The haemoglobin (Hb) was 7·6 g/dl, Hb A2 5·4% and Hb F 14·6% of the total Hb. The Hb A2 of both parents was 3·5%. The Hb F level in the mother and father were 0·9, 1·2% and the mean cell volume (MCV) value was 70 and 72 fl respectively. DNA analysis of the ,-gene region of the propositus revealed homozygosity for a G,A substitution at nucleotide +22 relative to the ,-gene cap site, within a functional downstream region that was referred to as the DCE (downstream core element). In addition to the data obtained previously from in vitro transcription assays, clinical findings and in vivo expression studies gave some valuable clues about the effect of +22 G,A mutation on the expression of ,-gene. Phenotypic expression of this homozygous patient is highly suggestive that G,A substitution at nt +22 confers a relatively mild (silent) ,+ -thalassaemia phenotype. [source] | |||||||||||||