scholarly journals Simple and inexpensive three-step rapid amplification of cDNA 5′ ends using 5′ phosphorylated primers

2013 ◽  
Vol 434 (1) ◽  
pp. 1-3 ◽  
Author(s):  
Kai Dallmeier ◽  
Johan Neyts
Keyword(s):  
Rapid Amplification

scholarly journals Single molecule counting coupled to rapid amplification enables detection of αSynuclein aggregates in cerebrospinal fluid of PD patients

2021 ◽  
Author(s):  
Akshay BHUMKAR ◽  
Chloe MAGNAN ◽  
Derrick LAU ◽  
Eugene Soh Wei Jun ◽  
Nicolas DZAMKO ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Single Molecule ◽  
Rapid Amplification

Rapid Amplification of Uncharacterized Transposon-tagged DNA Sequences from Genomic DNA

Yeast ◽  
1997 ◽  
Vol 13 (3) ◽  
pp. 233-240 ◽  
Author(s):  
KRISTIN T. CHUN ◽  
HOWARD J. EDENBERG ◽  
MARK R. KELLEY ◽  
MARK G. GOEBL
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Rapid Amplification

scholarly journals Molecular cloning of mouse intestinal trefoil factor and its expression during goblet cell changes

1995 ◽  
Vol 311 (1) ◽  
pp. 293-297 ◽  
Author(s):  
M Tomita ◽  
H Itoh ◽  
N Ishikawa ◽  
A Higa ◽  
H Ide ◽  
...  
Keyword(s):  
Goblet Cell ◽  
Goblet Cells ◽  
Recovery Phase ◽  
Nippostrongylus Brasiliensis ◽  
Trefoil Factor ◽  
Intestinal Trefoil Factor ◽  
Cell Hyperplasia ◽  
Reverse Transcription Pcr ◽  
Rapid Amplification ◽  
Mitf Expression

A cDNA encoding mouse intestinal trefoil factor (mITF) was successfully cloned and sequenced from the small intestine of C57BL/6 mouse by using the combination of reverse transcription-PCR and rapid amplification of cDNA ends methods. The gene was, similar to rat and human ITFs, mainly expressed in the small and large intestine. The mITF expression was up-regulated during the recovery phase after depletion of goblet cells in acetic acid-induced colitis. On the other hand, the expression in the jejunum was not altered, while goblet cell hyperplasia was induced by Nippostrongylus brasiliensis infection. These results suggest that the mITF expression did not simply correlate with the number of goblet cells. The mITF may play an important role in the maintenance and repair of mucosal function of the rectum. Additionally, the mITF in the jejunum may play a role in alteration of the physicochemical nature of goblet cell mucins, thereby affecting the establishment of intestinal helminths.

Improved rapid amplification of cDNA ends (RACE) for mapping both the 5′ and 3′ terminal sequences of paramyxovirus genomes

2005 ◽  
Vol 130 (1-2) ◽  
pp. 154-156 ◽  
Author(s):  
Zhuo Li ◽  
Meng Yu ◽  
Hong Zhang ◽  
Hai-Yan Wang ◽  
Lin-Fa Wang
Keyword(s):  
Rapid Amplification

Cloning and functional expression of α-galactosidase cDNA from Penicillium janczewskii zaleski

Biologia ◽  
2011 ◽  
Vol 66 (2) ◽  
Author(s):  
Bo Zhang ◽  
Yiqun Chen ◽  
Zhimin Li ◽  
Wenqing Lu ◽  
Yunhe Cao
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Functional Expression ◽  
Recombinant Enzyme ◽  
Methanol Induction ◽  
Potential Applications ◽  
Prospective Application ◽  
Polymerase Chain ◽  
Penicillium Janczewskii ◽  
Rapid Amplification ◽  
Different Levels

AbstractIn recent years, α-galactosidase has been attracting more and more attention because of its potential applications in many aspects. Using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, a full-length cDNA sequence composed of 2,439 bp was cloned from Penicillium janczewskii zaleski and was subcloned into pPICZαA and transformed into Pichia pastoris strain X-33. In a 10-L fermentor, the recombinant yeast expressed α-galactosidase with a yield of 254 U/mL by methanol induction for 120 h. The recombinant enzyme showed the optimal activity at 40°C and pH 5.2. The K m values of the recombinant enzyme using p-nitrophenyl-α-D-galactopyranoside (pNPG), melibiose, raffinose and stachyose as substrates were 1, 16, 17.8 and 5.3 mM, respectively. V max values were 227.3, 116.7, 104.8, and 80.6 μM/min using pNPG, melibiose, raffinose and stachyose as substrates, respectively. The α-galactosidase exhibited no sensitivity to various metal ions and ethylenediaminetetraacetic acid, and hydrolyzed melibiose, raffinose and stachyose with different levels of galactose release. The biochemical characteristics of the α-galactosidase suggest that the enzyme may have a prospective application in feed industry as an additive.

Characterization of an A-type cyclin-dependent kinase gene from Dendrobium candidum

Biologia ◽  
2012 ◽  
Vol 67 (2) ◽  
Author(s):  
Gang Zhang ◽  
Chao Song ◽  
Ming-Ming Zhao ◽  
Biao Li ◽  
Shun-Xing Guo
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Postembryonic Development ◽  
Kinase Domain ◽  
Dimensional Structure ◽  
Pcr Analysis ◽  
Kinase Gene ◽  
Multiple Sequence ◽  
Dendrobium Candidum ◽  
Rapid Amplification

AbstractCyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and postembryonic development of organisms. To better understand the molecular mechanisms of CDKs involved in embryogenesis regulation in the endangered medicinal plant Dendrobium candidum Wall. ex Lindl., a 1229-bp full-length cDNA of an A-type CDK gene, Denca;CDKA;1, was identified using 3′ rapid amplification of cDNA end (RACE) PCR. Denca;CDKA;1 was predicted to encode a 294 amino acid residue-long protein of 33.76 kDa with an isoelectric point of 7.72. The deduced Denca;CDKA;1 protein contained a conserved serine/threonine-protein kinase domain (S-TKc) and a canonical cyclinbinding “PSTAIRE” motif. Multiple sequence alignment indicated that members of CDKA family from various plants exhibited a high degree of sequence identity ranging from 82% to 93%. A neighbor-joining phylogenetic tree showed that Denca;CDKA;1 was clustered into the plant group and was distant from the animal and fungal groups. The modeled three-dimensional structure of Denca;CDKA;1 exhibited the similar functional structure of a fold consisting of β-sheets and α-helices joined by discontinuous random coils forming two relatively independent lobes. Quantitative real-time PCR analysis revealed that Denca;CDKA;1 transcripts were the most abundant in protocorm-like bodies with 4.76 fold, followed by that in roots (4.19 fold), seeds (2.57 fold), and stems (1.57 fold). This study characterized the novel Denca;CDKA;1 gene from D. candidum for the first time and the results will be useful for further functional determination of the gene.

scholarly journals Functional Characterization of IS1999, an IS4 Family Element Involved in Mobilization and Expression of β-Lactam Resistance Genes

2006 ◽  
Vol 188 (18) ◽  
pp. 6506-6514 ◽  
Author(s):  
Daniel Aubert ◽  
Thierry Naas ◽  
Claire Héritier ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Resistance Genes ◽  
Consensus Sequence ◽  
Functional Characterization ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Transposition Frequency ◽  
Transposase Gene ◽  
Reverse Transcription Pcr ◽  
Mutant Derivative ◽  
Rapid Amplification

ABSTRACT IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla VEB-1 and bla OXA-48. 5′ Rapid amplification of cDNA ends experiments revealed that expression of these β-lactamase genes was driven by the outward-directed promoter, Pout, located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

scholarly journals The genomic organization of the region containing the Drosophila melanogaster rpL7a (Surf-3) gene differs from those of the mammalian and avian Surfeit loci.

1995 ◽  
Vol 15 (5) ◽  
pp. 2367-2373 ◽  
Author(s):  
N Armes ◽  
M Fried
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Cluster ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Gene Organization ◽  
Genomic Region ◽  
Divergent Evolution ◽  
Close Proximity ◽  
Rapid Amplification ◽  
Rna Blots

The Surf-3 gene of the unusually tight mouse Surfeit locus gene cluster has been identified as the highly conserved ribosomal protein gene L7a (rpL7a). The topography and juxtaposition of the Surfeit locus genes are conserved for the 600 million years of divergent evolution between mammals and birds. This suggests cis interaction and/or coregulation of the genes and suggests that, within this locus, gene organization plays an important role in gene expression. The further evolutionary conservation of the organization of the Surfeit locus was investigated. A cDNA encoding the Drosophila melanogaster homolog of the Surf-3/rpL7a gene was cloned, was shown to be present as a single copy, and was expressed constitutively at high levels throughout development. Genomic cosmid clones encompassing the gene and its surrounding DNA were isolated. The gene was determined to have five introns, of which two were located in the 5' untranslated region of the gene. The remaining three introns had splice sites at positions equivalent to those found in the Surf-3/rpL7a mammalian homologs. S1 analysis and 5' rapid amplification of cDNA ends both confirmed the start of transcription to occur in a polypyrimidine tract in the absence of a TATA box in the promoter. The genomic region around the Surf-3/rpL7a gene was analyzed by low-stringency hybridization with murine Surfeit gene probes, by partial sequence analysis, and by hybridization of fragments to Northern (RNA) blots. No homologs of other members of the Surfeit gene cluster were detected in close proximity to the D. melanogaster Surf-3/rpL7a gene. However, a gene which was detected directly 3' to the Surf-3/rpL7a gene was shown to encode a homolog of a mammalian serine-pyruvate aminotransferase.

scholarly journals The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 923-935 ◽  
Author(s):  
Nicole Hansmeier ◽  
Andreas Albersmeier ◽  
Andreas Tauch ◽  
Thomas Damberg ◽  
Robert Ros ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Direct Repeat ◽  
Peptide Mass Fingerprinting ◽  
Gene Encoding ◽  
Force Microscopy ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Rapid Amplification

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

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