Search results for "Cloning"

T-cell cloning in human type I diabetes.

1992

Multiplex Ligation-dependent Probe Amplification (MLPA)

2011

The Multiplex Ligation-dependent Probe Amplification (MLPA) is a PCR-based method. The procedure relies on sequence-specific probe hybridization of genomic DNA, followed by multiplex-PCR amplification of the hybridized probe and a semiquantitative analysis of the resulting PCR products. MLPA allows the analysis of around 40 loci in the same reaction, and is a sensitive and relatively fast technique. Only a small amount of DNA is required and results are available within 2 days.The critical factors when performing MLPA analyses from formalin-fixed paraffin-embedded (FFPE) tissues are DNA integrity and purity; for this reason, a suitable DNA extraction method must be chosen.The MLPA protocol …

Gene cloning and characterization of an alkane hydroxylase system in the long chain n-alkane- degrader Gordonia sp. SoCg

2008

Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin s…

1994

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol…

Maturation of Epidermal Langerhans Cells In Vitro Is Accompanied by Downregulation of 4F2 (CD98) as Determined by Differential Display

1998

Following short-term culture, Langerhans cells mature morphologically and functionally into potent immunostimulatory cells. As regulation of gene expression accompanies this maturation process, it is likely that differentially expressed genes are involved in the maturation events. Using the recently described method of differential display, we generated cDNA expression patterns starting with mRNA of murine epidermal Langerhans cells isolated either directly (fLC) or following 3 d cultivation (cLC). Five hundred putative differentially expressed cDNA fragments were recovered from the gel. For a part of the fragments differential expression was confirmed by dot blot and Southern hybridization…

Double copies of blaKPC-3::Tn4401a on an IncX3 plasmid in Klebsiella pneumoniae successful clone ST512 from Italy

2015

ABSTRACT A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of bla KPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the bla KPC-3 gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of bla KPC-3 ::Tn 4401a caused by intramolecular transposition events were detected in the plasmid.

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery

2004

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a ver…

Genomic structure and promoter analysis of pathogen-inducedrepatgenes fromSpodoptera exigua

2009

The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5'-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions a…