Search results for "cDNA"
showing 10 items of 67 documents
Characterisation of a Cryptosporidium parvum-specific cDNA clone and detection of parasite DNA in mucosal scrapings of infected mice.
1998
A cDNA library was constructed using total RNA extracted from oocysts and sporozoites of the protozoan parasite Cryptosporidium parvum. The expression library was screened with an anti-C. parvum antiserum and a clone, Cp3.4, with a 2043 bp insert, was extracted. Southern blot analysis demonstrated a single copy gene that was located on a 1.6 Mb chromosome. The gene was found to be C. parvum specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan parasites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino acids. Overall, the polypeptide has a low isoelectric point (pI) of 3.…
Specific bovine antibody response against a new recombinant Cryptosporidium parvum antigen containing 4 zinc-finger motifs
2002
A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calv…
Peritoneal cavity phagocytes from the teleost sea bass express a glucocorticoid receptor (cloned and sequenced) involved in genomic modulation of the…
2005
Abstract To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL …
Molecular cloning of rat G-protein-coupled receptor kinase 6 (GRK6) from brain tissue, and its mRNA expression in different brain regions and periphe…
1997
The rat G-protein-coupled receptor kinase 6 (GRK6) cDNA was cloned from rat brain tissue by a combination of reverse-transcription polymerase chain reactions (RT-PCR), based on homology to the cloned human GRK6, and rapid amplification of cDNA ends (RACE-PCR). We obtained a clone of 2817 bp with an open reading frame of 1731 bp encoding for a protein of 576 amino acids that is 96.7% identical and 97.9% similar to its human counterpart. mRNA was detectable in all brain areas examined. In addition, GRK6 was expressed in skeletal muscle, small intestine, aorta, liver, heart, lung, thymus, stomach, uterus and kidney.
Cloning and expression of two novel aldo-keto reductases fromDigitalis purpurealeaves
2002
The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly the reduction of carbonyl groups or carbon–carbon double bonds of a wide variety of substrates including steroids. Such types of reactions have been proposed to occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were isolated from a D. purpurea cDNA library using the rat Δ4-3-ketosteroid 5β-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4% identity. DpAR proteins present high identities (68–80%) with four Arabidopsis clones and a 67% identity with the aldose/aldehyde reductase from Medi…
Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
2016
Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed s…
Agr system of Listeria monocytogenes EGD-e: role in adherence and differential expression pattern.
2007
ABSTRACT In this study, we investigated the agrBDCA operon in the pathogenic bacterium Listeria monocytogenes EGD-e. In-frame deletion of agrA and agrD resulted in an altered adherence and biofilm formation on abiotic surfaces, suggesting the involvement of the agr system of L. monocytogenes during the early stages of biofilm formation. Real-time PCR experiments indicated that the transcript levels of agrBDCA depended on the stage of biofilm development, since the levels were lower after the initial attachment period than during biofilm growth, whereas transcription during planktonic growth was not growth phase dependent. The mRNA quantification data also suggested that the agr system was a…
Identification of Metastasis Associated Antigen 1 (MTA1) by Serological Screening of Prostate Cancer cDNA Libraries
2008
Over the past 10 years the serological analysis of recombinant cDNA expression libraries (SEREX) has proved to be an effective method for the identification of tumour antigens. In the present study, two prostate cancer libraries were constructed and screened using autologous sera. Fifty five genes were isolated, including 46 known genes and 9 previously uncharacterised genes. Among the known genes, a metastasis-associated gene, MTA1, previously identified by differential cDNA hybridisation, was preferentially expressed in a panel of malignant tissues compared with normal tissues, as analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). MTA1 transcripts were observed to be ov…
Sequence of a novel cytochrome CYP2B cDNA coding for a protein which is expressed in a sebaceous gland, but not in the liver
1992
The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 R…
Different La/SS-B mRNA isoforms are expressed in salivary gland tissue of patients with primary Sjogren's syndrome
1996
Recently we isolated a La/SS-B mRNA isoform from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sjögren's Syndrome. In the La/SS-B mRNA isoform the exon 1 was replaced. The alternative exon was termed exon 1'. Genomic analysis showed that the exon 1' La mRNA was the result of a promoter-switch in combination with alternative splicing. Due to the unusual structure of the exon 1' La/SS-B mRNA, the function and the behaviour under physiological and pathophysiological conditions in tissue of patients with primary Sjögren's syndrome or Systemic Lupus Erythematosus remained obscure. Therefore assays were established allowing a qualitative and quantitative estimati…