Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBDI.
J Mol Graph Model 1998 Apr
PMID:Topological model of membrane domain of the cystic fibrosis transmembrane conductance regulator. 987 57

An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, primers for single copy genes also generated paired nuclear spots in a primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed primer and template is recognized by DNA polymerase. Using normal and mutant primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS primers, it was shown that amplicon accumulates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to primer dimer formation, especially in the absence of true template DNA. The impact of primer dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.
Diagn Mol Pathol 1998 Oct
PMID:Adapting in situ polymerase chain reaction for genotyping of cells in suspension. 999 Apr 81

It has previously been shown that defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are largely responsible for the condition of congenital bilateral absence of the vas deferens (CBAVD), without associated renal abnormalities, in Caucasian populations. To assess the involvement of the CFTR in CBAVD in a population with presumed low cystic fibrosis (CF) frequency, we have analysed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T, known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one deltaF508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 43.7%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.
Mol Hum Reprod 1999 Jan
PMID:Molecular analysis of the cystic fibrosis gene reveals a high frequency of the intron 8 splice variant 5T in Egyptian males with congenital bilateral absence of the vas deferens. 1087 56

The ATP binding cassette (ABC) transporter protein Yor1p was identified on the basis of its ability to elevate oligomycin resistance when it was overproduced from a high-copy-number plasmid. Analysis of the predicted amino acid sequence of Yor1p indicated that this protein was a new member of a subfamily of ABC transporter proteins defined by the multidrug resistance protein (MRP). In this work, Yor1p is demonstrated to localize to the Saccharomyces cerevisiae plasma membrane by both indirect immunofluorescence and biochemical fractionation studies. Several mutations were generated in the amino-terminal nucleotide binding domain (NBD1) of Yor1p to test if the high degree of sequence conservation in this region of the protein was important for function. Deletion of a phenylalanine residue at Yor1p position 670 led to a mutant protein that appeared to be retained in the endoplasmic reticulum (ER) and that was unstable. As shown by others, deletion of the analogous residue from a second mammalian MRP family member, the cystic fibrosis transmembrane conductance regulator (CFTR), also led to retention of this normally plasma membrane-localized protein in the ER. Changes in the spacing between or the sequences flanking functional motifs of Yor1p NBD1 led to defective trafficking or decreased activity of the mutant proteins. Analyses of the degradation of wild-type and DeltaF670 Yor1p indicated that the half-life of DeltaF670 Yor1p was dramatically shortened. While the vacuole was the primary site for turnover of wild-type Yor1p, degradation of DeltaF670 Yor1p was found to be more complex with both proteasomal and vacuolar contributions.
Mol Cell Biol 1999 Apr
PMID:Mutational disruption of plasma membrane trafficking of Saccharomyces cerevisiae Yor1p, a homologue of mammalian multidrug resistance protein. 1008 67

Keratinocyte growth factor (KGF) is mitogenic for epithelial cells and induces cystic dilation of fetal lung explants through cystic fibrosis transmembrane conductance regulator-independent chloride channels. One candidate fetal lung chloride channel that is highly expressed on the apical surface of the respiratory epithelium and markedly downregulated after birth is CLC-2. We hypothesized that KGF regulates CLC-2 expression in the fetal lung. Primary fetal rat distal lung epithelial cell monolayers were grown in medium containing 10 ng/ml KGF for 48 h. CLC-2 protein was increased by Western blot analysis of whole-cell lysates in KGF-treated cultures. Similarly, KGF stimulated CLC-2 messenger RNA (mRNA) by Northern blot analysis. This enhanced expression was dose-dependent and maximal at 48 h with 10 ng/ml KGF. Promoter-reporter gene experiments demonstrated that KGF did not stimulate gene transcription. By inhibition of new mRNA synthesis with actinomycin D, evidence was obtained that KGF stabilizes CLC-2 mRNA. We speculate that KGF may positively influence pulmonary chloride and fluid secretion by a secondary pathway affecting CLC-2 degradation.
Am J Respir Cell Mol Biol 1999 Apr
PMID:Keratinocyte growth factor stimulates CLC-2 expression in primary fetal rat distal lung epithelial cells. 1010 Oct 18

Internalization of Pseudomonas aeruginosa by epithelial respiratory cell lines has been suggested to be dependent on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Because we have observed intracellular (IC) P. aeruginosa only in cells that do not express apical CFTR, we addressed the question of whether bacterial internalization by epithelial cells depends on the degree of cell differentiation and polarity. Internalization of piliated P. aeruginosa PAO-1 and PAK by human epithelial respiratory cells in primary culture and by the 16 human bronchial epithelial 14o- cell line cultured either on thick collagen gels or on thin collagen films was evaluated by the gentamicin exclusion assay. Cells cultured on thick gels were differentiated, polarized, and tight. They exhibited CFTR at their apical membranes, expressed beta1 integrins at their basal membranes, excluded lanthanum nitrate, and uniformly expressed ZO-1 protein. In contrast, in cells cultured on thin films, CFTR was present mainly in the cytoplasm, whereas beta1 integrins were detected at apical membranes. Most cells cultured on thin films did not exclude lanthanum nitrate and rarely expressed ZO-1 protein. Cells grown on thick and thin collagen substrates differed markedly in bacterial internalization: no IC bacteria could be detected in cells cultured on gels, whereas high IC bacterial concentrations were isolated from cells cultured on thin films. Treatment of cells cultured on thin films with ethylenediaminetetraacetic acid, to disrupt intercellular junctions further, significantly enhanced P. aeruginosa internalization. Our results suggest that P. aeruginosa internalization by epithelial respiratory cells does not depend on CFTR protein expression at the epithelial cell surface but rather on cell polarity and junctional complex integrity.
Am J Respir Cell Mol Biol 1999 May
PMID:Pseudomonas aeruginosa internalization by human epithelial respiratory cells depends on cell differentiation, polarity, and junctional complex integrity. 1022 58

The present study was undertaken to evaluate the frequency and nature of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in infertile patients undergoing intracytoplasmic sperm injection. A total of 90 patients were screened for a panel of 10 mutations in the CFTR gene frequently involved in congenital absence of the vas deferens (CAVD); the patients included 14 with azoospermia and CAVD, 39 patients with azoospermia without CAVD (n = 39) and 37 patients with severe oligozoospermia. The length of the polymorphic polypyrimidine tract (allele 5T, 7T and 9T) in the intron 8/exon 9 splice-acceptor site was also determined. In 10 out of 14 patients with CAVD, CFTR mutations were found; nine patients had one DeltaISOdiaDeltaF508 mutation and one patient had two CFTR mutations (N1303K/R117H). Allele 5T was present in eight of these patients. In six patients, 5T was the non-DeltaISOdiaDeltaF508 allele and in two patients there was no known CFTR mutation. None of the CFTR mutations were observed in patients with azoospermia without CAVD or with severe oligozoospermia and the frequency of allele 5T was 3.6% (three out of 78 alleles) and 1.35% (one out of 74 alleles) respectively. Our observation suggests that the CFTR gene is not involved in either spermatogenesis or in the pathology of the genital tract, except for CAVD.
Mol Hum Reprod 1999 Jun
PMID:Screening for cystic fibrosis transmembrane conductance regulator gene mutations in men included in an intracytoplasmic sperm injection programme. 1034 Oct 8

In this article we introduce a strategy of preannealing labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous delta F508 and heterozygous delta F508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, delta I507, delta F508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant alleles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with 32P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base stacking interactions between the "capture probe" and the auxiliary oligonucleotide. The presence of the delta F508 mutation was detected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mer probes was improved by increasing the temperature of hybridization from 15 to 25 degrees C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis: 1. A convenient means of introducing label into the target DNA molecule. 2. Disruption of interfering short-range secondary structure in the region of analysis. 3. Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target). 4. Tandem hybridization with the capture probe (providing contiguous stacking) as a means for achieving efficient mismatch discrimination at the terminal position of the capture probe (adjacent to the auxiliary oligonucleotide). By use of multiple auxiliary oligonucleotides, all of the above benefits can be derived simultaneously.
Mol Biotechnol 1999 Feb
PMID:Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides. 1036 78

Many cystic fibrosis transmembrane conductance regulator (CFTR) mutants are recognized as aberrant by the quality control apparatus at the endoplasmic reticulum (ER) and are targeted for degradation. The mechanism whereby nascent chains are distinguished as either competent or incompetent for ER export has not been elucidated. Here we show that export-incompetent chains display multiple arginine-framed tripeptide sequences like the one recently identified in ATP-sensitive K+ channels. Replacement of arginine residues at positions R29, R516, R555, and R766 with lysine residues to inactivate four of these motifs simultaneously causes delta F508 CFTR, present in approximately 90% of CF patients, to escape ER quality control and function at the cell surface. Interference with recognition of these signals may be helpful in the management of CF.
Mol Cell 1999 Jul
PMID:Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis. 1044 36

Background: More than 600 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described; however, at least 50% of the disease-associated mutations in the African-American population remain unknown. Reported here is a novel missense mutation, R1283S, in a 47-year-old African-American patient with mild cystic fibrosis. Methods and Results: The patient was screened for 27 common and less common CFTR mutations and 2 mutations were detected. Direct sequencing confirmed the presence of the DeltaF508 mutation and revealed the presence of a novel missense mutation, R1283S. Conclusions: R1283S appears to be a cystic fibrosis mutation associated with mild disease, and adds to the number of known mutations in African-Americans. R1283S can be confused with the more common mutation, W1282X, when polymerase chain reaction-restriction fragment length polymorphism analysis is used for detection.
Mol Diagn 1997 Sep
PMID:A Novel Missense Mutation, R1283S, of the Cystic Fibrosis Transmembrane Conductance Regulator Gene in a 47-Year-Old African-American Patient. 1046 11


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