Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prenatal diagnosis of cystic fibrosis (CF) was made in a Turkish family whose first born child was diagnosed at necropsy as having CF. Two consecutive pregnancies followed. The fetus of the second pregnancy was diagnosed as having CF by the microvillar enzyme assay and was aborted. The diagnosis was verified by the DNA polymerase chain reaction analysis using chorionic villi from the abortus. In the third pregnancy, amniocentesis was performed in the 17th week, and KM19 polymorphism linked to CF was used to assess the status of the fetus. Since the fetus was determined to be a carrier, the family was advised to continue with the pregnancy.
...
PMID:Prenatal diagnosis of cystic fibrosis in a Turkish family. 184 85

In genomic diagnosis the ensemble of techniques has been recently expanded by the powerful method of the polymerase chain reaction (PCR). Using pairs of synthetic oligonucleotides for priming of synthesis and a thermoresistant DNA polymerase a millionfold amplification of target DNA sequences from patients provides DNA fragments for following investigations such as electrophoresis. The paper presents some examples of PCR application for diagnosis in cystic fibrosis and Duchenne muscular dystrophy.
...
PMID:[The polymerase chain reaction PCR and its use in genetic diagnosis]. 192 Nov 62

Conditions for assessing KM-19 probe detected by Pst-1 restriction fragment length polymorphism (RFLP) by means of polymerase chain reaction were provided. Computer controlled mechanical arm with waterbaths and cloned heat-stable DNA polymerase was used. Results of KM-19 allelic frequencies on 90 cystic fibrosis chromosomes are presented. Allele two frequency was -0.833.
...
PMID:Cystic fibrosis marker testing in Bohemia with polymerase chain reaction. 213 Jun 70

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight fragments by altering its functional mechanism from the native single-stranded nicking pathway to a much more efficient one which results in increased double-stranded scission. By introducing positively charged amino acids at DNase I positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA, we have created a hyperactive variant with approximately 35-fold higher DNA-degrading activity relative to wild type. This enhancement can be attributed to both a decrease in Km and an increase in Vmax. Furthermore, unlike wild-type DNase I, the hyperactive variants are no longer inhibited by physiological saline. Replacement of the same positions with negatively charged amino acids greatly reduced DNA cleavage activity, consistent with a repulsive effect with the neighboring DNA phosphates. In addition, these variants displayed similar activities toward a small synthetic substrate, p-nitrophenyl phenylphosphonate, suggesting that the difference in DNA cleavage activity is due to the interaction of the engineered charged residues with the DNA phosphate backbone rather than any change in catalytic machinery. Finally, experiments involving the repair of DNase I digested DNA with T4 DNA ligase and the Klenow fragment of DNA polymerase I suggest that single-stranded gaps are introduced by the hyperactive variants. Thus, the increased functional activity of the hyperactive variants may be explained in part by a shift toward a processive DNA nicking mechanism, which leads to a higher frequency of double-stranded breaks.
...
PMID:Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism. 918 42

Background: Many genetic loci exhibit substantial heterogeneity: the human leukocyte antigen (HLA) DRB loci include 139 alleles and the cystic fibrosis transmembrane regulator gene more than 500 known mutations. Identification of alleles at these loci is cumbersome with typical molecular diagnostic methods such as hybridization assays or restriction enzyme analysis. Direct DNA sequencing of polymerase chain reaction (PCR) products is a general approach to complex loci that allows detection of any allele within the nucleotide sequence analyzed. However, direct DNA sequence-based unambiguous identification of heterozygous nucleotide positions using PCR templates is a challenging problem. Methods and Results: The ability of direct DNA sequencing methods to accurately identify HLA DRB alleles was assessed. The authors evaluated the performance of modified T7 and Taq DNA polymerases in isothermal and thermal cycle sequencing of PCR products derived from HLA DRB genes in 235 individuals who were potential donors or recipients of bone marrow transplants. The uniformity of peak intensity and ability to identify heterozygous nucleotide positions was similar when either AmpliTaq FS- or Sequenase DNA polymerase-derived electropherograms were prepared. The modified Taq DNA polymerase allowed the use of unpurified, double-stranded PCR templates. Furthermore, this enzyme could be used in less laborious, less costly cycle sequencing assays coupled with automated fluorescent detection methodology. Direct sequencing performed with either enzyme allowed unambiguous identification of DRB1 alleles, resolution of difficult heterozygous combinations, and recognition of new alleles. Conclusions: The direct DNA sequencing methods employed here for HLA allele identification are relatively efficient and semiautomated, and may be reasonably considered as a general approach to other complex molecular diagnostic problems, especially when coupled to simplified sequencing chemistries allowing cycle sequencing.
...
PMID:Strategies for Unambiguous Detection of Allelic Heterozygosity via Direct DNA Sequencing of PCR Products: Application to the HLA DRB1 Locus. 1033 Feb 4

Recombinant adeno-associated serotype 2-based vectors (rAAV2) possess a number of theoretical advantages for cystic fibrosis (CF) gene therapy because they elicit little or no inflammatory response and generally result in stable expression. rAAV2 vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have previously been shown to mediate stable correction of the CF defect in CF bronchial epithelial cells and stable expression of CFTR in rabbit and nonhuman primate models. Here we report the results of the first trial initiated with rAAV in humans, a phase I study in 25 adult and adolescent CF patients with mild to moderate lung disease. Doses of the rAAV-CFTR vector (tgAAVCF) ranging from 3 x 10(1) to 1 x 10(9) replication units (RU), which is equivalent to approximately 6 x 10(4) to 2 x 10(12) DNase resistant particles (DRP), were administered to one side of the nose and to the superior segment of the lower lobe of the right lung. Several adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of the bronchoscopic procedures. Only one of the serious events was judged to be possibly vector-related (based on temporal association), and this event was a pulmonary exacerbation very similar to several others experienced by the same subject in the three months preceding vector delivery. Vector shedding was minimal throughout the study, and serum-neutralizing antibodies were detected after vector delivery to subjects in the highest dosage cohorts. Gene transfer as measured by DNA polymerase chain reaction (PCR) was not observed until cohort 10 in nasal and bronchial epithelia. Sporadic low-level copy numbers suggested gene transfer of anywhere from 0.002 copies per cell up to 0.5 copies per cell was possible; however, DNA PCR was positive in lungs prior to direct dosing suggesting aspiration from the nasal dosing. These data indicate the need for continued evaluation of rAAV-CFTR vectors in additional clinical trials.
...
PMID:Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study. 1288 47

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.
...
PMID:Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. 1532 24

A study was conducted to assess health care worker exposure to tgAAVCF during the aerosolized administration of this experimental gene transfer agent in clinical trials for the treatment of cystic fibrosis (CF). tgAAVCF is a recombinant adeno-associated virus (AAV) genetically engineered to contain the human CF transmembrane conductance regulator cDNA. Study subjects included eight health care workers involved in the administration of tgAAVCF in a phase II study and 12 control health care workers who were involved with the treatment of CF patients, but not administration of the study drug. The exposure assessment entailed the determination of personal and area airborne tgAAVCF concentrations. In addition, serologic status of the health care workers was evaluated throughout the study for the presence of antibodies to AAV. A symptom survey was also completed by both the active and control health care workers. Air samples were analyzed by an infectivity assay (active vector) and a DNA polymerase chain reaction amplification procedure (vector DNA). Air monitoring was conducted during 13 tgAAVCF and seven placebo administrations. Active vector and vector particles were detected in four of 51 and 48 of 51 air samples collected during the administration of tgAAVCF, respectively. Based on the airborne vector particle concentration, the workers' exposure was estimated to be 0.0006% of the administered dose. At this level of exposure, the prevalence of symptoms was very low, the spectrum was similar in both study groups and did not result in any reported negative health effects.
...
PMID:Evaluation of exposure and health care worker response to nebulized administration of tgAAVCF to patients with cystic fibrosis. 1550 60

Pseudomonas aeruginosa is a human opportunistic pathogen that chronically infects the lungs of cystic fibrosis patients and is the leading cause of morbidity and mortality of people afflicted with this disease. A striking correlation between mutagenesis and the persistence of P. aeruginosa has been reported. In other well-studied organisms, error-prone replication by Y family DNA polymerases contributes significantly to mutagenesis. Based on an analysis of the PAO1 genome sequence, P. aeruginosa contains a single Y family DNA polymerase encoded by the dinB gene. As part of an effort to understand the mechanisms of mutagenesis in P. aeruginosa, we have cloned the dinB gene of P. aeruginosa and utilized a combination of genetic and biochemical approaches to characterize the activity and regulation of the P. aeruginosa DinB protein (DinB(Pa)). Our results indicate that DinB(Pa) is a distributive DNA polymerase that lacks intrinsic proofreading activity in vitro. Modest overexpression of DinB(Pa) from a plasmid conferred a mutator phenotype in both Escherichia coli and P. aeruginosa. An examination of this mutator phenotype indicated that DinB(Pa) has a propensity to promote C-->A transversions and -1 frameshift mutations within poly(dGMP) and poly(dAMP) runs. The characterization of lexA+ and DeltalexA::aacC1 P. aeruginosa strains, together with in vitro DNA binding assays utilizing cell extracts or purified P. aeruginosa LexA protein (LexA(Pa)), indicated that the transcription of the dinB gene is regulated as part of an SOS-like response. The deletion of the dinB(Pa) gene sensitized P. aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB(Pa) in translesion DNA synthesis over N2-dG adducts. Finally, P. aeruginosa exhibited a UV-inducible mutator phenotype that was independent of dinB(Pa) function and instead required polA and polC, which encode DNA polymerase I and the second DNA polymerase III enzyme, respectively. Possible roles of the P. aeruginosa dinB, polA, and polC gene products in mutagenesis are discussed.
...
PMID:Role of Pseudomonas aeruginosa dinB-encoded DNA polymerase IV in mutagenesis. 1704 Oct 45

Pseudomonas aeruginosa is a frequent cause of respiratory infections in cystic fibrosis (CF) patients. P. aeruginosa strains isolated from these patients have often a mucoid phenotype at advanced disease. This mucoid structure contains a dense amount of alginate type polysaccharide which facilitates bacterial attachment to lung epithelia and provides protection from the immune system due to biofilm formation. The aims of this study were to investigate the biofilm formation and the relation of this property with genotype and antibiotic susceptibilities of P. aeruginosa strains isolated from CF patients. The biofilm formation was determined by using the Congo Red agar and Christensen methods. RAPD-PCR (Random amplification of polymorphic DNA polymerase chain reaction) and disc diffusion methods were used for genotyping and antibiotic susceptibility testing, respectively. Biofilm production was found positive in 33.3% (20/60) of P. aeruginosa tested. While 9 of these 20 isolates were of mucoid colony morphotype, among the 40 biofilm negative isolates mucoid colony was detected in 16 of them. RAPD genotyping based on 70% similarity yielded 19 (A-S) clusters and subtypes related to five of these clusters (K1, K2, N1, N2, Q1, Q2, R1, R2, S1, S2) making up a total of 24 genotypes. Nine of these genotypes composed of biofilm positive isolates and 15 were biofilm negative ones. Most of the biofilm positive strains belonged to K1 (n = 5) and K2 (n = 6) genotypes while biofilm negative isolates were in the L (n = 8) and O (n = 7) genotypes. The comparison of antibiotic susceptibilities in both groups revealed no statistically significant difference (p > 0.0%). However, highest rate of resistance was detected for tobramycin and lowest rate for piperacillin/tazobactam. The data obtained from this study indicated that biofilm negative and positive P. aeruginosa isolates clustered in different groups. These results should be supported with larger scale multi-center studies which may provide information about P. aeruginosa dynamics in CF lungs.
...
PMID:[Investigation of biofilm formation and relationship with genotype and antibiotic susceptibility of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis]. 2008 9


1 2 Next >>

---