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PubMed2005

Exploring blocker binding to a homology model of the open hERG K+ channel using docking and molecular dynamics methods.

Binding of blockers to the human voltage-gated hERG potassium channel is studied using a combination of homology modelling, automated docking calculations and molecular dynamics simulations, where binding affinities are evaluated using the linear interaction energy method. A homology model was constructed based on the available crystal structure of the bacterial KvAP channel and the affinities of a series of sertindole analogues predicted using this model. The calculations reproduce the relative binding affinities of these compounds very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. These results are consistent with recent alanine scanning mutation experiments on the blocking of the hERG channel by other compounds.

Amino Acid Sequence↗

PubMed2005

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2.

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

Alternative Splicing↗

PubMed2006

Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability.

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.

Alanine↗

PubMed2004

Structure analysis of the protein transduction domain of human Period1 and its mutant analogs.

Human Period1 (hPer1) has been proved to be able to translocate into cells in a protein transduction manner. The segment of amino acids 830-845 of hPer1 is its protein transduction domain (PTD). In order to explore the membrane penetrating mechanism of hPer1-PTD and the physico-chemical properties necessary in the process, Ala scanning mutation method was used to investigate the variation in the peptide internalization. To further investigate the related physico-chemical requirements, the three dimensional structures of hPer1-PTD and its mutant analogs were simulated by Rosetta method. The electrostatic potentials and energies of these structures were calculated using the Delphi algorithm to solve Poisson-Boltzman equation. The hydrophobicity was assessed by the percentage of the nonpolar area in SAS (solvent accessible surface (SAS)). It has been proved that the Arg836 was the key residue for peptide internalization. When this Arg mutated into Ala, the peptide could not cross the membrane. The large enough area with positive charge was the decisive factor for hPer1-PTD. The alpha-helical structure seemed to play an assistant role so as to enable the positive charge connected in spatial arrangement.

Amino Acid Sequence↗

PubMed2004

An electrophoretic tool for the genetic characterisation and delineation of lungworms.

In the present study, PCR-based single-strand conformation polymorphism (SSCP) analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was applied to the genetic characterisation of Dictyocaulus from red deer from New Zealand and to its differentiation from species of lungworm from cattle and other hosts. Based on SSCP profiles, Dictyocaulus individuals from red deer from different geographical localities in New Zealand could be readily distinguished from those representing other lungworms examined, irrespective of low-level sequence variability in the ITS-2 (0.4-2.6%) detectable among individuals. The ITS-2 of Dictyocaulus from red deer differed in sequence by approximately 7-35% from congeners from other cervid hosts, demonstrating that this parasite is genetically distinct from other species of Dictyocaulus for which ITS-2 sequence data are presently available. The results emphasize the need for a large-scale molecular systematic study of Dictyocaulus specimens from various species of cervid and other ruminant hosts and the usefulness of mutation scanning for taxonomic, epidemiological and population genetic investigations.

Animals↗

PubMed2006

Candidate gene analysis of IP-10 gene in patients with Alzheimer's disease.

Interferon-gamma-inducible Protein-10 (IP-10) is supposed to play a role in Alzheimer's disease (AD) development, as demonstrated by increased levels in cerebrospinal fluid from patients with AD. A mutation scanning of IP-10 exonic region was carried out in 10 patients with AD and 10 age-matched controls, demonstrating the presence of two previously reported single nucleotide polymorphisms (SNPs) in exon 4 (G-->C and T-->C) as well as a novel SNP in exon 2 (C-->T). Exon 4 G-->C and T-->C allelic variants were next evaluated in a population of 279 AD patients and 251 controls, in order to determine whether their presence could influence the susceptibility towards the development of the disease. These two SNPs were in complete linkage disequilibrium. No differences in haplotype frequencies were found in AD patients as compared with controls, even stratifying according to the presence of Apolipoprotein E varepsilon4 allele, gender or age at onset. A new protocol was developed to easily determine the C-->T SNP in exon 2. A preliminary analysis revealed a very low frequency of this allelic variant (1%). Therefore, the complete association study was not carried out because the size of our population was not sufficient to draw reliable conclusions. According to these results, IP-10 does not seem to be a risk factor for AD. However, a novel rare polymorphism has been identified, which could exert a role in AD susceptibility. Thus, further studies on larger populations are needed before confidently excluding IP-10 as a susceptibility gene for AD.

Age of Onset↗

PubMed2003

Anaerobic regulation of Actinobacillus actinomycetemcomitans leukotoxin transcription is ArcA/FnrA-independent and requires a novel promoter element.

The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces a 116-kDa leukotoxin that appears to help the bacterium evade the innate host immune response. The expression of leukotoxin is induced when cells are grown anaerobically, a condition found in the subgingival crevice. This regulation most likely occurs at the transcriptional stage since the levels of leukotoxin RNA are induced by hypoxic growth. In order to map the leukotoxin promoter element(s) responsible for oxygen regulation, deletion and linker-scanning mutations were cloned into a transcriptional reporter gene plasmid and then tested in A. actinomycetemcomitans grown aerobically or anaerobically. A 35-bp DNA element, at position -36 to -70, was found to be responsible for the repression of leukotoxin synthesis in aerobically grown A. actinomycetemcomitans. The sequence of this oxygen response element (ORE) does not match the consensus binding sites for known DNA binding proteins, not even Fnr or ArcA which play major roles in oxygen regulation in other bacteria. However, since sequence analysis alone cannot disprove a role for the Fnr or ArcAB pathways in leukotoxin regulation, the genes for the Fnr and ArcA homologues in A. actinomycetemcomitans were identified, mutated by targeted insertional mutagenesis and assessed for loss of oxygen regulation. Deletion of either fnr or arcA altered the expression of numerous A. actinomycetemcomitans proteins, but leukotoxin expression was still repressed by oxygen. These results, coupled with the promoter mutation analyses, lead to the conclusion that A. actinomycetemcomitans employs a novel pathway in the aerobic/anaerobic regulation of leukotoxin synthesis.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

cis-Acting packaging motifs of porcine adenovirus type 3.

The cis-acting packaging domain is required for selective encapsidation of adenovirus DNA into preformed empty capsids late in the viral life cycle. Earlier, it was demonstrated that the cis-acting packaging domain of porcine adenovirus type (PAdV)-3 is located between nucleotide position (nt) 212 and 531 at the left end genome which contains six AT/GC rich motifs. Removal of packaging domain from left end to the right end of the genome produced a viable mutant virus suggesting that the identified cis-acting packaging domain represents the DNA sequences required for selective packaging of PAdV-3 DNA, whose position and orientation appear to be flexible. Here, by constructing and analyzing a panel of virus mutants carrying deletions or linker scanning mutations in AT/GC rich sequences, we examined the significance of the continuous A/T or G/C sequences individually in the viral packaging process. In contrast to consensus bipartite structure (5'-TTTGN8CG-3') described for most of packaging motifs of human adenovirus type 5 (HAdV-5), the packaging motifs I, II, III, and IV of PAdV-3 displayed a tripartite structure in which the continuous A/T nucleotides were flanked by G/C-rich sequences. Mutations in both continuous A/T nucleotides and its flanking GC-rich sequences reduced the packaging efficiency of mutants to varying degrees. In addition, although the continuous A/T sequences were present in all of the packaging motifs, their significance in the packaging process appears to vary within each packaging motif.

Adenoviruses, Human↗

PubMedDate not supplied

DHPLC in clinical molecular diagnostic services.

A high-capacity low-cost mutation scanning method based on denaturing high-performance liquid chromatography (DHPLC) has been recently introduced. We have implemented an automated and cost-effective strategy using DHPLC. To facilitate the semi-automated analysis of multiple exons, two steps were taken. The first step was the development of a PCR protocol for the amplification of multiple exons under the same conditions. Primer sets, which amplify each exon in the entire gene, were aliquoted to and air-dried on a 96-well format PCR plate. In this way, all the exons in a gene can be simultaneously amplified on a single PCR machine. The second step was the serial DHPLC analysis of multiple amplicons under conditions optimal for each amplicon. We named the 96-well plate containing the primer pairs and the corresponding computer file used to analyze each amplicon under the pre-determined optimal conditions as the "Condition-Oriented-PCR primer-Embedded-Reactor plate," or the COPPER plate. We have developed COPPER plate systems for more than 20 congenital disorders including classic congenital syndromes like Marfan syndrome (FBN1: 65 amplicons), CHARGE syndrome (CHD7: 39 amplicons), de Lange syndrome (NIPBL: 46 amplicons), Sotos syndrome (NSD1: 30 amplicons), and Rubinstein-Taybi syndrome (CREBBP: 41 amplicons). Using the COPPER plate system, we are functioning as a reference laboratory for the clinical molecular diagnosis of congenital malformation syndromes and are presently analyzing more than 200 samples annually from all over Japan.

Automation↗

PubMed1998

Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism.

Echinococcosis, a disease caused by infection with the larval stage of a tapeworm parasite of the genus Echinococcus, is of major socio-economic importance, and studying genetic variability within and between Echinococcus populations has important implications for disease control and epidemiology. Various DNA approaches have been used to study Echinococcus genetics, but most methods do not allow the accurate display or definition of mutational/allelic variation. To overcome this limitation, we established a mutation scanning approach. Single-strand conformation polymorphism (SSCP) of two different enzymatically amplified mitochondrial (mt) DNA regions was evaluated using seven different genotypes of Echinococcus (defined as G1, G4, G6, G8, O, V and M2). The NADH dehydrogenase 1 gene (ND1) or the cytochrome c oxidase subunit 1 (CO1) were amplified by polymerase chain reaction from parasite DNA, denatured and directly subjected to electrophoresis in a non-denaturing gel matrix. Each of the seven genotypes examined could be delineated from one another based on characteristic and reproducible banding patterns. The results demonstrate the usefulness of SSCP for the direct visual display of sequence variation in mtDNA of Echinococcus without the need for DNA sequencing or restriction analyses, and indicate its potential for studying allelic variability in a range of other genes.

Animals↗

PubMed2003

Human IL-21 and IL-4 bind to partially overlapping epitopes of common gamma-chain.

Interleukin 21 (IL-21) is a recently identified novel cytokine that plays an important role in the regulation of B, T, and NK cell functions. Its effects depend on binding to and signaling through an IL-21 receptor complex consisting of the IL-21 receptor (IL-21R) and the common gamma-chain (gamma(c)). In this study using biosensor technique, the ligand-binding properties of IL-21R and gamma(c), which are presently poorly understood on a molecular level, were analyzed employing recombinant ectodomains of IL-21R and gamma(c). The formation of a binary complex between IL-21 and immobilized IL-21R (K(D) 70pM), gamma(c) and immobilized IL-21 (K(D) 160 microM) and a ternary complex between gamma(c) and IL-21 saturated immobilized IL-21R (K(D) 160nM) could be analyzed. The gamma(c) residues involved in IL-21 binding were defined by alanine-scanning mutational analysis. The epitope comprises gamma(c) residues N44, Y103, N128, L161, E162, and L208. It is not identical but partially overlapping with the previously established gamma(c) epitope for IL-4 binding. These results open the way to understand the molecular recognition mechanism in the IL-21 receptor system and also the promiscuous binding properties of gamma(c).

Amino Acid Sequence↗

PubMed2000

Genetic variation in the B7-1 gene in patients with multiple sclerosis.

Co-stimulation of T cells by B7-1, a protein that is expressed on antigen presenting cells, favors a T helper type1 (Th1) cellular response. Th1/Th2 bias may influence disease susceptibility or course of MS. We screened the entire coding sequence as well as the 5'- and 3'-untranslated regions of the B7-1 gene using a mutation scanning technique, dideoxyfingerprinting, in DNA from 111 patients with MS from Olmsted County, Minnesota. We identified five genetic variants. None alter protein structure nor have apparent functional significance. Selected variants of sufficient frequency were tested for an association with course and severity of MS and one was tested for an association with susceptibility; none of the association tests were positive.

3' Untranslated Regions↗

PubMed1998

Microplate-array diagonal-gel electrophoresis (MADGE) and melt-MADGE: tools for molecular-genetic epidemiology.

Microplate-array diagonal-gel electrophoresis (MADGE) was invented for molecular-genetic epidemiological studies. It combines direct compatibility with microplates, convenient polyacrylamide-gel electrophoresis and economy of time and reagents at minimal capital cost, and enables one user to run up to several-thousand gel lanes per day for the direct assay of single-base variations. Melt-MADGE adds temporal-thermal-ramp apparatus to achieve similar throughput for de novo mutation scanning.

Electrophoresis↗

PubMed2001

Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene.

Multiple transcription start sites were identified in the human gonadotropin releasing hormone receptor (hGnRHR) gene. Recently, an upstream promoter residing at -1727/-1674, in vicinity of a CAP site at -1673, was characterized. In this report, we elucidated the underlying mechanisms for the regulation of this promoter. Functionally, this promoter was constitutively suppressed by a silencer element (-1673/-1351) situated immediately downstream to it. On the other hand, pituitary adenylate cyclase-activating polypeptide (PACAP), via the cAMP pathway, was found to be the extracellular cue to control the upstream promoter. Following PACAP-27, PACAP-38 (30 nM) and forskolin (25 microM) treatment, there were significant increases in the reporter gene activities. By deletion analysis, the region residing at -1727 to -1577, containing the distal promoter and 97 bp of the silencer was subsequently found to be responsible for PACAP/cAMP induction. To localize the PACAP-dependent cis-acting element(s) within the silencer, block replacement scanning mutation was performed and a hGnRHR gene PACAP-responsive element (GPRE) was identified at -1676/-1648. The actions of PACAPs and forskolin on the GPRE were further evidenced by gel mobility shift assays. There was an increase in protein binding onto this element only after peptide treatment. As GnRH receptor number on gonadotrope cell surface is a key factor in regulating gonadotropin release, the present study provides an insight into the interplay between PACAP and GnRH receptors on pituitary gonadotropes to control human reproductive functions.

Base Sequence↗

PubMed1999

PCR-based technology in veterinary parasitology.

DNA technology is having a major impact in many areas of veterinary parasitology. In particular, the polymerase chain reaction (PCR) has found broad applicability because its sensitivity permits enzymatic amplification of gene fragments from minute quantities of nucleic acids derived from limited amounts of parasite material. This paper discusses some recent applications of PCR-based methods to parasites and highlights their usefulness or potential for those of veterinary importance. The focus is on PCR tools for the accurate identification of parasites and their genetic characterisation, the diagnosis of infections, the isolation and characterisation of expressed genes, the detection of anthelmintic resistance, and mutation scanning approaches for the high resolution analysis of PCR products.

Animals↗

PubMed1997

Functional characterization of the 5'-flanking region of the gene encoding human 2-oxoglutarate dehydrogenase.

The 5'-flanking region of the gene (OGDH) encoding human 2-oxoglutarate dehydrogenase (OGDH) does not contain TATA or CAAT boxes, but contains an inverted GC box. To identify a functional OGDH promoter, systematic transient expression analysis of the 5'-flanking region (wild type, -3276/+212) of the OGDH was performed using serially nested deletions and linker-scanning mutations. The OGDH (wild type)-luciferase (LUC) reporter vector (pGV-E) construct and its deletion or linker-mutant constructs were transfected into BHK-21 cells and LUC activity was assessed. The OGDH-LUC construct expressed reproducibly >12-fold more LUC activity than did a control pGV-E vector. The promoter activity was up-regulated by treatment with 2-oxoglutarate and 2-oxoglutarate/glutamate. Deletions of sequences between nt -563 and -62 (M6 and M7) resulted in a 2-fold increase in LUC activity. Further deletion of the sequence between nt -61 and +212 (M8-M10) abolished LUC activity. High resolution mutagenesis within the -113 to -14 region indicated that the -53 to -44 and -33 to -24 sequences were required for positive regulation and the -93 to -84 sequence for negative regulation. We have identified a nuclear factor that binds to nt -63 to -24 including two cis-acting sites.

Animals↗

PubMed1999

The role of NF-1 factors in regulation of elastin gene transcription.

The -195- to -500-bp region of the human elastin promoter has been shown to convey high activity in neonatal rat aortic smooth muscle cell and pulmonary fibroblast cell cultures. In addition, this region has been implicated in controlling the differential basal level of elastin transcription in these two cell types. The overall goal of this study was to define the positive element(s) within the -195- to - 500-bp region and to identify the trans-acting factors binding to this sequence. A combination of deletion and linker scan mutational analyses localizes the positive element between -401 and -415 bp. Gel shift analyses demonstrate that the positive element binds NF-1 family members. Co-transfection of a CTF1 expression vector in Drosophila Schneider cells shows the ability of an NF-1 family member to activate elastin promoter activity through this site. Comparative Western and Southwestern blot analyses of nuclear extracts isolated from SMC and lung fibroblasts lay the foundation for possible differential regulation of elastin transcriptional levels via cell specific expression of different NF-1 family members.

Animals↗

PubMed1995

A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen.

BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗