Screening for mutations in the antithrombin III gene causing recurrent venous thrombosis by single-strand conformation polymorphism analysis.
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Antithrombin III↗
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Results for “Conformational analysis”
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Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.
The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.
DNA Mutational Analysis↗
Gene mapping in zebrafish using single-strand conformation polymorphism analysis.
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Animals↗
Technical note: use of PCR-single-strand conformation polymorphism analysis for detection of bovine beta-casein variants A1, A2, A3, and B.
We have optimized the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique to screen the most frequent variants (A1, A2, A3, and B) of the bovine beta-casein gene. Five partly overlapping PCR products (233, 234, 265, 466, and 498 bp) of Exon VII of the beta-casein gene that encompass the target point mutations were heat-denatured, separated on nondenaturing polyacrylamide gels, and silver-stained. Simultaneous detection of all variants in reference samples of known genotypes (A1A2, A2A2, A1A3, A1B, and A2B) was best achieved on 17% polyacrylamide (100:1 acrylamide:bis-acrylamide ratio) gels with the PCR product of 234 bp. These results were confirmed by sequencing the allele-specific SSCP bands directly excised from polyacrylamide gels. A population of 65 anonymous samples belonging to various breeds was then analyzed twice, without discrepancies in a blind trial. Routine beta-casein genotyping using PCR-SSCP is proposed as a cost-effective, fast, and sensitive technique.
Animals↗
SSCP (single strand conformation polymorphism) analysis of PCR fragments.
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DNA, Single-Stranded↗
Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.
Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.
Animals↗
Molecular determinants of recognition for the intestinal peptide carrier.
Computer-aided conformational analysis was used to characterize the pharmacophore for the intestinal peptide carrier. The active analog approach to pharmacophore building was applied as implemented in the SYBYL software package. Conformational analysis and MOPAC calculations were used to determine the lowest energy conformation of carrier substrates, as well as the conformations of compounds that displayed a common pharmacophoric geometry (i.e., inhibitors and inactive structural analogs). A pharmacophore map was calculated, and based on structural mutualities and functional topology, three substrate groups were suggested: compounds that bind to the transporter and are transferred across the membrane; compounds that show affinity for the peptide carrier (i.e., known to inhibit transport of substrates) but are not transferred across the membrane; and compounds that contain the pharmacophoric geometry but show no affinity for the carrier. Affinity for the peptide transporter can be diminished or abolished in either of three ways: esterification of the free carboxylic acid moiety; introduction of a second negative group; and intramolecular steric hindrance of the free carboxylic acid by either side chains with a positively charged nitrogen function or groups capable of hydrogen bond formation.
Administration, Oral↗
Computational carbohydrate chemistry: what theoretical methods can tell us.
Computational methods have had a long history of application to carbohydrate systems and their development in this regard is discussed. The conformational analysis of carbohydrates differs in several ways from that of other biomolecules. Many glycans appear to exhibit numerous conformations coexisting in solution at room temperature and a conformational analysis of a carbohydrate must address both spatial and temporal properties. When solution nuclear magnetic resonance data are used for comparison, the simulation must give rise to ensemble-averaged properties. In contrast, when comparing to experimental data obtained from crystal structures a simulation of a crystal lattice, rather than of an isolated molecule, is appropriate. Molecular dynamics simulations are well suited for such condensed phase modeling. Interactions between carbohydrates and other biological macromolecules are also amenable to computational approaches. Having obtained a three-dimensional structure of the receptor protein, it is possible to model with accuracy the conformation of the carbohydrate in the complex. An example of the application of free energy perturbation simulations to the prediction of carbohydrate-protein binding energies is presented.
Carbohydrate Conformation↗
Advanced pairwise structure alignments of proteins and analysis of conformational changes.
MOTIVATION: Comparing the 3D structures of two proteins or analyzing the structural changes undergone by a protein upon ligand binding or when it crystallizes under different conditions, can be both tricky and tedious, especially when the two proteins are distantly related, or when the structural changes are complex. Readily accessible tools for performing these tasks automatically and reliably should therefore be welcome. RESULTS: We describe a web interface to several automatic procedures for performing pairwise structure superposition in a flexible manner, for detailed analyses of conformational changes and for displaying the results in a pictorial fashion. AVAILABILITY: This interface can be accessed at the Brussels and Cuba Web sites, respectively: http://www.ucmb.ulb.ac.be/SCMBB/Tools.htmland http://bio.cigb.edu.cu.
Algorithms↗
Conformational studies of human [15-2-aminohexanoic acid]little gastrin in sodium dodecyl sulfate micelles by 1H NMR.
Human little gastrin is a 17 amino acid peptide that adopts a random conformation in water and an ordered structure in sodium dodecyl sulfate (SDS) micelles as well as in trifluoroethanol (TFE). The circular dichroism spectra in these two media have the same shape, indicative of a similar preferred conformation [Mammi, S., Mammi, N. J., Foffani, M. T., Peggion, E., Moroder, L., & Wünsch, E. (1987) Biopolymers 26, S1-S10]. We describe here the assignment of the proton NMR resonances and the conformational analysis of [Ahx15]little gastrin in SDS micelles. Two-dimensional correlation techniques form the basis for the assignment. The conformational analysis utilized NOE's, NH to C alpha H coupling constants, and the temperature coefficients of the amide chemical shifts. The NMR data indicate a helical structure in the N-terminal portion of the peptide. These results are compared with the conformation that we recently proposed for a minigastrin analogue (fragment 5-17 of [Ahx15]little gastrin) in TFE.
Amino Acid Sequence↗
[Raman analysis of conformation changes of insulin solvent after being exposed to ELF pulsed electric field].
Raman spectra of insulin solvents are presented before and after being exposed to the pulsed electric field with extremely low frequency 50 Hz (ELF). The covalences of the molecule were not affected and the changes of some secondary bonds such as hydrogen bonds and salt bonds were observed. Detailed analysis of these spectra indicates that the alpha-helix of insulin molecule was destroyed after the exposure, which is proved by the shift of the peak of the amide I region toward higher wave number and by the appearance of several new peaks: 1561 and 1594 cm(-1). The disulfides were affected by the weaken alpha-helix, and their vibrational modes were changed. Meanwhile the hydrogen bonding between the dimer are broken down which leads to the increase in the peak intensities at 1002 and at 1602 cm(-1).
Electricity↗
Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface.
The interaction of phloretin with single lipid bilayers on a spherical support and with multilamellar vesicles was studied by differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR). The results indicated that phloretin interacts with the lipid layer and changes its structural parameters. In DSC experiments, phloretin in its neutral form strongly decreased the lipid phase transition temperature and slightly reduced the cooperativity of the phase transition within the lipid layer. In NMR measurements, phloretin led to an increase of the transverse relaxation time constant but had no effect on the spin-lattice relaxation time constant. The overall dipole moment of phloretin was experimentally determined and was found to be roughly 40% lower than has been published previously. This result suggested that the size of the dipole moment of phloretin does not provide such a high contribution to the effect of phloretin on the dipole potential of monolayers and bilayers as has been published previously. To understand the discrepancy between phloretin adsorption and dipole potential change, we performed computational conformational analysis of phloretin in the gas phase. The results showed that a wide distribution of the dipole moments of phloretin conformers exists, which mainly depends on the orientation of the OH moieties. The adsorption of phloretin as determined from its binding to solid supported bilayers differed from the one determined from dipole potential measurements on black lipid membranes. The difference between the phloretin dissociation constants of both types of experiments suggested a change of its dipole moment normal to the membrane surface in a concentration-dependent manner, which was in agreement with the results of the computational conformational analysis.
Adsorption↗
Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing.
A series of 152 human bladder tumors, 14 bladder tumor cell lines, and 1 immortal urothelial cell line were examined by single-strand conformation polymorphism (SSCP) and designed restriction fragment.length polymorphism analyses for mutations in exons 1 and 2 of the H-ras gene. Nine tumors (6%) contained mutations. There was complete concordance between SSCP and restriction fragment length polymorphism analyses. Six mutations in exon 1 and three in exon 2 were identified by SSCP analysis. Subsequent restriction fragment length polymorphism analysis showed that of the exon 1 mutations, four were in codon 12 and two in codon 13, and all three exon 2 mutations were in codon 61. Eight mutations were confirmed by direct sequencing. One codon 13 mutation could not be identified by direct sequencing. Distinct strand mobility shifts detected by SSCP analysis identified specific point mutations, and in all cases, strands containing different mutations migrated differently. The base substitutions identified in these bladder tumors were diverse and included four transversions (three G-->T and one A-->T) and four transitions (two G-->A and two A-->G). This pattern of base substitutions is compatible with interactions of the urothelium with more than one class of environmental agent during bladder tumor development. No correlation was found between tumor grade and/or stage and the presence of H-ras mutation. We conclude that H-ras mutation does not play a role in the development of the majority of transitional cell tumors of the bladder.
Base Sequence↗
The glial glutamate transporter complementary DNA in patients with amyotrophic lateral sclerosis.
Here, we report a mutation screening by single-stranded conformational analysis of the astroglial human brain glutamate transporter (HBGT) II complementary DNA in patients with amyotrophic lateral sclerosis. The conformational analysis data indicate a lack of sequence variations in the HBGT II coding region in 6 patients with amyotrophic lateral sclerosis and the same number of nonneurological control subjects. In both groups, three variants of the HBGT II 5' untranslated region were isolated. We have no evidence that the reported complementary DNA variants are disease specific.
ATP-Binding Cassette Transporters↗
An in vitro investigation into conformational aspects of gabapentin.
Conformational analysis of constrained cyclohexane systems was pioneered fifty years ago by Barton and Hassel. We now report an investigation based on a conformational analysis of a number of novel cyclohexane based Gabapentin analogues coupled with their in vitro evaluation at the Gabapentin binding site. These data are used to propose a possible binding conformation for Gabapentin.
Acetates↗
1H NMR conformational study of a variety of alpha-anomers of C5-substituted 2'-deoxyuridines: comparison to their antiherpetic beta counterparts.
Although alpha-nucleosides are not found in nucleic acid, they do occur as constituents of smaller molecules in living cells, e.g., in vitamin B(12). There are now several examples of alpha-nucleosides exerting a biological activity in some instances equal to, or even exceeding, that of the corresponding beta-anomer. Examples include growth inhibitory properties against mouse leukemia cells and antitumor activity. From stereochemical point of view, alpha-anomers serve as references for studying of interaction of the base with the sugar moiety in beta-anomers and may help in better understanding of structure-activity relationships. One important problem preventing conformational analysis of alpha nucleosides is uncertainty in the determination of vicinal coupling constants from simulation of overlapping sugar proton resonances of strongly coupled spin systems. A successful resolution of near-isochronous H3' and H4' resonances made possible a full conformational analysis for a series of alpha-anomers C5-substituted 2'-deoxyuridines, including methyl, ethyl, isopropyl, fluor, vinyl, and bromovinyl, in comparison to their beta counterparts. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). A thorough analysis of chemical shifts as well as conformation of the sugar ring and C4'-C5' rotamers made possible determination of conformational preferences in equilibrium about the glycosidic bond between two regions, anti and syn. This work provides insights into the role of anomeric configuration of the base in conformational behavior of the sugar moiety, a link in the backbone of nucleic acids.
Antiviral Agents↗
Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing.
Rifampin susceptibility of 32 rifampin-resistant and 26 rifampin-susceptible Mycobacterium tuberculosis strains was analyzed by PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing within the 157-bp region of the rpoB gene (Ala500 to Val550). Two false-positive PCR-SSCP results were observed among the susceptible strains due to the silent mutation Gln513 (CAA-->CAG) and the deletion mutation Thr508 and Ser509. Another silent mutation [Leu511 (CTG-->CTA)], combined with the mutation Ser531-->Leu, was observed in a resistant strain. These results suggest that to rule out false-positive PCR-SSCP results, sequencing of the target DNA is required.
DNA-Directed RNA Polymerases↗
Single-strand conformation polymorphism analysis on the delta-sarcoglycan gene in Japanese patients with hypertrophic cardiomyopathy.
To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the delta-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the delta-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C-->T substitution at nucleotide residue 84 (TAC-->TAT) with no amino acid alteration (Tyr-->Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in delta-SG may not be a common cause of HC in Japanese patients.
Adult↗