The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Peggy Hsieh - One of the best experts on this subject based on the ideXlab platform.
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oligomerization of a muts mismatch repair protein from thermus aquaticus
Journal of Biological Chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Peggy HsiehAbstract:Abstract The MutS DNA mismatch protein recognizes Heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to Heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair Heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
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Photocross-linking of the NH2-terminal region of Taq MutS protein to the major groove of a Heteroduplex DNA.
The Journal of biological chemistry, 1997Co-Authors: Indranil Biswas, R D Camerini-otero, Peggy HsiehAbstract:Next Section Abstract The MutS DNA mismatch repair protein recognizes Heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the Heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing Heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25–49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a Heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for Heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for Heteroduplex DNA binding by Taq MutS protein.
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Interaction of MutS Protein with the Major and Minor Grooves of a Heteroduplex DNA
The Journal of biological chemistry, 1997Co-Authors: Indranil Biswas, Peggy HsiehAbstract:Abstract Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to Heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of TaqMutS protein to a Heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24–28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish thatTaq MutS protein makes contacts with the major groove of the Heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1,10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the Heteroduplex DNA.
G A Ottersom - One of the best experts on this subject based on the ideXlab platform.
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RT-PCR Heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal model.
BioTechniques, 2002Co-Authors: W Duan, H Ding, W G Zhu, K Srinivasan, G A Otterson, M A Villalona-calero, G A OttersomAbstract:In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR Heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR Heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR Heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR Heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.
Indranil Biswas - One of the best experts on this subject based on the ideXlab platform.
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oligomerization of a muts mismatch repair protein from thermus aquaticus
Journal of Biological Chemistry, 1999Co-Authors: Indranil Biswas, Jeffrey W Lary, David A. Yphantis, Karen G. Fleming, Wei Yang, Peggy HsiehAbstract:Abstract The MutS DNA mismatch protein recognizes Heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to Heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair Heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.
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Photocross-linking of the NH2-terminal region of Taq MutS protein to the major groove of a Heteroduplex DNA.
The Journal of biological chemistry, 1997Co-Authors: Indranil Biswas, R D Camerini-otero, Peggy HsiehAbstract:Next Section Abstract The MutS DNA mismatch repair protein recognizes Heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the Heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing Heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25–49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a Heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for Heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for Heteroduplex DNA binding by Taq MutS protein.
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Interaction of MutS Protein with the Major and Minor Grooves of a Heteroduplex DNA
The Journal of biological chemistry, 1997Co-Authors: Indranil Biswas, Peggy HsiehAbstract:Abstract Thermus aquaticus MutS protein is a DNA mismatch repair protein that recognizes and binds to Heteroduplex DNAs containing mispaired or unpaired bases. Using enzymatic and chemical probe methods, we have examined the binding of TaqMutS protein to a Heteroduplex DNA having a single unpaired thymidine residue. DNase I footprinting identifies a symmetrical region of protection 24–28 nucleotides long centered on the unpaired base. Methylation protection and interference studies establish thatTaq MutS protein makes contacts with the major groove of the Heteroduplex in the immediate vicinity of the unpaired base. Hydroxyl radical and 1,10-phenanthroline-copper footprinting experiments indicate that MutS also interacts with the minor groove near the unpaired base. Together with the identification of key phosphate groups detected by ethylation interference, these data reveal critical contact points residing in the major and minor grooves of the Heteroduplex DNA.
Paola Carrera - One of the best experts on this subject based on the ideXlab platform.
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Seven Novel Additional Small Mutations and a New Alternative Splicing in the Human Dystrophin Gene Detected by Heteroduplex Analysis and Restricted RT-PCR Heteroduplex Analysis of Illegitimate Transcripts
European journal of human genetics : EJHG, 1996Co-Authors: Anna Maria Barbieri, Nadia Soriani, Alessandra Ferlini, Anna Michelato, Maurizio Ferrari, Paola CarreraAbstract:Around 35% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients cannot be identified by techniques which identify major DMD rearrangements in the dystrophin gene. In order to characterize the gene defect in these patients, we screened 40 exons of the dystrophin gene by Heteroduplex analysis on genomic DNA in 50 affected Italian males. Using conventional Heteroduplex analysis and a modified Heteroduplex analysis on restricted RT-PCR products of illegitimate transcripts, restricted RT-PCR Heteroduplex analysis, we were able to identify 7 novel small mutations and a new alternative splicing involving exon 25 of the dystrophin gene in peripheral blood lymphocytes and skeletal muscle transcripts.
Ayfer Günalp - One of the best experts on this subject based on the ideXlab platform.
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Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis Isolates by Heteroduplex Analysis and Determination of Rifamycin Cross-Resistance in Rifampin-Resistant Isolates
Journal of Clinical Microbiology, 2003Co-Authors: Zeynep Saribas, Tanil Kocagöz, Alpaslan Alp, Ayfer GünalpAbstract:Direct Heteroduplex analysis and a universal Heteroduplex generator assay were performed to detect rifampin resistance rapidly in Turkish Mycobacterium tuberculosis isolates. Cross-resistance to rifapentine, rifabutin, and rifalazil was investigated. A relationship between specific mutations and resistance patterns, which can guide the choice of an appropriate therapeutic regimen for tuberculosis patients, was identified.