Biomedical subjects

M Sip

Publications and source records attributed to M Sip.

6 recordsLinked to original sources

PubMed2005

Oligonucleotide-based microarray: a new improvement in microarray detection of plant viruses.

Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY(NTN) and PVY(O) strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.

Oligonucleotide Array Sequence Analysis↗

PubMed2003

Mitochondrial DNA D-loop hypervariable regions: Czech population data.

In order to identify polymorphic sites and to find out their frequencies and the frequency of haplotypes, the complete D-loop of mitochondrial DNA (mtDNA) from 93 unrelated Czech Caucasians was sequenced. Sequence comparison showed that 85 haplotypes were found and of these 78 were unique, 6 were observed twice and 1 was observed three times. Genetic diversity (GD) was estimated at 0.999 and the probability of two randomly selected sequences matching (random match probability, RMP) at 1.2%. Additionally these calculations were carried out for hypervariable regions 1, 2 (HV1, HV2), for the area between HV1 and HV2 and for the area of the hypervariable region HV3. The average number of nucleotide differences (ANND) was established to be 10.2 for the complete D-loop. The majority of sequence variations were substitutions, particularly transitions. Deletions were found only in the region where HV3 is situated and insertions in the same place and in poly-C tracts between positions 303 and 315 in HV2. A high degree of length heteroplasmy was found especially in the regions of poly-C tracts between positions 16184 and 16193 in HV1 and between positions 303 and 315 in HV2. Position heteroplasmies were found in two cases.

Complementarity Determining Regions↗

PubMed1993

Instability of the monofunctional adducts in cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl](2+)-modified DNA: rates of cross-linking reactions in cis-platinum-modified DNA.

Single- and double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adduct have been studied at two NaCl concentrations. In 50 mM and 1 M NaCl, the adducts within the single-stranded oligonucleotides are stable. In contrast, they are unstable within the corresponding double-stranded oligonucleotides. In 50 mM NaCl, the bonds between platinum and guanine or N-methyl-2,7-diazapyrenium residues are cleaved and subsequently, intra- or interstrand cross-links are formed as in the reaction between DNA and cis-DDP. In 1 M NaCl, the main reaction is the replacement of N-methyl-2,7-diazapyrenium residues by chloride which generates double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)Cl]+ adduct. The rates of closure of these monofunctional adducts to bifunctional cross-links have been studied in 60 mM NaClO4. Within d(TG.CT/AGCA), d(CG.CT/AGCG) and d(AG.CT/AGCT) (the symbol.indicates the location of the adducts in the central sequences of oligonucleotides), the half-lifes (t1/2) of the cis-[Pt(NH3)2(dG)Cl]+ adducts are respectively 12, 6 and 2.8 hr and the cross-linking reactions occur between guanine residues on the opposite strands. Within d(AG.TC/GACT), d(CG.AT/ATCG) and d(TGTG./CACA) or d(TG.TG/CACA) t1/2 are respectively 1.6, 8 and larger than 20 hr and the intrastrand cross-links are formed at the d(AG), d(GA) and d(GTG) sites, respectively. The conclusion is that the rates of conversion of cis-platinum-DNA monofunctional adducts to minor bifunctional cross-links are dependent on base sequence. The potential use of the instability of cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adducts is discussed in the context of the antisense strategy.

Base Sequence↗

PubMed1992

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).

Base Sequence↗

PubMed1990

Formation of a DNA monofunctional cis-platinum adduct cross-linking the intercalating drug N-methyl-2,7-diazapyrenium.

Our purpose was to better understand the mutual influence of cis-diamminedichloroplatinum (II) (cis-DDP) and intercalating drugs in their interactions with DNA. The present study deals with the intercalating drug N-methyl-2,7-diazapyrenium (MDAP). Two sets of experiments have been performed. In one set, the reaction between cis-DDP and nucleic acid was carried out in the presence of MDAP. The main adduct is a guanine residue chelated by platinum to a MDAP residue. It has the same spectroscopic properties as the synthesized compound cis-[Pt (NH3)2 (N7-d-guanosine) (N7-MDAP)] , the structure of which has been determined by 1H NMR. This adduct was only formed with double-stranded nucleic acids which reveals the importance of DNA matrix in orienting favorably the reactants. In the second set of experiments, the triamine complex cis-[Pt(NH3)2 (MDAP)CI]++ was reacted with the nucleic acids. At molar ratios drug over nucleotide residue equal or less than 0.10, all the added triamine complexes bind by covalent coordination to double-stranded nucleic acids. With natural DNA, the major adduct is cis-[Pt(NH3)2(d-guanosine) (MDAP)] . Thus the same adduct is formed on one hand in the reaction between DNA, MDAP and cis-DDP and on the other hand in the reaction between the triamine complex and DNA. The triamine complex offers the possibility to study the biological role of the new adduct.

Cisplatin↗

PubMedDate not supplied

Quantitative and sequence-specific analysis of DNA-ligand interaction by means of fluorescent intercalator probes.

A novel method of analysis of double-stranded DNA-ligand interaction is presented. The interaction is monitored by the fluorescence of a DNA bis-intercalator oxazole homodimer YoYo-3. The fluorescence intensity or its decay time reflects the modification of the DNA double helix. The DNA sequence is scanned by hybridization with short oligomers having consecutively overlapping complementary sequences to analyse the sequence specificity of binding. In our experiments we used as ligands the minor groove binders netropsin, SN6999 (both with AT-preference), the GC-specific ligand chromomycin A3 as well as the derivative SN6113 (non-specific interaction), which displace the bis-intercalator YoYo-3 or influence the duplex structure in such away that the fluorescence intensity and lifetime decrease in comparison to a ligand-free screening. The changes of fluorescence emission clearly define the binding motif and indicate minor groove interactions with a reduced DNA binding site. Titration of the ligand quantitatively characterizes its binding by determining the dependence of the binding constant on the oligonucleotide sequence.

Binding Sites↗