Biomedical subjects
Publications and source records attributed to W T Markiewicz.
MOTIVATION: A formulation of a new problem of the restriction map construction based on a simplified digestion experiment and a development of an algorithm for solving both ideal and noisy data cases of the introduced problem. RESULTS: A simplified partial digest problem and a branch and cut algorithm for finding the solution of the problem.
The application of synthetic oligonucleotide combinatorial libraries is described in the studies of triplex DNA. A new method of selection of dispersed (beaded) oligonucleotide combinatorial libraries based on the use of streptavidin magnetic beads is presented. A combinatorial chemistry approach is also proposed for studies of polyaminooligonucleotides.
The problem addressed in this paper is concerned with DNA sequencing by hybridization. An algorithm is proposed that solves a computational phase of this approach in the presence of both positive and negative errors resulting from the hybridization experiment. No a priori knowledge of the nature and source of these errors is required. An extensive set of computational experiments showed that the algorithm behaves surprisingly well if only positive errors appear. The general case, where positive and negative errors occur, can be also solved satisfactorily for an error rate up to 10%.
The aim of the study was to determine the frequency of additional prothrombotic defect in family members of 14 symptomatic, heterozygous carriers of factor V Leiden mutation (FV Leiden). The FV Leiden was found in fifty-five from among 127 persons (43%). Thirty-two from 53 (68%) family carriers of FV Leiden had venous thromboembolic disease at the time of examination. Combined defects were found in 6 out of 14 families (in 4 families coexistence of FV Leiden mutation with protein S deficiency, in 1 with G20210 A mutation of prothrombin gene and in 1 with antithrombin III deficiency). Until now 63% (7/11) of combined defect carriers (mean age 48.5 years) and 59% (24/42) of heterozygous FV Leiden carriers (mean age 52.2 years) have been symptomatic. The thrombosis-free survival (Kaplan-Maier analysis) was comparable in combined and in single defect carriers groups.
A new approach to the chemical synthesis of oligodeoxynucleotides bearing reporter functional groups at base residues of 3'-end nucleosides is reported. Applications of the 3'-end fluorescently labelled primers for automated DNA sequencing are shown.
The recently described point mutation of nucleotide 1691 of the factor V gene is responsible for the factor V resistance to a cleavage with an activated protein C (APC-resistance). Despite the high sensitivity and specificity of the APC-resistance test as a screening method for the detection of the factor V APC-resistance, there is still a necessity to develop a simple, cheap and accurate DNA-based assay. Herein, two different ASO-PCR methods were used for the detection of the Leiden mutation. The first involved a direct ASO-PCR with a consensus FV N1 primer and a sequence specific primer for the 1691 bp "G" normal allele (FV G1) or a specific primer for the 1691 bp "A" mutant allele (FV A1). The second method consisted of direct ASO-PCR by using the consensus FV N1 primer and one of two primers with an additional T-->G mismatch at the penultimate position from the 3'-end. One primer was specific for the 1691 bp "G" normal allele (FV G2) and the other was specific for the 1691 bp "A" mutant allele (FV A2). These permit clear and easy distinctions between homozygous normal and heterozygous and homozygous mutant probands. We also tested a T-->A mismatched primer to compare our method with that recently reported by Bellisimo et al. We found that within a large range of PCR conditions, T-->G mismatched oligonucleotides discriminated better than T-->A mismatched between three factor V 1691 position genotypes. We therefore recommend our method for the screening of a single 1691G-->A nucleotide mutation in the factor V gene.
MOTIVATION: Reconstruction of the original DNA sequence in the sequencing by the hybridization approach (SBH) requires computational support due to a large number of possible combinations. One can notice a lack of algorithms admitting false-negative data and giving in addition all possible solutions. RESULTS: In this paper, a new method of sequencing has been proposed. An algorithm based on its idea (for the general case, when some data are missing, like in the real experiment) has been implemented and tested. Authentic DNA sequences have been used for testing. A parallel version of the algorithm has also been implemented and tested. The quality of the reconstruction is satisfactory for the library of oligonucleotides of length between 8 and 12, and 100, 200 and 300 bp long sequences. A way to a further decrease in the computation time is also suggested.
The presence of point mutation G-->A of nucleotide 1691 of Factor V gene (Leiden mutation) is responsible for the resistance of factor Va to activated protein C (APC-resistance) and is associated with an increased risk for thrombosis. Herein, we reported on a case of 20 year male with a two years history of recurrent, extensive deep vein thrombosis. His family history showed grand-mother from mother side, who died from thromboembolic disease many years ago. His laboratory investigation reveals abnormal results of APC-resistance test (R = 1.80) and normalized APC-resistance test sensitivity ratio (0.57). Moreover, on the basis of a sequence specific primer polymerase chain reaction (SSP-PCR) a heterozygous from (G/A at 1691 position) of Leiden mutation was found. Family study showed two between 8 others asymptomatic persons with abnormal results of APC-resistance test and heterozygous genotype.
The aim of the study was to evaluate the thrombosis free survival in unrelated 24 heterozygous carriers for Leiden mutation and in unrelated 26 patients with "idiopathic" thromboembolic disease. The subjects with coexisting antithrombin III, protein C, S deficiency, obesity, antiphospholipid antibodies, cancer or taking oral contraceptives were excluded from analysis. The clinical manifestations of thrombosis were comparable in both investigated group, similarly the mean age of the first episode occurrence (37.2 vs 41.5 years). The probability for development of thrombosis in Kaplan-Meier analysis was not different between two groups. Moreover, during six months of observation period thrombosis reoccurrence rate was similar (1/24 vs 1/26).
A series of E. coli promoters made of the consensus -35 and -10 hexamers separated by 17 bp spacer with variously located bending dTn.dAn, n = 5 or 6, sequences was constructed and cloned into the plasmid pDS3. Electrophoretic gel mobilities of restriction fragments containing these promoters correlated with the number of the T tracts encoded in the promoter sequences. The open complexes formed by E. coli RNA polymerase on promoters containing the T5(-34...-38) tract exhibited gel retardation indicative of their different gross geometry. The strength of these promoters measured in vivo in relation to an internal transcriptional standard was shown to be significantly lower than that of the group without the T5(-34...-38) tract. Within both these groups the promoters with two T6 tracts in the spacer, aligned in phase with the B-DNA helix repeat, had lower transcriptional activity, while the T6 tract encoded in the -7...-2 promoter region apparently had no influence on the strength of the respective promoters.
We have demonstrated that synthetic oligonucleotide representing glucocorticoid responsive element (GRE I) of MMTV inserted into the enhancerless early promoter of SV40 in p delta SVE-CAT expression vector, enhances transient expression of chloramphenicol acetyltransferase gene in HeLa and hepatoma cells cultivated in the presence of dexamethasone. The following changes in the structure of the core sequences (GTTACAAACTGTTCT) of the synthesized GRE eliminated its enhancing ability: i, changes in the left end of the core sequences from GTTACAAAATGTTCT to TCTTCAAACTGTTCT or to TACTCAAACTGTTCT; ii, the increase of gap between TGTTCT and the inverted repeat of this sequence. The above changes did not eliminate specific binding of glucocorticoid receptor to the synthetic oligonucleotides studied.
The preparation of two types of supports based on controlled pore glass (CPG) is presented. These supports are compatible with established phosphoroamidite chemistry of oligonucleotides synthesis giving rise to an oligonucleotide with terminal 3'-phosphate function during final deprotection. CPG was modified with: (i) methacrylic acid derivatives and 2-mercaptoethanol (1) or (ii) aminoalkylsilane, succinic anhydride and benzidine (2). Support 2 can be also used for the synthesis of partially protected oligonucleotide 3'-phosphates.
Three E.coli promoters with the consensus sequences in the -35 and -10 regions and the 17 bp spacer made of random, heteronomous, and of both these classes of AT DNA simultaneously were constructed and cloned into plasmid pDS3. Electrophoretic gel mobilities of restriction fragments containing these promoters indicated that bending of the latter was proportional to the number of heteronomous AT DNA tracts. The strength of these promoters in vivo measured in relation to an internal transcriptional standard was shown to correlate well with gel mobilities of the respective restriction fragments and to decrease with the number of potential DNA bending sites encoded in the promoter structure.
Tetra-t-butoxydisiloxane-1,3-diyl (TBDSi) group is introduced into nucleoside chemistry as an analogue of tetraisopropyldisiloxane-1,3-diyl (TIPDSi) and an example of a new type of bifunctional silyl protective group. Its introduction and properties of derivatives of ribonucleosides are presented.
The influence of 2'-O-substituents (i.e. methyl, tetrahydropyranyl, t-butyldimethylsilyl) on the chemical synthesis of oligoribonucleotides by the phosphoroamidite approach on solid support is studied and compared with 2'-deoxynucleosides. Strong dependence on the steric hindrance of 2'-substituents both for the 3'-phosphitylation and condensation steps is described.
Recent results concerning the synthesis of oligoribonucleotides via the phosphotriester method, such as functionalization of ribonucleosides, new phosphorylating agents, 5'-O-sulfonylation and chromatography on Sephadex LH-20 for monitoring the removal of internucleotide phosphotriester groups, are presented. To show that efficiency of a new approach to the synthesis of oligoribonucleotides the pentamer /Up/4U was obtained.
In this work, the first example of chemical synthesis of oligoribonucleotide containing the hypermodified nucleoside N6-/N-threonylcarbonyl/-adenosine /t6A/ is presented. Synthesis of the heptamer C-C-C-A-U-t6A-A IX, the sequence of which is related to the anticodon loop of the initiator tRNA from yellow lupine, was achieved by: /i/ phosphotriester block synthesis of suitably protected heptamer VI containing an adenosine unit with a free exo-NH2 group, /ii/ highly effective "one-flask" procedure for the transformation of the free exo-NH2 group of adenosine unit of heptamer VI into a N,N'-disubstituted urea system of t6A of heptamer VII /hypermodification/, and /iii/ final deprotection of VIII /32% total yield/ with the use of a new approach for simultaneous hydrogenolysis /PdO-hydrogen-pyridine/ of the p-nitrobenzyl group and 2,2,2-trichloroethyl groups from carboxyl function of t6A and internucleotide phosphates respectively.
Zinc/acetylacetone/pyridine treatment has been designed as a very efficient method for removal of 2,2,2,-trichloroethyl group from phosphoesters. Internucleotide and terminal 2,2,2-trichloroethylphosphotriesters were transformed to corresponding diesters quantitatively. Much less reactive 2,2,2-trichloroethylphosphodiesters produced monoesters with ca. 90% yield. 31P NMR spectroscopy has been proposed as a new tool for analysis of removal of internucleotide phosphate protecting groups-a crucial step in oligonucleotides synthesis via phosphotriester approach.