Biomedical subjects

T T Nikiforov

Publications and source records attributed to T T Nikiforov.

5 recordsLinked to original sources

PubMed1995

Determination of sequence specificity between a plasmid replication initiator protein and the origin of replication.

Staphylococcal plasmids of the pT181 family replicate by a rolling circle mechanism, requiring the activities of a plasmid-specified Rep protein. The initiation event involves site-specific phosphodiester bond cleavage by Rep within the replication origin, ori. In vitro the Rep proteins also display type-I topoisomerase activity specific for this plasmid family. Although the single site of bond cleavage, ICR II, is conserved among all members of the pT181 family, the plasmid-specific Rep proteins are able to discriminate between family members in vivo, initiating replication only from the cognate origin. The basis of such specificity is believed to be due to a non-covalent binding interaction between Rep and a DNA sequence adjacent to the site of phosphodiester bond cleavage. Using the RepD protein specified by plasmid pC221, we present data for the physical parameters of RepD:oriD complex formation. Quantification of the relative strengths of the non-covalent interactions for different but related ori target sequences, measured by gel mobility shift experiments, has yielded data that are in accord with the known specificity of the protein in vivo. Oligonucleotide competition experiments demonstrate that this interaction is indeed attributable to the specificity determinant, ICR III. Protein-DNA crosslinking methods show that a carboxyl-terminal proteolytic fragment of RepD makes a specific interaction with the ICR III region of its cognate replication origin. Analysis of topoisomerase rates indicates that the interaction between ICR III and the carboxyl terminus of the protein is required before a productive interaction, namely the phosphodiester bond cleavage at the ICR II, can occur.

Bacterial Proteins↗

PubMed1995

The use of 96-well polystyrene plates for DNA hybridization-based assays: an evaluation of different approaches to oligonucleotide immobilization.

A reliable, simple, and cost-effective method for the immobilization of relatively short (12-30 mer) oligonucleotide probes to 96-well polystyrene plates was required in our laboratory for use in DNA hybridization-based assays. We compared three different approaches to achieve this immobilization. Two of them are modifications of previously published procedures, requiring the use of modified oligonucleotides and/or modified plates. These were compared to a method developed in our laboratory, whereby passive immobilization occurs by incubation in the presence of salt or a cationic detergent. While all methods resulted in the productive binding of the DNA probes and could therefore be used for hybridization, only the passive immobilization approach met our strict performance criteria and was implemented for use in our DNA genotyping laboratory. It was found that the immobilization in the presence of cationic detergents takes place via a unique mechanism.

Base Sequence↗

PubMed1994

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Autoanalysis↗

PubMed1994

The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.

The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.

Base Sequence↗

PubMed1992

Oligodeoxynucleotides containing 4-thiothymidine and 6-thiodeoxyguanosine as affinity labels for the Eco RV restriction endonuclease and modification methylase.

4-Thiothymidine and 6-thiodeoxyguanosine were incorporated into synthetic dodecamers containing the recognition site d(GATATC) of the enzymes Eco RV endonuclease and Eco RV methyltransferase. Upon irradiation with long wavelength UV light (340-360 nm), these oligodeoxynucleotides were photochemically crosslinked to both enzymes. The yields were up to 35% with the methyltransferase, but lower (up to 6%) with the endonuclease. Oligodeoxynucleotides containing 4-thiothymidine generally gave higher yields of crosslinking than those containing 6-thiodeoxyguanosine. Although both specific (i.e. those containing the d(GATATC) sequence) and non-specific (lacking this sequence) photoreactive oligodeoxynucleotides gave rise to crosslinked products, the use of a non-reactive, competitive substrate oligodeoxynucleotide suppressed the crosslinking, indicating that the reaction takes place at the enzymes' active sites. Oligodeoxynucleotides containing 4-thiocyanatothymidine or 6-thiocyanatodeoxyguanosine were also prepared by treatment of the title oligomers with CNBr and KCN. The dodecamers containing 4-thiocyanatothymidine were found to covalently modify both enzymes under study, with levels of crosslinking reaching up to 42% with the endonuclease and up to 12% with the methyltransferase. No crosslinking was observed with oligodeoxynucleotides containing 6-thiocyanatodeoxyguanosine.

Affinity Labels↗