ABSTRACT
For monitoring retroviral infection on the gene level, we propose the use of quantitative PCR with two internal standards: one for a fragment of the viral genome and the other for the host cell marker gene. The standards (one for HIV and the other for a human DNA marker gene HLA-DQ alpha) were constructed by PCR-mediated joining of DNA fragments and were found to be effective in quantitative PCR despite rather different structures of amplified fragments in target and standard DNAs. The number of HIV DNA copies was found to be 2-500 per 1000 lymphocytes in blood from HIV-infected patients and up to 5000+ per 1000 cells in chronically infected cell lines. The degree of infection thus measured was found to change over the course of treatment.
Subject(s)
HIV Infections/virology , HIV-1/genetics , HLA-DQ Antigens/genetics , Polymerase Chain Reaction , Base Sequence , Cell Line , DNA Primers , DNA, Viral , HIV Infections/blood , Humans , Lymphocytes/virology , Molecular Sequence DataABSTRACT
Polymerase-mediated recombination based on DNA polymerase chain reactions (PCRs) has been used to carry out directed joining at a present point of two DNA fragments initially contained in a plasmid and a single-stranded synthetic DNA. The process includes copying of these fragments by PCR with generation of an overlapping homologous region. Such overlap of 12 base pairs in length was found to be sufficient to provide further DNA joining also by use of PCR.
Subject(s)
DNA, Recombinant/metabolism , Gene Amplification , Oligodeoxyribonucleotides/metabolism , Polymerase Chain Reaction , Base Sequence , Molecular Sequence Data , PlasmidsABSTRACT
Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA, Single-Stranded/metabolism , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific , Base Sequence , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Substrate SpecificityABSTRACT
The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.
Subject(s)
DNA Restriction Enzymes/metabolism , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific , Base Sequence , DNA-Binding Proteins/metabolism , Kinetics , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Protein Binding , Structure-Activity RelationshipABSTRACT
Eco RII restriction endonuclease cleaves synthetic DNA-duplexes in which the recognition sites of this enzyme (5'...CCATGG...) are repeated every 9 base pairs with the alternating orientation of the central AT pair. It operates in a processive mode, i.e. the bound enzyme molecule slides along the substrate toward neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained point to the capability of Eco RII endonuclease to recognize and cleave the substrate under both possible orientations of the central AT-pair of the recognition site with respect to the bound enzyme molecule. These data also show the close similarity of DNA structures in a complex with the enzyme and without.
Subject(s)
DNA Restriction Enzymes/metabolism , Deoxyribonucleases, Type II Site-Specific , DNA , In Vitro Techniques , Nucleic Acid Heteroduplexes , Substrate SpecificityABSTRACT
Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT-pair in the recognition site for a non-complementary TT-or AA-pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5'-terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.
