Biomedical subjects

A A Melnikov

Publications and source records attributed to A A Melnikov.

5 recordsLinked to original sources

PubMed1991

Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs.

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.

Animals↗

PubMed1986

DNA topoisomerase I from rat brain neurons.

The DNA topoisomerase I has been isolated from neurons of rat cerebral cortex. The most homogeneous fraction purified contains only one polypeptide of Mr approx. 100 000. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. The optimum monovalent cation concentration for the relaxation of superhelical DNA under conditions of DNA excess is found to be 175-200 mM. The neuron enzyme is similar to other mammalian type I DNA topoisomerases in that it links to the 3' ends of the broken DNA strands. Like calf thymus DNA topoisomerase I, the neuron topoisomerase can be selectively inhibited by poly(dG) but not by other homopolymerical deoxyribonucleotides.

Animals↗

PubMed1986

Genetic selection of lambda phage clones from a gene clonotheque.

A genetic procedure for selection of specific lambda clones, by homologous recombination between lambda clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of alpha-interferon-specific sequences from the human gene clonotheque.

Bacteriophage lambda↗

PubMed1984

Alternate domains of neuron DNA topoisomerase I in developing rat brain.

The DNA topoisomerase found in rat brain neurons relaxes supercoiled DNA in the absence of ATP or Mg2+. The estimated content of the active enzyme per nucleus of nerve cell is constant during development from a fetal proliferating neuroblast at the embryonic stage of 18 days to the terminally differentiated neuron (postnatal age of 60 days). The salt stability of DNA topoisomerase association with chromatin varies with the stage of development of nerve cells: at 300 mM NaCl most of the enzyme activity (greater than 90% of the removed activity) elutes from differentiated neuron chromatin, whereas only approx. 25% of the enzyme activity elutes from neuroblast chromatin.

Aging↗

PubMed1984

Lambda phagemids and their transducing properties.

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.

Bacteriophage lambda↗