ABSTRACT
Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.
Subject(s)
Bacteriophages/genetics , DNA, Complementary/biosynthesis , DNA, Neoplasm/biosynthesis , DNA, Recombinant/biosynthesis , Gene Library , Genetic Vectors , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , RNA-Directed DNA Polymerase/metabolism , Transcription, Genetic/geneticsABSTRACT
Atlow concentrations, dimethyl sulfoxide (DMSO), stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40 percent stimulation was obtained in the presence of 5 percent (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15 percent inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100 percent was found in the presence of 20 percent DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 µg/ml
Subject(s)
Animals , Dimethyl Sulfoxide/pharmacology , RNA-Directed DNA Polymerase/metabolism , Avian Myeloblastosis Virus/enzymologyABSTRACT
Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide
Subject(s)
Avian Myeloblastosis Virus/enzymology , Guanosine Triphosphate/metabolism , Poly C/metabolism , RNA-Directed DNA Polymerase/metabolism , Binding Sites , Magnesium/metabolism , Manganese/metabolismABSTRACT
O objetivo da presente revisäo é mostrar alguns aspectos relacionados com o reverso da transcriçäo, incluindo descriçäo do retrovírus, ciclo de vida do vírus, mecanismo do reverso da transcriçäo, propriedades cinéticas e imunológicas da transcritase reversa, e inibidores da enzima viral. Estäo também incluídas algumas referências sobre o retrovírus AIDS. O reverso da transcriçäo parece näo ser restrito apenas aos vírus de RNA. Alguns vírus de DNA, como o vírus da hepatite B, e o vírus do mosaico da couve-flor, e outros elementos genéticos, como pseudogenes e elementos de transposiçäo, parecem utilizar o reverso da transcriçäo para suas existências
Subject(s)
Humans , Retroviridae/enzymology , RNA-Directed DNA Polymerase/metabolism , Transcription, Genetic , DNA, Viral/genetics , Retroviridae/genetics , RNA, Viral/genetics , RNA-Directed DNA Polymerase/antagonists & inhibitorsABSTRACT
Poly (2'-O-methyladenylic acid), poly (Am), is recognized as template in the reaction catalyzed by avian myeloblastosis virus, AMV, reverse transcriptase better in the presence of Mn than Mg as divalent cation. An apparent KM value of 2.5 x 10 M in relation to TTP and an inhibition of the reaction at poly (Am concentration higher than 5 microng/ml have been observed. Ethidium bromide, EtBr, tetramethyl ethidium bromide, TMEtBr, dideoxy TTP, and novobiocin are inhibitors of the poly (Am)-directed reverse transcriptase reaction in the presence of Mn