RESUMO
Polyalkylating derivatives of single-stranded polynucleotides (30-200-mers) complementary to the long E1 oncogene sequences of simian adenovirus SA7 cause inherited normalization of SH2 and G11 cells transformed with adenovirus SA7; certain deletions in the integrated proviral E1A oncogene were observed in several cases during this process. The transformed cells are indifferent to reagents noncomplementary to the E1 region. Thus polyalkylating derivatives of single-stranded 30-200-mers act as addressed mutagenes which react in a specific way with the integrated complementary DNA sequences of E1 oncogene in transformed rodent cells and realize oncogene-directed mutagenesis in vivo. During this treatment temporary normalized cells reverting to the initial transformed phenotype are also produced.
Assuntos
DNA de Cadeia Simples/genética , Mutagênese Sítio-Dirigida , Oncogenes , Adenovirus dos Símios , Alquilantes , Animais , Linhagem Celular Transformada , Transformação Celular Viral , DNA de Cadeia Simples/metabolismo , Hibridização de Ácido Nucleico , Fenótipo , Reação em Cadeia da Polimerase , RoedoresRESUMO
Using oligonucleotide probes, sequences containing the Mbcr locus involved in chromosome translocation t(9:22) were cloned form the library of human genes in the Charon 4A vector. The recombinant clone lambda BCR 1.1 obtained contained Mbcr sequences, but the 3' region of the Mbcr locus in lambda BCR 1.1 clone was strongly altered. Subcloning of a fragment of the altered region and blot hybridization analysis using it as a DNA probe revealed recombination in the 3' region of the Mbcr locus in clone lambda BCR 1.1 which resulted in insertion of unknown sequences into the region. A modified system is suggested for chromosome 22 breakpoint identification using restriction analysis of genome DNA with four restriction endonucleases and one 5'-DNA probe.
Assuntos
Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Translocação Genética , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Recombinação Genética , Mapeamento por RestriçãoRESUMO
The ss-DNA of the (+) and (-) chains of Ela DNA fragment was obtained by hydrolysis of the recombinant bacteriophages M13 mp8G and mp9G (where G is 1-1750 bp:, E1a region of oncogene SA7) in complexes with the 16 bp oligonucleotides containing AluI and BspRI sites of restriction and sequences complementary to E1a SA7. The obtained fragments overlap the E1a zones associated with the immortalizing potential of SA7.
Assuntos
Proteínas E1A de Adenovirus/genética , Adenovirus dos Símios/metabolismo , Bacteriófago M13/genética , DNA de Cadeia Simples/isolamento & purificação , DNA Viral/metabolismo , Oligonucleotídeos/metabolismo , Sequência de Bases , Hidrólise , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
New systems are proposed for the PCR analysis of HindIII polymorphic sites in the gamma A and gamma G globin genes and of TaqI polymorphic site in the human factor IX gene of blood population. DNA fragments amplified according to the systems described contain constant restriction site of the appropriate endonuclease, in addition to the polymorphic one, which significantly improves the reliability of the RELP analysis. The systems proposed are highly specific and may be used for DNA diagnosis of beta-thalassemia and haemophilia B.
Assuntos
Fator IX/genética , Globinas/genética , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Desoxirribonuclease HindIII , Desoxirribonucleases de Sítio Específico do Tipo II , Marcadores Genéticos , Humanos , Hidrólise , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
A new variant of the PCR test system is discussed which allows one to detect Bcl I and Hind III polymorphic sites of FVIII gene. It can be used for rapid and effective diagnosis of hemophilia A, especially, in combination with the blot-hybridization technique that detects other polymorphic variants of FVIII gene. The method proposed is highly accurate, reliable and simple. It allows one to analyze submicrogram quantities of DNA without using radiolabeled probes. The whole procedure takes several hours. The variant discussed can, possibly, be the part of the general scheme of hemophilia diagnosis completely based on the effective PCR test.