RESUMEN
The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 3 , ADN de Neoplasias/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias del Cuello Uterino/genética , Clonación Molecular , Metilación de ADN , Femenino , Humanos , Hibridación Fluorescente in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Mapeo RestrictivoRESUMEN
New human and mouse cDNAs which are homologous to human intersectin gene (ITSN) mapped on a q22.1-22.2 region of human chromosome 21 have been obtained. ITSN gene structure has been determined using nucleotide sequences of human chromosome 21 presented in nucleotide's data bases. The analysis of human and mouse ITSN gene transcripts revealed that their pre-mRNA splicing could occur in different ways. New form of ITSN gene transcripts with alternatively spliced SH3C domain (exon 25 and 26) was detected in different human and mouse tissues. The other splice form with absence of exons 6-14 that results in reading frame shift and stop-codon formation was identified in the mouse adult lung and kidney. In addition, we showed alternative splicing of exons 20, 25 and part of exon 6.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Empalme Alternativo/genética , Proteínas Portadoras/genética , Transcripción Genética , Animales , Secuencia de Bases , Cromosomas Humanos Par 21/genética , ADN Complementario/genética , Humanos , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Subgroup C avian sarcoma viruses efficiently infect and transform but poorly replicate in duck cells. Nucleotide sequence analysis of Prague strain of Rous sarcoma virus adapted by numerous passages on duck embryonic fibroblasts (daPr-RSV-C) showed that adaptation of originally chicken virus to duck cells correlated with changes in viral genome, first of all in gp85-coding domain of env-gene. Besides, changes in LTR and src-gene sequences could play a role in widening of host range for this virus. The major changes of daPr-RSV-C in comparison with original Pr-RSV-C appeared to be the result of homologous recombinations with corresponding regions of chicken endogenous retroviruses.
Asunto(s)
Virus del Sarcoma Aviar/genética , Secuencia de Aminoácidos , Animales , Virus del Sarcoma Aviar/fisiología , Secuencia de Bases , Células Cultivadas , Patos , Genes env , Genes gag , Genes src , Genoma Viral , Datos de Secuencia Molecular , Recombinación Genética , Replicación Viral/genéticaRESUMEN
The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.
Asunto(s)
Adaptación Fisiológica , Virus del Sarcoma Aviar/genética , Patos/microbiología , Animales , Virus del Sarcoma Aviar/fisiología , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Datos de Secuencia Molecular , Especificidad de la Especie , Replicación ViralRESUMEN
For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.
Asunto(s)
Virus del Sarcoma Aviar/genética , Transformación Celular Viral/genética , Productos del Gen env/genética , Genes env , Proteínas Oncogénicas Virales/genética , Proteínas del Envoltorio Viral/genética , Animales , Secuencia de Bases , Células Cultivadas , Pollos , Patos , Fibroblastos , Datos de Secuencia MolecularRESUMEN
Inverse transcriptase of bird myeloblastosis virus is a unique instrument for artificial synthesis of structural genes of viruses, plants, animals. Methods for the virus production in preparative amounts are developed due to selection of the corresponding line of chickens, conditions of their maintenance, diet infection methods and myeloblastosis diagnostics. Main demands to the inverse transcriptase preparations (their high activity, absence of nuclease impurities, high concentration of the enzyme preparation solutions and their stability in storage) are ensured by zonal centrifugation purification of the virus in a sucrose density gradient, described methods of inverse transcriptase isolation and purification as well as conditions of its storage.
Asunto(s)
Virus de la Leucosis Aviar/enzimología , Virus de la Mieloblastosis Aviar/enzimología , ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Animales , Leucosis Aviar/diagnóstico , Virus del Sarcoma Aviar/enzimología , Pollos , Cromatografía DEAE-Celulosa , Escherichia coli/enzimología , Leucemia Experimental/microbiología , Ratones , Técnicas Microbiológicas , Fosforilación , ADN Polimerasa Dirigida por ARN/metabolismo , Virus Rauscher/enzimologíaAsunto(s)
Clonación Molecular , ADN/metabolismo , Proinsulina/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enzimas de Restricción del ADN , Insulina , Plantas/metabolismo , Poli A/genética , Biosíntesis de Proteínas , ARN/genética , ARN Mensajero/genética , Salmón , Triticum/metabolismoRESUMEN
cDNA synthesized on rabbit bone marrow erythroid cells pre-mRNA was cloned in bacterial plasmids. Cold phenol extracted pre-mRNA was a several times more effective template in the reaction of reverse transcription without oligo(dT) 10-primer than hot phenol extracted pre-mRNA. There was no yield increase of DNA-product on hot phenol extracted pre-mRNA in the reaction of reverse transcription with the oligo(dT)10-primer addition. The "hot phenol" poly (A)+-pre-mRNA was used to obtain the representative, full-sized cDNA. The double-stranded form of this cDNA, obtained with the help of DNA-polymerase I, was inserted into the PstI-site of pBR322 plasmid. About 25% E. coli JC5183 clones, transformed with this hybrid plasmid, were found to contain the globin sequences.
Asunto(s)
Clonación Molecular , ADN/metabolismo , Globinas/genética , Precursores de Ácido Nucleico/genética , ARN Mensajero/genética , Animales , Secuencia de Bases , Médula Ósea/metabolismo , ADN Polimerasa I/metabolismo , Escherichia coli/genética , Plásmidos , Precursores del ARN , ADN Polimerasa Dirigida por ARN/metabolismo , ConejosRESUMEN
DNA-products synthesized on pre-mRNA's from rat liver and rabbit erythroidal bone marrow cells, on rabbit globin mRNA and the RNA-templates themselves have been examined by electron microscopy. In spreading conditions providing extension of molecules the sizes of DNA-products corresponded to sizes of RNA templates. Globin mRNA, pre-mRNA as well as cDNA synthesized on these templates in the presence of actinomycine D were seen as single-stranded molecules by electron microscopy. The preparations of cDNA synthesized in the absence of actinomycine D on the pre-mRNA template were represented by double-stranded molecules along side with sigle-sranded. Up to 10% of double-stranded DNA-product appeared to be branched; all branches of such molecules had the thickness and rigidity of double-stranded structures. The possible ways of formation of branched structures are discussed.
Asunto(s)
ADN , ADN Polimerasa Dirigida por ARN , Fenómenos Químicos , Química Física , Globinas/genética , Microscopía Electrónica , Conformación de Ácido Nucleico , ARN MensajeroRESUMEN
Total poly(A)-mRNA from polyribosomes of MOPC 21 mouse myeloma were investigated. Poly(A)-mRNA was released by two successive chromatography on oligo (dT)-cellulose. A 14S fraction of total poly(A)-mRNA was obtained and partially purified by sucrose gradient centrifigation followed by acrylamide gel electrophoresis. As estimated from the electrophoretic analysis, the 14S mRNA has three components, one of which appears to be 18S rRNA and two others--mRNAs with molecular weight of 5.2.10(5) and 3.8.10(5), respectively. Total poly(A)-mRNA and partially purified 14S mRNA were active when employed as a template in a reverse transcription and cell-free system from wheat germ. DNA complementary to the 14S mRNA was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA was heterogeneous in size with the average size of about 800 nucleotides when analyzed by gel electrophoresis in 98% formamide. The maximal length was about 1100 nucleotides that consistent with full template length. About half of the translation product directed by the 14S mRNA migrated as mature L-chain Ig (upon polyacrylamide gel electrophoresis in sodium dodecylsulfate). The presented data suggested that 14S mRNA species contain mRNA L-chain Ig.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Transcripción Genética , Animales , Línea Celular , Ratones , Peso Molecular , Plantas/metabolismo , Plasmacitoma , Poli A/metabolismo , Triticum/metabolismoAsunto(s)
Núcleo Celular/metabolismo , ADN/biosíntesis , ARN Nuclear Heterogéneo/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/biosíntesis , Animales , Virus de la Mieloblastosis Aviar/enzimología , Sitios de Unión/efectos de los fármacos , Médula Ósea/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Células HeLa/metabolismo , Técnicas In Vitro , Desnaturalización de Ácido Nucleico/efectos de los fármacos , ConejosRESUMEN
Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.