RESUMEN
Data on the molecular arrangement of viruslike particles (VLPs) of yeast and Drosophila retrotransposons are presented. Two methods for identifying VLPs from specific retrotransposon families have been offered. The first method is based on VLPs fractionation by electrophoresis in agarose gel under strictly controlled conditions. VLPs of the Drosophila melanogaster retrotransposon families copia and gypsy and D. virilis retrotransposon Tv1 were identified by this method. The method based on heterologous induction of retrotransposons in cells of the mutant spt3 strain of Saccharomyces cerevisiae was used to identify VLPs of yeast retrotransposon Tyl and D. melanogaster gypsy retrotransposon.
Asunto(s)
Drosophila melanogaster/genética , Drosophila/genética , Polimorfismo Genético , Retroelementos/genética , Saccharomyces cerevisiae/genética , Virión/genética , Animales , Fraccionamiento Químico , Drosophila/citología , Drosophila melanogaster/citología , Electroforesis en Gel de Agar , Microscopía Electrónica , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/citología , Transformación GenéticaRESUMEN
A new method was developed to study the mechanism of initiation of the retrotransposition cycle: retrotransposons of Drosophila melanogaster, gypsy, copia, and 17.6 were expressed in yeast under the control of potent yeast promoters. Expression of retrotransposons induced formation of viruslike particles (VLPs) associated with full-length Ty1 RNA and DNA sequences. This phenomenon was termed heterologous induction. When the gene for reverse transcriptase of human immunodeficiency virus (HIV) was expressed in yeast, the same results were obtained. These data allowed us to assume the excess of active reverse transcriptase to play the central role in induction of transposition. Possible mechanisms of induction of Ty1 transposition by homologous and heterologous elements are discussed.
Asunto(s)
ADN Polimerasa Dirigida por ARN/metabolismo , Retroelementos , Animales , Clonación Molecular , Drosophila melanogaster/genética , Vectores Genéticos , Transcriptasa Inversa del VIH , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
A new method for isolation and cloning of functionally active retrotransposons was proposed and tested. The method is based on the hypothesis about the universal character of retrotransposition through reverse transcription. A new retrotransposon family (Tvl) was coned from the Drosophila virilis genome. A full-length Tvl copy was 7 kb. The cell line under consideration contained 500-1000 Tvl copies per cell, i.e., it accounted for 0.3% of the genome. Tvl RNA, detected in the cell culture, was almost absent in cells of adult flies. A 7-kb polyadenylated RNA was the main transcript of Tvl. Extrachromosomal circular and linear Tvl copies were cloned and characterized. The following features of Tvl structural organization were studied: (1) element was flanked by perfect direct repeats of 453 bp size (long terminal repeats, LTRs); (2) element sequences adjacent to the LTRs were homologous to binding sites of primer serine tRNA and the polypurine blocks of retrotransposons 17.6 and 297; and (3) expected transcription of LTRs occurred as for known retrotransposons, --producing RNA with a direct terminal repeat.
Asunto(s)
Elementos Transponibles de ADN , Drosophila/genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Cartilla de ADN , ADN Circular , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.
Asunto(s)
Baculoviridae/genética , Expresión Génica/genética , Luciferasas/genética , Regiones Promotoras Genéticas/genética , Transfección/genética , Animales , Línea Celular , Escarabajos , Vectores Genéticos , PlásmidosAsunto(s)
Elementos Transponibles de ADN/efectos de los fármacos , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Elementos Transponibles de ADN/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/genética , VIH-1/genética , Poli A/genética , ARN/genética , ARN Mensajero , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/aislamiento & purificación , Saccharomyces cerevisiae/genética , Transformación Genética/efectos de los fármacos , Transformación Genética/genéticaRESUMEN
The yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe transformed by plasmids containing retrotransposon from yeast or Drosophila under the control of a strong promoter show the remarkable reverse transcriptase activity. The activity results in the impaired yeast growth and decreased mitotic stability of the plasmids. The phenotypic expression of the reverse transcriptase activity is observed within 30 days.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fenotipo , ADN Polimerasa Dirigida por ARN/genética , Saccharomyces cerevisiae/enzimología , Elementos Transponibles de ADN , Escherichia coli/genética , Regulación Fúngica de la Expresión Génica , Plásmidos , Saccharomyces cerevisiae/genética , Transformación BacterianaRESUMEN
Yeast cells Saccharomyces cerevisiae were transformed by the recombinant plasmids containing the Yeast retrotransposon Ty and the Drosophila mobile element gypsy under the control of a strong Yeast promoter. The exogenous Ty-element induces the complete cycle of Ty-retrotransposition including the TyRNA synthesis, formation of virus-like particles, synthesis of all reverse transcriptase intermediates in the virus-like particles with the subsequent circles formation and transposition. The Drosophila mobile element gypsy is capable of inducing the formation of the virus-like particles containing RNA, DNA and proteins of the Ty-retrotransposon only. The Ty-circles and induction of transposition were not observed. The obtained data demonstrates the existence of the multistep repression system for Ty-transposition cycle. The possibility and efficiency of using the model to study the mechanism for retrotransposon transposition is discussed.
Asunto(s)
Elementos Transponibles de ADN , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , Hibridación de Ácido Nucleico , Plásmidos , ARN de Hongos/genética , Mapeo RestrictivoRESUMEN
DNA methylase activity has been studied in partially purified extracts from cultured cells, embryos, and adult Drosophila flies. No significant level of transfer of methyl groups from S-adenosylmethionine with formation of 5-methylcytosine and 6-methyladenine was observed. Methylase activity in Drosophila cells as compared to bovine lymphocytes and rat liver is either absent or at least 5000-15,000 times lower and hence cannot be detected using the present method.
Asunto(s)
Metilasas de Modificación del ADN/análisis , Drosophila/enzimología , Animales , Bovinos , Células Cultivadas/enzimología , Metilasas de Modificación del ADN/aislamiento & purificación , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/veterinaria , Hígado/enzimología , Regeneración Hepática/fisiología , Linfocitos/enzimología , Masculino , RatasRESUMEN
The recombinant plasmids pPTX and pPGX were constructed, containing the sequences of yeast Ty retrotransposon and Drosophila element mdg4, correspondingly. Transformation of yeast by these plasmids lead to induction of reverse transcriptase activity associated with virus-like particles, containing only the sequences of Ty. The data obtained show that mdg4 is capable of expression in yeast and the products of its expression are used to form the yeast virus-like particles. The system described may be used to study the expression of different retrotransposons from various cells in yeast.
Asunto(s)
Elementos Transponibles de ADN , Drosophila/genética , Regulación de la Expresión Génica , Saccharomyces cerevisiae/genética , Animales , Genes Fúngicos , Plásmidos , ADN Polimerasa Dirigida por ARN/genéticaAsunto(s)
Secuencia de Bases , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Homología de Secuencia de Ácido Nucleico , Virión/genética , Animales , Células Cultivadas , Drosophila melanogaster/microbiología , ADN Polimerasa Dirigida por ARN/genética , Retroviridae/enzimología , Retroviridae/genética , Virión/enzimologíaRESUMEN
In Drosophila melanogaster cultured cells, RNA reverse transcription intermediate forms connected with initiation of minus and plus DNA strand synthesis (minus and plus strong-stop DNA) are detected for mobile dispersed genetic elements MDG1, MDG3 and MDG4 (gypsy). A comparative analysis of intermediate forms has proved that mdg elements pass the same stages of reverse transcription as retroviruses, revealing a complete similarity between intermediate products. It has also been established that these three mdg elements possess a common mechanism of reverse transcription, despite their structural differences. The length of the minus strong-stop DNA, that gives the position of the RNA start site, coincides with the data obtained from SI nuclease analysis of transcription initiation. SI mapping has also revealed that mdg RNA carries a repeated sequence R on its ends, similar to retroviral RNA molecules, and that mdg LTRs have a U3-R-U5 structure analogous to that of proretroviral LTRs. Transcription of mdg1, mdg3 and mdg4 is initiated within or immediately after the same sequence TCAGTPy. Neither TATA box nor CAAT box can be found in their characteristic positions upstream of the 5' ends of mRNA.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , ADN Polimerasa Dirigida por ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Animales , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , ADN/genética , ARN/genética , Factores de Transcripción/genéticaRESUMEN
Poly(A)+RNA-DNA hybrids complementary to mobile dispersed genetic elements MDG 1 and MDG 3 have been isolated from Drosophila melanogaster culture cells. DNA and RNA in these complexes are represented by perfect hybrids. Three types of single-stranded DNA are detected in these hybrids: full-length DNA molecules of MDG containing one and two long terminal repeats (minus strand) and the long terminal repeat itself (plus strand). The results are consistent with the model of reverse transcriptional replication of MDG similar to that of retroviruses.
Asunto(s)
ADN Circular/genética , Drosophila melanogaster/genética , Hibridación de Ácido Nucleico , ARN/genética , Transcripción Genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Elementos Transponibles de ADN , ADN Circular/análisis , Electroforesis en Gel de Agar , Herencia Extracromosómica , Poli A/análisis , ARN/análisis , ADN Polimerasa Dirigida por ARN/genéticaRESUMEN
In a system containing isolated HeLa cell nuclei the release of RNA from the nuclei may be paralleled with the antagonistic process, i. e., RNA translocation into the nuclei. The RNA release from the nuclei depends on incubation time, pH, Mg2+ and nucleoside triphosphate concentration. The rate of reverse transport depends on pH, size of RNA to be translocated and the physiological state of the nuclear membrane. Low molecular weight RNAs (less than 10 S) are translocated into the nuclei most effectively. The nuclei of synchronized HeLa cells in the G1-period are more "permeable" for translocated RNA as compared with the S-phase HeLa cell nuclei.
Asunto(s)
Núcleo Celular/metabolismo , ARN/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Sistema Libre de Células , Células HeLa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Uridina Trifosfato/metabolismoRESUMEN
Regulation of the rate of RNA synthesis in Drosophila melanogaster diploid cells with 1, 2, 3 and 4 nucleolar organizers is studied. The data indicate that in flies with the redundant nucleolar organizer (NO) content extra NOs are eliminated by the 18th generation and the level of rRNA synthesis is typical for control wild stock flies. In flies bearing single NO on the Y chromosome, extra rDNA replication does not occur and rRNA genes deficiency by the increased rRNA synthesis is compensated. It is suggested that in genotypes studied the rate of rRNA synthesis depended on sex chromosome quantity per genome.