RESUMEN
A wide spectrum of hop 7SL RNA-encoding sequences was detected by temperature gradient-gel electrophoresis. Four hop 7SL RNA genes were cloned and characterized. A new subvariant of the upstream sequence element (USE) 5'TCCCACATGG 3' and two distinct variants of TATA signal were found at positions characteristic for RNA polymerase III-driven transcription in plants. In addition, a more distant conserved sequence element 5' CATGTATAAACTTTCTGC 3' was present in all cloned genes, about 160 bp upstream of the 7SL RNA coding sequence. Consensus secondary structures calculated for hop 7SL RNAs revealed characteristic features, although some structure differences from formerly published models were predicted. Specific in-vitro transcription of plant 7SL RNA genes was observed in a heterologous system (HeLa extract). This in-vitro transcription assay showed significant differences among individual clones in transcription rates, suggesting the requirement of complexity of 7SL RNA sequence for its efficient transcription in HeLa extract. Southern blot analysis of hop DNA revealed 12 7SL-specific signals corresponding to HindIII fragments ranging from 0.45 to 7.8 kb. Several 7SL RNA-encoding sequences and various intergenic spacers were amplified from the individual HindIII fragments of about 1.3 and 2.8 kb. These facts suggest that at least some of the hop 7SL RNA genes are organized in genomic clusters.
Asunto(s)
Genoma de Planta , ARN Citoplasmático Pequeño/genética , Rosales/genética , Partícula de Reconocimiento de Señal/genética , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN de Plantas/química , ADN de Plantas/genética , Genes de Plantas/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Filogenia , Rosales/química , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
We have performed an analysis of transcription of hop 7SL RNA genes in heterologous human extract from HeLa cells. Several variants of the Hl7SL-1 gene with a truncated or mutated 5' non-transcribed part revealed a crucial importance of TATA box for transcription. The USE element was found important but dispensable for transcription. Transcription of mutants in the A-like box and experiments with hop 7SL RNA pseudogenes E44 and G32 revealed the importance of internal elements. The A-like box and possibly CG doublet at position +15/+16 described by Bredow et al. (1990a) are according to our results indispensable for in vitro transcription of plant 7SL RNA genes in human extract.
Asunto(s)
Humulus/genética , ARN Citoplasmático Pequeño/genética , Partícula de Reconocimiento de Señal/genética , Transcripción Genética , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de SecuenciaAsunto(s)
Acrilatos/síntesis química , Antineoplásicos/síntesis química , Benzoatos/síntesis química , Acrilatos/farmacología , Animales , Antineoplásicos/farmacología , Bromo , Química Farmacéutica , Femenino , Rayos Infrarrojos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , EspectrofotometríaRESUMEN
We have previously shown that heat treatment of hop plants infected by hop latent viroid (HLVd) reduces viroid levels. Here we investigate whether such heat treatment leads to the accumulation of sequence variability in HLVd. We observed a negligible level of mutated variants in HLVd under standard cultivation conditions. In contrast, the heat treatment of hop led to HLVd degradation and, simultaneously, to a significant increase in sequence variations, as judged from temperature gradient-gel electrophoresis analysis and cDNA library screening by DNA heteroduplex analysis. Thirty-one cDNA clones (9.8%) were identified as deviating forms. Sequencing showed mostly the presence of quadruple and triple mutants, suggesting an accumulation of mutations in HLVd during successive replication cycles. Sixty-nine percent of base changes were localised in the left half and 31% in the right half of the secondary structure proposed for this viroid. No mutations were found in the central part of the upper conserved region. A "hot spot" region was identified in a domain known as a "pathogenicity domain" in the group representative, potato spindle tuber viroid. Most mutations are predicted to destabilise HLVd secondary structure. All mutated cDNAs, however, were infectious and evolved into complex progeny populations containing molecular variants maintained at low levels.