RESUMO
In Arabidopsis thaliana, nuclear multisubunit RNA polymerase IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) are required for the biogenesis of 24-nucleotide small interfering RNAs (siRNAs) that direct DNA methylation and transcriptional silencing at target loci transcribed by nuclear multisubunit RNA polymerase V (Pol V). Pol IV and RDR2 physically associate and RDR2's polymerase activity in vitro is dependent on Pol IV. RDR2 transcription of nascent Pol IV transcripts might result in discontinuous second strands, analogous to lagging-strand Okazaki fragments generated during DNA replication. In vitro, Pol V is unable to displace nontemplate DNA during transcriptional elongation. This suggests a need for DNA duplex unwinding by helper proteins, perhaps analogous to the helicase-mediated duplex unwinding that occurs at replication forks to enable leading strand synthesis by DNA polymerase ε. A multiprotein complex (DRD1, DMS3, DMS11, RDM1) known to enable Pol V transcription might facilitate duplex unwinding via ATP-dependent DNA translocase, single-stranded DNA binding, and cohesin-like strand capture activities. These considerations are discussed and incorporated into a "transcription fork" model for Pol IV and Pol V-dependent RNA-directed DNA methylation.
Assuntos
Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Genéticos , RNA de Plantas/metabolismo , Transcrição Gênica , Transporte ProteicoRESUMO
Double target in situ hybridization to root tip metaphase and interphase cells of Silene cintrana and Silene rothmaleri was used to allocate the position of 18S-5.8S-25S and 5S rRNA genes. In both species, the 18S-5.8S-25S rDNA probe labelled four sites located on the short arms of two submetacentric chromosomes. Only one locus for 5S rDNA was mapped adjacent to 18S-5.8S-25S genes in a subterminal position on the centromere side: in S. rothmaleri the 5S rDNA locus was adjacent to the small 18S-5.8S-25S locus while in S. cintrana it was near the large one. The NOR activity analysed by Ag-staining in metaphase cells revealed proportionality between in situ labelling dimensions and Ag-NORs. In both species all rDNA loci were potentially active, although in S. rothmaleri a tendency for the expression of only one locus was observed. Interphase organisation analysis of rDNA showed some differences between both species that were correlated with NOR activity.
Assuntos
DNA Ribossômico/ultraestrutura , Interfase , Mapeamento Físico do Cromossomo , Raízes de Plantas/genética , Plantas/genética , Centrômero/ultraestrutura , Hibridização In Situ , Metáfase , Região Organizadora do Nucléolo/ultraestrutura , RNA Ribossômico 18S/ultraestrutura , RNA Ribossômico 5,8S/ultraestrutura , RNA Ribossômico 5S/ultraestrutura , Coloração pela PrataRESUMO
The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to rebuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid.
Assuntos
Aphthovirus/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Sequência de Aminoácidos , Aphthovirus/enzimologia , Sequência de Bases , Clonagem Molecular , Códon/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Oligonucleotídeos/química , Plasmídeos , TransfecçãoRESUMO
The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid
