RESUMO
Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.
Assuntos
Macronúcleo/metabolismo , Micronúcleo Germinativo/metabolismo , Paramecium tetraurellia/metabolismo , Proteínas de Protozoários/metabolismo , Autofertilização , Regulação da Expressão Gênica no Desenvolvimento , Macronúcleo/genética , Micronúcleo Germinativo/genética , Paramecium tetraurellia/genética , Paramecium tetraurellia/crescimento & desenvolvimento , Proteínas de Protozoários/genéticaRESUMO
cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.
Assuntos
Endopeptidases/metabolismo , Luteovirus/enzimologia , Mutação/genética , Solanum tuberosum/virologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência de Bases , Capsídeo/biossíntese , Capsídeo/genética , Caulimovirus/genética , Endopeptidases/química , Endopeptidases/genética , Teste de Complementação Genética , Genoma Viral , Luteovirus/genética , Luteovirus/fisiologia , Fases de Leitura Aberta/genética , Folhas de Planta/microbiologia , Folhas de Planta/virologia , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , RNA Viral/biossíntese , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Rhizobium/genética , Serina/genética , Serina/metabolismo , Solanum tuberosum/microbiologia , Replicação ViralRESUMO
Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.