RESUMO
In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.
Assuntos
Adenosina Trifosfatases , Arabidopsis/genética , Proteínas de Ciclo Celular , Meiose/genética , Animais , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis , Sequência de Bases , Cromossomos/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Genes de Plantas , Teste de Complementação Genética , Camundongos , Mutagênese Insercional , Mutação , Proteínas Nucleares , Fenótipo , Proteínas de Ligação a Fosfato , Proteínas de Plantas/genética , Recombinases Rec ARESUMO
By using RT-PCR and degenerate oligonucleotides based on the sequence homology between the yeast RAD51 and DMC1 genes, two genes belonging to the RAD51 and DMC1 families were isolated from Arabidopsis thaliana ecotype Columbia. A RAD51 genomic DNA was also sequenced which is almost identical to its Landsberg erecta counterpart, except for a few translationally silent substitutions and for the presence of a 527-bp element downstream of the polyadenylation site. This element is repeated in the genome of Arabidopsis. Northern analyses were conducted to characterize the expression pattern of both these genes. AtRAD51 and AtDMC1 are expressed in flower buds, but also in the mitotically active cells from a suspension culture. AtRAD51, but not AtDMC1, transcript level increases after gamma irradiation of the cells. Finally, a synchronisation experiment conducted with the suspension culture indicated that not only AtRAD51 but also AtDMC1 are regulated during the cell cycle, with S-phase-specific induction. Since DMC1 genes have always been regarded as being specifically meiotic, we discuss the significance of this mitotic transcriptional regulation in Arabidopsis.
Assuntos
Arabidopsis/genética , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Genes de Plantas/genética , Arabidopsis/citologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis , Ciclo Celular/genética , Elementos de DNA Transponíveis/genética , DNA Complementar/análise , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/efeitos da radiação , Raios gama , Regulação da Expressão Gênica , Meiose , Rad51 Recombinase , Recombinases Rec ARESUMO
To understand the mechanisms involved in the regulation of the mitotic cyclin B Nicta; CycB1;1 expression, we have cloned the Nicotiana sylvestris cyclin gene, Nicsy; CycB1;1, whose coding sequence is homologous to that of Nicta;CycB1;1 cDNA. The structure and the function of its 5'-flanking region, 1149 bp upstream of the first start codon, was analysed. By producing stably transformed cells of a synchronized culture with the Nicsy;CycB1;1 promoter/beta-glucuronidase (gus) reporter gene fusion, we demonstrate that the 1149 bp promoter fragment mediates a gus transcriptional oscillation, indistinguishable from that of endogenous Nicsy;CycB1;1 cyclin B transcripts. Transient GUS activity in BY-2 protoplasts reveals that promoter activity is considerably reduced by shortening the 5'-flanking region to 538 or 320 bp. Furthermore, the 320 bp fragment no longer mediates the observed transcriptional regulation of the 1149 bp Nicsy;CycB1;1 promoter in BY-2 protoplasts isolated from synchronized cells.