RESUMO
The transposition of Mu elements underlying Mutator activity in maize requires a transcriptionally active MuDR element. Despite variation in MuDR copy number and RNA levels in Mutator lines, transposition events are consistently late in plant development, and Mu excision frequencies are similar. Here, we report previously unsuspected and ubiquitous MuDR homologs that produce both RNA and protein. MuDR transcript levels are proportional to MuDR copy number, and homolog transcript levels increase in active Mutator lines. A subset of homologs exhibits constitutive transcription in MuDR(-) and epigenetically silenced MuDR lines, suggesting independent transcriptional regulation. Surprisingly, immunodetection demonstrated nearly invariant levels of MuDR and homolog protein products in all tested Mutator and non-Mutator stocks. These results suggest a strict control over protein production, which might explain the uniform excision frequency of Mu elements. Moreover, the nonfunctional proteins encoded by homologs may negatively regulate Mutator activity and represent part of the host defense against this transposon family.
Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas/biossíntese , Homologia de Sequência do Ácido Nucleico , Zea mays/genética , Zea mays/metabolismo , Sequência de Bases , Replicação do DNA , Genes Reguladores , Mutação , Filogenia , Proteínas de Plantas/genética , Pólen , Processamento Pós-Transcricional do RNA/genética , RNA de Plantas/biossíntese , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
To select for Ds transposition in transgenic tomato plants a phenotypic excision assay, based on restoration of hygromycin phosphotransferase (HPT II) gene expression, was employed. Some tomato plants, however, expressed the marker gene even though the Ds had not excised. Read-out transcriptional activity of the Ds element is responsible for the expression of the HPT II gene. Transcription initiation was mapped to multiple positions spanning about 300 bp in the subterminal part of the Ds element. In this respect Ds in tomato resembles the maize element Mu1, which also promotes transcription outward from the element. Transposon read-out transcription might thus supply an additional general mechanism for controlling plant gene expression.
Assuntos
Cinamatos , Elementos de DNA Transponíveis , Regulação Enzimológica da Expressão Gênica , Genes de Plantas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transcrição Gênica , Verduras/genética , Processamento Alternativo , Sequência de Bases , Primers do DNA , Resistência a Medicamentos/genética , Marcadores Genéticos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Ativação Transcricional , Verduras/enzimologia , beta-Glucosidase/genéticaRESUMO
We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.
Assuntos
Clonagem Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Biotina , DNA/isolamento & purificação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Plasmídeos , Rhizobium/genéticaRESUMO
We describe the use of plasmid rescue to facilitate studies on the behaviour of Ds and Ac elements in transgenic tomato plants. The rescue of Ds elements relies on the presence of a plasmid origin of replication and a marker gene selective in Escherichia coli within the element. The position within the genome of modified Ds elements, rescued both before and after transposition, is assigned to the RFLP map of tomato. Alternatively to the rescue of Ds elements equipped with plasmid sequences, Ac elements are rescued by virtue of plasmid sequences flanking the element. In this way, the consequences of the presence of an (active) Ac element on the DNA structure at the original site can be studied in detail. Analysis of a library of Ac elements, rescued from the genome of a primary transformant, shows that Ac elements are, infrequently, involved in the formation of deletions. In one case the deletion refers to a 174 bp genomic DNA sequence immediately flanking Ac. In another case, a 1878 bp internal Ac sequence is deleted.
