RESUMO
Fluorescent in situ hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of 'off-target' hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350-1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated k-mers, we provide genome-wide annotations of k-mer-uniqueness at http://cbio.mskcc.org/ approximately aarvey/repeatmap.
Assuntos
Hibridização in Situ Fluorescente/métodos , Sondas RNA/química , RNA Mensageiro/análise , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/química , Proteínas Nucleares/genética , RNA Mensageiro/química , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/genéticaRESUMO
We tested whether Artemia abd-A could repress limbs in Drosophila embryos, and found that although abd-A transcripts were produced, ABD-A protein was not. Similarly, developing Artemia epidermal cells showed expression of abd-A transcripts without accumulation of ABD-A protein. This finding in Artemia reveals a new variation in Hox gene function that is associated with morphological evolution. In this case, a HOX protein expression pattern is completely absent during early development, although the HOX protein is expressed at later stages in the central nervous system in a "homeotic-like" pattern. The combination of an absence of ABD-A protein expression in the Artemia limb primordia and the weak repressive function of Artemia UBX protein on the limb-promoting gene Dll are likely to be two reasons why homonomous limbs develop throughout the entire Artemia trunk.
Assuntos
Artemia/genética , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Animais , Artemia/crescimento & desenvolvimento , Artemia/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Inativação Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Fosforilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Functional assays in Drosophila melanogaster with orthologous transcription factors from other species suggest that changes in the protein-coding sequence may play a larger role in the evolution of transcription factor pathways than was previously believed. Interestingly, recent studies provide evidence that changes in transcription factor protein sequence can affect the regulation of only a subset of target genes, even in the same cells of a developing animal.