RESUMO
The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pugD transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pugD pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pugD eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pugD limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Olho/metabolismo , Genes Dominantes , Genes de Insetos , Proteínas de Membrana Transportadoras/metabolismo , Repetições de Microssatélites/genética , Mutação/genética , Animais , Baculoviridae/metabolismo , Sequência de Bases , Posicionamento Cromossômico/genética , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis/genética , Dosagem de Genes , Vetores Genéticos/metabolismo , Injeções , Dados de Sequência Molecular , Fenótipo , Pigmentação , Transporte Proteico , Pteridinas/metabolismo , Pupa/metabolismo , Transgenes , Proteína Wnt1/metabolismoRESUMO
Ring chromosomes are of basic interest to the geneticist and cell biologist who study their behavior in meiotic and mitotic divisions. In addition, the mitotic instability associated with some ring-X chromosomes has proven useful in Drosophila as a means to produce gynandromorphs for developmental studies. We describe a method to construct ring-X chromosomes in Drosophila via I-CreI-mediated exchange in rDNA, and then rapidly diagnose the recovery of ring chromosomes via FLP-mediated sister chromatid exchange within the ring. The method we describe provides a ready means to tailor the genetic content of ring-X chromosomes, making it suited to produce ring-X chromosomes for a variety of experimental purposes.
Assuntos
Cromossomos de Insetos/genética , Drosophila melanogaster/genética , Técnicas Genéticas , Cromossomos em Anel , Cromossomo X/genética , Animais , Enzimas de Restrição do DNA/metabolismo , DNA Ribossômico/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Masculino , Mitose , Troca de Cromátide IrmãRESUMO
We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.
