RESUMO
The homeotic gene Antennapedia (Antp) controls determination of many different cell types in the thorax and abdomen of Drosophila melanogaster. The spontaneous mutant allele Nasobemia (AntpNs) and its revertants have been widely used to infer normal Antp gene function but have not themselves been thoroughly characterized. Our analysis reveals that AntpNs consists of an internal 25-kb partial duplication of the Antp gene as well as a complex insertion of > 40 kb of new DNA including two roo transposons. The duplication gives the mutant gene three Antp promoters, and transcripts from each of these are correctly processed to yield functional ANTP proteins. At least two of the promoters are ectopically active in the eye-antenna imaginal discs, leading to homeotic transformation of the adult head. A molecular and genetic description of several AntpNs revertants shows them to be diverse in structure and activity, including a restoration of the wild type, rearrangements separating two of the AntpNs promoters from the coding sequences, and protein nulls and hypomorphs affecting expression from all three of the promoters. Finally, one revertant has a suppressing lesion in the osa locus far away from Antp. These features explain the unusual homozygous viable nature of AntpNs, suggest a mechanism by which its homeotic transformation occurs, and exemplify the diversity of ways in which mutational reversion can take place.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Genes Homeobox , Proteínas de Homeodomínio , Mutação , Proteínas Nucleares , Fatores de Transcrição , Animais , Proteína do Homeodomínio de Antennapedia , DNA , Proteínas de Drosophila , Expressão Gênica , Genes Letais , Teste de Complementação Genética , Transcrição GênicaRESUMO
In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho/genética , Proteínas de Insetos/genética , Animais , Sequência de Bases , Primers do DNA , Feminino , Rearranjo Gênico , Genes Dominantes , Masculino , Sequências Repetitivas de Ácido NucleicoRESUMO
The variegating mutation brownDominant (bwD) of Drosophila melanogaster is associated with an insertion of heterochromatin into chromosome arm 2R at 59E, the site of the bw gene. Mutagenesis produced 150 dominant suppressors of bwD variegation. These fall into two classes: unlinked suppressors, which also suppress other variegating mutations; and linked chromosome rearrangements, which suppress only bwD. Some rearrangements are broken at 59E, and so might directly interfere with variegation caused by the heterochromatic insertion at that site. However, most rearrangements are translocations broken proximal to bw within the 52D-57D region of 2R. Translocation breakpoints on the X chromosome are scattered throughout the X euchromatin, while those on chromosome 3 are confined to the tips. This suggests that a special property of the X chromosome suppresses bwD variegation, as does a distal autosomal location. Conversely, two enhancers of bwD are caused by translocations from the same part of 2R to proximal heterochromatin, bringing the bwD heterochromatic insertion close to the chromocenter with which it strongly associates. These results support the notion that heterochromatin formation at a genetic locus depends on its location within the nucleus.
Assuntos
Drosophila melanogaster/genética , Cor de Olho/genética , Regulação da Expressão Gênica , Genes de Insetos , Genes Supressores , Heterocromatina/ultraestrutura , Animais , Elementos Facilitadores Genéticos , Genes Dominantes , Ligação Genética , Mutagênese , Translocação Genética , Cromossomo XRESUMO
We examined the behavior of the brownDominant (bwD) heterochromatic insertion moved to different locations relative to centric heterochromatin. Effects were measured as the degree of silencing of a wild-type brown eye pigment gene by bwD across a tandem duplication. A series of X-ray-induced effects were recovered at high frequency. Cis-acting enhancers were obtained by relocation of the duplication closer to autosomal heterochromatin. Enhancers were also recovered on the homologous chromosome when it was similarly rearranged, revealing a novel interhomologue effect whereby interactions occur between genetic elements near opposite ends of a chromosome arm rather than between paired alleles. Cis-acting suppressors were obtained as secondary rearrangements in which the duplication was moved farther away from heterochromatin. Suppression was correlated with loss of cytological association between bwD and the polytene chromocenter. Surprisingly, the distance from bwD to the chromocenter was not correlated with the strength of enhancement or suppression. We propose that bwD fails to coalesce with the chromocenter when its position along the chromosome places it beyond a threshold distance from heterochromatin, and this threshold depends upon the configuration of both the chromosome carrying bwD and its paired homologue.
Assuntos
Mapeamento Cromossômico , Drosophila/genética , Heterocromatina/fisiologia , Alelos , Animais , Cruzamentos Genéticos , Drosophila/citologia , Drosophila/fisiologia , Feminino , Genes Dominantes , Heterocromatina/ultraestrutura , Heterozigoto , Masculino , Mutagênese , Supressão Genética , Raios XRESUMO
Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho , Heterocromatina/metabolismo , Proteínas de Insetos/genética , Ativação Transcricional , Transgenes , Animais , Genes DominantesRESUMO
The form of seed plants is determined by the growth of a number of meristems including apical meristems, leaf meristems and cambium layers. We investigated five recessive mutant alleles of a gene REVOLUTA that is required to promote the growth of apical meristems and to limit cell division in leaves and stems of Arabidopsis thaliana. REVOLUTA maps to the bottom of the fifth chromosome. Apical meristems of both paraclades (axillary shoots) and flowers of revoluta mutants frequently fail to complete normal development and form incomplete or abortive structures. The primary shoot apical meristem sometimes also arrests development early. Leaves, stems and floral organs, in contrast, grow abnormally large. We show that in the leaf epidermis this extra growth is due to extra cell divisions in the leaf basal meristem. The extent of leaf growth is negatively correlated with the development of a paraclade in the leaf axil. The thickened stems contain extra cell layers, arranged in rings, indicating that they may result from a cambium-like meristem. These results suggest that the REVOLUTA gene has a role in regulating the relative growth of apical and non-apical meristems in Arabidopsis.
Assuntos
Arabidopsis/genética , Genes de Plantas , Meristema/genética , Folhas de Planta/genética , Caules de Planta/genética , Alelos , Divisão Celular/genética , Meristema/crescimento & desenvolvimento , Mutagênese , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Caules de Planta/crescimento & desenvolvimentoRESUMO
The dam DNA methyltransferase gene from Escherichia coli was introduced into Drosophila in order to probe chromatin structure in vivo. Expression of the gene caused no visible defects or developmental delay even at high levels of active methylase. About half of each target site was found to be methylated in vivo, apparently reflecting a general property of chromatin packaged in nucleosomes. Although site-specific differences were detected, most euchromatic and heterochromatic sites showed comparable degrees of methylation, at least at high methylase levels. Methylase accessibility of a lacZ reporter gene subject to position-effect variegation throughout development was only slightly reduced, consistent with studies of chromatin accessibility in vitro. Silencing of lacZ during development differed from silencing of an adjacent white eye pigment reporter gene in the adult, as though chromatin structure can undergo dynamic alterations during development.
Assuntos
Cromatina , DNA/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Animais , Sequência de Bases , Extratos Celulares , DNA/análise , Drosophila melanogaster/citologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Olho , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Genes Bacterianos/genética , Genes Reporter/genética , Masculino , Metilação , Dados de Sequência Molecular , Fenótipo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , beta-Galactosidase/genéticaRESUMO
Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli.