RESUMO
Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.
Assuntos
Reprogramação Celular , Cromossomos Artificiais de Mamíferos , Fibroblastos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Transfecção/métodos , Animais , Células CHO , Técnicas de Cocultura , Cricetinae , Cricetulus , Regulação da Expressão Gênica no Desenvolvimento , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.
Assuntos
Drosophila melanogaster/enzimologia , Peptídeo Hidrolases/química , Complexo de Endopeptidases do Proteassoma , Ubiquitinas/metabolismo , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/enzimologia , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/ultraestrutura , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Ubiquitina TiolesteraseRESUMO
Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.
Assuntos
Cromossomos Artificiais de Mamíferos/genética , Terapia Genética/métodos , Leucodistrofia de Células Globoides/terapia , Transplante de Células-Tronco/métodos , Animais , Quimera , Vetores Genéticos/uso terapêutico , Hibridização in Situ Fluorescente , Cariotipagem , Camundongos , Camundongos Transgênicos , Células-Tronco Pluripotentes , Transfecção , Transgenes/genéticaRESUMO
In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.
Assuntos
Cromatina/química , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Animais , Sítios de Ligação/fisiologia , Células Cultivadas , Cromatina/metabolismo , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA Ribossômico/genética , Drosophila/genética , Histonas/genética , Novobiocina/farmacologia , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , RNA de Transferência/genética , Teniposídeo/farmacologiaRESUMO
We have examined the in vivo sites of action for topoisomerases II in the 87A7 heat shock locus as a function of gene activity. When the hsp70 genes are induced, there is a dramatic redistribution of topoisomerase II in the locus which parallels many of the observed alterations in chromatin structure. In addition to changes in the topoisomerase II distribution within the locus, we find topoisomerase II localized around the putative domain boundaries scs and scs'. During recovery, when the chromatin fiber of the locus recondenses, the major sites of action for topoisomerase II appear to be located within the two hsp70 genes and in the intergenic spacer separating the two genes.
Assuntos
Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Choque Térmico/genética , Animais , Mapeamento Cromossômico , DNA Ribossômico/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genes de Insetos , Temperatura Alta , Nucleossomos/metabolismoRESUMO
We have examined the chromatin organization of the 87A7 heat-shock locus (which contains two hsp 70 genes transcribed in opposite orientation) as a function of the time of heat induction and during the course of recovery from heat shock. Our studies show that both induction and recovery from heat shock are accompanied by highly specific alterations in the nucleoprotein structure of this locus. Moreover, these changes parallel the transcriptional activity of the hsp 70 heat-shock genes. We have also examined the effect of inhibitors of transcription and translation. Cycloheximide, an inhibitor of translation, blocks both the attenuation of the heat-shock response (which occurs after a long-term incubation at elevated temperatures) and the re-establishment of the pre-induced chromatin organization of the locus during recovery from heat shock. Actinomycin D, an inhibitor of transcription, prevents some but not all of the alterations in chromatin structure which normally accompany heat induction.
Assuntos
Cromatina/ultraestrutura , Proteínas de Choque Térmico/genética , Animais , Autorradiografia , Cicloeximida/farmacologia , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Dactinomicina/farmacologia , Desoxirribonuclease EcoRI , Drosophila , Eletroforese em Gel de Ágar , Nuclease do Micrococo/metabolismo , Fatores de TempoRESUMO
In vitro transcription of ribosomal RNA was studied on the DNA of the transducing bacteriphage lambdarifd 18, which carries an rRNA transcription unit from Escherichia coli. rRNA synthesis was preferential at all polymerase/DNA ratios tested, and at the optimal 0.3 weight ratio nearly 60% of the transcript was rRNA. At this ratio the principal product of transcription comigrated in acrylamide-agarose electrophoresis with authentic 30-S rRNA precursor synthesized in vivo. In the presence of rifampicin more than one equivalent of rRNA was synthesized, thus suggesting the existence of two initiation sites for rRNA on the phage DNA. Similar results were obtained on E. coli DNA. Preincubation with heparin virtually eliminated the transcription of rRNA, in sharp contrast with the results of similar experiments on bacterial DNA where rRNA genes were transcribed 4--5 times in the presence of heparin. The possible explanation of this difference between rRNA promotors on the phage and the bacterial DNA are discussed.
Assuntos
Colífagos/metabolismo , DNA Viral/metabolismo , RNA Ribossômico/biossíntese , Transcrição Gênica , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Heparina/farmacologia , RNA/biossíntese , Rifampina/farmacologia , Moldes GenéticosRESUMO
The promoter region of two bacterial rRNA genes was investigated by electron-microscopic analysis of polymerase binding, transcription initiation and nitrocellulose filtration of RNA-polymerase-DNA complexes, using restriction endonuclease generated fragments of recombinant plasmids and a transducing phage. The following observations have been made: 1. Two transcription initiation sites have been located approximately 200 and 300 base pairs upstream from the beginning of the sequence coding for mature 16 S rRNA. 2. Polymerase binding at these sites can be observed electronmicroscopically and a 360 base-pair fragment containing these sites binds to nitrocellulose in the presence of RNA-polymerase. This complex dissociates even at moderately high (0.1-0.2 M) salt concentrations. Although transcription initiation is reported to be more frequent at the first of these sites, the binding is much stronger at the second site. 3. In the case of the rrnD gene, BamHI cleaves a few base pairs upstream from the first transcription start site. This cleavage destroys polymerase binding at this site but does not influence binding at the second site. 4. At higher polymerase/DNA ratio four weak but distinct and regularly spaced binding sites can be observed preceding the two initiation sites at approximately 1000, 820, 640 and 440 base pairs before the mature 16 S rRNA sequence. 5. An extremely strong binding site is located about 1300 base pairs upstream from the beginning of the 16 S rRNA sequence. Very little (if any) initiation occurs at this site. The possibility is discussed that the noninitiating binding sites preceding the two transcription start points might functionally belong to the promoter region.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Óperon , RNA Bacteriano/genética , RNA Ribossômico/genética , Sítios de Ligação , Enzimas de Restrição do DNA , Escherichia coli/metabolismo , Microscopia Eletrônica , Transcrição GênicaRESUMO
The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Genes , Óperon , RNA Ribossômico/genética , Transcrição Gênica , Sequência de Bases , Enzimas de Restrição do DNA , DNA Recombinante , PlasmídeosRESUMO
We have used the enzyme micrococcal nuclease and the methylating reagent dimethyl sulfate to examine the structural properties of eukaryotic DNAs. Our studies demonstrate extensive structural polymorphism in the DNA double helix. Moreover, we find that the distribution of helical variants is in some instances correlated with the functional organization of the DNA. These observations raise the possibility that eukaryotic DNAs may be organized into discrete functional units having characteristic structural properties. In addition, we find that boundaries between different functional units are typically marked by DNA segments having unusual conformational properties. Such structural perturbations could serve as signals in the utilization of genetic information in eukaryotes, and may be important in a variety of different protein-DNA interactions.
Assuntos
DNA , Polimorfismo Genético , Animais , Sequência de Bases , Desoxirribonucleotídeos/análise , Drosophila melanogaster , Eletroforese em Gel de Ágar , Histonas , Metilação , Nuclease do Micrococo/metabolismo , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Cloreto de Sódio , Ésteres do Ácido Sulfúrico/farmacologiaRESUMO
We have examined the chromatin structure of the hsp 70 gene complex at the 87A7 heat shock locus of Drosophila melanogaster. Our results indicate that this locus has a complex chromatin organization. Heat induction causes highly specific alterations in the chromatin throughout the locus. There are major changes within the heat shock gene transcription units, and in both the upstream and downstream flanking spacers.
Assuntos
Cromatina/análise , Mapeamento Cromossômico , Proteínas de Choque Térmico/genética , Animais , DNA , Enzimas de Restrição do DNA , DNA Recombinante , DNA Ribossômico , Drosophila melanogaster/genética , Eletroforese em Gel de Ágar , GenesRESUMO
We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the hsp 83 heat shock gene from the Drosophila melanogaster cytogenetic locus 63 BC. Both of these nucleases generate double strand breaks in chromatin at specific sites close to the 5' end of the hsp 83 gene. With N. crassa nuclease we observe one major 5' fragment which is derived from nuclease cleavage in a DNA segment mapping approximately 120 base-pairs from the beginning of the transcription unit. With S1 nuclease we observe one major fragment which overlaps the transcription start site. In addition to the major hypersensitive sites at the beginning of the gene, the hsp 83 transcription unit is also sensitive to attack by these nucleases both before and after heat shock; however, the yield of cleavage products from within the gene is considerably greater after heat induction.
Assuntos
Cromatina/análise , Endonucleases , Proteínas de Choque Térmico/genética , Nuclease do Micrococo , Neurospora crassa/enzimologia , Neurospora/enzimologia , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Fúngico , Drosophila melanogaster/genética , Eletroforese em Gel de Ágar , Genes , Temperatura Alta , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
In the studies reported here we show that the antibiotic novobiocin, an in vitro inhibitor of topoisomerase II, blocks the Drosophila heat shock response. If novobiocin is added prior to induction, there is no detectable expression of the Drosophila heat shock genes. Moreover, analysis of the chromatin organization of the 87A7 heat shock locus indicates that the antibiotic prevents the structural alterations which normally accompany heat induction. When novobiocin is added after induction, transcription appears to be rapidly turned off, and the chromatin organization of the 87A7 locus is "fixed" in an "active" configuration. Novobiocin also prevents the re-establishment of the pre-induced 87A7 chromatin organization which occurs during recovery from heat shock. We have also presented data suggesting that this antibiotic blocks transcription at 25 degrees C. These findings raise the possibility that topoisomerase II may be required in eukaryotes for both gene activation and deactivation.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Novobiocina/farmacologia , Autorradiografia , Cromatina/efeitos dos fármacos , Desoxirribonucleases , Drosophila/genética , Endonucleases , Genes/efeitos dos fármacos , Temperatura Alta , Nuclease do Micrococo , Neurospora crassa/enzimologia , RNA Mensageiro/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Ativação TranscricionalRESUMO
The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.
Assuntos
Cromatina , Cromossomos/análise , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Desoxirribonuclease I/metabolismo , Drosophila melanogaster/genética , Endonucleases/metabolismo , Exonucleases/metabolismo , Proteínas de Choque Térmico/genética , Substâncias Macromoleculares , Nuclease do Micrococo/metabolismo , Neurospora crassa/enzimologia , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the 87A7 heat shock locus of Drosophila melanogaster. Both of these nucleases generate single and double-strand breaks in chromatin at specific sites in the 87A7 locus. Before heat induction, we find that the 5' ends of the two 87A7 hsp 70 genes contain N. crassa and S1 nuclease hypersensitive sites, while there are only a few cleavage products from elsewhere in the locus. With N. crassa nuclease, we observe one major 5' fragment, and this is derived from cleavage in a DNA segment mapping about 90 to 115 base-pairs from the beginning of the transcription unit. With S1 nuclease, we find two 5' cleavage products. The first maps about 120 to 130 base-pairs from the beginning of the gene. Interestingly, this site is also sensitive to S1 nuclease in supercoiled but not linear naked DNA. The other fragment maps very close to the transcription start site (approximately 0 to -15 base-pairs). After heat induction, there is a transition in the chromatin architecture of 87A7. First, there is a marked reduction in the yield of the prominent 5' N. crassa and S1 nuclease fragments. Second, the entire hsp 70 gene, as well as the spacer DNA just downstream from the 3' end of the gene, becomes highly sensitive to both of these nucleases.
Assuntos
Cromatina , Endonucleases , Transcrição Gênica , Animais , Cromatina/genética , Mapeamento Cromossômico , DNA , Enzimas de Restrição do DNA , DNA de Cadeia Simples , DNA Super-Helicoidal , Drosophila melanogaster , Eletroforese em Gel de Ágar , Genes , Proteínas de Choque Térmico/genética , Temperatura Alta , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The sequence dependence of Drosophila topoisomerase II supercoil relaxation and binding activities has been examined. The DNA substrates used in binding experiments were two fragments from Drosophila heat shock locus 87A7. One of these DNA fragments includes the coding region for the heat shock protein hsp70, and the other includes the intergenic non-coding region that separates two divergently transcribed copies of the hsp70 gene at the locus. The intergenic region was previously shown to have a much higher density of topoisomerase cleavage sites than the hsp70 coding region. Competition nitrocellulose filter binding assays demonstrate a preferential binding of the intergene fragment, and that binding specificity increases with increasing ionic strength. Dissociation kinetics indicate a greater kinetic stability of topoisomerase II complexes with the intergene DNA fragment. To study topoisomerase II relaxation activity, we used supercoiled plasmids that contained the same fragments from locus 87A7 cloned as inserts. The relative relaxation rates of the two plasmids were determined under several conditions of ionic strength, and when the plasmid substrates were included in separate reactions or when they were mixed in a single reaction. The relaxation properties of these two plasmids can be explained by a coincidence of high-affinity binding sites, strong cleavage sites, and sites used during the catalysis of strand passage events by topoisomerase II. Sequence dependence of topoisomerase II catalytic activity may therefore parallel the sequence dependence of DNA cleavage by this enzyme.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Drosophila/enzimologia , Plasmídeos , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Eletroforese em Gel de Ágar , Cinética , Concentração OsmolarRESUMO
We have examined the chromatin organization of the Drosophila melanogaster ribosomal RNA genes using both micrococcal nuclease and DNase I. Several findings are of interest. First, the transcribed DNA segments of the rRNA repeat unit appear to be packaged into an unstable or "multiphasic" nucleosome structure. Second, the 5' end of the transcription unit is preferentially exposed to nuclease attack. Third, the non-transcribed spacer immediately upstream from the transcription start site has a novel chromatin organization with micrococcal nuclease and DNase I cleavage sites spaced at intervals of about 240 base-pairs. This unusual fragment distribution appears to reflect the underlying sequence organization of the spacer DNA segment, which consists of a series of tandemly repeated 239 base-pair sequence blocks. We have also examined the chromatin structure of the rRNA repeat unit after extraction of nuclei with different concentrations of salt. Our results suggest that the higher order structures may be of importance in determining the novel chromatin organization of the rRNA repeat unit.
Assuntos
Cromatina , Genes , RNA Ribossômico/genética , Animais , Autorradiografia , Desoxirribonuclease I , Drosophila melanogaster/genética , Endodesoxirribonucleases , Nuclease do Micrococo , Concentração Osmolar , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.
Assuntos
Cromatina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Autorradiografia , Cromatina/efeitos dos fármacos , DNA Super-Helicoidal/metabolismo , Drosophila/enzimologia , Ácido Edético/farmacologia , Teniposídeo/farmacologia , Inibidores da Topoisomerase IIRESUMO
The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.
Assuntos
Drosophila melanogaster/genética , Mitose/genética , Mutação , Fosfoproteínas Fosfatases/genética , Animais , Northern Blotting , Mapeamento Cromossômico , Elementos de DNA Transponíveis , Feminino , Teste de Complementação Genética , Heterozigoto , Masculino , Mosaicismo , Proteína Fosfatase 1 , Proteínas Repressoras/genética , Transformação GenéticaRESUMO
The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.