RESUMEN
Nod, a nonmotile kinesin-like protein, plays a critical role in segregating achiasmate chromosomes during female meiosis. In addition to localizing to oocyte chromosomes, we show that functional full-length Nod-GFP (Nod(FL)-GFP) localizes to the posterior pole of the oocyte at stages 9-10A, as does kinesin heavy chain (KHC), a plus end-directed motor. This posterior localization is abolished in grk mutants that no longer maintain the microtubule (MT) gradient in the oocyte. To test the hypothesis that Nod binds to the plus ends of MTs, we expressed and purified both full-length Nod (Nod(FL)) and a truncated form of Nod containing only the motor-like domain (Nod318) from Escherichia coli and assessed their interactions with MTs in vitro. Both Nod(FL) and Nod318 demonstrate preferential binding to the ends of the MTs, displaying a strong preference for binding to the plus ends. When Nod318-GFP:MT collision complexes were trapped by glutaraldehyde fixation, the preference for binding to plus ends versus minus ends was 17:1. Nod(FL) and Nod318 also promote MT polymerization in vitro in a time-dependent manner. The observation that Nod is preferentially localized to the plus ends of MTs and stimulates MT polymerization suggests a mechanism for its function.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Proteínas de Microtúbulos/metabolismo , Proteínas de Microtúbulos/fisiología , Microtúbulos/metabolismo , Animales , Cromosomas/ultraestructura , Técnicas In Vitro , Cinesinas , Oocitos/ultraestructura , Polímeros/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Moduladores de Tubulina/metabolismoRESUMEN
NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD(DTW) and NOD("DR2")) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Adenina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Drosophila/genética , Cinesinas , Cinética , Proteínas de Microtúbulos/genética , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/metabolismoRESUMEN
The meiotic mutant c(3)G (crossover suppressor on 3 of Gowen) abolishes both synaptonemal complex (SC) formation and meiotic recombination, whereas mutations in the mei-W68 and mei-P22 genes prevent recombination but allow normal SC to form. These data, as well as a century of cytogenetic studies, support the argument that meiotic recombination between homologous chromosomes in Drosophila females requires synapsis and SC formation. We have cloned the c(3)G gene and shown that it encodes a protein that is structurally similar to SC proteins from yeast and mammals. Immunolocalization of the C(3)G protein, as well as the analysis of a C(3)G-eGFP expression construct, reveals that C(3)G is present in a thread-like pattern along the lengths of chromosomes in meiotic prophase, consistent with a role as an SC protein present on meiotic bivalents. The availability of a marker for SC in Drosophila allowed the investigation of the extent of synapsis in exchange-defective mutants. These studies indicate that SC formation is impaired in certain meiotic mutants and that the synaptic defect correlates with the exchange defects. Moreover, the observation of interference among the residual exchanges in these mutant oocytes implies that complete SC formation is not required for crossover interference in Drosophila.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genes de Insecto/genética , Complejo Sinaptonémico/química , Complejo Sinaptonémico/genética , Animales , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas/química , Cromosomas/genética , Cromosomas/metabolismo , Clonación Molecular , Proteínas de Drosophila/química , Femenino , Mutación de Línea Germinal/genética , Masculino , Meiosis/genética , Microscopía Fluorescente , Modelos Moleculares , Mutación/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Oocitos/metabolismo , Ovario/citología , Ovario/metabolismo , Conformación Proteica , Transporte de Proteínas , Recombinación Genética/genética , Transformación GenéticaRESUMEN
The Segregation Distorter System in Drosophila is one of the best-known and genetically characterized systems of meiotic drive. A recent paper by Kusano et al. (2001) provides a key molecular insight into the molecular mechanism by which one chromosome can ensure the destruction of its partner.
Asunto(s)
Núcleo Celular/metabolismo , Segregación Cromosómica , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Activadoras de GTPasa/metabolismo , Meiosis , Transporte Activo de Núcleo Celular , Animales , Drosophila melanogaster/fisiología , Femenino , Proteínas Activadoras de GTPasa/genética , Genes de Insecto , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
We present the cloning and characterization of mei-P26, a novel P-element-induced exchange-defective female meiotic mutant in Drosophila melanogaster. Meiotic exchange in females homozygous for mei-P26(1) is reduced in a polar fashion, such that distal chromosomal regions are the most severely affected. Additional alleles generated by duplication of the P element reveal that mei-P26 is also necessary for germline differentiation in both females and males. To further assess the role of mei-P26 in germline differentiation, we tested double mutant combinations of mei-P26 and bag-of-marbles (bam), a gene necessary for the control of germline differentiation and proliferation in both sexes. A null mutation at the bam locus was found to act as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P26(1); bam(Delta)(86)/+ females is also severely decreased in comparison to mei-P26(1) homozygotes, indicating that bam affects the meiotic phenotype as well. These data suggest that the pathways controlling germline differentiation and meiotic exchange are related and that factors involved in the mitotic divisions of the germline may regulate meiotic recombination.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Drosophila , Drosophila/genética , Células Germinativas/citología , Células Germinativas/fisiología , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Meiosis/genética , Alelos , Animales , División Celular , Clonación Molecular , Drosophila/citología , Drosophila/fisiología , Elementos de Facilitación Genéticos/genética , Femenino , Genes Dominantes , Infertilidad/genética , Masculino , Modelos Genéticos , Mutagénesis , No Disyunción Genética , Fenotipo , Plásmidos/genética , Recombinación Genética , Transcripción Genética , Transformación Genética , Cromosoma X/genética , Dedos de ZincRESUMEN
Homologous chromosomes initially undergo weak alignments that bring homologous sequences into register during meiosis. These alignments can be facilitated by two types of mechanisms: interstitial homology searches and telomere-telomere alignments. As prophase (and chromatin compaction) proceeds, these initial pairings or alignments need to be stabilized. In at least some organisms, such as Saccharomyces cerevisiae and S. pombe, these pairings can apparently be maintained by the creation of recombination intermediates. In contrast, synapsis during zygotene may be able to facilitate and/or maintain chromosome pairing even in the absence of exchange in several higher organisms. It thus seems possible that the synaptonemal complex plays a role both in maintaining homolog adhesion during meiotic prophase and, more speculatively, in facilitating meiotic exchange.
Asunto(s)
Emparejamiento Base/genética , Segregación Cromosómica/genética , Cromosomas/genética , Meiosis/genética , Recombinación Genética/genética , Homología de Secuencia de Ácido Nucleico , Animales , Bombyx , Caenorhabditis elegans , ADN/genética , Drosophila , Modelos Genéticos , Profase/genética , Saccharomyces cerevisiae , Schizosaccharomyces , Telómero/genéticaRESUMEN
This paper reports on a new role for mei-41 in cell cycle control during meiosis. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41 mei-218 double mutants, however, metaphase arrest was restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect.
Asunto(s)
Anafase/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila , Drosophila/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Rotura Cromosómica/genética , Proteínas del Huevo/genética , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacología , Péptidos y Proteínas de Señalización Intracelular , Meiosis/genética , Metafase/genética , Mutagénesis Sitio-Dirigida , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Recombinación Genética , Transducción de Señal/genética , Huso Acromático/genéticaRESUMEN
Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Endonucleasas/genética , Proteínas Nucleares , Proteínas/genética , Alelos , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Drosophila melanogaster/enzimología , Endodesoxirribonucleasas/genética , Femenino , Endonucleasas de ADN Solapado , Mutación del Sistema de Lectura , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción , Técnicas del Sistema de Dos HíbridosRESUMEN
Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3epsilon, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene.
Asunto(s)
Daño del ADN/genética , Drosophila/embriología , Fase G2/genética , Mitosis/genética , Animales , Reparación del ADN/genética , Embrión no Mamífero/anomalías , Ojo/citología , Ojo/embriología , Femenino , Pérdida de Heterocigocidad , Meiosis/genética , MutaciónRESUMEN
Drosophila melanogaster oocytes heterozygous for mutations in the alpha-tubulin 67C gene (alphatub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the alphatub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase.
Asunto(s)
Segregación Cromosómica/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto/genética , Mutación/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Animales , Centrómero/genética , Centrómero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Dosificación de Gen , Genes Dominantes/genética , Genes Dominantes/fisiología , Cinesinas , Meiosis/genética , Proteínas de Microtúbulos/genética , Proteínas de Microtúbulos/metabolismo , Modelos Genéticos , No Disyunción Genética , Oocitos/citología , Oocitos/metabolismo , Huso Acromático/genética , Huso Acromático/fisiología , Cromosoma X/genética , Cromosoma X/metabolismoRESUMEN
Members of the RecQ helicase superfamily have been implicated in DNA repair, recombination and replication. Although the genome of the budding yeast Saccharomyces cerevisiae encodes only a single member of this family, there are at least five human RecQ-related genes: RecQL, BLM, WRN, RecQ4 and RecQ5. Mutations in at least three of these are associated with diseases involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability. Metazoan RecQ helicases are defined by a core region with characteristic helicase motifs and sequence similarity to Escherichia coli RecQ protein. This core region is typically flanked by extensive, highly charged regions, of largely unknown function. The recently reported human RecQ5, however, has only the core RecQ-homologous region. We describe here the identification of the Drosophila RecQ5 gene. We recovered cDNAs corresponding to three alternative splice forms of the RecQ5 transcript. Two of these generate nearly identical 54 kDa proteins that, like human RecQ5, consist of the helicase core only. The third splice variant encodes a 121 kDa isoform that, like other family members, has a C-terminal extension rich in charged residues. A combination of RACE and cDNA analysis of human RECQ5 demonstrates extensive alternative splicing for this gene also, including some forms lacking helicase motifs and other conserved regions.
Asunto(s)
Empalme Alternativo/genética , ADN Helicasas/genética , Drosophila melanogaster/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada/genética , ADN Helicasas/química , ADN Helicasas/metabolismo , Drosophila melanogaster/citología , Etiquetas de Secuencia Expresada , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , RecQ Helicasas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In contrast to the more typical mock grant proposals or literature reviews, we describe the use of the creative essay as a novel tool for teaching human genetics at the college level. This method has worked well for both nonmajor and advanced courses for biology majors. The 10- to 15-page essay is written in storylike form and represents a student's response to the choice of 6-8 scenarios describing human beings coping with various genetic dilemmas. We have found this tool to be invaluable both in developing students' ability to express genetic concepts in lay terms and in promoting student awareness of genetic issues outside of the classroom. Examples from student essays are presented to illustrate these points, and guidelines are suggested regarding instructor expectations of student creativity and scientific accuracy. Methods of grading this assignment are also discussed.
Asunto(s)
Genética/educación , Humanos , Universidades , EscrituraRESUMEN
The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.
Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Genes de Insecto , Meiosis/genética , Animales , Cromosomas/genética , ADN/genética , Femenino , Heterocromatina , Masculino , Metafase , Mutación , No Disyunción Genética , Fenotipo , Recombinación Genética , Proyectos de Investigación , Cromosoma X/genéticaRESUMEN
Flavonoid plant pigments are an integral part of the human diet. Although potentially negative mitotic effects of flavonoids have been observed in model organisms, investigation into meiotic effects of flavonoids has been neglected. As flavonoids affect cell signalling and DNA replication, and because the flavonoid content of the human food supply is being increased, determining the effects of flavonoids on meiotic fidelity is important. Here, the effect of the human food supply's most prevalent flavonoid, quercetin, on the level of meiotic recombination and the amount of X and 4th chromosome non-disjunction in Drosophila melanogaster females was determined. This model organism was chosen since Drosophila melanogaster and Homo sapiens share a remarkable number of commonalities in the meiotic processes of oogenesis and because genetic techniques allow a detailed analysis of meiotic processes in Drosophila. No significant effect on either non-disjunction levels or the percentage distribution of exchange bivalents was observed. A significant effect was observed on the number of offspring; F1 and F2 generations of flies raised on a quercetin diet produced over 10% more progeny than flies raised on a control diet. In this investigation, high quercetin consumption by Drosophila melanogaster females did not pose a threat to meiotic fidelity.
Asunto(s)
Dieta , Drosophila melanogaster/efectos de los fármacos , Meiosis/efectos de los fármacos , No Disyunción Genética , Quercetina/toxicidad , Cromosoma X/efectos de los fármacos , Animales , Drosophila melanogaster/genética , Femenino , Genes de Insecto/efectos de los fármacos , Masculino , Mutagénesis/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Reproducción/efectos de los fármacos , Cromosoma X/genéticaAsunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas Nucleares , Animales , ADN Polimerasa I/genética , Enzimas Reparadoras del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN , Meiosis , Antígeno Nuclear de Célula en Proliferación/genética , Recombinación GenéticaRESUMEN
Although in Saccharomyces cerevisiae the initiation of meiotic recombination, as indicated by double-strand break formation, appears to be functionally linked to the initiation of synapsis, meiotic chromosome synapsis in Drosophila females occurs in the absence of meiotic exchange. Electron microscopy of oocytes from females homozygous for either of two meiotic mutants (mei-W68 and mei-P22), which eliminate both meiotic crossing over and gene conversion, revealed normal synaptonemal complex formation. Thus, synapsis in Drosophila is independent of meiotic recombination, consistent with a model in which synapsis is required for the initiation of meiotic recombination. Furthermore, the basic processes of early meiosis may have different functional or temporal relations, or both, in yeast and Drosophila.