Spontaneous X chromosome MI and MII nondisjunction events in Drosophila melanogaster oocytes have different recombinational histories.

Recent studies of human oocytes have demonstrated an enrichment for distal exchanges among meiosis I (MI) nondisjunction events and for proximal exchanges among meiosis II (MII) events. Our characterization of 103 cases of spontaneous X chromosome nondisjunction in Drosophila oocytes strongly parallels these observations. The recombinational histories of MI (97/103) and MII (6/103) nondisjunctional ova were strikingly different. MI nondisjunction occurred primarily in oocytes with non-exchange X chromosomes; of the new nondisjoining exchange bivalents, most carried distal crossovers. Thus, spontaneous MI nondisjunction reflects the failure of the achiasmate segregation systems. MII nondisjunction occurred only in oocytes with proximal exchanges. We propose several models to explain how very proximal exchanges might impair proper segregation.

Altered nuclear transport and the most selfish of genes.

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.

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

Identification of the chromosome localization domain of the Drosophila nod kinesin-like protein.

The nod kinesin-like protein is localized along the arms of meiotic chromosomes and is required to maintain the position of achiasmate chromosomes on the developing meiotic spindle. Here we show that the localization of ectopically expressed nod protein on mitotic chromosomes precisely parallels that observed for wild-type nod protein on meiotic chromosomes. Moreover, the carboxyl-terminal half of the nod protein also binds to chromosomes when overexpressed in mitotic cells, whereas the overexpressed amino-terminal motor domain binds only to microtubules. Chromosome localization of the carboxyl-terminal domain of nod depends upon an 82-amino acid region comprised of three copies of a sequence homologous to the DNA-binding domain of HMG 14/17 proteins. These data map the two primary functional domains of the nod protein in vivo and provide a molecular explanation for the directing of the nod protein to a specific subcellular component, the chromosome.

Mutations in the alpha-tubulin 67C gene specifically impair achiasmate segregation in Drosophila melanogaster.

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.

Chromosomal control of meiotic cell division.

Chromosomes have multiple roles both in controlling the cell assembly and structure of the spindle and in determining chromosomal position on the spindle in many meiotic cells and in some types of mitotic cells. Moreover, functionally significant chromosome-microtubule interactions are not limited to the kinetochore but are also mediated by proteins localized along the arms of chromosomes. Finally, chromosomes also play a crucial role in control of the cell cycle.

Induction of metaphase arrest in Drosophila oocytes by chiasma-based kinetochore tension.

In normal Drosophila melanogaster oocytes, meiosis arrests at metaphase I and resumes after oocyte passage through the oviduct. Thus, metaphase arrest defines a control point in the meiotic cell cycle. Metaphase arrest only occurs in oocytes that have undergone at least one meiotic exchange. Here it is shown that crossovers between homologs attached to the same centromere do not induce metaphase arrest. Hence, exchanges induce metaphase arrest only when they physically conjoin two separate kinetochores. Thus, the signal that mediates metaphase arrest is not the exchange event per se but the resulting tension on homologous kinetochores.

Meiotic synapsis in the absence of recombination.

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.

Drosophila Nod protein binds preferentially to the plus ends of microtubules and promotes microtubule polymerization in vitro.

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.

Requiem for distributive segregation: achiasmate segregation in Drosophila females.

The segregation of achiasmate chromosome pairs at meiosis I is not brought about by a single 'distributive system' as previously thought, but rather by two separate mechanisms. One system uses the pairing of proximal heterochromatic sequences to mediate the segregation of achiasmate homologs-an observation that, at long last, defines a function for heterochromatin. The other system facilitates the segregation of heterologous chromosomes, by an as yet undiscovered mechanism.

Orphan kinesin NOD lacks motile properties but does possess a microtubule-stimulated ATPase activity.

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.

Drosophila and human RecQ5 exist in different isoforms generated by alternative splicing.

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.

Chromosomal sites necessary for normal levels of meiotic recombination in Drosophila melanogaster. I. Evidence for and mapping of the sites.

Meiotic exchange was measured in females heterozygous for a normal sequence X chromosome and for each of eleven T(1;4)s and each of sixteen T(1;Y)s. The results indicate that the X chromosome can be divided into five intervals, such that heterozygosity for a breakpoint in one interval strongly suppresses exchange within that interval, but has little or no effect on exchange in other intervals. The boundaries between these intervals are identified and mapped to regions 3C4-6/7, 7A-7E, 11A and proximal to 18C on the standard salivary map; each boundary is located at (or within a small region containing) a major constriction (i.e., a block of intercalary heterochromatin).--Exchange was examined in females heterozygous for translocations broken within the constriction at 11A. The results imply that a boundary occupies only a subregion of the entire constriction and is subdivisible by translocation breakpoints. Several other properties of boundaries have been elucidated. Finally, the relationship of these data to a simple model of meiotic pairing proposed by I. Sandler (1956) and to the role of intercalary heterochromatin in the meiotic process is discussed.

The genetic analysis of distributive segregation in Drosophila melanogaster. I. Isolation and characterization of Aberrant X segregation (Axs), a mutation defective in chromosome partner choice.

We describe the isolation and characterization of Aberrant X segregation (Axs), a dominant female-specific meiotic mutation. Although Axs has little or no effect on the frequency or distribution of exchange, or on the disjunction of exchange bivalents, nonexchange X chromosomes undergo nondisjunction at high frequencies in Axs/+ and Axs/Axs females. This increased X chromosome nondisjunction is shown to be a consequence of an Axs-induced defect in distributive segregation. In Axs-bearing females, fourth chromosome nondisjunction is observed only in the presence of nonexchange X chromosomes and is argued to be the result of improper X and fourth chromosome associations within the distributive system. In XX females bearing a compound fourth chromosome, the frequency of nonhomologous disjunction of the X chromosomes from the compound fourth chromosome is shown to account for at least 80% of the total X nondisjunction observed. In addition, Axs diminishes or ablates the capacity of nonexchange X chromosomes to form trivalents in females bearing either a Y chromosome or a small free duplication for the X. Axs also impairs compound X from Y segregation. The effect of Axs on these segregations parallels the defects observed for homologous nonexchange X chromosome disjunction in Axs females. In addition to its dramatic effects on the X chromosome, Axs exerts a similar effect on the segregation of a major autosome. We conclude that Axs defines a locus required for proper homolog disjunction within the distributive system.

Tales from the front lines: the creative essay as a tool for teaching genetics.

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.

The effect of mei-41 on rDNA redundancy in Drosophila melanogaster.

The recombination and repair defective mutant, mei-41, exhibits three rather striking effects on the genetic properties and chromosomal stability of rDNA in Drosophila. First, mei-41 inhibits rDNA magnification. However, mei-9, another recombination and repair defective mutation has no similar effect. This indicates that magnification requires some, but not all, of the gene products necessary for meiotic exchange. Second, under magnifying conditions, mei-41 induces interchanges between the X rDNA and either arm of the Ybb- chromosome. These interchanges occur at high frequency and are independent of rDNA orientation. Third, in mei-41 bb+/Ybb+ males, bobbed mutants in the X, but not the Y, also arise at high frequency. Evidence suggests that these events involve the rDNA type I insertion. The recombination and repair defective properties of mei-41 together with our results regarding its unusual and specific effects involving rDNA are explained in a simple model that has general implications for chromosome structure.

The genetic analysis of distributive segregation in Drosophila melanogaster. II. Further genetic analysis of the nod locus.

In Drosophila melanogster females the segregation of nonexchange chromosomes is ensured by the distributive segregation system. The mutation noda specifically impairs distributive disjunction and induces nonexchange chromosomes to undergo nondisjunction, as well as both meiotic and mitotic chromosome loss. We report here the isolation of seven recessive X-linked mutations that are allelic to noda. As homozygotes, all of these mutations exhibit a phenotype that is similar to that exhibited by noda homozygotes. We have also used these mutations to demonstrate that nod mutations induce nonexchange chromosomes to nondisjoin at meiosis II. Our data demonstrate that the effects of noda on meiotic chromosome behavior are a general property of mutations at the nod locus. Several of these mutations exhibit identical phenotypes as homozygotes and as heterozygotes with a deficiency for the nod locus; these likely correspond to complete loss-of-function or null alleles. None of these mutations causes lethality, decreases the frequency of exchange, or impairs the disjunction of exchange chromosomes in females. Thus, either the nod locus defines a function that is specific to distributive segregation or exchange can fully compensate for the absence of the nod+ function.

A two-stage model for the control of rDNA magnification.

Males of the genotype bb/Ybb- have been shown to produce both magnified (bbm+) and, less frequently, reduced (bbrl) X chromosomes. An analysis of the progeny of single magnifying bb/Ybb- males reveals that bbm+ revertants may be recovered either as rare single events or, more frequently, in large clusters. To analyze the role of the bb phenotype in the induction of rDNA magnification we have constructed a series of bb and bb+ derivatives of Ybb-. Males carrying an X chromosomal bb allele and one of these derivatives (bb/bbYbb- or bb/bb+Ybb-) produce small numbers (one to two) of bbm+ progeny at a frequency similar to that observed for bb/Ybb- males but do not produce large clusters of bbm+ revertants. In addition, bb/bb+Ybb- males produce essentially equal numbers of magnified (bbm+) and reduced (bbrl) X chromosomes. These data, together with a consideration of the growth properties of the male germline in Drosophila, suggest that magnification/reduction may occur at two different times during development. Those events that give rise to large clusters, and, thus, necessarily arise early in germ cell development, appear to be dependent on the bb phenotype. However, those events that give rise to single bbm+ chromosomes arise late in spermatogenesis, probably at meiosis, and are independent of the bb phenotype.

Cloning of the Drosophila melanogaster meiotic recombination gene mei-218: a genetic and molecular analysis of interval 15E.

The mei-218 gene product is required for both meiotic crossing over and for the production of recombination modules, suggesting that these organelles are required for meiotic exchange. In this study the null phenotype of mei-218 was defined through the analysis of three preexisting and five new alleles. Consistent with previous studies, in homozygous mei-218 mutants meiotic crossing over is reduced to < 10% of normal levels. A molecular analysis of mei-218 was initiated with the isolation and mapping of lethal mutations and genome rearrangements in the region containing mei-218, polytene interval 15E on the X chromosome. This high resolution genetic map was aligned with a physical map constructed from cosmid and P1 clones by genetically mapping restriction fragment length polymorphisms and localizing rearrangement breakpoints. Within a region of 65 kb, we have identified seven transcription units, including mei-218 and the Minute(1)15D gene, which encodes ribosomal protein S5. The mei-218 mutant phenotype has been rescued by germline transformation with both a genomic fragment and a cDNA under the control of the hsp83 promoter. The mei-218 gene is predicted to produce an 1186-amino acid protein that has no significant similarities to any known proteins.