R. Scott Hawley

Direct evidence of a role for heterochromatin in meiotic chromosome segregation.

We have investigated the mechanism that enables achiasmate chromosomes to segregate from each other at meiosis I in D. melanogaster oocytes. Using novel cytological methods, we asked whether nonexchange chromosomes are paired prior to disjunction. Our results show that the heterochromatin of homologous chromosomes remains associated throughout prophase until metaphase I regardless of whether they undergo exchange, suggesting that homologous recognition can lead to segregation even in the absence of chiasmata. However, partner chromosomes lacking homology do not pair prior to disjunction. Furthermore, euchromatic synapsis is not maintained throughout prophase. These observations provide a physical demonstration that homologous and heterologous achiasmate segregations occur by different mechanisms and establish a role for heterochromatin in maintaining the alignment of chromosomes during meiosis.

The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene

The D. melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes. Here we show that the predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins. There is also extensive functional overlap between mei-41 and ATM. Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability. We also demonstrate that mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells. Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene.

Homologous chromosome interactions in meiosis: diversity amidst conservation

Proper chromosome segregation is crucial for preventing fertility problems, birth defects and cancer. During mitotic cell divisions, sister chromatids separate from each other to opposite poles, resulting in two daughter cells that each have a complete copy of the genome. Meiosis poses a special problem in which homologous chromosomes must first pair and then separate at the first meiotic division before sister chromatids separate at the second meiotic division. So, chromosome interactions between homologues are a unique feature of meiosis and are essential for proper chromosome segregation. Pairing and locking together of homologous chromosomes involves recombination interactions in some cases, but not in others. Although all organisms must match and lock homologous chromosomes to maintain genome integrity throughout meiosis, recent results indicate that the underlying mechanisms vary in different organisms.

DNA binding and meiotic chromosomal localization of the drosophila nod kinesin-like protein

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. We report that the nonmotor domain of the nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed nod protein, we show that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motor-based chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force.

Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ∼70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.

Heterochromatic Threads Connect Oscillating Chromosomes during Prometaphase I in Drosophila Oocytes

In Drosophila oocytes achiasmate homologs are faithfully segregated to opposite poles at meiosis I via a process referred to as achiasmate homologous segregation. We observed that achiasmate homologs display dynamic movements on the meiotic spindle during mid-prometaphase. An analysis of living prometaphase oocytes revealed both the rejoining of achiasmate X chromosomes initially located on opposite half-spindles and the separation toward opposite poles of two X chromosomes that were initially located on the same half spindle. When the two achiasmate X chromosomes were positioned on opposite halves of the spindle their kinetochores appeared to display proper co-orientation. However, when both Xs were located on the same half spindle their kinetochores appeared to be oriented in the same direction. Thus, the prometaphase movement of achiasmate chromosomes is a congression-like process in which the two homologs undergo both separation and rejoining events that result in the either loss or establishment of proper kinetochore co-orientation. During this period of dynamic chromosome movement, the achiasmate homologs were connected by heterochromatic threads that can span large distances relative to the length of the developing spindle. Additionally, the passenger complex proteins Incenp and Aurora B appeared to localize to these heterochromatic threads. We propose that these threads assist in the rejoining of homologs and the congression of the migrating achiasmate homologs back to the main chromosomal mass prior to metaphase arrest.

Hanging on to your homolog: the roles of pairing, synapsis and recombination in the maintenance of homolog adhesion.

Abstract.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 Saccharomycescerevisiae 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.

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

The Molecular Control of Meiotic Chromosomal Behavior: Events in Early Meiotic Prophase in Drosophila Oocytes

We review the critical events in early meiotic prophase in Drosophila melanogaster oocytes. We focus on four aspects of this process: the formation of the synaptonemal complex (SC) and its role in maintaining homologous chromosome pairings, the critical roles of the meiosis-specific process of centromere clustering in the formation of a full-length SC, the mechanisms by which preprogrammed double-strand breaks initiate meiotic recombination, and the checkpoints that govern the progression and coordination of these processes. Central to this discussion are the roles that somatic pairing events play in establishing the necessary conditions for proper SC formation, the roles of centromere pairing in synapsis initiation, and the mechanisms by which oocytes detect failures in SC formation and/or recombination. Finally, we correlate what is known in Drosophila oocytes with our understanding of these processes in other systems.

Comparing Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases for Gene Targeting in Drosophila

Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.