Normal Segregation of a Foreign-Species Chromosome During Drosophila Female Meiosis Despite Extensive Heterochromatin Divergence

The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. Here we use a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D. melanogaster. These two species differ by ∼12–13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. We find a visible imbalance in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, we find that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females and does not experience elevated nondisjunction. We conclude that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence. Animal chromosomes typically contain large amounts of noncoding repetitive DNA that nevertheless varies widely between species. This variation may potentially induce non-Mendelian transmission of chromosomes. We have examined the meiotic properties and transmission of a highly diverged chromosome 4 from a foreign species within the fruitfly Drosophila melanogaster. This chromosome has substantially less of a simple sequence repeat than does D. melanogaster 4, and we find that this difference results in altered positioning when chromosomes align during meiosis. Yet this foreign chromosome segregates at normal frequencies, demonstrating that chromosome segregation can be robust to major differences in repetitive DNA abundance.     

The Influence of Chromosome Content on the Size and Shape of Sperm Heads in DROSOPHILA MELANOGASTER and the Demonstration of Chromosome Loss during Spermiogenesis

The volumes of sperm heads were estimated from three-dimensional reconstructions of serially sectioned bundles of nearly mature spermatid nuclei. Cysts from males in which all sperm are expected to have comparable amounts of chromatin (X/Y and In(3LR)/+) show unimodal frequency distributions of nuclear volumes, whereas cysts from males in which meiotic segregation is expected to deliver unequal amounts of chromatin material to spermatid nuclei show two (XY/O and XY/Y) or more (T(2;3)/⁺ and C(2L);C(2R)) modes. The mean volumes of the subpopulations in these cases are related in the same proportions as the metaphase lengths of their chromosomal complements. Thus the volumes of sperm nuclei are proportional to their DNA content. Sperm head shape, on the other hand, does not appear to be very sensitive to chromosomal constitution, as heads of different size do not vary greatly in shape.—The numbers of sperm heads in the various size classes in a cyst depart from mendelian expectations; these departures are caused by the elimination, during individualization, of chromosomes contained within micronuclei that are formed in spermatids at the end of the second meiotic division. The effect of this chromosome loss is to increase the proportion of nullosomic gametes in the sperm pool.—The relative frequencies of XY-bearing and nullo-X, nullo-Y sperm in XY/O males were estimated from the volume measurements. Taking this estimate as a measure of the fertilizing population, it is possible to infer from the change in sex ratio over time following insemination, that XY-bearing sperm have an advantage of 1.5 over nullo-X, nullo-Y sperm in leaving the seminal receptacle of the female for fertilization of ova.     

Loss of Pol32 in Drosophila melanogaster Causes Chromosome Instability and Suppresses Variegation

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32’s participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32^rd1 and pol32^rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.     

Sperm competition in the Drosophila female

Summary Modified B ^S translocation males were developed at 26.0° C where univalentbearing gametes are recovered with less than half the frequency than at 18.0° C. Upon eclosion the males were stored for definite time periods at either temperature before mating individually to single y free-X females. the transfer cultures of the females show a higher frequency of recovery of univalent-bearing progeny regardless of the temperature or storage treatment of the male. In addition, postmeiotic temperature treatment does not appear to fundamentally alter the overall frequency of recovery of univalent-bearing gametes which is presumably determined by the developmental temperature of the male. A similar trend is observed for matings of y females to single X.Y^SY^L/O males in which the males were developed and stored at 26.0° C; namely, a higher frequency of recovery of attached-XY gametes in the transfer cultures.     

Somatic Instability of a Drosophila Chromosome

A mitotically unstable chromosome, detectable because of mosaic expression of marker genes, was generated by X-ray mutagenesis in Drosophila. Nondisjunction of this chromosome is evident in mitotic chromosome preparations, and premature sister chromatid separation is frequent. The mosaic phenotype is modified by genetic elements that are thought to alter chromatin structure. We hypothesize that the mitotic defects result from a breakpoint deep in the pericentric heterochromatin, within or very near to the DNA sequences essential for centromere function. This unique chromosome may provide a tool for the genetic and molecular dissection of a higher eukaryotic centromere.     

Female Sterile Mutations on the Second Chromosome of Drosophila Melanogaster. I. Maternal Effect Mutations

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 529 chromosomes were isolated which allow the homozygous females to survive, but cause them to be sterile. In 136 of these lines, mutant females produce morphologically normal eggs which cannot support normal embryonic development. These "maternal-effect" mutations fall into 67 complementation groups which define 23 multiply hit and 44 singly hit loci. In eggs from 14 complementation groups development is blocked before the formation of a syncytial blastoderm. In eggs from 12 complementation groups development is abnormal before cellularization, 17 complementation groups cause abnormal cellularization, 12 complementation groups cause changes in cellular morphology in early gastrulation stages, and 12 complementation groups seem to affect later embryonic development.     

Mitotic Chromosome Loss in a Disomic Haploid of SACCHAROMYCES CEREVISIAE

Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed. The selective methods employed utilize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The frequency of spontaneous chromosome loss in the disome is of the order 10^-4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid.     

Y Chromosome Loops in Drosophila Melanogaster

Primary spermatocyte nuclei of fixed testes of Drosophila melanogaster exhibit three large clusters of thread-like structures, each consisting of two long, continuous, loop-shaped filaments. No comparable intranuclear structures are observed in spermatogonia, secondary spermatocytes or spermatids. The threads begin to form in young spermatocytes, grow throughout spermatocyte development, reach their maximum size in mature spermatocytes and disintegrate prior to meiotic metaphase I. The presence of each cluster of threads depends upon the presence of a specific region of the Y chromosome; when this region is deleted the cluster is absent, and when it is duplicated the cluster is also duplicated. Together these observations strongly suggest that these structures represent giant Y chromosome lampbrush-like loops analogous to those described in Drosophila hydei. Two antibodies, one polyclonal and one monoclonal, differentially react with the three loops of D. melanogaster. Moreover, two of these loops are specifically stained by Giemsa at pH 10. By indirect immunofluorescence with these antibodies followed by Giemsa staining, each loop can be unambiguously identified and its presence and normality readily assessed. This enabled us to perform fine mapping experiments to determine the relationships between the Y chromosome fertility factors and the loops. The loop-forming sites map within the kl-5, kl-3 and ks-1 fertility factors. Regions h3 and h21 of the Y chromosome correspond to the loop-forming sites of kl-5 and ks-1, respectively. Each of these regions contains about 1300 kb of DNA and spans about one-third of its locus. The loop-forming site of the kl-3 locus is coextensive with region h7-h9 which contains about 4300 kb of DNA and corresponds to the minimum physical size of this locus. These data suggest that each loop is an integral part of a different fertility factor, representing the cytological manifestation of its activity in primary spermatocytes. The kl-2, kl-1 and ks-2 fertility regions do not produce any visible intranuclear structure and do not affect the kl-5, kl-3 and ks-1 loops. Thus, these loci may either not form loops at all or produce loop-like structures that we are unable to see because they are physically minute, destroyed by our fixation procedure, or both.     

Evolution of a Distinct Genomic Domain in Drosophila: Comparative Analysis of the Dot Chromosome in Drosophila melanogaster and Drosophila virilis

The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.EUKARYOTIC genomes are packaged into two major types of chromatin: euchromatin is gene rich and has a diffuse appearance during interphase, while heterochromatin is gene poor and remains densely packaged throughout the cell cycle (Grewal and Elgin 2002). The distal 1.2 Mb of the fourth chromosome of Drosophila melanogaster, known as the dot chromosome or Muller F element, is unusual in exhibiting an amalgamation of heterochromatic and euchromatic properties. This domain has a gene density that is similar to the other autosomes (Bartolomé et al. 2002; Slawson et al. 2006). However, it appears heterochromatic by many criteria, including late replication and very low levels of meiotic recombination (Wang et al. 2002; Arguello et al. 2010). It exhibits high levels of association with heterochromatin protein 1 (HP1) and histone H3 di- and trimethylated at lysine 9 (H3K9me2/3), as shown by immunofluorescent staining of the polytene chromosomes (Riddle and Elgin 2006; Slawson et al. 2006). This association with heterochromatin marks has recently been confirmed by the modENCODE Project [N. C. Riddle, A. Minoda, P. V. Kharchenko, A. A. Alekseyenko, Y. B. Schwartz, M. Y. Tolstorukov, A. A. Gorchakov, C. Kennedy, D. Linder-Basso, J. D. Jaffe, G. Shanower, M. I. Kuroda, V. Pirrotta, P. J. Park, S. C. R. Elgin, G. H. Karpen, and the modENCODE Consortium (http://www.modencode.org), unpublished results]. To understand this unique domain and to examine the evolution of a region with very low levels of recombination, we have undertaken a comparative study using the dot chromosome of D. virilis, a species that diverged from D. melanogaster 40–60 million years ago (Powell and Desalle 1995). We sequenced and improved the assembly of the D. virilis dot chromosome and created a manually curated set of gene models to ensure that both the assembly and the gene annotations are at a quality comparable to those in D. melanogaster. We then compared the sequence organization and gene characteristics of the distal portion of the D. virilis dot chromosome with the corresponding region from the D. melanogaster dot chromosome.In addition to examining the long-term dot chromosome evolution, we also investigated the short-term dot chromosome evolution by comparing the genomic sequences from two different strains of D. virilis. Agencourt Biosciences (AB) has previously produced a whole genome shotgun assembly of Tucson strain 15010–1051.87, while we have sequenced Tucson strain 15010–1051.88 of D. virilis [the Genomics Education Partnership (GEP) assembly]. The AB assembly has been improved by the Drosophila 12 Genomes Consortium and released as part of the comparative analysis freeze 1 (CAF1) assembly (Drosophila 12 Genomes Consortium et al. 2007).Using the GEP and CAF1 assemblies from D. virilis, and the high-quality D. melanogaster assembly and its gene annotations from FlyBase (Crosby et al. 2007), we compared the gene properties and sequence organization of the dot chromosomes and reference euchromatic and heterochromatic domains. The dot chromosomes from D. melanogaster and D. virilis are distinct from the heterochromatic and euchromatic regions of the two genomes, both in organization (e.g., repeat density) and in characteristics of the genes (e.g., size, codon bias). The two dot chromosomes resemble each other by most criteria and differ only in the types of repetitive sequences present and in relative gene order and orientation. Despite the very low rate of meiotic recombination, comparison of the two D. virilis strains shows that dot chromosome genes are under weak purifying selection. Our analysis of genes that are present in a euchromatic chromosome arm in one species and on the dot chromosome in the other (the “wanderer” genes) shows that this set of genes evolves to maintain function while responding to the changes in the local chromosomal environment.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Quantitative Genetics of Sperm Precedence in Drosophila Melanogaster

To assess the genetic basis of sperm competition under conditions in which it occurs, I estimated additive, dominance, homozygous and environmental variance components, the effects of inbreeding, and the weighted average dominance of segregating alleles for two measures of sperm precedence in a large, outbred laboratory population. Both first and second male precedence show significant decline on inbreeding. Second male precedence demonstrates significant dominance variance and homozygous genetic variance, but the additive variance is low and not significantly different from zero. For first male precedence, the variance among homozygous lines is again significant, and dominance variance is larger than the additive variance, but is not statistically significant. In contrast, male mating success and other fitness components in Drosophila generally exhibit significant additive variance and little or no dominance variance. Other recent experiments have shown significant genotypic variation for sperm precedence and have associated it with allelic variants of accessory-gland proteins. The contrast between sperm precedence and other male fitness traits in the structure of quantitative genetic variation suggests that different mechanisms may be responsible for the maintenance of variation in these traits. The pattern of genetic variation and inbreeding decline shown in this experiment suggests that one or a few genes with major effects on sperm precedence may be segregating in this population.