Drosophila Chromosomes Research Articles

A simple and rapid method for constructing ring-X chromosomes in Drosophila melanogaster

Ring chromosomes are of basic interest to the geneticist and cell biologist who study their behavior in meiotic and mitotic divisions. In addition, the mitotic instability associated with some ring-X chromosomes has proven useful in Drosophila as a means to produce gynandromorphs for developmental studies. We describe a method to construct ring-X chromosomes in Drosophila via I-CreI-mediated exchange in rDNA, and then rapidly diagnose the recovery of ring chromosomes via FLP-mediated sister chromatid exchange within the ring. The method we describe provides a ready means to tailor the genetic content of ring-X chromosomes, making it suited to produce ring-X chromosomes for a variety of experimental purposes.

Dosage compensation in drosophila: Sequence-specific initiation and sequence-independent spreading of MSL complex to the active genes on the male X chromosome

For the dosage compensation to occur, genes on the single male X chromosomes in Drosophila must be selectively bound and acetylated by the ribonucleoprotein complex called MSL complex. It remained unknown how such exquisite specificity is achieved, and whether specific DNA sequences were involved. In the present work we demonstrate that it is transcription of the gene on the X chromosome that is important for MSL targeting, irrespective of gene origin and DNA sequence.

Shop talk: Annual Drosophila Research Conference, 2010

Shop talk: Annual Drosophila Research Conference, 2010

Dosage Compensation and Demasculinization of X Chromosomes in Drosophila

The X chromosome of Drosophila shows a deficiency of genes with male-biased expression, whereas mammalian X chromosomes are enriched for spermatogenesis genes expressed premeiosis and multicopy testis genes. Meiotic X-inactivation and sexual antagonism can only partly account for these patterns. Here, we show that dosage compensation (DC) in Drosophila may contribute substantially to the depletion of male genes on the X. To equalize expression between X-linked and autosomal genes in the two sexes, male Drosophila hypertranscribe their single X, whereas female mammals silence one of their two X chromosomes. We combine fine-scale mapping data of dosage compensated regions with genome-wide expression profiles and show that most male-biased genes on the D. melanogaster X are located outside dosage compensated regions. Additionally, X-linked genes that have newly acquired male-biased expression in D. melanogaster are less likely to be dosage compensated, and parental X-linked genes that gave rise to an autosomal male-biased retrocopy are more likely located within compensated regions. This suggests that DC contributes to the observed demasculinization of X chromosomes in Drosophila, both by limiting the emergence of male-biased expression patterns of existing X genes, and by contributing to gene trafficking of male genes off the X.

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.

Fragile regions and not functional constraints predominate in shaping gene organization in the genus Drosophila

During evolution, gene repatterning across eukaryotic genomes is not uniform. Some genomic regions exhibit a gene organization conserved phylogenetically, while others are recurrently involved in chromosomal rearrangement, resulting in breakpoint reuse. Both gene order conservation and breakpoint reuse can result from the existence of functional constraints on where chromosomal breakpoints occur or from the existence of regions that are susceptible to breakage. The balance between these two mechanisms is still poorly understood. Drosophila species have very dynamic genomes and, therefore, can be very informative. We compared the gene organization of the main five chromosomal elements (Muller's elements A-E) of nine Drosophila species. Under a parsimonious evolutionary scenario, we estimate that 6116 breakpoints differentiate the gene orders of the species and that breakpoint reuse is associated with approximately 80% of the orthologous landmarks. The comparison of the observed patterns of change in gene organization with those predicted under different simulated modes of evolution shows that fragile regions alone can explain the observed key patterns of Muller's element A (X chromosome) more often than for any other Muller's element. High levels of fragility plus constraints operating on approximately 15% of the genome are sufficient to explain the observed patterns of change and conservation across species. The orthologous landmarks more likely to be under constraint exhibit both a remarkable internal functional heterogeneity and a lack of common functional themes with the exception of the presence of highly conserved noncoding elements. Fragile regions rather than functional constraints have been the main determinant of the evolution of the Drosophila chromosomes.

Evolutionary Biology: The Origins of Two Sexes

Evidence is accumulating that in the green algae the evolution of female and male gametes differing in size--anisogamy--involves genes linked to the mating-type locus, as was predicted theoretically.

Drosophila SAF-B Links the Nuclear Matrix, Chromosomes, and Transcriptional Activity

Induction of gene expression is correlated with alterations in nuclear organization, including proximity to other active genes, to the nuclear cortex, and to cytologically distinct domains of the nucleus. Chromosomes are tethered to the insoluble nuclear scaffold/matrix through interaction with Scaffold/Matrix Attachment Region (SAR/MAR) binding proteins. Identification and characterization of proteins involved in establishing or maintaining chromosome-scaffold interactions is necessary to understand how the nucleus is organized and how dynamic changes in attachment are correlated with alterations in gene expression. We identified and characterized one such scaffold attachment factor, a Drosophila homolog of mammalian SAF-B. The large nuclei and chromosomes of Drosophila have allowed us to show that SAF-B inhabits distinct subnuclear compartments, forms weblike continua in nuclei of salivary glands, and interacts with discrete chromosomal loci in interphase nuclei. These interactions appear mediated either by DNA-protein interactions, or through RNA-protein interactions that can be altered during changes in gene expression programs. Extraction of soluble nuclear proteins and DNA leaves SAF-B intact, showing that this scaffold/matrix-attachment protein is a durable component of the nuclear matrix. Together, we have shown that SAF-B links the nuclear scaffold, chromosomes, and transcriptional activity.

CHROMOSOMES AS INTERDEPENDENT ACCOUNTING UNITS: THE ASSIGNED ORIENTATION OFC. ELEGANSCHROMOSOMES MINIMIZES THE TOTAL W-BASE CHARGAFF DIFFERENCE

DNAs of individual chromosomes violate, albeit perhaps by only one in a thousand bases, Chargaff's second parity rule, which is that Chargaff's first parity rule for duplex DNA (A = T, G = C) applies, to a close approximation, to single stranded DNA. If the "top" strand of one chromosome has A > T and the "top" strand of another has T > A, can they complement to approach even parity (A = T)? Assignment of orientation to the six chromosomes of Caenorhabditis elegans is said to have been arbitrary and, of 26(= 64) possible combinations of top (T) and bottom (B) strands, the GenBank orientation (designated "TTTTTT") is but one. Yet, for the W bases (A and T) the chromosomes in the GenBank orientation complement to reduce the Chargaff difference (A–T) to only 200 bases (i.e. only one in 323,658 bases does not have a potential Watson-Crick pairing partner). This suggests that the assignment was not arbitrary. However, the GenBank orientation for the S bases (G and C) allows an approach to even parity less well than many other orientations, the best of which is BBBBTT (indicating a disparity between the GenBank orientations of the first four autosomes and those of chromosomes V and X). Although only the euchromatic regions of Drosophila melanogaster chromosomes have been sequenced, there are orientations that allow an approach to even parity. We conclude that, with respect to their Chargaff differences, the chromosomes of C. elegans have the potential to engage in interdependent base accounting. Since this might also apply to D. melanogaster, even when heterochromatin-associated DNA rich in tandem repeats (microsatellite DNA) is excluded, then heterochromatic DNA might not normally participate in the hypothetical accounting process.

Functional Copies of the Mst77F Gene on the Y Chromosome of Drosophila melanogaster

The Y chromosome of Drosophila melanogaster has <20 protein-coding genes. These genes originated from the duplication of autosomal genes and have male-related functions. In 1993, Russell and Kaiser found three Y-linked pseudogenes of the Mst77F gene, which is a testis-expressed autosomal gene that is essential for male fertility. We did a thorough search using experimental and computational methods and found 18 Y-linked copies of this gene (named Mst77Y-1-Mst77Y-18). Ten Mst77Y genes encode defective proteins and the other eight are potentially functional. These eight genes produce approximately 20% of the functional Mst77F-like mRNA, and molecular evolutionary analysis shows that they evolved under purifying selection. Hence several Mst77Y genes have all the features of functional genes. Mst77Y genes are present only in D. melanogaster, and phylogenetic analysis confirmed that the duplication is a recent event. The identification of functional Mst77Y genes reinforces the previous finding that gene gains play a prominent role in the evolution of the Drosophila Y chromosome.

Multiple Roles of the Y Chromosome in the Biology ofDrosophila melanogaster

The X and Y chromosomes of Drosophila melanogaster were the first examples of chromosomes associated with genetic information. Thanks to the serendipitous discovery of a male with white eyes in 1910, T.H. Morgan was able to associate the X chromosome of the fruit fly with a phenotypic character (the eye color) for the first time. A few years later, his student, C.B. Bridges, demonstrated that X0 males, although phenotypically normal, are completely sterile. This means that the X chromosome, like the autosomes, harbors genes that control several phenotypic traits, while the Y chromosome is important for male fertility only. Notwithstanding its long history – almost 100 years in terms of genetic studies – most of the features of the Y chromosome are still a mystery. This is due to the intrinsic nature of this genetic element, namely, (1) its molecular composition (mainly transposable elements and satellite DNA), (2) its genetic inertia (lack of recombination due to its heterochromatic nature), (3) the absence of homology with the X (with the only exception of the nucleolar organizer), (4) the lack of visible phenotypes when it is missing (indeed, except for their sterility, X0 flies are normal males), and (5) its low density as for protein-coding sequences (to date, only 13 genes out of approximately 14,000 have been mapped on this chromosome in D. melanogaster, i.e., ~0.1% of the total). Nonetheless, a more accurate analysis reveals that this chromosome can influence several complex phenotypes: (1) it has a role in the fertility of both sexes and viability of males when over-represented; (2) it can unbalance the intracellular nucleotide pool; (3) it can interfere with the gene expression either by recruiting proteins involved in chromatin remodeling (PEV) or, to a higher extent, by influencing the expression of up to 1,000 different genes, probably by changing the availability of transcription factors; (4) it plays a major role (up to 50%) in the resistance to heat-induced male sterility; (5) it affects the behavior; and (6) it plays a role in genetic imprinting. In the present paper, all these Y-related phenotypes are described and a potential similarity with the human Y chromosome is drawn.

WD40 Repeats Arrange Histone Tails for Spreading of Silencing

Direct binding of WD40 repeat of Embryonic Ectoderm Development (EED) in the Polycomb Repressor Complex 2 (PRC2) to the histone H3 tail regulates the H3K27 methyltrasnferase activity of PRC2. The binding activity is required for the methylation of H3K27 over long distance of a Drosophila chromosome from the UBX (Hox gene) promoter to the PBX upstream enhancer region, implying EED initiates propagation of this repressive mark.

X Chromosome: Expression and Escape

Although there are some obvious differences between the X chromosome and the autosomes—such as the X chromosome being present in only one copy in males—the two types of chromosome are remarkably similar in their cytological appearance and gene density [1]. Genomic and transcriptomic studies of Drosophila, however, have revealed two major differences in gene content between the X chromosome and the autosomes. First, there is an excess of functional, duplicate genes that have moved from the X chromosome to the autosomes (Figure 1A) [2]. Second, there is a paucity of genes with male-biased expression on the X chromosome (Figure 1B) [3]. A hypothesis that could explain these observations was proposed by Betran and colleagues in 2002 [2] and is based on the phenomenon of meiotic sex chromosome inactivation (MSCI, see below). Since then, there has been controversy about whether or not MSCI occurs in Drosophila and, if it does, what role it plays in shaping the gene content of the X chromosome. In this issue of PLoS Genetics, Vibranovski et al. [4] present a detailed analysis of gene expression across three stages of Drosophila spermatogenesis. Their results support the occurrence of MSCI and suggest that its effect on germline expression of X-linked genes promotes the selective maintenance of autosomal duplicates arising from X-linked genes. Figure 1 The X chromosome and autosomes of Drosophila melanogaster differ in retrogene and male-biased gene content.

Rapid increase in viability due to new beneficial mutations in Drosophila melanogaster

It is usually assumed that new beneficial mutations are extremely rare. Yet, few experiments have been performed in multicellular organisms that measure the effect of new beneficial mutations on viability and other measures of fitness. In most experiments, it is difficult to clearly distinguish whether adaptations have occurred due to selection on new beneficial mutations or on preexisting genetic variation. Using a modification of a Dobzhansky and Spassky (Evolution 1:191-216, 1947) assay to study change in viability over generations, we have observed an increase in viability in lines homozygous for the second and third chromosomes of Drosophila melanogaster in 6-26 generations due to the occurrence of new beneficial mutations in population sizes of 20, 100 and 1,000. The lines with the lowest initial viability responded the fastest to new beneficial mutations. These results show that new beneficial mutations, along with selection, can quickly increase viability and fitness even in small populations. Hence, new advantageous mutations may play an important role in adaptive evolution in higher organisms.

Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA–immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

HMOF, a KAT(8) with Many Lives

Dynamic lysine acetylation regulates proteins involved in diverse cellular processes, with individual enzymes often acetylating multiple substrates. Here, Li et al. (2009) show that the substrate specificity of hMOF/MYST1/KAT8 is controlled by differential interaction with two mutually exclusive partners.

Uncovering the Footprint of Positive Selection on the X Chromosome of Drosophila melanogaster

A usual approach to detect the spatial footprint left by recent adaptive events has been to follow up putative candidates emerging from multilocus scans of variation by sequencing additional fragments. We have used a similar experimental and analytical approach to study variation at 15 independently evolving and randomly chosen regions of the X chromosome of Drosophila melanogaster. These incompletely sequenced regions, each extending over approximately 40 kb, were subjected to two tests of positive selection that take into account the spatial distribution of nucleotide variation. Our analysis of variation at these genomic regions in a European population of D. melanogaster has allowed us to uncover a candidate region for positive selection and to empirically evaluate the comparative performance of the two tests of selection under a bottleneck scenario. Moreover, the boundaries here estimated for both the rate of adaptive substitution (delta) and the average selection coefficient (s) would support previous estimates obtained by maximum likelihood that suggest rather strong but uncommon positive selection.

Lack of detectable interaction between the Curly inversions and direction of pairing in the X chromosome of Drosophila melanogaster.

The interchromosomal effect of In(2L+ 2R)Cy, that are known to enhance recombination in the X chromosome distally, on a heterozygous duplication/normal system of the wch site was tested. This system shows unequal recovery of left and right halves of the duplication, which has previously been interpreted as an indication of polarized pairing. Assuming this view is correct, effects of Cy on the polarization would have permitted a tentative explanation of the unusual action of Cy distally in X. However, no other effect than a general enhancement of recombination was observed.

THE RELATION BETWEEN X-RAY SENSITIVITY AND STAGES OF DEVELOPMENT OF TREATED CELLS IN SPERMATO- AND SPERMIOGENESIS OF DROSOPHILA MELANOGASTER

By using a dual-purpose stock it was possible to study the frequency of induced XO males and induced non-disjunction between the paternal X and Y chromosomes in Drosophila after 0 to 1 and 3 to 4 day old y/sup 16/; sc/ssup 8/Y males were irradiated (1100 r) and mated to virgin y w sn females in successive mating periods. The first mating period was delimited to 0 to 4 hours after irradiation but from the 4th day and on each mating period consisted of 24 hours. lt is shown that irradiation of Drosophila males causes non-disjunction between the paternal X and Y chromosomes. Hence, the occurrence of an increase in the frequency of non-disjunction in sperm ejaculated during a limited period after irradiation must be taken as an indication that those spermatozoa were in meiosis at the time of treatment. Factors which may influence this cell stage/ sensitivity relationship are discussed.

Laboratory Exercises To Examine Recombination &amp; Aneuploidy inDrosophila

[ILLUSTRATION OMITTED] Chromosomal aneuploidy, a deviation from an exact multiple of an organism's haploid chromosome number, is a difficult concept for students to master. Aneuploidy arising from chromosomal non-disjunction (NDJ) is particularly problematic for students, since it arises in the context of meiosis, itself a challenging subject. Students learning NDJ are forced to actively apply their knowledge of normal meiosis, often revealing previously unapparent deficits in their understanding. Teaching NDJ is thus an important opportunity for students to review and apply their skills in modeling meiosis. Despite this opportunity, standard methods for teaching NDJ are passive. While experiments illustrating recombination mapping, sex-linkage, and other core genetics concepts are readily available for high school and undergraduate genetics courses, simple exercises demonstrating NDJ are not. This is regrettable, for non-disjunction is not only medically important but also historically significant as the original proof for the chromosomal theory of inheritance (Bridges, 1916). Presented here is a straightforward method to detect NDJ of the sex chromosomes in Drosophila in the context of laboratory exercises to examine recombination of sex-linked loci. The exercises presented here allow for detecting maternal and paternal NDJ events within a single cross, an advantage since the use of standard markers obscures paternal NDJ events (Bridges, 1916). An additional advantage of this method is that it easily allows for recombination mapping with recessive alleles in trans as well as in cis. This method allows students to simultaneously investigate recombination and aneuploidy with live organisms, and as such is complementary to theoretical methods of teaching recombination and NDJ. The exercises presented here are based on a Drosophila virginizing system that has recently been developed as a method to perform teaching crosses without the need for collecting virgins manually (Venema, 2006). This system employs a modified Y chromosome that harbors a conditional, dominant lethal mutation: a regulatory sequence that is activated in response to heat connected to a protein called head involution defective, or hid that activates programmed cell death. Since this modified Y chromosome (abbreviated as Y{hs-hid}) is present only in males, temporarily raising the temperature of a Y{hs-hid} Drosophila culture during larval stages will cause all males to undergo systemic apoptosis and die. Female larvae, however, survive the heat shock and remain virgin after hatching since there are no surviving males to mate with (Venema, 2006). The use of this system is of great advantage for educators in that large numbers of virgins can be obtained without scoring or segregating young female flies. This ability to use large numbers of flies in pedagogical crosses also facilitates finding the results of rare genetic events in the progeny. (1) Venema (2006) describes a scheme for recombination mapping between autosomal loci using this system; however, recombination mapping of sex-linked loci in Drosophila is more convenient for high school or introductory-level university courses. Additionally, examining recombination between sex-linked loci allows for the simultaneous detection of chromosomal NDJ events in the progeny, since some Drosophila sex chromosome aneuploids are viable (Table 1). Four possible exercises are presented: two versions of a dihybrid testcross and two versions of a three-point testcross. The dihybrid testcross exercises map sex-linked loci with the recessive mutant alleles either together as a parental genotype (i.e., recessive alleles in cis) or with recessive mutations inherited from both parents (recessive alleles in trans). Similarly, the options for the three-point testcross are mapping the three loci with mutant alleles all in cis, or with two in cis, and the third in trans. The dihybrid or three-point testcross options allow for instructors to choose the exercises of appropriate complexity for their particular grade level and learning objectives. …