Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of ³H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.     

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.     

Dosage compensation of a retina-specific gene in Drosophila miranda

The X1R chromosome of Drosophila miranda and the 3L autosome of Drosophila melanogaster are thought to have originated from the ancestral D chromosomal element and therefore may contain the same set of genes. It is expected that these genes will be dosage compensated in D. miranda because of their X linkage. To test these possibilities and to study evolution of the dosage compensation mechanism, we used the 3L-linked autosomal head-specific gene 507ml of D. melanogaster to isolate the homologous gene (507 mr) from a D. miranda genomic library. In situ hybridization showed that gene 507 is located at the 12A region of the X1R chromosome of D. miranda, indicating that the chromosomal homology deduced by cytogenetic means is correct. Restriction analysis and cross-specific DNA and RNA blot hybridization revealed the presence of extensive restriction pattern polymorphism and lack of sequence similarity in some areas of the 507 mr and 507 ml DNA, including the 3 portion of the transcribed region. However, the 5 portion of the transcribed region and the DNA sequences, located approximately 0.8 kb upstream and 3 kb downstream from the 507 ml gene showed a high degreee of similarity with the DNA sequences of comparable regions of the 507 mr gene. In both species gene 507 codes for a highly abundant 1.8 kb RNA which is expressed in the retina of the compound eye. Although in D. miranda the males have one and the females have two copies of the 507 gene, the steady-state levels of the 507 mRNA in both sexes were found to be similar, indicating that gene 507 is dosage compensated in D. miranda. Thus, along with the disparate rates of evolution in different areas of the DNA associated with gene 507, in D. miranda this gene has come under the regulation of the X chromosomal dosage compensation mechanism.by M.L. Pardue     

Dosage Compensation of Genes on the Left and Right Arms of the X Chromosome of DROSOPHILA PSEUDOOBSCURA and DROSOPHILA WILLISTONI

We have investigated the occurrence of dosage compensation in D. willistoni and D. pseudoobscura, two species whose X chromosome is metacentric with one arm homologous to the X and the other homologous to the left arm of chromosome 3 of D. melanogaster. Crude extracts were assayed for isocitrate dehydrogenase (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?), and α-glycerophosphate dehydrogenase (chromosome 2) in D. willistoni, and for esterase-5 (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?) and amylase (chromosome 3) in D. pseudoobscura. Our results indicate that a mechanism for dosage compensation is operative in both arms of the X chromosome of these two species.     

Evolutionary changes in the organization of the major LCP gene cluster during sex chromosomal differentiation in the sibling speciesDrosophila persimilis,D. pseudoobscura andD. miranda

The major larval cuticle protein (LCP) genes I–IV ofDrosophila melanogaster are clustered on the right arm of the second chromosome. By cross-hybridization we cloned the corresponding genes from three different members of theobscura group:D. persimilis, D. pseudoobscura andD. miranda. InD. pseudoobscura andD. persimilis the gene cluster maps to autosome3. In contrast, inD. miranda it was found on theX2 andY sex chromosome. Hence, this exceptional karyotypic situation offers a unique opportunity to analyse the molecular processes underlying the phenomenon of chromosome degeneration. Comparison of LCP genes I–IV in theX2 andY chromosomal region inD. miranda revealed extensive DNA rearrangements at the latter. TheY chromosomal LCP cluster is characterized by DNA insertions which are absent in the correspondingX2 chromosomal DNA, suggesting that these DNA sequences must have invaded this area. In addition, part of the analysedY chromosomal region is duplicated.     

Restricting Dosage Compensation Complex Binding to the X Chromosomes by H2A.Z/HTZ-1

Dosage compensation ensures similar levels of X-linked gene products in males (XY or XO) and females (XX), despite their different numbers of X chromosomes. In mammals, flies, and worms, dosage compensation is mediated by a specialized machinery that localizes to one or both of the X chromosomes in one sex resulting in a change in gene expression from the affected X chromosome(s). In mammals and flies, dosage compensation is associated with specific histone posttranslational modifications and replacement with variant histones. Until now, no specific histone modifications or histone variants have been implicated in Caenorhabditis elegans dosage compensation. Taking a candidate approach, we have looked at specific histone modifications and variants on the C. elegans dosage compensated X chromosomes. Using RNAi-based assays, we show that reducing levels of the histone H2A variant, H2A.Z (HTZ-1 in C. elegans), leads to partial disruption of dosage compensation. By immunofluorescence, we have observed that HTZ-1 is under-represented on the dosage compensated X chromosomes, but not on the non-dosage compensated male X chromosome. We find that reduction of HTZ-1 levels by RNA interference (RNAi) and mutation results in only a very modest change in dosage compensation complex protein levels. However, in these animals, the X chromosome–specific localization of the complex is partially disrupted, with some nuclei displaying DCC localization beyond the X chromosome territory. We propose a model in which HTZ-1, directly or indirectly, serves to restrict the dosage compensation complex to the X chromosome by acting as or regulating the activity of an autosomal repellant.     

Genome assembly,sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.     

Cytogenetic mapping of marker genes on the chromosome elements C and E of Drosophila pseudoobscura and D. subobscura

Enzyme loci, visible marker genes and -cloned DNA-sequences from a D. miranda library were mapped cytologically on the chromosome elements C and E of D. pseudoobscura and D. subobscura. New data are incorporated into the linkage maps of the two species. Homologous segments can now be localized in the polytene chromosomes with these markers. A comparison of the chromosome elements E of D. melanogaster and D. subobscura shows 12 conserved subsections which have been rearranged by paracentric inversions in the evolution of the two lineages.     

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.     

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

Background  

The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.     

The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation

Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.     

Inviability of hybrids between D. melanogaster and D. simulans results from the absence of simulans X not the presence of simulans Y chromosome

Interspecific crosses between D. melanogaster and D. simulans or its sibling species result in unisexual inviability of the hybrids. Mostly, crosses of D. melanogaster females X D. simulans males produce hybrid females. On the other hand, only hybrid males are viable in the reciprocal crosses. A classical question is the cause of the unisexual hybrid inviability on the chromosomal level. Is it due to the absence of a D. simulans X chromosome or is it due to the presence of a D. simulans Y chromosome? A lack of adequate chromosomal rearrangements available in D. simulans has made it difficult to answer this question. However, it has been assumed that the lethality results from the absence of the D. simulans X rather than the presence of the D. simulans Y. Recently I synthesized the first D. simulans compound-XY chromosome that consists of almost the entire X and Y chromosomes. Males carrying the compound-XY and no free Y chromosome are fertile. By utilizing the compound-XY chromosome, the viability of hybrids with various constitutions of cytoplasm and sex chromosomes has been examined. The results consistently demonstrate that the absence of a D. simulans X chromosome in hybrid genome, and not the presence of the Y chromosome, is a determinant of the hybrid inviability.     

Analysis of chromosomal homologies between two species of the subgenus sophophora: D. miranda and D. melanogaster using cloned DNA segments

Chromosomal homology between two species of the subgenus Sophophora, D. miranda and D. melanogaster, belonging to the obscura and the melanogaster group respectively, was probed by DNA in situ cross hybridizations. A set of recombinant plasmids with inserts derived from the D. melanogaster genome were cross hybridized to the D. miranda karyotype. Vice versa, recombinant Lambda phages isolated from a genomic D. miranda library were localized in D. miranda and probed for localization in D. melanogaster. In the main, the results support the homology relations proposed on the basis of cytogenetic data. However, the location of both tandemly repetitive genes tested, 5S RNA genes and the histone genes, is not in accordance with expectation. The 5S RNA genes, when probed with the D. melanogaster plasmid 12D8 (Artavanis-Tsakonas et al., 1977), were found to occur at two sites in both, D. miranda and D. pseudoobscura.