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.     

Enhancing effect of lucanthone (miracil D) on the frequency of X-ray-induced chromosome loss in drosophila male germ cells

The effect of lucanthone (miracil D), an inhibitor of RNA synthesis, plus X-irradiation on Drosophila melanogaster males X^c2yB/Ysc⁸y⁺ and on the subsequent production of chromosome loss via breakage has been investigated. Lucanthone feeding plus 495 R induced a significantly higher frequency of chromosome loss when irradiation was given in three equal fractionated doses at 3-h intervals than the same dose given acutely. On the other hand, the difference in frequency of non-disjunctional females was not significant. The enhancing effects of this chemical were found only in the fractionated series but were absent in acute X-irradiation series. This effect was found primarily in those cells in spermatid and spermatocyte stages at the time of irradiation. A pertinent point of interest presented was that not only protein synthesis but also RNA synthesis may play a significant role in the development of radiation damage and in postradiation repair processes at the chromosomal level, since inhibition of RNA synthesis may eventually inhibit protein synthesis.     

Increase in viability due to the accumulation of X chromosome mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> males

To increase our understanding of the role of new X-chromosome mutations in adaptive evolution, single-X Drosophila melanogaster males were mated with attached-X chromosome females, allowing the male X chromosome to accumulate mutations over 28 generations. Contrary to our hypothesis that male viability would decrease over time, due to the accumulation and expression of X-linked recessive deleterious mutations in hemizygous males, viability significantly increased. This increase may be attributed to germinal selection and to new X-linked beneficial or compensatory mutations, possibly supporting the faster-X hypothesis.     

Rates of RNA synthesis during early embryogenesis in Drosophila melanogaster

We determined the absolute rates of RNA synthesis during embryogenesis in Drosophila melanogaster by measuring the incorporation of ³H-5-orotic acid into RNA, and the specific activity of the UTP pool. Initially (preblastoderm) the rate of RNA synthesis is relatively high, but declines to a lower level by gastrulation. The data suggest that RNA synthesis is initiated during very early embryogenesis.     

Modulation of Heterochromatin by Male Specific Lethal Proteins and roX RNA in Drosophila melanogaster Males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila melanogaster males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Drosophila melanogaster has different ribosomal RNA sequences on S and Y chromosomes.

The primary structures of ribosomal RNAs transcribed from the nucleolus organizers on X and Y chromosomes of Drosophila melanogaster were compared by RNase T₁ fingerprints made with two different systems; i.e. homochromatography on DEAE-cellulose, and polyethyleneimine-cellulose thin-layer chromatography.Ribosomal RNA derived from the X-linked nucleolus organizer was obtained from a strain producing only female larvae and ribosomal RNA derived from the Y-linked nucleolus organizer was isolated from a mutant lacking the X-linked nucleolus organizer.No difference was detected between the fingerprints of 28 S RNA from these animals.In 18 S RNA, however, one oligonucleotide showed a remarkable difference in mobility. The structure of the X-linked organizer-specific oligonucleotide was 5′ U-C-U-U-U-U-U-U-C-C-U-A-U-G 3′, and that of the Y-linked organizer-specific oligonucleotide was 5′ U-C-U-C-U-U-U-U-C-C-U-A-U-G 3′, indicating one base substitution (U á3 C) between them.The absence of 5′-temninal phosphate in this oligonucleotide and available sequence data also suggest that these oligonucleotides did not come from either the 5′ or 3′ terminus of 18 S RNA.D. simulans, whose Y chromosome has no nucleolus organizer (Ritossa &; Atwood, 1966), showed an 18 S RNA fingerprint having only the X-linked organizer-specific oligonucleotide.We conclude from these results that in Drosophila the ribosomal RNA gene sequences are different for the two nucleolus organizers located on the X and Y chromosomes. The implications of those findings concerning the parallel evolution of these genes are discussed.     

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.     

Drosophila dosage compensation

Dosage compensation of X-linked genes is a phenomenon of concerted, chromosome-wide regulation of gene expression underpinned by sustained and tightly regulated histone modifications and chromatin remodeling, coupled with constrains of nuclear architecture. This elaborate process allows the accomplishment of regulated expression of genes on the single male X chromosome to levels comparable to those expressed from the two X chromosomes in females. The ribonucleoprotein Male Specific Lethal (MSL) complex is enriched on the male X chromosome and is intricately involved in this process in Drosophila melanogaster. In this review we discuss the recent advances that highlight the complexity lying behind regulation of gene expression by just two-fold.     

Nature of the ribosomal RNA transcribed from the X and Y chromosomes of Drosophila melanogaster

In Drosophila melanogaster there is one nucleolar organizer (NO) on each X and Y chromosome. Experiments were carried out to compare the ribosomal RNAs derived from the two nucleolar organizers. ³²PO₄-labelled ribosomal RNA was isolated from two strains of D. melanogaster, one containing only the X chromosome NO, the other containing only the Y chromosome NO. 28 S and 18 S RNA from the two strains were subjected to a variety of “fingerprinting” and sequencing procedures. Fingerprints of 28 S RNA were very different from those of 18 S RNA. Fingerprints of “X” and “Y” 28 S RNA were indistinguishable from each other, as also were fingerprints of “X” and “Y” 18 S RNA. In combined “T₁ plus pancreatic” RNAase fingerprints several distinctive products were characterized and quantitated. Identical products were obtained from X and Y RNA, and the molar yields of the products were indistinguishable. Together these findings imply that the rRNA sequences encoded by the X and Y NOs are closely similar and probably identical to each other.Two further findings were of interest in “T₁ plus pancreatic” RNAase fingerprints: (1) in 28 S (as well as in 18 S) fingerprints several distinctive products were recovered in approximately unimolar yields. This indicates that 28 S RNA does not consist of two identical half molecules, though it does consist of two non-identical half molecules together with a “5.8 S” fragment. (2) Several methylated components in Drosophila rRNA also occur in rRNA from HeLa cells and yeast. This suggests that certain features of rRNA structure involving methylated nucleotides may be highly conserved in eukaryotic evolution.     

The ribosomal DNA of Drosophila melanogaster is organized differently from that of Drosophila hydei

On the X chromosome of Drosophila melanogaster there is a single tandem array of 240 ribosomal RNA genes. The majority of these contain an insertion, known as type I, in the 28 S coding region. Previous genetic and electron microscopic studies indicated that genes bearing the type I insertion (ins⁺) are interspersed at random with those lacking it (ins^?). In contrast, Renkawitz-Pohl et al. (1981) have analyzed the restriction pattern of X chromosomal ribosomal DNA in Drosophila hydei and demonstrated that in this case ins⁺ genes are segregated from ins^?. This suggests either that the rDNA is organized differently in these two species or that the restriction enzyme technique reveals significant clustering not detected by previous methods. By using an appropriate restriction enzyme, we demonstrate that ins⁺ and ins^? genes are intermingled at random in D. melanogaster. These experiments also indicate that genes containing the short form of the insertion are flanked by a larger spacer upstream than downstream.     

Genetics of Catalase in DROSOPHILA MELANOGASTER: Rates of Synthesis and Degradation of the Enzyme in Flies Aneuploid and Euploid for the Structural Gene

A screen for allelic variants of the enzyme catalase indicated that the Cat⁺ locus is essentially monomorphic in D. melanogaster. Segmental aneuploidy was used to screen the genome for a dosage-sensitive region for catalase activity. One region, 75D–78A on the polytene chromosome map of 3L, exhibited a hyperploid/euploid ratio of enzyme activity of 1.5. Further dissection localized the region to 75D–76A. We suggest that this region contains the structural locus for catalase in D. melanogaster.

Simple methods have been developed using the specific inhibitor, 3-amino-1,2,4-triazole, for the direct analysis of rates of synthesis and degradation of the Cat⁺ gene product. Based on kinetic studies of catalase synthesis in flies aneuploid and euploid for region 75D–76B, we suggest that these techniques can be readily applied to an examination of mutants that control the expression of the structural gene for catalase in Drosophila.

     

Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.