Polytenization of the Ribosomal Genes on the X and Y Chromosomes of DROSOPHILA MELANOGASTER

It has previously been shown (Endow and Glover 1979), that polytenization of the ribosomal genes in D. melanogaster Ore-R X/Y cells and in hybrid X/X cells (Endow 1980) involves replication of genes predominantly from one of the cell's two nucleolus organizers. This analysis takes advantage of strain-specific differences in X and Y chromosome rDNA hybridization patterns detected using the Southern blotting technique. In this report, I extend the previous observations by examining polytene rDNA patterns in wild-type and hybrid X/Y cells. A dominance hierarchy for the X and Y chromosomes from three strains of D. melanogaster is presented and possible mechanisms of replicative dominance are discussed.     

Sex-specific Trans-regulatory Variation on the Drosophila melanogaster X Chromosome

The X chromosome constitutes a unique genomic environment because it is present in one copy in males, but two copies in females. This simple fact has motivated several theoretical predictions with respect to how standing genetic variation on the X chromosome should differ from the autosomes. Unmasked expression of deleterious mutations in males and a lower census size are expected to reduce variation, while allelic variants with sexually antagonistic effects, and potentially those with a sex-specific effect, could accumulate on the X chromosome and contribute to increased genetic variation. In addition, incomplete dosage compensation of the X chromosome could potentially dampen the male-specific effects of random mutations, and promote the accumulation of X-linked alleles with sexually dimorphic phenotypic effects. Here we test both the amount and the type of genetic variation on the X chromosome within a population of Drosophila melanogaster, by comparing the proportion of X linked and autosomal trans-regulatory SNPs with a sexually concordant and discordant effect on gene expression. We find that the X chromosome is depleted for SNPs with a sexually concordant effect, but hosts comparatively more SNPs with a sexually discordant effect. Interestingly, the contrasting results for SNPs with sexually concordant and discordant effects are driven by SNPs with a larger influence on expression in females than expression in males. Furthermore, the distribution of these SNPs is shifted towards regions where dosage compensation is predicted to be less complete. These results suggest that intrinsic properties of dosage compensation influence either the accumulation of different types of trans-factors and/or their propensity to accumulate mutations. Our findings document a potential mechanistic basis for sex-specific genetic variation, and identify the X as a reservoir for sexually dimorphic phenotypic variation. These results have general implications for X chromosome evolution, as well as the genetic basis of sex-specific evolutionary change.     

不同温度条件下果蝇染色体结构的变化

将黑腹果蝇Drosophila melanogaster置于不同温度环境下培养,应用改良苯酚品红染色法制片.发现其唾腺染色体在不同的温度培养条件下,分布于染色体上的膨松区域有明显变化,在15℃和27℃下染色体的变化区域明显较19℃和23℃下的多,这些区域都与机体调控和表达的基因相关.这可能是机体对不适生存条件采取的保护措施所引起更多的染色体或基因发生变化的结果.对不同温度条件下部分染色体膨松区域的特殊基因作了简单讨论.     

Ribosomal DNA organization before and after magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.     

DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster

DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.     

Surprising Differences in the Variability of Y Chromosomes in African and Cosmopolitan Populations of Drosophila melanogaster

The nonrecombining Drosophila melanogaster Y chromosome is heterochromatic and has few genes. Despite these limitations, there remains ample opportunity for natural selection to act on the genes that are vital for male fertility and on Y factors that modulate gene expression elsewhere in the genome. Y chromosomes of many organisms have low levels of nucleotide variability, but a formal survey of D. melanogaster Y chromosome variation had yet to be performed. Here we surveyed Y-linked variation in six populations of D. melanogaster spread across the globe. We find surprisingly low levels of variability in African relative to Cosmopolitan (i.e., non-African) populations. While the low levels of Cosmopolitan Y chromosome polymorphism can be explained by the demographic histories of these populations, the staggeringly low polymorphism of African Y chromosomes cannot be explained by demographic history. An explanation that is entirely consistent with the data is that the Y chromosomes of Zimbabwe and Uganda populations have experienced recent selective sweeps. Interestingly, the Zimbabwe and Uganda Y chromosomes differ: in Zimbabwe, a European Y chromosome appears to have swept through the population.     

Variation in Y Chromosome Segregation in Natural Populations of Drosophila melanogaster

Functional variation among Y chromosomes in natural populations of Drosophila melanogaster was assayed by a segregation study. A total of 36 Y chromosomes was extracted and ten generations of replacement backcrossing yielded stocks with Y chromosomes in two different genetic backgrounds. Eleven of the Y chromosomes were from diverse geographic origins, and the remaining 25 were from locally captured flies. Segregation of sexes in adult offspring was scored for the four possible crosses among the two backgrounds with each Y chromosome. Although the design confounds meiotic drive and effects on viability, statistical partitioning of these effects reveals significant variation among lines in Y chromosome segregation. Results are discussed in regards to models of Y-linked segregation and viability effects, which suggest that Y-linked adaptive polymorphism is unlikely.     

Unequal exchanges and the coevolution of X and Y rDNA arrays in Drosophila melanogaster

We have examined the molecular basis of the response of individuals of D. melanogaster to artificial selection for high and low abdominal bristles. By monitoring the fate of particular rDNA spacer length variants associated with individually isolated X and Y chromosomes, we show that flies from the low bristle number selection lines have undergone an unequal exchange between the X and Y rDNA arrays. Such exchanges result in translocations between X and Y chromosomes, visualised as X.Y compound chromosomes at mitosis. Transfer of few copies of a length variant between X and Y indicates a clustering of variants. Flies that have reverted back to wild-type seemingly have undergone a second unequal exchange, giving rise to a compound X.Y chromosome containing Y rDNA of normal amounts. Unequal exchanges between X and Y rDNA arrays could contribute to the observed coevolution of rDNA sequences on these chromosomes. The biological significance of this outcome is discussed.     

A New Construct for Cloning DNA and Modeling the Structure of Drosophila melanogaster Polytene Chromosomes

Modification of P-element-based transformation vector pCaSpeR3 yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5 and 3 regulatory regions of hsp70, pUC19, and two tandem FRTs. Owing to the hsp70 promoter, the pICon insertion site may be located on polytene chromosomes after heat shock by light or electron microscopy. The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3 end of pICon by P-target rescue. Functional FRTs allow insertion or deletion of various DNA fragments. The construct is large (22,046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation. To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.     

X-Y Exchange and the Coevolution of the X and Y Rdna Arrays in Drosophila melanogaster

The nucleolus organizers on the X and Y chromosomes of Drosophila melanogaster are the sites of 200-250 tandemly repeated genes for ribosomal RNA. As there is no meiotic crossing over in male Drosophila, the X and Y chromosomal rDNA arrays should be evolutionarily independent, and therefore divergent. The rRNAs produced by X and Y are, however, very similar, if not identical. Molecular, genetic and cytological analyses of a series of X chromosome rDNA deletions (bb alleles) showed that they arose by unequal exchange through the nucleolus organizers of the X and Y chromosomes. Three separate exchange events generated compound X·Y ^L chromosomes carrying mainly Y-specific rDNA. This led to the hypothesis that X-Y exchange is responsible for the coevolution of X and Y chromosomal rDNA. We have tested and confirmed several of the predictions of this hypothesis: First, X· Y^L chromosomes must be found in wild populations. We have found such a chromosome. Second, the X·Y^L chromosome must lose the Y^L arm, and/or be at a selective disadvantage to normal X⁺ chromosomes, to retain the normal morphology of the X chromosome. Six of seventeen sublines founded from homozygous X·Y^Lbb stocks have become fixed for chromosomes with spontaneous loss of part or all of the appended Y^L. Third, rDNA variants on the X chromosome are expected to be clustered within the X⁺ nucleolus organizer, recently donated (" Y") forms being proximal, and X-specific forms distal. We present evidence for clustering of rRNA genes containing Type 1 insertions. Consequently, X-Y exchange is probably responsible for the coevolution of X and Y rDNA arrays.     

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.     

Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome

The structural analysis of a yeast artificial chromosome clone from Drosophila melanogaster enriched in dodecasatellite sequences has led us to find a new retrotransposon that we have called Circe. Moreover, this retrotransposon has allowed the isolation of a contig encompassing ∼200 kb near the centromere of the Y chromosome, providing an entry point into a region from which very little sequence information has been obtained to date. The molecular characterization of the contig has shown the presence of HeT-A telomeric retrotransposons close to the centromere of the Y chromosome, suggesting a telocentric origin for this submetacentric chromosome. Received: 19 November 1996 / Accepted: 30 May 1997