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.     

Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster

A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

RNA synthesis in Drosophila melanogaster polytene chromosomes indications of simultaneous dosage compensation and dosage effect in X chromosomes

It is shown that the apparent incompleteness of dosage compensation when RNA synthesis is measured autoradiographically is not due to the existence of contiguous dosage compensated and non-dosage compensated genes. Rather this seems to be the result of peculiarities in the coordination of RNA synthesis between the X chromosomes and autosomes. The slope of the line defined by \([\bar X]_i \) and \([\overline {2R} ]_i \) (number of grains over the X and autosomal segments averaged over the different nuclei assayed in each gland) is indistinguishable in males and females (apparent complete dosage compensation). An average of the slopes obtained for different individual glands (from [X] and [2R], the grain counts over each nucleus belonging to a particular gland), on the other hand, has a value in males which is approximately half of the value attained by females (a value of one half, in males, indicates dosage effect since males have one X and females have two).     

R1 and R2 retrotransposition and deletion in the rDNA loci on the X and Y chromosomes of Drosophila melanogaster

The non-LTR retrotransposons R1 and R2 insert into the 28S rRNA genes of arthropods. Comparisons among Drosophila lineages have shown that these elements are vertically inherited, while studies within species have indicated a rapid turnover of individual copies (elimination of old copies and the insertion of new copies). To better understand the turnover of R1 and R2, 200 retrotranspositions and nearly 100 eliminations have been scored in the Harwich mutation-accumulation lines of Drosophila melanogaster. Because the rDNA arrays in D. melanogaster are present on the X and Y chromosomes and no exchanges were detected in these lines, it was possible to show that R1 retrotranspositions occur predominantly in the male germ line, while R2 retrotranspositions were more evenly divided between the germ lines of both sexes. The rate of elimination of elements from the Y rDNA array was twice that of the X rDNA array with both chromosomal loci containing regions where the rate of elimination was on average eight times higher. Most R1 and R2 eliminations appear to occur by large intrachromosomal events (i.e., loop-out events) that involve multiple rDNA units. These findings are interpreted in light of the known abundance of R1 and R2 elements in the X and Y rDNA loci of D. melanogaster.     

Molecular evolution of two paralogous tandemly repeated heterochromatic gene clusters linked to the X and Y chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.     

Evolution of transcribed and spacer sequences in the ribosomal RNA genes of Drosophila.

Examination of the ribosomal RNA (rRNA) gene of six sibling species that make up the D. melanogaster subgroup reveals that the nontranscribed spacer is highly conserved during evolution. Indeed, the spacer is at least as conserved as the transcribed rRNA sequence in four of the six species and only slightly less conserved in the others. These data support the hypothesis previously suggested (Tartof and Dawid, 1976) that selection has a significant role in maintaining the parallel evolution of genetically separate but homologous redundant gene clusters.     

Alternative pathways in the processing of ribosomal RNA precursor in Drosophila melanogaster

The size of RNA molecules that are intermediates in the processing of ribosomal RNA in Drosophila melanogaster has been determined by gel electrophoresis under fully denaturing conditions. These molecules have been characterized by transfer from agarose gels to diazobenzyloxymethyl-paper and hybridization with restriction fragments derived from cloned ribosomal DNA. Five cleavage sites leading to the production of 18 S and 28 S RNA have been mapped in the precursor. The first cleavage in the precursor molecule occurs at one of two different sites. Therefore, we propose two alternative pathways for the processing of D. melanogaster ribosomal RNA. A precursor molecule to 2 S and 5.8 S ribosomal RNA has been identified in nuclear RNA.     

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.     

Characterization of the Drosophila melanogaster ribosomal proteome

We have combined high-resolution two-dimensional (2-D) gel electrophoresis with mass spectrometry to identifying proteins represented in a 2-D gel database of Drosophila melanogaster ribosomes. First, we purified ribosomes from third instar Drosophila larvae and constructed a high-resolution 2-D gel database containing 58 Coomassie blue stained polypeptides. Next, we carried out preparative 2-D PAGE to isolate some of the polypeptides and characterize them by MALDI-TOF. Using this strategy we identified 52 ribosomal spots in the database, and in each case confirmed their identity by MALDI-TOF/TOF. The database can be used to analyze Minute mutants of Drosophila.