Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Natural selection drives Drosophila immune system evolution

Evidence from disparate sources suggests that natural selection may often play a role in the evolution of host immune system proteins. However, there have been few attempts to make general population genetic inferences on the basis of analysis of several immune-system-related genes from a single species. Here we present DNA polymorphism and divergence data from 34 genes thought to function in the innate immune system of Drosophila simulans and compare these data to those from 28 nonimmunity genes sequenced from the same lines. Several statistics, including average K(A)/K(S) ratio, average silent heterozygosity, and average haplotype diversity, significantly differ between the immunity and nonimmunity genes, suggesting an important role for directional selection in immune system protein evolution. In contrast to data from mammalian immunoglobulins and other proteins, we find no strong evidence for the selective maintenance of protein diversity in Drosophila immune system proteins. This may be a consequence of Drosophila's generalized innate immune response.     

Natural selection on the Drosophila antimicrobial immune system

The evolutionary dynamics of immune defenses have long attracted interest because of the special role the immune system plays in mediating the antagonistic interaction between hosts and pathogens. The antimicrobial immune system of the fruit fly Drosophila melanogaster is genetically well characterized and serves as a valuable model for studying insect and human innate immune defenses. I review here evolutionary and comparative genomic analyses of insect antimicrobial immune genes, with an emphasis on Drosophila. Core signal transduction pathways in the immune system are orthologously conserved across long evolutionary distances, but genes in these pathways evolve rapidly and adaptively at the amino acid sequence level. By contrast, families of genes encoding antimicrobial peptides are remarkably dynamic in genomic duplication and deletion, yet individual genes show little indication of adaptive sequence evolution. Pattern recognition receptors that trigger humoral immunity are evolutionarily rather static, but receptors required for phagocytosis show considerable genomic rearrangement and adaptive sequence divergence. The distinct evolutionary patterns exhibited by these various classes of immune system genes can be logically connected to the functions of the proteins they encode.     

The ribosomal RNA genes of Drosophila mitochondrial DNA.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba which contains the A+T-rich region and the small and large rRNA genes separated by the tRNAval gene has been determined. The 5' end of the small rRNA gene was located by S1 protection analysis. In contrast to mammalian mtDNA, a tRNA gene was not found at the 5' end of the D. yakuba small rRNA gene. The small and large rRNA genes are 20.7% and 16.7% G+C and contain only 789 and 1326 nucleotides. The 5' regions of the small rRNA gene (371 nucleotides) and of the large rRNA gene (643 nucleotides) are extremely low in G+C (14.6% and 9.5%, respectively) and convincing sequence homologies between these regions and the corresponding regions of mouse mt-rRNA genes were found only for a few short segments. Nevertheless, the entire lengths of both of the D. yakuba mt-rRNA genes can be folded into secondary structures which are remarkably similar to secondary structures proposed for the rRNAs of mouse mtDNA. The replication origin-containing, A+T-rich region (1077 nucleotides; 92.8% A+T), which lies between the tRNAile gene and the small rRNA gene, lacks open reading frames greater than 123 nucleotides.     

Characterization of cloned ribosomal DNA from Drosophila hydei.

The structure of ribosomal genes from the fly Drosophila hydei has been analyzed. EcoRI fragments, cloned in a plasmid vector, were mapped by restriction enzyme analysis. The lengths of the regions coding for 18S and 28S rRNA were defined by R-loop formation. From these data a physical map of the rRNA genes was constructed. There are two major types of rDNA units in D. hydei, one having a size of 11 kb and the other a size of 17 kb. The 17 kb unit results from an intervening sequence (ivs) of 6.0 kb, interrupting the beta-28S rRNA coding region. Some homology between th D. hydei ivs and D. melanogaster type 1 ivs has been described previously (1). However, the restriction sites within these ivs show considerable divergence. Whereas D. hydei rDNA D. melanogaster rDNA, the nontranscribed spacer has little, if any, sequence homology. Despite difference in sequence, D. hydei and D. melanogaster spacers show structural similarities in that both contain repeated sequence elements of similar size and location.     

Positive and negative selection on noncoding DNA in Drosophila simulans

There is now a wealth of evidence that some of the most important regions of the genome are found outside those that encode proteins, and noncoding regions of the genome have been shown to be subject to substantial levels of selective constraint, particularly in Drosophila. Recent work has suggested that these regions may also have been subject to the action of positive selection, with large fractions of noncoding divergence having been driven to fixation by adaptive evolution. However, this work has focused on Drosophila melanogaster, which is thought to have experienced a reduction in effective population size (N(e)), and thus a reduction in the efficacy of selection, compared with its closest relative Drosophila simulans. Here, we examine patterns of evolution at several classes of noncoding DNA in D. simulans and find that all noncoding DNA is subject to the action of negative selection, indicated by reduced levels of polymorphism and divergence and a skew in the frequency spectrum toward rare variants. We find that the signature of negative selection on noncoding DNA and nonsynonymous sites is obscured to some extent by purifying selection acting on preferred to unpreferred synonymous codon mutations. We investigate the extent to which divergence in noncoding DNA is inferred to be the product of positive selection and to what extent these inferences depend on selection on synonymous sites and demography. Based on patterns of polymorphism and divergence for different classes of synonymous substitution, we find the divergence excess inferred in noncoding DNA and nonsynonymous sites in the D. simulans lineage difficult to reconcile with demographic explanations.     

Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster.

The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers and analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy.     

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.     

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster

Summary Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.     

Analysis of differences in the structure of ribosomal DNA from two sibling species: Drosophila melanogaster and Drosophila simulans

Southern-blot hybridization studies have revealed structural differences within the ribosomal DNA clusters of two sibling species, Drosophila melanogaster and Drosophila simulans. The approach used was shown to be suitable for taxonomic identification of the species examined.     

Localization of ribosomal DNA insertion elements in polytene chromosomes of Drosophila simulans,Drosophila mauritiana and their interspecific hybrids

The locations of the ribosomal DNA (rDNA) insertion elements type I and type II along the polytene chromosomes of three Drosophila species of the melanogaster subgroup-D. simulans, D. mauritiana and D. melanogaster-have been compared. In situ hybridization has shown that the intragenomic distribution of type I as well as of type II insertions is different for these related species. In particular, we have revealed rDNA-free autosomal sites, containing type II element sequences within the D. simulans and D. mauritiana chromosomes. This finding confirms the ability of this type of insertion to transpose, as was demonstrated earlier for Bombyx mori. The appearance of the rDNA not associated with the nucleolar organizers, evident by additional nucleoli, occurred with species-specific frequency. At the same time, for all three species the pattern of such changes (an attachment of the nucleoti to varying sites of the chromosomes and the presence of ectopic contacts between them, a composition of the rDNA repeats in the nucleolar material not integrated at the nucleolar organizer) was similar. The number of additional nucleoti in the hybrid polytene nuclei corresponded to the value of the parental species exhibiting nucleolar replicative dominance.     

Nonselective amplification of ribosomal DNA repeat units in compensating genotypes of Drosophila melanogaster

Compensation is a mechanism by which the X-chromosome nucleolus organizer region of Drosophila melanogaster can increase its ribosomal DNA content up to twofold. It occurs in somatic cells under specific genetic conditions and is mediated by a defined genetic site, the compensatory response locus. The In and various type I ribosomal DNA repeat units were separated by restriction endonuclease digestion. Comparison of the percentages of these repeat unit types between compensating and noncompensating genotypes showed the same distribution. Therefore no selective amplification of these repeat unit types occurs during ribosomal DNA compensation. These results demonstrate that two processes of rDNA amplification in somatic cells, compensation and independent rDNA polytenization, are exclusive events.This research was supported by NIH Grant GM 28008.