Chromosomal homologies betweenDrosophila lebanonensis andD. melanogaster determined by in situ hybridization

Twelve biotin-labelled recombinant DNA probes were hybridized to polytene chromosomes ofDrosophila melanogaster andD. lebanonesis. Probes were chosen in order to cover the whole chromosomal complement. Six probes correspond to known genes fromD. melanogaster (RpII215, H3–H4, MHC, hsp28/23, hsp83, hsp70), four probes are clones isolated from aD. subobscura library (Xdh, DsubS3, DsubG3, DsubG4) and the remaining two probes correspond to the Adh gene ofD. lebanonensis and to one sequence (262), not yet characterized, from the same species. The chromosomal homologies obtained from the in situ hybridization results allow us to determine that Muller's C and D chromosomal elements are fused in the karyotype ofD. lebanonensis and constitute the large metacentric chromosome. Single pericentric inversions in theE andB elements have generated the medium and small metacentric chromosomes, respectively. No great changes are detected in Muller'sA element, which remains acrocentric. The changes detected in the karyotypic evolution ofD. lebanonensis are frequently observed inDrosophila evolution, as deduced from chromosomal homologies of severalDrosophila species. The results are also consistent with Muller's proposal that chromosomal elements have been conserved during the evolution ofDrosophila.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

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.     

Preadult competition between Drosophila subobscura and Drosophila pseudoobscura

The Palaearctic species Drosophila subobscura has recently colonized a large area of North America where it coexists with Drosophila pseudoobscura. The viability and developmental rate of these species were studied at 13 d?C, 18d?C and 23 d?C and at densities of 10, 50, 100 and 200 eggs per vial. The two species were differently affected by density and temperature in the ranges studied. Both intra- and interspecific cultures showed that D. pseudoobscura was best adapted to 23 d?C, where it was clearly the dominant species. On the other hand, at 18 d?C and especially at 13 d?C, although D. subobscura was less viable than D. pseudoobscura, its developmental time was shorter, which may give advantage to this species. Results reported here agree with the observed distribution of these species in North America.     

Chromosomal locations of actin genes are conserved between the melanogaster and obscura groups of Drosophila

The actin genes of D. subobscura and D. madeirensis were mapped by in situ hybridization, using a D. melanogaster probe. Six loci were detected, and they were strikingly similar in chromosomal location to the six actin loci previously characterized in D. melanogaster.     

Comparative mapping of cosmids and gene clones from a 1.6 Mb chromosomal region of Drosophila melanogaster in three species of the distantly related subgenus Drosophila

The successful hybridization of cosmid clones from Drosophila melanogaster (Sophophora subgenus) to the salivary gland chromosomes of other species as distantly related as those in the Drosophila subgenus attests their great potential for unravelling genome evolution. We have carried out, using 28 cosmids and 13 gene clones, a study of the organization of the D. melanogaster 95A-96A chromosomal region in three Drosophila subgenus species: D. repleta, D. buzzattii and D. virilis. These clones were first used to built an accurate map of this 1.6 Mb region of D. melanogaster chromosome 3R (Muller’s element E). Then, they were hybridized and mapped to the homologous chromosome 2 of the other three distantly related species. The studied region is disseminated over 13 different sites of chromosome 2 in the Drosophila subgenus species, which implies a minimum of 12 inversion breakpoints fixed between the two subgenera. Extrapolation to the entire chromosome gives 90 fixed inversions. The D. melanogaster Pp1-96A-Acr96Aa segment conserved in D. repleta and D. buzzatii is longer than previously thought and is also conserved in D. virilis. In addition, three other D. melanogaster segments conserved in the three Drosophila subgenus species were found. Finally, our data indicate significant statistical differences in the evolution rate of Muller’s element E among lineages, a result that agrees well with the previous cytogenetic data. Received: 22 July 1998; in revised form: 11 November 1998 / Accepted: 12 November 1998     

A Genomic Comparison of Faster-Sex, Faster-X, and Faster-Male Evolution Between Drosophila melanogaster and Drosophila pseudoobscura

A genomic comparison of Drosophila melanogaster and Drosophila pseudoobscura provides a unique opportunity to investigate factors involved in sequence divergence. The chromosomal arrangements of these species include an autosomal segment in D. melanogaster which is homologous to part of the X chromosome in D. pseudoobscura. Using orthologues to calculate rates of nonsynonymous (d_N) substitutions, we found genes on the X chromosome to be significantly more diverged than those on the autosomes, but it is not true for segment 3L-XR which is autosomal in D. melanogaster (3L) and X-linked in D. pseudoobscura (XR). We also found that the median d_N values for genes having reproductive functions in either the male, the female, or both sexes are higher than those for sequences without reproductive function and even higher for sequences involved in male-specific function. These estimates of divergence for male sex-related sequences are most likely underestimates, as the very rapidly evolving reproductive genes would tend to lose homology sooner and thus not be included in the comparison of orthologues. We also noticed a high proportion of male reproductive genes among the othologous genes with the highest rates of d_N. Reproductive genes with and without an orthologue in D. pseudoobscura were compared among D. melanogaster, D. simulans, and D. yakuba and it was found that there were in fact higher rates of divergence in the group without a D. pseudoobscura orthologue. These results, from widely separated taxa, bolster the thesis that sexual system genes experience accelerated rates of change in comparison to nonsexual genes in evolution and speciation. [Reviewing Editor: Dr. Willie J. Swanson]     

Sex‐ratio in natural and experimental populations of Drosophila subobscura from North America

The sex‐ratio (SR), defined as the proportion of males, has been studied in three North American colonizing populations of Drosophila subobscura (Eureka, Davis and Gilroy). The proportion of sexes under laboratory conditions was studied using the one‐generation serial transfer technique in one‐ and two‐species populations, to infer whether biased SR affects the outcome when competing with Drosophila pseudoobscura, another member of the same group now in sympatry with D. subobscura in North America. The wild samples of D. subobscura yielded a significantly higher number of males than females during those months where the species is more abundant. However, there was no significant deviation in the 1 : 1 proportion of sexes in the descendants of D. subobscura at any of the experimental conditions. On the contrary, D. pseudoobscura produced a higher proportion of females which could be responsible for the exclusion of D. subobscura in laboratory competition experiments with overlapping generations. Thus, if sexes are equal at birth and survival is similar, the preponderance of males of D. subobscura in our wild collections could indicate greater activity and probably greater chance of dispersal of males versus females especially under favourable conditions.     

An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

Enzyme activity and dynamics of Drosophila development

The length of preadult development is negatively correlated with the activity of a majority of studied enzymes, in adult D. melanogaster and D. subobscura flies. This has been shown when activities of seven enzymes (G6PD, 6PGD, GPD, ADH, HK, ME & IDH) were estimated per mg of protein, or of body mass, in four groups of 6-days old males (50 individuals each, with 3 replications), that had an extremely different preadult development rate. The average activity of studied enzymes is for c. 25% decreased in synchronously grown flies with the longest egg-to-adult development at 21°C and optimal laboratory conditions, compared with those of the same species with the fastest growth. When the group of slowest growing D. subobscura males (20.4±0.1 days) is compared with the fastest D. melanogaster flies (10.6±0.03 days), a decrease of 47% in enzyme activity was observed. Among studied gene-enzyme loci, four in D. subobscura (Gpd, Adh, Me & idh) and one in D. melanogaster (Idh) are monomorphic, which implies an involvement of regulatory genes. Among those of D. melanogaster which are polymorphic, specific combinations of alleles have been determined in fast and slow developed flies, suggesting that interactions of structural genes are also of great importance in the control of two studied fitness characteristics.     

Historical effective size and the level of genetic diversity in Drosophila melanogaster and Drosophila pseudoobscura

We report the results of a sequential gel electrophoretic study of protein variation in Drosophila melanogaster and its comparison with D. pseudoobscura. The number of alleles and mean heterozygosity were lower in D. melanogaster than in D. pseudoobscura. On the other hand, geographical populations of Drosophila melanogaster have been shown to be much more differentiated than those of D. pseudoobscura. The results suggest that in D. melanogaster low-frequency alleles have been lost during the colonization process and that major alleles have become differentiated among populations. Population bottlenecks, due to various causes, appear to have played a significant role in the shaping of genetic variation in natural populations of many species. It is proposed that a comparison of genetic variation at homologous gene loci between related species can bring out effects of historical bottlenecks and provide an alternative approach for analyzing causes of genetic variation in natural populations.We thank the Natural Science and Engineering Research Council of Canada for financial support (Grant A0235 to R.S.S.).     

INTERSPECIFIC LABORATORY COMPETITION OF THE RECENTLY SYMPATRIC SPECIES DROSOPHILA SUBOBSCURA AND DROSOPHILA PSEUDOOBSCURA

Abstract.—Drosophila subobscura and D. pseudoobscura are closely related species coexisting on the West Coast of North America, which was recently colonized by D. subobscura. In competition experiments with overlapping generations, D. subobscura is eliminated by D. pseudoobscura in a few generations at all four temperatures and two initial frequencies tested. Yet in one-species cultures, D. subobscura thrives at all experimental conditions. Single-generation competition experiments reveal lower survivorship and productivity of D. subobscura at all temperatures and frequencies. Productivity per female is dependent on the initial frequencies: greater for D. subobscura as its initial frequency becomes higher, but lower for D. pseudoobscura as its frequency becomes higher. Strains of D. subobscura from three disparate geographic origins yield similar results.     

Ultrastructure of regions containing homologous loci in polytene chromosomes of Drosophila melanogaster and Drosophila subobscura

We have used a new approach involving in situ hybridisation and electron microscopy to establish ultrastructural homologies between polytene chromosome regions of Drosophila melanogaster and Drosophila subobscura. Twelve probes were chosen to cover all the chromosomal elements: the myospheroid gene, the collagen type IV gene, the collagen-like gene, the w26 homeobox gene, the β3 tubulin gene, the kinesin heavy chain gene, the tryptophan hydrolase gene, the Hsp82, Hsp22–26 and Hsp23–28, Hsp68, Hsp70 genes and the β unit of the F₀–F₁ ATPase gene. Most of these loci were previously undescribed in D. subobscura and imprecisely located in D. melanogaster. We have demonstrated here, by an ultrastructural analysis of each chromosomal region, that homologous genetic loci tend to show a similar ultrastructure in the two species. With a few exceptions, the structural homology extends to the chromosomal regions surrounding the loci. In some cases, however, no structurally recognisable homology can be seen either in the locus or in its flanking regions. Received: 15 December 1996; in revised form: 15 October 1997 / Accepted: 28 January 1998     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

Molecular characterization of the zerknüllt region of the Antennapedia complex of D. subobscura

We have characterized at the molecular level the zerknüllt (zen) region of the Drosophila subobscura Antennapedia complex. The sequence comparison between D. subobscura and D. melanogaster shows an irregular distribution of the conserved and diverged regions, with the homeobox and a putative activating domain completely conserved. Comparisons of the promoter sequence and pattern of expression of the gene during development suggest that the regulation of zen has been conserved during evolution. The conservation of zen expression in a subpopulation of the polar cells indicates the existence of an important role in such cells. We describe a transitory segmented pattern of expression of zen in both species, suggesting the existence of interactions with a pair rule gene. Some indirect clues indicate that the z2 gene might be absent from the D. subobscura genome. A chromosome walk initiated to reach the proboscipedia gene of D. subobscura reveals that the distance between pb and zen is at least four times the one described for D. melanogaster and for D. pseudoobscura. Finally, we present cytological evidence showing that the ANT-C is inverted in D. subobscura as compared to D. melanogaster.     

Analysis of a swallow homologue from Drosophila pseudoobscura

We analyzed a functional homologue of the swallow gene from Drosophila pseudoobscura. The swallow gene of D. melanogaster plays an essential role in localizing bicoid mRNA in oocytes, and swallow mutant embryos show anterior pattern defects that result from the lack of localization of the bicoid morphogen. The pseudoobscura homologue rescues the function of swallow mutants when introduced into the genome of D. melanogaster, and its expression is similar to that of the melanogaster gene. The predicted pseudoobscura and melanogaster proteins are 49% identical and 69% conserved. The coiled-coil domain previously identified in the melanogaster swallow protein is strongly conserved in the pseudoobscura homologue, but the weak similarity of the melanogaster swallow protein to the RNP class of RNA-binding proteins is not conserved in the pseudoobscura homologue. These and other observations suggest a structural role for swallow in localizing bicoid mRNA, perhaps as part of the egg cytoskeleton. Received: 3 August 1999 / Accepted: 29 September 1999     

Cytological localization and organization of dispersed middle repetitive DNA sequences of Drosophila subobscura

To study the middle repetitive fraction of the Drosophila subobscura genome, 26 phage clones containing repetitive sequences were examined by Southern DNA blot analysis and by in situ hybridization to polytene chromosomes. These results led to a classification of the clones according to five different types of hybridization patterns. Two types, each containing seven clones, are characterized by hybridization at 100 to 300 sites dispersed over the euchromatic parts of the chromosomes, and in addition by one prominently labelled chromosome band. One of these two classes also showed strong labelling of the chromocentre. The remaining types of hybridization pattern lacked a prominent band but showed hybridization either to the euchromatic regions or to the chromocentre or both. Chromosome A (=X) was the preferred location of prominently labelled bands and it also showed an excess of labelling by some clones. Some of the cloned dispersed sequences were localized cytologically on chromosomes of larvae from crosses between different strains of D. subobscura and between two closely related species, in order to detect heterozygosity at hybridization sites. Comparisons of the chromosomal distribution of labelling sites showed differences in number and location, indicating the possibility of transposition events.     

Repetitive DNA sequences in Drosophila

The satellite DNAs of Drosophila melanogaster and D. virilis have been examined by isopycnic centrifugation, thermal denaturation, and in situ molecular hybridization. The satellites melt over a narrow temperature range, reassociate rapidly after denaturation, and separate into strands of differing buoyant density in alkaline CsCl. In D. virilis and D. melanogaster the satellites constitute respectively 41% and 8% of the DNA isolated from diploid tissue. The satellites make up only a minute fraction of the DNA isolated from polytene tissue. Complementary RNA synthesized in vitro from the largest satellite of D. virilis hybridized to the centromeric heterochromatin of mitotic chromosomes, although binding to the Y chromosome was low. The same cRNA hybridized primarily to the -heterochromatin in the chromocenter of salivary gland nuclei. The level of hybridization in diploid and polytene nuclei was similar, despite the great difference in total DNA content. The centrifugation and hybridization data imply that the -heterochromatin either does not replicate or replicates only slightly during polytenization. Similar but less extensive data are presented for D. melanogaster. — In D. melanogaster cRNA synthesized from total DNA hybridized to the entire chromocenter (- and -heterochromatin) and less intensely to many bands on the chromosome arms. The X chromosome was more heavily labeled than the autosomes. In D. virilis the X chromosome showed a similar preferential binding of cRNA copied from main peak sequences.—It is concluded that the majority of repetitive sequences in D. virilis and D. melanogaster are located in the - and -heterochromatin. Repetitive sequences constitute only a small percentage of the euchromatin, but they are widely distributed in the chromosomes. During polytenization the -heterochromatin probably does not replicate, but some or all of the repetitive sequences in the -heterochromatin and the euchromatin do replicate.     

Nucleotide sequence comparison of the Adh gene in three drosophilids

Summary The alcohol dehydrogenase (Adh) gene has been isolated fromDrosophila simulans andD. mauritiana by screening clone libraries of each with a previously cloned Adh gene fromD. melanogaster. The isolated clones were subcloned and partially sequenced to determine the relatedness of these species and to examine details of evolutionary change in the structure of the Adh gene. We report the sequence of the first 704 nucleotides of each gene as well as 127 bases in the 5 untranslated region. When these sequences are compared,D. melanogaster differs fromD. simulans andD. mauritiana by 2.8% and 3.1%, respectively.D. simulans andD. mauritiana differ by only 1.8%, implying that they are more closely related to each other than either is toD. melanogaster. This is consistent with phylogenetic relationships established by a variety of genetic, biochemical, and morphological means and illustrates that DNA sequencing of a single gene may be used to assess the evolutionary relationships of species.