Complementary DNA-DNA hybridization in Drosophila

Summary We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.On leave from the Dipartimento di Biologia, II Università di Roma Tor Vergata, Rome, Italy     

Localization of genes ecs,dor and swi in eight Drosophila species

A cluster of genes corresponding to the early ecdysone stimulated puff 2B of the Drosophila melanogaster X chromosome has been localized using in situ hybridization in eight Drosophila species. Genes ecs, dor and swi from this cluster have been mapped in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum and D. paranaensis to the telomeric region of the X chromosome, in D. kanekoi to the distal region, and in D. pseudoobscura, to the proximal region of the X chromosome. It is assumed that organization of this cluster in these species is conserved. In D. hydei, multiple hybridization sites of certain DNA probes from this region were found.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

Identification and Characterization of the Cecropin Antibacterial Protein Gene Locus in Drosophila virilis

Cecropin is a type of antibacterial peptide that is synthesized in response to infection and has been characterized in many insect species and one mammal. The Cecropin locus of Drosophila melanogaster also contains the gene Andropin, which has been identified only in this species and encodes a male-specific antibacterial peptide. As a first step in studying the molecular evolution of the cecropin and andropin genes among Drosophila species, we have isolated genomic clones that cover the Cecropin locus in Drosophila virilis. The cloned region totals approximately 25 kb, within which a 9-kb fragment contains four cecropin genes and one pseudogene. All four genes have a high level of sequence homology to D. melanogaster Cecropin, about 80% identity in the coding regions, and the intron positions are conserved. As in D. melanogaster and other insects, κB-related cis-regulatory elements are found upstream of these cecropin genes. An Andropin-related sequence was not identified in D. virilis; however, genome Southern hybridizations suggest that Andropin-related sequences are present in at least the melanogaster species subgroup. Analysis of 19 insect cecropin genes identifies a common ancestral Cecropin before the divergence of Diptera and Lepidoptera. In addition, D. melanogaster and D. virilis can be identified by monophyletic clades for Cecropin. In contrast, the Lepidopteran species show polyphyletic relationships for duplicated cecropin genes. Received: 12 August 1996 / Accepted: 18 October 1996     

Radiation of the ,,Drosophila“ subgenus (Drosophilidae,Diptera) in the Neotropics

We present a phylogenetic hypothesis for 72 ,,Drosophila“ species, constructed through analysis from the paralogous alpha methyldopa (amd) and dopa decarboxylase (ddc) nuclear genes (encompassing a total of 2015 base pairs). Our data support the subdivision of the paraphyletic subgenus ,,Drosophila“ into three main radiations (the immigrans‐tripunctata, the virilis‐repleta and the Hawaiian Drosophilidae), each of which is further subdivided to originate monophyletic ‘sub‐radiations’. Moreover, this study raises the possibility that the Zaprionus/Liodrosophila species encompass a fourth radiation within the subgenus ,,Drosophila“ phylogeny and provides temporal estimates for each of the postulated divergence events.     

On the Evolution of Dopa decarboxylase (Ddc) and Drosophila Systematics

We have sequenced most of the coding region of the gene Dopa decarboxylase (Ddc) in 24 fruitfly species. The Ddc gene is quite informative about Drosophila phylogeny. Several outstanding issues in Drosophila phylogeny are resolved by analysis of the Ddc sequences alone or in combination with three other genes, Sod, Adh, and Gpdh. The three species groups, melanogaster, obscura, and willistoni, are each monophyletic and all three combined form a monophyletic group, which corresponds to the subgenus Sophophora. The Sophophora subgenus is the sister group to all other Drosophila subgenera (including some named genera, previously considered outside the Drosophila genus, namely, Scaptomyza and Zaprionus, which are therefore downgraded to the category of subgenus). The Hawaiian Drosophila and Scaptomyza are a monophyletic group, which is the sister clade to the virilis and repleta groups of the subgenus Drosophila. The subgenus Drosophila appears to be paraphyletic, although this is not definitely resolved. The two genera Scaptodrosophila and Chymomyza are older than the genus Drosophila. The data favor the hypothesis that Chymomyza is older than Scaptodrosophila, although this issue is not definitely resolved. Molecular evolution is erratic. The rates of nucleotide substitution in 3rd codon position relative to positions 1 + 2 vary from one species lineage to another and from gene to gene. Received: 2 June 1998 / Accepted: 3 September 1998     

A framework physical map of Drosophila virilis based on P1 clones: applications in genome evolution

The analysis of patterns of genome evolution may help to evaluate the evolutionary forces that shape the composition and organization of the genome. Comparisons between the physical maps of divergent species can be used to identify conserved blocks of closely linked genes whose synteny is possibly under selective constraint. We have used in situ hybridization to determine the genomic position of 732 randomly selected clones from a bacteriophage P1 library of Drosophila virilis. The resulting map includes at least one clone in each of 69% of the subdivisions into which the D. virilis polytene chromosomes are divided. A subset of these clones was used to carry out a comparative physical analysis of chromosome 2 from D. virilis and from Drosophila montana. A number of discrepancies with the classical scenario of chromosome evolution were noted. The D. virilis P1 clones were also used to determine the physical relations between ten genes that are located in the X chromosome of Drosophila melanogaster between the markers crn (2F1) and omb (4C5-6). In this region, which is approximately 2 Mb in length, there have been at least six breakpoints since the divergence of the species, and six of the genes are found at widely scattered locations in the D. virilis X chromosome. However, a block of four functionally unrelated genes, including white, roughest, Notch, and dunce, seems to be conserved between the two species. Received: 1 March 1996 / Accepted: 8 February 1997     

Molecular phylogeny of the subgenus Sophophora of Drosophila derived from large subunit of ribosomal RNA sequences

RNA sequencing has been used to assess the relationships among species of the subgenus Sophophora of the genus Drosophila. Two divergent domains, D1 and D2, of the large ribosomal RNA (28S), totalling 550 nucleotides have been sequenced using the rRNA direct sequencing method. A tree has been reconstructed from the neighbor-joining algorithm and the confidence intervals were evaluated by the bootstrap procedure. Results have shown that the branching of the willistoni and saltans groups of the subgenus Sophophora is very ancient and probably predates that of the subgenus Drosophila. The other groups and subgroups of Sophophora are clustered in three main lineages: 1) the melanogaster and oriental subgroups; 2) the montium subgroup; 3) the ananassae subgroup of the melanogaster group clustered with the fima and obscura groups. Thus, in comparison with our results, several taxa of various ranks appear paraphyletic (the genus Drosophila, the subgenus Sophophora and the melanogaster group). Our biochemical phylogeny is only in partial agreement with the pattern of Throckmorton's radiations as well as with classical taxonomy, both based on morphological data.     

Karyotype variation in Drosophila birchii

Drosophila birchii, a member of the melanogaster species group of the subgenus Sophophora, is common in the tropical rain forests of the Australia-New Guinea areas. Chromosome squashes are easily prepared from the larval ganglion cells and the sex chromosomes are readily recognizable. The species exhibits a remarkable karyotype variation. The metaphase plate figures, in general, show two pairs of V's, one pair of dots and one pair of sex chromosomes. Variations in metaphase chromosome morphology are found in the X (with four types), the Y (with three types) and chromosome IV (with two types). Chromosomal interchanges between X- and Y-chromosomes Type I are postulated to be involved in the differentiation of sex chromosome morphology while the modification of chromosome IV seems likely to be a result of the acquisition of extra heterochromatin. These chromosome types form seven distinct metaphase plate figures, all encountered in wild populations, thus giving D. birchii the most variable karyotype in the genus Drosophila.     

High Incidence of Interchromosomal Transpositions in the Evolutionary History of a Subset of Or Genes in Drosophila

In insects, the odorant receptor (Or) multigene family is an intermediate-sized family with genes present in all chromosomes, indicating that duplication followed by interchromosomal transposition played an important role in the early stages of the family evolution. Here, we have explored the occurrence of interchromosomal transpositions in more recent stages through the comparative analysis of a subset of Or genes in Drosophila, where the gene content of chromosomal arms is highly conserved. The studied subset consisted of 11 Or genes located on the left arm of chromosome 3 (Muller’s D element) in D. melanogaster. Our study focused on the number and chromosomal arm location of these members of the family across the 12 Drosophila species with complete genome sequences. In contrast to previous results from in situ hybridization comparative mapping that were mainly based on single-copy genes, our study, based on members of a multigene family of moderate size, revealed repeated interchromosomal transposition events and a complex history of some of the studied genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Divergence of the Regulation of alpha-Amylase Activity in Drosophila melanogaster, Drosophila funebris, and Drosophila saltans

The regulation of amylase activity in threeDrosophila species, D. melanogaster,D. funebris and D. saltans, wasanalyzed by measuring the specific activity levels infour dietary environments, cornmeal, glucose, 5% starch, and 10% starch, at threedevelopmental stages, i.e., the third-instar larval,pupal, and 2-day-old adult stages. The developmentalprofiles of amylase activity for the threeDrosophila species showed that the level of activity washigh at the larval and adult stages but substantiallylow at the pupal stage, suggesting thatDrosophila does not utilize starch at the pupalstage. Divergence in the regulation of amylase was observed amongthe three Drosophila species on the followingpoints. (1) The order of amylase specific activity wasD. melanogaster > D. funebris >D. saltans. (2) The response pattern to the dietary environment varied amongthe species and changed during development. (3) Thetiming of the switch in the response pattern to thedietary environment during development was before pupation in D. funebris and D.saltans but after pupation in D.melanogaster. The significance of the divergence inthe regulation of amylase activity for adaptation to astarch environment in Drosophila is discussed.     

Distribution of the Transposable Element Minos in the Genus Drosophila

We analyzed 28 species of the genus Drosophilafor the presence of the Tc1-like transposable element Minosusing Southern blot hybridization under high stringency conditions. The Minostransposon was found in members of both the Drosophilaand the Sophophorasubgenus showing a distribution that is wider if compared to other well-studied Drosophilatransposons such as the Pelement, hoboand mariner. The presence of Minos-hybridizing sequences was discontinuous in the Sophophorasubgenus, especially in the melanogasterspecies group. Using the Polymerase Chain Reaction we amplified a portion corresponding to the putative Minostransposase from different Drosophilaspecies. Cloning and sequence analysis of randomly selected Minoscopies from D. mojavensisis, D. saltansand D. willistonisupports the idea that event(s) of horizontal transfer may have contributed to the spreading of this transposon in the Drosophilagenus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Conservation of the 93D puff of Drosophila melanogaster in different species of Drosophila

Temperature shock (TS) results in activation of a specific set of puffs in polytene nuclei of D. melanogaster. Earlier studies in this species from several laboratories revealed certain unique features of the major TS puff at 93D locus, which is also specifically induced by benzamide (BM) and by incubation of glands in heat shocked glands' homogenate (HSGH). We have now extended studies on TS response to several other species of Drosophila to ascertain whether loci homologous to 93D puff of D. melanogaster are present in other species. In polytene nuclei of two closely related (D. ananassae, D. kikkawai) and in two distantly related species (D. hydei, D. nasuta), six to nine puffs are induced by TS. Interestingly, in each species one of the major TS puffs, viz., 2L-2C in D. ananassae, E-11BC in D. kikkawai, 2R-48A in D. nasuta and 2-48C in D. hydei, is also specifically induced by BM, autologous species' HSGH and vitamine-B₆ (vit-B₆) treatment. HSGH of a different species fails to induce these puffs. These puffs thus resemble the 93D locus of D. melanogaster, although the 93D puff does not respond to vit-B₆. These observations are discussed in relation to the conservation of 93D puff locus in different species of Drosophila.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a λ vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes. This paper is dedicated to Professor W. Beermann on the occasion of his sixtieth birthday     

The Adh in Drosophila: Chromosomal location and restriction analysis in species with different phylogenetic relationships

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.by H. Jäckle     

The Suppressor of fused gene, involved in Hedgehog signal transduction in Drosophila, is conserved in mammals

 The Suppressor of fused [Su(fu)] gene of Drosophila melanogaster encodes a protein containing a PEST sequence [sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] which acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. We show that these two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. We have also isolated mammalian homologs of Su(fu). The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, we show that the murine Su(fu) protein can interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. These data are discussed in the light of their evolutionary relationships. Received: 11 September 1998 / Accepted: 9 December 1998     

The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement

The restriction endonuclease pattern of a number of hsp70-homologous clones isolated from a library of heat shock cDNA from Drosophila auraria, a species belonging to the montium subgroup of the melanogaster species group, reveals two types of clones, A and B, differing in a single restriction site. Both types, as well as hsp70-specific probes derived from both hsp70 loci of Drosophila melanogaster, hybridize in situ with a single band at region 32 A of the 2L polytene arm, indicating a clustered organization of the hsp70 gene copies in D. auraria. The longest type B clone was sequenced and it was found that one strand contains an open reading frame (ORF) exhibiting great identity with a previously described hsp70 gene of D. auraria (now denoted as type A) and with its counterparts of D. melanogaster, while its second strand, unlike the type A clone, does not contain a long antiparallel coupled ORF (LAC ORF) because of a base substitution resulting in a premature stop codon. After additional data had been derived from isolation and characterization of hsp70-homologous genomic clones, together with Southern analysis of genomic DNA, we found that two hsp70 gene copies are present at the above locus of D. auraria with an inverted tandem repeat organization, while the presence of a third hsp70 gene is not clearly evident. The above results are compared with those observed at the homologous loci of some melanogaster subgroup species (D. melanogaster and its sibling species), in which, however, the hsp70 locus is duplicated, and with the more distantly related Dipteran Anopheles albimanus. Received: 22 May 1998; in revised form: 18 September 1998 / Accepted: 21 September 1998