Elvira Juan

Fate of dot chromosome genes in Drosophila willistoni and Scaptodrosophila lebanonensis determined by in situ hybridization

One modification of the primitive karyotype of the Drosophilidae is the absence of dot chromosomes. The origin of this modification is diverse. In some cases, the fate of the dot chromosomes can be directly inferred from cytogenetic analysis but in other cases a genetic or a combined molecular and cytogenetic analysis is needed, as occurs in Drosophila willistoni and Scaptodrosophila lebanonensis. We determined the location of four dot chromosome sequences in D. willistoni and S. lebanonensis using in situ hybridization. Drosophila melanogaster and D. virilis, which possess dot chromosomes, were used as a control. The in situ hybridization results show that dot chromosome genes of D. melanogaster and D. virilis are closely linked in chromosome 3 of D. willistoni and in chromosome X of S. lebanonensis. These results suggest an autosome--dot fusion in D. willistoni and an X--dot fusion in S. lebanonensis, two different paths in the evolution of dot chromosomes.

Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae

There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities.

ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

SummaryIncreasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10−9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.

Nucleotide Sequence of the Genomic Region Encompassing Adh and Adh-Dup Genes of D. lebanonensis (Scaptodrosophila): Gene Expression and Evolutionary Relationships

The region of the genome of D. lebanonensis that contains the Adh gene and the downstream Adh-dup gene was sequenced. The structure of the two genes is the same as has been described for D. melanogaster. Adh has two promoters and Adh-dup has only one putative promoter. The levels of expression of the two genes in this species are dramatically different. Hybridizing the same Northern blots with a specific probe for Adh-dup, we did not find transcripts for this gene in D. lebanonensis. The level of Adh distal transcript in adults of D. lebanonensis is five times greater than that of D. melanogaster adults. The maximum levels of proximal transcript are attained at different larval stages in the two species, being three times higher in D. melanogaster late-second-instar larvae than in D. lebanonensis first-instar larvae. The level of Adh transcripts allowed us to determine distal and proximal initiation transcription sites, the position of the first intron, the use of two polyadenylation signals, and the heterogeneity of polyadenylation sites. Temporal and spatial expression profiles of the Adh gene of D. lebanonensis show qualitative differences compared with D. melanogaster. Adh and Adh-dup evolve differently as shown by the synonymous and nonsynonymous substitution rates for the coding region of both genes when compared across two species of the melanogaster group, two of the obscura group of the subgenus Sophophora and D. lebanonensis of the victoria group of the subgenus Scaptodrsophila. Synonymous rates for Adh are approximately half those for Adh-dup, while nonsynonymous rates for Adh are generally higher than those for Adh-dup. Adh shows 76.8% identities at the protein level and 70.2% identities at the nucleotide level while Adh-dup shows 83.7% identities at the protein level and 67.5% identities at the nucleotide level. Codon usage for Adh-dup is shown to be less biased than for Adh, which could explain the higher synonymous rates and the generally lower nonsynonymous substitution rates in Adh-dup compared with Adh. Phylogenetic trees reconstructed by distance matrix and parsimony methods show that Sophophora and Scaptodrosophila subgenera diverged shortly after the separation from the Drosophila subgenus.

Sequences Upstream of the Homologous cis-elements of the Adh Adult Enhancer of Drosophila Are Required for Maximal Levels of Adh Gene Transcription in Adults of Scaptodrosophila lebanonensis

The evolution of cis-regulatory elements is of particular interest for our understanding of the evolution of gene regulation. The Adh gene of Drosophilidae shows interspecific differences in tissue-specific expression and transcript levels during development. In Scaptodrosophila lebanonensis adults, the level of distal transcripts is maximal between the fourth and eighth day after eclosion and is around five times higher than that in D. melanogaster AdhS. To examine whether these quantitative differences are regulated by sequences lying upstream of the distal promoter, we performed in vitro deletion mutagenesis of the Adh gene of S. lebanonensis, followed by P-element-mediated germ-line transformation. All constructs included, as a cotransgene, a modified Adh gene of D. melanogaster (dAdh) in a fixed position and orientation that acted as a chromosomal position control. Using this approach, we have identified a fragment of 1.5 kb in the 5′ region, 830 bp upstream of the distal start site, which is required to achieve maximal levels of distal transcript in S. lebanonensis. The presence of this fragment produces a 3.5-fold higher level of distal mRNA (as determined by real time quantitative PCR) compared with the D. melanogaster dAdh cotransgene. This region contains the degenerated end of a minisatellite sequence expanding farther upstream and does not correspond to the Adh adult enhancer (AAE) of D. melanogaster. Indeed, the cis-regulatory elements of the AAE have been identified by phylogenetic footprinting within the region 830 bp upstream of the distal start site of S. lebanonensis. Furthermore, the deletions Δ-830 and Δ-2358 yield the same pattern of tissue-specific expression, indicating that all tissue-specific elements are contained within the region 830 bp upstream of the distal start site.

Chromosomal Homologies Between Drosophila Melanogaster and D. Funebris Determined By In-Situ Hybridization

Seventeen biotin-labeled DNA sequences were hybridized to polytene chromosomes of Drosophila melanogaster and D. funebris in order to establish chromosomal homologies between these species. Ten probes correspond to cloned DNA sequences from D. melanogaster (RpII 215, MHC, H3-H4, Tor, hsp 68, hsp 28/23, hsp 83, PP1α, RpII 140, and ey), four are clones isolated from a D. subobscura genomic library (Xdh, DsubS3, DsubG3, and DsubG4), two are clones from D. funebris (F2 and Adh) and one from D. virilis (ci). The probes were chosen in order to cover all the autosomes, since X-chromosome homologies have already been studied by linkage analysis of morphological mutants. Most probes gave a unique hybridization signal; consequently, our results allow unambiguous inferences about chromosomal homologies. The results show extensive gene rearrangement within all chromosomal elements, probably due to paracentric inversions, but are consistent with Mullers proposal that chromosomal elements have conserved their genetic content during the evolution of Drosophila.

Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny

The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.