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Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

In this study, we compare the variability of the regulatory region of the Lim3 gene, which plays a key role in the development of the nervous system, in two populations of Drosophila melanogaster inhabiting the cities of Aleksandrov (Russia) and Raleigh (United States). The two population areas are located in different geographic regions and differ in their ecology. A comparison of nucleotide sequences of 16 (2010) and 20 (2011) alleles from the Alexandrov population showed that in both cases the variability level of the untranslated Lim3 region was considerably lower than that of the 5' regulatory region adjacent to the transcription start site. According to quantitative and quantitative parameters of the variability, there was no difference between samples of different years, which indicates the stability of the population inhabiting the northern border of the species areal. The patterns of polymorphic sites are similar in both populations, which suggest a neutral character of the variability found with respect to environmental factors, as well as the importance of nucleotide substitutions in a number of sites of the Lim3 regulatory region with respect to the control of this gene expression.

The effects of suppressors of position-effect variegation were studied in a set of euchromatin-heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl. The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin-heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.

Adenosine diphosphate-ribosylation factor, ARF1, regulates membrane traffic and structure in the endoplasmic reticulum-Golgi and endosomal systems. The ARF activity, in turn, is regulated by the guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have cloned by transposon tagging a novel Drosophila gene, Gap69C, coding for a putative homolog of ARF1 GTPase-activating protein. The GAP69C protein shares an extensive similarity within its N-terminal zinc-finger domain with the rat and yeast homologs. This domain is known to be required for ARF-GAP activity. The Gap69C is a single-copy gene producing a major 2.1-kb mRNA throughout development, but its amount is decreased in larvae. The eye pigmentation produced by the reporter mini-white gene inserted into the 5' UTR of Gap69C suggests that the expression of Gap69C is nonuniform. In situ hybridization revealed a high level of Gap69C transcripts in the morphogenetic furrow of the eye imaginal disc, where cells are arrested in G(1). Generated by the excision of the P-element, the null allele of Gap69C was found to be viable and fertile and showed no apparent abnormal phenotype, indicating that Gap69C is not essential for fly development. Analysis of the Drosophila genome sequence revealed the presence of other genes related to Gap69C. We propose that the absence of a distinctive phenotype in Gap69C null mutants is attributable to redundancy with other homologs.

In Drosophila, insertions of the P¿lacW¿ transposon led to uncommon expression patterns of the reporter genes white and lacZ: gene activity was gradually reduced in the neighboring cells along the dorsal-ventral or proximal-distal axes. Using in situ hybridization, the gradual gene expression was shown to be caused by P¿lacW¿ insertions in regions 40A (lines G1 and G2) and 69C (line G3) of the Drosophila genome. The expression patterns of genes lacZ and white were similar in eye cells of each line. In salivary gland polytene cells of G3 flies, the lacZ expression was also gradually reduced, which indicates the existence of a mechanism maintaining the gene activity gradient during endomitosis.

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A-2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes.

Vinculin is thought to be an important cytoskeletal protein in the linkage between actin cytoskeleton and integrin transmembrane receptors. We identified Vinculin (Vinc) gene in the X chromosome of D. melanogaster. Drosophila vinculin is highly homologous in its N- and C-terminal domains both to mammalian and nematode vinculins, and contains internal repeats and proline-rich region typical for vinculins. The X chromosome rearrangement In(1LR)pn2a was found to disrupt Vinc so that the coding sequence is interrupted by the (AAGAG)n satellite DNA. Northern analysis revealed that the Vinc transcript is completely absent in the In(1LR)pn2a homozygous flies. Surprisingly, these Vinc flies are viable and fertile. This finding highlights plasticity and adaptive capacity of cellular cytoskeletal and anchorage system.

A new cytochrome P450 gene was found within the wap1-prune genetic region of the Drosophila melanogaster X chromosome. It encodes a putative cytochrome P450 of subfamily 4D and has been designated CYP4D2. CYP4D2 has a complex exon-intron structure. Both the heme-binding motif and a region diagnostic for family 4 are not interrupted by introns. CYP4D2 lies 20 kb proximal to the previously described CYP4D1 gene (Gandhi et al., 1992). In addition to CYP4D1 and CYP4D2, two other DNA fragments homologous to P450 genes are detected within the wap1-prune region.

A full-length 1.8 kb cDNA of the Drosophila melanogaster gene prune together with the corresponding genomic sequence was cloned and sequenced. Comparison of the two sequences revealed the existence of the intron in the prune primary transcript. The putative prune protein is likely to have a single transmembrane domain. The nature of several prune mutant alleles was analysed: the size and quantity of prune mRNA were found to be decreased in pn1 and pn3 mutations. Mutations pn1 and pn54c were shown to be accompanied by insertions of the same novel mobile repeated element.

Using Northern analysis three transcription areas are detected in the gene prune region from which three different mRNAs 1.8, 1.75 and 1.0 kb in length transcribe. Corresponded cDNAs are cloned. Only 1.8 kb mRNA is shown to be changed in size and quantity in the case of the pn1 and pn3 mutations. This mRNA seems to be a gene prune transcript. From the analysis of the portion of cDNA c1.1 nucleotide sequence corresponded 1.75 kb mRNA follows that its putative amino acid product contains a heme-binding site which is characterized for P450 cytochrome superfamily. On the basis of presented results and published data by Teng et al [1] it may be concluded that at least five different mRNAs transcribe in the gene prune region in area spanning about 10 kb.

Analysis of transcription of the gene prune being undergone position effect variegation in the inversion In(1LR)pn2a is presented. Using Northern hydridization with following densitometric analysis of blots it was shown that the quantity of the prune mRNA is decreased 1.8 +/- 0.4 times in comparison with normal. Transcripts differing from wild type sizes are not detected. On the basis of the results obtained of inactivation propagation in the case of the position effect.

Eight plasmids containing the satellite DNA of Drosophila melanogaster, density of 1,688 g/cm3, and DNA neighbouring sequences were selected from gene cosmid library. The Doc mobile element appeared to be present in three cosmids, together with the satellite DNA. Two Doc copies in the opposite orientations are present in one of these cosmids. One monomer of the satellite DNA is located between their 3'-ends, which indicates selectivity of the mobile element insertion into the monomer of this satellite DNA.

DNA fragments carrying the genes affected by position effect variegation in the In(1LR)pn2a chromosome were revealed. In situ hybridizations demonstrate preservation of specific DNA sequences in the polytene chromosome regions containing the inactivated genes. It was shown that both euchromatic-heterochromatic boundaries in the In(1LR)pn2a chromosome contain the mobile element B104 (roo).