On the evolution of Yeti, a Drosophila melanogaster heterochromatin gene

Abstract

Constitutive heterochromatin is a ubiquitous and still unveiled component of eukaryotic genomes, within which it comprises large portions. Although constitutive heterochromatin is generally considered to be transcriptionally silent, it contains a significant variety of sequences that are expressed, among which about 300 single-copy coding genes have been identified by genetic and genomic analyses in the last decades. Here, we report the results of the evolutionary analysis of Yeti, an essential gene of Drosophila melanogaster located in the deep pericentromeric region of chromosome 2R. By FISH, we showed that Yeti maintains a heterochromatin location in both D. simulans and D. sechellia species, closely related to D. melanogaster, while in the more distant species e.g., D. pseudoobscura and D. virilis, it is found within euchromatin, in the syntenic chromosome Muller C, that corresponds to the 2R arm of D. melanogaster chromosome 2. Thus, over evolutionary time, Yeti has been resident on the same chromosomal element, but it progressively moved closer to the pericentric regions. Moreover, in silico reconstruction of the Yeti gene structure in 19 Drosophila species and in 5 non-drosophilid dipterans shows a rather stable organization during evolution. Accordingly, by PCR analysis and sequencing, we found that the single intron of Yeti does not undergo major intraspecies or interspecies size changes, unlike the introns of other essential Drosophila heterochromatin genes, such as light and Dbp80. This implicates diverse evolutionary forces in shaping the structural organization of genes found within heterochromatin. Finally, the results of dS - dN tests show that Yeti is under negative selection both in heterochromatin and euchromatin, and indicate that the change in genomic location did not affected significantly the molecular evolution of the gene. Together, the results of this work contribute to our understanding of the evolutionary dynamics of constitutive heterochromatin in the genomes of higher eukaryotes.

Figure 1. Cytogenetic mapping of heterochromatin genes of chromosome 2.

The map was modified from that shown in previous papers , . The diagram shows the essential genes defined by mutational analyses (below) and annotated genes defined by the heterochromatin genome project (above). Shades of blue correspond to the intensity of DAPI staining, with the darkest blue blocks representing regions with strong fluorescence intensity and open blocks representing non fluorescent regions. The different cytological regions are numbered.

Figure 2. Examples of FISH mapping of Yeti probes to polytene chromosomes of Drosophila species.

Salivary gland polytene chromosomes were stained with DAPI and pseudocolorated in blue; fluorescent signals were pseudocolorated in red. In D. melanogaster (A) and in the closely related D. simulans (B) and D. sechellia (C) species, the Yeti cDNA probe maps to 2Rh at the base of polytene division 41. The large and diffuse morphology of the Yeti signal found in these species, reflects the disorganized and poorly banded structure of the heterochromatin in the chromocenter. The arrows point the base of 2Rh. In D. pseudobscura, the hybridization signal of Yeti PCR probe maps to region 63C in the proximal euchromatin (D). In D. virilis the Yeti hybridization signal maps to region 53E, in the distal euchromatin of chromosome 5 (E).

Figure 3. Alignment of the Yeti ortholog sequences encoding the BCNT-C domain.

The grey area corresponds to the intron present in the Drosophila species.

Figure 4. Alignment of the BCNT domain of YETI proteins among species.

The arrow points the intron position in the corresponding coding region of Drosophila species.

Figure 5. Comparison of the Yeti gene structure among sequenced genomes.

Only the coding regions are showed. Exons are in boxes and numbers refer to nucleotides. Symbols: §, annotated genes; ≠, this study; **, defective ORF. The grey area at the 3′ end represents the conserved BCNT-C domain in the protein.

Figure 6. PCR amplification of the genomic region containing the Yeti intron in Drosophila species.

A single PCR product of about 180 bp was found, in D. melanogaster (A) and D. simulans, (B) D. sechellia (C) and D. tessieri (C) related species. M =  Marker; Frib  =  Friburgo; Mal  =  Mali; Scan  = Scansano; Iso  =  y¹; cn¹ bw¹sp¹ isogenic strain; OR  =  Oregon-R; Bej  =  Bejin; Chi  =  Chicharo; DV  =  Death Valley; Gen  =  Genoa; Mor  =  Moruya; Kyo  =  Kyogle; Arm  =  Armidale; Can  =  Canaries; Sech  =  D. Sechellia; tes  =  D. tessieri. Molecular weight is in bps.

Figure 7. Sequencing of the purified PCR products from Drosophila species.

Sequence alignments from Iso and Scansano (D. melanogaster), Chicharo and Death Valley (D. Simulans), D. sechellia and D. tessieri. Sequence analysis confirmed that they correspond to the Yeti intron containing region. The Yeti intron is shown in normal text, the flanking exons are in bold. The D. simulans and D. sechellia intron lacks a 7 bp stretch (see the gap), in agreement with the genome sequence data (see results and Figure 5). The D. Tessieri intron sequence is identical to that of D. simulans and D. sechellia.

Figure 8. Evolutionary repositioning of the Yeti gene.

Schematic representation of Yeti gene transition of from euchromatin to heterochromatin. The arrows point the chromosomal position of Yeti. It appears that Yeti has been resident on the Muller C chromosomal element, but over evolutionary time it progressively approached to pericentric heterochromatin and in D. melanogaster it is found in the deep portion of 2Rh.