Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

Abstract

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5' and 3' flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.

Figure 1

Southern blot of genomic DNA from D. melanogaster probed with the entire Amyrel gene (³²P-labeled). To eliminate signal from Amy, washing was stringent (63°C, 0.25 × standard saline citrate, 1%SDS). The size and number of fragments suggest that Amyrel is single copy. For PstI, the band is double (see text).

Figure 2

Distance tree from an alignment of the proteins by clustalw (Neighbor joining method) showing the position of Amyrel relative to Amy. The Amyrel branch is the bold line. The tree was rooted with a Streptomycete (6). Numbers along branches are bootstrap values (1,000 replicates). [GenBank accession numbers: S. limosus, M18244; A. merus (mosquito), U01210; A. gambiae, L04753; D. virilis, U02029; D. pseudoobscura, X76240; D. melanogaster, X04569; O. nubilalis (moth), U04223; H. sapiens, P04746; M. musculus, P00688; and G. gallus, U63411.]

Figure 3

Putative AMYREL proteins of D. melanogaster (melrel), D. ananassae (anarel), and D. subobscura (subrel) and AMY proteins from D. melanogaster (melano) and pancreatic amylase of man (HumPancr) were aligned by clustalw and edited with seqapp. Shading indicates invariant residues. Vertical arrows show positions where AMYREL sequences share common residues that are different from conserved residues of animal AMY proteins. Asterisks mark the positions of cysteines involved in disulfide bridges in the pig and putatively in other animal AMY proteins. Plus signs mark additional cysteines in AMYREL.

Figure 4

(A) Alignment of 5′ regions of Amyrel in D. melanogaster (mel), D. ananassae (ana), D. pseudoobscura (pse), and D. subobscura (sub). Shading indicates invariant nucleotides. Long conserved sequences are numbered i, ii, and iii (see text); iv is the putative TATA box. clustalw alignment was corrected manually. (B) Alignment of the 3′ regions (except D. pseudoobscura, which was not available). The putative polyadenylation site is boxed.

Figure 5

In situ hybridizations and chromosomal localizations of Amyrel genes. (A) D. melanogaster. (B) D. ananassae. (C) D. subobscura. (D) Amy localization of D. subobscura.

Figure 6

RT-PCR analysis of Amyrel and Amy in D. melanogaster. The primers used are indicated in Table 1. Each time point was repeated on several individuals, one of which was representative and was selected for both genes. The sizes of amplified cDNAs are 344 bp for Amyrel and 352 bp for Amy. (E, embryo; L1, larva, first instar; L2, larva, second instar; 72h, L2/L3 molt time at 25°C; wL, wandering larva; wp, white pupa; bp, black pupa; em, emerging adult; ++, control (96-h larva) treated with DNase and RTase; +−, control treated with DNase and without RTase; − −, control with no DNase and no RTase; −, negative control.)