On the basis of chromosomal homology, the Amylase gene cluster in Drosophila miranda must be located on the secondary sex chromosome pair, neo-X (X2) and neo-Y, but is autosomally inherited in all other Drosophila species. Genetic evidence indicates no active amylase on the neo-Y chromosome and the X2-chromosomal locus already shows dosage compensation. Several lines of evidence strongly suggest that the Amy gene cluster has been lost already from the evolving neo-Y chromosome. This finding shows that a relatively new neo-Y chromosome can start to lose genes and hence gradually lose homology with the neo-X. The X2-chromosomal Amy1 is intact and Amy2 contains a complete coding sequence, but has a deletion in the 3′-flanking region. Amy3 is structurally eroded and hampered by missing regulatory motifs. Functional analysis of the X2-chromosomal Amy1 and Amy2 regions from D. miranda in transgenic D. melanogaster flies reveals ectopic AMY1 expression. AMY1 shows the same electrophoretic mobility as the single amylase band in D. miranda, while ectopic AMY2 expression is characterized by a different mobility. Therefore, only the Amy1 gene of the resident Amy cluster remains functional and hence Amy1 is the dosage compensated gene.
DROSOPHILA MIRANDA shows an unusual karyotype due to the fusion of an autosome to the Y chromosome (Dobzhansky 1935; MacKnight 1939; Steinemann 1982). The rearrangement led to a neo-Y chromosome, which already shows signs of genetic degeneration (Steinemann and Steinemann 1992), and initiated the transformation of the remaining, unfused partner chromosome into a neo-X chromosome, designated X2 (Steinemannet al. 1996). Thus, in D. miranda, formerly autosomal genes are now located on a pair of sex chromosomes. It is generally assumed that X and Y chromosomes have evolved from a pair of originally homologous autosomes (for review, see Rice 1996). A so-called “primitive type” (White 1973) is represented, for instance, by the X and Y sex chromosome pair in Chironomids (Kraemer and Schmidt 1993). In Megaselia scalaris the sex chromosome pair is determined by the presence or absence of a male-determining factor M whereas the X and Y chromosomes are morphologically indistinguishable (cf. Traut 1994). A very early stage in the evolution of a neo-Y, neo-X chromosome pair is found in Drosophila americana americana. In D. a. americana the neo-Y shows no obvious signs of degeneration (Charlesworthet al. 1997) and no molecular evidence is found indicating dosage compensation of the neo-X chromosomal genes (Bone and Kuroda 1996; Marinet al. 1996). While the chromosome rearrangement in D. a. americana took place a few hundred thousand years ago (Throckmorton 1982), the separation of D. miranda from its next relatives D. pseudoobscura and D. persimilis occurred about 2 mya (Barrioet al. 1992). Population genetic theory predicts degeneration for a nonrecombining Y chromosome (neo-Y chromosome) via several mechanisms, such as sampling drift, genetic hitchhiking, background-trapping, Muller's ratchet, and mutational overload (for review, see Rice 1996). In addition to accumulating point mutations on the Y, a second phenomenon occurs, the conformational change from a euchromatic chromosome state into a heterochromatic one. Evolutionary changes during the process of sex chromosome differentiation in D. miranda are associated with massive DNA rearrangements on the neo-Y (Steinemann and Steinemann 1992; Steinemannet al. 1993; Lucchesi 1994) and changes in the X2-chromosomal chromatin (Bone and Kuroda 1996; Marinet al. 1996; Steinemannet al. 1996). We have chosen the species D. miranda as a model system to analyze the molecular bases of the evolutionary processes of Y chromosome degeneration and dosage compensation of the X chromosome (for review, see Steinemann and Steinemann 1998). Both evolutionary processes have led to dramatic structural changes in the sex chromosomes.
The Amy locus is on chromosome 2R in D. melanogaster, 54A1-B1 (Bahn 1971a; Gemmillet al. 1985; Boer and Hickey 1986). On the basis of chromosomal homology the Amy genes must be located on the X2 and neo-Y in D. miranda (Steinemannet al. 1986). Crosses between D. miranda lines showing different electrophoretic variants of amylase revealed no active amylase on the neo-Y chromosome (Steinemannet al. 1986; Norman and Doane 1990). Measurements of the α-amylase activity in males and females indicated that the X2-chromosomal locus is dosage compensated (Steinemannet al. 1986; Norman and Doane 1990). Autoradiographic studies (Strobelet al. 1978) and immunofluorescent staining with antibodies against H4.Ac16, a histone H4 isoform, which in Drosophila is preferentially located on the dosage compensated X chromosome in males, and antibody staining of the gene products of maleless (mle) and male-specific lethal 1 (msl-1) suggest that the X2 of D. miranda is already dosage compensated in great part (Bone and Kuroda 1996; Marinet al. 1996; Steinemannet al. 1996). We have cloned the Amy gene cluster from the X2 chromosome of D. miranda and in addition from chromosome 3 of the two sibling species, D. pseudoobscura and D. persimilis. In their most recent article Da Lage et al. (1998) describe a gene from D. melanogaster related to the Amy gene. This gene was designated Amyrel. From D. miranda and its sibling species, D. pseudoobscura and D. persimilis, we obtained sets of Amyrel sequences, which are localized outside the Amy locus near the tip of the X2 chromosome and chromosome 3, respectively. From sequencing the X2-chromosomal Amy gene cluster it became clear that the Amy3 gene, containing two large deletions, is structurally eroded. Amy2 contains a complete coding sequence, but has a deletion in the 3′-flanking region. Amy1 is structurally intact. Functional analysis of transgenic lines with ectopically integrated X2-chromosomal Amy1 and Amy2 regions allows us to address the question of whether each of the resident Amy1, Amy2 genes is active or inactive.
We were unable to isolate clones derived from the neo-Y chromosome. In situ hybridizations reveal no in situ signal on the neo-Y. Genomic Southern analysis of the Lcp gene cluster from the neo-X/neo-Y chromosome pair showed, due to several insertions on the neo-Y, size differences between homologous male and female DNA fragments (Steinemann and Steinemann 1990, 1992). Long-range PCR and genomic Southern analyses from the Amy region with male and female DNA revealed no evidence for fragments of expected different sizes corresponding to a neo-Y copy. These results strongly suggest that the Amy gene cluster is already deleted from the neo-Y chromosome. Thus a relatively new neo-Y chromosome can start to lose genes even after only a couple of million years of evolution.
MATERIALS AND METHODS
Fly strains and cloning of the Amy genes from D. miranda and the two sibling species: D. miranda MPI, D. pseudoobscura ST, and D. persimilis ST flies were cultured on standard Drosophila food at 18°. High-molecular-weight DNA from D. miranda and the two sibling species, D. pseudoobscura and D. persimilis, were isolated from a mixture of male and female flies according to Steinemann (1982). Genomic EMBL4 λ libraries from partial Sau3A (Boehringer Mannheim, Mannheim, Germany) digests were established and screened as detailed in Steinemann and Steinemann (1990). The filters were probed with pOR-M7, a cDNA clone from D. melanogaster OR, described in Figure 3 of Benkel et al. (1987). In earlier experiments we had screened a D. melanogaster library using the cDNA clone pMSa104 from mouse, described in Schibler et al. (1980, 1982). For detailed restriction mapping, the regions containing the Amy genes of the X2 from D. miranda and of chromosome 3 from D. persimilis were subcloned into pUC18. Agarose gel electrophoresis and Southern blots were performed as detailed in Steinemann and Steinemann (1990). Selected regions of genomic male and female DNAs from D. miranda were amplified with the Expand Long Template PCR System (Boehringer Mannheim), as described by the manufacturers. The Amy region of the X2 chromosome from D. miranda was sequenced on both strands from M13mp18/19 subclones by the dideoxy chain termination method according to the protocol supplied with sequenase (United States Biochemical, Cleveland). Cloning and standard DNA techniques were carried out according to Sambrook et al. (1989).
Constructs and germ-line transformation: DNA fragments of interest were inserted into the polylinker of the CaSpeR vector (Pirrotta 1988). Cloning and germ-line transformations into D. melanogaster OR w snw (cf. Lindsley and Zimm 1992) embryos were performed as detailed in Steinemann et al. (1993). For embryo injections, constructs containing the following orientation of the Amy1 and Amy2 genes were chosen. The dmirAmy1 region used in AX1 starts at an artificial EcoRI site about 1.8 kb 5′ to the TATA-box motif and ends about 1.6 kb after the inferred poly(A) site with the EcoRI site left of the 3′ end of Amy2, total length 5085 bp (see Figure 1B). The dmirAmy2 region used in AX2 starts with the EcoRI site about 3.5 kb 5′ to the TATA-box motif, and extends 3′ to the EcoRI site, bordering the AX1 fragment, about 0.7 kb from the inferred poly(A) site, total length 5853 bp (see Figure 1B).
Computer analysis: The DNA database screening with BLASTN (Altschulet al. 1990) and the Genetics Computer Group Sequence Analysis Package (Devereuxet al. 1984) was done using the updated GenBank/EMBL nucleotide Sequence Data Library (EMBL, Heidelberg, Germany; GenBank, NCBI, Washington, DC). DNA sequences were aligned using either MacMolly (Softgene, Berlin) or pileUp (GCG package) alignment programs. Critical sections were aligned by visual inspections. To clarify the arrangement of the Amy3 gene from D. miranda we aligned the DNA sequence with the sequence from D. pseudoobscura Standard (ST) arrangement. For the alignment with the D. miranda Amy2 deletion we used the Amy2 DNA sequence from the D. pseudoobscura Chiricahua (CH) arrangement. The published sequence size from D. pseudoobscura ST was too short to match the end of the D. miranda Amy2 deletion. In D. pseudoobscura and D. persimilis the third chromosome is polymorphic for gene arrangements that are the results of overlapping, paracentric inversions. On the basis of the breakpoints of the inversions these gene arrangements can be arranged in a phylogeny (Sturtevant and Dobzhansky 1936). Standard (ST), Santa Cruz (SC), and Chiricahua (CH) are representatives of different gene arrangements. Cladograms were performed using the PAUP program of Swofford (1993).
Chromosome in situ hybridization: λDmir1785 (containing the complete Amy region of D. miranda) and λDmir1792 (representative of the Amy-related clones) were labeled with Biotin16-dUTP (Boehringer Mannheim) by the nick translation reaction and hybridized at 58° overnight to alkali denatured chromosome squashes (Steinemann and Steinemann 1992). The slides were washed three times in 2× SSC at 53°. Signal detection followed the protocol for immunoperoxidase staining supplied with the DETEK I-hrp kit used (Enzo Diagnostics, New York). The intensity and contrast of the diaminobenzidine precipitate were enhanced using a silver diaminobenzidine enhancement kit (Amersham, Buckinghamshire, U.K.). Photomicrographs of the chromosome squashes were made with an Agfa Pan 25 film.
Fly homogenates and gel electrophoresis: Preparation of the fly homogenate, separation of the homogenate, and the visualization of the amylase enzyme activity were performed as detailed in Steinemann et al. (1986). For one sample, three flies were frozen in solid CO2 and homogenized in a volume of 25 μl sample buffer (stock solution: mix 1 ml of 0.47 m Tris solution, adjusted with H3PO4 to pH 6.9, with 4 ml of 40% sucrose solution, 3 ml distilled water, and 40 μl of a 1% bromphenol blue solution). The homogenate was centrifuged for 30 min at 4° in an Eppendorf minifuge. A total of 20 μl of the supernatant was mixed with 3 μl 100% glycerol and applied directly to the gel. Polyacrylamide gel electrophoresis was performed according to Davis (1964). Crude extracts were separated on a 3.75% stacking and a 7.5% resolving gel (anode pole at the bottom) in the multiphasic buffer system A (Davis 1964). After the run, the gels were equilibrated for 10 min in 0.5 m Tris-HCl, pH 7.1 and then incubated for 90 min in a starch solution (1 g soluble starch and 220 mg CaCl2, dissolved in 100 ml of boiling 0.1 m Tris-HCl, pH 7.5). The gel was washed twice in distilled water and stained for 2-5 min in a staining solution (300 mg KI and 130 mg iodine dissolved in 100 ml distilled water). As the AMY proteins from D. miranda and D. melanogaster OR could be clearly separated in the gel system that was used, we were able to monitor the expression of both genes in the same separation lane. We thus preferred the analysis of the expression of the Amy genes at the protein to the RNA level. Under the electrophoretic conditions used here, AMY1 and AMY2 revealed different mobilities. Zymograms were photographed with an Agfa Ortho 25 ASA film.
Amylase loci in D. miranda and the two sibling species D. pseudoobscura and D. persimilis: From crosses between D. miranda lines, carrying Amy isozymes with different electrophoretic mobilities, it became clear that in D. miranda males only the Amy gene(s) from the X2-chromosomal locus encodes an active amylase enzyme. Estimates of the α-amylase activity in crude homogenates of male and female flies strongly suggest that the Amy gene(s) is dosage compensated in D. miranda (Steinemannet al. 1986; Norman and Doane 1990). In all other species, the Amy genes are autosomally inherited and hence not dosage compensated. We have cloned about 30 kb of the Amy region from the X2 chromosome of D. miranda. The X2-chromosomal Amy region contains three stretches which reveal cross-hybridization with the D. melanogaster pOR-M7 probe, Figure 1A. As the 3′-fragments in these experiments always show stronger hybridization with this probe, the orientation of the Amy genes in the cluster could be derived from the intensity of the hybridization signals. The third stretch consists of a shorter, strongly labeled fragment indicating the observed deletions (see below). In addition, in D. miranda and the sibling species examined, D. pseudoobscura and D. persimilis, we obtained a second class of clones that showed a different restriction pattern when compared with the Amy gene cluster (not shown). These Amyrel clones are localized at a more distal site on the X2 chromosome or chromosome 3, respectively (see below).
In in situ hybridization experiments, clones of the Amy region from D. miranda labeled one site on the X2 chromosome, Figure 2A. The label is restricted to one band in subdivision 27A using the cytogenetic map of Das et al. (1982). Using different λ clones and different in situ hybridization conditions we were unable to detect any signal on the neo-Y. On the other hand, in situ hybridizations with the cloned Krüppel (Kr) gene, which in D. miranda is located as well on the neo-X/neo-Y chromosome pair, show labeling, on both the neo-X and neo-Y loci (M. Steinemann and S. Steinemann, unpublished results). The Amyrel clones labeled one band at the distal tip of the X2, subdivision 22B, Figure 2B. A combination of both clones from D. miranda showed labeling of the Amy gene cluster and the Amyrel locus in D. miranda (Figure 3A) and the sibling species D. pseudoobscura and D. persimilis (Figure 3, B and C, respectively). Immunofluorescent stainings with antibodies against a histone H4 isoform, H4.Ac16, typically associated with the dosage compensated X chromosome in Drosophila males (Steinemannet al. 1996), and against the gene products of the maleless and the male-specific lethal 1 genes MLE and MSL-1 (Bone and Kuroda 1996; Marinet al. 1996) stain chromosome regions along the X2. The Amy gene cluster is located in an H4.Ac16-enriched region of the X2 chromosome. Anti-H4.Ac16 antibodies do not label the distal tip region of about 10% of the X2 chromosome length (Steinemannet al. 1996). The Amyrel locus lies within this distal tip region. Thus we expect that, if these Amyrel sequences were transcribed in D. miranda, they should be autosomally regulated. In D. pseudoobscura ST, the Amy cluster is on chromosome 3, division 73 and the Amyrel locus is at the distal tip, division 81 [Figure 3B; using the photographic map of Kastritsis and Crumpacker (1966)]. In D. persimilis ST, the Amy cluster is located on chromosome 3, division 73 and the Amyrel locus at the distal tip, division 81 [Figure 3C; using the photographic map of Moore and Taylor (1986)]. Because of the resolution of the maps, a closer localization is not feasible. The Amy gene cluster and the Amyrel sequences are thus located at comparable positions along the neo-X (X2) of D. miranda and chromosome 3 of D. pseudoobscura ST and D. persimilis ST, respectively. Similar locations were reported in Norman et al. (1991). The D. melanogaster clone Dm1B, which was isolated during a screen of a genomic D. melanogaster library using the Amy clone pMSa104 from mouse (Schibler et al. 1980, 1982) as a probe, labels one site on D. melanogaster 2R, division 53, and not the Amy locus. In cross-hybridizations to D. miranda this clone labels only one band in subdivision 22B on the tip of the X2 (not shown), exactly the same locus that is labeled with the set of Amyrel clones. We conclude that these loci are related on the basis of sequence similarities in both species.
DNA sequence analysis of the X2-chromosomal D. miranda Amylase gene cluster: Sequencing of the D. miranda Amy region revealed the structure of three genes (Figure 4A). For comparison they are aligned with the Amy1-3 of D. pseudoobscura ST (Brownet al. 1990; Popadic and Anderson 1995) and Amy-d and Amy-p from D. melanogaster (GenBank accession numbers X04569 and L22724). The D. miranda Amy gene (dmirAmy) sequences are deposited in the EMBL gene bank, accession numbers Amy1, Y15603 DMAMY1; Amy2, Y15604 DMAMY2; and Amy3, Y15605 DMAMY3. Amy1 and Amy2 are arranged tail-to-tail (Figure 1A). Amy3 is localized with respect to Amy2 in a head-to-tail orientation. To determine the arrangement of the deletions in Amy2 and Amy3 in detail, we performed alignments with DNA sequences from D. pseudoobscura arrangements (cf. materials and methods). The Amy1 gene is structurally intact. Alignment of the DNA sequences of Amy2 and Amy1 reveals for the Amy2 gene, apart from an intact coding region, intact CAAT- and TATA-box motifs and an ATCAG motif for transcription initiation. Both genes are interrupted by an intron of 67 bp. The introns are located at identical positions, but diverge by the substitution of 4 bp. The intron size reported here corresponds to the result reported from Da Lage et al. (1996) using PCR-amplified fragments. Amy2 is flanked on its 3′-side by a deletion of 273 bp that starts 1 bp in front of the inferred poly(A) signal (Figure 4A). The size of this deletion is given with respect to the Amy2 sequence of D. pseudoobscura CH (GenBank accession number U20336). Alignment of the D. miranda Amy1 gene with the D. pseudoobscura ST Amy1 gene shows extended similarities in the 5′- and 3′-flanking sequences (Figure 4A). Beside the CAAT- and TATA-box motifs, the 5′-upstream sequences of Amy1 and Amy2 from D. miranda are different (Figure 4A). A reasonable alignment of the 3′-flanking sequences after the deletion of Amy2 is not possible with Amy1, but we obtain good sequence similarity with the corresponding 3′-flanking Amy2 region of D. pseudoobscura (Figure 4A). It became obvious that the 3′-flanking sequences of Amy1 and the putative corresponding sequences of Amy2 from D. miranda already diverge 8 bp downstream of the poly(A) signal. In principle, Amy1 and Amy2 share only the transcribed sequences. The maximum parsimony analysis of the Amy1 and Amy2 genes using only the transcribed sequences (based on an alignment of 1658 nucleotides) from D. miranda and D. pseudoobscura (three arrangements: ST, SC, and CH) reveals that they cluster with one another and not with the putative orthologues (Figure 4B), thus indicating that the transcribed sequences have been homogenized within the species (cf. Shibata and Yamazaki 1995). However, the maximum parsimony analysis of the Amy1 and Amy2 genes including the 5′- and 3′-flanking sequences (based on an alignment of 2600 nucleotides) shows that the genes are clustered within each locus (Figure 4C). This clustering of species within genes indicates that the Amy duplications occurred before the divergence of the D. pseudoobscura and D. miranda species and that mechanisms of concerted evolution have not homogenized the Amy1 and Amy2 flanking sequences.
Amy3 reveals two large deletions, one of 445 bp and a second one of 872 bp in size, with respect to Amy1 of D. miranda (Figure 4A). The arrangements of the Amy3 deletions were clarified from the alignment of D. miranda Amy3 with Amy3 from D. pseudoobscura ST (Brownet al. 1990; Popadic and Anderson 1995; Figure 4A). This alignment shows that the 5′-nondeleted Amy3 sequences from D. miranda, the CAAT- and TATA-box motifs, are conserved. The first deletion starts after the TATA-box and ends within exon 2. Exon 1 and the intron are completely deleted. The second deletion starts within exon 2 and ends shortly after the inferred poly(A) signal (Figure 4A). From exon 2 only a small part of 361 bp is left. Thus the Amy3 gene of D. miranda is structurally hampered by missing regulatory motifs and coding sequences.
In contrast to the alignment of Amy1 and Amy2, the sequence similarity shared between Amy2 and Amy3 of D. miranda extends further into the 5′-flanking (about 860 bp, not shown) and into the 3′-flanking sequences (about 180 bp; for Amy2 the sequences are inferred from the alignment with D. pseudoobscura CH; see materials and methods). Upstream and downstream from this shared region the similarity drops off very quickly. Beyond this point the sequence similarities in the 3′-flanking regions are restricted to the Amy2 genes (brick pattern) and the Amy3 genes (checkerboard pattern) of D. miranda and D. pseudoobscura (Figure 4A). The intraspecific divergence between Amy2 and Amy3 in the compared 5′-flanking region is only 1.49%; the interspecific divergence is 4.17% (Table 1). Thus, the duplication that involves Amy2 and Amy3 of D. miranda encompassed, in addition to the transcribed region, several hundred base pairs of 5′- and 3′-flanking sequences. The extended flanking sequence similarity between Amy2 and Amy3 of D. miranda parallels the arrangements found in D. pseudoobscura (Brownet al. 1990; Popadic and Anderson 1995).
Expression of the X2-chromosomal Amy genes: Amylase is a monomeric protein. Thus in heterozygotes with alleles coding for different electrophoretic variants, two bands are found. When the Amy locus of D. miranda is made homozygous and tested in different lines, from the pseudoobscura subgroup, D. miranda, D. persimilis, and D. pseudoobscura, only a single amylase isozyme was found (Norman and Prakash 1980). These observations suggest that only one Amylase gene is active. Therefore the question arises as to whether the Amy1 or the Amy2 gene in D. miranda produces an active amylase enzyme. DNA fragments containing the X2-chromosomal Amy1 or Amy2 gene from D. miranda, together with flanking 5′- and 3′-sequences (see materials and methods), were cloned into the P-element derived CaSpeR vector (Figure 1B). Transgenic lines of D. melanogaster with single insertions were tested. In seven tested transgenic lines containing the Amy1 region (dmirAmy1) from the X2 chromosome of D. miranda (AX1 construct), the Amy1 gene is expressed, showing the same mobility as the D. miranda band (Figure 5). Two of the lines show a reduced D. miranda amylase activity (not shown). In contrast, five transgenic lines transformed with the AX2 construct containing the Amy2 region (dmirAmy2) from the X2 of D. miranda indicate no Amy2 gene expression (not shown). However, in two independent lines, the Amy2 gene from D. miranda is expressed, but is characterized by a slightly different mobility than the amylase band in D. miranda (Figure 5). Therefore, only the Amy1 gene of the resident Amylase cluster is expressed in D. miranda and hence the Amy1 must be the dosage compensated gene.
Neo-Y chromosomal Amy locus: From 35 analyzed clones, no neo-Y-chromosomal Amy clone was detected. In contrast to this finding, analyzing about the same amount of clones from the Lcp region, we detected several neo-Y chromosomal clones. In situ hybridization experiments showed no labeled site on the neo-Y using different clones from the X2-chromosomal Amy cluster. We thus wondered whether the Amy region is still present on the former homologous neo-Y chromosome, as we have found for the Lcp1-4 cluster (Steinemann and Steinemann 1992; Steinemannet al. 1993). Long-range PCR, amplifying a fragment of about 9.9 kb from the Amy gene cluster in genomic male and female D. miranda DNAs, reveals a single band of the same size in both sexes, respectively (not shown). In Southern blots, equal amounts of restricted genomic male and female DNA from D. miranda, hybridized with the 32P-labeled pOR-M7 probe from D. melanogaster, show two bands of the same size in males and females (Figure 6). Despite equally loaded lanes the signal strength in the male lanes is about half the intensity as in the female lanes. These results strongly suggest that the Amy cluster is deleted from the evolving neo-Y chromosome.
DNA sequences of Amy1, Amy2, and Amy3 from D. miranda: In this article we present the complete DNA sequences of the small multigene family Amy1, Amy2, and Amy3 from D. miranda. The described sequences are cloned from the evolving X2 chromosome. Database screens of the GenBank/EMBL gene bank revealed partial sequences from D. miranda. The reported sequence fragments of D. miranda Amy2 (GenBank accession number U51236) and Amy3 (GenBank accession number U51237) aligned with the sequences presented here appear to be a mixture of intermingled partial Amy2 and Amy3 sequences, respectively. The partial coding sequences of clone miranda1 (GenBank accession number AB003769) and clone miranda2 (GenBank accession number AB003770) seem to be sequences from D. pseudoobscura (intron length of 71 bp and base substitutions), and not from D. miranda. Alignment of the three Amy genes from D. miranda reveals a structurally intact Amy1 gene and an Amy2 gene with a complete coding sequence and a deletion in the 3′-region. Amy1 and Amy2 in D. miranda are arranged tail-to-tail and Amy2 and Amy3 head-to-tail, while Amy-p and Amy-d in D. melanogaster show a head-to-head orientation (Figure 7). Of interest is the observation that the short conserved flanking CAAT-box sequences (between CAAT- and TATA-box) of Amy-d and Amy-p show a more extended similarity to Amy2 and Amy3 than do those of Amy1 (Figure 4A). The intergenic regions between Amy-p and Amy-d of D. melanogaster and D. teissieri contain open reading frames with sequence similarities to the serpin gene (Okuyamaet al. 1997). Alignment of the serpin gene with the intergenic regions of Amy1-Amy2 and Amy2-Amy3 reveals no sequence similarity. Either the serpin gene is deleted from this position in D. miranda or it was inserted into D. melanogaster after the separation of the melanogaster and obscura groups.
The DNA sequence analysis reveals two large deletions within the Amy3 gene, including the start of transcription and several hundred base pairs of coding sequences. Thus Amy3 on the X2 chromosome from D. miranda is structurally impaired and cannot be expressed. In Amy2, the 273-bp deletion starts 1 bp in front of the inferred poly(A) signal. Without the correctly positioned poly(A) signal, Amy2 RNA might not be processed. On the other hand, further downstream sequences showing a poly(A) signal motif might be used (see below). In the coding region, there are 11 nucleotide differences among the D. miranda Amy1 and Amy2; 6 represent synonymous substitutions and 5 nonsynonymous substitutions. The five replacement changes relative to the six silent ones could indicate an acceleration of replacement changes due to an inactive Amy2 at its resident locus.
Expression and nonexpression of the resident Amy genes: The structural data provide no decisive answer as to whether in D. miranda the Amy2 gene can produce a functional AMY2 enzyme or not. The Amy3 gene cannot be expressed. Thus only the Amy1 and Amy2 genes are candidates as potentially active genes. In all tested germ-line transformed lines containing the X2 chromosomal Amy1 region from D. miranda, the Amy1 gene is expressed with the same mobility as the amylase encoded by the resident Amy gene in D. miranda. In two lines carrying the AX1 construct, the D. miranda Amy1 gene reveals low-level expression. It was shown by Bahn (1971b) that the Amy locus is sensitive to position-effect variegation (PEV). The reduced activities observed in these lines could be due to PEV effects induced from the ectopic sites of integration (cf. Henikoff and Matzke 1997). A more detailed analysis of these epigenetic effects has to be done. Five transgenic lines, containing the Amy2 region, do not express the Amy2 gene from the X2 in D. miranda. However, in two independent lines, the Amy2 gene is expressed. AMY2 reveals a slightly faster mobility compared with the AMY band in D. miranda. In all transgenic D. melanogaster lines AMY1 shows the same mobility as in D. miranda; therefore posttranscriptional modifications, being responsible for the slight mobility difference of AMY2, could be excluded. The five nonsynonymous substitutions must be responsible for the slight mobility difference observed between AMY1 and AMY2. Thus, only the Amy1 gene of the resident small multigene family is active in D. miranda and hence must be the dosage compensated gene. The reason for nonexpression of the Amy2 gene at its resident position is unclear. Either it cannot make use of a further downstream poly(A) signal motif (see above), or the 3′-deletion has removed a necessary regulatory element. If it can make use of a further downstream poly(A) signal motif, the Amy2 gene might be silenced by epigenetic effects at the resident Amy locus. The remaining two transgenic lines that do express the construct may be due to fortuitous positive effects compensating the putative missing regulatory element or due to chromatin effects at the ectopic integration sites.
Basic arrangement of Amy gene cluster in the pseudoobscura subgroup: The number of structurally intact Amy genes can vary between different D. pseudoobscura arrangements. In the D. pseudoobscura ST arrangement the structure of all three Amy genes is intact (Brownet al. 1990; Figure 7). The Amy3 gene, however, has a stop codon at amino acid 157 that shortens the deduced protein to 31.6% of its normal length. In the D. pseudoobscura SC arrangement, Amy1 and Amy2 are structurally intact and Amy3 has a large deletion including 5′-flanking and coding sequences. In the D. pseudoobscura TL arrangement, Amy2 and Amy3 show large deletions upstream from the start of transcription reaching into the coding region (Popadicet al. 1996). The basic arrangement of three Amy genes in the pseudoobscura group (Figure 7) seems to be prone to deletions in the Amy3 and Amy2 genes, while selection pressure keeps the Amy1 gene structurally intact. For mechanisms (unequal crossing over and/or gene conversion) that may be homogenizing the gene family, the extended shared flanking regions of Amy2 and Amy3 could have some relevance for the apparent instability of the Amy locus in the pseudoobscura group.
Deletion of the neo-Y Amy gene cluster: D. miranda females carry two X2 chromosomes, while males have an X2 and a neo-Y chromosome. Detailed analysis of the neo-Y chromosomal Lcp1-4 loci revealed a large tandem duplication that is several kilobases in length (Steinemann and Steinemann 1993), short deletions, a massive accumulation of transposable elements, and silencing of flanking Lcp genes (Steinemann and Steinemann 1992; Steinemannet al. 1993). The fate of the three Amy genes on the degenerating neo-Y is different from Lcp1-4. In situ hybridizations, long-range PCR, and Southern blot analysis of genomic male and female DNA strongly suggest that the Amy gene cluster has been deleted during the degeneration process in progress. This finding shows that a relatively new neo-Y chromosome can start to lose genes and hence gradually lose homology with the neo-X, even after about 2 million years of evolution. The molecular mechanism(s) generating the deletion is unclear. On the basis of the target site duplication associated with the 221-bp deletion we have detected in the neo-Y chromosomal Lcp4, we concluded that the null allele there is generated by insertion/excision mutagenesis (Steinemann and Steinemann 1992). This might be one possible mechanism responsible for the generation of the deletion of the Amy loci on the neo-Y. The mechanisms discussed in the context of neo-Y chromosome degeneration in D. miranda could be relevant to studies of mammalian Y chromosome evolution. In the addition-attrition hypothesis, the original X and Y have been enlarged by cycles of autosomal addition to one partner, recombination onto the other, and continuing attrition of the compound Y (for review, see Graves 1995). Drosophila males have achiasmate meiosis. Because of the absence of recombination in D. miranda males we assume that the degeneration process of the neo-Y will be faster than the progressive degradation of the pseudoautosomal region in mammals. Plant sex chromosomes have evolved recently on a geological time scale. For example, Silene latifolia, a dioecious plant, shows a heteromorphic sex chromosome pair. The X-linked MROS3 gene has a homologue in the nonpairing region of the Y chromosome that has degenerated as a result of nucleotide deletion and accumulation of repetitive sequences (Guttman and Charlesworth 1998). These findings corroborate the described mechanisms for Y chromosome degeneration in D. miranda (Steinemann and Steinemann 1992; Steinemannet al. 1993). Investigating the enigma of Y chromosome degeneration we could demonstrate in our neo-Y/neo-X D. miranda model system, apart from point mutations, three mechanistic principles involved in Y chromosome degeneration: (1) accumulation of transposable elements and silencing of flanking resident genes, (2) tandem duplications, and (3) deletions of loci. This greatly strengthens the classic argument that true Y chromosomes have evolved from ancestors that were originally homologous to the X by a process of gradual erosion.
We thank Donal A. Hickey for the pOR-M7 clone, U. Schibler for the pMSa104 clone, C. Kraemer for support with the long-range PCR, and Mrs. K. Wildhagen for preparing the photomicrographs. We thank the anonymous reviewers for constructive criticism of this article. This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Ste 266/4-1.
Communicating editor: S. Henikoff
- Received July 6, 1998.
- Accepted September 14, 1998.
- Copyright © 1999 by the Genetics Society of America