Evolution of Nucleotide Substitutions and Gene Regulation in the Amylase Multigenes in Drosophila kikkawai and its Sibling Species

Nobuyuki InomataGo, and Tsuneyuki Yamazaki

Laboratory of Molecular Population Genetics, Department of Biology, Kyushu University, Fukuoka, Japan

Abstract

In order to determine evolutionary changes in gene regulation and the nucleotide substitution pattern in a multigene family, the amylase multigenes were characterized in Drosophila kikkawai and its sibling species. The nucleotide substitution pattern was investigated. Drosophila kikkawai has four amylase genes. The Amy1 and Amy2 genes are a head-to-head duplication in the middle of the B arm of the second chromosome, while the Amy3 and Amy4 genes are a tail-to-tail duplication near the centromere of the same chromosome. In the sibling species of D. kikkawai (Drosophila bocki, Drosophila leontia, and Drosophila lini), sequencing of the Amy1, Amy2, Amy3, and Amy4 genes revealed that the Amy1 and Amy2 gene group diverged from Amy3 and Amy4 after duplication. In the Amy1 and Amy2 genes, the divergent evolution occurred in the flanking regions; in contrast, the coding regions have evolved in concerted fashion. The electrophoretic pattern of AMY isozymes was also examined. In D. kikkawai and its siblings, two or three electrophoretically different isozymes are encoded by the Amy1 and Amy2 genes (S isozyme) and by the Amy3 and Amy4 genes (F (M) isozymes). The S and F (M) isozymes show different patterns of band intensity when larvae and flies were fed in different media. Amy1 and Amy2, which encode the S isozyme, are more strikingly regulated than Amy3 and Amy4, which encode the F (M) isozyme. The GC content and codon usage bias were higher for the Amy1 and Amy2 genes than for the Amy3 and Amy4 genes. Although the ratio of synonymous and replacement substitutions within the Amy1 and Amy2 gene group was not significantly different from that within the Amy3 and Amy4 gene group, the synonymous substitution rate in the lineage of Amy1 and Amy2 was lower than that of Amy3 and Amy4. In conclusion, after the first duplication but before speciation of four species, the synonymous substitution rate between the two lineages and the electrophoretic pattern of the isozymes encoded by them changed, although we do not know whether there was any evolutionary relationship between the two.

Introduction

In the progressive change of organisms, gene duplication with subsequent diversification is considered to have played very important roles (Ohno 1970Citation ; Ohta 1988, 1991Citation ). Functional diversification among members of a multigene family results from amino acid substitutions and/or changes in gene regulation. There are examples that positive Darwinian selection acts on the amino acid substitutions after gene duplication (e.g., Zhang, Rosenberg, and Nei 1998Citation ), although it is usually difficult to discriminate whether the acceleration of amino acid substitutions results from fixation of advantageous mutations or relaxation of functional constraints because of redundancy between the duplicated genes. On the other hand, regulatory changes are also thought to be important for adaptive evolution (King and Wilson 1975Citation ; Yamazaki and Matsuo 1984Citation ; Dickinson 1991Citation ). Recently, there has been much evidence of this (e.g., Averof and Patel 1997Citation ). However, little is known about the relationship between the functional diversification related to regulatory changes and the nucleotide substitution pattern.

The Drosophila amylase system is one of the best model systems with which to study evolutionary significance of multigene families. Enzymatically, amylase breaks starch into maltose and glucose. Amylase isozymes are polymorphic in natural populations and can be readily distinguished by electrophoresis (Kikkawa 1964Citation ; Doane 1969Citation ). Amylase activity is repressed by dietary glucose (Hickey and Benkel 1982Citation ) and induced by dietary starch (Inomata et al. 1995aCitation ). Changes in activity are correlated with changes in the amount of mRNA (Benkel and Hickey 1986Citation ; Yamate and Yamazaki 1999Citation ). Moreover, the difference in activity among the different AMY isozymes is mostly attributed to that in the amount of mRNA (Yamate and Yamazaki 1999Citation ). In the 5'-flanking regions of the Amy coding regions, there are cis-acting regulatory elements responsible for amylase gene expression (Magoulas et al. 1993Citation ; Choi and Yamazaki 1994Citation ). These results indicate that the amylase activity is mainly regulated at the transcriptional level. There is also genetic variation in the response ability to dietary carbohydrates (inducibility), and this inducibility was found to be important for the fitness (Matsuo and Yamazaki 1984Citation ; Yamazaki and Matsuo 1984Citation ). The Drosophila amylase system examined is a multicopy gene system (Brown, Aquadro, and Anderson 1990Citation ; Da Lage et al. 1992Citation ; Shibata and Yamazaki 1995Citation ; Popadic et al. 1996Citation ; Inomata, Tachida, and Yamazaki 1997Citation ; Steinemann and Steinemann 1999Citation ). Duplication events seem to have occurred independently in the subgenus Sophophora lineage (Da Lage, Wegnez, and Cariou 1996Citation ; Inomata, Tachida, and Yamazaki 1997Citation ). Recently, Da Lage et al. (1998)Citation found a new paralogous amylase gene, Amyrel, although its function is unknown.

We systematically investigated the response pattern to dietary carbohydrates of amylase (AMY) activity, which is likely to reflect the level of the transcription, in addition to nucleotide substitution patterns of the amylase genes (Amy), in various Drosophila species (Inomata et al. 1995aCitation ; Inomata, Tachida, and Yamazaki 1997Citation ). We inferred a general picture of evolution of regulatory changes and nucleotide substitution patterns separately in the Drosophila amylase system. However, the relationship between them remains to be determined. In order to focus on the evolutionary relationship between the functional diversification caused by regulatory changes and the nucleotide substitution patterns in a multigene family, we selected the amylase system as a model system, since amylase activity of Drosophila kikkawai was highly affected not only by the dietary carbohydrates, but also by developmental stages (Inomata et al. 1995aCitation ). In the context of gene expression patterns represented by electrophoretic patterns of the isozymes and nucleotide substitution patterns of amylase multigenes in D. kikkawai and its sibling species, the evolutionary relationship between regulatory changes and nucleotide substitution patterns is discussed.

Materials and Methods

Drosophila Species
We used four Drosophila species from the kikkawai complex, which belongs to the montium species subgroup of the melanogaster species group. The species used were D. kikkawai (Naha-1 strain, Okinawa Island, Japan, 1982), Drosophila bocki (A65 strain, Taiwan, 1979), Drosophila leontia (RGN210-8 strain, Rangoon, 1982), and Drosophila lini (14028-0581.0 strain, Yun-Shui, Taiwan). The first three species were supplied by the Tokyo Metropolitan University. The last one was obtained from the National Drosophila Species Resource Center at Bowling Green State University.

Medium Composition
The glucose medium consisted of 10% glucose (w/v), 5% killed yeast (w/v), 0.6% agar (w/v), and 0.4% propionic acid (v/v) in distilled water. The starch medium consisted of 10% soluble starch (w/v), 5% killed yeast (w/v), 0.6% agar (w/v), and 0.4% propionic acid (v/v) in distilled water. Two media had identical compositions except for the addition of a specific carbohydrate (glucose or starch).

Sample Collection for AMY Protein Electrophoresis
Adult flies were transferred to a new vial containing either glucose or starch medium and allowed to lay eggs at 22°C. After 3 days, 10 adult flies were randomly collected without distinguishing sexes and frozen at -70°C. Ten third-instar larvae grown on either glucose or starch medium were also randomly collected without distinguishing sexes. They were washed with distilled water and then stored at -70°C. These constituted one replicate of the sample. Four replicates were prepared on each medium.

AMY Protein Electrophoresis
The samples were homogenized by sonication in a buffer (pH 8.9) (0.1 M Tris-borate, 5 mM MgCl2, and 10% sucrose [w/v]). Before electrophoresis, the protein content of each sample was measured by the BCA protein assay reagent (Pierce). Then, the samples with equal protein contents were applied to the polyacrylamide gels (5% acrylamide [w/v], 0.2% Bis-acrylamide [w/v], 20 mM CaCl2, and 0.1 M Tris-borate) in a 0.1 M Tris-borate (pH 8.9) buffer. A quarter of the other samples was applied in D. kikkawai. After running for 3 hours at 4°C and 300 V, the gels were incubated in starch solution (1% soluble starch [w/v], 0.1 M Tris-HCl [pH 7.4], and 20 mM CaCl2) for 1 h at 37°C. They were washed with water briefly and incubated again in a solution without starch (0.1 M Tris-HCl [pH 7.4] and 20 mM CaCl2) for 30 min at 37°C (see Benkel and Hickey 1986Citation ). They were then washed with water and stained in I2-KI solution. The band mobility was measured with reference to that of standard marker strains of Drosophila melanogaster (Inomata et al. 1995bCitation ).

Molecular Cloning and Sequencing
A genomic DNA library of each species was constructed according to Frichauf et al. (1983)Citation using the commercially available packaging extract GigaPack III Gold (Stratagene). The molecular cloning described below was conducted according to Maniatis, Fritsch, and Sambrook (1982)Citation . The lambda dash II phage clones containing the Amy region were isolated from the genomic libraries by plaque hybridization using either a recombinant plasmid with a 3.8-kb EcoRI fragment containing the Amy gene of the Canton-S strain of D. melanogaster (Levy, Gemmill, and Doane 1985Citation ) or a recombinant plasmid with the PCR product containing the partial Amy gene of D. kikkawai as a probe. The PCR primers and PCR conditions were described in Inomata, Tachida, and Yamazaki (1997)Citation . The latter probe was confirmed to be an Amy homolog by sequencing. For the screening of the Amy3 and Amy4 genes of the sibling species, a recombinant plasmid with only about 430 bp of the 5'-flanking region of the Amy3 gene of D. kikkawai was used as a probe. DNA fragments of phage clones were digested by EcoRI or HindIII. Then, each of the DNA fragments with an Amy gene was subcloned into a phagemid vector, pBlueScript SK or KS (Stratagene). The Amy4 gene of D. leontia was obtained by PCR with the Amy3 (Amy4) specific primers (kik3f5: 5'-TCGCGATGTGGGGGCTGAGTG-3' and kik3f3: 5'-GCTCCACAAAGTATACTTGTCC-3'), in which an extracted DNA fragment after EcoRI digestion of a genomic DNA was used as a template. PCR conditions were as follows: 50 µl of the reaction mix was preheated at 95°C for 3 min. The reaction condition for 32 cycles was denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and polymerization at 72°C for 1 min. The DNA was directly sequenced. The sequencing was performed with an ABI model 377 automated sequencer using a DNA sequencing kit (BigDye terminator cycle sequencing ready reaction, ABI) with the synthetic oligonucleotide primers. In this study, 5'-flanking and coding regions were sequenced.

In Situ Hybridization to Polytene Chromosomes
The probes used in the screening of phage clones were labeled with a commercially available kit (BioNick labeling system, GibcoBRL). Hybridization was done at 37°C overnight to alkali-denatured chromosomes. Signals were detected using the BluGENE nonradioactive nucleic acid detection system kit (GibcoBRL).

PCR Amplification
Long and accurate (LA) PCR was performed using the TaKaRa LA PCR kit, version 2. Specific primers for LA PCR are k3RUP (5'-AGATTAGTGGCCTGATTAGC-3') and k6FUP (5'-GTGCATTCCGCTGATATCTG-3'). This primer set amplified an intermediate region of the Amy1 and Amy2 genes with a head-to-head structure. In order to amplify an intermediate region of the Amy3 and Amy4 genes with a tail-to-tail structure, a specific primer (k46for: 5'-GCTTCCTGGCCTTGGCTACT-3') for the Amy3 and Amy4 exon was used. Fifty microliters of the reaction mix was preheated at 94°C for 1 min. The reaction condition for 25 cycles was denaturation at 98°C for 20 s and annealing and polymerization at 65°C for 20 min. Following 25 cycles of the reaction, a further polymerization step at 72°C for 10 min was added.

Data Analysis
In noncoding regions, the DNA sequences were initially aligned with the CLUSTAL W program (Thompson, Higgins, and Gibson 1994Citation ) and then further aligned by eye. A neighbor-joining (NJ) tree (Saitou and Nei 1987Citation ) with the bootstrap probability based on nucleotide substitutions was constructed with the CLUSTAL W program. In the tree, the Amy genes of D. melanogaster (L22730) and D. pseudoobscura (X76240) were added, and that of D. virilis (U02029) was used as an outgroup. NJ trees of the synonymous and replacement substitutions estimated by the method of Nei and Gojobori (1986)Citation and an NJ tree of the 5'-flanking regions were constructed with the ODEN system (Ina 1994Citation ). The numbers of synonymous and replacement substitutions within each gene group for the independence test of substitution type and gene group were estimated by summing all branch lengths of the trees. G+C content (%) at the third positions of codons was also calculated using the ODEN system (Ina 1994Citation ). Codon bias was measured by the scaled {chi}2 ({chi}2/n) (Shields and Sharp 1987Citation ; Shields et al. 1988Citation ). Quantitation of isozyme activity observed on gels was performed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health). The average activity of four replicates was expressed by relative activity standardized to the band intensity of the S isozyme in larvae of D. kikkawai on glucose medium. The activity of D. kikkawai was multiplied by 4, since the samples applied were a quarter in D. kikkawai. The nucleotide sequences obtained in this study were deposited in the DDBJ (DNA Data Bank of Japan) with the accession numbers AB035055AB035069.

Results

Amylase Gene Structure in D. kikkawai and its Sibling Species
The genomic Southern blot analysis indicated that there were multiple amylase copies in D. kikkawai and the sibling species, D. bocki, D. leontia, and D. lini (data not shown). We cloned all of the Amy genes from a genomic library of D. kikkawai. Drosophila kikkawai has four amylase gene copies in a genome. For convenience, we call them Amy1 through Amy4, respectively. We also obtained the Amy genes of D. bocki, D. leontia, and D. lini from genomic libraries. Consequently, we found that D. bocki and D. leontia had four Amy genes and that D. lini had at least three Amy genes in their genomes. All of the Amy genes obtained were sequenced and analyzed in this study. Based on the restriction maps of phage clones and subsequent sequencing, two copies from each species were assigned to Amy1 and Amy2 of D. kikkawai. Since other copies could not be assigned to the Amy3 and Amy4 genes corresponding to those of D. kikkawai, the numbering of Amy3 and Amy4 in the sibling species is arbitrary. For D. lini, we also performed PCR with the Amy3 and Amy4 specific primer set and directly sequenced the product. We found that the sequence obtained was not heterogeneous. This indicates that there is no fourth copy or that there are more than two copies with the same sequence. Then, we concluded that there were at least three amylase gene copies in D. lini. All amylase genes were encoded by two exons interrupted by a putative intron whose position was the same as that in other Drosophila species (Brown, Aquadro, and Anderson 1990Citation ; Magoulas et al. 1993Citation ; Da Lage, Wegnez, and Cariou 1996Citation ; Inomata, Tachida, and Yamazaki 1997Citation ). Their exon length was 1,482 bp, encoding 494 amino acid residues. Note that the members of the melanogaster species subgroup and several Drosophila species have no intron (Shibata and Yamazaki 1995Citation ; Da Lage, Wegnez, and Cariou 1996Citation ; Inomata, Tachida, and Yamazaki 1997Citation ).

A restriction map of the Amy region in D. kikkawai is shown in figure 1 . The chromosomal locations of the Amy loci were examined by in situ hybridization to polytene chromosomes (see fig. 1 ). The Amy1 and Amy2 genes were about 20 kb apart and were in a head-to-head orientation. They were located in the middle of an arm, named the B arm (Roy and Lakhotia 1979Citation ), of the second chromosome. The head-to-head orientation is the same as that of the melanogaster species subgroup (Shibata and Yamazaki 1995Citation ), but the distance between the duplicated genes is about four times as long. The Amy3 and Amy4 genes of D. kikkawai were a tail-to-tail duplication. They were located near the centromere of the second chromosome, the same chromosome upon which the Amy1 and Amy2 genes are located. In order to examine the structure of the duplicated genes, LA PCR was performed (data not shown). In addition to D. kikkawai, D. bocki and D. leontia showed single 20-kb bands when the k3RUP and k6FUP primers were used. This indicates that the structure of D. bocki and D. leontia was the same as that of D. kikkawai in the duplicated Amy1 and Amy2 genes. In D. lini, the structure of Amy1 and Amy2 may not be in a head-to-head orientation, or the distance between them may be too long to amplify the DNA fragments by PCR. For Amy3- and Amy4-specific primer, a positive band was found only in D. kikkawai. The structure of the Amy3 and Amy4 genes of D. bocki and D. leontia may not be in a tail-to-tail orientation, or the distance may be too long for effective PCR. Note that we could not find a fourth amylase copy in D. lini.



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Fig. 1.—Restriction maps and chromosomal locations by in situ hybridization of the linked Amy genes in Drosophila kikkawai. E, H, S, and Xb indicate EcoRI, HindIII, SalI, and XbaI, respectively. The fine lines show the phage clones from a genomic library

 
Nucleotide Difference Between the Paralogous Genes Within Species
The numbers of nucleotide difference per site between the paralogous genes within each species were estimated by the method of Nei and Gojobori (1986)Citation (table 1 ). The nucleotide difference per synonymous site between the Amy1 and Amy2 genes ranged from 0.034 ± 0.0101 (D. kikkawai) to 0.0686 ± 0.0145 (D. leontia). The nucleotide difference per nonsynonymous site between the Amy1 and Amy2 genes ranged from 0 (D. lini) to 0.0044 ± 0.0020 (D. kikkawai). The Amy1 and Amy2 gene group was greatly diverged from the Amy3 and Amy4 gene group. The nucleotide difference per synonymous site between the Amy1 (Amy2) and Amy3 (Amy4) genes ranged from 0.6540 ± 0.0633 (D. lini) to 0.7976 ± 0.0775 (D. leontia). The nucleotide difference per nonsynonymous site between the Amy1 (Amy2) and Amy3 (Amy4) genes ranged from 0.0316 ± 0.0054 (D. lini) to 0.0450 ± 0.0065 (D. kikkawai). The degree of variation in nonsynonymous substitutions between the Amy1 (Amy2) and Amy3 (Amy4) genes was comparable to that in synonymous substitutions between the Amy1 and Amy2 genes.


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Table 1 Nucleotide Differences Between the Paralogous Genes Within Species

 
Nucleotide Divergence Between Species in the Orthologous Genes
The numbers of nucleotide divergences between species in the orthologous genes were also estimated by the method of Nei and Gojobori (1986)Citation (table 2 ). The nucleotide divergence per synonymous site ranged from 0.0812 ± 0.0158 (D. bocki vs. D. leontia) to 0.1169 ± 0.0194 (D. leontia vs. D. lini) in the Amy1 gene and from 0.0561 ± 0.0130 (D. kikkawai vs. D. bocki) to 0.1168 ± 0.0194 (D. leontia vs. D. lini) in the Amy2 gene. Table 3 shows all comparisons of the Amy3 and Amy4 genes, since we could not say which ones were orthologous to the gene described above. The nucleotide divergence per synonymous site ranged from 0.0204 ± 0.0077 (D. kikkawai Amy3 [Amy4] vs. D. leontia Amy4) to 0.1348 ± 0.0211 (D. bocki Amy4 vs. D. lini Amy3) among the Amy3 and Amy4 genes.


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Table 2 Nucleotide Divergence Between Species in the Orthologous Genes

 

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Table 3 Nucleotide Divergence Between Species Between the Amy3 and Amy4 Genes

 
Molecular Phylogeny of the Amy Genes
In order to examine the phylogenetic relationship of the Amy genes, an NJ tree (Saitou and Nei 1987Citation ) was constructed (fig. 2 ). The tree apparently shows multiple duplication events and recombination such as gene conversion during the evolution of D. kikkawai and the sibling species. The Amy1 and Amy2 genes from kikkawai and its siblings clustered together with a high probability (100%). The Amy3 and Amy4 genes also clustered together with a high probability (100%). This indicates that the first duplication separated the lineage of the Amy1 and Amy2 genes from that of the Amy3 and Amy4 genes. As a result of the first duplication, the Amy3 and Amy4 genes are outside of the Amy gene of D. melanogaster. On the first look, the tree appears to support the hypothesis that the second duplication, which generated Amy1 and Amy2 genes, independently occurred in each species lineage after speciation. However, this hypothesis is rejected by the fact that the gene structure of the Amy1 and Amy2 genes of these four species is well conserved. In conclusion, we propose that the second duplication occurred only once and predated speciation of the four species. That is, the topology of the tree is a result of one duplication event and subsequent recombinations such as gene conversion. The copy number of the Amy genes and the clustering pattern seem to suggest that the third duplication took place after diversification of D. kikkawai, D. bocki, and D. leontia from D. lini but before speciation of the three species. The Amy3 and Amy4 genes in D. kikkawai were completely identical in the coding region and almost the same in the flanking region (99.8%). Thus, interlocus recombination such as gene conversion probably occurred quite recently or frequently.



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Fig. 2.—A neighbor-joining tree with bootstrap probability. Over 85% of the bootstrap probability from 1,000 replications is shown along each branch. Drosophila virilis was used as an outgroup

 
Concerted Evolution in the Coding Region Between the Amy1 and Amy2 Genes
The second duplication predated speciation of the four species. If there are no genetic exchanges between the duplicated genes after the duplication, the nucleotide difference between the duplicated Amy1 and Amy2 genes within a species is expected to exceed the nucleotide divergence between species in the orthologous gene. As described above, however, the nucleotide difference between the duplicated genes within a species (0.0349 ~ 0.0686) was smaller than the nucleotide divergence between species in the orthologous genes (0.0561 ~ 0.1002). The bootstrap probabilities of clustering of the Amy1 and Amy2 genes in D. kikkawai, D. leontia, and D. lini were high (fig. 2 ). These observations suggest that there were genetic exchanges between the duplicated genes. In other words, the duplicated genes have experienced concerted evolution.

Divergent Evolution in the 5'-Flanking Region Between the Amy1 and Amy2 Genes
The Amy1 and Amy2 genes are divergent from the Amy3 and Amy4 genes in the 400-bp upstream region, such that a multiple alignment of this region from Amy1 to Amy4 was impossible. An NJ tree of the Amy1 and Amy2 genes of this region was constructed (fig. 3 ). Two clusters were clearly recognized, although the tree was unrooted. One consisted of the Amy1 gene cluster, and another consisted of the Amy2 gene cluster. This clustering pattern suggests that the 5'-flanking region divergently evolved after the second duplication. In contrast to concerted evolution of the coding region, divergent evolution of the 5'-flanking region was also observed in amylase of the melanogaster species subgroup (Hickey et al. 1991Citation ; Shibata and Yamazaki 1995Citation ; Okuyama et al. 1996Citation ) and Drosophila pseudoobscura (Popadic and Anderson 1995Citation ).



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Fig. 3.—A neighbor-joining tree based on an alignment shown in figure 4A. The numbers of nucleotide substitutions per site were estimated by Jukes and Cantor's (1969) method

 
Putative Regulatory Elements in the 5'-Flanking Region
Putative regulatory motifs were observed in the 5'-flanking region (fig. 4A ). In D. melanogaster, the motif sequences of initiation of transcription are different between the duplicated (proximal and distal) genes (Shibata and Yamazaki 1995Citation ; Okuyama et al. 1996Citation ). In D. kikkawai and the siblings, the motif sequence in the Amy1 and Amy2 genes was the same as that of the distal gene (ATCAG) of D. melanogaster except for that of the Amy1 gene of D. lini (TTCAG). The transcription initiation motif and TATA box (TATATAA) were found at similar positions to those of D. melanogaster. The other motifs, are a CAAT motif (CAAAT), a putative midgut specific regulator (GATAAG; Magoulas et al. 1993Citation ), a similar sequence (CCAGTCAGTCCGTCTGC) to a putative glucose repressible element (CCAGTCAATAC/GGTCTGC; Bore and Hickey 1986Citation ), and a TCACGC sequence, were also observed in the same order as those of D. melanogaster (Okuyama et al. 1996Citation ). The TCACGC sequence is similar to a motif sequence found in moth chorion genes (Mitsialis et al. 1987Citation ) and is necessary for the full expression of the amylase gene in D. melanogaster (Choi and Yamazaki 1994Citation ). Interestingly, the Amy2 genes had CAAT motifs downstream of the putative TATA boxes. The motif sequence of transcription initiation in the Amy3 and Amy4 genes (ATCAA) was different from that of the Amy1 and Amy2 genes (fig. 4B ). The TATA box, the CAAT motif, and a sequence (GATAAC) similar to a putative midgut-specific regulator sequence (GATAAG) were also observed. However, a putative glucose repressible element (CCAGTCAGTCCGTCTGC) and TCACGC sequences were not detected in the upstream region sequenced.



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Fig. 4.—Multiple alignment of the 5'-flanking region of the Amy1 and Amy2 genes (A) and that of the Amy3 and Amy4 genes (B) in D. kikkawai and its sibling species. Met indicates the initiation codon. Putative regulatory sequences are boxed. A nucleotide common to all of the Amy1 sequences or all of the Amy2 ones is denoted by an asterisk at the top or bottom of the sequences, respectively. A nucleotide common to all the Amy sequences is denoted by an asterisk between the Amy1 sequences and the Amy2 ones. The dashes indicate deletions

 
G+C Content and Codon Usage Bias
The Amy genes of most Drosophila species show high G+C contents at the third positions of codons and high codon usage bias, and they show positive correlation between them (Inomata, Tachida, and Yamazaki 1997Citation ). The codon usage bias in D. kikkawai and the siblings was high for the Amy1 and Amy2 genes (1.04 ~ 1.10) and low for the Amy3 and Amy4 genes (0.51 ~ 0.56). Similarly, the G+C content at the third codon position was higher for Amy1 and Amy2 (87.0% ~ 88.5%) than for Amy3 and Amy4 (71.3% ~ 72.7%). For comparison, the codon usage bias of D. melanogaster is ~1.2 and the G+C content is ~90%. Figure 5 clearly shows that there are two groups: the Amy1, Amy2 genes and the Amy3, Amy4 genes.



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Fig. 5.—The relationship between codon bias and G+C content. Codon bias is represented by the scaled {chi}2. 3rd GC (%) is the G+C content (%) at the third positions of codons

 
Nucleotide Substitution Pattern of the Duplicated Genes
We tested the molecular evolutionary clock hypothesis by Tajima's (1993)Citation 1D method using D. pseudoobscura as an outgroup. When the Amy1 gene of D. kikkawai was compared with the Amy3 gene, the rate constancy of nucleotide substitutions was rejected ({chi}2 = 6.97, df = 1, P < 0.01). For detailed analysis, NJ trees were constructed based on replacement and synonymous substitutions (fig. 6 ). Under the strict neutral model of molecular evolution (Kimura 1983Citation ), the relative numbers of replacement and synonymous substitutions should be the same between the two categories in any clade of phylogenetic trees. We counted the number of replacement and synonymous substitutions on branch a (the Amy1 and Amy2 lineage) and branch b (the Amy3 and Amy4 lineage) (shown in fig. 6 ). Their branch lengths represent the number of substitutions after the first duplication and prior to speciation of the four species. The null hypothesis was tested by the chi-square test. The result is presented in table 4 . There was heterogeneity between substitution type and substitution rate in the two lineages (0.01 < P < 0.05). This observation seems to suggest that the rates of synonymous substitution were accelerated in the lineage leading to the Amy3 and Amy4 genes. We also counted the number of substitutions after speciation summing all branch lengths of the tree. For example, the number of replacement substitutions of the Amy3 and Amy4 gene group was counted summing all branch lengths presented by bold lines in figure 6A. We performed the chi-square test to examine the null hypothesis described above. However, it was not rejected (table 5 Go ).



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Fig. 6.—Neighbor-joining trees based on replacement (A) and synonymous (B) substitutions. The numbers of synonymous substitutions were estimated by Nei and Gojobori's (1986) method. Within-gene group variation was estimated summing all branch lengths represented by bold lines

 

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Table 4 A 2 x 2 Contingency Table for Testing Independence of Substitution Type and Gene Lineage

 

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Table 5 A 2 x 2 Contingency Table for Testing Independence of Substitution Type and Gene Group

 

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Table 6 The Relationship Between Relative Mobility and Charge in Mature AMY Isozymes

 
Electrophoretic Pattern of AMY Isozymes on the Different Media
Drosophila kikkawai and its sibling species had two or three electrophoretically different isozymes. Here, for convenience, we call the faster, middle, and slower bands the F, M, and S isozymes, respectively, in each species. In D. melanogaster, the mobility of AMY isozymes is mostly determined by the charge differences of proteins (Inomata et al. 1995bCitation ; Matsuo, Inomata, and Yamazaki 1999Citation ). The first 18 amino acid residues from the N-termini were predicted to be cut off after translation, and mature amylase proteins would be then produced (Bore and Hickey 1986Citation ). We inferred and assigned which gene encodes an AMY isozyme of D. kikkawai and its sibling species based on the net charge differences of putative mature proteins (476 amino acid residues). The net charge of mature proteins was determined by scoring +1, 0, or -1 for positive (Lys, Arg), neutral, or negative (Asp, Glu) amino acid residues, respectively, in this electrophoretic condition. For example, for D. kikkawai, although there were several amino acid substitutions between the AMY proteins encoded by the Amy1 and Amy2 genes, their net charge was the same (-7). On the other hand, the AMY proteins encoded by the Amy3 and Amy4 genes showed the same charge (-10) due to no difference in coding sequences. Therefore, we assigned the Amy1 and Amy2 genes to the S isozyme and the Amy3 and Amy4 genes to the F one. Table 6 lists the net charge and relative mobility on polyacrylamide gel.

In order to visualize the electrophoretic pattern of isozymes on different media (glucose and starch media) at larval and adult stages, we performed electrophoresis of AMY proteins. A representative result of four replicates is shown in figure 7 . The average relative activity of each isozyme at the two stages on the two media is shown in figure 8 . At face value, there was a quantitative difference, although statistical significance could not be determined, since variance in activity was large. Although in figure 7 the S isozyme of larvae of D. lini appears to have higher activity than the F isozyme on the starch medium, on average, the F isozyme has higher activity than the S isozyme (fig. 8 ). Activity was highest in D. kikkawai and lowest in D. leontia. The F isozymes of the four species and the M isozyme of D. leontia were observed at both larval and adult stages, although their activity appeared to be lower on glucose medium and their levels were different among species. In contrast, the activity of the S isozyme was highest on starch medium in larvae of all species. The activity level of the S isozyme markedly changed in both the developmental stage and the food environment. Amylase activity is mostly determined by the amount of mRNA in D. melanogaster (Benkel and Hickey 1986Citation ; Yamate and Yamazaki 1999Citation ). Therefore, the electrophoretic pattern of AMY isozymes observed in this study is likely to reflect the expression pattern at the transcriptional level.



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Fig. 7.—Electrophoretic pattern of AMY isozymes at the two stages on two test media in Drosophila kikkawai and its sibling species. G = glucose medium; S = starch medium; L = larvae; A = adult flies. The AMY1–AMY6 isozymes of Drosophila melanogaster were used as mobility markers (M)

 


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Fig. 8.—Average of relative activity of AMY isozymes at two stages on two test media in Drosophila kikkawai and its sibling species. Combinations of three letters along the X axis are as follows. The first letter represents stage (L = larvae; A = adult flies), the second one represents medium (G = glucose; S = starch), and the last one represents isozyme (S = slow; M = middle; F = fast). Note that the M isozyme is only in D. leontia.

 
Discussion

The coding regions of the duplicated Amy1 and Amy2 genes have evolved in a concerted fashion. Concerted evolution of the Amy genes was also reported for other Drosophila species, and gene conversion is suggested to be a homogenizing mechanism between the duplicated Amy genes (Hickey et al. 1991Citation ; Inomata et al. 1995bCitation ; Popadic and Anderson 1995Citation ; Shibata and Yamazaki 1995Citation ; Popadic et al. 1996Citation ). Thus, although the two linked (Amy1 and Amy2) genes of D. kikkawai and its sibling species are separated by a longer distance (about 20 kb) than those of other Drosophila (4 kb in the melanogaster species subgroup, about 2 kb in D. pseudoobscura), gene conversion is considered to act on the coding region of the duplicated genes to homogenize them. In contrast, the two linked genes divergently evolved in the 5'-flanking region (figs. 3 and 4A ). This suggests that some selective forces acted on the 5'-flanking region to diverge their sequences after the origination of the duplicated genes. On the other hand, the Amy3 and Amy4 genes have divergently evolved from the Amy1 and Amy2 genes in both the coding and the 5'-flanking regions. The 5'-flanking sequence of Amy genes is relatively conserved among Drosophila species, although it is more diverged than the coding one. Some putative regulatory sequences of the Amy3 and Amy4 genes were also conserved, although they were slightly modified. For example, the transcription initiation and midgut-specific regulator sequences were ATCAA and GATAAC instead of ATCAG and GATAAG. However, the TCACGC sequence, which is similar to that found in moth chorion genes (Mitsialis et al. 1987Citation ) and is necessary for the full expression of amylase in D. melanogaster (Choi and Yamazaki 1994Citation ), and a putative glucose repressible element (CCAGTCAATAC/GGTCTGC; Bore and Hickey 1986Citation ) were not found in the upstream regions of the Amy3 and Amy4 genes. This observation suggests that the Amy3 and Amy4 genes are expressed in midgut but are less affected by dietary glucose. Modification and loss of regulatory sequences is likely to change not only the level of expression, but also the pattern of expression.

In D. melanogaster, the level of amylase activity depends on that of mRNA abundance (Benkel and Hickey 1986Citation ; Yamate and Yamazaki 1999Citation ), and differences in activity among the AMY isozymes are also attributed to those in the amount of mRNA (Yamate and Yamazaki 1999Citation ), suggesting transcriptional regulation. In D. kikkawai and its sibling species, the relationship between the two is plausible. Therefore, the electrophoretic pattern observed in this study is likely to represent the gene expression pattern at the transcriptional level.

Divergent evolution between paralogous Amy genes within species was also reported based on partial sequences (Da Lage, Wegnez, and Cariou 1996Citation ). Recently, a new paralogous Amy gene, Amyrel, was found (Da Lage et al. 1998Citation ). However, it was highly diverged from usual Amy genes, and its function remains to be determined. The new paralogous Amy genes reported in this study were suggested to encode active amylase isozymes, and therefore we referred to the relationship between nucleotide substitutions and expression pattern.

There are two plausible evolutionary histories of gene duplication events. One is that a linked gene pair occurred by the first duplication, which was followed by the second duplication of this pair and subsequent complex changes of gene structure such as an orientation of the duplicated genes at the different chromosomal location. Taking the conserved gene structure among species, the topology of the phylogenetic tree, and the electrophoretic pattern into consideration, we suggest another evolutionary history: that three duplication events have occurred during the Amy gene evolution of the kikkawai and the siblings. The first duplication separated the lineage of the Amy1 (Amy2) gene from that of the Amy3 (Amy4) gene. The Amy1 (Amy2) gene could develop a regulatory diversification between developmental stages and in the response to dietary carbohydrates. On the other hand, Amy3 (Amy4) seems to retain an original function as amylase. A second duplication produced a head-to-head structure of the Amy1 and Amy2 genes before speciation of the four species. The third duplication probably took place before the speciation of D. kikkawai, D. bocki, and D. leontia and, at least in D. kikkawai, resulted in a tail-to-tail structure of Amy3 and Amy4. Since there were no substitutions in the coding region of D. kikkawai, gene conversion seems to have happened recently or frequently. Minor modification of gene regulation seems to have taken place in or after speciation.

We found (1) that the Amy1 and Amy2 genes had high G+C content at the third positions of codons and a low synonymous substitution rate compared with the Amy3 and Amy4 genes, (2) that the difference in activity of the S isozyme encoded by Amy1 and Amy2 was remarkably high at the larval stage between the different environments and electrophoretic pattern of the S isozyme in larvae is different from that in adults, and (3) that the F isozyme was observed throughout all stages and the difference in activity between environments was not high. In conclusion, after the first duplication but before speciation of four species, the synonymous substitution rate between the two lineages and the electrophoretic pattern of the isozymes encoded by them changed, although we do not know whether there was any evolutionary relationship between the two.

The capacity for gene regulation is thought to be important for adaptation. In the case of amylase, it is considered the response ability, or inducibility, for different food environments (Yamazaki and Matsuo 1984Citation ). Actually, the inducibility was positively correlated with fitness of individuals in D. melanogaster (Yamazaki and Matsuo 1984Citation ; Matsuo and Yamazaki 1984Citation ). The inducibility is likely to be determined at the transcriptional level, since amylase activity is mostly determined by the amount of mRNA. In D. kikkawai and its sibling species, the inducibility of the Amy1 and Amy2 genes, which encode the S isozyme, would be enhanced by natural selection to adapt to severe environments. On the other hand, the Amy3 and Amy4 genes, which encode the F isozyme, would be continuously expressed through the developmental stages to survive. In other words, natural selection acting on the 5'-flanking region of the Amy1 and Amy2 genes is different from that for the Amy3 and Amy4 genes. The diversification of the regulatory systems, which include cis- and trans-acting elements, rather than that of amino acid sequences is important for adaptive evolution (King and Wilson 1975Citation ; Dickinson 1991Citation ). Our findings are evidence that the regulatory system of the Amy multigenes in D. kikkawai and its sibling species has been divergently evolving to rapidly adapt to environments in which the respective species live. In order to elucidate the adaptive significance of the Amy multigenes, further molecular genetic analyses are needed in future studies. In order to assess the model of selection on the 5'-flanking region described above, the regulatory elements in the 5'-flanking region must be characterized. The role of trans-acting factors for the expression of amylase genes must also be examined for a complete understanding of this system. Characterizations and comparisons of activity levels, transcription levels, and the regulatory sequences required for expression between orthologous genes between species and between paralogous genes within species would deepen our understanding of evolution in multigene family and regulatory systems.

Acknowledgements

We are grateful to Drs. H. Tachida, Y. Matsuo, and T. S. Takano for their helpful discussions. This work was supported by research grants to N.I. and T.Y. from the Ministry of Education, Science and Culture of Japan.

Footnotes

Wolfgang Stephan, Reviewing Editor

1 Keywords: gene duplication gene expression codon bias adaptive evolution amylase (Amy) gene Drosophila. Back

2 Address for correspondence and reprints: Nobuyuki Inomata, Laboratory of Molecular Population Genetics, Department of Biology, Kyushu University, Fukuoka 812-8581, Japan. E-mail: ninomscb{at}mbox.nc.kyushu-u.ac.jp Back

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Accepted for publication December 14, 1999.