j. f. aquadrot and wyatt w. anderson**'sequence, or that amy2 has recently undergone a gene...

<:opyrighc 0 1990 by the Genetics Society of America

DNA Sequence Evolution of the Amylase Multigene Family in Drosophila pseudoobscura

Celeste J. Brown,* Charles F. Aquadrot and Wyatt W. Anderson**'

*Department of Genetics, University of Georgia, Athens, Georgia 30602, ?Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, and Section of Genetics and Development, Cornell

University, Ithaca, New York 14853 Manuscript received January 13, 1989

Accepted for publication May 24, 1990

ABSTRACT The a-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies

on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amyl ) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amyl gene of D. pseudoobscura differs from the Amy-p' gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 f 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 & 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amyl. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amyl sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amyl; and that Amy3 is nonfunctional and subject to random genetic drift.

THE a-amylase enzyme (EC 3.2.1.1, a-l,4-glucan- 4-glucanohydrolase) breaks down starch into

maltose by cleaving a-l,4-glucosidic linkages (BERN- FELD 1955). In Drosophila, it is a stable monomer with a molecular mass of 54,500 daltons (DOANE et al. 1975). Phenotypes from acrylamide gel electrophoresis indicate that a-amylase in some strains of D. melanogaster is encoded by a duplicated gene (DOANE 1969a,b), and linkage studies indicate that the duplications are very tightly linked (BAHN 1967). The duplication has been confirmed at the molecular level by the isolation of a genomic clone (GEMMILL, LEVY and DOANE 1985) carrying two regions of DNA ca- pable of producing amylase in an in vitro translation system (LEVY, GEMMILL and DOANE 1985). Recent studies of restriction site variation in D. melanogaster indicate that all strains carry the duplication (GEM- MILL, SCHWARTZ and DOANE 1986; LANGLEY et al. 1988).

In D. pseudoobscura, Amylase is located on the third chromosome, an autosome polymorphic for some 50 gene arrangements that are the results of overlapping, paracentric inversions. These gene arrangements can be arranged in a phylogeny on the basis of the breakpoints of the inversions that generated them (STUR- TEVANT and DOBZHANSKY 1936), assuming that each inversion arose only once (Figure 1). Crosses among amylase phenotypes in D. pseudoobscura give no evi-

' To whom requests for reprints should be addressed.

Genetica 126: 131-1 38 (Septenlber, 1990)

dence for a functional duplication (POWELL 1979). Molecular cloning of the Amylase region in D. pseudoobscura and restriction map analyses of lines homozygous for several common inversions revealed up to three regions of sequence similarity to the D. melanogaster Amylase gene (AQUADRO et al. 1990). Each gene arrangement has a characteristic number of amylase homologous regions: either one, two, or three (Figure 1). The physical map of the three putative genes on a chromosome with the Standard gene arrangement is illustrated in Figure 2a. One gene, A m y l , was consid- ered the best candidate for the functional amylase gene because it was the only amylase homologous region found in all lines, both of the other regions being absent in some of the gene arrangements. Suppression of recombination in heterozygotes for different gene arrangements appears to have pre- served the evolutionary history of the Amylase gene duplications, affording us a unique opportunity to study the evolution of the members of a multigene family.

In this paper, we present the nucleotide sequences of three Amylase genes in D. pseudoobscura and show them to be recently diverged members of an Amylase multigene family. Comparisons of the three sequences with each other and with a previously published amylase sequence from D. melanogaster allow us to infer the evolutionary history of the members of this multigene family.

132 C. J. Brown, C. F. Aquadro and W. W. Anderson

TREELINE (1 ) CHlRlCAHUA ( 2 ) a. AC2 AC1

Amyl Amy2 Amy3 - c_

ti B S S S E SB E S E E S SB z I 1 1 1 1 I I, I , , I , , I

I 1 I pFA4 pAC2 + >

D rnlranda (2)

SANTA CRUZ (2)

HYPOTHETICAL

STANDARD (3)

ARROWHEAD (2) KLAMATH (3 ) 0 pc',sllrllI!s

FIGURE 1 .-Phylogeny of third chromosome gene arrangements i n D. pseudoobscura based on inversion breakpoints. Numbers in parentheses are numbers of complete Amylase genes.

MATERIALS AND METHODS

Materials: Enzymes were purchased from Bethesda Re- search Laboratories, Boehringer Mannheim Biochemicals, International Biotechnologies Incorporated, and Promega Corporation. Enzymes were used according to manufactur- er's specifications and standard procedures. Cloning vectors pUC8, mp18 and mp19 were purchased from Bethesda Research Laboratories. Nucleotides and m13 sequencing primers were purchased from Pharmacia LKB.

Methods: Two overlapping clones (AC1 and AC2) were isolated from an EMBL4 library of genomic DNA from a strain of D. pseudoobscura containing the Standard gene arrangement (AQUADRO et al. 1990) (Figure 2a). From these phage clones, each of the three putative Amylase genes was subcloned into pUC8 (shown in Figure 2a) and subsequently into mp18 or mp19. Nested deletions of pUC or M13 subclones were made using the methods of DALE, MCCLURE and HOUCHING (1985) or PONCZ et al. (1982) and sequenced using the dideoxy-chain termination method (SANGER, NICKLEN and COULSON 1977). Sequence information was also obtained using three synthetic oligonucleotides as primers: (5'-GGTGGGAGCGCTACCAG, +221-237 in Figure 3), (5'-AACTGCTCCTCGTTGCC, +284-268 in Figure 3), (5'-GGCAAGACCGTCACCGT, + 1384-1400 in Figure 3).

Detection of amylase homologous regions: In order to determine whether other regions homologous to amylase exist in the genome, we hybridized the plasmid clone pFA4 (Figure 2a) to filters of genomic DNAs digested with EcoRI. The genomic DNAs were prepared from fly strains made homozygous for the third chromosome and represent the Treeline, Santa Cruz and Standard gene arrangements. There are EcoRI sites on each side of the three Amylase gene regions. The filters were hybridized at 42" in 50% form- amide, 5X SSC, 1X Denhardt's solution, 20 mM sodium phosphate (pH 6.5), 1 mg denatured and sonicated salmon

- ~ Z k b

b Amyl

4 A A A AR E RAS R GGA SA S I I I I II I Ill I 111 I I I

""c

" + - " c_

"c_

Amy2 S B I I - c__ " t "" -""

Amy3 S B I I -"" -" """ -

c- " c- -

500 bp

FIGURE 2.-Restriction maps of the Amylase gene region in D. pseudoobscura. (a) Map of phage clones ACI and AC2 from the Standard gene arrangement, showing the relationships of the three Amylase genes. Arrows are 5' to 3'. (b) Sequencing strategies for each of the Amylase genes. Arrows beginning from dots indicate sequences obtained from oligonucleotide primers described in the text. (A = A l u l , B = BamH1, E = EcoR1, G = Bgl l l , H = HindI11, R = RsaI, S = SalI, Z = XhoI).

sperm DNA, and 10% dextran sulfate (1X SSC = 150 mM NaC1, 15 mM Na-citrate). The filters were washed at moderate stringency (42 O , 0.1 % sodium dodecyl sulfate), autoradiographed, rewashed at high stringency (65O, 0.1 % sodium dodecyl sulfate), and autoradiographed again.

Analysis of DNA sequences: Sequences were aligned with each other and with the D. melanogaster amylase sequence (BOER and HICKEY 1986) using the ARNOLD et al. (1986) package of programs on a PDP 11/34 computer, or the IBI/Pustell (PUSTELL and KAFATOS 1984) package on an IBM XT computer, or the STADEN (1982) package on a VAX computer.

The percent nucleotide differences per site were calculated directly for all pairwise comparisons of Amyl, Amy2 and Amy3, using the formula p = nd/nt , where nd is the number of nucleotides which differ between the two sequences, and n, is the total number of nucleotides compared between the two sequences. The standard errors of p were calculated as SE = [ p ( 1 - p)/n,]". No correction for multiple substitutions is needed for differences as small as those among these three genes (NEI 1987).

For comparisons of Amyl and the Amy-p' gene from D. melanogaster, the percent nucleotide substitutions per site for synonymous and nonsynonymous substitutions, corrected for multiple substitutions, were estimated by the method of LI, Wu and Luo (1 985). The percent nucleotide

FIGURE 3.-Nucleotide sequences of D. pseudoobscura Amyl (Al) , Amy2 (A2), and Amy3 (A3) genes, numbered from the initiation codon. Differences of Amy2 (A2) and Amy3 (A3) from the homologous sequences of Amyl from -41 to + I 548 are indicated above the Amyl sequence. Differences between Amyl (Al ) and the D. melanogaster Amy-p' from the TATA box to the stop codon are indicated below A1 on the line marked M . Differences which result in an amino acid change are underlined. Differences between Amy2 and Amy3 in the flanking sequences are designated by an * above the sequences. Regulatory motifs (a, b) are underlined. The putative introns are numbered il-i71 ( A m y l ) or i 1 -i8 1 (Amy2 and A m y 3 ) to nlaintain the alignment with the D. melanogaster sequence; this region is indicated as a gap in the D. melanoguster sequence.

Amylase Gene Family Evolution 133

- 6 4 4 A3 TATGCCCCGC TTATATCAAC GGAAAGACCA TTCAAAATAG TTTGATTGGG CCTGCCTTTC

- 5 8 4 A I AATAATTATC ATTATCATGC TGGACTATGA TTTCCTGTCA GAGAAAATCT ACGAAAACAC CACCACTCAT CAATAATCAG CTAATATTAT ACATITGGCA TTCCAGGGGT TCCACTTTTC

h 3 ACCATTATCG GATGATCTGC TAGGCGGGAG TCACCGATCA CGCTCTGAGT AGCATGCAGC CAGGAGAGGG ACCATAAATC AGGGCCCATG AGGCGATATC AGGGAAATGC AGTGTCCCGA CCGA - 4 6 4 A2

A3 TGACTACGTT CAGATTCCGG AATCGCCCCC GAAZTAGTCT GATTAGTAAT TCAAATAATT AGCCACTTAA CTAATCATCA G-CGCCAGTT CGCAAGC-AC CGG-----CC CAGTCCCCTG - 1 1 4 A2 TGACTACTTT CAGATTCCGC AATCGCCCCC GAATTAGTCT GATTAGTAAT TCAAATAATT AGCCACTTAA CTAATCATCA GGCGCCAGTT CGCAAGCCAC CGGTCCCACC CAGTCCCCTG

A3 CCAAAATACC CGCAATCAGC CACAACGGCT GGCCCGGCTT TCACTAATCA GCCGGCGTGATGCCCCACAC CCAGAGAGAA AGAAGTGAGT GAAGCCTGCC GATAAGATCG A A T C G W A2 CCAAAATACC CGCAATCAGC CACAACGTCT GGCCCGGCTT TCACTAATCA GCCGGTGTGATGCCCCACAC GCAGAGAGAA AGAAGTGAGT GAAGCCTGCC GATAAGATCG A A T C G W

- 2 2 4 A 1 CAC GGCACCCCAA GCGATCCGAT AAGGTCTCAA TTTGAATCGG S C A C T C A GGCAGCCAAA ACACCGTCCG

h h3 CACTCCCCGC GAAGTCAACG CGAAGTCAAC GGCGACCCCT ATLAAGCAG CCCCAGAAAT CTCAA C A G A2 CACTCCCCGC GAAGTCAACG ---------- -GCGACCCCT ATAAMGCAG CCCCAGAGATCT-AA C A G

- 1 0 4 A 1 ATGACGCCGA TTCTCTCTTT CCAGGCACTC CAGACACCGT ATATAAGGCG CAGCAACAGC CGGATTGGAC TCAGAGTGAA ACATAGGTTC CATCTAGCAT CAAC M T ATATAA A GGCTCTGAG TAG- CCGA C TG AC G A T

A I A2

+ l ~1 ATG TTC CTG ACA AAG ACC CTC GGG TGC ATC GCC TTC CTG GCG ATC GCC AGC GCC CAG TTC AAC ACC ARC TAC GTT GGT GGC CGC AGC GGC ATG GTC M T C _ C G_ & A T_ C c C G A_ A C C_A TCC T T T A

A3 A2

t97 ~1 CAC CTC m c GAG TGG AAG TGG GAC GAC ATC GCC GCC GAG TGC GAG AAC TTC CTG GGC CCC CAG GGC TAC GCC GGT CTT CAG GTGAGATCCG TCCGGAGA M T A T A & T G """"" """"

A3 AAGGATGTGC A2 AAGGATGTGC

T 119 GCTTCCCTCCACCGTCACC----------CAATCCTCATCTCCCGCCTCTACCTCCTTCCCAG GTG TCG CCC GTC AAC GAG AAC GCC GTG AAG AGT GGG CGT CCG TGG TGG

_______"."_"__"_"""""""""""""""""""--"- C C T G c % C & C c c

A3 A2

+ 2 2 6 ~1 GAG CGC TAC CAG ccc ATC TCC TAC AAG CTG ACC ACC CGT TCC GGC AAC GAG GAG CAG TTC GCC AGC ATG GTC AGG CGC TGC AAC AAC GCC GGA GTG M A T G&G C A A - A S T

A A A2

A3

+ ) 2 2 CGC ACC TAC GTG GAC GTC ATc TTC AAC CAC ATG GCA GCC GAT GGC GGC ACC TAC GGC ACA GCC GGC AGC ACC GCC AGC CCC AGC TCC AAG AGC TTC M G G & C C A T G AG AT

A3 - G

M C_ G G C T + 4 1 8 AI ccc GCA GTG ccc TAC TCC TCC CTG GAC l T C AAC CCC ACC TGC GCC ATC AGC AAC TAC AAC GAC GCC ARC CAG GTG CGC AAC TGC GAG CTG GTC GGC

A2

A 3 A2

E A ! ? P

+j14 ~1 CTG CGT GAC CTC AAC CAG GGC AAC TCG TAC GTC CAG GAC AAG GTT TCC GAT 'PTC CTG AAC CAC TTG ATC GAT CTG GGT GTG GCC GGA TTC CGC GTG M C T C G G E C_ C_ T C T c c

A3 A A

+610 GAC GCT GCC AAG C A ~ A ~ G TGG ccc GCC GAT CTG GGC GTC ATC TAT GGCCGC c"c AAG M C c T G Mc GAT GGC TTc GCG TCC GGA TCC AGG A2

M C c c C G R

A3 A2

r 9 9 4 A 1 GCC TTC ATG CTG GCC CAT CCC TTC GGC ACT Ccc CGT GTG ATG TCC TCCTTC TCC TPC ACC GAC ACC GAC CAG GGC CCA G CCC ACC ACC GAC GGA CAG H G C C G T c c

+1090 Al GCC TCT ccc ACC ~ p c AAC AGC GAC M G TCC TGC GGC GGC GGATGG GTG GAG cAc CGC CGC TAC lUIc GTG GCC TTc . .- A2

M C G T T - C A C

A3 T GA CT C T A2

t t

GAGCAT A G C M M C G C A C T C m A G C AAAGAGGTTA T G A C T - C T T T GAGCAT A G C U I U C G C A C T C m A G C AAAGAGGTTA

+147.q ~1 GCC ATG 'FrG TAA GGGACTCAGC ATGTGAGA" -CCCCAACAA TCMTCGAGA TTATTACTAT TMATACACA ATGATATTAT GCMTGCTCG TCGACTCTGA GTTGGGACTG M B

A) GGGG-TTCm CGTCAAGTAG GGCITMGCA C-CACGA TATCCTGCCG GAGTTOTTOT ToTpMTooI ACTCCCOTGA ATATOCCGCC GGCTGACACC T G T G m G G C TTCACTcTCC A2 GGGGTXTCTT CGTCMGTAG GGCT?AAGCA CTLTTCACGA TATCCTGCCG G A G W T T G T l W l T " T M A ACTCCCGTGA TTATGCCGCC GGCTGACACC TGTCTITGGC TlTACTCTCC

+I586 ~l CTGGMACGA TAMGGGTTA GAGCAGGTOC CTGGGCATTA CCAGATMjTC AGTGATATCC GTATTOTATT GCGCCAAGlT GGAGAGMGC CTCCATTCAA TCCGGMGGA CGAGCAGTGC

.*.. . f *...* A 3 CCCGCAGAAC CTGGTGAGAT lTRG A2 TCCGCAGAAC CGCTCGCGAG GCCAC

t1706 A I GGAACGTGGA AA


substitutions by which A m y l and Amy-p' differ were estimated for the total sequence and the noncoding regions from the percent nucleotide differences per site, corrected for multiple substitutions by the method ofJUKEs and CAN- TOR (1 969).

RESULTS

The sequencing strategy for each of the three Am- ylase genes is depicted in Figure 2b. As shown in Figure 2b, both strands were sequenced over Amyl, Amy2 and Amy3. We have included data from sequencing of one strand for the outside edges of the flanking regions of Amy2 and Amy3. The nucleotide sequence of Amyl is presented in Figure 3 together with site changes within the coding regions of Amy2, Amy3, and the Amy-p' sequence from D. melanogaster (BOER and HICKEY 1986). Figure 3 also presents the aligned 5' and 3' flanking sequences, respectively, for Amyl, Amy2 and Amy3. There are 7 1 bp in Amyl (il-i7 1) and 8 1 bp i n Amy2 and Amy3 (il-i81) that have no counterpart in the D. melanogaster amylase sequence. There are consensus splice junction signals at the 5' and 3' ends of the inserted sequences, and we tenta- tively designate the inserted bases as intron sequences.

In the coding region, there are 11 nucleotide differences among the three D. pseudoobscura amylase sequences; 3 represent synonymous substitutions (#'s 570, 636, 1320) and 8 are nonsynonymous substitutions (#'s 178, 416, 474, 552, 575, 800, 817, 1434). Note that the substitution at position #474 in Amy3 generates a stop codon. The percent nucleotide substitutions per site among the three coding sequences are presented in Table 1.

In the flanking sequences, we infer that Amyl shares a 99 bp region of homology with Amy2 and Amy3. In the 3' flanking sequences, Amy2 and Amy3 share 240 bp of homology; in the 5' flanking sequences they share more than 340 bp. The percent nucleotide substitutions per site among the 3' and 5' flanking sequences of the three genes are presented in Table 1. Figure 3 shows that the 5' flanking sequence of all three D. pseudoobscura genes and the D. melanogaster gene contain the TATA control sequence at -65 (b in Figure 3). Amy2 and Amy3 have a CAAT control sequence at -109 and Amyl has a CAAT sequence at - 135 (a in Figure 3).

Nucleotide differences between the functional Amyl gene and the Amy-p ' gene of D. melanogaster (BOER and HICKEY 1986) have accumulated at 180 of the 1482 sites in the coding sequence. The estimated divergence between these sequences is 13.3%. The corrected rate of synonymous substitutions is 0.398 at 330.5 possible synonymous sites. The corrected rate of nonsynonymous substitutions is 0.068 at 1148.5 possible nonsynonymous sites. The number of amino acid differences is 64 of a total of 494, or 12.9%.

Three regions of homology to putative Amylase genes could be detected by hybridization of clone pFA4 (containing Amyl) to Southern blots of genomic DNA digests from strains homozygous for Standard, Treeline and Santa Cruz gene arrangements (Figure 4). For each chromosome, the three homologous regions were at the sites of the three Amylase genes in the clones, which were derived from a Standard chromosome. The Santa Cruz chromosome has a 1.6-kb deletion in the fragment where Amy3 is located, and Treeline has I .6-kb deletions in each of the two fragments where Amy2 and Amy3 are located. Hybridiza- tion to the fragments carrying the deletions was faint but distinguishable. No more than three Amylase genes were detected by hybridization to pFA4 under conditions of moderate stringency (data not shown).

DISCUSSION

Our sequence data indicate that the three amylase homologous regions from a Drosophila pseudoobscura chromosome carrying the Standard gene arrangement contain the complete coding sequence for an Amylase gene. Using the sequence data for the three genes, we can infer the evolutionary history of the duplication events and the history of the nucleotide sequences themselves.

Evolution of the amylase duplications: The evolutionary relationships that we infer from the coding sequence data for the amylase duplications are presented in Figure 5. There are two pieces of evidence that indicate that the second duplication involved Amy2 and Amy3, but did not involve Amyl. First, while Amy2 and Amy3 share only 100 bases of homology to the 5' and 3' flanking sequences of Amyl, these two genes share more than 400 bases outside of the area of homology to the Amyl flanking sequences. Second, there are 3.9% nucleotide differences between the homologous flanking sequences of Amy2 and Amy3, us. 7.0% divergence between Amyl and Amy3 and 9% between Amyl and Amy2.

The amylase duplications apparently predate the split of D. pseudoobscura and its sibling species D. miranda. D. miranda has two Amylase genes in the same orientation and location as Amyl and Amy2 in D. pseudoobscura (DOANE et al. 1987; AQUADRO et al. 1990). The D. melanogaster Amylase gene also hybrid- izes to a third region in D. miranda, represented by a fragment of approximately 1.0 kb. This third region maps to the location of Amy3 in D. pseudoobscura (DOANE et al. 1987), but has a 0.8-kb deletion relative to the D. pseudoobscura phage clones. In addition, the nucleotide sequence divergence between these two species, as calculated from restriction site variation over more than 20 kb of flanking and coding sequence, is only 3.6% for the Amylase gene region (AQUADRO et al. 1990) and 4.0% for the Alcohol dehy-


TABLE 1

Nucleotide substitutions per site for coding and flanking sequences of Amyl, Amy2 and Amy3 from D. pseudoobscura

A my I A my2 Amy3

A my I - 0.47 f 0.18% 0.54 f 0.19%

:\m$? 9.0 f 3.0% - 0.47 f 0.18%

AmT3 7.0 & 3.0% 3.9 & 1 . 0 %

( 1 482) ( 1 482)

(99) ( I 482)

(99) (.5 H 0 ) -

Nuclrotidr suI>stitutiolls per site are presented as percent f stantlard error. Data for coding sequences are above the diagonal and data for flanking sequences are below it. Numbers in parentheses are the nunllxr of nucleotides compared.

TREELINE kbl SANTA CRUZ kbl STANDARD

- 7.0 -5.6

.- 3.7

2.1

FIGURE 4.-Autot;1diogr;1phs o f the hybridization of clone pFA4 containing Amyl I O genomic DNAs fronl strains made homozygous for Standard (ST), Santa Cruz (SC), and Trttline (TL) gene arrangements. Numbers indicate size of DNA fragments in kilobases, and kbl is the kilobase I;ldtler used a s ;I size reference.

Amy.p Amyl Amy2 Amy3

D melanogasler D. pseudoobscura

FIGURE 5.--lnferred evolutionary relationships o f the four amylase sequences shown in Figure 3. Branch lengths are not propor- tioned to distances. Nunlbers represent average numbers o f substitutions between the Amyl and D. melanogaster Amy-p'. R = nonsy- nonytnous substitutions. S = synonymous substitutions, and R* = nonsynonymous substitutions creating a stop codon.

drogenase gene region (SCHAEFFER, AQUADRO and AN- DERSON 1987). The homologous flanking sequences of Amyl and Amy2, however, have diverged at 9.0% of their nucleotides, indicating that the duplication is perhaps twice as old as the divergence of D. miranda and D. pseudoobscura. The 3.9% divergence in the flanking sequences of Amy2 and Amy3 indicates that the second duplication occurred at about the time that D. miranda and D. pseudoobscura diverged.

If it is true that the three Amylase genes existed

prior to the divergence of D. miranda and D. pseudoobscura, then it is also true that the three genes existed in the ancestor of the extant gene arrangements of D. pseudoobscura. Hybridization of pFA4 to DNA fragments which carry 1.6-kb deletions relative to the phage clones is evidence that remnants of Amylase genes or their flanking sequences may exist in Treeline and Santa Cruz chromosomes. The gene arrangements, therefore, may preserve the history of the loss, rather than the gain, of the members of the Amylase multigene family.

Evolution of the amylase nucleotide sequences: Eleven nucleotide substitutions have occurred among the three D. pseudoobscura amylase coding sequences. We have used a cladistic analysis, with the D. melanogaster gene (Amy-p') as the outgroup, to allocate these substitutions to the different branches of the tree depicted in Figure 5 . Four of the substitutions are unique to Amy3 and are placed on the Amy3 branch after the second duplication event. Three of the substitutions are unique to Amy2 and are placed on the Amy2 branch after the second duplication. Four of the substitutions are unique to Amyl. By comparing the D. pseudoobscura amylase sequences to the D. melanogaster amylase sequence, we can attribute each substitution to either the Amyl branch or the A m y 2 3 branch prior to the second duplication. When Amyl and the Amy-p' share the same base, the substitution is placed on the Amy2-3 branch. When the D. melanogaster gene, Amy2, and Amy3 all share the same base, the substitution is placed on the Amyl branch. By this process, one substitution is attributed to the Amyl branch and three substitutions are attributed to the Amy2-3 branch. There is one site (#1320) where Amyl and Amy3 share the same base, and Amy2 and the D. melanogaster sequence share another base. This pattern could be due to convergent evolution or to a recent gene conversion event between the 3' ends of Amyl and Amy3.

As shown in Figure 5 , the distribution of nucleotide substitutions among the three genes is not equal. Amyl appears to be more functionally constrained than the other two genes because Amyl has only one nucleotide substitution relative to the other two genes. Interest- ingly, this substitution is nonsynonymous. T w o of the three substitutions that occurred prior to the divergence of Amy2 and Amy3 are synonymous, suggesting that the antecedent gene also may have been functionally constrained. The predominance of nonsynonymous substitutions in Amy2 and Amy3 after their divergence suggests that they are no longer functionally constrained.

The low level of divergence among the coding regions of the three genes is very different from the divergence among their flanking sequences. The divergence in the flanking sequences of Amy2 and Amy3


is eight times greater than the divergence in their coding regions (3.9% us. 0.5%). The estimated divergence between Amyl and Amy2, and between Amyl and Amy3, is 0.5% in the coding region and 9.0% and 7.0%, respectively, in their flanking sequences. From these differences, we suggest that either Amy2 and Amy3 have been functionally constrained in their coding regions until quite recently in their evolutionary history, or else recent gene conversions have occurred which included only the coding sequences of the genes. The evidence for gene conversion in the Amy- lase genes of D. pseudoobscura does not seem as com- pelling as the evidence for gene conversion in D. erecta (PAYANT et al. 1988).

Prior to the molecular cloning of the Amylase gene region, there was no evidence from acrylamide gel electrophoresis for the duplication of amylase in D. pseudoobscura (POWELL 1979). All three genes have CAAT and TATA control sequences in appropriate positions for initiating transcription. Immediately up- stream of the TATA box, however, the homology between Amyl and the other two sequences ends. The flanking sequences of Amy2 and Amy3 may not contain other sequences that are important for the expression of significant levels of amylase.

Amy2 and Amy3 may also be inactivated due to mutations within their coding sequences. All four substitutions in the Amy3 gene relative to Amyl and Amy2 are nonsynonymous. One of the nonsynonymous changes (#474) produces a TAG stop codon at amino acid 157, thus shortening the protein to 3 1.6% of its normal length. Three of the six substitutions in Amy2 relative to Amyl are replacement substitutions. The amino acid changes do not occur in regions which are apparently important for amylase activity (NAKA- JIMA, IMANAKA and AIBA 1986).

Microinjection of the Amy3 gene into eggs of an Amylase null strain of D. melanogaster failed to produce a functional amylase protein, whereas protein was produced when Amyl and Amy2 were injected under identical conditions (HAWLEY et al. 1990). Although we cannot formally exclude the possibility that Amy3 is expressed at some life stage in tissues or at levels which were undetectable in our microinjection experiments, it seems reasonable to regard Amy3 as a pseudogene. In the microinjection experiments, the Amy2 enzyme activity was barely detectable, due to either reduced transcription, translation or protein function.

The Amyl gene appears to be strongly constrained at synonymous sites relative to other genes compared between the melanogaster and obscura species groups. Table 2 presents the expected rate of nucleotide substitutions per site at synonymous and nonsynonymous sites for five loci: (Amylase (Amy); Alcohol dehydrogenase (Adh) (SCHAEFFER and AQUADRO 1987); the ORF that is 3' to Adh (SCHAEFFER and AQUADRO 1987); heat

TABLE 2

Expected number of nucleotide substitutions per site in comparisons between pairs of Drosophila species

Substitutionsb

Locus Species" Nonsynotiymous Synonymous

Amy ps, me 6.8 k 0.80 39.8 k 4.3 Ad h ps, ma 4.9 k 0.90 70.2 k 9.0* 3'ORF ps, ma 2 .8 k 0.60* >100.0 k 21.0* rp4Y su, me 2 .5 k 0.90* 67.0 k 12.4 hspCl2 ps, me 1.6 _t 0.40* 64.5 k 7.6*

a ps = D. pseudoobscura, me = D. melanogaster, ma = D. mauritiana (a sibling species o f D. melanogaster), su = D. subobscura (a European member ofthe obscura group). ' Expected number of nucleotide substitutions are presented as

percent 2 standard error. References are in the text. * Difference from Amy value is statistically significant (P < 0.05).

shock protein 86 (hsp86) (BLACKMAN and MESELSON 1986); and ribosomal protein 4 9 ( r p 4 9 ) (AGUADE 1988). The common ancestor for the comparisons is the ancestor of D. pseudoobscura and D. melanogaster. The comparisons in Table 2 show that the Amylase genes have significantly fewer silent substitutions than the other genes, except rp49.

Amylase also shows a high degree of codon bias as calculated by the method of SHIELDS et al. (1988). The inverse relation between silent substitution rate and codon bias has led several authors to infer the presence of selection against silent substitutions (GRANTHAM et al. 1980, 1981 ; GOUY and GAUTIER, 1982). In yeast, genes that show high codon bias are also highly expressed (SHARP, TUOHY and MOSURSKI 1986). Strong codon bias in highly expressed genes is also found in Drosophila, although the relationship of expressivity to codon bias may be more complicated in multicellular eukaryotes (SHIELDS et al. 1988). Expression of Amyl in D. pseudoobscura is repressed by the presence of glucose in the diet (D. A. HICKEY, personal communication). In its derepressed state, Amyl is a highly expressed gene. In D. melanogaster, amylase mRNA is >1% of the total polyA mRNA in a derepressed fly (BENKEL and HICKEY 1986). Thus the low rate of silent substitutions at the Amylase locus may be due to selection related to strong codon bias and the high level of Amylase expression.

In strong contrast to synonymous substitutions, the frequency of nonsynonymous substitutions in amylase is higher than in any of the other genes sequenced in D. pseudoobscura and the melanogaster subgroup (Table 2). The high frequency of amino acid substitutions is also associated with a high level of intraspecific polymorphism. There are eight amylase electromorphs in D. melanogaster (DOANE et al. 1983), at least five electromorphs in D. pseudoobscura (POWELL 1979; NORMAN and PRAKASH 1980), and at least three electromorphs in D. miranda (PRAKASH 1977). Within the electromorphs there may also be additional polymor-


phisms. BOER and HICKEY (1986) showed that the Amy-p' allozymes from two strains of D. melanogaster differ at two amino acids. This level of polymorphism contrasts with Drosophila Adh, which has very little amino acid polymorphism (KREITMAN 1983; S. W. SCHAEFFER, unpublished results). The sequence data indicate a low degree of constraint of the amylase protein sequence relative to some other Drosophila genes. The divergence between insect amylase and mammalian amylase, however, is quite low (HICKEY et al. 1988) and is on the order of the divergence between mammalian and reptilian genes (DICKERSON 197 1).

We conclude by noting that there are few pseudogenes in Drosophila compared to the large number of pseudogenes in mammals. Pseudogenes for a tRNA (SHARP et al. 1981) and a cuticle protein (SNYDER et al. 1982) in D. melanogaster, and for Adh in D. mulleri (FISCHER and MANIATIS 1985) and D. mojavensis (AT- KINSON et al. 1988) have been reported. The presence of the Amylase pseudogene Amy3 is the first documen- tation of a pseudogene in D. pseudoobscura.

We wish to acknowledge CHARLES LANGLEY, BURKE JUDD, and other staff members of the Laboratory of Genetics at the National Institutes of E:nvironmental Health Sciences for their kindness in providing encouragement and support for the initiation of this research. We wish to tlxrnk JONATHAN ARNOLD, JOHN AVISE, WIN- IFRED DOANE, DONAL HICKEY, %rKVE KARL, JOHN MCDONALD, RICHARD MEAGHER, STEVE SCHAEFFER, KATHY SPINDLER and CHUNG-I. WU for critically reading the manuscript. WEN-HSIUNG LI kindly provided h i s computer program for estimating nucleotide substitutions. This research was supported by National Science Foundation grant BSR-8516188, to W.W.A.; U.S. Public Health Service grant GM-36431 to C.F.A.; and U.S. Public Health Service training grant in genetics 5-T32-GM07103-9 to C.J.B.

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Communicating editor: C. C. LAURIE

j. f. aquadrot and wyatt w. anderson**'sequence, or that amy2 has recently undergone a gene...

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