the structure of the adh locus of drosophila mettleri - molecular

The Structure of the Adh locus of Drosophila mettleri: An Intermediate in the Evolution of the Adh locus in the Repleta Group of Drosophila1

Jungsun Yum, William T. Starmer, and David T. Sullivan Department of Biology, Syracuse University

Members of species of the mulleri and hydei subgroups of the repleta group of Drosophila have duplicate Adh genes. The Adh regions of D. mojavensis, D. mulleri, and D. hydei contain three genes-a pseudogene, Adh-2, and Adh-l--arranged 5’ to 3’. To understand the evolution of the triplicate Adh structure, we have cloned and sequenced the Adh locus of D. mettleri. This region consists of a 5 ’ pseudogene and a 3’ functional Adh gene. On the basis of the structure and nucleotide sequence comparisons of Adh genes of D. mettleri and other species, we propose that an initial duplication of the ancestral Adh gene generated two Adh genes arranged in tandem. The more 5’ Adh gene became a pseudogene, while the more 3’ gene remained functional through all the developmental stages. A second duplication of this 3’ gene resulted in Adh regions with three genes-a pseudogene, Adh-2, and Adh-I.

Introduction

Species of the repleta group of the genus Drosophila have been shown to contain isozymes of alcohol dehydrogenase (ADH), (Batterham et al. 1982; Oakeshott et al. 1982), suggesting that a duplication of the Adh locus occurred during the evolution of these species. Analysis of the hucleotide sequence of the Adh regions of D. mulleri (Fischer and Maniatis 1985), D. mojavensis (Atkinson et al. 1988), and D. hydei (Menotti-Raymond et al. 199 1) confirmed the presence of two functional genes and also revealed the presence of a pseudogene at the locus. The three genes are tandemly arranged and span -9 kb of DNA. Therefore, it is likely that at least two independent duplication events occurred during the evolution of the Adh genes in the repleta group. On the basis of the amount of divergence between the Adh genes of D. mojavensis and between the D. mojavt+s and D. mulleri Adh genes, Atkinson et al. ( 1988) proposed a model for the evolution of the Adh locus. The model assumes that the ancestral Adh gene had a structure similar to that found in both D. melanogaster (Benyajati et al. 1983) and all other Drosophila species which have been analyzed. This structure consists of a coding region interrupted by two small introns and two promoter regions, one close to the translation start and the other located several hundred nucleotides upstream and whose use requires splicing of an intron from the 5’ un- translated region of the transcript. This upstream promoter is designated a distal promoter and contains an Adh distal TATA box, TATTTAA. The other one, a proximal promoter, contains an Adh proximal TATA box, TATAAATA. The distal promoter

1. Key words: Drosophila, Adh, gene duplication.

Address for correspondence and reprints: David T. Sullivan, Department of Biology, Syracuse University, 130 College Place, Syracuse, New York 13244.

Mol. Biol. Evol. 8(6):857-867. 1991. 0 1991 by The University of Chicago. All rights reserved. 07374038/91/0806-008$02.00

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is used for A& expression in adults, while the proximal promoter is used for larval expression.

The phylogenetic distribution of the Adh duplication has been studied by ex- amining species for the presence of ADH isozymes (Oakeshott et al. 1982; Batterham et al. 1984), by analysis of the developmental profile of ADH forms and by Southern blot analysis of genomic DNAs (D. T. Sullivan, unpublished data). No species outside the repleta group shows any evidence of having an Adh duplication. The repleta group has four subgroups, hydei, mercatorum, repleta, and mulleri (Wasserman 1982). Each species of the hydei subgroup has ADH isozymes, and the sequence of the D. hydei Adh locus reveals two coding genes and a pseudogene ( Menotti-Raymond et al. 199 1) . Species of the mercatorum and repleta subgroups do not contain ADH isozymes and are currently being analyzed at the DNA level. The mulleri subgroup is composed of several species complexes, the largest being the mulleri species complex. All species of the mulleri species complex have two coding genes whose pattern of expression is similar to that reported for D. mulleri (Fischer and Maniatis 1985 ) and D. mojavensis (Batterham et al. 1983a; 1983b). However, members of other species complexes of the mulleri subgroup have ADH patterns that differ from those of D. mulleri and D. mojavensis. Some of these-e.g., members of the anceps and meridiana species complexes-have ADH expression patterns that resemble those of D. hydei more than they resemble those of D. mulleri. The principal difference between these species complexes is that the mulleri species complex first expresses Adh-2 during the late third instar, while species of the hydei subgroup, as well as the meridiana and anceps species complexes, express Adh-2 throughout development. Members of the eremophila and stalkeri species complexes have no ADH isozymes. Therefore, it is likely that one Adh duplication originally occurred during the early evolution of the repleta group. Sub- sequently, several events have occurred in different lineages within the repleta group to result in extant species having varied structure and expression of their Adh genes.

In an attempt to identify, in the evolution of the Adh locus, intermediate predicted by the model of Atkinson et al. ( 1988), we have focused on species in complexes of the mulleri subgroup that, with respect to the structure and expression of the Adh locus, appear to differ from other species in the mulleri complex. Here we report the sequence of the Adh region of D. mettleri, a member of the eremophila species complex of the mulleri subgroup. Neither D. mettleri nor its close relatives-D. micromettleri and D. eremophila, the other species of the eremophila complex-have multiple forms of ADH. The sequence of the Adh region from D. mettleri reveals only two tandemly arranged Adh sequences. One of these is a pseudogene, while the other has coding potential. Comparisons of the sequence divergence among the Adh genes of the repleta group suggest that the locus structure found in D. mettleri represents an intermediate stage in the evolution of the Adh region and that the presently understood relationships within and between the mulleri and hydei subgroups may require modification.

Material and Methods

A genomic library was constructed using DNA prepared from adult Drosophila mettleri and the vector EMBL-4 (Frischauf et al. 1983). DNA was partially digested with MboZ and 15-20-kb fragments were isolated and ligated to BamHI-digested EMBL4. Recombinant molecules were packaged using lambda packaging extracts obtained from Amersham. Plaques were screened by the method of Benton and Davis ( 1977 ) by using as a probe a fragment from the plasmid pLM 19 (Mills et al. 1986))

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Adh Locus of Drosophila mettleri 859

which contains the D. melunoguster Adh gene. Three clones that hybridized to an Adh probe were isolated from the genomic library. One clone, A&-mtt 1, which appeared to contain the entire A& region, was analyzed. The restriction map of this clone is shown in figure 1. Restriction fragments-2.3-kb SmaI-HindIII, 1.75kb HindIII- EcoRI, and 1.8-kb EcoRI-EcoRI-were ligated into m 13 sequencing vectors mp 18 and mp19 (Norrander et al. 1983). The nucleotide sequence of the 4.6-kb region, indicated in figure 1, was determined.

Nucleotide sequences were determined using the chain-termination method with “S-ATP (Hong 1982) and buffer gradient gels (Biggin et al. 1983). Gels were read and sequences were determined using a digitizer and computer programs from DNAS- TAR (Madison, WI).

Sequence alignment of Adh genes between and within species was performed by the algorithm devised by Wilbur and Lipman ( 1983 ). Exon sequences were compared by the method of Li et al. ( 1985), which corrects for multiple fixations at a site. A dendrogram was generated by the Fitch and Margoliash method ( 1967), to infer phylogenies from sequences by using the KITSCH program of PHYLIP.

Results

Analysis of genomic DNA by Southern blots reveals a set of Adh hybridizing fragments; for example, in the ClaI digestion shown in figure 2 two bands, 2.5 kb and 4.7 kb, hybridized to the A& probe. These and all other fragments are the same as those included in the clone, A&-mtt 1, as indicated in figure 1. Since no additional fragments were found, we concluded that this clone contains the entire Adh locus of Drosophila mettleri.

Figure 3 shows the nucleotide sequence and the conceptual translation of the single Adh gene with coding potential. The region contains a second Adh-like gene. This 5’ gene has both a putative translation start codon at position 862 and sequences similar to the expected Adh splice sites at positions 957, 10 11, 14 16, and 1480. However, this gene must be a pseudogene, because there is a series of alterations to the translation reading frame that are generated by the addition of nucleotides at positions 865 and 866 and by the deletion of a nucleotide at position 1386.

Downstream of the pseudogene, the second Adh gene is capable of being translated. Its start codon is at position 2990, and in all respects it is a typical Adh gene. The Adh coding gene has a sequence identical to a TATA box of an Adh proximal promoter, TATAAATA, beginning at 2920. The Adh coding gene does not have a sequence similar to a distal Adh TATA box. However, at position 654 there is a sequence, TATTTAA, which is identical to a distal promoter TATA box. In all repletu-group Drosophila Adh regions that have been sequenced, a distal promoter-like region has been found upstream of the pseudogene and has been interpreted to be the remnant

7 S HFN CE P NE B C H B

1Kb

FIG. 1 .-Restriction map of Drosophila mettleri Adh locus cloned into EMBL4. Shaded boxes indicate areas that hybridize to the Adh coding fragment of D. melanogaster. The dark line below the map represents the extent of genomic DNA that was sequenced. Restriction enzymes used are BgZII (B), C/a1 (C), EcoRI (E), Hind111 (H), NcoI (N), PstI (P), and SmaI (S).

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Table 1 Nucleotide Substitution Comparisons of Drosophila mettleri Adh Exons

SPECIES AND GENE

D. mettleri: Y Adh

D. mojavensis: Y Adh-2 Adh-I

D. hydei: Y Adh-2 . . . . . . Adh-I ..__...

Adh-Y Adh

0.575(0.078)/0.082(0.055) 0.575(0.078)/0.082(0.055)

0.631(0.083)/0.068(0.043) 0.651(0.086)/0.093(0.074) 0.621(0.083)/0.105(0.068)

1.096(0.158)/0.082(0.071) 0.581(0.079)/0.084(0.059) 0.583(0.080)/0.092(0.064)

0.690(0.092)/0.105(0.066) 0.470(0.065)/0.043(0.042) 0.506(0.069)/0.052(0.039)

0.940(0.127)/0.101(0.064) 0.408(0.059)/0.0 18(0.020) 0.435(0.061)/0.027(0.029)

NOTE.-The number of sites compared was 765. ’ SE = standard error, calculated as the square root of the variances estimated according to eq. (20)

(for Ks) and (22) (for K,,) of Li et al. (1985).

We also tested the branch lengths between the nodes leading to the two D. mojavensis genes and the node connecting the monophyletic group (D. hydei Adh-1, D. hydei Adh-2, and D. mettleri Adh ) by a likelihood-ratio test ( Felsenstein 1988 ) . The branch was significantly positive (P < 0.01). Thus, both the bootstrap and maximum-likelihood evaluation support the placement of D. mettleri Adh in the lineage leading to D. hydei after the D. mojavensis lineage diverged. Therefore, it is likely that the lineage leading to D. hydei contained species that had not yet had a second duplication at the Adh locus.

Discussion

Two discoveries-( a) that the Adh locus of Drosophila mettleri has two genes and (b) the position that these two genes occupy on a gene tree-offer important clues to the evolutionaryhistory of the Adh duplication in the repleta group of Drosophila. The initial model proposed by Atkinson et al. ( 1988) postulated that two duplication events occurred, leading to the structure of the Adh loci now found in D. mulleri and D. mojavensis. Drosophila mettleri has two Adh genes and appears to represent a species derived from a lineage that diverged following the first duplication but prior to the second duplication event. The gene tree based on the sequence of the Adh genes of the repleta-group Drosophila locates D. mettleri at a point consistent with this species having preserved an intermediate Adh locus structure. However, since the Adh gene of D. mettleri branches from the tree later than does the lineage to D. mojavensis and earlier than does the lineage to D. hydei, and since both D. hydei and D. mojavensis have three Adh genes, we are required to postulate that the second duplication events in their lineages are independent. An analysis of the orthologous and parologous sequence divergence between the Adh genes of D. hydei and D. mojavensis (Menotti- Raymond et al. 199 1) came to the same conclusion. Since the most plausible mech- anism for the generation of additional copies of a gene which is tandemly repeated is by means of unequal crossing-over, and since unequal crossing-over does not appear

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Table 2 Comparison of Nucleotide Similarity Distance among Drosophila Species

NUCLEOTIDE SIMILARITY DISTANCE IN [ 1 - (TO similarity/lOO)]

SPECIES AND GENE

D. hydei D. mojavensis D. mettleri

Adh-I Adh-2 Y Adh-I Adh-2 Y Adh Y

D. hydei: Adh-I Adh-2 Y

D. mojavensis: Adh-1 Adh-2 Y

D. mettleri: Adh Y

0.027 0.189 0.116 0.111 0.172 0.101 0.162 0.027 0.189 0.118 0.110 0.165 0.090 0.157 0.199 0.199 0.207 0.202 0.199 0.204 0.199

0.118 0.118 0.199 0.067 0.157 0.128 0.175 0.118 0.118 0.199 0.067 0.155 0.118 0.171 0.165 0.165 0.198 0.165 0.165 0.184 0.152

0.094 0.094 0.199 0.118 0.118 0.165 0.156 0.165 0.165 0.198 0.165 0.165 0.152 0.165

Nom.-Above the diagonal are the observed dissimilarity distances. Dissimilarity is the fraction of sites that differ when gapped positions are excluded. Below the diagonal are the distances based on the output of the KITSCH version of the Fitch and Margoliash (1967) method.

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0.099

0.076 0.0% 1

0.076

D. hydei Y

D. mettleri Y

D. mojavensis Y

D. hydei 2

D. hydei 1

D. mettleri

D. mojavemis 2

D. mojavensis I

FIG. 4.-Dendrogram ofAdh sequences of D. mettleri, D. mojavensis, and D. hydei, based on dissimilarity shown in table 2. The dendrogram was constructed by the Fitch and Margoliash ( 1967) method using the KITSCH program of PHYLIP. The consensus tree constructed using the bootstrap program of PHYLIP was identical in topology to the dendrogram. The number at each node shows the number of times the set of those descendent taxa was found in 1,000 bootstrap replicates. Y = pseudogene; 2 = Adh-2; and 1 = Adh-1.

to be rare, it is not surprising that a number of independent duplications could follow the initial duplication event.

Atkinson et al. ( 1988) noticed an unexpectedly high degree of sequence conser- vation between the pseudogene of D. mulleri and that of D. mojavensis, and this was taken to indicate that the pseudogene was a coding gene during a substantial fraction of its evolutionary history in the repleta group. However, the pseudogenes of D. mulleri, D. mojavensis, and D. hydei all have a common deletion of the fourth nucleotide in what would be the second codon, suggesting that the pseudogene was translationally inactivated early in the lineage, before the divergence leading to the mulleri and hydei subgroups. Since the pseudogene of D. mettleri has several nucleotide differences in what would be codon 2, it is not possible to be certain that the same nucleotide is also deleted as in the other species. The sequence that is found in this region is consistent with this possibility.

Since the pseudogene was translationally inactivated before the second duplication, a constraint has been placed both on the pattern of expression and on evolution of the active Adh genes. Habitats of repleta-group species include rots in stems, cladodes, and fruit of cactus. D. mettleri uses as a breeding site the pools of efflux which collect, in the soil, from rots of giant columnar cacti (e.g., saguaro). These habitats have high concentrations of many organic volatiles, including alcohols (Fogleman and Heed 1989). Consequently, it is unlikely that a species of Drosophila that did not have ADH at all stages of development could persist. Therefore, it is not surprising that the single Adh gene is expressed at all stages of development in D. mettleri. However, the promoter region of the Adh coding gene of D. mettleri is homologous to a proximal type Adh promoter which is usually expressed only during larval stages. It is likely that adult Adh expression is accomplished through the action of an enhancer located between basepair positions 300 and 500, a region which is -450 bp 5’ of the beginning of the pseudogene. This enhancer has been shown to be responsible for the expression of the Adh-2 gene in adults of D. mulleri (Fischer and Maniatis 1986) and D. mojavensis (Bayer 1989 ) . This enhancer is also homologous to a region which is utilized to promote adult expression from the distal promoter of D. melanogaster (Ayer and Benya- jati 1990).

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Adh Locus of Drosophila mettleri 865

On the basis of the above evidence, we propose a modified version of the model for the series of steps in the evolution of the A&I genes in the repletu group (fig. 5 ). This model assumes that the ancestral A&r gene had a structure which was similar to the A& gene currently found in D. melanugaster, with a proximal promoter used for expression in larval stages and a distal promoter used for expression in adults (fig. 5A). The model proposes that an initial duplication of the ancestral A& gene generated a locus with two genes. The two genes are arranged in tandem. The more 3’ gene has a single promoter, while the more 5’ gene maintains the ancestral arrangement (fig. 5B). Subsequently, the 5’ gene has become a pseudogene, and this is accompanied by a deletion of -600 nucleotides which extends from a point downstream of the distal promoter through the 5’ intron and into the proximal region. After the translational inactivation of the 5’ gene, the downstream gene must have been expressed in both larvae and adults, since ADH activity is likely to be essential at all stages. The promoter labeled “P*,” which is homologous to a proximal promoter, is able to direct larval and adult Ad/z expression that is probably facilitated by enhancers (fig. 5C). Second duplications of the 3’ gene resulted in regions with three genes-one pseudogene and two coding genes. One of these coding genes, A&-I, has a proximal promoter and is expressed in larvae; the other, A&-2, retains a P* promoter and is expressed in larvae and adults. Finally, we propose, mutations in P* served to generate P* *, such that larval expression from this promoter is no longer possible. Figure 5 represents species which have an Adh locus structure and expression patterns of the indicated loci.

D

Y-Adh Adh-2 Adh-I

FIG. 5.-Model of evolution of Adh locus of Drosophila. A, Ancestral gene with dual promoter structure. B, Adh locus after initial duplication-5 ancestral gene and 3’ gene with single proximal promoter. C, Adh locus as result of inactivation of 5’ gene-5’ pseudogene and 3’ functional Adh gene with P* promoter. The Adh locus of D. mettleri represents this structure, and the 3’ gene with P* promoter is expressed in both larval and adult stages. D, Adh locus after second duplication-pseudogene, Adh-2, and Adh-1, arranged 5’ to 3’. The Adh loci of D. mojavensis and D. hydei contain this structure. The Adh-I with a proximal promoter is expressed in larvae. The Adh-2 of D. mojavensis with P* * promoter is expressed only in adults, while the Adh-2 of D. hydei with P* promoter is expressed in both larval and adult stages.

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Acknowledgment

This work was supported by NIH grant GM 31857 to D.T.S.

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MARTIN KREITMAN, reviewing editor

Received January 18, 199 1; revision received April 15, 199 1

Accepted April 26, 199 1

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the structure of the adh locus of drosophila mettleri - molecular

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