*Institute of Molecular Biology and Biotechnology, Research Center of Crete, Foundation for Research and Technology, Heraklion, Crete, Greece; and
Division of Medical Sciences, University of Crete Medical School, Heraklion, Crete, Greece
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Abstract |
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Introduction |
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From an evolutionary perspective, no other gene among the Diptera has been studied as extensively as the Adh gene of Drosophila. Duplications of a preexisting locus have occurred in the Adh region, often with subsequent functional differentiation (Powell 1997
). The origin of the Adh gene in Drosophila may be partially explained by the observation that it is fairly similar to another Drosophila gene coding for the fat body protein P6 (Rat, Veuille, and Lepesant 1991
), which has the features of the short-chain dehydrogenases. The proteins of this group, nevertheless, are too distantly related to Drosophila ADH to provide any information on how and when it acquired its catalytic activity. However, it has recently been suggested that Drosophila ADH coevolved with fleshy fruits of angiosperms and fermenting yeast (Ashburner 1998
).
The acquisition of nucleotide sequence information from the Adh gene in a number of Drosophila species has increased substantially in recent years, allowing the interpretation of the genetic events which occurred over evolutionary time and the establishment of phylogenetic relationships between species and divergence times within this family (Sullivan, Atkinson, and Starmer 1990
; Jeffs, Holmes, and Ashburner 1994
; Russo, Takezaki, and Nei 1995
). In addition, the question of whether the Drosophila ADH protein differentiation during evolution is associated with speciation through adaptation to new feeding niches or if it is an outcome of the time separation between species has been approached by Atrian et al. (1998)
, who analyzed the amino acid variability of 57 ADH sequences.
However, only one non-Drosophila Adh gene has been sequenced so far, that of the flesh fly Sarcophaga peregrina (Horio, Kubo, and Natori 1996
), a higher dipteran quite divergent from Drosophila. We decided to extend the molecular study of ADH to two non-Drosophila fruit flies, the Mediterranean fruit fly (medfly) Ceratitis capitata, a major agricultural pest in tropical and subtropical regions, and the olive fly Bactrocera oleae, the major pest of olives. Both species belong to the family Tephritidae, which is estimated to have diverged from the family Drosophilidae between 80 and 100 MYA, an estimate based on immunological and molecular data (Beverley and Wilson 1984
; Kwiatowski et al. 1994
). In addition to being interesting in evolutionary studies, the fruit fly Adh genes are also of practical interest because they can be used as selectable markers in genetic engineering schemes for biological control (Robinson, Savakis, and Louis 1988
).
Ceratitis capitata has two ADH isozymes, ADH-1 and ADH-2, which are expressed in different tissues (Gasperi et al. 1992
) and are encoded by a pair of tightly linked genes (Malacrida et al. 1992
). The two isozymes have been purified to homogeneity, and, although they also belong to the short-chain dehydrogenase family, they show no immunological cross-reactivity to Drosophila ADH (Gasperi et al. 1994
). However, they cross-react to olive fly ADH protein (Gasperi et al. 1994
), which has also been purified to homogeneity (Mazi et al. 1998b
).
Here, we report the cloning and sequencing of cDNAs encoding the two medfly ADH isozymes and the tissue distribution of the corresponding mRNAs. We also report the sequence of an ADH cDNA of the olive fly B. oleae, which was cloned by functional complementation in yeast (Benos et al. 2000
). We found that, contrary to expectation, the flesh fly ADH was more closely related to the tephritid than to the drosophilid ADH sequences. We hypothesize that the Drosophila Adh gene may not be orthologous with the other known Adh genes of higher diptera.
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Materials and Methods |
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Nucleic Acid Preparation and cDNA Library Construction
Medfly genomic DNA and total RNA were prepared essentially as described previously (Holmes and Bonner 1973
). Poly(A)+ RNA was separated from total RNA as described in Holmes and Bonner (1973)
using an oligo(dT) cellulose column from New England Biolabs. cDNA synthesis and library construction were done from poly(A)+ RNA from third-instar larvae using the lambdaZap-cDNA synthesis kit from Stratagene. Approximately 1 x 106 primary clones, more than 80% containing cDNA inserts, were recovered. All subsequent work was done with an amplified library.
Generation of an ADH-1Specific cDNA Probe by Polymerase Chain Reaction
Medfly third-instar larva cDNA was synthesized from 20 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (Stratagene) and oligo(dT) primer (Pharmacia) as described by the manufacturer (Stratagene). cDNA was amplified by the polymerase chain reaction (PCR) (30 cycles of 94°C for 1 min, 47°C for 1 min, and 72°C for 30 s). The three degenerate primers that were used (A, 5'-ta(tc)(tc)tNatgaa(tc)aa(tc)ga(tc)(tc)tNgc-3'; B, 5'-atNgc(tc)tcNgg(tc)tt(ag)tg-3'; C, 5'-taNgtNgg(tc)tccca(ag)taNac-3') were derived from reverse translation of the ADH-1 peptides (see Results).
Medfly cDNA Library Screening
Phages (2 x 105 PFU) were screened as described in Green and Struhl (1988)
, using 32P-labeled DNA as probe. The positive clones were isolated, and pBluescript phagemids containing the insert were excised in vivo using the R408 helper phage, as described in the ZAP-cDNATM synthesis kit manual from Stratagene.
Cloning of the 5' End of the ADH-1 cDNA
The 5' end of the ADH-1 cDNA was amplified directly from the lambdaZap cDNA library by PCR using an antisense primer specific for ADH-1 (5'-ACTTGCAGGCTTCATAAG-3') and a vector primer (the -20M13 universal primer). A ladder of fragment sizes was obtained. The longest fragment (approximately 230 bp) was cloned into Bluescript, and two clones were sequenced.
S1 Protection
A mixture of two oligonucleotides of different lengths, A1 (5'-CATATTTTATGCACTACTTTTTCGA-TTTGAACACTCACAGtctca-3') and A2 (5'-CTCAG-TCTCTATATATATTTACTTTCGTTCGCTAATGTGT-TTTTCTGCCTGtctca-3'), complementary to the untranslated region of ADH-1 and ADH-2, respectively, were used as probes for a quantitative analysis of the two transcripts according to the protocol described in Sambrook, Fritsch, and Maniatis (1989)
. Each of the two oligonucleotides contained a 5-nt-long tail at its 3' end (shown in lowercase) in order to distinguish the protected fragment from the probe. Total RNA (approximately 20 µg per reaction) was used in all experiments.
Cloning of an ADH cDNA from B. oleae by Complementation in Yeast
A cDNA library was constructed in the yeast expression vector pDB20 (Fikes et al. 1990
) from RNA isolated from B. oleae adult flies using the cDNA synthesis reagents provided by Stratagene (Lambda ZAP) according to the kit's instructions. The ADH-deficient yeast strain MC892-1C (MAT-alpha, adh1, adh2, adh3, ura3-52, trp1, leu2, his3), kindly provided by Dr. M. von Ciriacy-Wantrup, was used for complementation. Complementation was performed by growing transformed yeast cells on minimal plates (2% glucose, 0.7% yeast nitrogen base without amino acids, and 2% agar) with the addition of the appropriate amino acids for 3 days, followed by replica plating on plates containing, additionally, 0.5 ppm antimycin A. The method for cloning ADH coding sequences by complementation in yeast is described in detail in Benos et al. (2000)
.
DNA and Protein Sequence Analysis
DNA and protein sequences were analyzed using the GCG programs in the Wisconsin Package, version 9.0, (Genetics Computer Group [GCG], Madison, Wis.). Multiple alignments, tree computation and bootstrap analysis were performed with the program CLUSTAL W (Thompson, Higgins, and Gibson 1994
).
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Results |
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The cloned 700-bp cDNA fragment was used to screen a cDNA library from third-instar larvae. More than 40 different positive clones were isolated, and six of them were sequenced. All six clones had an open reading frame (ORF) of 258 codons, a 58-bp 5' untranslated region (UTR), and a 143-bp 3' UTR. These clones could be divided into two sequence classes, each probably representing a polymorphic allele of the gene: three clones carried seven synonymous substitutions relative to the other three (data not shown). Surprisingly, the nucleotide sequence of these clones was only about 80% identical to the ADH-1 probe used for screening, showing many nonsynonymous codon differences. This suggested that the cDNA clones recovered probably corresponded to ADH-2 rather than ADH-1 cDNAs, a result not totally unexpected, since ADH-2 is the most abundant of the two isozymes.
Because of the low abundance of ADH-1 cDNA clones, the missing 5' end of ADH-1 was amplified by PCR from pooled DNA of the entire cDNA library using a combination of an ADH-1specific primer and a primer corresponding to vector sequences. A full-length ADH-1 cDNA clone was recovered using this 5' end as probe and showed a 39-bp 5' untranslated sequence, an ORF encoding 257 amino acids, and a 3' UTR of 146 bp. The coding regions of the two genes were considerably divergent, with a total of 167 nucleotide differences (21.7%), of which 16% were synonymous substitutions and 5.7% resulted in amino acid replacements. The replacements accounted for 45 amino acid differences (17%) between the two protein sequences. Both protein sequences contained the "short-chain dehydrogenase" motif (PROSITE accession number PS00061; ADH_SHORT). The noncoding regions were considerably more divergent. It is notable, though, that the 3' UTRs of the two mRNAs were the same size (except for a 3-nt difference due to ADH-2 having one extra codon at the carboxy terminus). No significant similarities could be detected in the 5' UTRs.
In summary, we have shown that in the medfly, the two ADH isozymes are encoded by two similar genes, sharing approximately 83% amino acid sequence identity. The sequence similarity strongly supports the notion that these genes are products of a gene duplication. The relatively high degree of divergence between the two genes and the observation that most species of the family Tephritidae have two or three isoenzymes for ADH (Matioli et al. 1992
) would support the view that this duplication is a rather early event in the radiation of Tephritidae.
Tissue Distribution of the Medfly ADH mRNAs
Previous studies have shown that the two ADH isozymes in the medfly have nonoverlapping tissue distributions. ADH-1 is found only in body muscles, and ADH-2 is in all other tissues in which ADH activity has been detected, mostly in the midgut, the fat body, and the ovary (Gasperi et al. 1992
). Therefore, to confirm the identity of the two cDNA clones, the pattern of expression of the corresponding mRNAs was determined. Because the two mRNAs were very similar in length and in sequence, an S1 nuclease protection assay was employed to detect specific mRNAs, using probes specific for the divergent 5' UTRs of the two sequences. As shown in figure 1
, ADH-1 transcripts are present only in muscle, while ADH-2 is present in all other tissues examined, with the fat body showing the highest abundance and ovary the lowest. These data confirm the identity of the two ADH sequences. ADH-2 seems to be more similar to the Drosophila enzyme, considering its tissue distribution, while the unusual expression of Adh1 in the muscle raises a puzzle as to this isozyme function.
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This is also schematically represented in the phylogenetic tree of figure 2 (top), which is based on the same protein sequences. The human prostaglandin dehydrogenase gene was used to root this tree, as it was the closest noninsect "relative" to the short-chain dehydrogenases. The bootstrap analysis performed on these data was further supportive of this "paradoxical" evolution of the Adh gene in Diptera. In particular, three distinct clusters comprise this tree, all having bootstrap values of 1,000/1,000 (confidence 100%): one with the Adhr genes from the two most divergent Drosophila species, another with the Adh genes of the same Drosophila species, and a third with the Sarcophaga together with the tephritid genes. This conclusion was further supported by the pattern of indels in the aligned sequences. As shown in figure 3 , there are three internal indels in the aligned sequences, each involving one or two amino acids; they are located after positions 66, 168, and 193 of the D. melanogaster ADH sequence. The Sarcophaga and the tephritid ADHs share the same three indels, one of which is also in the ADH-related but not the ADH protein of Drosophila. This is additional evidence that the tephritid and Sarcophaga ADHs are more closely related to each other than to Drosophila ADH. The apparent lack of congruence between the tree based on the species and the tree based on protein phylogenies is illustrated in figure 2 (bottom).
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Discussion |
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We cloned and sequenced cDNAs for the two ADH isozymes of the medfly C. capitata and for one ADH of the olive fly B. oleae. These represent the only known fruit fly Adh genes cloned outside of the family Drosophilidae. Because of the unexpectedly high degree of sequence divergence (<40% identity at the amino acid level between the known Drosophila sequences and medfly ADH), cloning of the medfly genes had to rely on partial sequencing of purified ADH-1 protein (Gasperi et al. 1994
). For easier cloning of distantly related Adh genes, we have developed an efficient method based on complementation of ADH activity in yeast for the olive fly ADH. This method, which is presented elsewhere in detail (Benos et al. 2000
), relies on the inability of yeast mutants lacking ADH activity to grow under anaerobic conditions, and it may be of more general use for cloning ADH from diverse sources.
Apart the evolutionary implication of the nondrosophilid ADH system, the availability of tephritid Adh genes clears the way for their use as selectable genetic markers in pest control schemes based on genetic engineering (Robinson, Savakis, and Louis 1988
). In the olive fly, dramatic changes are observed in ADH allele frequencies when natural populations are introduced in the laboratory, and the ADH alleles involved differ in specific activity and enzymatic properties, suggesting that selection may be taking place (Mazi et al. 1998a
). Cloning of olive fly ADH clears the way for investigating this interesting adaptive phenomenon.
Adh Duplications in Tephritids
The sequences and tissue distribution of the two cDNA classes from C. capitata confirm the existence of duplicated Adh genes in this species. It is known that the Adh locus has undergone multiple duplication events in insects. Russo, Takezaki, and Nei (1995)
proposed a scenario according to which as many as four independent duplications occurred in the last 180 Myr, resulting in the present status of the Adh region in drosophilid species.
Our data show that there has been at least one duplication event in tephritids, which resulted in medfly ADH-1 and ADH-2. These genes are tightly linked in the medfly genome (Malacrida et al. 1992
) and have identical intron/exon structures (unpublished data). The observation that most tephritids have two ADH isozymes (Matioli et al. 1992
), combined with the relatively high degree of sequence divergence between the two copies of the medfly ADH (17.5% amino acid substitutions), indicates that the duplication of the Adh gene in this family is old. The B. oleae ADH sequence is more closely related to that of medfly ADH-2; a second ADH isozyme is also electrophoretically detectable in the olive fly (unpublished data), although more work will be required to fully characterize the ADH system of this species.
Evolutionary Inferences from the ADH Sequences in Diptera
The finding that S. peregrina ADH is more closely related to the tephritid, rather than being equidistant to the Drosophila and tephritid sequences, is intriguing. The flesh fly S. peregrina is placed in the Calyptrata series of the division Schizophora (infraorder Cychlorrapha, suborder Brachycera, order Diptera), while the drosophilid and tephritid fruit flies belong to the Acalyptrata series of the Schizophora (Hennig 1958
; Griffiths 1972
). It would be expected, therefore, that the Sarcophaga ADH sequence is equally distant to Drosophila and to tephritid ADH sequences. In contrast, Sarcophaga ADH branches together with the tephritid sequences, while the Drosophila ADH sequences form a separate cluster. This conclusion is strongly supported by (1) bootstrap analysis of the distance-based phylogenetic tree and (2) the presence of three shared indels in the Sarcophaga and tephritid sequences. The Drosophila ADHs are actually as distant from the Sarcophaga/tephritid ADHs as from the ADH-related sequences, with the three branches forming a virtual trifurcation (fig. 2
).
Two evolutionary scenarios may account for this unexpected finding; either the rate of evolution of ADH in the Drosophila lineage is (or has been) significantly faster than that in the other two lineages, or the Drosophila Adh genes are not orthologous to the tephritid and Sarcophaga genes. These two models are not, of course, mutually exclusive.
An acceleration in the rate of ADH evolution in drosophilids cannot be ruled out. If acceleration occurred, it would appear from figure 2
that it must have commenced or occurred in the ancestral Drosophila lineage. A temporal change in the rate of amino acid substitutions has been invoked to explain the apparently erratic evolution of glycero-3-phosphate dehydrogenase in higher Diptera (Kwiatowski et al. 1997
). In this case, the rates of amino acid replacements appear to be slightly different between the two drosophilid taxa, Drosophila and Chymomyza, but higher when species of these two genera are compared with the medfly enzyme. Analysis of ADH sequences from diverse tephritid species will be required to test the possibility that Adh may have evolved at different rates in tephritids than in drosophilids.
However, being members of a multigene family, the tephritid/Sarcophaga and Drosophila Adh genes may be paralogous and therefore not connected to each other by the same phylogenetic tree as the corresponding lineages. It is possible that the ancient lineage that gave rise to the Calyptrata and the Acalyptrata was already endowed with two duplicated and already divergent genes and that each of the three lineages (Drosophilidae, Tephritidae, and Sarcophagidae) subsequently retained a different copy of these genes; under this scenario, the copy retained by tephritids would be the same as that used by the distantly related sarcophagids (fig. 4
). This hypothesis is in agreement with the observation that Adh has undergone multiple duplication events during evolution. A related, "co-optation," scenario is that the ADH activity in both the Calyptrata and the tephritid lineages is associated with an orthologous gene (which has duplicated relatively recently, at least in the tephritid lineage), but this ortholog was lost in the drosophilid line in parallel with the de novo evolution of ADH activity from a different member of the preexisting multigene family of short-chain dehydrogenases. This scenario is also in agreement with the observation of emergence of new functions in duplicated members of the Adh family (Long and Langley 1993
; Begun 1997
). In either case, the Adh genes of, for example, Drosophila and the medfly would be paralogous, and their evolutionary distance would have no direct relation to that of the species lineages. No cross-hybridization could be detected between medfly Adh probes and Drosophila DNA or, reciprocally, between Drosophila Adh and medfly DNA (unpublished data). Moreover, the absence of regions of synteny between the two chromosomal Adh regions in the medfly and D. melanogaster may be an additional criterion for proposing a high evolutionary differentiation between the Adh systems of C. capitata and those of Drosophila (Malacrida et al. 1992
). These data suggest that under the paralogy scenario, the ADH orthologs becoming pseudogenes may have diverged in nucleotide sequence to a degree that does not allow detection.
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Conclusions |
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, Massachusetts.
2 Present address: Department of Genetics, Medical School, Washington University, St. Louis, Missouri.
3 Present address: Department of Animal Biology, University of Pavia, Pavia, Italy.
4 Keywords: Adh,
ADH
ADH evolution
Ceratitis capitata,
Bactrocera oleae,
Sarcophaga peregrina.
5 Address for correspondence and reprints: Charalambos Savakis, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, P.O. Box 1527, Vasilika Vouton, Heraklion 71110, Greece. E-mail: savakis{at}imbb.forth.gr
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