Isolation and Characterization of the Genomic Region from Drosophila kuntzei Containing the Adh and Adhr Genes

Jantien E. Oppentocht, Wilke van Delden and Louis van de Zande

Population Genetics, Center for Ecological and Evolutionary Studies, Biological Center, University of Groningen, The Netherlands


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among Drosophila. This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2–20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8–44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity ({pi}) was 0.0139, which is two times higher than that in D. melanogaster.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The role of alcohol dehydrogenase (ADH: EC 1.1.1.1) in conferring tolerance to environmental alcohols in Drosophila has been the object of research for many years. In most Drosophila species, ADH activity is positively correlated with alcohol tolerance, but the exact role of ADH in providing alcohol tolerance is not clear, and its function may not be restricted to alcohol metabolism alone.

The alcohol dehydrogenase (Adh) gene is one of the best-studied Drosophila genes. Sequencing of the genomic region of the Adh gene of many Drosophilid species has revealed a large uniformity in the architecture of the protein-coding region, which is organized in the last three exons, whereby both exon and intron lengths are similar across species, with the exception of D. willistoni, which has the last intron precisely removed from the gene (Anderson, Carew, and Powell 1993Citation ). The genomic region flanking the structural gene sequences, however, shows a striking diversity among all species studied. Species of the subgenus Sophophora, to which D. melanogaster belongs, and the subgenus Scaptodrosophila have a single Adh gene, temporally regulated by two tandemly arranged promoters, with an Adhr gene immediately downstream of the Adh gene. The function of Adhr is still unknown (it is not an ADH), but the high level of sequence conservation between Adhr genes of Drosophila species indicates that this gene is preserved by natural selection and thus is likely to be functional (Kreitman and Hudson 1991Citation ). In some species of the Drosophila subgenus, duplication of the Adh gene has occurred. Drosophila virilis and some other members of the virilis group have a duplicated Adh gene, and both genes are regulated by two tandemly arranged promoters (Nurminsky et al. 1996Citation ). In members of the repleta group (for example D. mettleri, D. hydei, and D. mulleri) two or three copies of the Adh gene are described. The most upstream copy is called Adh-P, {psi}Adh or Finnegan, which was originally thought to be a nonfunctional pseudogene, but there is some evidence that this gene encodes a functional protein which is not an ADH (Begun 1997Citation ). The Adh gene(s) in these species are controlled by a single promoter each, and recently Adhr has been identified in D. buzzatii (Betrán and Ashburner 2000Citation ). In D. funebris and in many other members of the funebris group, nonfixed duplication of the complete Adh gene and a part of the Adhr gene has been described. Here, the Adh gene is under the control of a single promoter (Amador and Juan 1999Citation ). This embedding of a conserved protein-coding region within highly variable flanking regions may reflect the evolution of adaptive changes in gene regulation and gene structure of the Adh gene in Drosophila. These adaptive changes should be most manifest when comparing the Adh genomic region of species where environmental alcohols do not provide a selective pressure with that of species that may be confronted with high concentrations of environmental alcohol like D. melanogaster. Furthermore, the availability of Adh genomic sequences for many Drosophila species allows the construction of phylogenetic relationships for Drosophila species for which few molecular data are available.

Here, we describe the cloning and sequencing of the Adh genomic region of D. kuntzei, a member of the quinaria group and a species with extremely low alcohol tolerance. The quinaria group belongs to the immigrans-HirtoDrosophila radiation of the subgenus Drosophila (Throckmorton 1975Citation ), comprises about 28 species that are endemic to the Nearctic and Palearctic regions and are known to feed either on mushrooms or on decaying (water)plants (Spencer 1942Citation ; Brown 1956Citation ; Shorrocks 1977Citation ; Jaenike 1978Citation ; Hummel, van Delden, and Drent 1979Citation ; Grimaldi and Jaenike 1983Citation , 1984Citation ; Lacy 1984Citation ; Jaenike and James 1991Citation ). These habitats do not contain the relatively high levels of alcohols as those found in the environments inhabited by D. melanogaster. In addition, it has been shown that in D. kuntzei, as little as 3% (v/v) ethanol kills all individuals within minutes (Oppentocht 2001Citation ). The ADH activity of D. kuntzei flies is about 2% of that of D. melanogaster ADHFF but displays the characteristic expression pattern with higher activity in the larval and the adult stage compared with the pupal stage (Oppentocht 2001Citation ).

In this study we have compared the Adh genomic region of D. kuntzei with that of other Drosophila species at three levels (1) the 5'-flanking region, comprising the regulatory motifs, (2) the structural Adh gene, and (3) the downstream detected Adhr gene. The structural Adh and Adhr gene sequences were used to establish phylogenetic relationships with other Drosophila species. In addition, we have determined sequence variation within the Adh gene of D. kuntzei in strains from different geographic origin, in order to allow for both inter- and intraspecific comparisons.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Drosophila Stocks and Rearing Conditions
Drosophila kuntzei flies, used for construction of the library, were from a strain caught in Switzerland. In addition, a Dutch strain collected in the province of Drenthe and two French strains from Paris and Brittany were used for comparison of Adh sequences. The D. melanogaster strain used to compare ADH activities was genotypically AdhFF (Van Delden, Boerema, and Kamping 1978Citation ).

DNA Isolations and Manipulations
Standard protocols were applied for all DNA and RNA manipulations (Ausubel et al. 1987Citation ). Genomic DNA of Drosophila was isolated by Proteinase K–sodium dodecyl sulfate (SDS) digestion followed by phenol and chloroform-isoamylalcohol (24:1 [v/v]) extraction and ethanol precipitation. Phage DNA was isolated by centrifugation and subsequent lysis of phage particles was performed according to Grossberger (1987)Citation . Plasmid DNA was isolated using the alkaline lysis method of Birnboim and Doly (1979)Citation .

Southern Hybridizations
After electrophoresis, DNA fragments were transferred to nylon membranes (Hybond-N, Amersham). Membranes were hybridized overnight at 65°C in 10% (w/v) polyethylene glycol (PEG), 1 M NaCl, and 1% (w/v) SDS to a probe labeled with [{alpha}32P]dCTP by random primed labeling (Boehringer Mannheim). After washing at medium to high stringency, autoradiography was performed by exposing Kodak X-OMAT X-ray film at -80°C using two intensifying screens.

Construction and Screening of the Genomic Library
For the construction of the genomic library, 200 µg high–molecular weight genomic DNA was partially digested with MboI, and DNA fragments ranging in size from 9 to 23 kb were used to establish a genomic library in {lambda} DASH II (Stratagene). For screening the library for Adh positive clones, approximately 5 x 105 recombinants were plaque-lifted onto nitrocellulose membranes (Amersham, England). Hybridization was performed using the same procedures as in the southern hybridization procedure.

PCR and Inverse PCR
The position and sequence of the primers that were used in PCR reactions are listed in figure 1A and table 1B . Primers residing within the protein-coding regions correspond to the conserved regions in the nucleotide sequences of all known Adh genes of Drosophila species. For all applications, PCR fragments were purified using the QIAquick PCR purification kit (Qiagen).



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Fig. 1.—A, Physical map of the D. kuntzei Adh genomic region. Arrows indicate regions covered by the inverse PCR (solid) and the phage clone (dashed), respectively. The gray box represents the adult enhancer (AAE), the white boxes represent noncoding transcribed regions, and the black boxes represent protein-coding regions. Numbered arrows indicate primers used for PCR, listed in table 1B. S = SalI, B = BamH1, E = EcoR1, P = promoter. B, Alignment of D. kuntzei (KUN) and D. melanogaster (MEL) AAE region. Numbers indicate distance from the TATA box of the proximal promoter. C, Alignment of D. kuntzei (KUN) and D. melanogaster (MEL) proximal promoter region. Numbers indicate distance from the TATA box of the proximal promoter

 

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Table 1 Drosophila Species and Sequences of Primers

 
Primers 5 and 6 (see fig. 1A ) were used to generate a PCR product containing upstream and downstream Adh sequences from circular, self closed fragments that were generated by digestion of chromosomal DNA of D. kuntzei with HindIII and subsequent overnight ligation (Ochman, Gerber, and Hartl 1988Citation ). This fragment was subsequently ligated into vector pGEM-T (Promega).

For comparison of Adh sequences, a 1.7-kb fragment containing the complete Adh gene was amplified using primers 7 and 8 (see fig. 1A ).

Nucleotide Sequencing and Data Analysis
All fragments were sequenced on an ABI 310 sequencer (PE Biosystems) using either T7 and M13-Reverse or specifically designed primers. The resulting sequence data were analyzed using Sequence Assembler and Sequence Navigator programs (PE Biosystems). From the nucleotide sequences, protein-coding regions were derived on the basis of similarity with other known Adh sequences of Drosophila species (see table 1 ). Codon bias was estimated by calculating the percentage of G+C in the third codon position, excluding methionine, tryptophan, and stop codons. The deviation from random synonymous codon usage (scaled chi-square or {chi}2/L) was calculated according to Shields et al. (1988)Citation . The effective number of alleles (Nc; Wright 1990Citation ) and the frequency of optimal codon use (Fop; Ikemura 1985Citation ) were calculated using the Codons program version 1.4 (Lloyd and Sharp 1992Citation ). A D. melanogaster optimal codon usage table was used as reference. The number of synonymous nucleotide substitutions per synonymous site (Ks) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (Ka) were calculated according to Li, Wu, and Luo (1985)Citation . On the basis of these Ks and Ka values, least squares distances between species were calculated using the FITCH program of PHYLIP version 3.5 (Felsenstein 1993Citation ). DNA parsimony dendrograms were constructed using the program DNAPARS from the PHYLIP package. Nucleotide diversity ({pi}) was calculated according to Nei (1987, pp. 256–273)Citation . The DnaSP program version 3.14 (Rozas and Rozas 1999Citation ) was used to calculate Ks and Ka values and {pi}.

RNA Extractions and Northern Hybridizations
Total RNA was isolated from third instar larvae and 1-week-old adult flies using the RNeasy kit from Qiagen. Approximately 10 µg RNA (as determined by spectrophotometry) was vacuum-dried and dissolved in 20 µl sample buffer. From each sample, 5 µl was loaded onto a 1% agarose gel for later calibration. The remaining 15 µl (7.5 µg) was separated through a 1% agarose gel containing 2.2 M formaldehyde and transferred onto a nylon membrane (Hybond-N, Amersham) in 20 x SSC. Membranes were hybridized overnight at 42°C to a [{alpha}32P]dCTP-labeled probe, obtained as the PCR product using primers 2 and 3 (fig. 1A ) from D. melanogaster and D. kuntzei genomic DNA, respectively. The nucleotide identity of these two probes is 79.7%. The filters were washed at medium to high stringency. Autoradiography was carried out for both 16 and 48 h at -80°C using intensifying screens. Filters were stripped by pouring a boiling solution of 0.1% (w/v) SDS onto the blot and allowing it to cool to room temperature.

ADH Activity Measurements
To determine ADH activity, three replicates of either 10 third instar larvae or 10 adult flies were homogenized in 1 ml 50 mM Tris-HCl (pH 8.5), 1 mM EDTA. Fifty microliters of the homogenate was assayed in 50 mM Tris-HCl (pH 8.5), 200 mM isopropanol, 5 mM NAD+ (1 ml reaction volume) by measuring the extinction at 340 nm caused by NADH formation ({varepsilon}NADH = 6,300 M-1 cm-1) for 2 min.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Cloning of the Adh Gene
The library was screened with a 230-bp D. kuntzei Adh-specific PCR fragment that was amplified using primers 2 and 3 (see fig. 1A ). One positive clone containing an insert of about 12 kb was isolated, containing Adh sequences corresponding to a part of exon III and the complete exon IV of the D. melanogaster Adh gene but without more upstream genomic Adh sequences. A second screening of the D. kuntzei library with an extreme 5' 500 bp Sau3A fragment from this clone (chromosome walk) yielded two different clones with average insert sizes of 20 kb. Restriction and Southern blot analysis with an 850-bp PCR probe (primers 1 and 4, fig. 1A ) revealed that these new clones did not contain more upstream genomic Adh sequences than the previous clone but differed only in downstream sequence lengths. Another screening of the library with a 180-bp probe (primers 1 and 5, fig. 1A ) did not yield any positive clones at all.

Southern analysis of total genomic DNA revealed that more upstream Adh sequences are contained within a 4-kb HindIII fragment, of which approximately 1.3 kb of the 3' part is represented in our isolated clones. This enabled us to design a primerset (primers 5 and 6, fig. 1A ) to be used in an inverse-PCR reaction to clone more upstream genomic Adh sequences. The amplified fragment was cloned into vector pGEM-T and sequenced. No further HindIII sites were found upstream, confirming the integrity of this fragment. The complete sequence was checked by amplification with the designed primers 7 and 8 and subsequent sequencing of the amplified 1.72 kb fragment containing the entire Adh gene (see fig. 1A ).

Gene Characterization
Structural Gene
The consensus sequence derived from all nucleotide sequences from the library, PCR, and inverse-PCR clones has been submitted to GenBank (accession number AF399012). Although the sequence contains some ambiguities caused by polymorphic sites, this did not interfere with the analysis of the sequence as described subsequently. The intron-exon boundaries of the structural Adh and Adhr genes were deduced by sequence similarity with other Drosophila species and by conformation to splice donor and acceptor consensus motifs. By assigning position +1 to the A of the initiating ATG triplet, the three protein-coding regions of Adh range from +1 to +94, +156 to +561, and +622 to +889. The open reading frame thus contains 255 codons (including the stop codon). The inferred protein has a predicted molecular weight of 27.6 kDa. Further sequencing revealed the presence of an Adhr gene downstream of the Adh gene. Its start codon is located 277 nucleotides from the stop codon of Adh, and the gene consists of three coding regions, ranging from +1 to +97, +330 to +735, and +792 to +1119. The inferred protein consists of 275 amino acids and has a predicted molecular weight of 30.7 kDa. Both Adh and Adhr genes are very similar in gene architecture to sequences of other species and have the same predicted protein weights. If amino acid identity is defined as exact matches of amino acids and similarity as both exact matches and conservative amino acid changes (see legend to fig. 4 ), then comparing D. kuntzei with D. funebris yields for Adh 88% identity and 94% similarity and for Adhr 96% identity and 98% similarity. Comparing D. kuntzei with D. immigrans yields for Adh 87% identity and 93% similarity and for Adhr 95% identity and 98% similarity (data not shown). The identity and similarity of D. kuntzei to D. melanogaster is 78% and 89% for AdhS and 85% and 91% for Adhr, respectively.



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Fig. 4.—Alignment of D. kuntzei (kun) and D. melanogaster (mel) ADH (A) and ADHR (B) proteins. "*" indicates full conservation of residues; ":" indicates conservation of strong groups (STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW); "." indicates conservation of weaker groups (CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, HFY)

 
Adh Genomic Region
Sequence analysis of the genomic region upstream of the coding region showed that the Adh gene of D. kuntzei has one promoter with high similarity to the proximal (larval) promoter of D. melanogaster (fig. 1C ). In agreement with this feature, no sequences were found that showed any correspondence to the first (noncoding) exon of species with a distal promoter. In addition, Northern analysis of D. kuntzei RNA with a probe spanning 613 bp of 5' upstream Adh sequences did not indicate the presence of additional exon sequences (data not shown). The Adh promoter of D. kuntzei has a TATA box (TATAAATA) at -107 to -100 from the ATG start codon and a highly conserved GATAA motif (box A–binding factor; Abel, Michelson, and Maniatis 1993Citation ) at -52 bp from the TATA box (fig. 1C ). In addition, possible binding sites reported for Adf-1 and Adf-2, Adh distal promoter factors, were identified in the upstream genomic region (fig. 1C ).

Further upstream of the D. kuntzei Adh gene, at -755 from the TATA box, an adult enhancer element (AAE) was found that is very similar to that described for other species (fig. 1A and B ). Its structure is strikingly similar to that of D. melanogaster, containing a box B–binding factor-2, a fat body–specific activator (Abel, Bhatt, and Maniatis 1992Citation ) in which a (weak) C/EBP binding is embedded (Falb and Maniatis 1992Citation ); an Adult enhancer factor-1 (AEF-1)–binding site, a negative element within the AAE (Falb and Maniatis 1992Citation ); and a common response element for two steroid hormone receptor superfamily members (Ayer and Benyajati 1992Citation ; Ayer et al. 1993Citation ): FTZ-F1, an activator, and Drosophila Hormone Receptor-39, a repressor (fig. 1B ). The distance of 755 bp of the adult enhancer to the TATA box in D. kuntzei is much shorter than that in D. melanogaster, where its distance is 1,242 bp. Alignment of the sequences upstream of the structural Adh gene of several Drosophila species invariably showed a similar pattern: a high similarity region corresponding to the AAE, a stretch of variable length with no significant similarity, followed by a second region of moderate to high similarity comprising the proximal promoter region (see fig. 2 ).



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Fig. 2.—Multiple clustal analysis of the Adh genomic region from the AAE to the ATG start codon for D. kuntzei (KUN), D. funebris (FUN), and D. immigrans (IMM)

 
Between the Adh and Adhr genes a polyA signal was found at +988 from the Adh start codon. Upstream of the Adhr gene, no obvious TATA box was found, indicating that in D. kuntzei, like in D. melanogaster, Adh and Adhr may also be dicistronic (Brogna and Ashburner 1997Citation ).

Adh Expression
Northern analysis of RNA extracted from third instar larvae and adult flies revealed that in both life stages of D. kuntzei, Adh mRNA is present at levels comparable to or slightly less than that in D. melanogaster (fig. 3 ). The result of the hybridization clearly demonstrated that the probes derived from the D. kuntzei or the D. melanogaster gene discriminate between the Adh mRNA of the two species. The fact that there is no conspicuous difference in Adh mRNA levels between both species for either life stage tested is in contrast to the difference in activity of the ADH protein between D. kuntzei and D. melanogaster (AdhFF) for both life stages (fig. 3 ).



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Fig. 3.—A, ADH activity in Units per gram body weight for third instar larval (LAR) and adult (ADU) D. melanogaster (MEL) and D. kuntzei (KUN) flies. B, Northern blot of total RNA isolated from third instar and adult D. melanogaster and D. kuntzei flies hybridized to a D. kuntzei-specific Adh probe (Top) or a D. melanogaster-specific Adh probe (Bottom). 1. Drosophila kuntzei third instar, 2. Drosophila kuntzei adult, 3. Drosophila melanogaster third instar, 4. Drosophila melanogaster adult, 5. Drosophila kuntzei third instar, 6. Drosophila kuntzei adult, 7. Drosophila melanogaster third instar, 8. Drosophila melanogaster adult

 
Protein Analysis
Although in the ADH protein sequence of D. kuntzei some differences in the N-terminal part, which forms the NAD-binding site, were found compared with other species, none of them were at strictly conserved places or at sites known to be important in NAD binding. The amino acids of the active site were all conserved, including the three amino acids that make up the catalytic triad, except for Thr189. In most species, valine is found at this position. The same amino acid change is also found in D. funebris, whereas in D. hawaiiensis glutamic acid is present. Also, at amino acid 191, corresponding to the ADHS-ADHF difference in D. melanogaster, Arg191 is found instead of Lys191, like in most species. In D. melanogaster it is shown that the amino acid difference Lys-Thr is associated with the difference in catalytic efficiency found between the two allozymes ADHS and ADHF. An arginine at this position is also found in D. flavomontana and D. borealis, whereas D. funebris contains histidine, D. hawaiiensis serine, and D. lebanonensis tyrosine at position 191. If, and to what extent, these amino acid changes truly cause differences in catalytic behavior of ADH is not known.

ADHR differs in its C-terminal length among the various species. In D. kuntzei the C-terminal end is unique because it possesses five glutamic acid residues in a row. Taken together with the other amino acids found, the C-terminus appears to be highly hydrophilic in both D. kuntzei and in other species. The alignment of the D. kuntzei and D. melanogaster ADH and ADHR protein is shown in figure 4 .

Codon Bias
The G+C percentages, Nc, {chi}2/L, and Fop values of the Adh and Adhr genes of D. kuntzei and a number of other species are presented in table 2 . For Adh of D. kuntzei, the G+C percentages, scaled chi-square values, and Fop are lower than that in all other species listed in table 2 . The only exception is D. funebris, which possesses a lower third position G+C percentage. In fact, the values that are found in D. kuntzei Adh are among the lowest reported until now for these genes. The same exceptional position is found for Nc, which is higher than that in all other species. All these values indicate that D. kuntzei Adh has a lower codon bias than most other species and resembles the low level of codon bias found in D. funebris (Amador and Juan 1999Citation ). In D. kuntzei Adhr, the percentage G+C is, like in Adh, among the lowest reported until now, but the other estimates are within the range of values found in other species. Codon bias in general is higher in the Adh genes compared with the Adhr genes. Codon bias in the two genes of D. kuntzei, on the other hand, was about the same (0.34 for Adh and 0.36 for Adhr, difference 5.6%).


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Table 2 Codon Bias in Adh and Adhr Genes of D. kuntzei and Several Other Species

 
Substitution Rates
The number of synonymous nucleotide substitutions per synonymous site (Ks) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (Ka) in the coding regions of the Adh and Adhr genes of D. kuntzei compared with other species are presented in table 3 . In both Adh and Adhr, numbers of synonymous substitutions are higher than the numbers of nonsynonymous substitutions. This has also been reported for other Drosophila species by Albalat, Marfany, and Gonzàles-Duarte (1994)Citation and Jeffs, Holmes, and Ashburner (1994)Citation and is consistent with a gene that is under the influence of functional constraints and which is conserved by natural selection.


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Table 3 Synonymous and Nonsynonymous Substitution Rates for Adh and Adhr of D. kuntzei Compared with Other Drosophila Species

 
For D. kuntzei, nonsynonymous substitution rates are higher in Adh than in Adhr. For the comparison of D. kuntzei with other species, the values of the ratio KaAdh/KaAdhr is always larger than 1. This shows that at nonsynonymous positions, Adh is evolving at a higher rate than Adhr. Synonymous substitution rates, however, are higher in Adhr than in Adh. For comparisons of D. kuntzei with other species, the ratio KsAdh/KsAdhr is always smaller than 1, indicating that Adhr evolves faster than Adh at synonymous positions. The observation that Adh and Adhr differ in their evolutionary pattern is in agreement with other reports (Kreitman and Hudson 1991Citation ; Albalat and Gonzàlez-Duarte 1993Citation ; Albalat, Marfany, and Gonzàles-Duarte 1994Citation ; Jeffs, Holmes, and Ashburner 1994Citation ; Amador and Juan 1999Citation ).

Phylogenetic Analysis
Figure 5B shows an unrooted consensus distance tree based on Ka or Ks substitution rates for Adh and Adhr, respectively. Figure 5A shows a dendrogram based on alignments of Adh sequences. Both trees show that D. kuntzei is most related to D. funebris, and both D. kuntzei and D. funebris cluster with D. immigrans. All three species are members of the Drosophila subgenus. Only the tree based on Adhr Ks values (not shown) places D. lebanonensis (subgenus Scaptodrosophila) in a cluster with the species of the Drosophila subgenus (D. immigrans, D. kuntzei, and D. funebris), whereas in the three other trees D. lebanonensis seems to be equally related to the Sophophora and the Drosophila subgenus species.



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Fig. 5.—A, Dendrogram constructed using Adh-coding sequences and parsimony analysis. Numbers before nodes represent the proportion of similar replicates out of 1,000 permutations conducted. B, Consensus distance tree based on Adh Ks, Adh Ka, Adhr Ks, and Adhr Ka values. Numbers before nodes indicate the number of occurrence out of four trees

 
Genetic Variation in the Adh Gene
To establish the level of Adh variation present in D. kuntzei flies of different geographic origin, the nucleotide sequence of the complete region, including most of the regulatory 5' part of 10 randomly chosen alleles for each strain was determined. Using primers 7 and 8 (fig. 1A ), a 1.7-kb PCR fragment was amplified and sequenced. The results are summarized in table 4 Go . Insertions and deletions were counted as one difference. In all strains, polymorphic sites were found. The Swiss strain appeared to be the least polymorphic of all, with an overall nucleotide diversity ({pi}) of 0.0005, whereas the three other strains varied from {pi} = 0.0055 to 0.0137. The reason for this low variability is probably because this strain was initially maintained in our laboratory on regular food which contained 1.25% ethanol. Because of the very low alcohol tolerance of this species, death rates were high and thus genetic drift may have caused the low genetic variation levels. The other strains were grown on food without ethanol from the start. The overall nucleotide diversity for all alleles was 0.0139. Most of the variation was found upstream and downstream of the coding region ({pi} = 0.0190 and {pi} = 0.0105, respectively). Here, in total, 10 sites were found with insertions-deletions of 1–12 nucleotides. Two of the insertions, at -627 and -336, respectively, were always linked and could be remnants of a transposable element. The most conserved region was exon II ({pi} = 0.0045). The overall sequence variability was larger in noncoding regions ({pi} = 0.0185) compared with coding regions ({pi} = 0.0088), and most differences within the coding regions were silent. In total, four amino acid polymorphisms were found: cysteine-arginine at position 28 (first exon), phenylalanine-tyrosine at 60, valine-isoleucine at 85, and isoleucine-leucine at position 145 (all second exon sequences). No amino acid variants were found in exon III.


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Table 4 Analysis of Polymorphic Sites in Protein-Coding Regions of Exons I, II, and III and Nonprotein–Coding Regions of Adh in Four Populations of D. kuntzei (10 alleles were sequenced for each population; see text for details)

 

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Table 4 Continued

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Adh Genomic Region
As in most Drosophila species examined so far, the protein-coding parts of the Adh gene of D. kuntzei are highly conserved, as well as its architecture. A more detailed discussion on the amino acid composition of D. kuntzei ADH will be given subsequently.

As has also been described for D. funebris (Amador and Juan 1999Citation ), the Adh gene of D. kuntzei is under the control of a single promoter, corresponding to the D. melanogaster proximal promoter. Indeed, all essential regulatory sequences that have been described for the proper functioning of this promoter are present in the D. kuntzei Adh genomic sequence, immediately upstream of the TATA box (fig. 1A ). Moreover, an AAE is present at -755 of the TATA box that is strikingly similar to the AAE of D. melanogaster. Interestingly, there is a difference with the D. melanogaster sequence at the putative binding site for the repressor AEF-1 and the activator C/EBP. Although a binding site for AEF-1 is in the AAE of D. kuntzei, no overlapping and competing (Falb and Maniatis 1992Citation ) site for C/EBP was detected (fig. 1B ). Similarly, an AEF-1 site in the initiator region of the D. kuntzei Adh gene does not show complete correspondence to that of the AEF-1 site in the initiator region of the proximal promoter of D. melanogaster (fig. 1C ). It has been shown that AEF-1 is involved in the Adh promoter switching during development by interfering with the transcription machinery (Ren and Maniatis 1998Citation ). The differences described for the AEF-1 sites in the AAE and initiator region might reflect the fact that no temporal promoter switching occurs in the one-promoter system of D. kuntzei. Nevertheless, the temporal Adh expression pattern of D. kuntzei is similar to that of D. melanogaster (fig. 3 ; Oppentocht 2001Citation ). Similarly, in the initiation region of D. funebris, a species which also has a one-promoter Adh gene, an imperfect AEF-1 site is present (Amador and Juan 1999Citation ). However, D. immigrans, that has a two-promoter system, does not contain a perfect AEF-1 site at this region (see fig. 2 ). Apparently, temporal regulation of Adh promoter utilization is dependent on more regulatory systems. Therefore, it is not clear if the absence of an C/EBP site overlapping the AEF-1 site in the AAE of D. kuntzei is a consequence of its one-promoter system or if this feature is involved in the low ADH activity in D. kuntzei, which is most pronounced in the adult stage when compared with D. melanogaster (fig. 3 ). Moreover, regions corresponding to neither the 5' (Laurie, Bridgham, and Choudhary 1991Citation : Laurie and Stam 1994Citation ) nor the 3' (Parsch, Stephan, and Tanda 1999Citation ) regulatory regions that have been shown to be involved in ADH expression in D. melanogaster were found in the Adh-flanking regions of D. kuntzei. These features, combined with the observation that in at least two different life stages levels of D. kuntzei Adh mRNA are comparable with those of D. melanogaster, indicate that the amino acid composition of the ADH protein of D. kuntzei is an important factor in the low ethanol tolerance of this species.

Adh-Coding Region
It has been reported that a high level of expression of genes in D. melanogaster is correlated with high codon bias (high G+C content in the third position of a codon, high scaled chi-square values, and low Nc values) (Shields et al. 1988Citation ; Sharp and Li 1989Citation ; Moriyama and Gojobori 1992Citation ; Moriyama and Hartl 1993Citation ). Ikemura and Ozeki (1983)Citation pointed out that Fop values decrease with reduction of protein production levels in Escherichia coli. Also Ks values are negatively correlated with codon bias (Shields et al. 1988Citation ). We showed that D. kuntzei Adh has a very low codon bias and also has high Ks values. In fact, together with the closely related species D. funebris, D. kuntzei has the lowest Adh codon bias among all species investigated until now. Accordingly, this would be consistent with a gene that is expressed at a low level. Codon bias in Adh and Adhr of D. kuntzei was about the same, suggesting equal levels of expression of the two genes, whereas in other species codon bias is higher in Adh than in Adhr genes, reflecting higher expression levels of Adh compared with Adhr (Shields et al. 1988Citation ; Jeffs, Holmes, and Ashburner 1994Citation ). However, assumptions on the level of expression of a gene based on codon bias should be taken with precaution because the correlation between codon bias and expression is not always found to be very robust in multicellular organisms (Ikemura 1985Citation ). In addition, Amador and Juan (1999)Citation also observed little difference in codon bias between the Adh and Adhr gene of D. funebris, whereas expression differed dramatically. The observation that the difference in codon bias between Adh and Adhr disappears in D. kuntzei, together with the observation that the Adh gene in this species evolves at a higher rate at nonsynonymous positions than Adhr suggests that at least some specific selective pressure on this gene has been relaxed. Although it is tempting to speculate that resistance to environmental alcohols is a part of such pressures, no direct evidence for this is apparent. Nevertheless, at the mRNA level, Adh is definitely expressed in D. kuntzei, to what end, is open for further investigation.

ADH Protein
The large amino acid sequence similarity with other species and the preservation of amino acids crucial to activity suggest that the ADH protein of D. kuntzei is probably active. In fact, we have measured substantial, but extremely low, ADH activities in this species (fig. 3 ; Oppentocht 2001Citation ). However, in the amino acid sequence we found a change in one of the amino acids in the R1 cavity of the active site. Val189 is changed to Thr189, which is also found in D. funebris but is otherwise unique within the Drosophila genus. The only other exception is D. hawaiiensis, which contains Glu189. Crystallization and modeling data showed that Val189 is placed in the R1 active site cavity in ADH of D. lebanonensis and D. melanogaster (Benach-Andreu 1999Citation ; Benach et al. 1999Citation ). A change to Thr189 will presumably cause the active site to become less hydrophobic. The high hydrophobicity of the active site is needed to allow binding of aliphatic alcohols. Replacement of this amino acid by threonine in D. kuntzei will probably result in either a loss of activity with alcohol substrates or will yield a different substrate usage pattern. In fact, this amino acid difference alone may account for the low activity of this protein on alcohol substrates. Because threonine has a polar group, the active site cavity will become more polar, and the protein may have a higher specificity for multi substituted alcohols containing hydroxyl groups or other polar groups in different positions of the molecular chain, like sugars and polyols (Benach-Andreu 1999Citation ). Maybe this amino acid change explains the difference in activity ratio found with primary (ethanol) and secondary (isopropanol) alcohols (E-I ratio). In D. kuntzei this ratio is higher than that in other species and may vary considerably among different species of the same quinaria group (Mercot et al. 1994Citation ; Oppentocht 2001Citation ). Because D. kuntzei feeds and oviposits on fungi and decaying plants, where alcohol formation by fermentation is unusual, it may be that this amino acid change represents a change in function of the protein from alcohol breakdown under stress conditions to a more general physiological function in the absence of alcohol stress.

Arg191 was also found in D. kuntzei, which is the S-F polymorphic site in D. melanogaster. Normally, this amino acid is not located near the active site. But upon substrate binding a conformational change of the molecule (closure of a loop in which amino acid 191 is located at a central position) may bring amino acid 192 closer to the NAD+-substrate complex and may thus have an influence on the catalytic properties of the enzyme (Smilda 1997Citation ).

Phylogeny
Both distance trees and parsimony analysis showed that D. kuntzei (quinaria group) is closest related to D. funebris (funebris group) and that both species cluster together with D. immigrans (immigrans group). All three species belong to the Drosophila subgenus. The higher Ks values in Adhr compared with Adh show that Adhr is evolving at a higher rate than Adh at synonymous positions, whereas the slightly lower Ka values indicate that the opposite is true for changes that modify the protein composition. So, the amino acid sequence in Adhr is more conserved than that in Adh. This was also reported by analyzing other Drosophilid species by Albalat and Gonzàlez-Duarte (1993)Citation , Albalat, Marfany, and Gonzàles-Duarte (1994)Citation , Jeffs, Holmes, and Ashburner (1994)Citation , and Amador and Juan (1999)Citation .

The time of divergence between D. kuntzei and D. funebris was calculated using two substitution rates and the mean Ks values of Adh and Adhr per species. With substitution rates of 1.43 x 10-8 substitutions per synonymous site per year according to Li (1993)Citation and 1.00 x 10-8 substitutions per synonymous site per year according to Tajima and Nei (1984)Citation , the divergence time for D. kuntzei and D. funebris was calculated to be 14.2 and 20.2 Myr, respectively. The time of divergence between the D. kuntzei-D. funebris lineage and D. immigrans was calculated analogous to the D. kuntzei-D. funebris divergence, using the mean of the Ks values of D. kuntzei and D. funebris Adh and Adhr genes. The D. kuntzei-D. funebris lineage was estimated to have split from the D. immigrans lineage 30.8–44.0 MYA. Amador and Juan (1999)Citation estimated the divergence times between D. funebris and D. immigrans to be 23.5–26.9 MYA.

Genetic Variation of the Adh Gene
We detected an overall nucleotide diversity of {pi} = 0.0139 in the Adh gene and flanking sequences in four European strains of D. kuntzei over a stretch of 1,615 nucleotides. The overall sequence variability in Adh of D. kuntzei is larger in the intron regions compared with the exons. This indicates that the Adh gene of D. kuntzei is to some extent preserved by natural selection and is therefore very likely to be functional. Kreitman (1983)Citation sequenced 2,379 nucleotides of Adh of 11 D. melanogaster individuals and found an overall nucleotide diversity of {pi} = 0.007. We sequenced 40 alleles derived from four geographically distinct strains and found {pi} = 0.0139, which is two times higher than that in D. melanogaster. In addition, Kreitman (1983)Citation only found one amino acid substitution in 11 individuals, whereas we found four substitutions in 40 alleles. This indicates that exact sequence preservation of the gene in D. kuntzei by natural selection is less strong than that in D. melanogaster.

Because of the higher levels of genetic variation levels found in D. kuntzei compared with D. melanogaster, combined with the observation of higher evolution of the Adh gene at nonsynonymous positions, it may be that Adh is less important for functioning in its natural environment in D. kuntzei than it is in D. melanogaster. Given the different habitats where these species occur, this may very well be the case. Drosophila melanogaster feeds and oviposits on fermenting fruit where considerable concentrations of alcohol can be found. Drosophila kuntzei, on the other hand, feeds and oviposits on plant material and mushrooms, where the production of alcohols because of fermentation processes is expected to be low. Therefore, the importance of ADH as an enzyme involved in ethanol tolerance is less important in this species. It may be, however, that ADH has a different function, which may not directly be related to alcohol metabolism. We have indeed found a change in one of the amino acids of the active site in D. kuntzei ADH, which could account for a change in substrate usage. Nevertheless, the high levels of genetic variation do not suggest high levels of selection; subsequently, together with the low enzyme activity, the relative importance of this gene to D. kuntzei may be less than in D. melanogaster. In this respect, the role of Adhr in this species may be relatively more prominent than that of Adh. Further investigation of the functional properties of ADH of D. kuntzei and other quinaria group species is needed with respect to the Thr189 change. Elucidation of differences in substrate usage pattern will possibly point to a shift in metabolic function of ADH of D. kuntzei.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Dr. G. Bächli, Zoological Museum, Zürich, Switzerland, J. Sevenster, University of Leiden, The Netherlands, and Prof. J. R. David, Gif sur Yvette, France for providing the Swiss, Dutch, and French strains of D. kuntzei, respectively, and an anonymous reviewer for constructive comments. This research was funded by the Netherlands Organization for Scientific Research (NWO), project number 436933P.


    Footnotes
 
Pierre Capy, Reviewing Editor

Keywords: Drosophila kuntzei quinaria group alcohol dehydrogenase genetic variation nucleotide diversity codon bias Back

Address for correspondence and reprints: Louis van de Zande, Population Genetics, Biological Center, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. zandelpw{at}biol.rug.nl Back


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 Abstract
 Introduction
 Materials and Methods
 Results
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Accepted for publication February 5, 2002.





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