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Originally published as Genetics Published Articles Ahead of Print on March 31, 2005.

Genetics, Vol. 170, 709-718, June 2005, Copyright © 2005
doi:10.1534/genetics.104.036483

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A High-Frequency Null Mutant of an Odorant-Binding Protein Gene, Obp57e, in Drosophila melanogaster

Aya Takahashi*,{dagger},1 and Toshiyuki Takano-Shimizu*,{dagger},{ddagger}

* Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japan
{dagger} Department of Genetics, Graduate University for Advanced Studies (Sokendai), Mishima 411-8540, Japan
{ddagger} School of Advanced Sciences, Graduate University for Advanced Studies (Sokendai), Shonan Village, Hayama, Kanagawa 240-0193, Japan

1 Corresponding author: Division of Population Genetics, National Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan.
E-mail: atakahas{at}lab.nig.ac.jp

Manuscript received October 7, 2004. Accepted for publication February 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
We have found a null mutant of an odorant-binding protein, Obp57e, in Drosophila melanogaster. This frameshift mutation, which is a 10-bp deletion in the coding region, is at a high frequency in the Kyoto population and is also present in Taiwan and Africa. We have sequenced a 1.5-kb region including the tandemly duplicated gene, Obp57d, from 16 inbred lines sampled in Kyoto, Japan. The analyses showed a peak of nucleotide diversity and strong linkage disequilibrium around this mutation. This pattern suggests an elevated mutation rate or an influence of balancing selection in this region. The level of nucleotide divergence between D. melanogaster and D. simulans does not support the former possibility. Thus, this presence/absence polymorphism may be due to balancing selection, which takes advantage of the relatively weak functional constraint in members of a large gene family. In addition, the Obp57d gene region showed an excess of high-frequency-derived mutants that is consistent with a pattern predicted under positive natural selection.

MANY gene families arise as a result of recurrent gene duplication events. Many of these genes would either acquire new functions or be degenerated because of decreased functional constraints. In the gene families of chemoreceptors, for example, there are many putative null mutants that are segregated in human and Drosophila populations (MENASHE et al. 2002, 2003; ROBERTSON et al. 2003; TAKANO-SHIMIZU et al. 2004; A. KAWABE and T. TAKANO-SHIMIZU, unpublished data). These null mutants are typically inferred from the annotated gene sequences, and information on their functional aspects is scarce. A population genetic approach is one way to gain insight into the function and evolutionary dynamics of this class of mutations. If the selective constraints on those duplicated genes are extremely weak due to functional redundancy, it is possible that null mutants act almost like neutral alleles and segregate in a population at some frequency. Alternatively, in some genes under certain conditions, both wild-type and null alleles could be subjected to balancing selection. This is the case with the disease resistance (R) genes of Arabidopsis. In the R genes, Rpm1 and RPS5, the presence/absence polymorphisms of the genes are shown to be under balancing selection (STAHL et al. 1999; TIAN et al. 2002). In these cases, a balance between selection for disease resistance and cost of resistance when pathogens are absent seems to exist. So far, a signature of balancing selection between wild-type and null alleles is rarely observed in genes other than those involved in disease resistance or immunity.

Recently, a large family of odorant-binding proteins (OBPs) was identified in Drosophila (GALINDO and SMITH 2001; GRAHAM and DAVIES 2002; HEKMAT-SCAFE et al. 2002; VOGT et al. 2002). The family contains up to 51 putative members that are expressed in olfactory and gustatory organs (GALINDO and SMITH 2001; HEKMAT-SCAFE et al. 2002). The exact function of these proteins is not known, but they seem to play an important role in odor detection by restricting the odorants accessible to specific receptors (VOGT et al. 1991). We expect to find a reasonable amount of null mutants in OBPs, considering that many such null mutants have been found in the receptor side.

We focused on one tandemly duplicated pair of odorant-binding proteins, Obp57d and Obp57e. These two genes are known to be expressed in the same four cells of tarsi (GALINDO and SMITH 2001) and thus may have an overlapping function that could have weakened the functional constraints on one or both of them. Our survey indeed found two types of null mutations (deletions that disrupt the transcript) in Obp57e. One of them was found at a fairly high frequency in the populations we examined. Our aim in this study was to analyze the surrounding DNA regions of these two genes and to investigate if there was any pattern that deviated from neutrality.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Fly samples and sequencing:
Drosophila melanogaster females were collected from a natural population in Kyoto, Japan, in 2002. The flies were subjected to >9 generations of sib mating to avoid ambiguity in sequencing heterozygotes. The number of generations is preceded by a "G" in each line of Figure 2. Sixteen inbred lines were established as population samples. One D. simulans line from Africa (Sim5G69), which went through 69 generations of sib mating, was used as an outgroup. In addition, 17 worldwide samples from D. melanogaster lines (13 from Africa) were provided by Kyoto Drosophila Genetic Resource Center for the Obp57e genotype survey. These lines are listed in Table 1.



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  		FIGURE 2.— Nucleotide variations detected in the Obp57d and Obp57e regions of 16 D. melanogaster lines from Kyoto, Japan. The first row indicates the nucleotide information of the sequenced D. simulans line. Nucleotide information of 7 other D. melanogaster lines is in the last rows. Numbers in rectangles and ovals indicate positions of amino acid changes and frameshift mutations, respectively. The presence and absence of the sequence at each indel position is indicated, respectively, by + and –.

 

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  		TABLE 1 Presence of Obp57e frameshift mutation (deletion) at positions 1151–1160

 
DNA was extracted from two males from each line using the GenElute Mammalian Genomic DNA kit (Sigma-Aldrich, St. Louis). PCR products were obtained from these genomic samples by the AmpliTaq Gold PCR kit (Applied Biosystems, Foster City, CA) and were purified using MultiScreen-FB filter plates (Millipore, Billerca, CA). These purified products were directly sequenced on both strands using the BigDye terminator cycle sequencing kit version 3 (Applied Biosystems) and run on an ABI 3100 automated sequencer (Applied Biosystems). The genotype survey of the Obp57e position 1151–1160 indel was based on restriction enzyme length polymorphism using HhaI enzyme. All sequences obtained in this study were deposited in the DDBJ/EMBL/GenBank database with the accession nos. AB189644, AB189645, AB189646, AB189647, AB189648, AB189649, AB189650, AB189651, AB189652, AB189653, AB189654, AB189655, AB189656, AB189657, AB189658, AB189659, AB189660, AB189661, AB189662, AB189663, AB189664, AB189665, AB189666, AB189667.

RT-PCR:
RNA was extracted from the adult tissues of KY02073 on the third day after eclosion. Wings, tarsi, labela, antennae, and maxillary palps were collected from 10 individual males and females and stored in RNAlater RNA stabilization reagent (QIAGEN, Valentia, CA). Poly(A)+ RNA was extracted with the Rneasy mini kit (QIAGEN) and cDNA was synthesized using the SuperScript first-strand synthesis system (Invitrogen, Carlsbad, CA). A BD Advantage 2 PCR kit (CLONTECH, BD Biosciences, San Jose, CA) was used to amplify each transcript. The reactions were hot started at 95° for 1 min, then at 95° for 30 sec, 64° for 1 min for 35 cycles, and finally 64° for 1 min. The primers used were as follows: Obp57d, 5'-TGTACCGCATCTGGCTTGTA-3' and 5'-ACTTGTGGGACCTTTTCACG-3'; Obp57e, 5'-TTGGACCAACTTACACTGTGTTT-3' and 5'-ACTGGCCAATTCTCCATCAC-3'. The primers for the ribosomal protein 49 gene (Rp49), 5'-AGATCGTGAAGAAGCGCACCAAG-3' and 5'-CACCAGGAACTTCTTGAATCCGG-3', were used as internal controls. All the primer pairs were selected in the coding regions and designed to span introns. The products from these genes were visualized on a 1.5% agarose gel by loading 10 µl from the 50-µl total reaction volume.

Data analyses:
DNA sequences were aligned using the Clustal W program (THOMPSON et al. 1994). The level of nucleotide diversity ({pi}), nucleotide divergence, population recombination rate (C = 4Ner, where Ne is effective population size and r is recombination rate per site per generation; HUDSON 1987), and linkage disequilibrium (LD) were estimated using the DnaSP program v.4.0 (ROZAS et al. 2003). The sliding-window analyses of {pi} and divergence were also performed by this program. The test of heterogeneity of {pi} in the sliding-window analyses was conducted by generating 100 random sequence data sets by coalescent simulation using ProSeq v.2.91 (FILATOV 2002). The parameters ({pi}, C, etc.) used to generate these sequences were estimated from the population samples. The estimation and permutation tests of FST levels (HUDSON et al. 1992) between the Obp57e indel types at positions 1151–1160 were performed using the ProSeq program. Tajima's test (TAJIMA 1989) and Fay and Wu's test (FAY and WU 2000) were performed using the DnaSP program. We performed 1000 coalescent simulations with the observed level of recombination, C = 0.001, for both regions estimated by HUDSON (1987), which are implemented in the DnaSP program, to determine the critical values of the test statistics. HKA tests (HUDSON et al. 1987) were also performed using the DnaSP program.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
We sequenced the 1585-bp region, including the two tandemly duplicated OBP genes, Obp57d and Obp57e, from 16 inbred lines that originated in Kyoto, Japan. The position of coding regions and variable sites are shown in Figures 1 and 2. One D. simulans line was also sequenced as an outgroup. A deletion caused a frameshift mutation in a coding region (positions 1151–1160) present in 11 of 16 lines, and another deletion (singleton) was at an intron-exon boundary (positions 1226–1241; Figures 1 and 2).



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  		FIGURE 1.— Physical map of the Obp57d and Obp57e regions (shown by arrows). Open rectangles, coding regions of the two genes. Open triangles, insertion/deletion. Numbers indicate the positions of the nucleotide variants detected in D. melanogaster from Kyoto, Japan. Numbers in rectangles and ovals indicate positions of amino acid changes and frameshift mutations, respectively.

 
It was necessary to check that the two OBP genes were functional to determine whether the above mutants are truly null alleles. We first confirmed that the two genes are transcribed in vivo. To corroborate the pattern of promoter fused reporter gene expression by GALINDO and SMITH (2001), we assayed the expression of the two genes in olfactory and gustatory organs using RT-PCR (Figure 3). The results showed Obp57d expression in the wings, tarsi, and labelum (Figure 3A), and Obp57e expression in the tarsi and labelum (Figure 3B). The amount of RNA extracted from 10 individuals, indicated by Rp49 intensities (Figure 3C), differed depending on the organ. We did not control for these differences. However, positive PCR bands indicated expression of these genes at organs other than tarsi, which is a different pattern from the previous study (GALINDO and SMITH 2001).



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  		FIGURE 3.— RT-PCR products from (A) Obp57d and (B) Obp57e transcripts. Lanes indicate cDNA templates derived from the following: W, wings; T, tarsi; L, labela; A, antennae; M, maxillary palps. RNA was extracted from tissues of 10 3-day-old adults of both sexes. (C) Products of ribosomal protein 49 (Rp49) gene served as the internal control. All the primer pairs were designed to span introns.

 
Second, we calculated the nucleotide diversity of nonsynonymous and synonymous sites, {pi}a and {pi}s, respectively, among the four lines with intact exons to determine if the amino acid changes are suppressed by functional constraints. In both genes, the finding that {pi}a < {pi}s (Obp57d—{pi}a = 0.011, {pi}s = 0.038; Obp57e—{pi}a = 0.0032, {pi}s = 0.027) suggests functional constraints in these genes. In addition, among the six amino acid changes in Obp57d and the two found in the four intact Obp57e lines (Figure 1), four were conservative in terms of charge, polarity, and volume using the criteria by ZHANG (2000). Only one site in each of the two genes (at nucleotide positions 624 and 1535) was radically changed in terms of charge. Another site in each of the genes (at positions 538 and 1559) was radically changed in terms of polarity and volume. These results suggest that these OBP genes are functional, because most of the observed amino acid changes were conservative. We also confirmed the exon-intron boundary predicted in the database by sequencing the RT-PCR products (data not shown) to make sure that the deletions found in the coding regions disrupt translation.

We surveyed 13 African isofemale lines and 3 non-African lines to investigate whether the frameshift mutation of Obp57e at positions 1151–1160 is present in other regions of the world. The results indicate that the mutation found in Kyoto is not a locally occurring new mutation, but exists in worldwide populations and thus could be quite old (Table 1).

This frameshift mutation (deletion at positions 1151–1160) could be merely drifting to high frequency due to weak functional constraint. Alternatively, if it is under some type of balancing selection, a sliding-window analysis should reveal elevated nucleotide diversity around this mutation. The analysis showed a peak of nucleotide diversity of silent sites at the window surrounding this mutation (Figure 4A). We tested whether this heterogeneity in nucleotide diversity levels was statistically significant. To be conservative, we used the concatenated sequence of introns and third-position amino acid sites. The concatenated sequence with a size of 998 bp also showed elevated {pi} level at four adjacent 100-bp windows with a step size of 25 bp (175 bp). The maximum {pi} value of 175-bp windows with a step size of 1 was 0.032. In this data set, {pi} = 0.012 and the estimated C = 4Ner = 0.001. We generated 100 sets of 16 random sequences using these parameters by coalescent simulation. The probability that the maximum {pi} value among 175-bp windows with a step size of 1 is ≥0.032 in these generated data sets was P = 0.04. Thus, heterogeneity of nucleotide diversity in this gene was significant at the 5% level. Nevertheless, it should be noted that because the simulations assume a constant panmictic population, we cannot exclude the possibility that the true variance of {pi} and P might be greater than that obtained by the simulation model.



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  		FIGURE 4.— Sliding-window analyses in the Obp57d and Obp57e regions. (A) Sliding-window profile of {pi}. (B) Sliding-window profile of nucleotide diversity within (dotted lines) and between (solid line) wild-type and frameshift mutant classes. (C) Sliding-window profile of nucleotide divergence (average number of nucleotide substitutions per site) between D. melanogaster (16 lines from Kyoto) and D. simulans. Window size, 100 bp; step size 25 bp. Alignment gaps are excluded from the analyses.

 
Another explanation for the high nucleotide diversity around this frameshift mutation is that it is a mutation hotspot. To investigate this possibility, we performed a sliding-window analysis of the divergence between D. melanogaster and D. simulans; Figure 4C shows a moderate elevation of divergence level around positions 1100–1200. However, it is not the highest peak in the diagram or beyond the normal divergence level. Therefore, an elevated mutation rate hypothesis is not supported from our data.

Sliding-window analyses of divergence between wild-type and deletion and that of nucleotide diversity within each of the two types were then performed to determine if the frameshift mutation (deletion at positions 1151–1160) is responsible for the heterogeneity of nucleotide diversity observed in Figure 4A. The former showed a similar peak pattern to the pooled sample, whereas the latter two did not. This indicates that the peak pattern of {pi} is due to divergence of these two alleles. The peak of divergence between the two alleles shown in Figure 4B (0.077 at the window surrounding position 1164) may be close to the divergence level between D. melanogaster and D. simulans, which is estimated to be ~0.05–0.10. For example, the divergence in the Obp57d region was estimated to be 0.10 including all sites, and 0.15 when using silent sites. Recent data by DUMONT et al. (2004) showed that the average divergence between D. melanogaster and D. simulans was 0.078 among 27 intron regions, and 0.124 among synonymous sites of 81 genes.

We also tested for a significant association of Obp57e sequence variation with the wild-type or null (deletion) variation at positions 1151–1160 by analyzing differentiation. We have utilized FST (HUDSON et al. 1992), a parameter generally used to measure population differentiation to test whether the differentiation between two different alleles at positions 1151–1160 is greater than expected from a random mixing of a set of sequences. The strain with another null mutant at positions 1226–1241 (KY02101) was excluded from the analysis. The sequence differentiation between the wild-type and null (deletion) alleles was significant in the Obp57e region (FST = 0.68, P < 0.001), but not in the proximal Obp57d region (FST = 0.17, P = 0.064).

The LD between pairs of all the polymorphic sites is shown in Figure 5A. After correcting for multiple tests, there were 21 significant pairs of sites in the Obp57e region surrounding the position of the frameshift mutation at positions 1151–1160, whereas in the Obp57d region, there was only one significant pair. No significant pairs were found after correction when all the sites from both regions were compared, indicating that a strong LD between the two regions did not exist (see also Figure 2 polymorphism table). There were more pairs of sites in LD in the Obp57e region than in the Obp57d region. To see if this difference was due to the longer range of LD in the Obp57e region, LD expressed by the square of correlation coefficient (R2) was plotted against distance between the pairs of sites (Figure 5B). The expected R2 = 1/(1 + k x distance) + 1/16 (WEIR and HILL 1986), where k is a constant in the unit of 4Ner. The best fit was k = 0.0049 for the Obp57d region and k = 0.0070 for the Obp57e region. R2 falls off more quickly in the latter region than in the former, indicating that the reason for more significant pairs in the latter region was probably not due to the smaller r.



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  		FIGURE 5.— (A) Linkage disequilibrium between polymorphisms in the Obp57d region (left) and the Obp57e region (right). All the phylogenetically informative sites (nonsingletons) are used for the analyses. Pairs of sites that are P < 0.05 by Fisher's exact test after Bonferroni correction are solid. (B) Linkage disequilibrium (R2) plotted against distance (in base pairs) between polymorphic sites in the Obp57d region (left) and the Obp57e region (right). Open circles represent pairs of sites that are P < 0.05 by Fisher's exact test after Bonferroni correction. The fitted lines are for the values of k that minimize the sums of squared deviations between the observed value of R2 and its expectation: R2 = 1/(1 + k x distance) + 1/16 (WEIR and HILL 1986). k is a constant in the unit of 4Nec. The best fit was k = 0.0049 for the Obp57d region and k = 0.0070 for the Obp57e region.

 
Frequency spectra of polymorphic sites in Obp57d and Obp57e regions are shown in Figure 6. There was an excess of high-frequency-derived variants in the Obp57d region (H = –11.75, P = 0.007; FAY and WU 2000; Tajima's D = –0.46, P > 0.10; TAJIMA 1989), indicating a signature of positive natural selection. However, despite the unusual pattern seen in the Obp57e region, the spectrum revealed no significant deviation from neutrality (H = –4.85, P > 0.10; Tajima's D = –0.79, P > 0.10).



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  		FIGURE 6.— Frequency spectrum of the Obp57d region (left) and the Obp57e region (right). Open and solid bars indicate amino acid replacement sites and silent sites, respectively.

 
If the Obp57d region is under positive directional selection and the Obp57e is under balancing selection, as suggested from the above analyses, a lower-than-neutral level of polymorphism is expected in the former and a higher-than-neutral level of polymorphism is expected in the latter. HKA tests were performed between the two regions to test for this relative difference, contrasting with the interspecific divergence levels (Table 2). Since there was an unalignable region between D. melanogaster and D. simulans in the Obp57e region (positions 815–939), it was excluded from the analysis. The test between the two regions was not significant (P = 0.090; Table 2); however, when we tested between the Obp57d region and the region directly flanking the site of frameshift mutation (exon 1 and intron 1 of Obp57e, positions 1108–1231), the result became significant (P = 0.021; Table 2). Because the effect of balancing selection appears only at a narrow region flanking the target site, the comparison of the whole region may not be sensitive enough for this rather conservative test.


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  		TABLE 2 HKA tests for silent-site differences in the Obp57d and Obp57e regions

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
We found a high frequency of a null mutation of Obp57e in a natural population of D. melanogaster. In a population sample from Kyoto, Japan, some aspects of the flanking DNA sequences deviate from the neutral expectation. A peak of nucleotide diversity around this mutation is caused by divergence between the wild-type and the null allele sequences. A sliding-window analysis of the partial sequences from six African lines revealed a similar peak observed among African samples as well (Figure 7). There is a large differentiation between the wild-type and the null alleles (FST = 0.68) and it is statistically significant in this gene region.



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  		FIGURE 7.— Sliding-window profile of nucleotide diversity within (dotted lines) and between (solid line) wild-type and frameshift mutant classes in African lines. The wild-type class consists of MEL6G59, MEL8, and KSA2 lines; the frameshift mutant class consists of MEL7, Algeria, and CA1 lines. Window size, 100 bp; step size, 25 bp, starting on nucleotide position 708. Alignment gaps are excluded from the analyses.

 
Although these observations are consistent with the pattern seen under balancing selection, an alternative possibility of population structure must be seriously considered. Demographic factors are not likely to affect just a portion of the genome, but should influence many regions concurrently. The finding that all of the above patterns are restricted to the region flanking the site that determines the presence/absence of the Obp57e gene and are not seen in the proximal Obp57d gene regions suggests that population structure is probably not the major force forming the haplotype structure in this region.

Another relevant data set is available from the same Kyoto population. INOMATA et al. (2004) analyzed DNA sequences of a gustatory receptor gene, Gr5a, in 152 samples collected at the same collection site in Kyoto in the same year. The authors rejected neutrality by Tajima's test and Fay and Wu's test and argued the possibility of both selection and demographic factors. Nevertheless, further analysis of their data (52 haplotypes with 59 segregating sites identified in 1786 bp) provided us with some information on the population structure. One thousand coalescent simulations on their data showed that the probability for having ≤52 haplotypes would be P = 0.183. Despite the large sample size in their study, the paucity of haplotype numbers, which is evidence of a strong recent population bottleneck, was not detected. Although we cannot deny a possibility of complicated population history affecting our gene region, a simple model of bottleneck-causing biallellism observed in Obp57e is not likely.

Although population bottleneck does not seem to be a large factor in this population, we should note that the level of nucleotide diversity within deletion strains is lower than that within wild-type strains (Figure 4B). Interestingly, this tendency can be seen in the African populations as well (Figure 7). Although many aspects of this gene region favor balancing selection, it may be sensible to consider additional factors beyond a simple selection model to explain the above pattern. One scenario could be that a small number of null mutants have recently spread in many populations by either directional or balancing selection after being maintained for a long time by balancing selection in a relatively restricted area.

Our data showed a higher number of significant pairs of sites in LD in the Obp57e region than in the Obp57d region (Figure 5A), but did not indicate a longer range of LD in the former (Figure 5B). The length of the region affected by balancing selection, characterized by elevated nucleotide diversity and strong LD, is influenced by the population recombination rate C = 4Ner. Several genes in Drosophila show patterns of nucleotide diversity compatible with balancing selection: Adh (KREITMAN and HUDSON 1991), Idgf1 (UROVCOVá and AYALA 2002), and Est-6 (AYALA et al. 2002), as indicated by elevated nucleotide variation around the sites predicted to have functional divergences. The widths of these peaks are all <500 bp, as seen in Obp57e in our study (Figure 4A). In Arabidopsis R genes, compatibility of the balancing selection model has been investigated by coalescent simulation using the estimate of 2Ner from the map distances (STAHL et al. 1999; TIAN et al. 2002; MAURICIO et al. 2003). The widths of the peaks in nucleotide diversity are longer (several kilobases) than those in Drosophila genes. This is not surprising since LD in Arabidopsis is known to be extremely large compared to that of Drosophila. A genomic scale analysis in Arabidopsis thaliana showed that LD decays within ~250 kb (NORDBORG et al. 2002), whereas in D. melanogaster, it typically decays within 1 kb (i.e., LONG et al. 1998). Despite the detailed experimental data on recombination frequencies (map distances), it is still difficult to obtain a reasonable estimate of population recombination rate in Drosophila. Indeed, there is an excess of LD relative to the standard neutral model in the non-African Drosophila population, given the estimated rate of crossing over from the map distances (ANDOLFATTO and PRZEWORSKI 2000; WALL et al. 2002). Therefore, we could not obtain the expected peak width in nucleotide diversity by coalescent simulations as in the studies of Arabidopsis R genes (STAHL et al. 1999; TIAN et al. 2002; MAURICIO et al. 2003).

Our data also could not reject selective neutrality using Tajima's test, which is a standard method for detecting balancing selection. With regard to another R gene in Arabidopsis, Rps2, CAICEDO et al. (1999) initially did not favor a balancing selection model to explain their data because of the nonsignificant Tajima's D. However, MAURICIO et al. (2003) reanalyzed the region with larger-scale data and succeeded in finding other statistical evidence supporting the selection hypothesis. Probably due to the small sample size, Tajima's test lacked power to detect linked variants to the alleles of frequencies 5/16 = 0.32 (wild type) and 11/16 = 0.68 (frameshift) in our data.

GALINDO and SMITH (2001) demonstrated that Obp57d and Obp57e coexpress exclusively in four cells associated with chemosensory bristles of tarsi. They also showed that expressing Grim, a proapoptotic factor that induces programmed cell death, in the cells expressing these genes results in decreased sensitivity to sucrose, which was determined by measuring the proboscis extension reflex. This suggests that the two genes are expressed in cells that are important for gustation. However, GALINDO and SMITH (2001) did not report expression of these genes in wings or labela (despite their investigation of these organs) as shown by RT-PCR in our study (Figure 3). A possible reason for this incongruence may be that they detected expression by fusing several kilobases of an upstream regulatory sequence for each gene to a reporter gene. This assay may have missed expression in some organs due either to regulatory elements further upstream or to a positional effect of the transgene. The null mutant of Obp57e found in our study could serve as a naturally occurring gene knockout for further understanding the function of these OBP genes since, to date, the only mutant available for OBPs in Drosophila is lush, which was generated by P-mutagenesis (KIM et al. 1998).

Although the molecular pattern favors the balancing selection hypothesis for Obp57e, it is not easy to infer the advantage and disadvantage of losing the gene function. One possibility may be something to do with the formation of dimers as pointed out by SáNCHEZ-GRACIA et al. (2003) regarding the two duplicated OSE and OSF genes that are coexpressed in the same cells. The formation of dimers in insect OBPs is demonstrated in physiological conditions (SANDLER et al. 2000). If Obp57d and Obp57e can form heterodimers in the coexpressed cells, the dosage of Obp57e may be important for determining sensitivity to particular odorants. If this is the case, there may be a slight heterozygous advantage of presence/absence alleles at this locus. This possibility has many assumptions that require further experimental investigation. In general, the high frequency of a null mutation may provide insights into the mode of selection as it relates to functional activity of a class of proteins.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Kimiko Suzuki for technical assistance and Masanobu Itoh, Rumi Kondo, and Naruya Saitou for comments and suggestions. We also thank Etsuko Moriyama and an anonymous reviewer for editorial assistance. This work was supported in part by a grant from The Nissan Science Foundation and Grants-in-Aid for Young Scientists (B) from The Ministry of Education, Culture, Sports, Science and Technology of Japan to A.T. and a grant from The Mitsubishi Foundation to T.T-S.


   FOOTNOTES
 
Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession nos. AB189644, AB189645, AB189646, AB189647, AB189648, AB189649, AB189650, AB189651, AB189652, AB189653, AB189654, AB189655, AB189656, AB189657, AB189658, AB189659, AB189660, AB189661, AB189662, AB189663, AB189664, AB189665, AB189666, AB189667.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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