Evidence for Positive Selection in the Superoxide Dismutase (Sod) Region of Drosophila Melanogaster

DNA sequence variation in a 1410-bp region including the Cu,Zn Sod locus was examined in 41 homozygous lines of Drosophila melanogaster. Fourteen lines were from Barcelona, Spain, 25 were from California populations and the other two were from laboratory stocks. Two common electromorphs, SOD(S) and SOD(F), are segregating in the populations. Our sample of 41 lines included 19 Sod(S) and 22 Sod(F) alleles (henceforward referred to as Slow and Fast alleles). All 19 Slow alleles were identical in sequence. Of the 22 Fast alleles sequenced, nine were identical in sequence and are referred to as the Fast A haplotypes. The Slow allele sequence differed from the Fast A haplotype at a single nucleotide site, the site that accounts for the amino acid difference between SOD(S) and SOD(F). There were nine other haplotypes among the remaining 13 Fast alleles sequenced. The overall level of nucleotide diversity (π) in this sample is not greatly different than that found at other loci in D. melanogaster. It is concluded that the Slow/Fast polymorphism is a recently arisen polymorphism, not an old balanced polymorphism. The large group of nearly identical haplotypes suggests that a recent mutation, at the Sod locus or tightly linked to it, has increased rapidly in frequency to around 50%, both in California and Spain. The application of a new statistical test demonstrates that the occurrence of such large numbers of haplotypes with so little variation among them is very unlikely under the usual equilibrium neutral model. We suggest that the high frequency of some haplotypes is due to natural selection at the Sod locus or at a tightly linked locus.     

Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region.     

Molecular and Phenotypic Variation of the Zw Locus Region in Drosophila Melanogaster

Restriction map polymorphism in a 13-kb region of the Zw locus in Drosophila melanogaster was investigated for 64 X chromosome lines with seven 6-cutter and ten 4-cutter restriction enzymes. A total of 203 restriction sites were scored, of which 20 were found to be polymorphic. The estimated nucleotide variation for this region for overall data (pi = 0.003 and 0.001, and theta = 0.003 and 0.002, for 4-cutter and 6-cutter studies, respectively) was smaller than that reported for most regions studied in D. melanogaster. It was found that the Slow allozyme has a larger nucleotide variation and haplotype diversity than the Fast allozyme. Results suggest the relatively recent divergence of the Fast allozyme from the Slow allozyme. Glucose 6-phosphate dehydrogenase (G6PD) activity was measured as a phenotype of the Zw locus. A significant difference in G6PD activity between allozymes was detected. The between-line effect was highly significant within the Slow allozyme, but was not significant within the Fast allozyme. Although a direct causative link could not be established, these results suggest an association between the amounts of quantitative and molecular genetic variation at the Zw locus region.     

The Effect of an Intronic Polymorphism on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

Several lines of evidence indicate that natural selection controls the frequencies of an allozyme polymorphism at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster. However, because of associations among sequence polymorphisms in the Adh region, it is not clear whether selection acts directly (or solely) on the allozymic site. This problem has been approached by using in vitro mutagenesis to distinguish among the effects on Adh expression of individual polymorphisms. This study shows that a polymorphism within the first Adh intron ( &1) has a significant effect on the level of ADH protein. Like the allozyme, & shows a geographic cline in frequency, indicating that it may also be a target of natural selection. These results suggest that multisite selection models may be required to understand the evolutionary dynamics of individual loci.     

The Coalescent Process in Models with Selection and Recombination

The statistical properties of the process describing the genealogical history of a random sample of genes at a selectively neutral locus which is linked to a locus at which natural selection operates are investigated. It is found that the equations describing this process are simple modifications of the equations describing the process assuming that the two loci are completely linked. Thus, the statistical properties of the genealogical process for a random sample at a neutral locus linked to a locus with selection follow from the results obtained for the selected locus. Sequence data from the alcohol dehydrogenase (Adh) region of Drosophila melanogaster are examined and compared to predictions based on the theory. It is found that the spatial distribution of nucleotide differences between Fast and Slow alleles of Adh is very similar to the spatial distribution predicted if balancing selection operates to maintain the allozyme variation at the Adh locus. The spatial distribution of nucleotide differences between different Slow alleles of Adh do not match the predictions of this simple model very well.     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

How intron splicing affects the deletion and insertion profile in Drosophila melanogaster

Studies of "dead-on-arrival" transposable elements in Drosophila melanogaster found that deletions outnumber insertions approximately 8:1 with a median size for deletions of approximately 10 bp. These results are consistent with the deletion and insertion profiles found in most other Drosophila pseudogenes. In contrast, a recent study of D. melanogaster introns found a deletion/insertion ratio of 1.35:1, with 84% of deletions being shorter than 10 bp. This discrepancy could be explained if deletions, especially long deletions, are more frequently strongly deleterious than insertions and are eliminated disproportionately from intron sequences. To test this possibility, we use analysis and simulations to examine how deletions and insertions of different lengths affect different components of splicing and determine the distribution of deletions and insertions that preserve the original exons. We find that, consistent with our predictions, longer deletions affect splicing at a much higher rate compared to insertions and short deletions. We also explore other potential constraints in introns and show that most of these also disproportionately affect large deletions. Altogether we demonstrate that constraints in introns may explain much of the difference in the pattern of deletions and insertions observed in Drosophila introns and pseudogenes.     

A Comprehensive Study of Genic Variation in Natural Populations of Drosophila Melanogaster. IV. Mitochondrial DNA Variation and the Role of History Vs. Selection in the Genetic Structure of Geographic Populations

Preliminary studies with restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) in natural populations of Drosophila melanogaster revealed considerable variation in terms of nucleotide sequence and overall size. In this report we present data from more isofemale lines and more restriction enzymes, and explore the utility of the data in inferring a colonization history of this species. Size variation in the noncoding A + T-rich region is particularly plentiful, with size variants occurring in all restriction site haplotypes in all populations. We report here classes of small-scale mobility polymorphisms (apparent range of 20 bp) in specific restriction fragments in the coding region. The variation in one such fragment appears to be generated even more rapidly than in the noncoding region. On the basis of the distribution of restriction site haplotypes, the species range can be divided into three major regions along longitudinal lines: Euro-African populations are the most diverse and are taken to be oldest; Far East populations have a complex distribution of haplotypes; Western Hemisphere populations are the least diverse and are interpreted to be the youngest. The history inferred from mtDNA alone is remarkably similar to one based on several nuclear markers. The mtDNA haplotype distribution is also very different from that of allozymes in these same populations. We interpret this as further evidence that natural selection is still the most parsimonious explanation for the parallel latitudinal allozyme clines in this species.     

The Rosy Region of Drosophila Melanogaster and Drosophila Simulans. I. Contrasting Levels of Naturally Occurring DNA Restriction Map Variation and Divergence

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.     

Molecular Population Genetics of the Distal Portion of the X Chromosome in Drosophila: Evidence for Genetic Hitchhiking of the Yellow-Achaete Region

We have estimated DNA sequence variation and differentiation within and between Drosophila melanogaster and its sibling species, Drosophila simulans, using six-cutter restriction site variation at yellow-achaete (y-ac), phosphogluconate dehydrogenase (Pgd), and period (per). These three gene regions are of varying distance from the telomere of the X chromosome and range from very low to moderate rates of recombination in D. melanogaster. According to Tajima's test of neutrality, the Pgd region has been influenced by balancing selection in D. melanogaster. This is consistent with previous data suggesting the allozyme polymorphism at this locus is visible to selection. The Hudson, Kreitman, Aguadé test of neutrality reveals a significant departure from neutrality for the y-ac region compared to the per or rosy regions in D. simulans. There is also a significant departure for the y-ac region compared to the Adh 5' flanking region in D. melanogaster. In both species the departure appears to be due to reduced variation at y-ac compared to that expected from divergence between D. simulans and D. melanogaster. We conclude that recent hitchhiking associated with the selective fixation of one or more advantageous mutants in the y-ac region is the best explanation for reduced variation at y-ac.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.