Natural variation of the Y chromosome suppresses sex ratio distortion and modulates testis-specific gene expression in Drosophila simulans.

X-linked sex-ratio distorters that disrupt spermatogenesis can cause a deficiency in functional Y-bearing sperm and a female-biased sex ratio. Y-linked modifiers that restore a normal sex ratio might be abundant and favored when a X-linked distorter is present. Here we investigated natural variation of Y-linked suppressors of sex-ratio in the Winters systems and the ability of these chromosomes to modulate gene expression in Drosophila simulans. Seventy-eight Y chromosomes of worldwide origin were assayed for their resistance to the X-linked sex-ratio distorter gene Dox. Y chromosome diversity caused males to sire ∼63% to ∼98% female progeny. Genome-wide gene expression analysis revealed hundreds of genes differentially expressed between isogenic males with sensitive (high sex ratio) and resistant (low sex ratio) Y chromosomes from the same population. Although the expression of about 75% of all testis-specific genes remained unchanged across Y chromosomes, a subset of post-meiotic genes was upregulated by resistant Y chromosomes. Conversely, a set of accessory gland-specific genes and mitochondrial genes were downregulated in males with resistant Y chromosomes. The D. simulans Y chromosome also modulated gene expression in XXY females in which the Y-linked protein-coding genes are not transcribed. The data suggest that the Y chromosome might exert its regulatory functions through epigenetic mechanisms that do not require the expression of protein-coding genes. The gene network that modulates sex ratio distortion by the Y chromosome is poorly understood, other than that it might include interactions with mitochondria and enriched for genes expressed in post-meiotic stages of spermatogenesis.

Evolution of allelic dimorphism in malarial surface antigens.

The extensive sequence variation in most surface antigens of Plasmodium falciparum is one of the major factors why clinical immunity to malaria develops only after repeated infections with the same species over several years. For some P. falciparum surface antigens, all observed alleles clearly fall into two allelic classes, with divergence between classes dwarfing divergence within classes. We discuss the ways in which such allelic dimorphism deviates from the expected shape of the genealogy of genes under either neutral evolution or standard balancing selection, and present a simple test, based on coalescent theory, to detect this deviation in samples of DNA sequences. We review previous hypotheses for the origin and evolution of allelic dimorphism in malarial antigens and discuss the difficulties of explaining the available data under these proposals. We conclude by offering several possible classes of explanations for allelic dimorphism, which are worthy of further theoretical and empirical exploration.

Genome clashes in hybrids: insights from gene expression.

In interspecific hybrids, novel phenotypes often emerge from the interaction of two divergent genomes. Interactions between the two transcriptional networks are assumed to contribute to these unpredicted new phenotypes by inducing novel patterns of gene expression. Here we provide a review of the recent literature on the accumulation of regulatory incompatibilities. We review specific examples of regulatory incompatibilities reported at particular loci as well as genome-scale surveys of gene expression in interspecific hybrids. Finally, we consider and preview novel technologies that could help decipher how divergent transcriptional networks interact in hybrids between species.

Sex-ratio segregation distortion associated with reproductive isolation in Drosophila.

Sex-ratio distortion is the most common form of non-Mendelian segregation observed in natural populations. It may occur even more frequently than direct observations suggest, because the dysgenic population consequences of a biased sex ratio are expected to result in the rapid evolution of suppressors, resulting in suppressed or "cryptic" segregation distortion. Here we report evidence for cryptic sex-ratio distortion that was discovered by introgressing segments of the genome of Drosophila mauritiana into the genome of Drosophila simulans. The autosomal suppressor of sex-ratio distortion, which is also associated with a reduction in hybrid male fertility, has been genetically localized to a region smaller than 80-kb pairs in chromosome 3.

Patterns of DNA sequence variation suggest the recent action of positive selection in the janus-ocnus region of Drosophila simulans.

Levels of nucleotide polymorphism in three paralogous Drosophila simulans genes, janusA (janA), janusB (janB), and ocnus (ocn), were surveyed by DNA sequencing. The three genes lie in tandem within a 2.5-kb region of chromosome arm 3R. In a sample of eight alleles from a worldwide distribution we found a significant departure from neutrality by several statistical tests. The most striking feature of this sample was that in a 1.7-kb region containing the janA and janB genes, 30 out of 31 segregating sites contained variants present only once in the sample, and 29 of these unique variants were found in the same allele. A restriction survey of an additional 28 lines of D. simulans revealed strong linkage disequilibrium over the janA-janB region and identified six more alleles matching the rare haplotype. Among the rare alleles, the level of DNA sequence variation was typical for D. simulans autosomal genes and showed no departure from neutrality. In addition, the rare haplotype was more similar to the D. melanogaster sequence, indicating that it was the ancestral form. These results suggest that the derived haplotype has risen to high worldwide frequency relatively recently, most likely as a result of natural selection.

Recent origin of Plasmodium falciparum from a single progenitor.

Genetic variability of Plasmodium falciparum underlies its transmission success and thwarts efforts to control disease caused by this parasite. Genetic variation in antigenic, drug resistance, and pathogenesis determinants is abundant, consistent with an ancient origin of P. falciparum, whereas DNA variation at silent (synonymous) sites in coding sequences appears virtually absent, consistent with a recent origin of the parasite. To resolve this paradox, we analyzed introns and demonstrated that these are deficient in single-nucleotide polymorphisms, as are synonymous sites in coding regions. These data establish the recent origin of P. falciparum and further provide an explanation for the abundant diversity observed in antigen and other selected genes.

Mitochondrial pseudogenes: evolution's misplaced witnesses.

Nuclear copies of mitochondrial DNA (mtDNA) have contaminated PCR-based mitochondrial studies of over 64 different animal species. Since the last review of these nuclear mitochondrial pseudogenes (Numts) in animals, Numts have been found in 53 of the species studied. The recent evidence suggests that Numts are not equally abundant in all species, for example they are more common in plants than in animals, and also more numerous in humans than in Drosophila. Methods for avoiding Numts have now been tested, and several recent studies demonstrate the potential utility of Numt DNA sequences in evolutionary studies. As relics of ancient mtDNA, these pseudogenes can be used to infer ancestral states or root mitochondrial phylogenies. Where they are numerous and selectively unconstrained, Numts are ideal for the study of spontaneous mutation in nuclear genomes.

Molecular evolution of the ocnus and janus genes in the Drosophila melanogaster species subgroup.

Genes involved in male fertility are potential targets for sexual selection, and their evolution may play a role in reproductive isolation and speciation. Here we describe a new Drosophila melanogaster gene, ocnus (ocn), that encodes a protein abundant in testes nuclear extracts. RT-PCR indicates that ocn transcription is limited to males and is specific to testes. ocn shares homology with another testis-specific gene, janusB (janB), and is located just distal to janB on chromosome 3. The two genes also share homology with the adjacent janusA (janA) gene, suggesting that multiple duplication events have occurred within this region of the genome. We cloned and sequenced these three genes from species of the D. melanogaster species subgroup. Phylogenetic analysis based on protein-encoding sequences predicts a duplication pattern of janA --> janA janB --> janA janB ocn, with the latter event occurring after the divergence of the D. melanogaster and Drosophila obscura species groups. We found significant heterogeneity in the rates of evolution among the three genes within the D. melanogaster species subgroup as measured by the ratio of nonsynonymous to synonymous substitutions, suggesting that diversification of gene function followed each duplication event and that each gene evolved under different selective constraints. All three genes showed faster rates of evolution than genes encoding proteins with metabolic function. These results are consistent with previous studies that have detected an increased rate of evolution in genes with reproductive function.

Genomic gigantism: DNA loss is slow in mountain grasshoppers.

Several studies have shown DNA loss to be inversely correlated with genome size in animals. These studies include a comparison between Drosophila and the cricket, Laupala, but there has been no assessment of DNA loss in insects with very large genomes. Podisma pedestris, the brown mountain grasshopper, has a genome over 100 times as large as that of Drosophila and 10 times as large as that of Laupala. We used 58 paralogous nuclear pseudogenes of mitochondrial origin to study the characteristics of insertion, deletion, and point substitution in P. pedestris and Italopodisma. In animals, these pseudogenes are "dead on arrival"; they are abundant in many different eukaryotes, and their mitochondrial origin simplifies the identification of point substitutions accumulated in nuclear pseudogene lineages. There appears to be a mononucleotide repeat within the 643-bp pseudogene sequence studied that acts as a strong hot spot for insertions or deletions (indels). Because the data for other insect species did not contain such an unusual region, hot spots were excluded from species comparisons. The rate of DNA loss relative to point substitution appears to be considerably and significantly lower in the grasshoppers studied than in Drosophila or Laupala. This suggests that the inverse correlation between genome size and the rate of DNA loss can be extended to comparisons between insects with large or gigantic genomes (i.e., Laupala and Podisma). The low rate of DNA loss implies that in grasshoppers, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than their loss through indel accumulation, whereas the reverse is true for Drosophila. The main factor contributing to the difference in the rates of DNA loss estimated for grasshoppers, crickets, and Drosophila appears to be deletion size. Large deletions are relatively rare in Podisma and Italopodisma.

Chromosomal effects of rapid gene evolution in Drosophila melanogaster.

Rapid adaptive fixation of a new favorable mutation is expected to affect neighboring genes along the chromosome. Evolutionary theory predicts that the chromosomal region would show a reduced level of genetic variation and an excess of rare alleles. We have confirmed these predictions in a region of the X chromosome of Drosophila melanogaster that contains a newly evolved gene for a component of the sperm axoneme. In D. simulans, where the novel gene does not exist, the pattern of genetic variation is consistent with selection against recurrent deleterious mutations. These findings imply that the pattern of genetic variation along a chromosome may be useful for inferring its evolutionary history and for revealing regions in which recent adaptive fixations have taken place.

Directional selection and the site-frequency spectrum.

In this article we explore statistical properties of the maximum-likelihood estimates (MLEs) of the selection and mutation parameters in a Poisson random field population genetics model of directional selection at DNA sites. We derive the asymptotic variances and covariance of the MLEs and explore the power of the likelihood ratio tests (LRT) of neutrality for varying levels of mutation and selection as well as the robustness of the LRT to deviations from the assumption of free recombination among sites. We also discuss the coverage of confidence intervals on the basis of two standard-likelihood methods. We find that the LRT has high power to detect deviations from neutrality and that the maximum-likelihood estimation performs very well when the ancestral states of all mutations in the sample are known. When the ancestral states are not known, the test has high power to detect deviations from neutrality for negative selection but not for positive selection. We also find that the LRT is not robust to deviations from the assumption of independence among sites.

Manifold anomalies in gene expression in a vineyard isolate of Saccharomyces cerevisiae revealed by DNA microarray analysis.

Genome-wide transcriptional profiling has important applications in evolutionary biology for assaying the extent of heterozygosity for alleles showing quantitative variation in gene expression in natural populations. We have used DNA microarray analysis to study the global pattern of transcription in a homothallic strain of Saccharomyces cerevisiae isolated from wine grapes in a Tuscan vineyard, along with the diploid progeny obtained after sporulation. The parental strain shows 2:2 segregation (heterozygosity) for three unlinked loci. One determines resistance to trifluoroleucine; another, resistance to copper sulfate; and the third is associated with a morphological phenotype observed as colonies with a ridged surface resembling a filigree. Global expression analysis of the progeny with the filigreed and smooth colony phenotypes revealed a greater than 2-fold difference in transcription for 378 genes (6% of the genome). A large number of the overexpressed genes function in pathways of amino acid biosynthesis (particularly methionine) and sulfur or nitrogen assimilation, whereas many of the underexpressed genes are amino acid permeases. These wholesale changes in amino acid metabolism segregate as a suite of traits resulting from a single gene or a small number of genes. We conclude that natural vineyard populations of S. cerevisiae can harbor alleles that cause massive alterations in the global patterns of gene expression. Hence, studies of expression variation in natural populations, without accompanying segregation analysis, may give a false picture of the number of segregating genes underlying the variation.

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.

Pseudogene evolution and natural selection for a compact genome.

Pseudogenes are nonfunctional copies of protein-coding genes that are presumed to evolve without selective constraints on their coding function. They are of considerable utility in evolutionary genetics because, in the absence of selection, different types of mutations in pseudogenes should have equal probabilities of fixation. This theoretical inference justifies the estimation of patterns of spontaneous mutation from the analysis of patterns of substitutions in pseudogenes. Although it is possible to test whether pseudogene sequences evolve without constraints for their protein-coding function, it is much more difficult to ascertain whether pseudogenes may affect fitness in ways unrelated to their nucleotide sequence. Consider the possibility that a pseudogene affects fitness merely by increasing genome size. If a larger genome is deleterious--for example, because of increased energetic costs associated with genome replication and maintenance--then deletions, which decrease the length of a pseudogene, should be selectively advantageous relative to insertions or nucleotide substitutions. In this article we examine the implications of selection for genome size relative to small (1-400 bp) deletions, in light of empirical evidence pertaining to the size distribution of deletions observed in Drosophila and mammalian pseudogenes. There is a large difference in the deletion spectra between these organisms. We argue that this difference cannot easily be attributed to selection for overall genome size, since the magnitude of selection is unlikely to be strong enough to significantly affect the probability of fixation of small deletions in Drosophila.

Identification of phylogenetic footprints in primate tumor necrosis factor-alpha promoters.

The human tumor necrosis factor-alpha (TNF-alpha) gene encodes a pleiotropic cytokine that plays a critical role in basic immunologic processes. To investigate the TNF-alpha regulatory region in the primate lineage, we isolated TNF-alpha promoters from representative great apes, Old World monkeys, and New World monkeys. We demonstrate that there is a nonuniform distribution of fixed human differences in the TNF-alpha promoter. We define a "fixed human difference" as a site that is not polymorphic in humans, but which differs in at least one of the seven primate sequences examined. Furthermore, we identify two human TNF-alpha promoter single nucleotide polymorphisms that are putative ancestral polymorphisms, because each of the human polymorphic nucleotides was found at the identical site in at least one of the other primate sequences. Strikingly, the largest conserved region among the primate species, a 69-nt "phylogenetic footprint," corresponds to a region of the human TNF-alpha promoter that forms the transcriptionally active nucleoprotein-DNA complex, essential for gene regulation. By contrast, other regions of the TNF-alpha promoter, which exhibit a high density of variable sites, are nonessential for gene expression, indicating that distinct TNF-alpha promoter regions have been subjected to different evolutionary constraints depending on their function. TNF-alpha is the first case in which a promoter region dissected by functional analyses can be correlated with nucleotide polymorphism and variability in primate lineages. The results suggest that patterns of polymorphism and divergence are likely to be useful in identifying candidate regions important for gene regulation in other immune-response genes.

Solvent accessibility and purifying selection within proteins of Escherichia coli and Salmonella enterica.

The neutral theory of molecular evolution predicts that variation within species is inversely related to the strength of purifying selection, but the strength of purifying selection itself must be related to physical constraints imposed by protein folding and function. In this paper, we analyzed five enzymes for which polymorphic sequence variation within Escherichia coli and/or Salmonella enterica was available, along with a protein structure. Single and multivariate logistic regression models are presented that evaluate amino acid size, physicochemical properties, solvent accessibility, and secondary structure as predictors of polymorphism. A model that contains a positive coefficient of association between polymorphism and solvent accessibility and separate intercepts for each secondary-structure element is sufficient to explain the observed variation in polymorphism between sites. The model predicts an increase in the probability of amino acid polymorphism with increasing solvent accessibility for each protein regardless of physicochemical properties, secondary-structure element, or size of the amino acid. This result, when compared with the distribution of synonymous polymorphism, which shows no association with solvent accessibility, suggests a strong decrease in purifying selection with increasing solvent accessibility.

Evidence for DNA loss as a determinant of genome size.

Eukaryotic genome sizes range over five orders of magnitude. This variation cannot be explained by differences in organismic complexity (the C value paradox). To test the hypothesis that some variation in genome size can be attributed to differences in the patterns of insertion and deletion (indel) mutations among organisms, this study examines the indel spectrum in Laupala crickets, which have a genome size 11 times larger than that of Drosophila. Consistent with the hypothesis, DNA loss is more than 40 times slower in Laupala than in Drosophila.

Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase.

Aberrant repair products of mariner transposition occur at a frequency of approximately 1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5' end of peach (40 alleles), in the 3' end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease "nibble" and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5' inverted repeat are smaller and more frequent than those in the 3' inverted repeat, but secondary mutations in target elements with a 5' lesion usually had 3' lesions resembling those normally found at the 5' end. We suggest that the mariner transposase distinguishes between the 5' and 3' ends of the element, and that the 5' end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.