Evolution of heat-shock protein expression underlying adaptive responses to environmental stress.

Heat-shock proteins (Hsps) and their cognates are primary mitigators of cell stress. With increasingly severe impacts of climate change and other human modifications of the biosphere, the ability of the heat-shock system to affect evolutionary fitness in environments outside the laboratory and to evolve in response is topic of growing importance. Since the last major reviews, several advances have occurred. First, demonstrations of the heat-shock response outside the laboratory now include many additional taxa and environments. Many of these demonstrations are only correlative, however. More importantly, technical advances in "omic" quantification of nucleic acids and proteins, genomewide association analysis, and manipulation of genes and their expression have enabled the field to move beyond correlation. Several consequent advances are already evident: The pathway from heat-shock gene expression to stress tolerance in nature can be extremely complex, mediated through multiple biological processes and systems, and even multiple species. The underlying genes are more numerous, diverse and variable than previously appreciated, especially with respect to their regulatory variation and epigenetic changes. The impacts and limitations (e.g., due to trade-offs) of natural selection on these genes have become more obvious and better established. At last, as evolutionary capacitors, Hsps may have distinctive impacts on the evolution of other genes and ecological consequences.

Phylogeny disambiguates the evolution of heat-shock cis-regulatory elements in Drosophila.

Heat-shock genes have a well-studied control mechanism for their expression that is mediated through cis-regulatory motifs known as heat-shock elements (HSEs). The evolution of important features of this control mechanism has not been investigated in detail, however. Here we exploit the genome sequencing of multiple Drosophila species, combined with a wealth of available information on the structure and function of HSEs in D. melanogaster, to undertake this investigation. We find that in single-copy heat shock genes, entire HSEs have evolved or disappeared 14 times, and the phylogenetic approach bounds the timing and direction of these evolutionary events in relation to speciation. In contrast, in the multi-copy gene Hsp70, the number of HSEs is nearly constant across species. HSEs evolve in size, position, and sequence within heat-shock promoters. In turn, functional significance of certain features is implicated by preservation despite this evolutionary change; these features include tail-to-tail arrangements of HSEs, gapped HSEs, and the presence or absence of entire HSEs. The variation among Drosophila species indicates that the cis-regulatory encoding of responsiveness to heat and other stresses is diverse. The broad dimensions of variation uncovered are particularly important as they suggest a substantial challenge for functional studies.

Locomotion in response to shifting climate zones: not so fast.

Although a species' locomotor capacity is suggestive of its ability to escape global climate change, such a suggestion is not necessarily straightforward. Species vary substantially in locomotor capacity, both ontogenetically and within/among populations, and much of this variation has a genetic basis. Accordingly, locomotor capacity can and does evolve rapidly, as selection experiments demonstrate. Importantly, even though this evolution of locomotor capacity may be rapid enough to escape changing climate, genetic correlations among traits (often due to pleiotropy) are such that successful or rapid dispersers are often limited in colonization or reproductive ability, which may be viewed as a trade-off. The nuanced assessment of this variation and evolution is reviewed for well-studied models: salmon, flying versus flightless insects, rodents undergoing experimental evolution, and metapopulations of butterflies. This work reveals how integration of physiology with population biology and functional genomics can be especially informative.

Contrasting patterns of transposable element insertions in Drosophila heat-shock promoters.

The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila.

Location of P element insertions in the proximal promoter region of Hsp70A is consequential for gene expression and correlated with fecundity in Drosophila melanogaster.

We compared a series of Drosophila strains with P element insertions from -28 to -144 nucleotides 5' to the transcription start site of the Hsp70A genes-corresponding to the range of naturally occurring P element insertion sites-to elucidate the consequences of insertion site for Hsp70A gene expression. Although all insertions reduced Hsp70A expression below that of a control strain, the magnitude of the reduction was inversely related to the number of nucleotides between the transcription start site and the insertion site. A pre-existing hypothesis is that naturally occurring transposable element insertions in Hsp promoters may be beneficial in some circumstances, which may account for their retention in natural populations. In the present study, in a control line heat shock reduced fecundity, whereas in lines with P element insertions heat shock typically increased fecundity. Finally, according to cluster-specific quantitative RT-PCR, expression of the Hsp70A cluster genes was typically greater than that of the Hsp70B gene cluster genes, although the latter are more numerous and, in this case, free of P element insertions.

Evidence-based appointment and promotion of academic faculty at the University of Chicago.

The authors report how one academic medical center (AMC) and associated nonclinical departments implemented evidence-based academic criteria and an evidence-based academic vetting process, which may be models for other institutions. In 2004-2005, The University of Chicago Division of the Biological Sciences and Pritzker School of Medicine reconceptualized its appointment, promotion, and tenure criteria to recognize all forms of scholarship as equally legitimate bases for academic tenure. The revised criteria also accommodate differences in academic effort consistent with varying clinical demands. Implementation of these criteria, however, necessitated revised practices in providing objective evidence and analysis of their satisfaction. Three complementary mechanisms now yield excellent evidence and analysis. The first, electronic forms (e-forms) comprise highly specific response items with embedded instructions, advice, and rationale. The e-forms encourage candidates and departments to provide the evidence that subsequent review needs to evaluate appointment or promotion proposals. Unexpectedly, the e-forms have been coopted as effective mechanisms for faculty development. Second, a faculty dean of academic affairs, a regular faculty member, was appointed to provide robust academic authority and perspective to the process. Third, the promotion and tenure advisory committee was restricted to evaluating academic criteria, and from considerations of institutional value. This change interposed a "firewall" between academic and institutional review. These changes have attenuated dissatisfaction with the appointments and promotions process both within and outside the AMC.

Key issues in achieving an integrative perspective on stress.

An integrative perspective on molecular mechanisms of stress resistance requires understanding of these mechanisms not just in vitro or in the model organism in the research laboratory - but in the healthy or diseased human in society,in the cultivated plant or animal in agricultural production,and in populations and species in natural communities and ecosystems. Such understanding involves careful attention to the context in which the organism normally undergoes stress,and appreciation that biological phenomena occur at diverse levels of organization (from molecule to ecosystem). Surprisingly,three issues fundamental to achieving an integrative perspective are presently unresolved: (i) Is variation in lower-level traits (nucleotide sequences, genes, gene products) seldom, commonly, or always consequential for stress resistance? (ii) Does environmental stress reduce or enhance genetic variation, which is the raw material of evolution? (iii) Is the present distribution of organisms along natural gradients of stress largely the result of organisms living where they can, or is adaptive evolution generally sufficient to overcome stress? Effective collaboration among disciplinary specialists and meta-analysis may be helpful in resolving these issues.

Evolvability of physiological and biochemical traits: evolutionary mechanisms including and beyond single-nucleotide mutation.

A longstanding challenge for biologists has been to explain not just how organisms are adapted to diverse environments, but how these adaptations arise. Although natural selection is clearly sufficient to act on heritable variation, is this heritable variation sufficient to yield complex adaptations and how does this variation itself arise? Much prior focus has been on mutation of single nucleotides in genes. This process is common and can have dramatic phenotypes, but could be limited in its ability to culminate in complex adaptations for two kinds of reasons: (i) because natural selection is powerful, it can purge genetic variation, and (ii) evolutionary transition from the absence to the presence of a complex adaptation seemingly requires multiple mutations at the right place and time and in the right sequence, with each intermediate stage having increased overall fitness; this seems highly improbable. Because the networks that organisms comprise are hierarchical and redundant and have modular structure, however, single-nucleotide mutations can have large and tolerable impacts. Diverse mechanisms, collectively evolutionary capacitors, can shield genetic variation from the purgative of selection. These features can enable evolution to proceed via single-nucleotide mutation. Importantly, single-nucleotide mutation usually only modifies existing genes rather than creating new ones, and numerous other mechanisms eclipse single-nucleotide mutation in creating genetic variation. These include gene duplication (both segmental and whole-genome), lateral gene transfer, hybridization, mobile genetic elements and symbiosis. Other processes can scramble and reassemble nucleotide sequence. The mechanisms beyond single-gene mutation offer considerable promise in detailing the evolution of complex physiological and biochemical traits, and have already done so for several morphological traits.

Heat-shock promoters: targets for evolution by P transposable elements in Drosophila.

Transposable elements are potent agents of genomic change during evolution, but require access to chromatin for insertion-and not all genes provide equivalent access. To test whether the regulatory features of heat-shock genes render their proximal promoters especially susceptible to the insertion of transposable elements in nature, we conducted an unbiased screen of the proximal promoters of 18 heat-shock genes in 48 natural populations of Drosophila. More than 200 distinctive transposable elements had inserted into these promoters; greater than 96% are P elements. By contrast, few or no P element insertions segregate in natural populations in a "negative control" set of proximal promoters lacking the distinctive regulatory features of heat-shock genes. P element transpositions into these same genes during laboratory mutagenesis recapitulate these findings. The natural P element insertions cluster in specific sites in the promoters, with up to eight populations exhibiting P element insertions at the same position; laboratory insertions are into similar sites. By contrast, a "positive control" set of promoters resembling heat-shock promoters in regulatory features harbors few P element insertions in nature, but many insertions after experimental transposition in the laboratory. We conclude that the distinctive regulatory features that typify heat-shock genes (in Drosophila) are especially prone to mutagenesis via P elements in nature. Thus in nature, P elements create significant and distinctive variation in heat-shock genes, upon which evolutionary processes may act.

Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster.

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions -96 and -97, and no insertions were into a coding or 3'-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides -96 and -97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act.

Reverse transcriptional profiling: non-correspondence of transcript level variation and proximal promoter polymorphism.

BACKGROUND: Variation in gene expression between two Drosophila melanogaster strains, as revealed by transcriptional profiling, seldom corresponded to variation in proximal promoter sequence for 34 genes analyzed. Two sets of protein-coding genes were selected from pre-existing microarray data: (1) those whose expression varied significantly and reproducibly between strains, and (2) those whose transcript levels did not vary. Only genes whose regulation of expression was uncharacterized were chosen. At least one kB of the proximal promoters of 15-19 genes in each set was sequenced and compared between strains (Oregon R and Russian 2b). RESULTS: Of the many promoter polymorphisms, 89.6% were SNPs and 10.4% were indels, including homopolymer tracts, microsatellite repeats, and putative transposable element footprints. More than half of the SNPs were changes within a nucleotide class. Hypothetically, genes differing in expression between the two strains should have more proximal promoter polymorphisms than those whose expression is similar. The number, frequency, and type of polymorphism, however, were the same in both sets of genes. In fact, the promoters of six genes with significantly different mRNA expression were identical in sequence. CONCLUSION: For these genes, sequences external to the proximal promoter, such as enhancers or in trans, must play a greater role than the proximal promoter in transcriptomic variation between D. melanogaster strains.

Unusual arrangement of the hsp68 locus in the virilis species group of Drosophila implicates evolutionary loss of an hsp68 gene.

Unlike all other Drosophila species studied to date, species in the virilis group of Drosophila have 2 complete copies of hsp68 arranged in inverted head-to-head orientation. Evidence for this conclusion includes Southern blots for D. virilis, D. lummei, and D. montana, PCR analysis of the former 2 species, in situ hybridization in D. virilis x D. lummei hybrids, and the complete nucleotide sequence of the locus in D. lummei. This organization resembles the primitive state of hsp70 in Diptera. Moreover, the Hsp68 peptide sequence for D. virilis and D. lummei is intermediate between that of Hsp70 and Hsp68 from other Drosophila spp. Therefore, we suggest that the hsp68 locus may have arisen via duplication of the hsp70 locus (or vice versa) early in the history of the genus Drosophila, with 1 hsp68 copy subsequently lost in most other Drosophila species groups.

Aims of undergraduate physiology education: a view from the University of Chicago.

Physiology may play an important, if not essential role, in a liberal arts education because it provides a context for integrating information and concepts from diverse biological and extra-biological disciplines. Instructors of physiology may aid in fulfilling this role by clarifying the core concepts that physiological details exemplify. As an example, presented here are the core principles that are the basis for an undergraduate physiology course taught at the University of Chicago. The first of these is: Evolution has resulted in organisms comprising mechanisms for maintenance, growth, and reproduction, despite perturbations of the internal and external environment. Such principles necessitate a coupling of physiology to diverse disciplines (i.e., "sciomics") and provide a basis for integrating discoveries in other disciplines.

What evidence is there for the existence of individual genes with antagonistic pleiotropic effects?

Classical evolutionary theory predicts the existence of genes with antagonistic effects on longevity and various components of early-life fitness. Quantitative genetic studies have provided convincing evidence that such genes exist. However, antagonistic pleiotropic effects have rarely been attributed to individual loci. We examine several classes of longevity-assurance genes: those involved in regulation of the gonad; the insulin-like growth factor pathway; free-radical scavenging; heat shock proteins and apoptosis. We find initial evidence that antagonistic pleiotropic effects are pervasive in each of these classes of genes and in various model systems--although most studies lack explicit studies of fitness components. This is particularly true of human studies. Very little is known about the early-life fitness effects of longevity loci. Given the possible medical importance of such effects we urge their future study.

Naturally occurring transposable elements disrupt hsp70 promoter function in Drosophila melanogaster.

Naturally occurring transposable element (TE) insertions that disrupt Drosophila promoters are correlated with modified promoter function and are posited to play a significant role in regulatory evolution, but their phenotypes have not been established directly. To establish the functional consequences of these TE insertions, we created constructs with either TE-bearing or TE-lacking hsp70 promoters fused to a luciferase reporter gene and assayed luciferase luminescence in transiently transfected Drosophila cells. Each of the four TEs reduces luciferase signal after heat shock and heat inducibility of the hsp70 promoter. To test if the differences in hsp70 promoter activity are TE-sequence dependent, we replaced each of the TEs with multiple intergenic sequences of equal length. These replacement insertions similarly reduced luciferase signal, suggesting that the TEs affect hsp70 promoter function by altering promoter architecture. These results are consistent with differences in Hsp70 expression levels, inducible thermotolerance, and fecundity previously associated with the TEs. That two different varieties of TEs in two different hsp70 genes have common effects suggests that TE insertion represents a general mechanism through which selection manipulates hsp70 gene expression.

Evolution and arrangement of the hsp70 gene cluster in two closely related species of the virilis group of Drosophila.

To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.

Dropping like flies: environmentally induced impairment and protection of locomotor performance in adult Drosophila melanogaster.

In Drosophila, heat shock (HS) during the pupal stage chronically hinders adult locomotor performance by disrupting wing development and cellular and/or tissue-level mechanisms that support walking and flight. Furthermore, heat pretreatment (PT) protects locomotor function against these disruptions. HS flies with abnormal wings were less able to alter trajectory in free fall relative to control, PT-only, and PT+HS wild-type flies. This deficit was less severe but still present in HS-only flies with wild-type wings. Transgenic increases in the copies of genes encoding the major inducible heat-shock protein of Drosophila melanogaster, Hsp70, also protected walking ability from disruption due to pupal HS. Walking velocity did not differ between excision (five natural hsp70 copies) and extra-copy (five natural and six transgenic hsp70 copies) flies in the control, PT, and PT+HS groups, nor did velocity vary among these thermal treatment groups. HS dramatically reduced walking velocity, however, but this effect occurred primarily in the excision flies. These results suggest that Hsp70 and other mechanisms protect against heat-induced locomotor impairment.