• "Nuclear-mitochondrial fitness interactions in Drosophila", NIH General Medicine, R01 GM067862, $1,095,301, 8/01/04 - 7/31/08, Dr. David Rand, PI; Dr. Bill Ballard, co Investigator. [read more]
   
• "Genetic architecture of thermal selection in Drosophila", NSF Population Biology, DEB 0343464, $536,000, 3/01/04 - 2/28/08, Collaborative Research Award with George Gilchirst at William and Mary ($281,000 to Brown University). [read more]  
   
• Brown University Seed Funds: Evolutionary Response to Nanomaterial Exposure in the Environment: Functional Genomics of C60-Resistance in Drosophila $55,000, 4/2007 - 4/2008. [read more]  
   
• "Rhode Island EPSCoR: Catalyzing a Research, Education and Innovation Network" NSF EPS 05-54548, $6,750,000, 7/06 - 6/09, Dr. Jeff Seeman, URI, P.I. Dr. D. Rand, Brown University Graduate Director ($375,000 in graduate fellowships. [read more]  
   

SUMMARY

Mitochondria consume ~90% of oxygen we breathe and produce ~90% of the ATP we need for normal daily functions. Over 100 genetic diseases are known to affect mitochondrial activity making this one of the most common classes of human pathologies. Mitochondrial function requires the coordinated expression of hundreds of nuclear-encoded genes and 37 mitochondrial-encoded genes, which has two important implications for the genetic basis of disease: 1) the genes regulating mitochondrial function are a large mutational target, and 2) the proper interaction between the two genomes is essential for normal physiological performance. Clinical studies have shown show that the same mtDNA mutations can cause very different phenotypes in different individuals. These effects have been attributed to interactions among linked mutations on the non-recombining mtDNA and to variable nuclear genetic backgrounds. The functional significance of this joint nuclear and mtDNA variation is very poorly understood given the complex interactions between these genomes. An important emerging generality is that mitochondrial diseases tend to be more common in males than in females. This has been attributed to weaker selection against male-specific mitochondrial defects due to the maternal inheritance of mtDNA. An effective test of this male dysfunction hypothesis requires an animal model where epistatic interactions among nuclear and mtDNA alleles can be manipulated in controlled genetic experiments. 

Over the past decade, the PIs have used Drosophila to examine the functional significance of mtDNA polymorphism and have played leading roles in identifying how selection acts on mtDNA and nuclear-mtDNA interactions. The aim of the current proposal is to develop a model in Drosophila to dissect the phenotypic consequences of nuclear and mitochondrial interactions in general populations. 'Mito-nuclear' phenotypes are considered as continuous, quantitative traits that can be dissected with the tools of quantitative genetics (e.g. disequilibrium and quantitative trait locus (QTL) mapping). We describe an approach where a graded series of intra- and interspecific mtDNA haplotypes - differing by a few base pairs up to more than 600 base pairs - can be crossed into diverse wild type and mutant nuclear backgrounds of D. melanogaster. MtDNA haplotypes are shown to have effects on fitness traits, oxygen consumption and mitochondrial enzyme activity, and to mediate epistatic interactions with nuclear genes. We will use this model to provide a robust test of the male mitochondrial dysfunction hypothesis, and to develop strategies for dissecting the genetics of nuclear-mitochondrial interactions. There are four specific aims:

1) Introgress all three of the divergent mtDNAs from D. simulans plus eight additional mtDNAs from D. melanogaster into three D. melanogaster wild type backgrounds and measure the effects of these introgressions on fitness traits, locomotor escape response, oxygen consumption and mitochondrial enzyme activities. This will establish the genotypes used in subsequent Aims, and test the hypothesis that the disruption of phenotypes is more severe in males and is correlated with degree of mtDNA sequence divergence.}

2) Map mito-nuclear QTL for fitness traits, locomotor escape response, oxygen consumption and mitochondrial enzyme activities using recombinant inbred lines (RILs) crossed into each of two mtDNA backgrounds: Oregon-R and D. simulans. Using explicit QTL x mtDNA interaction terms in the analyses, this will enable the mapping of nuclear QTL that show epistatic interactions with mtDNA haplotypes. Traits will be measured in both sexes to test the male dysfunction hypothesis.

3) Test the hypothesis that mtDNAs act epistatically with nuclear backgrounds that complement lethal mutants of nuclear proteins that function in the mitochondria. A quantitative complementation assay will be conducted using 80 wild 2nd chromosomes paired with null mutant alleles of nuclear subunits of mitochondrial enzymes. These genotypes will be placed in a range of increasingly diverged mtDNA backgrounds from D. melanogaster and D. simulans. This design will be used to determine the quantitative effects of mtDNA changes on mito-nuclear interactions for enzyme activities, fitness and performance traits in both sexes.

4) Test the hypothesis that mtDNAs interact genetically with homozygous viable mutations in nuclear genes with known defects in mitochondrial function. This provides a stringent test of intraspecific mtDNA variation in sensitized nuclear backgrounds that serve as models of mitochondrial disease.}

"Genetic architecture of thermal selection in Drosophila", NSF Population Biology, DEB 0343464, $536,000, 3/01/04 - 2/28/08, Collaborative Research Award with George Gilchirst at William and Mary ($281,000 to Brown University).

SUMMARY

For small insects, temperature is one of the most important environmental factors affecting overall fitness. Most biological processes are affected by temperature, which has immediate consequences for the performance of organisms that are isothermal with the environment. In this proposal, we use adaptation to latitudinal temperature patterns on two continents to ask the question: What is the genetic basis of the adaptive response to thermal selection? In previous studies we have shown that Drosophila melanogaster responds rapidly to thermal selection and that the genetic basis of this response maps largely to the X chromosome. Here we combine three approaches to dissect the genomic architecture of thermal selection: 1) artificial selection at distinct temperatures, 2) fine scale molecular genetic mapping of the chromosomal regions associated with the selection response, and 3) corroboration of these quantitative trait loci (QTL) with clinal analysis and association studies of recombinant genotypes from the lab and field. Drosophila melanogaster is ideal for this approach given the abundance of evidence about patterns of thermal adaptation in nature and the wealth of genetic and genomic tools available.

We focus on two thermal traits: knockdown temperature (tempKD), the temperature at which the fly loses the muscle control needed to cling to a vertical surface, and recovery time from cold exposure (chill-coma). In nature, flies exhibit inverse latitudinal clines in knockdown temperature and cold recovery: flies from low latitudes exhibit greater tolerance of high temperatures, but recover less rapidly from cold stress, whereas those from high latitudes are more sensitive to high temperature but have a shorter chill-coma recovery. We have artificially selected flies for high and low tempKD and mapped at least one QTL to bands 3-4 on the X chromosome. This region is of special interest because other lines of flies independently selected for the ability to live and mate at elevated temperatures also show QTL in the same chromosomal region implicating a major gene or genes with significance of thermal adaptation.

We have three specific aims: 1) Fine scale mapping of the X-linked QTL. We will use i) single nucleotide polymorphism (SNP) mapping to narrow the region of interest, and ii) deficiency mapping with Drosophila lines carrying deletions or mutants in the mapped regions to identify candidate genes. 2) Clinal analysis of tempKD, chill-coma recovery, and SNPs associated with the thermal QTL in both North American and Australia. This will test the adaptive significance of our thermal QTL in natural populations and establish the genetic correlations of these complimentary thermal phenotypes in independent clines. 3) Disruptive thermal selection and association analyses of mid-cline populations from North America and Australia. These studies will test the hypothesis that the same underlying genes and alleles are responsible for the variation in thermal phenotypes from independent populations.

The intellectual merit of this research lies in our addressing of two fundamental problems in biology: 1) How does one dissect the genomic basis of a complex adaptive trait? And 2) Are there specific quantitative trait loci that are critical for adaptation to the natural thermal environment? The findings of this study can improve our understanding of how natural populations can adapt to a changing climate.

The broader impacts of the project include the active participation of undergraduates in primary research and the training of graduate students and postdoctoral fellows. The PI's will continue to offer outreach lectures on how scientific research links genetics with environmental issues of international concern. High school students will participate in the research through summer enhancement programs at William & Mary and at Brown.

Brown University Seed Funds: Evolutionary Response to Nanomaterial Exposure in the Environment: Functional Genomics of C60-Resistance in Drosophila $55,000, 4/2007 - 4/2008

SUMMARY

This proposal addresses the ecotoxicological effects of Buckminster fullerene (C60) exposures through an evolutionary, developmental and genomic analysis of Drosophila. A rapidly growing literature has begun to address the toxic effects of nanoparticles (NPs) but no studies have examined the long term environmental effects on whole organisms. We will employ experimental evolution in Drosophila to evolve strains of flies resistant to C60. The genetic and developmental basis of this toxicity will be dissected using the powerful model system of Drosophila. Fruit fly larvae will be exposed to defined nanoscale clusters of C60 (nano-C60 or nC60) in a manner that avoids existing problems with toxic solvent residues and the delivery of hydrophobic C60 to biological tissues. A Darwinian selection regime will be imposed where the top 25% most-resistant flies will be bred as a distinct population, relative to a control strain. The genetic basis of this evolved response will be identified using hybridization of DNA to whole-genome tiled arrays, and subsequent analysis of chromosomal mutations. The developmental bases of these changes will be assessed using mutants with known cellular defects in gut and respiratory morphology, and the ecological effects will be assessed through studies of longevity and fecundity of the evolved strains. Successful completion of this project will put the team in competitive position to apply for grants from the NSF, EPA, and NIH and will strengthen Brown's existing position as an important center for nanotoxicology research by adding a novel ecological and evolutionary component. It will also bring two new biomedical researchers (Rand and Wharton) into Brown's emerging cross-departmental center, the Alliance for Molecular and Nanoscale Innovation. 

"Rhode Island EPSCoR: Catalyzing a Research, Education and Innovation Network" NSF EPS 05-54548, $6,750,000, 7/06 - 6/09, Dr. Jeff Seeman, URI, P.I. Dr. D. Rand, Brown University Graduate Director ($375,000 in graduate fellowships.