John Logsdon, Ph.D. - US grants

NSF has sponsored a series of science policy seminars for over a decade for the science policy community. PRA wishes to continue the seminar series for an additional three years. Since the current contract for conducting, planning and managing the seminars lapses in November, 1987, PRA issued an RFP to ensure that the new contractor will be in place by then. The purpose of the seminars is to bring together members of the science policy community on a periodic basis in a venue that allows for discussion of science policy issues that are of general interest and concern. Speakers and topics for each of the seven annual seminars are chosen, with NSF input and concurerence, by the contractor, who also manages and conducts the seminars. The seminar series serves to keep PRA, science policy professionals, and local graduate students in the field, abreast of the issues facing the science policy community.

genetic regulation; gene expression; eukaryote; biochemical evolution; meiosis; molecular biology; gene duplication; cell parasexuality; molecular genetics; yeast two hybrid system; polymerase chain reaction; gene complementation;

Much progress has been made toward understanding how meiosis works, but in only a few organisms. It is, thus, unclear which aspects and functions in meiosis are most fundamental. Nonetheless, the data from these organisms provide a solid basis for initiating an evolutionary investigation of meiosis--more specifically, meiotic genes and their encoded proteins--in a more diverse sampling of eukaryotes. The central goal of this project is to better understand the evolution and function of the meiotic process and its molecular machinery. To accomplish this, the investigator will expand his ongoing evolutionary studies of the eukaryotic recA gene family (RAD51 and DMC1) in protists by (a) isolating these genes from additional protist species
representing major eukaryotic lineages, and (b) investigating possible cases of gene loss in the recA family, some of which are associated with putatively asexual species. To complement and support the proposed
experimental studies, he will employ and develop appropriate computational tools to carry out a systematic and comprehensive bioinformatic analysis of additional meiotic genes and proteins. This bioinformatic "data-mining" should result in the identification and initial characterization of key genes encoding meiotic proteins from all available eukaryotic (especially protist) species. In addition to providing information on the function and evolution of meiotic proteins, a major priority of this work is to establish when meiosis evolved, be it during extant eukaryotic evolution (leaving some surviving asexual eukaryotes that lack meiosis because they never evolved it), or alternatively, prior to the diversification of eukaryotes (making contemporary asexual eukaryotes simply the descendants of sexual species which have lost this ability). Phylogenetic analyses of the meiotic genes identified from protists will be used to directly address this issue, especially those genes that have duplicated and diverged from prior non-meiotic functions. The results from this project will guide future experimental efforts to isolate and study particular meiotic genes from other relevant eukaryotic lineages.

Meiosis is the specialized cellular division cycle in which diploid cells are "reduced" to haploid cells (such as eggs and sperm), which then fuse to generate new (diploid) individuals. Meiosis is, thus, central to sexual
reproduction and has been crucial in the evolution and success of eukaryotes. However, the origin and evolution of sex remains one of the major enigmas in biology. Developing a clearer evolutionary understanding of key meiotic mechanisms will not only broadly illuminate our understanding of the sexual process, but also lend insight into the evolution of proteins and the macromolecular assemblages in which they operate. Finally, these studies should clearly demonstrate the importance of combining information from model eukaryotic genetic systems with data from less well-studied organisms in a comparative evolutionary framework.

Abstract for NSF-MRI#0320786

A grant has been awarded to Emory University under the direction of Dr. Justin Gallivan to support acquisition of instruments within the Center for Fundamental and Applied Molecular Evolution. Evolution is the central organizing principle of biology, and is responsible for the intricate complexity of living systems. By combining advances in chemistry, molecular biology, genetics, computation and robotics, it is now possible not only to understand molecular evolution, but also to recapitulate evolutionary processes in the laboratory to solve problems in chemistry, biotechnology, and materials science. The Center for Fundamental and Applied Molecular Evolution (FAME), a joint initiative between Emory and Georgia Tech, was established to seize new opportunities to develop an understanding of the fundamental principles of molecular evolution, and to apply these principles towards the production of new molecules and materials. Researchers within the FAME Center address problems in both fundamental and applied aspects of molecular evolution, such as determining how enzymes evolve to recognize new substrates, altering enzyme substrate specificity, increasing enzyme stability, and evolving enzymes to synthesize new molecules and materials. Solutions to these problems have many practical benefits to society, ranging from understanding the mechanisms of antibiotic resistance to developing new environmentally friendly syntheses of pharmaceuticals using directed molecular evolution. The instrumentation funded by the NSF, including a gene sequencer, high-throughput liquid handling system, colony picker, and plate-reading spectrophotometer, will support two critical elements in the discovery process-the ability to rapidly distinguish evolutionary winners from losers in Darwinian selection, and the ability to understand the molecular basis of the outcome through gene sequencing. The instrumentation will provide researchers within the Center the ability to observe, understand, and direct evolutionary processes in the laboratory in timeframes measured in hours, rather than billions of years.

Acquisition of these instruments will have an immediate and lasting impact on the research and training environment at both Emory and Georgia Tech. The instrument center will serve as a fertile breeding ground to complement the existing intellectual infrastructure. Additionally, these shared instruments will directly support the research efforts of the principal investigators, senior personnel, and their research groups-of these researchers, half are women and several are under-represented minorities. The instruments fill a critical infrastructural need, and will ultimately support the research and training of nearly 100 researchers in the area of molecular evolution over the next three years.

In addition to serving the Emory and Georgia Tech communities, the NSF supported instrumentation will be available to molecular evolution researchers throughout the region. Furthermore, the Center will serve as an important resource in educating the community at large about molecular evolution. Finally, the instruments will aid the quest to understand the basic molecular evolutionary principles that underlie life on earth, and to harness them to solve problems in chemistry, biotechnology, and materials science.

To truly understand biological diversity on Earth requires that microorganisms be correctly placed on the tree of life. Life on Earth exists in two forms: (1) prokaryotes (Bacteria and Archaea), all of which are microbial, and (2) eukaryotes, cells with nuclei. Eukaryotes themselves were exclusively microbial for about one billion years before the evolution of the more familiar macroscopic eukaryotic groups: plants, animals, and fungi. Microbial eukaryotes, or protists, are characterized by tremendous cellular diversity and play an essential role in ecosystems (e.g. carbon fixation in marine systems). Moreover, some microbial eukaryotes are the causative agents of prevalent infectious diseases (e.g. malaria) that impact the social and economic fortunes of many countries. This collaborative project, part of the NSF-funded effort "Assembling the Tree of Life" will elucidate relationships among eukaryotes by analyzing DNA sequences of nine genes from 200 species of predominantly free-living protists. Analyses of the resulting data will combine existing phylogenetic approaches with those developed for this project. These analyses are essential for: 1) unifying the universal tree of life that includes both prokaryotes and eukaryotes, 2) understanding the multiple origins of multicellular eukaryotes, and 3) interpreting the origins of disease-causing protists.

The study will invigorate protist research in the U.S. while answering fundamental questions about the eukaryotic Tree of Life. At least three postdoctoral fellows and three graduate students will be integrated into the proposed research. Undergraduates will also be trained in all aspects of the research, and the principal investigators will maintain their commitment to recruiting students from traditionally underrepresented groups. The project also includes the development of a workshop on collection and identification (by light microscopy) of protists. Finally, micro*scope, a web-based tool for exploring eukaryotic diversity (http://www.mbl.edu/microscope) will be expanded to include educational materials at multiple levels.

DESCRIPTION (provided by applicant): Sexual reproduction is prevalent in eukaryotes and plays a key role in their fitness and survival. Asexual species often arise, but regularly go extinct due, in part, to deleterious mutation accumulation and the inability to adapt to changing environments. Bdelloid rotifers are microscopic animals comprised entirely of females that reproduce asexually by parthenogenesis. Sexual reproduction and meiosis are completely unknown in bdelloids, even though related rotifers are capable of sex. Fossil evidence and molecular genetic studies suggest that bdelloids are "ancient asexuals", persisting for up to 100 million years without sex. Since meiosis is central to sexual reproduction, the central goal of this research is to determine the presence and study the evolution of meiosis-specific genes in genomes of sexual and bdelloid rotifers. Organisms possessing meiotic genes may be capable of meiotic sex. Our approach employs PCR to amplify meiosis- specific genes. We have already amplified partial sequences of four meiotic genes (MND1, DMC1, SPO11, MSH5) from bdelloid rotifers. We will survey the genomes of multiple bdelloid and related sexual species (eight in total) for the presence of these and five additional meiotic genes (MSH4, HOP1, HOP2, REC8, RDH54). This set of genes defines our "meiosis detection kit" that we will use to look for evidence of meiosis in bdelloids. Partial gene sequences will be used as probes to screen genomic (fosmid/cosmid) libraries of bdelloids and their sexual relatives (monogonont and acanthocephalan rotifers). Some libraries are available and we will construct four others. Library screens will allow us to isolate complete genes and to detect additional gene copies not amplified by PCR. Cosmid clones of meiotic genes will be used in subsequent FISH analyses to verify the presence and to document the chromosomal distribution of meiotic genes in rotifer genomes. Rigorous evolutionary analyses of rotifer meiotic genes will assess phylogenetic relationships and evolutionary rates, compared to mitotic genes and with homologs in sexual taxa. These analyses will allow functional inferences for the isolated genes. RNA expression studies using RT-PCR methods will be used to examine the functionality of these genes in sexual and bdelloid rotifers. The results from this project will either support the remarkable status of bdelloid rotifers as ancient asexuals or they will provide genetic evidence suggesting the bdelloids are capable of meiosis and possibly, sexual reproduction. Reproductive modes are crucial to the biology and population genetics of all organisms, and this is especially true for pathogens. Thus, the methods developed here have applications relevant to human health and disease;our approach to infer the presence of meiosis will be relevant to studies of parasite resistance, epidemiology, disease treatment and management.

Mixing of genetic material coming from different individuals is central for the generation of new genetic combinations, and plays a major role in evolution. Most evolutionary models implicitly assume that genetic mixing has a uniform rate at all times. In contrast, this research will investigate an alternative hypothesis: that genetic mixing is plastic, its rate depending on the state of the organism, so that less fit individuals have a higher tendency for mixing. The research will concentrate on three mechanisms of mixing: sexual reproduction, outcrossing and dispersal. The investigator will develop and analyze mathematical models and simulations of fitness-associated mixing, identify conditions favoring the evolution of fitness-associated mixing, and predict its implications for adaptation.

This research is a necessary step towards understanding the evolutionary basis and the implications of plastic genetic variation. By improving our understanding of the way genetic variation acts and evolves, the results of this research will affect many evolutionary and ecological models, and guide future experimental efforts. In addition to its direct theoretical significance, it would improve our understanding of how populations react to a changing environment--an issue of critical importance in conservation biology. It may also provide new insights regarding the accelerated evolution of drug-resistant pathogen strains in response to drug treatment.

The Genomics Core will be housed in the already-established Roy J. Carver Center for Genomics (CCG). This core will support technologically-advanced molecular biological research approaches, including genome-level analyses, needed for the study of inner ear development, physiology and disease. The goal of this core is to provide necessary expertise, technical resources and advanced instrumentation to allow users to identify and assess the underlying molecular processes and mechanisms of relevant inner ear biology. The scientific and technical expertise of the Director and Deputy Director is complementary and the half-time Research Assistant is an accomplished molecular biologist. Users will consult with the Director to determine appropriate methods and will work directly with the RA to develop and implement these procedures. Use of the extensive array of equipment in the CCG will be extended to all users of the Genomics Core. This includes: 2 ABI capillary DNA sequencers, an Experion electrophoresis system, microarray hybridization modules, a microarray scanner, 2 real-time PCR instruments (Roche & ABI), a flow-cytometer, and a Biomek robotic system with a microplate reader. The CCG can provide all necessary technical preparation work for next generation high-throughput DNA sequencing using available instruments at U. Iowa & Iowa State U. (Roc he Genome Sequencer FLX System (aka 454) and an lamina (aka Sol ex a) Genome Analyzer II). The specific aims of the Genomics Core are: 1) To provide access to advanced molecular biology techniques and high-throughput instrumentation for auditory research. 2) To develop and facilitate research using quantitative analysis of spatio-temporal expresssion patterns of selected sets of auditory genes by quantitative RT-PCR and microarray methods. 3) To implement and support genome-wide approaches for auditory research to include high density microarrays and deep transcriptome sequencing. The Genomics Core will work closely with the Histology and Imaging Core to prepare, organize and store molecular probes for in situ analyses. In sum, the Genomics Core will provide key resources for conducting molecular aspects of hearing research at the University of Iowa.

Meiosis is the specialized cell division that halves cell DNA content to generate gametes, which then fused during sexual reproduction. Although meiosis is widespread, some species undergo meiosis despite lacking genes that are required for meiosis in other species. Little is known about why some species have lost meiosis genes but not meiosis. This project will investigate whether lichens, mutually beneficial coexisting fungi and algae, harbor changes in meiosis genes associated with this form of symbiosis. DNA from multiple species of fungi and algae that form lichens will be searched for gene sequences. Evolutionary analyses will compare 17 meiotic genes from multiple lichens to determine whether lichenization is associated with loss or rapid sequence change of meiosis genes.

By characterizing the effects of symbiotic coexistence upon lichenized fungal and algal reproduction, this project will provide an important comparison for other symbioses (e.g. pathogens, mutualists). This project will also further our understanding of how meiosis genes vary while the overall process remains intact. Aspects of this work will be incorporated into on-going educational activities, including high school and undergraduate student research mentoring and annual public-access seminars.

Intellectual Merit: Sexual reproduction is more costly than asexual reproduction, yet nearly all organisms reproduce sexually at least some of the time. Why is sexual reproduction so common despite its costs? Are there significant evolutionary consequences of asexual reproduction? What are the effects of sexual reproduction or its absence on the evolution of genes and genomes? Despite decades of study, these and related questions remain unanswered. Established genetic model systems such as fruit flies and yeast have provided important insights into the genetic and genomic consequences of sex and recombination. However, these systems are limited because they do not offer the ability to make direct comparisons between sexual and asexual organisms--and their genomes--from the same natural populations with similar genetic background and environmental history. This project will use a different organism, Potamopyrgus antipodarum, a New Zealand snail, which has both sexual and independently-derived asexual lineages that make it ideally suited to address fundamental evolutionary questions of how genes and genomes evolve in the absence of sexual reproduction. This research will take advantage of the unique strengths of P. antipodarum and extend them to the genomic level, generating novel insights into the genetic consequences of sexual reproduction and its absence. Analyzing sexual and asexual lineages will make it possible to catch mutation accumulation and gene loss in the act. This research will also provide key steps forward in developing P. antipodarum into a powerful model system for many important biological questions, from host-parasite dynamics to ecotoxicology. Since a key unique element of sexual reproduction is the rapid generation of genetically diverse offspring, research outcomes will also illuminate the extent to which the preservation of genetic diversity within populations, species, and ecological communities is integral to the preservation of biological diversity.


Broader Impacts: The research will provide numerous opportunities for student training and career development in evolutionary biology, molecular genetics, genomics, and bioinformatics. Effort will be focused on the Biosciences Advantage and SROP/McNair programs, which serve students from historically underrepresented and underserved minority groups with interest in a research career. Collaborations will be initiated with two high schools in inner-city Minneapolis serving primarily underrepresented student groups. Science outreach efforts will be directed towards support and expansion of the local Darwin Day civic group, which is dedicated to organizing events aimed at increasing public scientific awareness, comfort, and literacy, and their efforts to initiate a multi-pronged media consortium.