David McCauley - US grants

Meta-populations are populations which are subdivided into many ephemeral, weakly interacting subcomponents. The genetic properties of metapopulations are determined, in large part, by genetic processes accompanying the extinction and recolonization of the local subcomponents. These processes have been the subject of recent theoretical analysis but are not well studied in nature. This study will investigate the population genetic properties of a meta-population of Silene alba, an herbaceous plant that has been the subject of the detailed, long-term demographic analysis required as background data for the study of meta-populations. The basic question to be asked in this project is whether the repeated extinction and recolonization of local demes acts to increase or to diminish genetic differentiation, relative to a similarly subdivided population but without extinction and recolonization. The work will include studies of the genetic properties of the meta-population, additional demographic studies geared towards understanding mechanisms of gene flow, and extension of the theoretical models. Experimental studies of pollen and seed flow, and recruitment from the seed bank, will evaluate sources of gene flow, both into established populations and as part of the process by which new populations are established. Extension of theory will be guided, in part, by the results of the empirical work. //

9707372 McCauley This project will investigate the genetic determinants of sex expression in natural populations of Silene vulgaris, a gynodioecious plant. In plants gynodioecy, or the presence of female and hermaphroditic individuals in the same population, is fairly common. Sex determination in gynodioecious plants is often a consequence of the interaction of genes in the cytoplasm that eliminate male function, and genes in the nucleus that restore it. The study of S. vulgaris will consist of two parts. First, a complex series of "tester" crosses will be conducted in order to characterize genetic diversity at the cytoplasmic and nuclear gene loci. Second, cytoplasmic molecular markers will be developed so that numerous individuals can be classified according to the male sterility types identified by the crosses. This will enable a rapid characterization of the genetic composition of populations. One observation that is often made of gynodioecious species is that the relative numbers of females and hermaphrodites (the sex ratio) can vary dramatically from place to place. One goal of the crosses and molecular studies is to understand the genetic basis for population to population variation in the sex ratio. This will be one of just a few investigations of the genetics of gynodioecy in natural plant populations. The use of male sterility factors to manipulate the gender of economically important crop plants is an important tool in plant breeding, yet the phenomenon is not well understood in nature. In addition to potential benefits to plant breeders, this study will also be of value to the field of basic population genetics, in that it will contribute to an understanding of how genetic diversity is maintained within species.

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McCauley

Gynodioecy is a condition in plant populations in which hermaphroditic and female individuals coexist. It arises when a mutation (called a cytoplasmic male sterility (CMS) factor) causes hermaphrodites to lose male function, with a concurrent increase in seed production. Understanding how gynodioecy persists is an ongoing question in the field of plant population biology. This project will combine molecular genetic markers and genetic crosses to ask how many different CMS factors exist in small natural populations of Silene vulgaris, and how those factors are distributed spatially. This will be combined with mathematical and computer models in order to understand how the extinction and recolonization of local populations influences the evolution of gynodioecy.

This project will be relevant to several broader scientific issues. First, CMS factors are an example of "selfish DNA". The studies of S. vulgaris will be one of the few to link molecular markers to selfish elements in nature. Second, the study of the genetics of small and fragmented populations is significant for conservation biology. This study of S. vulgaris will yield general information about genetic processes in small populations. Finally, gynodioecy is important for agriculture because it is used as a tool by plant breeders for increasing crop yield.

Abstract for NSF Proposal 0140522 - "Equipment for Automated Acquisition of DNA Sequence and DNA Fragment Size Data"

A grant has been awarded to Dr. David E. McCauley at Vanderbilt University to support the purchase of an automated DNA sequencer, a DNA extraction system, and supporting computer hardware. The BaseStation gathers DNA sequence data from slab gels and stores it in a form that can be interpreted by sequence analysis software. The machine can also gather and process other types of DNA fragment size data such as microsatellite DNA genetic markers. In the case of both DNA sequence and microsatellite analysis, products of polymerase chain reaction (PCR) are labeled with a florescent dye and separated by size during electrophoresis. The advantage of the BaseStation is that its 96 well capacity and automated loading allows for high throughput, and its configuration makes it equally suited for gathering sequence and microsatellite data. The associated DNA extraction system will allow for rapid extraction of genomic DNA from a large number of samples. The data gathered by these machines will be used by five P.I.'s (D. McCauley, J. Burke, D. Funk, C. Johnson and N.O. Pellmyr) in a variety of studies in evolutionary and population biology which rely on large quantities of DNA sequence and/or microsatellite data.
Some of the specific research projects that will make use of data generated by the equipment include the following. 1) A study designed to use both DNA sequence information and polymorphic microsatellite markers to investigate the population genetics of Silene vulgaris, a plant invasive in North America. Comparison of the genetic characteristics of the plant in North America and in its native Europe should indicate the mechanism or route of invasion. 2) A comparative molecular systematics study of several species of yucca plants and of the moths that pollinate them. This phylogenetic study will help document how highly coevolved plant-pollinator systems develop. 3) A study using polymorphic microsatellite loci that will help to map genes associated with the domestication of sunflowers from their wild relatives. 4) A DNA sequence based study of adaptive radiation and host plant shifts in the Neochlamisus bebbianae beetle complex. 5) A study of the adaptive significance of biological clock genes that follows changes in the frequency of molecular markers in model laboratory populations of cyanobacteria.
All of these studies will contribute to understanding basic biological processes such as range expansions, coevolution, host plant shifts and the evolution of the biological clock. However, knowledge coming from these studies will also contribute to the solution of more applied problems such as how invasive species might be controlled, how insects shift feeding from native plant species to plants of economic value, how the genetic manipulation of domesticated species might be made more efficient, or how we might overcome health problems associated with biological rhythm disorders. Finally, the results of all of the studies will contribute to the emerging field of bioinformatics, in that each study will require the P.I. to refine methods of gathering, storing, and analyzing a large volume of genetic information.

Plants and animals carry DNA in their mitochondria, in addition to the majority of genes that are carried on nuclear chromosomes. In flowering plants the mitochondrial DNA (mtDNA) is often inherited from the mother only, such that the copies of mtDNA within an individual are identical. Recent studies of the plant Silene vulgaris showed that inheritance of mtDNA is sometimes from both the mother and father, resulting in a heterogeneous mix of mtDNA molecules in the offspring (heteroplasmy). This study will investigate the frequency of non-maternal inheritance of mtDNA through experimental crosses of S. vulgaris conducted in the greenhouse, and document the frequency and magnitude of heteroplasmy in natural populations, using the tools of molecular biology.

The results will be of value to population biologists because mtDNA is used as a genetic marker in many types of studies, under the assumption of maternal inheritance. This assumption has only been tested directly in relatively few species, and the consequences of non-maternal inheritance are not well understood. The results will also interest applied biologists whose goal is to develop lines of economically important plants that produce a high proportion of females for breeding purposes, given that sex determination in plants is often traced to mitochondrial genes.

[unreadable] DESCRIPTION (provided by applicant): All vertebrates, including humans, contain chondrocytes that secrete a cartilaginous matrix, but the origin of chondrogenic mechanisms within the vertebrates is not well understood. Sox9 is an HMG-box transcription factor expressed in the neural crest that regulates expression of Type II collagen (Col2a1), the major matrix protein in vertebrate cartilage. Sox9 is a member of the SoxE subfamily of Sox genes. Recent research indicates SoxE genes play an essential role in proper development of branchial arch cartilage and formation of pharyngeal pouches in lower vertebrates. The research described in this proposal will elucidate the function of SoxE genes, SoxE1, SoxE2 and SoxE3, during chondrogenesis of the branchial arches in the sea lamprey Petromyzon marinus. The current project has two Specific Aims: (1) to determine if SoxE genes cross-regulate their own expression in the developing pharynx; (2) to determine if SoxE genes regulate expression of Col2a1 in the sea lamprey. A combination of molecular techniques, including overexpression, morpholino knock-down, and in situ hybridization will be used. This research will contribute to two goals. The primary goal is to determine if Col2a1 is regulated by multiple SoxE genes. People who suffer from haploinsufficiency of Sox9 are characterized by campomelic dysplasia, a semilethal osteochondrodysplasia characterized by skeletal abnormalities, and mice haploinsufficient for Sox9 develop cleft palate prior to perinatal death, suggesting two functioning copies of Sox9 are required for normal craniofacial and skeletal development in mammals, including humans. This research will form the background for understanding if the need for expression of multiple copies of the single Sox9 gene during chondrogenesis may have arisen from multiple SoxE factors expressed in the chondrogenic neural crest in early vertebrates. The secondary goal is to determine if SoxE genes regulate the expression of other SoxE transcription factors in the lamprey. In vertebrate models studied to date, Sox9 and Sox10 have partially overlapping expression domains and overexpression or loss of function of Sox10 can affect the expression of Sox9, suggesting regulatory interaction between these related genes. This research will determine if the requirement of multiple copies of SoxE genes for proper chondrogenes may have arisen early in vertebrate history following duplication of a single ancestral SoxE gene, and will form the basis for further investigation to uncover the origin of the control of chondrogenesis and pharyngeal patterning mechanisms by SoxE transcription factors. This project seeks to determine in a primitive vertebrate, how duplication of SoxE transcription factors may be important for proper control of cartilage development in all vertebrates. It will be valuable to understand how the regulation of collagen expression by obligate multiple functioning copies of a SoxE gene (Sox9) arose since other roles of Sox9 do not depend on multiple copies. Since a greater understanding of the control of chondrogenesis by Sox9 depends on understanding this role where it may have arisen in early vertebrates, this study serves the greater public good because it will shed light on the origin of chondrogenic pathologies in humans, such as campomelic dysplasia, that depend on multiple functioning Sox9 alleles for proper expression of type II collagen, and chondrogenesis. [unreadable] [unreadable] [unreadable]

Cartilage is an important component of the vertebrate skeleton, but its evolutionary origin is poorly understood. Neural crest cells evolved in vertebrates and are required for the development of numerous vertebrate-specific characters, including cartilage within the head. The central goal of Dr. McCauley's research is to determine how the evolution and development of cartilage in vertebrates is related to the duplication of a single Sox E gene that is also present in invertebrates. The duplicated SoxE genes, Sox8, Sox9, and Sox10 play an important role in neural crest specification, but the importance of this gene duplication to the evolution of facial cartilage is not known. Dr. McCauley will use a molecular approach to interfere with the function of individual Sox proteins in a primitive vertebrate, the sea lamprey, to determine their roles in the development of cartilage from neural crest. The lamprey was chosen because of its primitive relationship to other vertebrate species. Results of this research are expected to reveal how lamprey SoxE genes regulate cartilage development and whether the fundamental developmental mechanisms of cartilage formation derived from neural crest cells are common to all vertebrates. This research will have broad impacts for the scientific and education communities. These studies will integrate research and higher education and will involve undergraduate students from diverse backgrounds in original laboratory research, in addition to training graduate students and post-doctoral investigators. Results will be of interest to a diverse audience since studies on the "primitive" lamprey are relevant to understanding the evolution and development of "advanced" model vertebrates. Techniques developed and used in this research will also be applied to a laboratory course currently under development by Dr. McCauley. Students in this course will learn how molecular, developmental, and bioinformatics tools are used to address questions related to the evolution of novel traits.

This research project will expand our understanding of the causes and consequences of an unusual form of inheritance in plants, the transmission of organelles through pollen. A defining feature of cells with nuclei is the existence of organelles that have separate genomes. The nuclear genome is generally inherited from two parents. By contrast, the genomes of organelles, such as those in mitochondria, are nearly always inherited from the mother only. However, we know little about the frequency and cause of deviations from this general rule. Understanding the inheritance of organelle genomes is therefore important for understanding evolutionary processes. Using the plant Silene vulgaris as a model system, this project will explore the relationship between the geographic origins of the nuclear genome and paternal mitochondrial transmission, and the relationship between the physical structure of the mitochondrial genome and the propensity for mitochondrial genes to recombine. This requires 1) sequencing several S. vulgaris mitochondrial genomes to document genome structure and identify genetic markers used to study recombination, 2) genotyping individuals from natural populations in order to estimate recombination, 3) conducting experimental crosses between individuals of varying nuclear and mitochondrial genotypes, and 4) genotyping their offspring to identify incidences of mitochondrial paternal inheritance.

In addition to furthering fundamental knowledge of plant mitochondrial genome biology, the results should be of applied significance. Plant breeders are concerned with developing CMS (cytoplasmic male sterility) resources because utilization of male-sterile individuals in breeding programs increases efficiency, and CMS genes usually reside in the mitochondrial genome. Studies of the inheritance of organellar genomes are also valuable because of concerns about gene escape from genetically modified organisms.

This Major Research Instrumentation award funds the acquisition of a multi-photon laser scanning confocal microscope for research and training in interdisciplinary fields on the University of Oklahoma (OU) Norman campus. The new instrument has higher sensitivity and faster speed and will significantly boost the capabilities for live and deep-tissue imaging by a diversity of life science and engineering researchers on the OU campus and at nearby research institutions. This new confocal microscope will be used by a number of NSF-sponsored laboratories that are already active in cell and molecular imaging as well as by new users who will incorporate confocal imaging in their research projects. The research topics include, but they are not limited to, neural crest migration in the primitive vertebrate lamprey, neurite outgrowth and synaptic vesicle trafficking in fruit flies, calcium imaging of deep nerve tissues, temporal and spatial patterns of protein complex formation in plants, and biofilm growth and morphology during corrosion of metallic surface. The new microscope will be an important part of education and research integration; students will be trained initially through a new upper undergraduate and graduate student course, Applied Confocal Imaging, taught collectively by the PIs. In addition, the PIs and their students will collaborate with rural and urban Oklahoma K-12 teachers in developing and implementing cutting-edge outreach and classroom projects. The results of these research, teaching, and outreach efforts will be broadly disseminated through participation of students and faculty at professional meetings and through peer-reviewed publications.