published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The study of protein polymorphism in natural populations has stimulated heated controversy over the effects of various evolutionary forces on the observed patterns of genetic variation. One viewpoint is that a majority of the mutations at a locus are selectively equivalent and that variations in gene frequencies in time and space are primarily a result of nonselective evolutionary forces. The opposing view is that most mutations have sufficient effect on individual fitness that variations in gene frequencies are adaptations resulting from the action of natural selection. I compared gene frequency distributions among various loci to assess the roles of selective and nonselective evolutionary forces in determining patterns of allozyme variation in populations of rodents. I used two versions of the Lewontin-Krakauer test on temporal variation in allozyme frequencies reported for populations of the prairie vole, Microtus ochrogaster. The tests revealed that the changes in gene frequency were homogeneous among loci which suggests that nonselective forces such as genetic drift and migration were the primary cause of gene frequency change within populations. I also compared the spatial gene frequency distributions reported for 17 species of rodents to assess which evolutionary factors account for the genetic differentiation of populations within each species. Most loci showed similar degrees of differentiation, a pattern expected if nonselective forces operated in population differentiation. I found a positive relationship between the amount of differentiation of populations and the magnitude of positive association among rare alleles. This result suggests an active role of genetic drift in population differentiation within rodent species. The analysis of allozyme distributions in populations of rodents indicates that nonselective evolutionary forces play a substantial role in determining patterns of genetic variation. According to Wright's Shifting Balance Theory, the random differentiation of populations may actually accelerate adaptive evolution, which may account for the rapid evolutionary rates found in rodents.

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Paramutation is an allele interaction that causes a heritable change in the expression of one allele. At the b1 locus an interaction between B' and B-I alleles results in a change of B-I to B', symbolized by B'*. A combination of fine-structure mapping and transgenic approaches have demonstrated that the tandem repeats located ~100 kb upstream of the b1 transcription start site are sufficient for both paramutation and high expression.Plants carrying transgenes with tandem repeats in ectopic locations (repeat-transgene) were able to change B-I into B'*. The B'* state induced by the repeat-transgene was heritable and paramutagenic when segregated from the repeat-transgene. In addition, the repeat-transgene induced B-I silencing was prevented by the trans-acting mutation required for paramutation mop1-1, which was recently found to encode a RNA-dependent RNA polymerase (RdRP). Transgenes containing seven tandem repeats of only the 5' half of the sequence were able to paramutate B-I. Taken together, these results demonstrate that the paramutation sequences are contained in the 5' half of the repeats and they can paramutate B-I from non-allelic positions. Because paramutation induced by the repeat-transgenes and the endogenous B' allele are both heritable and depend on a functional RdRP, they likely involve a similar mechanism of RNA-mediated chromatin modification.Furthermore, we found that the tandem repeats are also sufficient for high expression of the b1 gene. When fused to a GUS reporter gene and introduced into maize, the tandem repeats enhanced GUS expression above the level observed for GUS transgenes that did not have the repeats. As observed with the endogenous B-I allele, the enhancer function of the repeats in the GUS transgenes is silenced by B' and the paramutagenic repeat-transgenes. After being with B' or the paramutagenic repeat-transgenes the repeats in the GUS constructs lost their ability to enhance gene expression.The identification of the tandem repeats as the sequences mediating paramutation suggest a new function for tandem repeats, mediating trans-interactions to establish heritable epigenetic states. Models are discussed for how alleles might communicate in trans to establish different epigenetic states and how the epigenetic state is maintained through mitosis and meiosis.

The normal aging process is accompanied by changes in cognitive function. One of the brain regions known to be an early target of the aging process is the hippocampus, a medial temporal lobe structure that is critically involved in spatial learning and memory function. The formation and maintenance of memory relies on rapid and sustainable synaptic modification, which requires new gene expression. Immediate-early genes are the first genes to be induced following relevant stimuli, and include genes that encode transcription factors, such as c-fos and zif268, and effector proteins that directly influence cellular function, such as Arc (activity-regulated cytoskeletal gene) and Homer1A. Blocking the expression of any one of these genes interferes with memory function, and thus, each of these genes is thought to have a memory enhancing effect. The hypothesis tested here was that aged animals would show a reduction in the expression of memory-promoting immediate-early genes within the hippocampus, and moreover, that these changes in expression would be subregion specific, based on the finding that the dentate gyrus is most vulnerable to the aging process.Potential age-related changes in immediate-early gene expression within the hippocampus was determined under basal conditions and after induction by a simple behavioral task. Of the genes under investigation, only c-fos did not show age-related changes under basal conditions, or following behavioral induction. The remaining genes, Arc, zif268 and Homer1A, each showed subregion specific patterns of change within the hippocampus under basal conditions or following induction (or both). The coordinate expression of immediate-early genes within the hippocampus was also investigated by assessing the extent to which Arc was expressed within the same neurons as c-fos, zif268 or H1a. The coordinate transcription of these genes was not significantly altered in the aged hippocampus, even though changes in the size of Arc and zif268 neural ensembles occurs within the aged denate gyrus.Taken together, these data indicate that age-related reductions in the basal and induced levels of immediate-early genes occur within the hippocampus, and that these changes are subregion specific.

Neutrophils, key cells of the innate immune system, are responsible for preventing bacterial infections. They are rapidly recruited to sites of infection where they eliminate bacteria through killing methods that require reactive oxygen dependent processes. It has recently been established that neutrophils are capable of rapid and complex changes in gene expression during inflammatory responses. The concept that neutrophils only directly kill bacteria has been replaced by the concept that activated neutrophils can influence the immune response through the secretion of a variety of cytokines and by acting as antigen-presenting cell (APC) expressing MHC Class II, allowing for activation of T cells. Recent advances in neutrophil biology demonstrated that neutrophils also have an active regulatory role in angiogenesis and tumoral fate. It has been noted that a number of diseases including arthritis, periodontitis and acute respiratory distress syndrome (ARDS) are associated with neutrophil hyperactivity that results in significant tissue damage. Our group has previously shown that for some periodontal diseases, neutrophil hyperactivity is a key determinant of disease progression and severity. However, it remains unclear what factors are responsible for a patient developing a hyperactive neutrophil mediated disease. I hypothesize that local gene expression changes in neutrophils are responsible for the hyperactive behaviour of these cells during an inflammatory response. In order to assess this, I characterized the neutrophil gene expression profile in various compartments (bone marrow, blood and peritoneum in mice and blood and oral cavity in humans) and then characterized this genetic and phenotypic profile during an inflammatory response. I hypothesize that the neutrophil has a characteristic set of genes that are normally activated when it enters a site of inflammation from the circulation and that neutrophils can be polarized into a different functional subset under certain conditions that result in inflammation mediated diseases. To identify changes in neutrophil gene expression in the circulation and inflamed tissue I used recent advances in neutrophil isolation, RNA amplification, and microarray technologies to characterize the specific transcriptome associated with neutrophil site-specific responses.

Identification and characterization of regions influencing the precise spatial and temporal expression of genes is critical to our understanding of gene regulatory networks. Connecting transcription factors to the cis-regulatory elements that they bind and regulate remains a challenging problem in computational biology. The rapid accumulation of whole genome sequences and genome-wide expression data, and advances in alignment algorithms and motif-finding methods, provide opportunities to tackle the important task of dissecting how genes are regulated. Genes exhibiting similar expression profiles are often regulated by common transcription factors. We developed a method for identifying statistically over-represented regulatory motifs in the promoters of co-expressed genes using weight matrix models representing the specificity of known factors. Application of our methods to yeast fermenting in grape must revealed elements that play important roles in utilizing carbon sources. Extension of the method to metazoan genomes via incorporation of comparative sequence analysis facilitated identification of functionally relevant binding sites for sets of tissue-specific genes, and for genes showing similar expression in large-scale expression profiling studies. Further extensions address alternative promoters for human genes and coordinated binding of multiple transcription factors to cis-regulatory modules. Sequence conservation reveals segments of genes of potential interest, but the degree of sequence divergence among human genes and their orthologous sequences varies widely. Genes with a small number of well-distinguished, highly conserved non-coding elements proximal to the transcription start site may be well-suited for targeted laboratory promoter characterization studies. We developed a “regulatory resolution” score to prioritize lists of genes for laboratory gene regulation studies based on the conservation profile of their promoters. Additionally, genome-wide comparisons of vertebrate genomes have revealed surprisingly large numbers of highly conserved non-coding elements (HCNEs) that cluster nearby to genes associated with transcription and development. To further our understanding of the genomic organization of regulatory regions, we developed methods to identify HCNEs in insects. We find that HCNEs in insects have similar function and organization as their vertebrate counterparts. Our data suggests that microsynteny in insects has been retained to keep large arrays of HCNEs intact, forming genomic regulatory blocks that surround the key developmental genes they regulate.

Neutrophils, key cells of the innate immune system, are responsible for preventing bacterial infections. They are rapidly recruited to sites of infection where they eliminate bacteria through killing methods that require reactive oxygen dependent processes. It has recently been established that neutrophils are capable of rapid and complex changes in gene expression during inflammatory responses. The concept that neutrophils only directly kill bacteria has been replaced by the concept that activated neutrophils can influence the immune response through the secretion of a variety of cytokines and by acting as antigen-presenting cell (APC) expressing MHC Class II, allowing for activation of T cells. Recent advances in neutrophil biology demonstrated that neutrophils also have an active regulatory role in angiogenesis and tumoral fate. It has been noted that a number of diseases including arthritis, periodontitis and acute respiratory distress syndrome (ARDS) are associated with neutrophil hyperactivity that results in significant tissue damage. Our group has previously shown that for some periodontal diseases, neutrophil hyperactivity is a key determinant of disease progression and severity. However, it remains unclear what factors are responsible for a patient developing a hyperactive neutrophil mediated disease. I hypothesize that local gene expression changes in neutrophils are responsible for the hyperactive behaviour of these cells during an inflammatory response. In order to assess this, I characterized the neutrophil gene expression profile in various compartments (bone marrow, blood and peritoneum in mice and blood and oral cavity in humans) and then characterized this genetic and phenotypic profile during an inflammatory response. I hypothesize that the neutrophil has a characteristic set of genes that are normally activated when it enters a site of inflammation from the circulation and that neutrophils can be polarized into a different functional subset under certain conditions that result in inflammation mediated diseases. To identify changes in neutrophil gene expression in the circulation and inflamed tissue I used recent advances in neutrophil isolation, RNA amplification, and microarray technologies to characterize the specific transcriptome associated with neutrophil site-specific responses.