The tempo of evolution and the causes of rate variation among lineages are central foci of evolutionary biology. I evaluated two hypothesized sources of variation in molecular evolutionary rate, and I applied a variable molecular clock to estimate the timescale of diversification in three families of Neotropical birds. First, I examined the phylogenetic evidence for molecular punctuated equilibrium, the hypothesis that speciation drives accelerated molecular evolution. Recent findings that rates of DNA evolution and speciation are linked implicate molecular punctuated equilibrium as an important cause of rate variation among lineages. I used phylogenetic simulations to test this reported link, and I found that it was entirely attributable to a methodological artifact. In a review of the topic, I found no unequivocal empirical evidence for molecular punctuated equilibrium and I concluded that its predicted phylogenetic consequences are theoretically implausible. Second, I tested the metabolic rate hypothesis, which holds that mutation rate in mtDNA is correlated with mass-specific metabolic rate. This hypothesis predicts that small-bodied lineages should evolve rapidly. Previous studies verified this prediction, but none utilized adequately large samples of independent contrasts among appropriate taxa. The use of many such contrasts from bird mtDNA sequences conspicuously failed to corroborate the link between metabolic and mtDNA rates. On the contrary, high rates of nonsynonymous substitution were associated with large body mass, implicating population size as a pervasive cause of evolutionary rate variation. Third, I developed molecular phylogenies for puffbirds, jacamars, and motmots to test hypothesized area relationships in the Neotropics. I used penalized likelihood to estimate node times while accommodating significant rate variation under a set of biologically realistic assumptions. Phylogenetic patterns in each family were consistent with expansion following the formation of the Central American Landbridge and subsequent vicariance across the Andes. I applied a calibration based on the final uplift of the Isthmus of Panama, 3.1 Ma. Average estimated rates were close to the commonly cited 2% sequence divergence/Myr. Concordant area relationships were found among co-distributed species complexes; however, the timescale of divergence was variable, suggesting that common dispersal corridors rather than common vicariant events may be driving co-phylogenetic patterns.

The Alu Ya and Yb-lineages are distinct groups of Short INterspersed Elements (SINEs) found exclusively in primates. These lineages are comprised of several distinct subfamilies, which contain elements that are evolutionarily-related because they share common diagnostic mutations. Some of these Alu subfamilies are still actively mobilizing in the human genome. We have recovered 4333 Ya and Yb-lineage elements that reside in the human genome draft sequence and focused our analyses on the 4051 autosomal Alu elements. A total of 2672 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual elements for insertion presence and absence. The polymorphism rate for the insertion presence/absence on the human autosomes was 22% and 20% for the Ya and Yb-lineages, respectively. Less than 0.5% (12/2672) of the loci analyzed by PCR displayed positive results for insertions in the orthologous loci of various non-human primate genomes. DNA sequence analysis of these inserts showed that the non-human primate orthologous loci contained older, pre-existing Y, Sc, Sg, Sq or Sx Alu subfamily elements. These PCR results were the result of various evolutionary processes within the human lineage. This is the largest analysis of human Alu mobile element lineages to date. The size, evolutionary age and variable allele insertion frequencies of the Alu Ya and Yb-lineage subfamilies, makes these elements useful tools for human population genetics, human identification and primate comparative genomics.

Retrotransposons are an active family of mobile elements within primate genomes and the Short INterspersed Element (SINE) Alu is the most abundant member. These non-autonomous elements are responsible for introducing genomic diversity on an intra- and inter- species level that is useful in studies of forensic identity, population genetics, and evolutionary biology. In a computational survey of the human sex chromosomes, 344 recently integrated Alu elements were detected and subjected to empirical testing by polymerase chain reaction to determine presence/absence polymorphism. Sixteen elements were found to be polymorphic on the X chromosome, and only one polymorphic element on the Y chromosome (previously termed YAP, Y chromosome Alu Polymorphism), across four geographically diverse populations. In line with previous research using other types of genetic markers, these results indicate a low Alu-associated diversity level on the human sex chromosomes, presumably due to reduced recombination rates and lower effective population sizes on the sex chromosomes. Alu elements often contribute to genomic instability via insertional and recombinational mutagenesis. Recently, a novel mechanism of retrotransposon-associated genomic instability was discovered, termed retrotransposition-mediated deletion. A computational search within the draft human and chimpanzee genomes found evidence of 33 retrotransposition-mediated deletion events that have eliminated approximately 9,000 nucleotides of genomic DNA. During the course of primate evolution, Alu retrotransposition may have contributed to over 3000 deletion events, eliminating approximately 900,000 bp of DNA in the process. Potential mechanisms for the creation of Alu retrotransposition-mediated deletions include L1 endonuclease-dependent retrotransposition, L1 endonuclease-independent retrotransposition, internal priming on DNA breaks, and promiscuous target primed reverse transcription (pTPRT). Approximately 0.27% of all human disease mutations are attributable to the activity of Long INterspersed Element (LINE) L1, Alu and SVA (SINE-R/VNTR/Alu) retrotransposons within our genomes. Although researchers in the field of human genetics have discovered many mutational mechanisms for retrotransposable elements, including retrotranspositional insertion, recombination, retrotransposition-mediated and gene conversion-mediated deletion, in addition to 3' transduction, their individual contribution to genetic variation within humans is still being resolved.

Long INterspersed Elements (LINEs or L1)and Short INterspersed Elements (SINEs) have played a critical role in shaping the human genomic landscape, making up about 33% of the human genomic mass. In this work, preTa, an L1 subfamily and the Ya-lineage of Alu SINEs were examined to determine their impact on the human genomic architecture and diversity. We analyzed 362 preTa L1 elements and 2318 Ya lineage Alu elements using computational methods and polymerase chain reaction (PCR) assays. PCR analysis on a geographically distinct panel of human populations indicated that 33 (14%) of preTa L1 elements and 313 (22%) of Ya-lineage Alu elements were polymorphic for insertion presence/absence. DNA sequence analysis of the preTa and the Ya-lineage subfamilies indicated an estimated average age of 2.34 and 2.27 million years respectively. This suggests that these elements began to amplify after the human lineage radiated from the non-human primates. None of the mobile elements analyzed were also located in orthologous non-human primate loci, supporting the fact that they are homoplasy free genetic characters. The continued amplification of mobile elements throughout evolutionary time and their variable insertion frequencies in diverse human populations make them robust tools for population genetics and comparative genomics.

This study is an examination of geographic variation and evolutionary history of Stelgidopteryx swallows. These swallows comprise two recognized species: Northern Rough-winged Swallow (S. serripennis) and Southern Rough-winged Swallow (S. ruficollis). A third species, Yucatan Rough-winged Swallow (S. ridgwayi)is also commonly recognized. The species are largely allopatric, except for a contact zone in Costa Rica. Using plumage and molecular variation, I examined the likelihood of two (or three) different species of rough-winged swallows, the genetic interrelationships among taxa, and their biogeographic history. Geographic plumage variation reveals a latitudinal cline across the genus from North to South America. Specimens from throughout the range of Stelgidopteryx show that most published subspecies should be synonymized as clinal variants. Molecular data (mitochondrial cytochrome-b sequences, microsatellite allele frequencies) show a pattern consistent with a species-level division between S. serripennis and S. ruficollis . The data also suggest that the Yucatan swallow is distinct. Phylogenetic trees from the sequence data divide Stelgidopteryx into northern and southern clades consistent with S. serripennis and S. ruficollis. The Yucatan clade, S. ridgwayi, is sister to the northern group. Microsatellite data indicate allelic frequency differences between the groups, but none fixed. Population genetic analyses among individuals within S. serripennis and S. ruficollis reveal genetic structure possibly worthy of taxonomic recognition. Measures of the population parameter theta indicate high allelic diversity in each group and suggest S. ruficollis is the younger population. The Northern Rough-winged Swallow exhibits broad clinal variation, but little subspecific subdivision. The Yucatan Rough-winged Swallow, is phenotypically and genetically dissimilar to the other groups, further supporting species-status. The Southern Rough-winged Swallow contains at least two subspecies, including decolor on the Pacific slope of Central America, and ruficollis throughout the rest of its range. The widespread distribution and contact zone in Costa Rica are consistent with secondary contact between the northern and southern swallows. The emergence of the Panamanian landbridge could have contributed to the formation of the contact zone. Pleistocene climatic shifts could also have played a role in isolating birds on opposite slopes of Central America allowing divergence between S. r. uropygialis and S. r. decolor.

The rapidly increasing amount of sequence data has brought about a new appreciation for the tremendous influence mobile elements have had in shaping eukaryotic genomes. Despite their ubiquity, however, the factors governing the proliferation of mobile elementsor, in some cases, the lack of proliferationacross diverse taxa remain poorly understood. Analysis of Alu activity in humans and chimpanzees since their divergence indicates a two-fold increase in human Alu activity compared to that of the chimpanzee. This human retrotransposition increase is accompanied by a roughly two-fold higher level of chimpanzee Alu diversity. We prepose a model, wherein smaller effective population sizes in humans brought about a shift in host-element dynamic, ultimately leading to increased Alu activity in humans. We also survey Alu-associated diversity on the human sex chromosomes in order to examine whether Alu elements behave similarly to genetic marker systems. Our results suggest that, comparable to other genetic systems, Alu elements exhibit reduced diversity on the sex chromosomes. Our data provide no evidence for retrotransposon targeted biology influencing Alu insertion frequencies. We go on to synthesize several recent advances in the mobile element field and propose a novel hypothesis concerning how retrotransposon lineages manage to largely lie below the radar of population-level negative selection.

The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds. This study presents a biochemical characterization of a novel MarR homolog, HucR (hypothetical uricase regulator), from the DNA damage-resistant eubacterium, Deinococcus radiodurans. Circular dichroism spectroscopy suggests that HucR has ~47% alpha-helix and 10% beta-strand conformation at 25 deg C, and undergoes a transition to a disordered state with Tm = 51.1 ± 0.0 deg C. HucR binds as a homodimer with high sequence-specificity to a single site in its promoter region (hucO) with an apparent Kd = 0.29 ± 0.02 nM. DNaseI and hydroxyl-radical footprinting indicate HucR binding site sizes of ~24 bp and 21 bp, respectively. The binding site contains a pseudopalindromic sequence comprised of 8 bp inverted repeats separated by 2 bp that overlaps predicted promoter elements for hucR and a putative uricase (dr1160). Specific phenolic weak acids, notably uric acid, antagonize HucR-hucO complex formation. In vivo, uric acid increases transcript levels of hucR and dr1160, ~1.6-fold, and stimulates uricase activity 1.5-fold. HucR-hucO complex formation involves protein conformational changes and a decrease in the helical twist of the DNA duplex. Intrinsic fluorescence measurements show that uric acid induces HucR conformational changes, and its apparent Kd = 11.6 ± 3.7 micromolar and Hill coefficient of 0.7 ± 0.1 suggest negative cooperativity. An amino acid substitution in the predicted HucR wing (HucR-R118A) reduces DNA-binding affinity ~5-fold (Kd = 1.60 ± 0.14 nM), whereas a substitution in the predicted recognition helix (HucR-S104A) does not significantly alter DNA-binding affinity (Kd = 0.23 ± 0.03 nM). Each mutation decreases complex stability on the gel, but does not affect sequence-specificity. Intrinsic fluorescence spectra suggest altered conformations of the HucR-variants and altered mechanisms of DNA association. The mutations at HucR positions 118 and 104 also alter a predicted weak ligand-binding site, as indicated by minor changes in uric acid affinities for HucR-R118A and HucR-S104A (Kd = 9.7 ± 3.2 micromolar and 7.4 ± 0.5 micromolar, respectively) and modest attenuations of protein-hucO complex formation in response to uric acid.

The level of torsion in double-stranded DNA regulates base-pair stability and DNA conformation. It is important in initiation and regulation of specific DNA metabolic processes as well as chromatin assembly. High mobility group proteins (HMGB) are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as supercoiled DNA and 4-way junctions. HMGB proteins play a role in transiently regulating or conserving DNA torsion. Topoisomerases regulate DNA supercoiling, which has been argued to provide a coherent explanation for the main modes of transcriptional control - stringent control, growth-rate control and growth-phase control during the normal cell growth. In this study, we have shown that HMO1, a Saccharomyces cerevisiae HMGB protein which is required for normal growth, plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease binds linear duplex DNA but has little preference for DNA with altered conformations. Divergent box A binds DNA and contributes structure-specific binding. Unlike most HMGB proteins, HMO1 does not supercoil relaxed DNA in the presence of topoisomerase. Casein Kinase II phosphorylates HMO1, altering its DNA binding properties. We have also shown that deletion of the highly basic C-terminal tail of HMO1 localizes this otherwise both nuclear and cytoplasmic protein only to the cytoplasm. As the C-terminally truncated HMO1 has been reported to rescue the hmo1 knockout phenotype, we conclude that the main function of HMO1 lies in the cytoplasm, and not in the nucleus. Vaccinia topoisomerase I relaxes supercoiled DNA. We have shown that it interacts with enrofloxacin, a fluoroquinolone antibiotic which otherwise targets DNA gyrase and topoisomerase IV. Enrofloxacin inhibits DNA relaxation by Vaccinia topoisomerase I. When presented with relaxed DNA, the enzyme executes the reverse reaction, supercoiling the DNA. Enrofloxacin does not interfere with the catalytic cleavage site of Vaccinia topoisomerase I or its ability to bind DNA. The mechanistic implication of these observations is that protein-DNA contacts downstream of the cleavage site must contribute to DNA supercoiling, contrary to the free rotation mechanism proposed for DNA relaxation.

The regulation of the photorespiratory pathway of Chlamydomonas reinhardtii during a shift from high to low CO2 conditions was investigated. To this end, a set of C. reinhardtii cDNA sequences for known photorespiratory enzymes was assembled using the C. reinhardtii EST data and primary sequencing data. The cDNA sequences of the proposed photorespiratory genes are presented. A subset of these genes was used for expression analyses under varied conditions. Expression data indicates that there was a rapid and coordinated induction of photorespiratory and carbon dioxide concentrating mechanism (CCM) gene expression during a time course switch from elevated CO2 conditions (5% [v/v]) to low CO2 conditions (0.038% [v/v]). While the expression of photorespiratory and CCM genes was coordinated during the initial change in CO2 level, the response of these two sets of genes to the CO2 level was not identical. Unlike the sustained high levels of CCM mRNAs seen under low CO2 conditions, photorespiratory mRNAs showed a transient increase in abundance in time course experiments. In addition, the expression of these photorespiratory genes was reduced in cia5, a C. reinhardtii strain that lacks a transcription factor required for the induction of genes involved in the CCM. From these observations, there appears to be coordination in the expression of the genes involved in the delivery of CO2 to Rubisco and the genes involved in the metabolism of the photorespiratory products that form when the CO2 level is low. Further expression analyses were carried out to gain a better understanding of the signal that induces CCM and photorespiration of C. reinhardtii. Low CO2 concentration appears to be the only signal for the induction of photorespiration and CCM under the tested conditions.

Metal-hydrocarbon mixtures are becoming increasingly prevalent in natural environments due to expanding industrial activity and urbanization. The ecotoxicology of metal-hydrocarbon mixtures in benthic environments is of particular concern because both classes of contaminants partition to sediments and can thereby exert toxic effects in benthic organisms. Mixtures of dissimilar chemicals (including metals and hydrocarbons) are broadly hypothesized to elicit independent toxic effects however; this hypothesis has little supporting data. The purpose of this dissertation was to test this hypothesis for metal-hydrocarbon mixtures using environmentally relevant exposures and to determine mechanisms for observed interactive effects. Sediment and water-only bioassays were conducted employing the toxic heavy metal cadmium (Cd) and the polynuclear aromatic hydrocarbon phenanthrene (Phen) as model toxicants. Lethal and sublethal effects of singular and combined contaminants were examined in two freshwater species, the epibenthic amphipod Hyalella azteca and the bulk deposit-feeding benthic oligochaete Ilyodrilus templetoni. When interactive toxicity was observed, mixture effects on contaminant bioavailability, bioaccumulation and elimination were tested. As well, mixture effects on bioenergetics parameters were investigated and kinetic modeling was conducted to establish the source of mixture-mediated changes in contaminant bioaccumulation in I. templetoni. Cadmium-Phen mixtures caused independent effects in water-only exposures, but when incorporated into sediments, elicited synergistic lethal effects in H. azteca and antagonistic lethal effects in I. templetoni. Interactive effects were likely caused by Phen-mediated alterations in Cd bioaccumulation that resulted from changes in exposure via feeding. The current basis for assessing ecotoxicological effects of contaminant mixtures in natural environments relies heavily on models derived from dosage-based mixture toxicology with considerably less emphasis on environmental science and biology. Understanding how contaminants interact toxicologically is important, but does not provide all the information necessary for assessing effects in natural populations that encounter contaminant mixtures in a diversity of natural environments. My experiments indicate that exposure source may be more important than dosage-based toxicological interactions in determining contaminant mixture effects in sediment environments. If this trend is widespread, understanding how species are exposed, determining the route of uptake and understanding how environmental characteristics affect exposure may be more important in determining mixture effects than mixture toxicology.