LINE-1 (Long INterspersed Element-1 or L1) and Alu elements are important sources of structural variation in primate genomes because they are highly active retrotransposons with copy numbers of ~520,000 and >1.2 million within the human genome, respectively. Although the bulk of these elements have resided in their respective host genomes for a long time, and have thus accumulated random mutations, overall these elements retain high levels of sequence identity among themselves. The presence of many nearly-identical retrotransposons located close to each other (e.g., Alu-Alu or L1-L1 pairs) disposes their host genomes to unequal homologous DNA recombination events that generate genomic deletions and inversions of varying sizes. Through computational comparisons of the human and chimpanzee genome sequences, and using rhesus macaque and orangutan genome sequences as outgroups, we have identified species-specific genomic variation. In the first analysis, we identified human and chimpanzee-specific L1s and examined their sequence evolution. We show that L1 retrotransposition activity is slightly higher in the human lineage, relative to the chimpanzee lineage, and that L1s have experienced different evolutionary fates in these two lineages, resulting from random variation or competition between L1 subfamily lineages. Next, we analyzed the magnitude of Alu recombination-mediated deletions (ARMDs) in the chimpanzee lineage subsequent to the human-chimpanzee divergence (~6 million years ago). We have identified 663 chimpanzee lineage-specific deletions (involving a total of ~771 kb of genomic sequence) attributable to this process. The RefSeq databases indicate that 13 exons in six genes are annotated as either demonstrably or putatively functional in the human genome, and 299 intronic regions have been deleted through ARMDs in the chimpanzee lineage. In the third analysis, we characterize chromosomal inversion events between the human and chimpanzee genomes caused by inverted L1-L1 or Alu-Alu pairs. We have identified 49 retrotransposon recombination-mediated inversion (RRMI) loci and, among them, three RRMI loci contain inverted exonic regions in known genes. Therefore, we suggest that L1 and Alu elements have contributed to the genomic and phenotypic diversity between humans and chimpanzees since the divergence of the two species.

Despite the theoretical link between the ecology and the population genetics of species, little empirical evidence is available that corroborates the association. Here, I examined genetic variation in 40 co-distributed species of lowland Neotropical rainforest birds that have populations isolated on either side of the Andes, Amazon River, and Madeira River. I found widely varying levels of genetic divergence among these taxa between the same biogeographic barriers. My investigation of the extent to which ecological traits predicted the level of cross-barrier divergence revealed a significant relationship between the forest stratum at which a species forages and the level of within-population and cross-barrier genetic differentiation. Canopy species had statistically lower divergence values across the Andes and two riverine barriers than did understory birds. I hypothesize that the association reflects an effect of dispersal propensity on the geographic structuring of genetic variation, and, consequently, on the ancestral and extant effective population sizes of each species. This is the first large-scale avian comparative study to document a significant association between ecological traits of a species and its level of genetic differentiation. I examined further the contrasting genetic patterns revealed previously by comparing the range-wide mitochondrial (mtDNA) phylogeography of two canopy and two understory species of lowland Neotropical rainforest birds. All species exhibited divergence between cross-Andean populations. Unlike canopy species, understory birds were structured at smaller spatial scales, particularly across riverine barriers of the Amazon basin. Surprisingly, estimates of isolation-by-distance, a proxy for dispersal propensity, are similar within areas of endemism for all taxa suggesting levels of gene flow are comparable through contiguous habitat in canopy and understory species. Lastly, I examined the multilocus phylogeography of three previously studied species with contrasting mtDNA patterns to investigate the role of historical demography in cross-Andean divergence. Demographic estimates using an isolation-with-migration model suggest among-taxa variance in cross-Andean divergences reflects a history of staggered isolation versus a simultaneous isolating event. Nuclear sequence data reveal asymmetrical gene flow in two species marked by relatively shallow cross-Andean divergence, further evidence of differential effectiveness of the Andes as a barrier to gene flow among co-distributed taxa.

Pseudomonas aeruginosa encodes a putative topoisomerase with sequence similarity to the type IB topoisomerase enzyme from vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA. The gene encoding the P. aeruginosa topoisomerase I (PAT) was cloned and expressed in E. coli. The enzyme relaxes supercoiled DNA, while a mutant of P. aeruginosa containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like vaccinia topoisomerase (VT), PAT relaxes DNA in the absence of ATP. Unlike VT, PAT does not relax supercoiled DNA without MgCl2 (or MnCl2) present. In addition, high concentration of NaCl is not able to substitute for MgCl2 as seen for VT. A truncated derivative of the topoisomerase lacking residues 1-98 relaxes DNA in the absence of the N-terminal domain, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations. Data shows that P. aeruginosa encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses. Fluoroquinolones are antibacterial agents in clinical use with activity against DNA gyrase and DNA topoisomerase IV. P. aeruginosa is an opportunistic pathogen causing life-threatening diseases. Sparfloxacin, enrofloxacin and norfloxacin fluoroquinolones, are able to inhibit the PAT at high concentrations but other drugs belonging to this family are unable to do so. VT is ¡Ö32 KDa and one-third the size of the human topoisomerase ¡Ö 100 KDa. It shares sequence and biochemical similarities with the human topoisomerase. The VT binds duplex DNA with stringent specificity for transesterification at 5`-(C/T) CCTT site, where the 3` phosphate of the incised strand is linked to the Tyr274 of the enzyme to form a covalent cleavage complex. The fluoroquinolone enrofloxacin inhibits relaxation of supercoiled DNA by VT in a Mg2+-dependent fashion. Further results indicate that the mechanism by which enrofloxacin inhibits VT is by preventing formation of the covalent complex which suggest that fluoroquinolones may be structurally optimized to target type IB topoisomerases.

Conjugation in Streptomyces invokes a unique mechanism involving few plasmid-encoded loci. Transfer of circular plasmid pIJ101 requires only a tra gene, and a cis-acting locus clt. Here, I investigated whether the transfer genes of pIJ101 could promote transfer of a linear plasmid. While pIJ101 Tra could transfer circularized versions of the linear plasmid containing clt, the linear plasmid itself could not be transferred efficiently by Tra. All plasmids isolated from transconjugants appeared to be circular regardless of the configuration of the plasmid in the initial donor cells. However the linear plasmid could be transferred in linear form from JW1, which contains the conjugative linear plasmid SLP2. Evidence that linear plasmids transferred from strains containing only SLP2 strongly suggest that TraSLP2 mediated the transfer of heterologous linear plasmid and pIJ101, and Tra is not responsible for transfer of the linear DNA molecules from JW1.These results indicate that Tra of pIJ101 shows specificity for the circular configuration and imply that efficient transfer of linear plasmids may require additional functions compared with circular plasmids. Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Here, I show that group III inhibitor ipomicin stably accumulates in culture supernatants of S. ipomoeae in a growth-regulated manner that does not coincide with the pattern of expression of the ipomicin structural gene ipoA. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the TTA codon in ipoA by the bldA leucyl tRNA. These results suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion, stably accumulates in the extracellular environment. An apparently inactive form of ipomicin protein is produced in most members of susceptible strains of S. ipomoeae, suggesting that further post-translational modification of ipomicin protein in the group III strains results in bacteriolytic activity.

Adaptation is among the most prominent subjects in evolutionary biology. Despite its ubiquity in nature, many details of how adaptation occurs in natural populations remain poorly understood. Of particular interest are the genes and biochemical pathways that underlie adaptive phenotypes and how plasticity in these systems contributes to adaptive evolution. In this dissertation, I address these questions by investigating the molecular genetic basis of high-altitude adaptation in the Rufous-collared Sparrow (Zonotrichia capensis), a species with a broad altitudinal distribution in the Andes. First, I examined the role that variable selection pressures along elevational gradients play in the population genetic structure of Z. capensis. I found that mitochondrial gene flow was severely reduced along elevational transects relative to latitudinal control transects. Nuclear gene flow, however, was not affected by the elevational gradient. These results suggest that natural selection constrains mitochondrial gene flow along elevational gradients. The mitonuclear discrepancy was consistent with local adaptation of mitochondrial haplotypes, highlighting the importance of metabolic pathways in high-altitude adaptation in Z. capensis. Second, I used a newly developed genomic tool, a zebra finch (Taeniopygia guttata) cDNA microarray, to measure variation in genome-wide patterns of gene expression between high- and low-elevation populations of Z. capensis. I found that nearly 200 genes, many of which were involved in metabolic processes, were differentially expressed when individuals were sampled at their native altitudes. A common garden experiment demonstrated substantial plasticity in gene expression, and these results suggest that plasticity in the biochemical pathways that underpin cold and hypoxia compensation in Z. capensis may mechanistically contribute to enabling its broad altitudinal distribution. Finally, I examined geographic variation in metabolic gene expression along an elevational gradient. Although metabolic adjustments are often involved in thermal stress response and temperature decreases linearly with elevation in the Andes, expression of metabolic genes was non-linearly related to elevation. These results suggest a decoupling of metabolic gene expression and local temperature regimes. This decoupling may have several explanations, but the most plausible seem to be related to either physiological tradeoffs between thermal stress and hypoxia compensation, or genetically encoded expression differences.

Speciation, geographic variation, and genetic differentiation are fundamental processes that generate diversity, and understanding these processes are major goals of evolutionary biology. Evolutionary phenomena may be more observable on islands as compared to continental landmasses as a result of small population sizes, unoccupied niches, and the relative simplicity of island systems and their populations: physical isolation, shorter (and often well documented) geologic time scale, reduced faunal diversity, and lack of outside faunal influence. Yet, despite their incredible diversity, Pacific island faunas have received little research attention relative to other tropical regions. Using molecular data from several species of scincid lizards in the genus Emoia, I test hypotheses related to the generation and maintenance of biodiversity in Pacific oceanic systems, examining historical patterns of colonization, dispersal, and differentiation for a member of a vertebrate family with a broad distribution in the islands of the Pacific. This research is primarily conducted within the Vanuatu Archipelago, an ideal island group in which to examine questions associated with the role of island systems in promoting diversification and speciation. Vanuatu is an oceanic archipelago and its fauna is derived either via over water dispersal or cladogenesis. As it is also a geologically young island group (most islands emergent < 2 mya) interpretation and analysis of intra-archipelago variation during the early stages of a radiation are possible from data collected in this system. Comparison of patterns of diversification and differentiation recovered from Emoia in Vanuatu with patterns recovered for species in other well-studied, older island radiations (such as the Hawaiian Islands) enables an understanding of the generality of factors promoting diversity and speciation in island systems.

A chemotopic map of biologically relevant odorants (that include amino acids, bile salts and nucleotides) exists in the olfactory bulb (OB) and forebrain (FB) of channel catfish, Ictalurus punctatus (Chapter one). Neurons processing bile salt odorant information lie medially within these bilaterally symmetric structures; however, information as to how single neurons discriminate and process this odorant information is lacking. Chapters two and three of the dissertation identify the range of odorant bile salt molecules that excite these neurons [i.e. the excitatory molecular receptive range (EMRR)] within the bile salt chemotopic zones of the OB and FB. The results of the investigations of single bile salt responsive neurons within the OB indicate that these neurons are selectively excited by combinations of molecular features found on the side-chain and the steroid nucleus of bile salt molecules. Further, the results of the investigations of single bile salt responsive neurons within the FB indicate that their EMRRs are virtually identical to that of OB neurons suggesting that little modification of the neural olfactory quality code for these molecules occurred between the OB and the FB. Bile salts are known olfactory stimuli to teleosts, but only a single report (Yamashita et al. 2006) indicated that the taste system of a fish was sensitive to this class of stimuli. Chapter four investigates the gustatory sensitivity of the facial taste system to bile salts in the channel catfish. Bile salts were shown to be highly effective facial taste stimuli with estimated electrophysiological thresholds of approximately 10-11M-10-10M. Multiunit cross-adaptation experiments indicate that bile salts and amino acids bind to relatively independent receptor sites; however, nerve twig data and a few single fiber recordings suggest that both independent and shared neural pathways exist for the transmission of bile salt and amino acid information to the primary gustatory nucleus of the medulla. The findings of the present report aid in understanding how bile salt molecules are detected and initially processed by the olfactory and gustatory systems in catfish and further suggest that bile salt odorant information is not greatly transformed by central olfactory neurons (Chapter five).

Dissociable Antibody Microarray (DAMA) staining is a novel technique that integrates protein microarrays with conventional immunostaining techniques. It can simultaneously determine the expression and subcellular localizations (SCLs) of hundreds of proteins in cultured cells. I optimized this technology for protein expression and SCL profiling, and generated expression profile data analysis program DAMAPEP, molecular image database management program ChipView and automatic SCL assignment program DAMASCL. We demonstrated the application of this technique in the identification of potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among ten breast cell lines and identified 10 differentially expressed proteins. Among those proteins, RAIDD, Rb p107, Rb p130, SRF and Tyk2 were confirmed by western blot and statistical analysis to have higher expression levels in cancer breast cells than in normal breast cells. We also compared the SCL profiles of 325 proteins among nine breast cell lines, and identified one protein, Cyclin B1, with different SCLs between two normal and seven cancer breast cell lines. With individual immunostaining, Cyclin B1 was confirmed to localize in the cytoplasm of seven cancer cells and in both cytoplasm and nuclei of two normal cells and to have higher expression levels in the seven cancer cell lines. We expanded the scale of DAMA staining to include 400 antibodies per array and surveyed SCL profiles of 400 antibodies in five prostate cell lines. Five proteins were identified to have altered SCL patterns between normal and cancer prostate cell lines. GRK2 was so far confirmed to localize ubiquitously in the cytosol of three normal and one cancer prostate cell lines while concentrating at certain regions right beneath plasma membrane in the other two cancer prostate cell lines. We also extended the application of DAMA staining to interrogate protein expression profiles in tissue samples and found 3 proteins with differential expression in two tissue samples from different breast cancer patients, demonstrating the potential use of DAMA staining in carcinoma characterization and classification. Database of annotated protein molecular images obtained from DAMA staining need to be created and shared for better understanding of cancer biology.

All animals face the same basic challenges in controling movement through their environment. The central nervous system must activate and effectively control muscle fibers capable of accomplishing the behavior. For multi functional muscles, an array of muscle fiber types must be selectively activated based on the behavioral task. In arthropods that have few motor neurons, it is not understod how the nervous system accomplishes this selective activation. If the selection cannot be achieved by recruitment of different motor neurons, then it most likely results from careful adjustment to the firing patterns of the motor neurons. I used the neuromuscular system of crab walking legs to pursue a detailed description of the relationship between neural firing patterns and the resultant muscle contraction, emphasizing how muscles with different walking use differ in physiological characteristics. Crabs use of a small number of identified motor neurons and their experimental tractability make them an attractive model system to investigate how nervous systems control behavioral movements. Experimentation was conducted on two brachyuran species Carcinus maenas and Libinia emarginata, the former an amphibious sideways walker and the latter an aquatic forward walker. Comparisons within and between the species showed that muscles that provide thrust in the animals most common walking direction had greater high frequency facilitation. Muscles that cycle more frequently during walking had shorter relaxation time constants across multiple behavioral criteria. These differences in pre- and postsynaptic kinetics reveal how the behavioral use of a muscle can constrain the array of physiological properties within the motor system. Computational models demonstrated that selective activation of postsynaptic muscle fibers can be accomplished by changing neural firing rates due to physiological differences described in previous chapters. Faster muscles were more sensitive to short-term changes in the excitatory firing pattern, while slower muscles were more sensitive to long-term changes. The ability to selectively recruit different muscle fiber types that are more sensitive to different aspects of the firing pattern of motor neurons allows the animal to have many functional motor units per muscle despite receiving excitatory innervation from as few as one motor neuron.

In western Amazonia, large numbers of frugivorous bats regularly visit natural forest clearings known locally as collpas (also called clay licks or mineral licks). Bats arrive at collpas to drink water that has accumulated in depressions created by larger mammals that consume soil. Although collpa visitation by bats appears relatively common in western Amazonia, little is known about its causes and its ecological implications. In this dissertation I describe general and seasonal patterns of collpa visitation by frugivorous bats in the Peruvian Amazon, and I investigate potential explanations for this unique behavior. Regardless of season, collpas seem to be activity hotspots for frugivorous bats, especially for reproductive females. Furthermore, collpas are visited almost exclusively by frugivorous species of the subfamily Stenodermatinae. Because some nutrients are found in low concentrations, a potential explanation for collpa visitation is to obtain key limited resources. Collpas are mineral-rich water sources. The content of selected minerals in collpa water, especially sodium, was significantly higher compared to other natural sources of water such as creeks, oxbow lakes, and rivers for both dry and rainy seasons. Thus, collpas may function as mineral sources for female reproductive frugivorous bats. Stenodermatine bats feed mostly on figs, whereas bats from the sub-family Carolliinae feed on Piper fruits, but also complement their diets with insects as well as other plant species. Thus, because stenodermatine species are extremely common at collpas, collpa visitation may be related to nutrient deficiencies in specific diets. Although there was a clear distinction in mineral and nitrogen content of Ficus and Piper fruits, they seem to provide frugivorous bats enough nitrogen (protein) and most minerals to meet their maintenance requirements. However, both fruit genera were very limited in sodium, which suggests sodium limitation for frugivorous bats in the southeastern Peruvian Amazon. Carolliine bats may be obtaining sodium from insects, whereas stenodermatine bats may use collpas as secondary sources of sodium, especially during reproduction. Additionally, I provide experimental evidence that demonstrates that stenodermatine bats have a strong preference for collpa water. Finally, because collpas are important mineral sources for frugivorous bats, they should be considered important conservation targets.