Thesis (Ph.D.)--University of New Hampshire (Dept. of Biochemistry and Molecular Biology), 2005. / Title from PDF title page. Available through UMI ProQuest Digital Dissertations. Includes bibliographical references. Also issued in print.

Several strain typing techniques are available to categorize Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) isolates into cattle, sheep, bison, and Intermediate groups. The majority of isolates studied were identified as members of the cattle associated group, regardless of sample host origin, suggesting that the cattle group of M. paratuberculosis isolates are very successful. This may be because host specificity is not critical for this group or the small differences required to demonstrate host specificity have not yet been found. A major limitation to the epidemiological study of M. paratuberculosis has been the difficulty associated with laboratory cultivation of this micro-organism. The new typing techniques described in this thesis do not require viable M. paratuberculosis bacteria and therefore open a door to novel typing practices. The new molecular techniques, single stranded conformation polymorphism (SSCP) analysis and satellite typing, were applied to M. paratuberculosis isolates (n=75) from a broad range of ruminant hosts and geographic locations. SSCP analysis and satellite typing were compared to currently accepted techniques (PCR-REA, RFLP, PFGE) for their ability to rapidly and reliably differentiate among M. paratuberculosis isolates. PCR-REA segregated isolates (n=75) into cattle (n=72), sheep (n=1) or bison (n=2) associated strain types. Two isolates from cattle in Canada were typed as RFLP-BstEII C5 by RFLP analysis. PFGE grouped a subset (n=8) of M. paratuberculosis isolates into 4 different PFGE types. Satellite typing resulted in 4 different satellite types (A, B, C, D). SSCP analysis identified 2 regions (IS900-2 and HSP70) where sequence polymorphisms could be targeted to display differences among M. paratuberculosis isolates.

Several strain typing techniques are available to categorize Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) isolates into cattle, sheep, bison, and Intermediate groups. The majority of isolates studied were identified as members of the cattle associated group, regardless of sample host origin, suggesting that the cattle group of M. paratuberculosis isolates are very successful. This may be because host specificity is not critical for this group or the small differences required to demonstrate host specificity have not yet been found. A major limitation to the epidemiological study of M. paratuberculosis has been the difficulty associated with laboratory cultivation of this micro-organism. The new typing techniques described in this thesis do not require viable M. paratuberculosis bacteria and therefore open a door to novel typing practices. The new molecular techniques, single stranded conformation polymorphism (SSCP) analysis and satellite typing, were applied to M. paratuberculosis isolates (n=75) from a broad range of ruminant hosts and geographic locations. SSCP analysis and satellite typing were compared to currently accepted techniques (PCR-REA, RFLP, PFGE) for their ability to rapidly and reliably differentiate among M. paratuberculosis isolates. PCR-REA segregated isolates (n=75) into cattle (n=72), sheep (n=1) or bison (n=2) associated strain types. Two isolates from cattle in Canada were typed as RFLP-BstEII C5 by RFLP analysis. PFGE grouped a subset (n=8) of M. paratuberculosis isolates into 4 different PFGE types. Satellite typing resulted in 4 different satellite types (A, B, C, D). SSCP analysis identified 2 regions (IS900-2 and HSP70) where sequence polymorphisms could be targeted to display differences among M. paratuberculosis isolates.

Given the distribution and probable evolutionary history of the Peromyscus maniculatus species group, an interspecific comparison of microsatellite variation among these species would be logically based (at least initially) on primers isolated from the genome of a geographically central population of P. maniculatus. Additionally, as the species in the group are recently diverged, reasonably informative microsatellite data are likely to require analysis of a rapid evolving category of microsatellite loci. The initial phase of this research involved the isolation, characterization and assessment of variation for a panel of DNA microsatellites containing perfect dinucleotide repeats from a geographically central population of P. maniculatus. Theoretical predictions and empirical studies indicate that phylogenetic analyses based on microsatellite primers isolated from a focal species may be subject to ascertainment biases that can be expected to degrade the efficacy of this approach with increasing phylogenetic depth between the species from which the microsatellites were isolated and those to which these loci are being compared. Results of an analysis of allelic variation at 12 pure, dinucleotide microsatellite loci (isolated from P. maniculatus) are reported for samples of all species in the P. maniculatus species group and the sister taxon P. leucopus. Examined for the species in the P. maniculatus species group for which there is an a priori highly corroborated phylogeny, evidence of ascertainment bias was apparent only for one locus that was unique to P. maniculatus. Genealogical analyses of the data over all loci yielded inferred relationships that were entirely concordant with the a priori corroborated phylogeny for P. maniculatus, P. keeni, P. polionotus, P. melanotis and P. leucopus. Genealogical analyses of the previously unresolved relationships of P. keeni and P. sejugis consistently placed these as an independent sister-group between P. maniculatus and P. polionotus. The geographically improbable sister-group association of P. keeni and P. sejugis may be the result of an historical ancestral continuity or may reflect large-scale lineage sorting rather than true phylogenetic propinquity. These data suggest that, given the choice of an appropriate focal species, even relatively small sets of pure dinucleotide microsatellites can provide reliable population genetic and systematic implications for taxa with divergence times dating to the Pleistocene.

This PhD dissertation is focused on short tandemly repeated nucleotide patterns which occur extremely often across DNA sequences, called microsatellites. The main characteristic of microsatellites, and probably the reason why they are so abundant across genomes, is the extremely high frequency of specific replication errors occurring within their sequences, which usually cause addition or deletion of one or more complete tandem repeat units. Due to these errors, frequent fluctuations in the number of repetitive units can be observed among cellular and organismal generations. The molecular mechanisms as well as the consequences of these microsatellite mutations, both, on a generational as well as on an evolutionary scale, have sparked debate and controversy among the scientific community. Furthermore, the bioinformatic approaches used to study microsatellites and the ways microsatellites are referred to in the general literature are often not rigurous, leading to misinterpretations and inconsistencies among studies. As an introduction to this complex topic, in Chapter I I present a review of the knowledge accumulated on microsatellites during the past two decades. A major part of this chapter has been published in the Encyclopedia of Life Sciences in a Chapter about microsatellite evolution (see Publication 1 in Appendix II). The ongoing controversy about the rates and patterns of microsatellite mutation was evident to me since before starting this PhD thesis. However, the subtler problems inherent to the computational analyses of microsatellites within genomes only became apparent when retrieving information on microsatellite distribution and abundance for the design of comparative genomic analyses. There are numerous publications analyzing the microsatellite content of genomes but, in most cases, the results presented can neither be reliably compared nor reproduced, mainly due to the lack of details on the microsatellite search process (particularly the program’s algorithm and the search parameters used) and because the results are expressed in terms that are relative to the search process (i.e. measures based on the absolute number of microsatellites). Therefore, in Chapter II I present a critical review of all available software tools designed to scan DNA sequences for microsatellites. My aim in undertaking this review was to assess the comparability of search results among microsatellite programs, and to identify the programs most suitable for the generation of microsatellite datasets for a thorough and reproducible comparative analysis of microsatellite content among genomic sequences. Using sequence data where the number and types of microsatellites were empirical know I compared the ability of 19 programs to accurately identify and report microsatellites. I then chose the two programs which, based on the algorithm and its parameters as well as the output informativity, offered the information most suitable for biological interpretation, while also reflecting as close as possible the microsatellite content of the test files. From the analysis of microsatellite search results generated by the various programs available, it became apparent that the program’s search parameters, which are specified by the user in order to define the microsatellite characteristics to the program, influence dramatically the resulting datasets. This is especially true for programs suited to allow imperfections within tandem repeats, because imperfect repetitions can not be defined accurately as is the case for perfect ones, and because several different algorithms have been proposed to address this problem. The detection of approximate microsatellites is, however, essential for the study of microsatellite evolution and for comparative analyses based on microsatellites. It is now well accepted that small deviations from perfect tandem repeat structure are common within microsatellites and larger repeats, and a number of different algorithms have been developed to confront the challenge of finding and registering microsatellites with all expectable kinds of imperfection. However, biologists have still to apply these tools to their full potential. In biological analyses single tandem repeat hits are consistently interpreted as isolated and independent repeats. This interpretation also depends on the search strategy used to report the microsatellites in DNA sequences and, therefore, I was particularly interested in the capacity of repeat finding programs to report imperfect microsatellites allowing interpretations that are useful in a biological sense. After analzying a series of tandem repeat finding programs I optimized my microsatellite searches to yield the best possible datasets for assessing and comparing the degree of imperfection of microsatellites among different genomes (Chapter III) During the program comparisons performed in Chapter II, I show that the most critical search parameter influencing microsatellite search results is the minimum length threshold. Biologically speaking, there is no consensus with respect to the minimum length, beyond which a short tandem repeat is expected to become prone to microsatellite-like mutations. Usually, a single absolute value of ~12 nucleotides is assigned irrespective of motif length.. In other cases thresholds are assigned in terms of number of repeat units (i.e. 3 to 5 repeats or more), which are better applied individually for each motif. The variation in these thresholds is considerable and not always justifiable. In addition, any current minimum length measures are likely naïve because it is clear that different microsatellite motifs undergo replication slippage at different length thresholds. Therefore, in Chapter III, I apply two probabilistic models to predict the minimum length at which microsatellites of varying motif types become overrepresented in different genomes based on the individual oligonucleotide frequency data of these genomes. Finally, after a range of optimizations and critical analyses, I performed a preliminary analysis of microsatellite abundance among 24 high quality complete eukaryotic genomes, including also 8 prokaryotic and 5 archaeal genomes for contrast. The availability of the methodologies and the microsatellite datasets generated in this project will allow informed formulation of questions for more specific genome research, either about microsatellites, or about other genomic features microsatellites could influence. These datasets are what I would have needed at the beginning of my PhD to support my experimental design, and are essential for the adequate data interpretation of microsatellite data in the context of the major evolutionary units; chromosomes and genomes.

Thesis (M.S.)--Brigham Young University. Dept. of Plant and Wildlife Sciences, 2007. / Includes bibliographical references (p. 74-83).

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 103-112).

Thesis (Ph. D.)--University of Canterbury, 2008. / Typescript (photocopy). Includes bibliographical references (leaves 192-230).

The pallid sturgeon (Scaphirhynchus albus) and shovelnose sturgeon (S. platorynchus) are benthic freshwater fishes that are sympatric throughout the range of the pallid sturgeon. Pallid sturgeon was listed as endangered under the Endangered Species Act in 1990, while shovelnose is common throughout the range. Previous abundance studies estimate the ratio of adult pallid to shovelnose sturgeon to be on the order of 1:82 in the middle Mississippi River, respectively. Despite adult abundances, reproduction and/or recruitment of pallid sturgeon larvae is undocumented in the middle Mississippi River. The current study aims to confirm the presence of pallid sturgeon reproduction and estimate the relative species abundances of larval pallid, shovelnose and hybrid sturgeon in the middle Mississippi River. Since larval pallid, shovelnose and hybrid sturgeon are virtually identical morphologically, the use of DNA markers was required for species designations; sixteen previously developed microsatellite loci were used in this study. Of the 583 larval Scaphirhynchus sturgeon collected from the middle Mississippi River, 581 were shovelnose, one was a hybrid and one was a pallid. This study was the first to genetically confirm the presence of pallid sturgeon reproduction in the middle Mississippi River. Differences in species ratios between adult and larval Scaphirhynchus sturgeon could be explained by three potential hypotheses; life history characteristics accentuate species ratios between adult and larval stages, pallid are experiencing low reproduction and/or low recruitment, or pallid larvae reside in different microhabitat locations compared to shovelnose.

Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-28Bitstream added on 2014-06-13T20:23:14Z : No. of bitstreams: 1 martins_afa_dr_botib.pdf: 1302290 bytes, checksum: f4a20eda14cf780d998f0e02bf28f074 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A malária é uma das principais parasitoses humanas do mundo, causando mais de um milhão de mortes, e quase 500 milhões de casos agudos da doença por ano. No Brasil, esta doença continua sendo uma das mais importantes do país, tendo sido registrados no ano de 2009 mais de 300 mil casos. O mosquito Anopheles darlingi é o principal vetor desta doença no Brasil e outros países da América do Sul. Devido à sua importância como vetor da malária humana, estudos sobre a estrutura populacional de An. darlingi tem sido objeto de vários estudos. A sua distribuição na Região Amazônica é ampla e dados recentes mostram alto grau de heterogeneidade, tanto genética como de comportamento. Grande parte desta variabilidade observada em An. darlingi pode estar relacionada com estratégias adaptativas para explorar nichos ecológicos distintos enquanto que a estrutura populacional e a diferenciação pode ser explicada por diferenças no tamanho efetivo da população, padrões de fluxo gênico e acontecimentos históricos e de colonização recente. De igual modo, alterações ambientais efetuadas pelo homem podem ter um impacto significativo na dinâmica de populações de vetores e, consequentemente, na transmissão da malária. Este projeto utilizou a genotipagem de 10 microssatélites para o estudo populacional de Anopheles darlingi coletados em sete localidades ao longo da extensão das Hidrelétricas de Jirau e Santo Antônio, às margens do Rio Madeira, em Porto Velho – RO. Estes métodos foram aplicados na caracterização de amostras coletadas nestas regiões no 1º e 2° semestres de 2007. O objetivo do trabalho foi analisar a estrutura populacional de An. darlingi ao longo do Rio Madeira, na área de influência das Hidrelétricas de Jirau e Santo Antônio. Os resultados mostraram alto fluxo gênico entre as populações, mesmo distando de 70 km. Foram encontradas diferenças... / Malaria is the major human parasitic diseases in the world, causing more than a million deaths and almost 500 million acute cases of disease per year. In Brazil, this disease remains one of the most important, having been recorded in the year of 2009 more than 300.000 cases. The mosquito Anopheles darlingi is the principal malaria vector in Brazil and other countries in South America. Due to its importance as a vector of human malaria, population structure of Anopheles darlingi has been the subject of several studies. Its distribution in the Amazon region is large and recent data show a high degree of heterogeneity, regarding genetic and behavioral aspects. Much of this observed variability may be related to adaptive strategies to exploit different ecological niches, while the population structure and differentiation can be explained by differences in effective population size, patterns of gene flow and historical events and recent colonization. Similarly, environmental changes made by man can take a significant impact on population dynamics of vectors and hence the transmission of malaria. This project genotyping 10 microsatellites for population-based study of Anopheles darlingi collected at seven locations along Rio Madeira at Porto Velho - RO. These methods were applied in the characterization of samples collected in these regions in the 1st and 2nd semesters of 2007. The objective was to analyze the population structure of Anopheles darlingi along the Madeira River, the area of influence of Hydroelectric of Jirau and San Antonio. The results showed high gene flow among populations, even at 70 km apart. We found significant genetic differences among populations when samples were compared seasonally. The samples collected in the first half of the year showed effective population size 10x greater than those collected in the second half. These differences may represent differences... (Complete abstract click electronic access below)