Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: David J. Pintel. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Committee on Evolutionary Biology, August 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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O veneno de Bothops jararaca contém uma série de componentes, entre eles as metaloproteinases hemorrágicas jararagina e bothropasina. Os cDNAs dessas toxinas mostram 97% de identidade. As diferenças, distribuídas ao longo de seus cDNAs, sugerem que estes mRNas não resultam de splicing alternativo. O objetivo deste trabalho foi caracterizar os genes codificadores da jararagina e bothropasina pela identificação de exons e introns no DNA genômico. DNA foi extraído do sangue de um exemplar de B. jararaca; os primers para PCR foram baseados nos cDNAs publicados. Os produtos de amplificação foram clonados e seqüenciados revelando a sequência dos genes TOX1 com 12535 pb e TOX2 com 12268 pb. Quatorze exons e treze introns foram identificados em ambos os genes. Comparação entre as sequências mostrou pontos de mutação, inserções e deleções nos exons, e principalmente nos introns dos dois genes. Este constitui o primeiro relato na literatura sobre a identificação de exons e introns nos genes codificadores de jararagina e bothropasina. / The Bothops jararaca venom contains a number of components, including hemorrhagic metalloproteinases as jararhagin and bothropasin. The cDNA of these toxins show 97% identity. The differences distributed along the cDNAs length suggest that these mRNAs do not result from alternative splicing. This study aimed to characterize the genes that encode for jararhagin and bothropasin through the identification of exons and introns in genomic DNA. DNA was extracted from the blood of a B. jararaca specimen; PCR primers were based on published cDNA sequences. Amplification products were cloned and sequenced revealing the TOX1 gene is about 12,535 bp long, and TOX2 is 12,268 bp. Fourteen exons and thirteen introns were identified in both genes. Comparison of the sequences showed point mutations, insertions and deletions in exons, and particularly in introns. This is the first report in the literature on the identification of exons and introns in genes encoding for jararhagin and bothropasin.

Terminado o sequenciamento do genoma humano, as atenções se voltaram para a determinação do conjunto completo de transcritos humanos. Diversos trabalhos sugerem que enquanto apenas uma pequena fração de mRNAs codificantes para proteína não é conhecida, existe um grande número de RNAs não-codificantes (ncRNAs) ainda não caracterizados. Nesse contexto, o presente trabalho visou explorar as informações de expressão gênica contidas em ESTs para identificar e caracterizar novos transcritos humanos. A busca genômica por membros de famílias gênicas relacionadas com câncer levou a identificação de novas pequenas GTPases, destacando uma subfamília que deve apresentar função supressora tumoral em próstata. Uma classe de ncRNAs longos, sem splicing, expressos antisenso a partir de regiões intrônicas foi descrita utilizando plataformas de microarrays, construídas pelo grupo, enriquecidas com seqüências sem anotação. O perfil de expressão de 23 ncRNAs intrônicos estava significativamente correlacionado com o grau de diferenciação de tumores de próstata (Gleason Score), e pode ser utilizado como candidato a marcador molecular de prognóstico. Um total de 39 ncRNAs intrônicos responderam à estimulação por andrógeno, apontando para um mecanismo regulatório da expressão intrônica por sinais fisiológicos hormonais. A biogênese da expressão intrônica parece ser complexa, pois uma fração não é transcrita pela RNA Polimerase II. A transcrição intrônica estava correlacionada com uso de exons em células tratadas com andrógeno. Assinaturas de expressão intrônica conservadas em tecidos humanos e de camundongos, e interações de transcritos intrônicos com proteínas regulatórias foram observadas. Este trabalho contribui com novas e originais evidências que dão apoio ao papel postulado para esses ncRNAs no controle fino do programa transcricional humano. / With the completion of the human genome sequence, attention has shifted towards determining the complete set of human transcripts. Multiple lines of evidence suggest that while only a small fraction of protein-coding mRNAs remains to be described, there is a huge amount of uncharacterized non-coding RNAs (ncRNAs). In this context, the present work sought to explore the gene expression information provided by ESTs to identify and characterize new human transcripts. A genomic-wide search for cancer related gene family members identified novel small GTPase genes, and highlighted an uncharacterized subfamily that may have a tumor suppressor role in prostate cancer. A class of long unspliced ncRNAs, expressed antisense from introns of protein-coding genes was described using custom-designed microarray platforms enriched with unannotated sequences. The expression profile of 23 intronic ncRNAs was significantly correlated to the degree of prostate tumor differentiation (Gleason Score), and could be used as a candidate prognostic molecular maker. A total of 39 intronic ncRNAs were responsive to androgen stimulation, poiting to a mechanism of intronic expression regulation by physiological hormone signals. Intronic ncRNA biogenesis seems to be complex, since a fraction of them is not transcribed by RNA Polymerase II. Intronic transcription was correlated to exon usage in androgen treated cells. Tissue expression signatures of intronic transcription were conserved in human and mouse, and intronic transcripts were found to interact with regulatory proteins. This work provides new and original contributions that support the postulated role of ncRNAs in the fine tunning of the human transcriptional program.

O veneno de Bothops jararaca contém uma série de componentes, entre eles as metaloproteinases hemorrágicas jararagina e bothropasina. Os cDNAs dessas toxinas mostram 97% de identidade. As diferenças, distribuídas ao longo de seus cDNAs, sugerem que estes mRNas não resultam de splicing alternativo. O objetivo deste trabalho foi caracterizar os genes codificadores da jararagina e bothropasina pela identificação de exons e introns no DNA genômico. DNA foi extraído do sangue de um exemplar de B. jararaca; os primers para PCR foram baseados nos cDNAs publicados. Os produtos de amplificação foram clonados e seqüenciados revelando a sequência dos genes TOX1 com 12535 pb e TOX2 com 12268 pb. Quatorze exons e treze introns foram identificados em ambos os genes. Comparação entre as sequências mostrou pontos de mutação, inserções e deleções nos exons, e principalmente nos introns dos dois genes. Este constitui o primeiro relato na literatura sobre a identificação de exons e introns nos genes codificadores de jararagina e bothropasina. / The Bothops jararaca venom contains a number of components, including hemorrhagic metalloproteinases as jararhagin and bothropasin. The cDNA of these toxins show 97% identity. The differences distributed along the cDNAs length suggest that these mRNAs do not result from alternative splicing. This study aimed to characterize the genes that encode for jararhagin and bothropasin through the identification of exons and introns in genomic DNA. DNA was extracted from the blood of a B. jararaca specimen; PCR primers were based on published cDNA sequences. Amplification products were cloned and sequenced revealing the TOX1 gene is about 12,535 bp long, and TOX2 is 12,268 bp. Fourteen exons and thirteen introns were identified in both genes. Comparison of the sequences showed point mutations, insertions and deletions in exons, and particularly in introns. This is the first report in the literature on the identification of exons and introns in genes encoding for jararhagin and bothropasin.

Many functional RNAs are required to fold into specific three-dimensional structures. A fundamental property of RNA is that its secondary structure and even some tertiary contacts are highly stable, which gives rise to independent modular RNA motifs and makes RNAs prone to adopting misfolded intermediates. Consequently, in addition to stabilizing the native structure relative to the unfolded species (defined here as stability), RNAs are faced with the challenge of stabilizing the native structure relative to alternative structures (defined as structural specificity). How RNAs have evolved to overcome these challenges is incompletely understood. Self-splicing group I introns have been used to study RNA structure and folding for decades. Among them, the Tetrahymena intron was the first discovered and has been studied extensively. In this work, we found that a version of the intron that was generated by in vitro selection for enhanced stability also displayed enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. Further dissection suggests that the increased specificity against misfolding arises from two point mutations, which strengthen a local tertiary contact network that apparently cannot form in the misfolded conformation. Our results suggest that the structural rigidity and intricate networks of contacts inherent to structured RNAs can allow them to evolve exquisite structural specificity without explicit negative selection, even against closely-related alternative structures. To explore further how RNAs gain stability from intricate architectures, we examined a novel group I intron from red algae (Bangia). Biochemical methods and computational modeling suggest that this intron possesses general motifs of group IC1 introns but also forms an atypical tertiary contact, which has been reported previously in other subgroups and helps position the reactive helix at the active site. In the Bangia intron, the partners have been swapped relative to known group I RNAs that include this contact. This result underscores the modular nature of RNA motifs and provides insight into how structured RNAs can arrange helices and contacts in multiple ways to achieve and stabilize functional structures. / text