Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine Molecular Virology Program. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 16, 2009). Includes bibliographical references.

The transcription efficiency of transfer RNA genes is modulated by sequences contained in their 5'-flanking region. For a tRNA val₄ gene a pentanucleotide with the sequence TCGCT was identified between positions -33 and -38. I have previously proposed that this sequence may be involved in specifically determining the rate of transcription of this gene. A general form of this sequence, TNNCT was found associated with other Drosophila tRNA genes which showed high ill vitro transcription efficiency. To further elucidate the role of TCGCT in tRNA transcription, single and double base-pair changes were created in the sequence TCGCT using site-specific mutagenesis. Mutations in the nucleotides -38T, -35C and -34T showed decreased levels of transcription whereas nucleotide changes at the nucleotides -37C and -36G did not reduce template activity. Therefore the sequence which modulates transcription of the tRNAVal₄ gene does have the general form TNNCT. Competition experiments between the Val₄ mutant -38G.-35A and a tRNASer₇ gene showed the TNNCT mutant to be a better competitor for transcription than the wild type template. Experiments analyzing the time-course of transcription, the effects of temperature and the effects of ionic strength indicated that TNNCT was not involved in determining the efficiency of stable complex formation. It is proposed that the pentanucleotide is probably responsible for influencing the rate of initiation of transcription. A sequence TGCCT contained in the anticodon stem/loop region of the Val₄ gene was also mutagenized and shown to be involved in complex stability or the elongation of Val₄ tRNAs. Using deletion analysis of the 5'-flanking sequences of a tRNASer₇ gene, a second positive transcription regulatory element was delimited. This sequence was also found in the 5'-flanks of the tRNAVal₄ and a tRNAArg gene. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The nucleotide sequence of the single Drosophila meianogaster tRNA gene contained in the recombinant plasmid, pDtl20R was determined by the Maxam and Gilbert method. This plasmid hybridizes to the 90 BC site on the Val Drosophila polytene chromosomes, a minor site of tRNA4 hybridization. The Val nucleotide sequence of the tRNA4 gene present in pDtl20R differs at four Val positions from the sequence expected from that of tRNA4 . The four differences occur at nucleotides 16, 29, 41 and 57 in the coding region. Comparison of the DNA sequence of pDtl20R to that of the plasmid pDt92R, which also hybridizes to the 90 BC site, indicates that the Drosophila fragments contained in these two plasmids are either alleles or repeats. The implications of these findings are discussed. An in vitro transcription system was developed from a Drosophila Schneider II cell line. This homologous cell-free extract support specific and accurate transcription of various Drosophila tRNA Val genes. The major product of transcription is a tRNA precursor which is processed to a tRNA sized species. Transfer RNA valine genes originating from different sites on the Drosophila chromosomes are transcribed at different rates. Comparison of the sequences in the internal promoter regions of the various genes indicates that the few differences within the coding regions may not be responsible for the observed difference in the rates of transcription. This conclusion is substantiated by studies with hybrid genes constructed during the course of this work. Preliminary evidence indicates that the Val tRNA gene which is transcribed at the highest rate may be preceded in its 5'-flanking region by a positively modulating sequence. Val The precursor RNAs directed by various tRNA genes are also processed at different rates. Transcription and processing experiments with hybrid genes suggest that nucleotide changes within the coding region, which do not affect the rate of transcription, influence the rate of processing. Time course and competition experiments demonstrate that at least two kinetic steps are required for the formation of a stable transcription complex. Studies with an in vitro constructed mutant missing in nucleotides 51-61 in the tRNA coding region suggests that this deleted region (which is highly conserved in eukaryotic tRNAs) may be involved in the primary interaction required for tRNA gene transcription. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Title from second page of PDF document. Includes bibliographical references (p. Xx-Xx).

No description available.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. / Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding. / Revisión por pares

A esquistossomose é uma doença importante em saúde pública. Dos genes selecionados como diferencialmente expressos em esquistossômulos a partir do transcriptoma do S. mansoni, 56% foram confirmados por RT-PCR em tempo real. Entre eles, a proteína Ly6.5, está presente no tegumento de esquistossômulos e vermes adultos por âncoras de GPI. Não foi detectada a função de inibir o sistema complemento, mas pode estar envolvido na manutenção do tegumento. O gene SmVal7 revelou transcritos nas glândulas esofágicas de vermes adultos por hibridização in situ, enquanto a localização da proteína não está definida. Anexina está associada ao tegumento de esquistossômulos e vermes adultos, de maneira dependente de cálcio. A supressão do gene por RNAi não resultou em alteração fenotípica significativa em esquistossômulos in vitro. Foi observada atividade parcial de inibição de coagulação e potencial atividade de endocitose de anticorpos ligados à superfície. A imunização com rLy6.5, rSmVal7, rAneI-II ou rAneII-III não levou a redução da carga parasitária após desafio. / Schistosomiasis is an important disease in public health. Genes selected from the S. mansoni transcriptome, 56% of them were confirmed as differentially expressed in schistosomula by real time RT-PCR. Among them, the protein Ly6.5 is present in the tegument of schistosomula and adult worms by GPI anchors. The function of inhibiting the complement system was not detected, but it may be involved in maintenance of the tegument. The gene SmVal7 revealed transcripts in the esophageal glands of adult worms by in situ hybridization, while the localization of the protein is not defined. Annexin is associated with the membranes of the schistosomula and adult worms tegument in a calcium-dependent manner. The suppression of the gene by RNAi did not resulted in a significant phenotypic change in schistosomula in vitro. Parcial inhibition of the coagulation activity and potential function of endocytosis of membrane-bound antibodies were observed. Immunization with the rLy6.5, rSmVal7, rAneI or rAneII-II-III did not show reduction in worm burden recovery after challenge.