Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.

Background: Mitochondrial genomes are a valuable source of data for analysing phylogenetic relationships. Besides sequence information, mitochondrial gene order may add phylogenetically useful information, too. Sipuncula are unsegmented marine worms, traditionally placed in their own phylum. Recent molecular and morphological findings suggest a close affinity to the segmented Annelida. Results: The first complete mitochondrial genome of a member of Sipuncula, Sipunculus nudus, is presented. All 37 genes characteristic for metazoan mtDNA were detected and are encoded on the same strand. The mitochondrial gene order (protein-coding and ribosomal RNA genes) resembles that of annelids, but shows several derivations so far found only in Sipuncula. Sequence based phylogenetic analysis of mitochondrial protein-coding genes results in significant bootstrap support for Annelida sensu lato, combining Annelida together with Sipuncula, Echiura, Pogonophora and Myzostomida. Conclusion: The mitochondrial sequence data support a close relationship of Annelida and Sipuncula. Also the most parsimonious explanation of changes in gene order favours a derivation from the annelid gene order. These results complement findings from recent phylogenetic analyses of nuclear encoded genes as well as a report of a segmental neural patterning in Sipuncula.

The work discussed in this thesis deals with the significance of initiator tRNA gene copy number in Escherichia coli. A summary of the relevant literature discussing the process of protein synthesis, initiator tRNA selection and gene redundancy is presented in Chapter 1. Chapter 2 describes the ‘Materials and Methods’ used in the experimental work carried out in this thesis. The next three chapters address the significance of initiator tRNA gene copy number in E. coli at three levels; at the level of the molecule (Chapter 3), at the level of the cell (Chapter 4) and at the level of the population (Chapter 5). At the end of the thesis are appended three publications, which include two papers where I have contributed to work not discussed in this thesis and one review article. A brief summary of chapters 3 to 5 is provided below: (i) Chapter 3: Can E. coli remain viable without the 3 G-C base pairs in initiator tRNA? Initiator tRNAs are distinguished from elongator tRNAs by several features key among which are the three consecutive and near universally conserved G-C base pairs found in the anticodon stem of initiator tRNAs. These bases have long been believed to be essential for the functioning of a living cell, both from in vitro and in vivo analysis. In this study, using targeted mutagenesis and an in vivo genetics based approach, we have shown that the 3 G-C base pairs can be dispensed with in E. coli, and the cell can be sustained on unconventional initiator tRNAs lacking the intact 3 G-C base pairs. Our study uncovered the importance of considering the relative amounts of molecules in a living cell, and their role in maintaining the fidelity of protein synthesis. (ii) Chapter 4: Can elongator tRNAs initiate protein synthesis? There are two types of tRNAs; initiator tRNA, of which there is one representative in the cell, and elongator tRNAs of which there are several representatives. In this study, we have uncovered initiation of protein synthesis by elongator tRNAs by depleting the initiator tRNA content in the cell. This raises the possibility that competition between initiator and elongator tRNAs at the P site of the ribosome occurs routinely in the living cell, and provides a basis for initiation at several 'start' sites in the genome that may not be currently annotated as such. We speculate that such a phenomenon could be exploited by the cell to generate phenotypic diversity without compromising genomic integrity. (iii) Chapter 5: How many initiator tRNA genes does E. coli need? E. coli has four genes that encode initiator tRNA, these are the metZWV genes that occur at 63.5 min in the genome, and the metY gene that occurs at 71.5 min in the genome. Earlier studies indicated that the absence of metY had no apparent impact on cell growth. In view of the importance of initiator tRNA gene copy number in maintaining the rate and fidelity of protein synthesis, we examined the fitness of strains carrying different numbers of initiator tRNA genes by competing them against each other in both rich and limited nutrient environments. Our results indicate a link between caloric restriction and protein synthesis mediated by the initiator tRNA gene copy number.

Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional. / Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called \"barcodes\". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.

Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional. / Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis, which may have adverse consequences, such as induction of proto-oncogenes. Such events have been described in clinical trials for the treatment of SCID-X1, chronic granulomatous disease and beta thalessemia with some retroviral vectors. Currently, there are few simple and quick methods that can reveal and quantify clonal expansion. Thus, we describe the construction of a vector library containing random markers, called \"barcodes\". The sequencing of the barcode could reveal, characterize and quantify the clonal expansion of a transduced cells population. This methodology will be valuable to test new arrangements of promoters and therapeutic genes, allowing the development of safer vectors, helping to reduce the probability of clonal proliferation events triggered by insertional mutagenesis.