Structure and expression of tRNA genes in Xenopus erythrocytes

Talwar, Sandeep (1984) Structure and expression of tRNA genes in Xenopus erythrocytes. University of Southampton, Doctoral Thesis.

Abstract

The use of nucleases to probe the higher-order organisation of genes and the development of cell-free transcription systems of high fidelity in combination with gene cloning and gene mapping techniques have greatly enhanced our comprehension of gene expression during cellular differentiation. This thesis concerns the structure and expression of tRNA genes in erythroid cells of Xenopus laevis. Cloned tRNA genes are found to be transcribed faithfully and accurately by RNA polymerase III in cell-free extracts from Xenopus oocytes. These cells are therefore an abundant source of polymerases and transcription factors which are necessary and sufficient for tRNA gene transcription in vitro. A calcium-activated deoxyribonuclease is detected endogenous to Xenopus erythrocytes. Nuclease digestion studies also show that tRNA genes are organised into a highly ordered chromatin structure and that some tDNA repeats contain sites which are hypersensitive to DNase I and endogenous nuclease. The methylation sensitive restriction enzymes Msp I, Hpa II and Hha I were used to investigate the methylation pattern of tRNA genes in germ line and somatic tissues of X. laevis. All tDNA repeats studied were found to be modified at these sites in sperm DNA. In somatic DNA, a fraction of repeats was found to contain specific, non-random demethylated sites which were conserved during development and differentiation. Although the results do not support a straight correlation between hypomethylation and gene transcription, they are in agreement with the hypothesis that DNase I hypersensitivity and demethylation are associated with tRNA gene expression during developmental processes.

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