Isolation And Characterization Of Human Ribosomal Protein Complementary-dnas

Ribosome biosynthesis serves as a model of organelle assembly and gene regulation in both prokaryotes and eukaryotes. In Escherichia coli the isolation and cloning of ribosomal RNA genes and ribosomal protein genes was a prerequisite to detailed investigations of expression of these genes. Research into ribosome biosynthesis in eukaryotes has lagged behind in part because of the lack of data on the structure of the genes and mRNAs for ribosomal proteins.;This thesis details experiments designed to identify genomic or cDNA clones that encode human ribosomal proteins. Two methods were employed to achieve the goal of obtaining full-length clones that would facilitate expression experiments in the future. The first, cross hybridization, used a ribosomal protein gene or cDNA from another species to probe a human genomic or cDNA library. In one case a fragment of yeast DNA containing the 3{dollar}spprime{dollar} end of one of the genes encoding yeast ribosomal protein S10 (the homolog of mammalian S6) was used to probe human genomic and cDNA libraries. This approach failed due to insufficient homology between the coding sequences of yeast ribosomal protein S10 and human ribosomal protein S6. In another set of cross hybridization experiments a rat ribosomal protein S11 cloned cDNA was used successfully to probe a human cDNA library and identify a full-length human cloned S11 cDNA.;The second method of isolation employed mixed sequence oligodeoxynucleotide probes to identify human ribosomal protein S6 cDNAs. Ribosomal protein S6 is the major substrate of protein kinases in eukaryotic ribosomes. Published amino acid sequence data for rat liver ribosomal protein S6 and yeast ribosomal protein S10 were used to design mixed oligodeoxynucleotide probes. Screening of several human cDNA libraries with these probes permitted the isolation of recombinant plasmids whose cDNA inserts encompass the entire coding sequence of S6 (249 aa residues), 27 bp of the 5{dollar}spprime{dollar} untranslated leader and all 39 bq of the 3{dollar}spprime{dollar} untranslated region. A comparison of the predicted amino acid sequence and the yeast rp S10 amino acid sequence shows highly conserved areas separated by regions of divergence

Name thesis:degree_name

Ph D

Year dc:date.issued

1989

Author dc:creator

• Lott, John Bryan

• Chemistry
• Biochemistry

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14721/21407

OAI identifier oai:identifier

oai:uwo.scholaris.ca:20.500.14721/21407