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dc.contributor.authorChakravarti, Debabrata
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 53-10, Section: B, page: 5072.;Advisors: Umadas Maitra.
dc.description.abstractEukaryotic translation initiation factor 5(eIF-5) catalyzes the hydrolysis of GTP bound to a 40S ribosomal initiation complex with the subsequent joining of a 60S ribosomal subunit to form an 80S ribosomal initiation complex. Using an assay that directly measures the formation of an 80S ribosomal initiation complex from a preformed 40S initiation complex, eIF-5 was purified to apparent electrophoretic homogeneity from total yeast cell lysates. Analysis of the size of the purified protein by Sephadex gel filtration as well as by SDS-polyacrylamide gel electrophoresis demonstrates that purified yeast eIF-5 is a monomeric protein of apparent Mr = 50,000-55,000. Monospecific polyclonal antibodies to purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80S initiation complex formation. When yeast cells were lysed directly in a denaturing buffer containing 3% SDS and analyzed by Western blot, a major immunoreactive polypeptide band was observed whose molecular weight corresponded to that of purified eIF-5.;By screening a yeast genomic {dollar}\lambda{dollar}gt 11 library using affinity-purified anti-yeast eIF-5 antibodies as probe, we have cloned the entire structural gene of eIF-5. The eIF-5 gene was sequenced, revealing an intron-free open reading frame encoding 405 amino acids predicting a protein of Mr = 45,400, in close agreement with the apparent molecular weight of purified yeast eIF-5. The open reading frame is preceded by an in-frame translational termination codon confirming that the 45.4 kDa protein is not part of a larger polypeptide chain. The identity of the eIF-5 gene was confirmed by several criteria. First, the partial amino acid sequences of three peptides generated by CNBr-digestion of yeast eIF-5 were present in the derived amino acid sequence of the eIF-5 gene. Second, the clone-encoded protein is immunologically related to purified yeast eIF-5. Finally, expression of the eIF-5 gene present in the recombinant {dollar}\lambda{dollar}gt 11 clone in Escherichia coli yielded full length eIF-5 protein that was catalytically active in mediating the joining of a 60S ribosomal subunit to a preformed 40S initiation complex to form the 80S initiation complex. Northern blot analysis indicates that eIF-5 transcript is about 1.75 kb long. Gene disruption and Southern hybridization experiments show that eIF-5 is encoded by an essential, single copy gene that maps on chromosome XVI. Search in the data bank failed to show any homology between eIF-5 and any previously characterized protein. Deduced amino acid sequence of yeast eIF-5 shares nearly 60% homology with the mammalian eIF-5 protein that has also been cloned in our laboratory. Both proteins contain sequence motifs characteristic of members of GTPase-superfamily.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleMolecular cloning and characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae

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