Graduate Thesis Or Dissertation
 

Attempted cloning of T4 phage ribonucleotide reductase

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/x920g108k

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  • Ribonucleotide reductase is an important enzyme in the control of DNA replication within the cell. Ribonucleotide reductase exerts its control through enzymatic reduction of nucleoside diphosphates. The bacteriophage T4 enzyme is an example of the class of iron-requiring reductases which also includes E. coli and mammalian ribonucleotide reductases. The two genes coding for the E. coli ribonucleotide reductase have at this time been cloned, sequenced and cloned into expression vectors. Many other ribonucleotide reductase genes have also been cloned. To date, however, only one of the two genes which code for Bacteriophage T4 ribonucleotide reductase has been cloned although fragments have been sequenced. It was the objective of this work to clone both of the genes for Bacteriophage T4 ribonucleotide reductase into high expression vectors The work of a different laboratory has demonstrated that at least one of the genes of T4 ribonucleotide reductase is contained on a 5.2 kb Hind Ill fragment. This thesis work describes the cloning of this 5.2 kb fragment into different plasmid vectors and the attempt to achieve over-expression of the T4 ribonucleotide reductase subunits. One recombinant plasmid containing the 5.2 kb fragment expressed a 60 kDa peptide. This peptide appears to be part of the large subunit of ribonucleotide reductase. Owing to its poor solubility in aqueous solutions, attempts to purify the 60 kDa peptide were not successful.
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