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Generation of discrete yeast DNA fragments by endonuclease RI

Naturevolume 257pages155157 (1975) | Download Citation



FINE structure genetic analysis has shown that the ilv1 gene of yeast, Saccharomyces cerevisiae, is multifunctional1–3. The ilv1 gene product, threonine deaminase, shows catalytic activity, as well as participating in multivalent repression of other enzymes involved in isoleucine–valine biosynthetic pathways1–3. A better understanding of the regulatory role of the ilv1 gene product and other proteins, such as isoleucyl-tRNA synthetase, which in addition to the ilv1 gene product seems to regulate ilv2 and ilv3 gene expression, could be obtained if a pure preparation of the various ilv genes were isolated in large quantities. One way of isolating the genes would be to use restriction enzymes to cleave yeast DNA which contains normal ilv genes and join the fragments to a bacterial plasmid. Because of the similarity of the isoleucine–valine pathways in yeast and Escherichia coli, the fused DNA could then be transformed into a strain of E. coli with a deletion in the particular ilv gene. Restriction enzyme-cleaved DNA from various sources has been amplified by growth in E. coli4–7. We have found that restriction enzyme EcoRI treatment of yeast DNA results in not only a reduction in the size of total DNA but also the generation of several distinct species of homogeneous size DNAs some of which are derived from ribosomal genes and others from mitochondrial genes.

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  1. Department of Biochemistry, University of Texas Health Science Center at Dallas, Dallas, Texas, 75235



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