HETERODUPLEX STUDIES ON THE PHYSICAL ORGANIZATION OF THE ILV GENE CLUSTER

TIMOTHY DELBERT LEATHERS, Purdue University

Abstract

Electron microscopic heteroduplex analysis was employed as the principal tool in the study of bacteriophages and plasmids containing DNA from the ilv gene cluster. A physical map of the ilv gene cluster was prepared in collaboration with the restriction endonuclease mapping studies of G. M. McCorkle. The six structural genes of the cluster were determined to lie tightly juxtaposed within an 8.7 kb region of DNA. Contributing to this map was the analysis of three plaque-forming transducing phages derived from a single prophage insertion into the ilvC gene. These results defined the orientation of a secondary lambda attachment site in ilvC, and suggested an excision scheme for these phages. Heteroduplex analysis of (lamda)pilv-lac1 and (lamda)pilv-lac3 was performed, these phages prepared by the Casadaban technique to include fusions of the lac genes to the ilvC and ilvD genes, respectively. Results confirmed the genetic assumptions involved in the preparation of these fusions, and provided physical specifications for these models. The organization of these phages confirmed that transcription proceeds in the direction of ilvE through ilvA, and that ilvC is transcribed in the same direction. Physical mapping was also performed for (lamda)p1(209) and (lamda)p123(209), the complementary pair of phages employed in the Casadaban technique of operon fusion. Physical analysis of the ilv-2130 deletion revealed that it extends from a point early in ilvE to a point in ilvG, covering 1.75 kb, and thus deleting the entire ilvO determinant region. Because ilv-2130 resulted in a coordinate elevation of ilvD and ilvA expression similar to that observed for ilvO strains, this study concluded that ilvO('+) exists as a site of natural polarity for ilvGEDA transcripts. Other physical mapping results further contributed to a new model for the organization of the ilv genes, in which ilvO('+) is postulated to be a polar mutation in the ilvG structural gene, contained in an ilvGEDA operon. Heteroduplex comparison of ilvO('+) and ilvO DNA revealed complete gross homology, providing presumptive evidence that ilvO('+) represents a point mutation. DNA from the ilv cluster of strain K-12 was compared to that from strain B by heteroduplex analysis. Complete homology indicated that the K-12 ilv operon organization is fundamentally identical to that of strain B, which exhibits the valine resistance characteristic of most enterics.

Degree

Ph.D.

Subject Area

Microbiology

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