Long-term growth in selective medium selects for integration of plasmid sequences.

<p>After 80 to 90 generations of selective growth in–uracil medium, single clones were isolated from the cultures of transformants 1, 3, 5, 7, and 8. DNA from the first day (F), the last day (L) and the clone (C) was isolated by smash-and-grab and by an agarose plug method and analyzed by conventional (A) and CHEF (B) gel electrophoresis. The fates of the transforming <i>URA3-ARS1</i> dog bones were determined by Southern blots using <i>URA3</i> as a probe (ethidium bromide images on left, Southern hybridization on right). The parental strain BY4741 (P) has a complete deletion of <i>URA3</i> on chromosome V. (A) Plasmids were identified in all samples (nicked circular (NC) and supercoiled (SC) molecules of two different sized plasmids), with the exception of the clone from transformant #7 (red box). In this sample, hybridization to chromosomal fragments suggested that the plasmid had integrated (marked with *). (B) CHEF analysis confirms the presence of <i>URA3</i> on chromosome IV. Increased hybridization of <i>URA3</i> sequences (over the background signal found in the parental strain (P)) on the last days of selective growth suggests that integration had occurred in other cultures, although the single clones did not reflect this event. (Note that small plasmids migrate very slowly under CHEF gel electrophoresis conditions and also are trapped in the wells. These molecules are indicated by brackets on the right side of the gel.) Both DNA sampling techniques also revealed that free plasmids were experiencing other changes as a consequence of long-term selective growth.