Investigation of yeast plasmid inheritance using techniques for the analysis of individual cells.

The 2Um plasmid of Saccharomyces cerevisiae is a stable multi-copy plasmid encoding copy number amplification and partitioning functions dedicated solely to its own maintenance. Although the role of these regions has been identified, the mechanism of partitioning and the precise relationship between amplification and partitioning is still unknown. At cell division plasmids may be distributed equally between mother and daughter cells, or there may be an unequal distribution resulting in a broad range of copy numbers. The amplification mechanism may play an important role in correcting any partitioning error. Investigations into the nature of partitioning and the co-ordination of partitioning and amplification require the measurement of copy number in individual cells. The aim of this work was to develop both indirect and direct methods to measure copy number in single cells, and to use these methods to analyse the distribution of plasmid molecules amongst individual cells. A feasibility study of the use of a gene product as a measurement of copy number suggested that an indirect enzyme assay would not be sensitive enough to detect low copy numbers. A direct measurement of nucleic acid sequences by in situ hybridization was developed for single yeast cells using both radio-labelled probes and non-radioactive probes detected by fluorescence. The method was specific, sensitive and provided the first direct evidence of a nuclear location for the 2Um plasmid. A significant difference was detected in the pattern of hybridization between budded and unbudded cells containing the wild-type 2Um plasmid and between wild-type cells and those containing a variant plasmid defective in the amplification mechanism. This indicates that the amplification mechanism may have a role in plasmid maintenance. Use of non-radioactive in situ hybridization revealed a distribution of fluorescence intensity amongst cells, suggesting a range of plasmid copy numbers amongst the population. In view of these results a proposed segregation method is discussed.