Recently we have observed occasional growth of cells from met15 strains on medium lacking methionine. This growth has only been observed when replica plating thick patches of cells, and is critically dependent on temperature: growth of met15 strains on -met medium is maximal at 22 degrees, drops dramatically at 25 degrees, and decreases approximately linearly with temperature from 25 to 37 degrees, at which point little if any papillation is observed. This phenomenon seems to be dependent on cell density, as most papillae lie near the edge of the patch which was replica plated; in addition, no growth of met15 strains on -met medium has ever been observed during transformation. Such cells are not stable revertants, as no growth is observed when these cells are struck for single colonies. These cells remain black when grown on lead nitrate containing medium. Pseudorevertants have been observed for two met15 alleles: the complete deletion (met15D0) as well as for the Ty-1 disruption of MET15 (strain 31-10c) described in Cost and Boeke (1996). In sum, the above observations are consistent with either or both of the following hypotheses:

• There exists a gene which, at low temperature, can function as a bypass suppressor of met15 in the methionine biosynthetic pathway.
• Cross feeding of methionine from dying cells can occur, and at high cell density, is sufficient to support papillation on met- medium.

Thus, the met15 marker may not be suitable for applications which require both replica plating and growth at ambient temperature or below.

Recently, we've even been able to distinguish MET15/met15 cells from MET15/MET15 cells (Connelly and Boeke, unpublished observation).

We've also noticed a more annoying phenomenon occurring at the MET15 locus: loss of heterozygosity. MET15 is distal to the RDN1 loci on chromosome VIII. When the rDNA repeat recombines, as it is prone to do, heterozygosity can be lost. Preliminary estimates are about a <3% frequency of LOH. (Connelly and Boeke, unpublished observation).