Plasmid pRS319 is plasmid pRS315 containing the CAN1 gene. It was
designed for plasmid shuffling. See Meth. Enzymol v 194, page 308.
We cloned our gene into pRS319 for a plasmid shuffle, but found that
it did not confer canavanine sensitivity. Restriction digests indicate
that our prep of the pRS319 vector is correct. In the Meth. Enzymol
article, it states in the Fig. 2 legend that a BamHI-SalI fragment that
lacks the last 31 amino acids of Can1 was used, and that this truncation
allele confers canavanine sensitivity.
My computer construction of pRS319 shows that the C-terminus of Can1
is fused in frame to 50 amino acids derived from the LEU2 fragment. Thus,
it is possible that this fusion makes the Can1 protein inactive.
My question: Has anyone else had problems with the canavanine
sensitivity of the pRS319 plasmid?
Thank you for your help.
Division of Molecular Biology and Genetics
Department of Oncological Sciences
University of Utah Heath Sciences Center
Salt Lake City, UT 84132
stillman at bioscience.utah.edu