The use of
centromeric gene library for identification of genes responsible for the [ISP +]
prion-like determinant maintenance.
Anna Aksenova, Kirill Volkov, Anton Svitin, Ludmila Mironova
Department of Genetics, St. Petersburg University, University emb. 7/9,
Saint-Petersburg, 199034, Russia (anton@btc.bio.pu.ru)
The
non-Mendelian determinant [ISP+] is presumably a new yeast prion
(Volkov et al.,
2002). Further elucidation of [ISP+] nature assumes identification of
its host gene as well as the other genes controlling its propagation. One of
approaches applied to detection of these genes is based on the obtaining of
spontaneous or UV-induced mutations eliminating [ISP+] and subsequent
identification of mutant genes. Selection of such mutations is possible, since
[ISP+]
is rather stable mitotically (the rate of its spontaneous loss is ~1,2 x 10-7).
We proposed that among mutations selected could be a class of dominant
mutations similar to PNM mutations in SUP35 gene eliminating the [PSI+] prion (Doel
et al.,
1994; De Pace et al., 1998). To avoid problems of their cloning we selected
these mutations in [ISP+] strain transformed with Ycp50 based gene
library. Among 40, 000 Ura+ transformants obtained, 450 clones that
altered their phenotype from [ISP+] to [isp-] were found.
In spite of the majority of these clones lost [ISP+]
spontaneously or contained chromosomal mutations, we have revealed four clones,
manifesting the [isp-] phenotype due to the plasmid. The results of
genetic and molecular characterization of these plasmids are presented. The
work is supported by RFBR 02-04-49699 and CRDF ST-012 grants.