Functional analysis of a 38 kilobase region on chromosome XVI in Saccharomyces cerevisiae.

Abstract

In this report we assess the functional importance of 16 open reading frames (ORFs) contained within a 38 780 base-pair region immediately adjacent to the centromere on the right arm of chromosome XVI in Saccharomyces cerevisiae. This analysis involved replacing one copy of each ORF in a diploid strain with a cassette encoding the green fluorescent protein from the jellyfish Aequorea victoria and HIS3. Each replacement cassette was generated by PCR using oligonucleotide pairs with 45-base extensions complementary to sequences immediately upstream and downstream of the target gene's coding region. After replacement of the targeted genes, each gene-replacement strain was subjected to a series of genetic and phenotypic tests to assess the functional importance of the deleted gene. This analysis showed that two ORFs were essential, one for spores to germinate and another for vegetative growth. A third gene encoded a copper-fist-like transcription factor that was required for proper bud-site selection. One of the 16 ORFs was duplicated, a situation not observed in the strain used to sequence the yeast genome (S288C). RNA analysis showed 11 of the 16 ORFs in this region expressed steady-state poly(A+) RNA levels that were greater than or equal to 2% of the level expressed from the yeast actin gene, ACT1.