Chromosomal localization of the HYP2‐gene in Saccharomyces cerevisiae and use of pulsed‐field gel electrophoresis for detection of irregular recombination events in gene disruption experiments

Abstract

In the hypusine‐containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4‐amino‐2‐hydroxybutyl‐lysine). The HP has been designated as an eucaryotic translation initiation factor—eIF‐5A—because of its stimulating effect in the methionyl‐puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1‐gene is identical to the AN B1‐gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2‐gene has not been elucidated. By using pulsed‐field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2‐gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.