Isolation, heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis.

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.