Research Article Marker construction and cloning of a cut1-like sequence with ARS activity in the fission yeast Schizosaccharomyces japonicus Aniko Bozsik 1,2 , Z. Szilagyi 1,2 , Z. Benko 1 and M. Sipiczki 1,2 * 1 Department of Genetics, University of Debrecen, Debrecen, Hungary 2 Research Group for Microbial Developmental Genetics, Hungarian Academy of Sciences, Debrecen, Hungary * Correspondence to: M. Sipiczki, Department of Genetics, University of Debrecen, PO Box 56, H-4010 Debrecen, Hungary. E-mail: lipovy@tigris.klte.hu Received: 22 October 2001 Accepted: 16 December 2001 Abstract The dimorphic fission yeast Schizosaccharomyces japonicus has proved to be an excellent experimental model for the investigation of the eukaryotic cell. Here we show that it has a haplontic life cycle, in which the diploid phase is confined to the zygote. To make it amenable to genetic and molecular analysis, we generated genetic markers and cloned a genomic sequence which acts as ars when integrated into a plasmid. Diploids suitable for testing complementation and recombination between markers can be formed by protoplast fusion. The complementation tests and the recombination frequencies determined in octads of spores identified 28 non-allelic groups (genes) of mutations of the auxotrophic and mycelium-negative mutants. Two groups of linked markers were also identified. The cloned fragment, which expresses ars activity, encodes a putative amino acid sequence highly similar to a conserved domain of proteins Cut1 (Schizosaccharomyces pombe), BimB (Aspergillus nidulans) and Esp1 (Saccharomyces cerevisiae). Copyright # 2002 John Wiley & Sons, Ltd. Keywords: fission yeast; dimorphism; auxotrophy; protoplast; octad; transformation; ars: Cut1 Introduction Fission yeasts (Schizosaccharomyces) are excellent models for the investigation of a wide spectrum of biological processes and structures. With a very few exceptions, most of the work has been done on Sz. pombe, which has become one of the best-studied eukaryotes by now. Its genome sequencing is prac- tically completed and sequence comparisons of its known and putative gene products with those of S. cerevisiae (Aravind et al., 2000) confirmed that these two yeasts are separated by an enormous phylogenetic gap. When combined with other mole- cular and cytological data, the sequence-based phylogenetic analyses demonstrated that Sz. pombe has a unique position among fungi (Sipiczki, 1995, 2001). Its peculiar phylogenetic position and the usefulness of the fission yeast cell as a model in the investigation of the eukaryotic cell, are also direct- ing attention to other species of the genus. Fission yeasts constitute a tiny genus of three species (Sz. japonicus, Sz. octosporus and Sz. pombe) classified into Archiascomycetes, which is an ancient phylo- genetic branch of Ascomycota (for review, see Sipiczki, 2000). Sz. japonicus, which is divided into two varieties (var. japonicus and var. versatilis), has recently attracted considerable attention from cytologists. Given its cylindrical (pombe-like) yeast cell morphology, large cells and yeast-mycelial dimorphism, Sz. japonicus appears to be a well- suited model for studying the organization of cytoskeletal structures, cell polarity, polar growth, and dimorphic transitions (e.g. Alfa and Hyams, 1990; Svoboda et al., 1995; Naehring et al., 1995; Svoboda and Slaninova, 1997; Gabriel et al., 1998; Sipiczki et al., 1998a,b, etc.). However, its genetic and molecular analysis has been hampered by the lack of genetic markers and a cloning system. Here we report on the generation of auxotrophy and morphology markers in Sz. japonicus var. japonicus Yeast Yeast 2002; 19: 485–498. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / yea.853 Copyright # 2002 John Wiley & Sons, Ltd.