Arch Microbiol (1985) 142:383- 388 Archives of Microbiology 9 Springer-Verlag 1985 Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains Gerhard Braus, Rolf Furter, Fransziska Prantl, Peter Niederberger, and Raft Hiitter Mikrobiologisches Institut, Eidgen6ssische Technische Hochsehule, ETH-Zentrum, CH-8092 Ziirich, Switzerland Abstract. The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180- IA, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains. Key words: Saccharomyces cerev&iae - Tryptophan bio- synthesis - General control - Systematic by Southern hybridization The arrangement of TRP genes encoding the invariable five biosynthetic steps from chorismic acid to tryptophan shows a wide variation in microorganism (Crawford 1975). One aspect of this variation is the existence of enzymes performing single, double or even triple functions, encoded for by one gene. These patterns have been studied for TRP genes of lower fungi by Hfitter and DeMoss (1967), and the results led to a phylogenetic scheme for these organisms. Of particular interest for the present work is the finding that in all fungi studied, and in particular also in the group of ascomycetes like Neurospora crassa, Aspergillus nidulans or the yeast Schizosaccharomyces pombe (Thuriaux et al. 1982) a trifunctional enzyme NHz-glutamine amidotransferase- InGPsynthase-PRAisomerase-COOH (involved in the first, fourth and third biosynthetic step) was found, whereas in the yeast S. cerevisiae only the two NH2-terminal functions are unified. The uniqueness of this arrangement was taken as an indication for a detachement of PRAisomerase from this bifunctional unit. At the DNA level this would reflect a translocation of a promotorless 3'-portion of the fused gene to another place on the DNA to form gene TRP1 (chromosome IV) and leaving behind a 5'-portion (with promotor) to form gene TRP3 (chromosome XI) in S. Offprint requests to: Gerhard Braus Non-standard abbreviations. InGP, Indole-3-glycerolphosphate; PRA, N(5'-phosphoribosyl)-anthranilate cerevisiae. This hypothetical development is supported by the observation that in N. crassa, as well as in A. nidulans the fused gene responds to a "cross pathway" regulation (Carsiotis and Lacy 1965; Piotrowska 1980), whereas this response is still found for TRP3 but no more for TRP1 in S. cerevisiae (Miozarri et al. 1978 a; Niederberger et al. 1981). Cloning of a number of genes of the commonly used labora- tory strain S. cerevisiae X2180-1A showing this "general control" response, in particular of HIS4, led to the detection of a consensus sequence upstream of the transcription start (Donahue et al. 1983). The promotor sequences of TRP1 (Tschumper and Carbon 1980; Dobson et al. t983) and TRP3 (Aebi et al. 1984; Zalkin et al. 1984) revealed that TRP3 carries the consensus sequence upstream of the trans- cription start, whereas TRP1 does not [a major transcription start for TRP1 has been mapped recently at position (-190); S. Kingsman, personal communication]. The exceptional situation observed for genes TRP1 and TRP3 in the strain S. eerevisiae X2180-1A prompted us to ask whether wild Saccharomyces strains of various origins would be identical in the arrangement, structure and regula- tory behaviour of this genes. We show by Southern hybridization technique and ge- netic complementation with gene libraries introduced into mutant strains of the X2180-1A cell line that in no case fusion of the two genes can be observed in S. cerevisiae wild type strains. The regulatory behaviour of gene TRP1 was found, however, to be different in some cases. A classifica- tion of the different S. cerevisiae, two S. uvarum (syn. cerevisiae) and a Kluyveromyes marxianus strains will be made according to their TRP1, TRP3, and partly also to their TRP2 and TRP4 Southern hybridization patterns. In two particular cases the usefulness of this technique as a tool to identify closely related but different strains is shown in more detail. Materials and methods Strains and plasmids. The haploid Saccharomyces cerevisiae laboratory strain X2180-1A MATa was compared with thirteen wild type yeast strains, twelve of which were a gift of P. Philippsen (Basel). The original sources of these strains are listed in Table 1. Kluyveromyces marxianus var. lactis strain CBS2359 was obtained from the Delft Yeast Collec- tion. For complementation analysis the following mutants of strain X2180-1A were used: RH 970 (trpl-218 leu2-31eu2- 112 his4-519 ade2-1), RH 964 (trp3A-56 leu2-3 leu2-112 his4- 519 ade2-1), RH 962 (trpl-218 trp3B-23).