Gene, 106 (1991) 129-133 0 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/91/$03.50 129 GENE 06045 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene ( pgkl) of Tricho- derma reesei (Recombinant DNA; filamentous fungus; heat-shock element; upstream activating sequence; CAMP regulatory element) S. Vanhanen *, A. Saloheimo, M. Ihnkn, J.K.C. Knowles * and M. PenttilSi VTT Biotechnical Laboratory, SF-021.51 Espoo (Finland) Received by J.R. Kinghorn: 15 February 1991 Revised/Accepted: 24 May/25 May 1991 Received at publishers: 12 July 1991 SUMMARY Transcription of the 3-phosphoglycerate kinase (PGK)-encoding gene (pgkl) of Trichoderma reesei results in two tran- scripts due to two main transcription start points (tsp) which are differentially regulated during the growth cycle. The nucleotide sequence of the promoter reveals a number of putative regulatory elements present also in the PGK promoter of Saccharom_vces cerevisiae: a 20-nt long sequence similar to the CTTCC-repeat region of the upstream activating sequence UAS, the eukaryotic heat-shock consensus sequence, HSE, and a putative eukaryotic CAMP regulatory sequence, The functionality of the putative HSE sequence was examined, but no clear effect could be seen on the total amount of pgkl mRNA at elevated temperatures nor on transcription initiation from the upstream tsp, preceded by the HSE sequence. INTRODUCTION Very little is known about promoter structure and regu- lation of gene expression in filamentous fungi and this is particularly true for T. reesei, a cellulolytic mesophilic imperfect fungus. To better understand the mechanisms regulating housekeeping genes in filamentous fungi, we have zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED Correspondence to: Dr. M. Penttill, VTT Biotechnical Laboratory, POB 202, SF-021 5 1 Espoo (Finland) Tel. (358-0)-4564504; Fax (35%O)-4552028. * Present addresses: (J.K.C.K.) Glaxo Institute for Molecular Biology S.A., 14, Chemin des Aulx, POB 674, CH-2 1228 Plan-les-Ouates, Geneva(Switzerland)Tel. (41-22)-7069930; Fax (41-22)-7946965; (S.V.). Department of Genetics, University of Melbourne, Parkville, Vict. 3052 (Australia) Tel. (61-3)-3445140; Fax. (61-3)-3445139. Abbreviations: aa, amino acid(s); ATF, activating transcription factor; bp, base pair(s); cbhl, gene encoding cellobiohydrolase I; CRE, CAMP regulatory element; gpdA, A. niduluns gene encoding glyceraldehyde-3- phosphate dehydrogenase; HSE, heat-shock element; HSF, heat-shock transcription factor; kb, kilobase or 1000 bp; nt, nucleotide(s); PGK, 3-phosphoglycerate kinase; pgkl, T. reesei gene encoding PGK; tsp, transcription start point(s); (/AS, upstream activating sequence. isolated the pgkl gene of T. reesei, encoding PGK (Vanhanen et al., 1989). The regulation of the PGK gene of S. cerevisiue, and the promoter regions involved, have been extensively investigated (e.g., Ogden et al., 1986; Piper et al., 1986; Chambers et al., 1988; Stanway et al., 1989). However, only limited information concerning promoter sequences and expression of the glycolytic genes, pgkA, tpiA and gpdA originating from filamentous fungi (Clements and Roberts, 1986; McKnight et al., 1986; Punt et al., 1990) has been reported. We report here on characterization of the promoter sequence of the T. reeseipgkl gene and the environmental factors affecting the expression of this gene. EXPERIMENTAL AND DISCUSSION (a) Expression of the pgkl gene The pgkl gene of T. reesei codes for two major mRNAs of 1.65 kb and 1.85 kb in size (Vanhanen et al., 1989). Unexpectedly, the relative amounts of the two pgk2 tran- scripts were shown to be differentially regulated during the