Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation. To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol. Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30 degrees C and undergo growth arrest in the G2 phase of the cell cycle at 35 degrees C. The ksg1 gene was isolated by functional complementation. It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues. The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid. Deletion of the gene is lethal for haploid cells. We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle.

We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellularly mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.

We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane-bound 54-kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72-kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54-kDa and 72-kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54-kDa acid phosphatase is glycosylated to the 72-kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72-kDa form. In cells grown in the minimal medium, however, all of the 54-kDa and 72-kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72-kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.Eucaryotic secretory proteins including those of Saccharomyces cerevisiae are synthesized at the endoplasmic reticulum, are transported to the Golgi apparatus and via vesicles to the cell surface. Many of them are N-glycosylated on their secretory pathway. In yeast the pathway of Asn-linked glycosylation starts with the transfer of the oligosaccharide precursor, (GlcNAc)2(Man)9(Glc)3, from the dolichyl diphosphate to appropriate asparagine residues [l, 21. This oligosaccharide is further processed by removal of the three glucose units and a mannose unit [l, 31. Glycoproteins that are secreted have large polymannose chains added to the oligosaccharide core units, whereas glycoproteins destined to remain in the cell have the core oligosaccharide increased by 1 -4 mannose residues [4 -61.Mainly by the use of temperature-sensitive mutants that are blocked at distinct steps in the export of cell-surface glycoproteins, it has been shown that the transfer of the (Glc)3(Man)9(GlcNAc)z core oligosaccharides to the protein occurs in the endoplasmic reticulum and that extension through the addition of outer polymannose chains takes place in Golgi-like organelles [7]. Little is known concerning the regulation of the Asn-linked glycosylation pathway in yeast. Data of Chu and Malley indicate that glucose impairs Nglycosylation of invertase and that the nonglycosylated polypeptide is rapidly degraded by an endogenous protease PI.Correspondence to M. E. Schweingruber, Institut fur Allgemeine Mikrobiologie der Universitat Bern, Baltzerstrasse 4, CH-3012 Bern, SwitzerlandAbbreviations. Endoglycosidase H, endo-N-acetylglucosaminidase H ; Tris/NaCl, 20 mM Tris/HCl, 150 mM NaC1, pH ...

The nucleotide sequence of the region preceding the transcrption initiation site of the tophan oeron of Escherichia co was determined. Essentially all of the trp operator precedes the transcribed portion of the operon. The deduced sequence contains the recognition site of endonuclease Hpa I. This site is protected from Hpa I cleavage by RNA polymerase and by tip repressor. Regions of 2-fold symmetry are present in the DNA sequence.In most 080 and X-+80 trp transducing phages, the trp operon replaces a segment of the phage N operon and is transcribed with normal orientation by read-through transcription initiated at the phage N promoter (1, 2). We prepared 32P-labeled read-through RNA in vio and in vitro and employed selective hybridization to the denatured DNA of appropriate tip transducing phages to isolate the segment of these transcripts that immediately precedes the 5' end of the normal trp operon messenger RNA.Here we report the sequence of 33 nucleotides preceding the transcription initiation site of the tip operon. We show that the corresponding segment of DNA contains sequences essential for promoter and operator function. MATERIALS AND METHODSPreparation of RNA. The read-through RNA transcript containing the sequence preceding the 5' end of tip mRNA was labeled either in vitro using &80tip DNA as a template or in vivo by infecting cells with 080tip phages (Fig. 1).In Vitro Synthesis. Synthesis was performed in a coupled transcription-translation system essentially as described (3, 4).As templates, DNAs of phages 4080trpALD102, 480trp-ALC145-2, and q80trpALC1415 were used (Fig. 1). a-32P-labeled nucleoside triphosphates (used at a concentration of 50 ,MM) were purchased from New England Nuclear Corp. (specific activity Ci/mmol) through the kind efforts of Dr.W. Salser. Incubations were for 60 min at 340. RNA was isolated (5) and sequences corresponding to the promoter-operator region were trapped by two consecutive liquid hybridizations to complementary separated DNA strands at 510 for 16-18 hr (6) and at 370 in 30% formamide (5) 00. The products were separated as described for a normal fingerprint except that electrophoresis was performed for 35 min at 5000 V and homochromatography mix "C-2" (9) was used for development in the second dimension. RESULTSCharacterization of RNA complementary to the promoter-operator region of the tip operon DNAs from several c80trp transducing phages were used as templates for the in vitro synthesis of 32P-labeled RNA in a coupled transcription-translation system (4). Promoter-operator RNA, purified as described in Materials and Methods, is defined at the 5'-terminus by the phage-bacterial fusion point of the template DNA, 080trpALD102, and at the 3' end by the end point of the LD102 deletion. This RNA, a fingerprint of which is shown ( Fig. 2A), exhibited the mobility expected for a molecule of 150 4 10 nucleotides when electrophoresed on a denaturing polyacrylamide gel. A RNase T1 fingerprint of the 150 nucleotide band was identical to that in Fig. 2A. The fin...