This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

We have prevlously cloned a gene for a zinc finger protein (EPF1) that is expressed specifically in petals and interacts with the promoter region of the 5-enolpyruvylshikimate-Sphosphate synthase gene in petunla. In an attempt to isolate genes encoding addltional factors that interact with this promoter, we cloned four nove1 genes encoding zinc finger proteins (EPF2-5a, EPF2-5b, EPF2-4, and EPF2-7). Sequence analyses revealed that overall similarity between the EPFl and the EPF2 protein family, except in the zinc finger motifs and the basic amino acid cluster, was very low, suggesting that the two groups belong to different subfamilies. DNA binding specificities of EPF1, EPF2-5, and EPF2-4 were very similar, as expected from the consewed zinc finger motifs. However, EPF2-7 showed no binding to the probes tested in spite of having the consewed motifs. DNA binding studies using a series of spacing mutant probes suggested a binding mechanism in which the EPF proteins recognize spacings in target DNA. RNA gel blot analyses and histochemical analyses with a promoter and B-glucuronidase fusion revealed that expression of the EPF2-5 gene (EPF2-5) was petal and stamen speciflc. Expression of the EPF2-7 gene (EPF2-7) was sepal and petal specific and localized in vascular tissues. The preferentia1 expression in two adjacent floral organs raises the possibility that these genes are downstream transcription factors of floral homeotic genes.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.

A glucoamylase gene has been cloned from a Rhizopus genomic DNAlibrary using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a CDNAlibrary prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the CDNAgene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The

We have previously cloned a gene for a zinc finger protein (EPF1) that is expressed specifically in petals and interacts with the promoter region of the 5-enolpyruvylshikimate-3-phosphate synthase gene in petunia. In an attempt to isolate genes encoding additional factors that interact with this promoter, we cloned four novel genes encoding zinc finger proteins (EPF2-5a, EPF2-5b, EPF2-4, and EPF2-7). Sequence analyses revealed that overall similarity between the EPF1 and the EPF2 protein family, except in the zinc finger motifs and the basic amino acid cluster, was very low, suggesting that the two groups belong to different subfamilies. DNA binding specificities of EPF1, EPF2-5, and EPF2-4 were very similar, as expected from the conserved zinc finger motifs. However, EPF2-7 showed no binding to the probes tested in spite of having the conserved motifs. DNA binding studies using a series of spacing mutant probes suggested a binding mechanism in which the EPF proteins recognize spacings in target DNA. RNA gel blot analyses and histochemical analyses with a promoter and beta-glucuronidase fusion revealed that expression of the EPF2-5 gene (EPF2-5) was petal and stamen specific. Expression of the EPF2-7 gene (EPF2-7) was sepal and petal specific and localized in vascular tissues. The preferential expression in two adjacent floral organs raises the possibility that these genes are downstream transcription factors of floral homeotic genes.

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