The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

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.

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.

Full length cDNA clones of flavonoid 3',5'-hydroxylase, dihydroflavonol 4-reductase and flavonoid 3-glucosyltransferase were cloned from petals of Gentiana triflora. Their sequences were homologous to counterparts from other plants. Flavonoid 3',5'-hydroxylase and flavonoid 3-glucosyltransferase were enzymatically characterized by expressing cDNAs in heterologous expression systems.