Adaptation of Saccharomyces cerevisiae to saline stress through laboratory evolution.

Most laboratory evolution studies that characterize evolutionary adaptation genomically focus on genetically simple traits that can be altered by one or few mutations. Such traits are important, but they are few compared with complex, polygenic traits influenced by many genes. We know much less about complex traits, and about the changes that occur in the genome and in gene expression during their evolutionary adaptation. Salt stress tolerance is such a trait. It is especially attractive for evolutionary studies, because the physiological response to salt stress is well-characterized on the molecular and transcriptome level. This provides a unique opportunity to compare evolutionary adaptation and physiological adaptation to salt stress. The yeast Saccharomyces cerevisiae is a good model system to study salt stress tolerance, because it contains several highly conserved pathways that mediate the salt stress response. We evolved three replicate lines of yeast under continuous salt (NaCl) stress for 300 generations. All three lines evolved faster growth rate in high salt conditions than their ancestor. In these lines, we studied gene expression changes through microarray analysis and genetic changes through next generation population sequencing. We found two principal kinds of gene expression changes, changes in basal expression (82 genes) and changes in regulation (62 genes). The genes that change their expression involve several well-known physiological stress-response genes, including CTT1, MSN4 and HLR1. Next generation sequencing revealed only one high-frequency single-nucleotide change, in the gene MOT2, that caused increased fitness when introduced into the ancestral strain. Analysis of DNA content per cell revealed ploidy increases in all the three lines. Our observations suggest that evolutionary adaptation of yeast to salt stress is associated with genome size increase and modest expression changes in several genes.

A novel RNA-binding protein from Triturus carnifex identified by RNA-ligand screening with the newt hammerhead ribozyme.

The newt hammerhead ribozyme is transcribed from Satellite 2 DNA, which consists of tandemly repeated units of 330 bp. However, different transcripts are synthesized in different tissues. In all somatic tissues and in testes, dimeric and multimeric RNA transcripts are generated which, to some extent, self-cleave into monomers at the hammerhead domain. In ovaries, primarily a distinct monomeric unit is formed by transcription, which retains an intact hammerhead self-cleavage site. The ovarian monomeric RNA associates to form a 12S complex with proteins that are poorly characterised so far. In this work we identified NORA, a protein that binds the ovarian form of the newt ribozyme. We show that the newt ribozyme binds to the Escherichia coli -expressed protein, as well as to a protein of identical size that is found exclusively in newt ovaries. Also NORA mRNA was detectable only in ovary, but in neither somatic tissues nor testes. The tissue-specific expression of NORA is analogous to the ovary-specific transcription of the newt ribozyme. Although NORA was identified by its ability to bind to the newt ribozyme in the presence of a vast excess of carrier RNA, it was able to interact with certain other RNA probes. This novel RNA-binding protein does not contain any motif characteristic for RNA-binding proteins or any other known protein domain, but it shares a striking similarity with a rat resiniferatoxin-binding protein.

Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library.

A screening assay for the detection of RNA-binding proteins was developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem-loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to beta-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA-protein interaction. Parameters influencing the specificity and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA-protein interaction could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10(-8) M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato.