Mso1p Regulates Membrane Fusion through Interactions with the Putative N-Peptide-binding Area in Sec1p Domain 1

Peer reviewe

High-precision mapping of protein–protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

Understanding networks of protein–protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein–protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1

Bacteriophage Mu integration in yeast and mammalian genomes

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed ‘transpososomes’. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells

Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice

Peer reviewe

Identification of the α-galactosidase<i>MEL</i>genes in some populations of<i>Saccharomyces cerevisiae</i>: a new gene<i>MEL11</i>

SummaryIn this report we mapped a newMEL11gene and summarize our population studies of the α-galactosidaseMELgenes ofS. cerevisiae. The unique family of structuralMELgenes has undergone rapid translocations to the telomeres of most chromosomes in some specificSaccharomyces cerevisiaepopulations inhabiting olive oil processing waste (alpechin) and animal intestines. A comparative study ofMELgenes in wine, pathogenic and alpechin populations ofS.cerevisiaewas conducted using genetic hybridization analysis, molecular karyotypingand Southern hybridization with theMEL1probe. Five polymeric genes for the fermentation of melibiose,MEL3, MEL4, MEL6, MEL7, MEL11, were identified in an alpechin strain CBS 3081. The newMEL11gene was mapped by tetrad analysis to the left telomeric region of chromosome I. In contrast, in wine and pathogenic populations ofS. cerevisiae, MELgenes have been apparently eliminated. Their rare Mel+strains carry only one of theMEL1, MEL2, orMEL8genes. One clinical strain YJM273 was heterozygotic on theMEL1gene; itsmell0allele did not have a sequence of the gene.</jats:p