We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae

Identifying the functional elements encoded in a genome is one of the principal challenges in modern biology. Comparative genomics should offer a powerful, general approach. Here, we present a comparative analysis of the yeast Saccharomyces cerevisiae based on high-quality draft sequences of three related species (S. paradoxus, S. mikatae and S. bayanus). We first aligned the genomes and characterized their evolution, defining the regions and mechanisms of change. We then developed methods for direct identification of genes and regulatory motifs. The gene analysis yielded a major revision to the yeast gene catalogue, affecting approximately 15% of all genes and reducing the total count by about 500 genes. The motif analysis automatically identified 72 genome-wide elements, including most known regulatory motifs and numerous new motifs. We inferred a putative function for most of these motifs, and provided insights into their combinatorial interactions. The results have implications for genome analysis of diverse organisms, including the human.

/pdf/sequencing-and-comparison-of-yeast-species-to-identify-genes-40xmgdglc4.pdf

Sequencing and comparison of yeast species to identify genes and regulatory elements

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we
report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000
protein-coding genes—more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer
than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology
including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism,
and important differences in Ca21 signalling as compared with plants and animals. Neurospora possesses the widest array of
genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced
point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the
creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related
genes.

/pdf/the-genome-sequence-of-the-filamentous-fungus-neurospora-2jw3s2832k.pdf

The genome sequence of the filamentous fungus Neurospora crassa

Most major systems that transport proteins across a membrane share the following features: an amino-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and a protein folding system on the trans side of the membrane. These transport systems are divided into two families: export systems that export proteins out of the cytosol, and import systems that transport proteins into cytosol-like compartments.

http://science.sciencemag.org/content/sci/271/5255/1519.full.pdf

Common Principles of Protein Translocation Across Membranes

Author(s): Amenta, Nina; Bern, Marshall; Kamvysselis, Manolis | Abstract: We describe our experience with a new algorithm for the reconstruction of surfaces from unorganized sample points in IR3. The algorthim is the first for this problem with provable guarantees. Given a ''good sample'' from a smooth surface, the output is guaranteed to be topologically correct and convergent to the original surface as the sampling sensity increases. The definition of a good sample is itself interesting: the required sampling density varies locally, rigorously capturing the intuitive notion that featureless areas can be reconstructed from fewer samples. The output mesh interpolates, rather than approximates, the input points. Our algorithm is based on the three-dimensional Voronoi diagram. Given a good program for thsi fundamental subroutine, the algorithm is quite easy to implement.

/pdf/a-new-voronoi-based-surface-reconstruction-algorithm-3z96n4eoxj.pdf

A new Voronoi-based surface reconstruction algorithm

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

S. Düsterhus

Papers

Complete Dna-Sequence Of Yeast Chromosome-Xi