An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

MCScan is an algorithm able to scan multiple genomes or subgenomes in order to identify putative homologous chromosomal regions, and align these regions using genes as anchors. The MCScanX toolkit implements an adjusted MCScan algorithm for detection of synteny and collinearity that extends the original software by incorporating 14 utility programs for visualization of results and additional downstream analyses. Applications of MCScanX to several sequenced plant genomes and gene families are shown as examples. MCScanX can be used to effectively analyze chromosome structural changes, and reveal the history of gene family expansions that might contribute to the adaptation of lineages and taxa. An integrated view of various modes of gene duplication can supplement the traditional gene tree analysis in specific families. The source code and documentation of MCScanX are freely available at http://chibba.pgml.uga.edu/mcscan2/.

MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity

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

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0061%28199807%2914%3A10%3C943%3A%3AAID-YEA292%3E3.0.CO%3B2-Y

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests ∼900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

/pdf/genome-duplication-in-the-teleost-fish-tetraodon-qw9iuo5jxx.pdf

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype

We have sequenced and annotated the genome of the filamentous ascomycete Ashbya gossypii. With a size of only 9.2 megabases, encoding 4718 protein-coding genes, it is the smallest genome of a free-living eukaryote yet characterized. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae. Analysis of this pattern revealed 300 inversions and translocations that have occurred since divergence of these two species. It also provided compelling evidence that the evolution of S. cerevisiae included a whole genome duplication or fusion of two related species and showed, through inferred ancient gene orders, which of the duplicated genes lost one copy and which retained both copies.

http://science.sciencemag.org/content/sci/early/2004/03/04/science.1095781.full.pdf

The Ashbya gossypii Genome as a Tool for Mapping the Ancient Saccharomyces cerevisiae Genome

Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1α. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UASrpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UASrpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UASrpg homologous sequences.

Sequence and Promoter Analysis of the Highly Expressed TEF Gene of the Filamentous Fungus Ashbya Gossypii

Publisher Summary This chapter discusses the different steps of the polymerase chain reaction (PCR)-based gene targeting method and its possible applications for basic and advanced gene analyses. It highlights the experience with the selection marker, kanMX , a hybrid gene expressing a bacterial aminoglycoside phosphotransferase under the control of a strong fungal promoter. The introduction of this marker led to two essential improvements. First, gene targeting was no longer dependent on the presence of auxotrophic markers in the host strain and, second, high backgrounds of false-positive transformants often obtained in PCR-targeting using Saccharomyces cerevisiae genes as selectable markers were virtually eliminated because kanMX lacks homology to yeast DNA. The chapter also summarizes the features of several new modules for PCR-targeting, including a heterologous HIS3 marker, called “ HIS3MX6, ” two green fluorescent protein (GFP) reporter modules with HIS3MX or kanMX as selection marker, which are useful for generating carboxy-terminal GFP fusions, a kanMX - GAL1 promotor module for making promotor exchanges, and a kanMX - GAL1 - GFP reporter module for generating amino-terminal fusions.

5 PCR-Based Gene Targeting in Saccharomyces cerevisiae

The rDNA cluster in the phytopathogenic fungus Ashbya gossypii consists of approximately 50 tandem repeat units of 8197 bp. Each unit carries a gene for the 35S pre-rRNA, processed into 18S, 5.8S and 25S rRNA, and a divergently transcribed gene for 5S rRNA. The well-characterized rDNA of the yeast Saccharomyces cerevisiae is the only other example of a completely sequenced rDNA unit (9137 bp) carrying both a 35S pre-rRNA and a 5S rRNA gene. The coding regions for the 5S, 5.8S, 18S and 25S rRNAs are 95–100% identical whereas transcribed and non-transcribed spacers show 43–66% sequence identity. Functionally characterized rDNA and rRNA elements of S. cerevisiae can be unambiguously recognized in the A. gossypii sequence, including the RNA polymerase-I transcription start site, two Reb1p enhancer binding sites and numerous recognition sequences for rRNA modification and processing. In addition to these functionally characterized sequences eight highly conserved elements from 10 to 71 bp were detected in the over 600-bp transcribed region upstream of the 18S rRNA gene which most likely play as yet uncharacterized functions at the DNA or RNA level. In addition to this work we started to identify A. gossypii homologs of S. cerevisiae nucleolar proteins involved in rDNA maturation.

Compact organization of rRNA genes in the filamentous fungus Ashbya gossypii.

Described is the promoter region of the A. gossypii gene which codes the translation elongation factor EF-1α. The promoter can be used in the synthesis of proteins.

Sabine Steiner

Papers

The Ashbya gossypii Genome as a Tool for Mapping the Ancient Saccharomyces cerevisiae Genome

Sequence and Promoter Analysis of the Highly Expressed TEF Gene of the Filamentous Fungus Ashbya Gossypii

5 PCR-Based Gene Targeting in Saccharomyces cerevisiae

Compact organization of rRNA genes in the filamentous fungus Ashbya gossypii.

New promoter region