Achim Wach

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

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 kanr 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 efficient 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.

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

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 ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae

A PCR‐method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH‐PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a first step, two several hundred base pairs long DNA fragments from the 5′‐ and 3′‐region of a S. cerevisiae gene were amplified in such a way that 26 base pairs extensions homologous to the kanMX6 marker were added to one of their end. In a second step, one strand of each of these molecules then served as a long primer in a PCR using kanMX6 as template. When such a LFH‐PCR‐generated disruption cassette was used instead of a PCR‐made disruption cassette flanked by short homology regions, transformation efficiencies were increased by at least a factor of thirty. This modification will therefore also help to apply PCR‐mediated gene manipulations to strains with decreased transformability and/or unpredictable sequence deviations.

Heterologous HIS3 Marker and GFP Reporter Modules for PCR-Targeting in Saccharomyces cerevisiae

We have fused the open reading frames of his3‐complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1·4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false‐positive His+ transformants seem to be due to non‐homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA‐plasmids with HIS3MX6 and kanMX markers. The 0·9 kb GFP reporters consist of wild‐type GFP or GFP‐S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR‐synthesized 2·4 kb‐long double modules flanked by 40–45 bp‐long guide sequences were successfully targeted to the carboxy‐terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter.

5 PCR-Based Gene Targeting in Saccharomyces cerevisiae

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.

Identification of a gene encoding a homocitrate synthase isoenzyme of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, most of the LYS structural genes have been identified except the genes encoding homocitrate synthase and α‐aminoadipate aminotransferase. Expression of several LYS genes responds to an induction mechanism mediated by the product of LYS14 and an intermediate of the pathway, α‐aminoadipate semialdehyde (αAASA) as an inducer. This activation is modulated by the presence of lysine in the growth medium leading to an apparent repression. Since the first enzyme of the pathway, homocitrate synthase, is feedback inhibited by lysine, it could be a major element in the control of αAASA supply.

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c–GFP) and the nucleus (Ynl075w–GFP) as the sites of action of two proteins. Ynl080c–GFP and Ynl081c–GFP fluorescence was visible in dot‐shaped and elongated structures, whereas the Ynl022c–GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C‐terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5′–GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP–Ynl020c and GFP–Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases. Copyright

- Cdc15: Cell division cycle protein 15 (S. cerevisiae)

The chapter discusses the Cdcl5 protein that is a serine/threonine PK. CDC15 gene arrest the cell cycle late in mitosis (post anaphase) at 36°C but not at 23°C. Upon return to permissive temperature, cells recover well from the mitotic block and resume normal growth passing through the next 1 to 2 cell cycles as a synchronized culture. However, at permissive temperature, CDC15 mutants have a slightly increased level of chromosome loss. The CDC 15 gene was isolated in an effort to clone all fragments of chromosome. The DNA sequence of the CDC 15 allele then revealed that CDC 15 encodes a 974 residue protein. Its first 270 N-terminal amino acids contain all 11 conserved motifs typically found in PK domains. The Cdc15 protein could be precipitated with antibodies directed against a C-terminal peptide. However, it was proposed that Cdcl5 is necessary (directly or indirectly) for Clb2/Cdc28 protein kinase destruction. In addition, Cdcl5 itself is autophosphorylated during in vitro assays. Cdc15 activity is assayed with immunoprecipitated Cdc15 by transfer of radioactivity from [γ32P]ATP to the artificial substrate casein. There is a low level of apparently constitutive CDC 15 mRNA. It is not known whether the Cdc15 protein kinase activity is regulated during the cell cycle.