A new type of gene‐disruption cassette with a rescue gene for Pichia pastoris

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene‐targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene‐disruption system with a rescue gene using an inducible Cre/mutant–loxP system. With only short homology regions, the gene‐disruption cassette of the system replaces its target–gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter‐driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant‐loxP sequences in the cassette is excised, leaving only the remaining mutant‐loxP sequence in the genome, and consequently a target gene‐disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color‐change of the colonies. Then, the system was applied for knocking‐out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene‐targeting methods. All three gene‐disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant–loxP system worked well to successfully isolate their knock‐out mutants. This study identified a new gene‐disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end‐joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201–1208, 2017     

3-Nitrocoumarin is an efficient inhibitor of budding yeast phospholipase-C.

3-Nitrocoumarin is described in the literature as a specific inhibitor of mammalian phospholipase-C and here we studied the effect of 3-nitrocoumarin on budding yeast phosphatidylinositol-specific phospholipase-C and its effect on yeast growth. 3-Nitrocoumarin is a powerful inhibitor in vitro of the yeast Plc1 protein with an IC(50) of 57 nM and it is also an inhibitor of yeast growth in minimal media at comparable concentrations. Moreover at the same concentration it inhibits the glucose-induced PI-turnover. Since the effects of 3-nitrocoumarin on yeast growth are superimposable on the growth phenotype caused by PLC1 gene deletion we can conclude that 3-nitrocoumarin is a specific and selective inhibitor of yeast phospholipase-C. In addition we show that 3-nitrocoumarin was also an effective inhibitor of the pathogenic yeast Candida albicans.     

Frequent and efficient use of the sister chromatid for DNA double-strand break repair during budding yeast meiosis

Recombination between homologous chromosomes of different parental origin (homologs) is necessary for their accurate segregation during meiosis. It has been suggested that meiotic inter-homolog recombination is promoted by a barrier to inter-sister-chromatid recombination, imposed by meiosis-specific components of the chromosome axis. Consistent with this, measures of Holliday junction-containing recombination intermediates (joint molecules [JMs]) show a strong bias towards inter-homolog and against inter-sister JMs. However, recombination between sister chromatids also has an important role in meiosis. The genomes of diploid organisms in natural populations are highly polymorphic for insertions and deletions, and meiotic double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter-sister recombination. Efforts to study inter-sister recombination during meiosis, in particular to determine recombination frequencies and mechanisms, have been constrained by the inability to monitor the products of inter-sister recombination. We present here molecular-level studies of inter-sister recombination during budding yeast meiosis. We examined events initiated by DSBs in regions that lack corresponding sequences on the homolog, and show that these DSBs are efficiently repaired by inter-sister recombination. This occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of JMs. Loss of the meiotic-chromosome-axis-associated kinase Mek1 accelerates inter-sister DSB repair and markedly increases inter-sister JM frequencies. Furthermore, inter-sister JMs formed in mek1Δ mutants are preferentially lost, while inter-homolog JMs are maintained. These findings indicate that inter-sister recombination occurs frequently during budding yeast meiosis, with the possibility that up to one-third of all recombination events occur between sister chromatids. We suggest that a Mek1-dependent reduction in the rate of inter-sister repair, combined with the destabilization of inter-sister JMs, promotes inter-homolog recombination while retaining the capacity for inter-sister recombination when inter-homolog recombination is not possible.     

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

A multisite integrative cassette for the yeast Saccharomyces cerevisiae.

We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Saccharomyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences. This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sigma elements by gene replacement. Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes. Consequently, these studies led to the genetical tagging of individual members of the sigma family.     

Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation.     

Heterologous gene expression in continuous cultures of budding yeast

Summary In a previous paper we have studied the expression of -galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements. Offprint requests to: D. Porro     

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan^r resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5⁺ from Schizosaccharomyces pombe and the dominant resistance marker ble^r from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP–marker gene–loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble^r as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.     

DNA damage checkpoint in budding yeast.

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.     

New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica

Yarrowia lipolytica is one of the most extensively studied nonconventional yeasts. Unfortunately, few methods for gene disruption have been reported for this yeast, and all of them are time-consuming and laborious. The functional analysis of unknown genes requires powerful disruption methods. Here, we describe such a new method for rapid gene disruption in Y. lipolytica. This knockout system combines SEP method and the Cre-lox recombination system, facilitating efficient marker rescue. Versatility was increased by using both auxotrophic markers like ylURA3 and ylLEU2, as well as the antibiotic resistance marker hph. The hph marker, which confers resistance to hygromycin-B, allows gene disruption in a strain lacking any conventional auxothrophic marker. The disruption cassette was shown to integrate at the correct locus at an average frequency of 45%. Upon expression of Cre recombinase, the marker was excised at a frequency of 98%, by recombination between the two lox sites. This new method for gene disruption is an ideal tool for the functional analysis of gene families, or for creating large-scale mutant collections in general.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

Selectable cassettes for simplified construction of yeast gene disruption vectors

Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of a Km^R-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker     

A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.

A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.