Gene Disruption Method Research Articles

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 yl URA3 and yl LEU2, 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.

PCR-based methods facilitate targeted gene manipulations and cloning procedures.

Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes. Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption. Gene replacement requires homologous recombination at the target locus. The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette. Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism. PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae. Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges. The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species. The high capacity of in vivo recombination of Sac. cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations. This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions. Furthermore, laborious cloning procedures, e.g. the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit.

Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae.

Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space. A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain. Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration. However, CCP1-disrupted cells were more sensitive to H2O2 than wild-type cells. We also constructed a CCP1-lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene. We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H2O2. These results hint that the biological function of CcP is to reduce H2O2 generated during aerobic respiratory process. Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.

バクテリアにおける遺伝子破壊株の作製方法 目的に合ったオーダーメイドの株を作る

Gene disruption methods are useful to construct bacteria lacking a specific gene especially when a gene’s function is unknown. In the 1980s, complementation techniques were used as the first step in cloning a gene. With the advances made through genome projects, gene identification and cloning have allowed easier construction of deficient bacterial strains with cloned genes. In this report, I describe three methods of disrupting specific genes on chromosomes in a strain of interest, namely linear transformation, preligation and the ‘one-step’ method. Moreover, several genetic techniques which are necessary for conducting these methods are also reviewed.

Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers.

Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed library of gene disruption strains has been produced by a consortium of yeast laboratories; however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces.

Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes

Background: The polyene macrolide amphotericin B is produced by Streptomyces nodosus ATCC14899. Amphotericin B is a potent antifungal antibiotic and has activity against some viruses, protozoans and prions. Treatment of systemic fungal infections with amphotericin B is complicated by its low water-solubility and side effects which include severe nephrotoxicity. Analogues with improved properties could be generated by manipulating amphotericin biosynthetic genes in S. nodosus. Results: A large polyketide synthase gene cluster was cloned from total cellular DNA of S. nodosus. Nucleotide sequence analysis of 113 193 bp of this region revealed six large polyketide synthase genes as well as genes for two cytochrome P450 enzymes, two ABC transporter proteins, and genes involved in biosynthesis and attachment of mycosamine. Phage KC515-mediated gene disruption was used to show that this region is involved in amphotericin production. Conclusions: The availability of these genes and the development of a method for gene disruption and replacement in S. nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.

Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast.

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.

Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination.

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP-mediated, site-specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P-FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP-mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.

Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions.

Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3. HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.

Gene disruption to evaluate the role of fungal candidate virulence genes

Gene disruption is a powerful genetic tool that can define pathogenic or virulence factors. In the past two years gene disruption approaches have been used to identify fungal virulence genes. The capsule genes, an α subunit of G protein and certain kinases of Cryptococcus neoformans have clearly been demonstrated to be associated with pathogenicity. In Candida albicans at least four genes involved in hyphal formation have been disrupted and tested for virulence. In other fungi, such as Histoplasma capsulatum, however, more efficient gene disruption methods need to be developed before such approaches can be regularly used for identifying virulence genes.

Optimization of gene-disruption methods for reverse genetics in the nematode C. elegants

Exposure in vitro of cultivated peripheral leukocytes to trimethylpsoralen and UV light (365nm) under conditions that are known to produce DNA cross-links caused a greater increase in the number of sister chromatid exchanges in cells from two patients with dyskeratosis congenita (DKC) than in normal cells. Absence of this enhancing phenomenon in an unaffected sibling of one patient as well as its presence to a partial degree in an obligate carrier suggest that the observation may be etiologically significant. The data suggest that DKC is associated with a heritable defect in the repair of DNA cross-links.

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri.

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G --> A nucleotide exchange in a 5'-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

Intergenic Flip Flop, a method for systematic gene disruption and cloning in yeast.

We have developed a strategy named Intergenic Flip Flop which, for each gene, allows us to produce in one experiment both a disrupting cassette and a plasmid for gap repair. The same method can also be used to insert a reporter gene downstream from the promoter. This approach extends the polymerase chain reaction (PCR)-based strategy proposed by Maftahi et al. 1996. Our method consists of PCR amplification of the two flanking intergenic regions of the open reading frame (ORF) of interest, using two sets of oligonucleotides. Each PCR product is flanked by two short defined nucleotidic sequences with a unique restriction site, allowing subsequent hybridization between them. The association of the two amplimers by the complementary sequences either in the same orientation as in genomic DNA or in the opposite orientation, allows the generation, after PCR, of two distinct cassettes which can be cloned into suitable vectors. When the amplimer in the head-to-tail orientation is cloned in a vector containing a selective marker for yeast such as G418 resistance, it provides a disrupting cassette after cleavage at the unique restriction site introduced by the PCR between the two intergenic amplimers. The amplimer with a direct orientation cloned into a yeast vector, after cleavage at the unique restriction site between the intergenic regions, permits cloning by gap repair of the gene of interest in yeast. Finally, a reporter gene can be inserted in the same plasmid. We report here the successful application of this strategy to an ORF of chromosome XIV of Saccharomyces cerevisiae: N1216.

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae

We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14–1 from chromosome XIV of S. cerevisiae.

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.

Breeding of a new type of baker's yeast by δ-integration for overproduction of glucoamylase using a homothallic yeast

With the aim of developing a new type of bread that can be produced from raw materials such as rice, new recombinant yeast strains producing glucoamylase were constructed. The yeast OC-2, which is used for wine making, was selected as a host strain in a screening test because of its superiority in flavor formation in bread. OC-2 is a homothallic and diploid strain, and has no auxotrophic marker. First, tryptophan auxotrophic mutants (Δtrp1/Δtrp1) were constructed from OC-2 by the gene disruption method. Conventional YEp- and YIp-type yeast expression plasmids containing glucoamylase cDNA from Aspergillus oryzae, and the TRP1 gene as a selective marker were introduced into the recipient strain. Both transformants with the YEp- or YIp-type plasmid showed low glucoamylase productivity in the culture broth, probably due to very low plasmid stability, and a low copy number, respectively. A new type of integrative plasmid was thus constructed by the δ-integration system using a δ sequence of the yeast retrotransposon Ty1 to obtain transformants with higher glucoamylase production ability. These transformants produced higher amounts of glucoamylase and could grow in synthetic medium containing starch as a carbon source. Furthermore, using the integrated transformants, duplication of copies of the integrated glucoamylase cDNA by spore formation and autodiploidization was carried out by utilizing the homothallic characteristics of the strain. By Southern blot analysis, the copy number was estimated to be about twice that of the original transformant, and the glucoamylase productivity was proportionally increased. The fact that the constructed strains could grow in synthetic medium containing soluble starch as a sole carbon source suggested that they will be useful for making new types bread.

The pcsA gene is identical to dinD in Escherichia coli

The pcsA68 mutant of Escherichia coli is a cold-sensitive mutant which forms long filaments with a large nucleoid in the central region at 20 degrees C. We here show that (i) the coding region for the pcsA gene is identical with orfY located upstream of pyrE and can be deleted without loss of viability; (ii) pcsA is also identical to dinD, a DNA damage-inducible gene, whose expression is regulated by the LexA-RecA system; (iii) the cold-sensitive phenotype of the pcsA68 mutation is suppressed by delta recA or lexA1 (Ind-) mutation, but not by sulA inactivation; (iv) overproduction of PcsA68 leads to inhibition of cell growth in recA+ and delta recA strains at 20 and 37 degrees C, but PcsA+ does not show such an effect at any temperature; (v) SOS response is induced in the pcsA68 mutant cells at 20 degrees C. We discuss the possible function of the pcsA gene, comparing it with the sulA or the dif-xerCD function. We also describe a new method for gene disruption with positive and negative selection.

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the "ura-blaster" protocol described for Saccharomyces [Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.

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.

New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535.

A new method for gene disruption in Salmonella typhimurium was developed. The key steps of this method are to produce restriction fragments with compatible ends, preligate to produce concatemers, and then transform by electrotransformation. We developed and used this method to construct a mutant of S. typhimurium TA1535 in which the resident ada-like (adaST) gene was replaced with a kanamycin resistance gene to produce an adaST-deletion mutant derivative. The S. typhimurium adaST-deletion strain did not exhibit a higher level of mutability upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine than did its wild-type parent strain. However, it did exhibit a higher sensitivity with respect to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The ability of AdaST to function as a transcriptional activator is discussed.