A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes

Summary Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34° C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms. Communicated by B. J. Kilbey     

Dissociation of meiotic and mitotic roles of the fission yeast cdc2 gene

Summary The fission yeastcdc2 gene is pleiotropic, functioning both in the cell division cycle and in meiosis. Here we show thatcdc2 is allelic totws1, a previously isolated meiotic gene. Dissociation of meiotic and mitotic roles of the gene is also demonstrated by finding mutant alleles specifically altered in only one of the two processes.     

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.     

Preparation of a whole-cell biocatalyst of mutated <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (mCALB) by a yeast molecular display system and its practical properties

To prepare a whole-cell biocatalyst of a stable lipase at a low price, mutated Candida antarctica lipase B (mCALB) constructed on the basis of the primary sequences of CALBs from C. antarctica CBS 6678 strain and from C. antarctica LF 058 strain was displayed on a yeast cell surface by α-agglutinin as the anchor protein for easy handling and stability of the enzyme. When mCALB was displayed on the yeast cell surface, it showed a preference for short chain fatty acids, an advantage for producing flavors; although when Rhizopus oryzae lipase (ROL) was displayed, the substrate specificity was for middle chain lengths. When the thermal stability of mCALB on the cell surface was compared with that of ROL on a cell surface, T _1/2, the temperature required to give a residual activity of 50% for heat treatment of 30 min, was 60°C for mCALB and 44°C for ROL indicating that mCALB displayed on cell surface has a higher thermal stability. Furthermore, the activity of the displayed mCALB against p-nitrophenyl butyrate was 25-fold higher than that of soluble CALB, as reported previously. These findings suggest that mCALB-displaying yeast is more practical for industrial use as the whole-cell biocatalyst.     

A long region upstream of the IME1 gene regulates meiosis in yeast

Summary Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.     

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full‐length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full‐length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark‐derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A‐GFP system. Yeast library screening of full‐length vNAR variants which were detected via GFP expression yielded the same high‐affinity binder that had previously been isolated by our group using the conventional epitope tag‐based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost‐friendly alternative to conventional epitope tag detections.     

Enhancement of copper resistance and CupI amplification in carcinogen-treated yeast cells

Summary Carcinogen-induced amplification at the CupI locus, coding for a metallothionein protein, was studied in the yeast Saccharomyces cerevisiae. Exposure of cells from three different haploid strains, 4939, DBY746 and 320, to chemical carcinogens such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS) and 4-nitroquinoline-N-oxide (4NQO) enhanced the frequency of copper-resistant colonies up to several hundred fold. Copper-resistant clones obtained from strains DBY746 and 320, which contain more than one copy of the CupI locus, displayed a four-to eightfold amplification of the CupI sequences. In these clones the amplified CupI sequences were organized in a tandem array. Carcinogen treatment of strain 4939 in which only one copy of the CupI gene is present produced resistant colonies without CupI amplification. The possible use of the yeast system to study gene duplication and amplification is discussed.     

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.     

Five novel elements involved in the regulation of mitosis in fission yeast

Summary Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 weel ^ts cdc25^ts triple mutant strains. These genes were designated wisl ⁺ –wis5⁺for win suppressing, and do not correspond to winl ⁺ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25 ^ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wisl ⁺ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4 ⁺ appears to be a specific suppressor of the winl-1 mutation. wis2 ⁺ and wis3 ⁺ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1 ^ts and cdc25 ^ts, suggesting that the wis2 ⁺ and wis3 ⁺ products may interact with elements central to the mitotic control.     

Multistage disruption of protoplasts by dikegulac

Dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-I-gulonate) is a growth regulator used to differentially kill terminal apices, and it analogously inhibits basic metabolic functions in dividing cells, but not stationary cells, in suspension culture. This report demonstrates an analogous situation in isolated tobacco protoplasts. At the lowest concentrations, dikegulac partially suppresses division of the protoplasts. Higher concentrations are required to produce visual cytoplasmic damage to the protoplasts, which probably first occurs at the level of the plasmalemma, as the vacuoles can be released intact. Later, tonoplast disruption occurs.Abbreviation FDA fluorescein diacetate     

Enhancement of maize transformation efficiency by the use of maize matrix attachment regions

Improving genetic transformation efficiency is a major concern in plant genetic engineering. While various strategies have been investigated, the enhancement of selectable marker gene expression has not been tried extensively. We used maize matrix attachment regions (MARs) to bracket an herbicide resistance transgene, bar. MARs have been reported to enhance transgene expression level and stability. We show here that MARs not only enhance transformation efficiency by 50%, but are also able to increase or decrease relative efficiencies of each step of the regeneration process depending on MAR sequence combinations. Furthermore, we assessed the trans-effect of MARs in co-bombardment experiments with two independent plasmids, one including the MAR sequences and the other one the bar gene. As for simple bombardment, MARs enhanced transformation efficiency by having a positive influence on organogenesis step in the regeneration process.     

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system

Summary A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1 ^s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30° C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4×10^-6 per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5×10^-6 per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.     

Production of therapeutic glycoproteins through the engineering of glycosylation pathway in yeast

The application of recombinant DNA technology to restructure metabolic networks can change metabolite and protein products by altering the biosynthetic pathways in an organism. Although some success has been achieved, a more detailed and thorough investigation of this approach is certainly warranted since it is clear that such methods hold great potential based on the encouraging results obtained so far. In last decade, there have been tremendous advances in the field of glycobiology and the stage has been set for the biotechnological production of glycoproteins for therapeutic use. Today glycoproteins are one of the most important groups of pharmaceutical products. In this study the attempt was made to focus on identifying technologies that may have general application for modifying glycosylation pathway of the yeast cells in order to produce glycoproteins of therapeutic use. The carbohydrates of therapeutic recombinant glycoproteins play very important roles in determining their pharmacokinetic properties. A number of biological interactions and biological functions mediated by glycans are also being targeted for therapeutic manipulationin vivo. For a commercially viable production of therapeutic glycoproteins a metabolic engineering of a host cell is yet to be established.