Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.     

Yeast vectors for integration at the HO locus

We have constructed new yeast vectors for targeted integration of desired sequences at the Saccharomyces cerevisiae HO locus. Insertion at HO has been shown to have no effect on yeast growth, and thus these integrations should be neutral. One vector contains the KanMX selectable marker, and integrants can be selected by resistance to G418. The other vector contains the hisG-URA3-hisG cassette, and integrants can be selected by uracil prototrophy. Subsequent growth on 5-FOA permits identification of colonies where recombination between the hisG tandem repeats has led to loss of the URA3 marker and return to uracil auxotrophy. We also describe several new bacterial polylinker vectors derived from pUC21 (ampicillin resistance) and pUK21 (kanamycin resistance).     

Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display

Homologous recombination in yeast can be exploited to reliably generate libraries of >10⁷ transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5′ homology to the cut vector and the second scFv PCR product lacked 3′ homology to the cut vector, or both PCR products had both 5′ and 3′ homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 10⁴–10⁵ transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.     

Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides

Here it is demonstrated that the yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp, and can be as long as 200 nucleotides in length. This straightforward scheme for assembling chemically-synthesized oligonucleotides could be a useful tool for building synthetic DNA molecules.     

Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae

Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.     

Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection

It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracycline-regulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast

In fission yeast the Weel kinase and the functionally redundant Mikl kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Weel and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate weel or mikl homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both weel and mikl. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Weel kinase function. The expression of this clone in a weel/mikl-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Weel kinase activity.     

Ultra-Low Background DNA Cloning System

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp^r). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp^r 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp^r 3′ UTR. This cassette allowed conversion of the Amp^r-containing vector into the yeast/E. coli shuttle vector through use of the Amp^r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.     

Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.     

Yeast engineering to express sex pheromone gland genes of the oriental fruit moth,Grapholita molesta

Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at ∆9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at ∆9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.     

GTPase Activity Plays a Key Role in the Pathobiology of LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson''s disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker''s yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human α-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.     

Assembly of long DNA sequences using a new synthetic <Emphasis Type="Italic">Escherichia coli</Emphasis>-yeast shuttle vector

Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

Open image in new window

     

Ablation of insulin-producing cells prevents obesity but not premature mortality caused by a high-sugar diet in Drosophila

Ageing can be modulated by genetic as well as nutritional interventions. In female Drosophila melanogaster, lifespan is maximized at intermediate concentrations of sucrose as the carbohydrate source, and yeast as the protein source. Dampening the signal through the insulin/IGF signalling (IIS) pathway, by genetic ablation of median neurosecretory cells (mNSCs) that produce insulin-like peptides, extends lifespan and counteracts the detrimental effects of excess yeast. However, how IIS reduction impacts health on a high-sugar diet remains unclear. We find that, while the ablation of the mNSCs can extend lifespan and delay the age-related decline in the health of the neuromuscular system irrespective of the amount of dietary sugar, it cannot rescue the lifespan-shortening effects of excess sugar. On the other hand, ablation of mNSCs can prevent adult obesity resulting from excess sugar, and this effect appears independent from the canonical effector of IIS, dfoxo. Our study indicates that while treatments that reduce IIS have anti-ageing effects irrespective of dietary sugar, additional interventions may be required to achieve full benefits in humans, where excessive sugar consumption is a growing problem. At the same time, pathways regulated by IIS may be suitable targets for treatment of obesity.     

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO^® expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD.Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).     

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ~2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ~60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.