Construction Of Cassettes Research Articles

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters thepre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Cassette Construction and Co-Expression of NA and F Proteins of H1N1 Influenza Virus and Newcastle Disease Virus in Saccharomyces cerevisiae

Cassette Construction and Co-Expression of NA and F Proteins of H1N1 Influenza Virus and Newcastle Disease Virus in Saccharomyces cerevisiae

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene.

Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB -which converts chorismate to trp-was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp- phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents.

GENOME SEQUENCE ANALYSIS, RT-PCR DIAGNOSTICS AND CONSTRUCTION OF VIRAL PROTEIN-EXPRESSION CASSETES FOR KAZAKH ISOLATE OF ORDINARY AND ANDEAN STRAINS OF POTATO VIRUS S

Potato virus S (PVS) belongs to the genus Carlavirus of family Betaflexiviridae with a positive-sense single-stranded RNA genome of 8.5 Kb. PVS is one of the most prevalent viruses of cultivated potatoes in Kazakhstan. Here we report phylogenetic analysis of complete genome sequences of Kazakh isolates of PVS from potato cultivars Fortuna and Ushkonyr, design of PVS strain-specific diagnostic PCR and construction of expression cassettes for conserved viral proteins from both isolates. The Fortuna and Ushkonyr isolates were found to share 79.9% nucleotide identity with each other and belong respectively to previously-defined ordinary and Andean strains of PVS. Based on analysis of conserved and variable regions of available PVS isolates, we designed primers for reverse transcription (RT)-duplex PCR to detect both strains in single and mixed infections and established their prevalence in Almaty region of Kazakhstan. Coding sequences of triple gene block proteins (25K, 12K, 7K), coat protein (34K) and cysteine-rich protein (11K) as well as methyltransferase, peptidase, helicase and RNA-dependent RNA polymerase domains of viral replicase were subcloned from both Fortuna and Ushkonyr genomic RNAs into the binary vector pBIN19 under the control of CaMV 35S promoter and nopaline synthetase terminator. These expression cassettes will be used to further investigate the biological properties and strain characteristics of viral proteins by their transient expression in plant cells and tissues or their stable expression in transgenic plants.

Effect of material properties on interaction of ceramic and glass flyer plates with hypervelocity jet

AbstractA study on the effect of material properties on the interaction of explosively driven ceramic and glass plates with shaped charge jets is performed in order to bolster future studies on the design of reactive cassettes with non‐steel flyer plates. A symmetric steel reactive cassette construction is taken as a base, and front and back plates are selectively changed with ceramic or glass plates keeping the areal densities similar. Plates manufactured from two different grades of alumina, silicon carbide, boron carbide, and borosilicate glass are employed to assess the ballistic protection performance of reactive cassettes. It is seen that a high density and fracture toughness is desired for the front plate and high bulk impedance for the back plate, respectively. Moreover, replacing the front plate of a steel cassette with an alumina or silicon carbide plate increases the ballistic protection efficiency close to 50%. Finally, the effect of material selection is investigated by hydrocode numerical simulations. The early phases of the interaction process are presented to reveal the basics of initial hole closure on the flyer plate and the determination of the length of the jet precursor.

Environmental design guidelines for residential NZEBs with liner tray construction

Liner tray, steel gauge, or cassette construction is a lightweight steel frame system (LSF) especially suited for low-rise buildings, which offers celerity and quality. It is a viable option for constructing affordable residential Nearly Zero Energy Buildings (NZEBs) in those areas where ‘craft-based’ house-building industries have proven unsustainable, as occurs in some countries of Southern Europe. From an environmental point of view, the continuous disposition of liner trays allows excellent adaptability to the microclimatic context and the particularities of the local site as well as the thermal homogeneity of the envelope. However, its lightweight can entail large thermal variations and overheating problems, especially in constructions with high thermal resistance. A careful design adapted to each location can help avoid these problems, but accurate energy calculations are not always feasible in detached house design. This paper studies the Proyectopía system's passive strategies, an innovative liner tray system made of extruded aluminum for sustainable, low-cost detached houses in Spain. Environmental design guidelines can be a powerful and suitable tool for addressing various house projects that share the same construction system. Several parameters with a substantial impact on the house energy demand, such as insulation thickness, window glazing type, window-to-wall ratio, solar shading, and thermal inertia, are analyzed in a house prototype. Designers can use these parametric study results to perform environmental assessments from the early stages of the design process.

Construction and Optimization of Herpes Simplex Virus Vectors for Central Nervous System Gene Delivery based on CRISPR/Cas9-mediated Genome Editing.

We aim to define parameters that affect the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Engineered, attenuated Herpes Simplex Virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and longterm transgene expression of attenuated HSV-1 vectors have not been fully understood. The study aimed at using CRISPR-Cas9 system to construct attenuated HSV-1 vectors and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and construct two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in mouse hippocampus gene transduction model. The in vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacking Poly (A) induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Our results indicated that the integrity of LAT transcripts was not necessary for establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, which suggests an important role of LAT in maintaining viral reactivation potential.

Strain Construction for Intracellular Metabolic Pathway Localization in Y. lipolytica.

Pathway localization by fluorophore or epitope tagging can be accomplished through a multi-staged DNA construct and confirmation process, to generate a series of successfully tagged protein targets. Prerequisite conditions for this process in Y. lipolytica are auxotrophic selection (leu2 or ura3), impaired non-homologous end joining by deletion or impairment of ku70, and plasmids or gene pieces for epitope-selection cassette construction. The general approach for gene tagging can work for C- or N-terminal tags. Gene overexpression from an episomal plasmid can be accomplished through transcript amplification and cloning. C-terminal tagging allows expression of a gene-GFP fusion to be regulated from the endogenous promoter. The epitope-selection cassette also includes a constitutive or highly expressed promoter driving the auxotrophic or other selectable marker gene such as one conferring antifungal or antibiotic resistance. Strains for pathway localization utilize overlap PCR, PEG-based transformation, and a fast DNA preparation for rapid colony screening. Successful transformants can be used for pathway localization and condition-specific response analysis.

Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease

<p>Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.</p>

Creation of Golden Gate constructs for gene doctoring

BackgroundGene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps.ResultsWe constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker.ConclusionsOur plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

Genetic Engineering by DNA Recombineering.

Recombineering inserts PCR products into DNA using homologous recombination. A pair of short homology arms (50 base pairs) on the ends of a PCR cassette target the cassette to its intended location. These homology arms can be easily introduced as 5' primer overhangs during the PCR reaction. The flexibility to choose almost any pair of homology arms enables the precise modification of virtually any DNA for purposes of sequence deletion, replacement, insertion, or point mutation. Recombineering often offers significant advantages relative to previous homologous recombination methods that require the construction of cassettes with large homology arms, and relative to traditional cloning methods that become intractable for large plasmids or DNA sequences. However, the tremendous number of variables, options, and pitfalls that can be encountered when designing and performing a recombineering protocol for the first time introduce barriers that can make recombineering a challenging technique for new users to adopt. This article focuses on three recombineering protocols we have found to be particularly robust, providing a detailed guide for choosing the simplest recombineering method for a given application and for performing and troubleshooting experiments. © 2019 by John Wiley & Sons, Inc.

Strategies for gene disruption and expression in filamentous fungi.

Filamentous fungi can produce many valuable secondary metabolites; among these fungi, endophytic fungi play an ecological role in mutualistic symbiosis with plants, including promoting plant growth, disease resistance, and stress resistance. However, the biosynthesis of most secondary metabolites remains unclear, and knowledge of the interaction mechanisms between endophytes and plants is still limited, especially for some novel fungi, due to the lack of genetic manipulation tools for novel species. Herein, we review the newly discovered strategies of gene disruption, such as the CRISPR-Cas9 system, the site-specific recombination Cre/loxP system, and the I-SceI endonuclease-mediated system in filamentous fungi. Gene expression systems contain using integration of target genes into the genome, host-dependent expression cassette construction depending on the host, a host-independent, universal expression system independent of the host, and reporter-guided gene expression for filamentous fungi. Furthermore, the Newly CRISPRi, CRISPRa, and the selection markers were also discussed for gene disruption and gene expression were also discussed. These studies lay the foundation for the biosynthesis of secondary metabolites in these organisms and aid in understanding the ecological function of filamentous fungi.

Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via athermal cycler-based microassay.

Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast Rhodosporidium toruloides.

The red yeast Rhodosporidium toruloide is a versatile host for production of lipids and carotenoids. Genetic tools are underdeveloped for red yeasts due to their unique genetics and physiology. Currently expression of a heterogonous gene in red yeasts is largely based on integration of the designed cassette by Agrobacterium mediated transformation, yet this method is somewhat restricted when multiple genes are required to be expressed due to the lack of functional genetic elements. Here we demonstrate that virus 2A sequence is effective to mediate co-expression of multiple enzymes in R. toruloides. Two different 2A sequences, Porcine teschovirus-1 2A (P2A) and foot-and-mouth disease virus 2A (F2A), were evaluated. It was found that P2A sequence was more effective for co-expression of two antibiotic selection markers. Co-expression of three antibiotic resistance proteins was successful from a single promoter mediated by P2A sequence. When three heterogeneous enzymes responsible for β-carotene biosynthesis were co-expressed, recombinant R. toruloides strains produced up to 4.5-fold more β-carotene than that of the parental one. The use of 2A sequence can facilitate cassette construction to engineer advanced cell factories for production of lipids and related oleochemicals.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors.

The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of novel RNA therapeutics. Transient induction of RNAi via transfection of synthetic small interfering RNAs can trigger the selective knockdown of a target mRNA. For durable silencing of gene expression, either artificial short hairpin RNA or microRNA encoding transgene constructs were developed. These miRNAs are based on the molecules that induce the natural RNAi pathway in mammals and humans: the endogenously expressed miRNAs. Significant efforts focused on the construction and delivery of miRNA cassettes in order to solve basic biology questions or to design new therapy strategies. Several viral vectors have been developed, which are particularly useful for the delivery of miRNA expression cassettes to specific target cells. Each vector system has its own unique set of distinct properties. Thus, depending on the specific application, a particular vector may be most suitable. This field was previously reviewed for different viral vector systems, and now the recent progress in the field of miRNA-based gene-silencing approaches using lentiviral vectors is reported. The focus is on the unique properties and respective limitations of the available vector systems for miRNA delivery.

Vascular expression driven by the promoter of a gene encoding a high-affinity potassium transporter HAK5 from Eucalyptus grandis

The construction of expression cassettes harboring tissue-specific promoters is a viable alternative to limit transgene expression to specific organs and cell types. In this study, we have functionally characterized the promoter of a Eucalyptus grandis gene encoding a putative high-affinity HAK5-like potassium (K+) transporter (designated EgHAK5) showing root-specific expression. The ability of the EgHAK 5′-flanking region (~1.3 kb) to drive root-specific expression of a reporter gene (β-glucuronidase; GUS) was examined using transgenic tobacco plants. Histochemical analysis revealed enhanced GUS staining in the vasculature of leaves, hypocotyls and roots, which was also confirmed in histological cross-sections. Moreover, the relative expression of GUS in the roots of the generated transgenic lines was increased in response to K+ starvation. Overall, our results indicate that, in a heterologous system, the EgHAK5 promoter shows expression in vascular tissues, mainly within the phloem, and is up-regulated upon potassium deprivation.

Abstract 4354: Methylation-mediated silenced PYCARD plays a key role in human prostate cancer

Abstract Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. In order to analyze effects of DNA methylation in prostate cancer, we constructed LNCaP-derived human prostate cancer cells that can induce global reactivation of hypermethylated genes by the methyl-CpG targeted transcriptional activation (MeTA) method. In MeTA, a cassette construct of MBD2-derived methyl-CpG binding domain (MBD) with NFκB-derived transcriptional activation domain is transfected into cancer cells. Then expressed MBD domain specifically binds to the hypermethylated promoter regions, and NFκB transcriptional activation domain recruits p300/CREB-binding protein (CBP) and reactivates hypermethylation-mediated silenced genes. A cell proliferation assay indicated that MeTA suppressed the growth of LNCaP cells. Furthermore, both flow cytometry and TUNEL assays demonstrated that MeTA induced apoptosis. In order to search genes responsible for apoptosis, we performed gene expression microarray analysis of MeTA-uninduced and -induced LNCaP cells: Five genes encoding apoptosis-inducing factors upregulated two-fold or more in accordance with the induction of MeTA; PYCARD (PYD and CARD domain containing), TNFRSF25 (tumor necrosis factor receptor superfamily 25), HRK (harakiri, BCL2 interacting protein), BIK (BCL2 interacting killer), and CIDEA (cell death-inducing DFFA-like effector a). These genes also contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site. We focused on PYCARD and TNFRSF25 because these two genes upregulated 10-fold or more in accordance with the induction of MeTA. We found that both genes were hypermethylated and showed low expression levels in LNCaP prostate cancer cells, whereas only PYCARD was unmethylated in RWPE-1 normal prostate epithelial cells. We further analyzed primary prostate cancer tissues and found tumor-specific promoter hypermethylation of PYCARD in 53.8% (7/13; P < 0.01). These results suggest that methylation-mediated silencing of PYCARD contributes to escape from apoptosis in human prostate cancer, and MeTA may provide important clues to analyze changes in cancer cell phenotypes by DNA methylation alterations. Citation Format: Shinichi Fukushige, Toshiya Miyauchi, Teppei Okubo, Koji Mitsuzuka, Yoichi Arai, Akira Horii. Methylation-mediated silenced PYCARD plays a key role in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2017-4354

Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning.

We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.