Insertion of a &lt;i&gt;lox&lt;/i&gt;P site in a size-reduced human accessory chromosome

Moralli, Daniela; Vagnarelli, Paola; Bensi, Mirella; Carli, L. De; Raimondi, Elena

doi:10.1159/000048801

Cited by 16 publications

(9 citation statements)

References 30 publications

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“…Applying the telomerator to chromosome engineering in mammalian cells will be more challenging because "regional" mammalian centromeres can extend for hundreds of kilobases and include repetitive sequences. Artificially generated human minichromosomes, existing as either linear or circular constructs, have been described and they may serve as a starting point for mammalian artificial chromosome engineering (33)(34)(35)(36)(37)(38)(39). We ultimately envision such artificial chromosomes as valuable platforms for gene targeting that will allow delivery of large DNA sequences to recipient cells and provide a means by which to investigate the incorporation of complex segments of DNA encoding networks and pathways.…”

Section: Discussionmentioning

confidence: 99%

Circular permutation of a synthetic eukaryotic chromosome with the telomerator

Mitchell

Boeke

2014

Proc. Natl. Acad. Sci. U.S.A.

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Chromosome engineering is a major focus in the fields of systems biology, genetics, synthetic biology, and the functional analysis of genomes. Here, we describe the "telomerator," a new synthetic biology device for use in Saccharomyces cerevisiae. The telomerator is designed to inducibly convert circular DNA molecules into mitotically stable, linear chromosomes replete with functional telomeres in vivo. The telomerator cassette encodes convergent yeast telomere seed sequences flanking the I-SceI homing endonuclease recognition site in the center of an intron artificially transplanted into the URA3 selectable/counterselectable auxotrophic marker. We show that inducible expression of the homing endonuclease efficiently generates linear molecules, identified by using a simple plate-based screening method. To showcase its functionality and utility, we use the telomerator to circularly permute a synthetic yeast chromosome originally constructed as a circular molecule, synIXR, to generate 51 linear variants. Many of the derived linear chromosomes confer unexpected phenotypic properties. This finding indicates that the telomerator offers a new way to study the effects of gene placement on chromosomes (i.e., telomere proximity). However, that the majority of synIXR linear derivatives support viability highlights inherent tolerance of S. cerevisiae to changes in gene order and overall chromosome structure. The telomerator serves as an important tool to construct artificial linear chromosomes in yeast; the concept can be extended to other eukaryotes.telomerator | chromosome engineering | telomeric silencing | Sc2.0 | synthetic chromosome

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Section: Discussionmentioning

confidence: 99%

Circular permutation of a synthetic eukaryotic chromosome with the telomerator

Mitchell

Boeke

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In some cases, transmission in families has also been observed. Human supernumerary marker chromosomes have been used as platforms for the construction of engineered minichromosomes (20,21,(27)(28)(29), because they display distinctive features that would make them suitable as gene therapy vectors.…”

Section: Non-integrating Large Capacity Vectorsmentioning

confidence: 99%

“…The authors developed a co-transformation procedure, based on the use of chromosome specific subcentromeric satellite DNA as a transformation carrier, to target the neo gene to the marker chromosome. This minichromosome, retrofitted with the selectable gene, was subsequently size reduced by X-ray exposure (smallest end product estimated size 4.7 Mb) and modified with a lox-P site (see Subheading 2.4), enabling the recombinogenic introduction of a reporter cassette (29). The molecular structure and the size of the deleted derivatives were determined by pulsed-field electrophoresis, their mitotic stability was checked by anaphase analysis and finally, the ability to bind centromeric proteins was verified by immunofluorescence.…”

Section: Size Reduction Of Natural Minichromosomesmentioning

confidence: 99%

“…An example of site-specific gene targeting is shown in (c). Two color FISH on chromatin fibers enables to demonstrate that a single copy of a reporter gene (bright fluorescent spots), is inserted into each of the loxP sites (darker fluorescent signals) carried by a small size-reduced supernumerary marker chromosome (29). In all, five copies of the loxP sequence and of the reporter gene are present (white arrows).…”

Section: Size Reduction Of Natural Minichromosomesmentioning

confidence: 99%

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Naturally Occurring Minichromosome Platforms in Chromosome Engineering: An Overview

Raimondi

2011

Methods in Molecular Biology

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Artificially modified chromosome vectors are non-integrating gene delivery platforms that can shuttle very large DNA fragments in various recipient cells: theoretically, no size limit exists for the chromosome segments that an engineered minichromosome can accommodate. Therefore, genetically manipulated chromosomes might be potentially ideal vector systems, especially when the complexity of higher eukaryotic genes is concerned. This review focuses on those chromosome vectors generated using spontaneously occurring small markers as starting material. The definition and manipulation of the centromere domain is one of the main obstacles in chromosome engineering: naturally occurring minichromosomes, due to their inherent small size, were helpful in defining some aspects of centromere function. In addition, several distinctive features of small marker chromosomes, like their appearance as supernumerary elements in otherwise normal karyotypes, have been successfully exploited to use them as gene delivery vectors. The key technologies employed for minichromosome engineering are: size reduction, gene targeting, and vector delivery in various recipient cells. In spite of the significant advances that have been recently achieved in all these fields, several unsolved problems limit the potential of artificially modified chromosomes. Still, these vector systems have been exploited in a number of applications where the investigation of the controlled expression of large DNA segments is needed. A typical example is the analysis of genes whose expression strictly depends on the chromosomal environment in which they are positioned, where engineered chromosomes can be envisaged as epigenetically regulated expression systems. A novel and exciting advance concerns the use of engineered minichromosomes to study the organization and dynamics of local chromatin structures.

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“…Thin smears of infected and uninfected erythrocytes from P. falciparum culture at the trophozoite stage on glass slides were fixed in 2 % paraformaldehyde, permeabilized as reported [13], probed with 1:800 diluted anti-PfPIG-B peptide antiserum followed by 1:400 of rat anti-GRP antibodies (MR4, ATCC). The slides were then treated with a mixture of DAPI, 1:400 diluted Alexa Fluor 568-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-rat IgG.…”

Section: Localization Of Pfpig-b By Immunofluorescence Analysismentioning

confidence: 99%

Plasmodium falciparum GPI mannosyltransferase-III has novel signature sequence and is functional

Basagoudanavar

Feng

Gowda

et al. 2007

Biochemical and Biophysical Research Communications

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The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum are indispensable for parasite survival since merozoite surface proteins-1, -2, -4, -5 and -10 crucial for erythrocyte invasion are GPI-anchored. Therefore, the GPI biosynthetic pathway can offer potential targets for novel antimalarial drugs. Here, we characterized the putative P. falciparum PIG-B gene (PfPIGB) that encodes mannosyltransferase-III of GPI biosynthesis. PfPIGB mRNA is transcribed in a developmental stage specific manner. A protein corresponding to the expected size of PfPIG-B is expressed by the parasite and is localized in the endoplasmic reticulum. Treatment of parasites with PfPIG-B specific siRNA caused reduced GPI synthesis, affecting the PIG-B specific GPI intermediate. These data demonstrate that PfPIG-B is functional and encodes for mannosyltransferase-III of the parasite GPI biosynthesis. The parasite PfPIG-B is novel in that its signature sequence HKEHKI is unique and is only partially conserved compared to HKEXRF signature motif of mammalian PIG-B enzymes. KeywordsPlasmodium falciparum; glycosylphosphatidylinositol anchors; biosynthesis; mannosyltransferase-III; characterization Malaria caused by Plasmodium falciparum is a major health problem in many countries. About 40% of the global population is at risk to malaria, and in sub-Saharan Africa, millions of deaths, mostly among children less than 5 years old, occur every year [1]. The death toll due to malaria has been rapidly increasing because of drug resistance. Development of novel drugs targeting the essential metabolic pathways of the parasite and therapeutics/vaccine that prevent the infection and/or illness are urgently needed. In this context, glycosylphosphatidylinositol (GPI) biosynthetic pathway can offer important targets [2].The GPIs of all organisms have a conserved glycan core, Manα1-2Manα1-6Manα1-4GlcNα1, covalently linked to myo-inositol of phosphatidylinositol (PI) at O-6 [3]. The mannose-3 of the glycan core is invariably substituted at O-6 with EtN-P, which is used for membrane anchoring of proteins through amide bond formation with the -COOH of the protein C-termini. GPIs from different organisms vary in the PI moiety in having acylated or nonacylated myo-inositol, and diacylglycerols, monoacylglycerols, alkyl/acylglycerols or ceramide [3]. In the case of P. falciparum GPIs, the conserved glycan core is substituted with an additional Man at O-2 of the third Man, and the PI comprised of diacylglycerol and O-2-acylated inositol moieties, both with variable fatty acids [4]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe biosynthesis of GPIs occurs in the ER by the sequential addition of sugars to the PI by a coordinated action of glycosyltransferases, N-deacetylase, inositol acylase, EtN-P transferase, and several accessory proteins [5]. The preassembled GPIs are transferred enbloc to the Ctermini of proteins that have GPI attachment signal sequence. In animals, hundreds of proteins including cell surface re...

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Insertion of a <i>lox</i>P site in a size-reduced human accessory chromosome

Cited by 16 publications

References 30 publications

Circular permutation of a synthetic eukaryotic chromosome with the telomerator

Circular permutation of a synthetic eukaryotic chromosome with the telomerator

Naturally Occurring Minichromosome Platforms in Chromosome Engineering: An Overview

Plasmodium falciparum GPI mannosyltransferase-III has novel signature sequence and is functional

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