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1.
Gene ; 748: 144697, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32325092

RESUMO

Artificial chromosomes were previously generated for use in bacteria, protists, yeast and human cells. A Tetrahymena thermophila artificial chromosome could serve as a versatile platform to study diverse aspects of Tetrahymena biology and beyond. Here, we placed a C3-type rDNA replication origin and telomere sequences from T. thermophila into a pNeo4 vector, producing the first T. thermophila macronuclear artificial chromosome (TtAC1). Circular or linear forms of TtAC1 can be stably transformed into both vegetative and conjugative T. thermophila cells. Linear TtAC1 was stably double in copy number under antibiotic selection, but its copy number was dropping without antibiotic selection pressure. Southern blot, Real-Time PCR and E. coli retransformation analyses together showed that TtAC1 vector did not integrate into the macronuclear genome, and was maintained as a linear or a circular chromosome in T. thermophila macronucleus under antibiotic selection. The use of TtAC1 for recombinant protein production was demonstrated by western blot analysis of a secreted 27 kDa TtsfGFP-12XHis protein. We present the first macronuclear artificial chromosome with species-specific chromosomal elements for use in T. thermophila studies and to aid broad recombinant biotechnology applications.


Assuntos
Cromossomos Artificiais , Tetrahymena thermophila/genética , Animais , Biolística , Telômero
2.
J Microbiol Biotechnol ; 29(12): 1931-1937, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693835

RESUMO

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.


Assuntos
Cromossomos Artificiais , Daptomicina/biossíntese , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Daptomicina/farmacologia , Furanos/metabolismo , Genes Bacterianos , Vetores Genéticos , Lipídeos , Macrolídeos/metabolismo , Peptídeo Sintases , Policetídeos/metabolismo , Staphylococcus aureus/efeitos dos fármacos
3.
G3 (Bethesda) ; 9(10): 3119-3127, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31506318

RESUMO

Oxytricha trifallax, like other ciliates, has separate germline and somatic nuclei. The diploid germline genome in the micronucleus is composed of long conventional chromosomes. The macronucleus contains a somatic genome which is naturally fragmented into thousands of kilobase-sized chromosomes. Here, we develop a method to stably incorporate artificial chromosomes into the macronucleus. We report two cases of successful transformation and demonstrate the use of somatic transformation to investigate gene regulation and gene function in Oxytricha We show that the transformed artificial chromosomes are maintained through multiple asexual divisions. Furthermore, they support the transcriptional regulation of the native chromosome from which they were derived and are translated to produce functional proteins. To test if transformed chromosomes are amenable to practical applications, we generated a tagged version of a representative gene (AL1) and used it to co-precipitate associated proteins. This revealed an association with nucleic acid binding proteins, specifically RNA-binding proteins, and RNA immunoprecipitation of AL1 revealed its association with multiple RNAs. The use of artificial chromosomes in Oxytricha enables an array of genetic and molecular biological assays, as well as new avenues of inquiry into the epigenetic programming of macronuclear development and genome rearrangement.


Assuntos
Cromossomos Artificiais , Oxytricha/genética , Imunofluorescência , Regulação da Expressão Gênica , Rearranjo Gênico , Genes de Protozoários , Genoma de Protozoário , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
4.
PLoS One ; 14(8): e0215882, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31386669

RESUMO

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.


Assuntos
Babesia bovis/enzimologia , Cromossomos Artificiais/genética , Técnicas de Inativação de Genes , Rad51 Recombinase/deficiência , Rad51 Recombinase/genética , Homologia de Sequência do Ácido Nucleico , Babesia bovis/genética , Centrômero/genética , Expressão Gênica , Modelos Moleculares , Fenótipo , Conformação Proteica , Rad51 Recombinase/química
5.
Nucleic Acids Res ; 47(11): 5998-6006, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31062017

RESUMO

A plasmid with a replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells at a random chromosomal location. A mouse artificial chromosome (MAC) vector can stably carry a large genomic region. In this study we combined these two technologies with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)9 strategy to achieve targeted amplification of a sequence of interest. We previously showed that the IR/MAR plasmid was amplified up to the extrachromosomal tandem repeat; here we demonstrate that cleavage of these tandem plasmids and MAC by Cas9 facilitates homologous recombination between them. The plasmid array on the MAC could be further extended to form a ladder structure with high gene expression by a breakage-fusion-bridge cycle involving breakage at mouse major satellites. Amplification of genes on the MAC has the advantage that the MAC can be transferred between cells. We visualized the MAC in live cells by amplifying the lactose operator array on the MAC in cells expressing lactose repressor-green fluorescent protein fusion protein. This targeted amplification strategy is in theory be applicable to any sequence at any chromosomal site, and provides a novel tool for animal cell technology.


Assuntos
Cromossomos Artificiais/genética , Replicação do DNA , Regiões de Interação com a Matriz , Plasmídeos/metabolismo , Origem de Replicação , Animais , Células CHO , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cricetinae , Cricetulus , Endonucleases/genética , Amplificação de Genes , Recombinação Homóloga , Camundongos
6.
Plant Mol Biol ; 97(6): 525-535, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30051252

RESUMO

KEY MESSAGE: Transgenomics for gene discovery in Populus euphratica. Transgenomics, a member of the omics family of methodologies, is characterized as the introduction of DNA from one organism into another on a genome-wide scale followed by the identification of recipients with altered phenotypes. This strategy allows investigators to identify the gene(s) involved in these phenotypic changes. It is particularly promising for woody plants that have a long life cycle and for which molecular tools are limited. In this study, we constructed a large-insert binary bacterial artificial chromosome library of Populus euphratica, a stress-tolerant poplar species, which included 55,296 clones with average insert sizes of about 127 kb. To date, 1077 of the clones have been transformed into Arabidopsis thaliana via Agrobacterium by the floral dip method. Of these, 69 transgenic lines showed phenotypic changes represented by diverse aspects of plant form and development, 22 of which were reproducibly associated with the same phenotypic change. One of the clones conferring transgenic plants with increased salt tolerance, 002A1F06, was further analyzed and the 127,284 bp insert in this clone harbored eight genes that have been previously reported to be involved in stress resistance. This study demonstrates that transgenomics is useful in the study of functional genomics of woody plants and in the identification of novel gene(s) responsible for economically important traits. Thus, transgenomics can also be used for validation of quantitative trait loci mapped by molecular markers.


Assuntos
Estudos de Associação Genética/métodos , Plantas Geneticamente Modificadas/genética , Populus/genética , Arabidopsis/genética , Cromossomos Artificiais/genética , Genoma de Planta/genética , Genômica/métodos , Fenótipo , Locos de Características Quantitativas/genética , Tolerância ao Sal/genética
7.
FEMS Microbiol Lett ; 365(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29718215

RESUMO

The exponential increase in available microbial genome sequences coupled with predictive bioinformatic tools is underscoring the genetic capacity of bacteria to produce an unexpected large number of specialized bioactive compounds. Since most of the biosynthetic gene clusters (BGCs) present in microbial genomes are cryptic, i.e. not expressed under laboratory conditions, a variety of cloning systems and vectors have been devised to harbor DNA fragments large enough to carry entire BGCs and to allow their transfer in suitable heterologous hosts. This minireview provides an overview of the vectors and approaches that have been developed for cloning large BGCs, and successful examples of heterologous expression.


Assuntos
Bactérias/genética , Cromossomos Artificiais/genética , Clonagem Molecular/métodos , Genômica/métodos , Proteínas de Bactérias/genética , Biologia Computacional , Vetores Genéticos/genética
8.
ACS Chem Biol ; 13(5): 1142-1147, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29631395

RESUMO

Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated CR) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.


Assuntos
Aspergillus/genética , Cromossomos Artificiais , Espectrometria de Massas/métodos , Morfolinas/metabolismo , Vias Biossintéticas/genética , Metabolômica
9.
Epigenetics Chromatin ; 11(1): 16, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29653589

RESUMO

BACKGROUND: The centromere is the specialized chromatin region that directs chromosome segregation. The kinetochore assembles on the centromere, attaching chromosomes to microtubules in mitosis. The centromere position is usually maintained through cell cycles and generations. However, new centromeres, known as neocentromeres, can occasionally form on ectopic regions when the original centromere is inactivated or lost due to chromosomal rearrangements. Centromere repositioning can occur during evolution. Moreover, de novo centromeres can form on exogenously transformed DNA in human cells at a low frequency, which then segregates faithfully as human artificial chromosomes (HACs). How centromeres are maintained, inactivated and activated is unclear. A conserved histone H3 variant, CENP-A, epigenetically marks functional centromeres, interspersing with H3. Several histone modifications enriched at centromeres are required for centromere function, but their role in new centromere formation is less clear. Studying the mechanism of new centromere formation has been challenging because these events are difficult to detect immediately, requiring weeks for HAC selection. RESULTS: DNA injected into the Caenorhabditis elegans gonad can concatemerize to form artificial chromosomes (ACs) in embryos, which first undergo passive inheritance, but soon autonomously segregate within a few cell cycles, more rapidly and frequently than HACs. Using this in vivo model, we injected LacO repeats DNA, visualized ACs by expressing GFP::LacI, and monitored equal AC segregation in real time, which represents functional centromere formation. Histone H3K9 and H4 acetylations are enriched on new ACs when compared to endogenous chromosomes. By fusing histone deacetylase HDA-1 to GFP::LacI, we tethered HDA-1 to ACs specifically, reducing AC histone acetylations, reducing AC equal segregation frequency, and reducing initial kinetochroe protein CENP-AHCP-3 and NDC-80 deposition, indicating that histone acetylations facilitate efficient centromere establishment. Similarly, inhibition of RNA polymerase II-mediated transcription also delays initial CENP-AHCP-3 loading. CONCLUSIONS: Acetylated histones on chromatin and transcription can create an open chromatin environment, enhancing nucleosome disassembly and assembly, and potentially contribute to centromere establishment. Alternatively, acetylation of soluble H4 may stimulate the initial deposition of CENP-AHCP-3-H4 nucleosomes. Our findings shed light on the mechanism of de novo centromere activation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Centrômero/metabolismo , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Acetilação , Animais , Animais Geneticamente Modificados , Cromossomos Artificiais/genética , Cromossomos Artificiais/metabolismo , Transcrição Genética
10.
PLoS One ; 13(3): e0193642, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29505588

RESUMO

Mouse artificial chromosome (MAC) vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration, transferability from donor cells to recipient cells via microcell-mediated chromosome transfer (MMCT), and the potential for loading a megabase-sized DNA fragment. Previously, a MAC containing a multi-integrase platform (MI-MAC) was developed to facilitate the transfer of multiple genes into desired cells. Although the MI system can theoretically hold five gene-loading vectors (GLVs), there are a limited number of drugs available for the selection of multiple-GLV integration. To overcome this issue, we attempted to knock out and reuse drug resistance genes (DRGs) using the CRISPR-Cas9 system. In this study, we developed new methods for multiple-GLV integration. As a proof of concept, we introduced five GLVs in the MI-MAC by these methods, in which each GLV contained a gene encoding a fluorescent or luminescent protein (EGFP, mCherry, BFP, Eluc, and Cluc). Genes of interest (GOI) on the MI-MAC were expressed stably and functionally without silencing in the host cells. Furthermore, the MI-MAC carrying five GLVs was transferred to other cells by MMCT, and the resultant recipient cells exhibited all five fluorescence/luminescence signals. Thus, the MI-MAC was successfully used as a multiple-GLV integration vector using the CRISPR-Cas9 system. The MI-MAC employing these methods may resolve bottlenecks in developing multiple-gene humanized models, multiple-gene monitoring models, disease models, reprogramming, and inducible gene expression systems.


Assuntos
Sistemas CRISPR-Cas/genética , Cromossomos Artificiais/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Animais , Células CHO , Cricetulus , Técnicas de Inativação de Genes , Camundongos , Mutação
11.
Biochemistry ; 57(23): 3237-3243, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29533658

RESUMO

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.


Assuntos
Aspergillus nidulans , Benzodiazepinas/metabolismo , Cromossomos Artificiais/genética , Cromossomos Fúngicos/genética , Proteínas Fúngicas , Peptídeo Sintases , Pirimidinonas/metabolismo , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Cromossomos Artificiais/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Domínios Proteicos
13.
Drug Metab Pharmacokinet ; 33(1): 17-30, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29398301

RESUMO

In the earliest stage of drug discovery/development, various cell-based models and animal models were used for the prediction of human pharmacokinetics and toxicokinetics. Unfortunately, drugs under development are often discontinued because their nonclinical results do not extrapolate to human clinical studies in relation to either safety or efficacy. Therefore, it is important to improve the time- and cost-effectiveness of drug development. This might be achieved by developing new technologies including pharmacokinetics and toxicokinetics models that use human and mouse artificial chromosome vectors (HACs/MACs). HACs/MACs are unique vectors with several advantages: 1) independent maintenance, 2) defined copy number and mitotically stable, 3) no silencing of the transgene, and 4) no limitation of DNA insertion size. This review provides information on the advantages and examples of the utility of various models based on the recent advances in HAC/MAC technologies, including multifunctional cell-based models for assaying drug-drug interactions, bidirectional permeability, and cytotoxicity, as well as fully genetically humanized mouse models. We also discuss the future prospects of these technologies to advance drug discovery. In summary, these technologies offer advantages over current conventional models and should improve the success rate of drug development related to efficacy and safety for humans.


Assuntos
Cromossomos Artificiais/metabolismo , Descoberta de Drogas/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/farmacocinética , Animais , Cromossomos Artificiais/genética , Cromossomos Artificiais Humanos/genética , Cromossomos Artificiais Humanos/metabolismo , Clonagem Molecular/métodos , Descoberta de Drogas/tendências , Técnicas de Transferência de Genes/tendências , Vetores Genéticos/genética , Humanos , Camundongos , Toxicocinética
15.
J Bacteriol ; 200(1)2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038254

RESUMO

Haloferax volcanii is polyploid and contains about 20 genome copies under optimal conditions. However, the chromosome copy number is highly regulated and ranges from two during phosphate starvation to more than 40 under conditions of phosphate surplus. The aim of this study was the characterization of the influence of two replication origins on the genome copy number. The origin repeats and the genes encoding origin recognition complex (ORC) proteins were deleted. The core origin oriC1-orc1 (ori1) deletion mutant had a lower genome copy number and a higher level of fitness than the wild type, in stark contrast to the oriC2-orc5 (ori2) deletion mutant. The genes adjacent to ori1 could not be deleted, and thus, at least two of them are probably essential, while deletion of the genes adjacent to ori2 was possible. Various fragments of and around the origins were cloned into a suicide plasmid to generate haloarchaeal artificial chromosomes (HACs). The copy number of the oriC1-orc1 HAC was much higher than that of the oriC2-orc5 HAC. The addition of adjacent genes influenced both the HAC copy number and the chromosome copy number. The results indicate that the origins of H. volcanii are not independent but that the copy number is regulated via a network of genes around the origins.IMPORTANCE Several species of archaea have more than one origin of replication on their major chromosome and are thus the only known prokaryotic species that allow the analysis of the evolution of multiorigin replication. The widely studied Haloferax volcanii H26 strain has a major chromosome with four origins of replication. Two origins, ori1 and ori2, were chosen for an in-depth analysis using deletion mutants and haloarchaeal artificial chromosomes. The analysis was not restricted to the core origin regions; origin-adjacent genes were also included. Because H. volcanii is polyploid, the effects on the chromosome copy number were of specific importance. The results revealed extreme differences between the two origins.


Assuntos
Cromossomos Artificiais , Deleção de Genes , Dosagem de Genes , Haloferax volcanii/genética , Origem de Replicação , Replicação do DNA , Regulação Bacteriana da Expressão Gênica , Aptidão Genética , Mutação , Complexo de Reconhecimento de Origem/genética , Plasmídeos
16.
G3 (Bethesda) ; 8(1): 173-183, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29150593

RESUMO

Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLα and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.


Assuntos
Sistemas CRISPR-Cas , DNA Fúngico/genética , Edição de Genes/métodos , Genes Fúngicos Tipo Acasalamento , Genoma Fúngico , Saccharomyces cerevisiae/genética , Engenharia Celular/métodos , Cromossomos Artificiais/química , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Loci Gênicos , Plasmídeos/química , Plasmídeos/metabolismo , Ploidias , RNA Guia/genética , RNA Guia/metabolismo , Saccharomyces cerevisiae/metabolismo
17.
J Genet Genomics ; 44(11): 531-539, 2017 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-29169922

RESUMO

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited. Here, telomere-mediated truncation was successfully performed in common wheat (Triticum aestivum) to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.


Assuntos
Cromossomos Artificiais/genética , Técnicas de Transferência de Genes , Engenharia Genética , Plantas Geneticamente Modificadas/genética , Recombinação Genética , Triticum/genética , Cromossomos de Plantas/genética , Rearranjo Gênico , Genes Reporter/genética , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Reação em Cadeia da Polimerase em Tempo Real , Plântula , Telômero/genética , Transgenes/genética , Translocação Genética
18.
Trends Genet ; 33(12): 897-898, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29107344

RESUMO

Artificial chromosomes are useful in making functional vectors for very large genes, studying chromosome physiology, and modeling chromosomal disorders. Shinohara et al. have succeeded for the first time in creating transchromosomic mice by manipulating spermatogonial stem cells (SSCs), which exhibited superior chromosomal stability compared with embryonic stem cells (ESCs).


Assuntos
Células-Tronco Adultas/fisiologia , Transtornos Cromossômicos/genética , Cromossomos Artificiais/genética , Células-Tronco Embrionárias/fisiologia , Espermatogônias/fisiologia , Animais , Masculino , Camundongos
19.
Stem Cell Reports ; 9(4): 1180-1191, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28943251

RESUMO

The introduction of megabase-sized large DNA fragments into the germline has been a difficult task. Although microcell-mediated chromosome transfer into mouse embryonic stem cells (ESCs) allows the production of transchromosomic mice, ESCs have unstable karyotypes and germline transmission is unreliable by chimera formation. As spermatogonial stem cells (SSCs) are the only stem cells in the germline, they represent an attractive target for germline modification. Here, we report successful transfer of a mouse artificial chromosome (MAC) into mouse germline stem cells (GSCs), cultured spermatogonia enriched for SSCs. MAC-transferred GSCs maintained the host karyotype and MAC more stably than ESCs, which have significant variation in chromosome number. Moreover, MAC-transferred GSCs produced transchromosomic mice following microinjection into the seminiferous tubules of infertile recipients. Successful transfer of MACs to GSCs overcomes the problems associated with ESC-mediated germline transmission and provides new possibilities in germline modification.


Assuntos
Cromossomos Artificiais , Técnicas de Transferência de Genes , Espermatogônias/citologia , Espermatogônias/metabolismo , Animais , Biomarcadores , Rastreamento de Células , Expressão Gênica , Genes Reporter , Instabilidade Genômica , Imunofenotipagem , Cariótipo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fenótipo , Espermatogênese
20.
Prog Mol Subcell Biol ; 56: 193-212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28840238

RESUMO

In recent years, various synthetic approaches have been developed to address the question of what directs centromere establishment and maintenance. In this chapter, we will discuss how approaches aimed at constructing synthetic centromeres have co-evolved with and contributed to shape the theory describing the determinants of centromere identity. We will first review lessons learned from artificial chromosomes created from "naked" centromeric sequences to investigate the role of the underlying DNA for centromere formation. We will then discuss how several studies, which applied removal of endogenous centromeres or over-expression of the centromere-specific histone CENP-A, helped to investigate the contribution of chromatin context to centromere establishment. Finally, we will examine various biosynthetic approaches taking advantage of targeting specific proteins to ectopic sites in the genome to dissect the role of many centromere-associated proteins and chromatin modifiers for centromere inheritance and function. Together, these studies showed that chromatin context matters, particularly proximity to heterochromatin or repetitive DNA sequences. Moreover, despite the important contribution of centromeric DNA, the centromere-specific histone H3-variant CENP-A emerges as a key epigenetic mark to establish and maintain functional centromeres on artificial chromosomes or at ectopic sites of the genome.


Assuntos
Proteína Centromérica A/metabolismo , Centrômero/genética , Centrômero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromossomos Artificiais/genética , Epigênese Genética
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