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1.
J Biotechnol ; 305: 43-50, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31505217

RESUMO

In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Escherichia coli/genética , Engenharia Metabólica/métodos , Cromossomos Artificiais Bacterianos/metabolismo , Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Biologia Sintética
2.
Mol Biotechnol ; 61(10): 783-790, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482466

RESUMO

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.


Assuntos
Flavivirus/metabolismo , Mardivirus/crescimento & desenvolvimento , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Cromossomos Artificiais Bacterianos/genética , Uso do Códon , Fibroblastos/citologia , Fibroblastos/virologia , Flavivirus/genética , Mardivirus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/metabolismo
3.
PLoS One ; 14(8): e0220818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393940

RESUMO

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, ß-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Sistema Digestório/metabolismo , Integrases/metabolismo , Queratina-19/metabolismo , Camundongos Transgênicos/metabolismo , Animais , Genes Reporter , Óperon Lac , Camundongos , Modelos Animais
4.
Hum Gene Ther Methods ; 30(4): 122-126, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31280610

RESUMO

The study of human cytomegalovirus (HCMV) has for long been challenging due to the inability of clinical strains to efficiently proliferate in vitro until adaptive mutations occur. These mutations lead to strains that differ considerably from clinical isolates, many of them showing altered cell tropism, a decrease in cell association and higher susceptibility to an innate immune response. These problems were recently solved by the use of bacterial artificial chromosome (BAC) vectors that allow for the conservation of an intact HCMV genome. Other characteristics that render HCMV difficult for in vitro study are related to its slow replication rate that leads to some constraints in its titration. During the cloning of HCMV into BAC vectors, many groups additionally inserted a fluorescent tag to facilitate the virus characterization. However, the methods used for titration of HCMV-BAC stocks are still relaying on the standard methods that are expensive and/or time consuming. In this study, we assessed the possibility of viral titration by fluorescence-activated cell sorting (FACS), making use of the fluorescent tags that many of the HCMV-BACs hold. We compared viral titers obtained by immunohistochemistry with FACS, a faster and inexpensive technique. We showed that viral titers are comparable using the techniques already mentioned, and that titration by FACS is an efficient, fast, and cost-effective method. The establishment of viral titration of BAC vectors by FACS can further simplify the study of HCMV.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Citomegalovirus/genética , Vetores Genéticos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética
5.
Plant J ; 100(2): 411-429, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31276249

RESUMO

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Engenharia Genética/métodos , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromossomos Artificiais Bacterianos/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde , Histonas/genética , Histonas/metabolismo , Proteínas Luminescentes , Mutagênese Insercional , Plantas Geneticamente Modificadas , Recombinação Genética
6.
BMC Genomics ; 20(1): 479, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185912

RESUMO

BACKGROUND: The mammalian major histocompatibility complex (MHC) harbours clusters of genes associated with the immunological defence of animals against infectious pathogens. At present, no complete MHC physical map is available for any of the wild ruminant species in the world. RESULTS: The high-density physical map is composed of two contigs of 47 overlapping bacterial artificial chromosome (BAC) clones, with an average of 115 Kb for each BAC, covering the entire addax MHC genome. The first contig has 40 overlapping BAC clones covering an approximately 2.9 Mb region of MHC class I, class III, and class IIa, and the second contig has 7 BAC clones covering an approximately 500 Kb genomic region that harbours MHC class IIb. The relative position of each BAC corresponding to the MHC sequence was determined by comparative mapping using PCR screening of the BAC library of 192,000 clones, and the order of BACs was determined by DNA fingerprinting. The overlaps of neighboring BACs were cross-verified by both BAC-end sequencing and co-amplification of identical PCR fragments within the overlapped region, with their identities further confirmed by DNA sequencing. CONCLUSIONS: We report here the successful construction of a high-quality physical map for the addax MHC region using BACs and comparative mapping. The addax MHC physical map we constructed showed one gap of approximately 18 Mb formed by an ancient autosomal inversion that divided the MHC class II into IIa and IIb. The autosomal inversion provides compelling evidence that the MHC organizations in all of the ruminant species are relatively conserved.


Assuntos
Antílopes/genética , Cromossomos Artificiais Bacterianos/genética , Genômica , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Mapeamento Físico do Cromossomo/métodos , Animais , Bovinos , Evolução Molecular , Masculino , Reação em Cadeia da Polimerase
7.
Methods Cell Biol ; 151: 197-218, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948008

RESUMO

To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Equinodermos/genética , Genômica/métodos , Microinjeções/métodos , Animais , Equinodermos/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Vetores Genéticos , Genoma/genética , Genômica/tendências , Microinjeções/tendências , Recombinação Genética , Sequências Reguladoras de Ácido Nucleico
8.
Viruses ; 11(3)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901892

RESUMO

Epstein⁻Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV BamHI S rightward reading frame 1 (BSRF1) encodes a tegument protein that, unlike its homologs herpes simplex virus unique long 51 (UL51) and human cytomegalovirus UL71, has not been extensively investigated. To examine the role of BSRF1, we prepared knockout and revertant strains using the bacterial artificial chromosome system. Unexpectedly, the disruption of the gene had little or no effect on EBV lytic replication and the transformation of primary B cells. However, the knockdown of BSRF1 in B95-8 cells decreased progeny production. An immunofluorescence assay revealed that BSRF1 localized to the Golgi apparatus in the cytoplasm, as did its homologs. BSRF1 also associated with BamHI G leftward reading frame 3.5 (BGLF3.5), BamHI B rightward reading frame 2 (BBRF2), and BamHI A leftward reading frame 1 (BALF1), and BALF1 was incorporated into the tegument fraction with BSRF1. Taken together, our results indicate that BSRF1 plays a role in secondary envelopment or virion egress in the cytoplasm, as do its homolog genes.


Assuntos
Complexo de Golgi/virologia , Herpesvirus Humano 4/genética , Proteínas Virais/genética , Liberação de Vírus , Animais , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Citoplasma/virologia , Imunofluorescência , Técnicas de Inativação de Genes , Células HEK293 , Herpesvirus Humano 4/fisiologia , Humanos , Fases de Leitura , Células Vero , Vírion/genética , Vírion/fisiologia , Montagem de Vírus , Replicação Viral
9.
Methods Mol Biol ; 1937: 177-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30706396

RESUMO

Herpes simplex virus (HSV) is one of the most extensively studied oncolytic virus platforms. The recent FDA approval of talimogene laherparepvec (T-VEC) has been accelerating translational research of oncolytic HSV (oHSV) as a promising therapeutic for refractory cancers such as glioblastoma, the deadliest primary malignancy in the brain. The large genome size of HSV readily allows arming of oHSV by incorporating therapeutic transgenes within the virus, as exemplified by T-VEC carrying GM-CSF, thereby enhancing the anticancer activity of oHSV. Here we describe a bacterial artificial chromosome-based method for construction of an oHSV expressing a transgene, which we routinely use in the laboratory to create a number of different recombinant oHSV bearing either therapeutic or reporter genes.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Herpesvirus Humano 1/genética , Vírus Oncolíticos/genética , Animais , Chlorocebus aethiops , Vetores Genéticos/genética , Humanos , Terapia Viral Oncolítica , Transfecção , Transgenes , Células Vero
10.
Mol Cells ; 42(3): 245-251, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30759972

RESUMO

Ependymal cells constitute the multi-ciliated epithelium, which lines the brain ventricular lumen. Although ependymal cells originate from radial glial cells in the perinatal rodent brain, the exact mechanisms underlying the full differentiation of ependymal cells are poorly understood. In this report, we present evidence that the Anks1a phosphotyrosine binding domain (PTB) adaptor is required for the proper development of ependymal cells in the rodent postnatal brain. Anks1a gene trap targeted LacZ reporter analysis revealed that Anks1a is expressed prominently in the ventricular region of the early postnatal brain and that its expression is restricted to mature ependymal cells during postnatal brain development. In addition, Anks1a-deficient ependymal cells were shown to possess type B cell characteristics, suggesting that ependymal cells require Anks1a in order to be fully differentiated. Finally, Anks1a overexpression in the lateral wall of the neonatal brain resulted in an increase in the number of ependymal cells during postnatal brain development. Altogether, our results suggest that ependymal cells require Anks1a PTB adaptor for their proper development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Epêndima/citologia , Epêndima/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Contagem de Células , Diferenciação Celular , Cromossomos Artificiais Bacterianos/genética , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos Transgênicos , Regulação para Cima
11.
Methods Mol Biol ; 1858: 27-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30414108

RESUMO

Isolation of high molecular weight (HMW) DNA from any organism can be daunting and populations of extremely small insects with hard exoskeletons are no exception. It can be challenging to isolate DNA that is clean enough to enzymatically label, digest or otherwise manipulate without sacrificing length. The method described here was originally developed to isolate HMW DNA for BAC library construction. More recently, it has proven reliable for long range optical mapping as well.


Assuntos
Cromossomos Artificiais Bacterianos/genética , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Genoma de Inseto , Insetos/genética , Animais , Peso Molecular
12.
Methods Mol Biol ; 1874: 43-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353507

RESUMO

Bacterial Artificial Chromosome (BAC) libraries are a valuable research resource. Any one of the clones in these libraries can carry hundreds of thousands of base pairs of genetic information. Often the entire coding sequence and significant upstream and downstream regions, including regulatory elements, can be found in a single BAC clone. BACs can be put to many uses, such as to study the function of human genes in knockout mice, to drive reporter gene expression in transgenic animals, and for gene discovery. In order to use BACs for experimental purposes it is often desirable to genetically modify them by introducing reporter elements or heterologous cDNA sequences. It is not feasible to use conventional DNA cloning approaches to modify BACs due to their size and complexity, thus a specialized field "recombineering" has developed to modify BAC clones through the use of homologous recombination in bacteria with short homology regions. Genetically engineered BACs can then be used in cell culture, mouse, or rat models to study cancer, neurology, and genetics.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Engenharia Genética/métodos , Recombinação Homóloga , Transgenes , Animais , Animais Geneticamente Modificados , Genes Reporter , Tamanho do Genoma , Genoma Bacteriano , Humanos
13.
Dev Biol ; 445(1): 68-79, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30392838

RESUMO

The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported "barcode" tag vectors of which> 100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Engenharia Genética/métodos , Análise de Sequência de DNA/métodos , Animais , Cromossomos Artificiais Bacterianos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia , Genes Reporter/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Biológicos , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Fatores de Transcrição/metabolismo
14.
Methods Mol Biol ; 1869: 207-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324526

RESUMO

To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.


Assuntos
Neoplasias Encefálicas/genética , Técnicas de Introdução de Genes/métodos , Engenharia Genética/métodos , Loci Gênicos , Mutação/genética , Oncogenes , Animais , Cromossomos Artificiais Bacterianos/genética , Modelos Animais de Doenças , Eletroporação , Vetores Genéticos/metabolismo , Genômica , Recombinação Homóloga/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutação Puntual/genética
15.
Biotechnol J ; 14(3): e1700686, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29701329

RESUMO

Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.


Assuntos
Anticorpos Monoclonais/genética , Técnicas de Cultura de Células/métodos , Animais , Biomassa , Células CHO , Linhagem Celular , Cromossomos Artificiais Bacterianos/genética , Cricetulus
16.
N Biotechnol ; 48: 12-19, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29526810

RESUMO

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere.


Assuntos
Cromossomos de Plantas/genética , Triticum/genética , Aegilops/genética , Centrômero/genética , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular , Genes de Plantas , Genoma de Planta , Hibridização Genética , Mapeamento Físico do Cromossomo/métodos , Melhoramento Vegetal
17.
Diabetes ; 68(2): 337-348, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30425060

RESUMO

The sustained expression of the MAFB transcription factor in human islet ß-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet ß-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet ß-cell population and that MafB expression is postnatally restricted in mouse ß-cells by de novo DNA methylation. To gain insight into how MAFB affects human ß-cells, we developed a mouse model to ectopically express MafB in adult mouse ß-cells using MafA transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse ß-cells, suggesting that the human adult ß-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet ß-cell defects in a mouse mutant lacking MafA in ß-cells. Of note, transgenic production of MafB in ß-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal ß-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet ß-cells.


Assuntos
Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Fator de Transcrição MafB/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Cromossomos Artificiais Bacterianos/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Feminino , Humanos , Técnicas In Vitro , Fatores de Transcrição Maf Maior/genética , Fator de Transcrição MafB/genética , Camundongos , Camundongos Transgênicos , Gravidez , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
18.
BMC Plant Biol ; 18(1): 307, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486783

RESUMO

BACKGROUND: Fluorescence in situ hybridization (FISH) is an efficient cytogenetic technology to study chromosome structure. Transposable element (TE) is an important component in eukaryotic genomes and can provide insights in the structure and evolution of eukaryotic genomes. RESULTS: A FISH probe derived from bacterial artificial chromosome (BAC) clone 299N22 generated striking signals on all 26 chromosomes of the cotton diploid A genome (AA, 2x=26) but very few on the diploid D genome (DD, 2x=26). All 26 chromosomes of the A sub genome (At) of tetraploid cotton (AADD, 2n=4x=52) also gave positive signals with this FISH probe, whereas very few signals were observed on the D sub genome (Dt). Sequencing and annotation of BAC clone 299N22, revealed a novel Ty3/gypsy transposon family, which was named as 'CICR'. This family is a significant contributor to size expansion in the A (sub) genome but not in the D (sub) genome. Further FISH analysis with the LTR of CICR as a probe revealed that CICR is lineage-specific, since massive repeats were found in A and B genomic groups, but not in C-G genomic groups within the Gossypium genus. Molecular evolutionary analysis of CICR suggested that tetraploid cottons evolved after silence of the transposon family 1-1.5 million years ago (Mya). Furthermore, A genomes are more homologous with B genomes, and the C, E, F, and G genomes likely diverged from a common ancestor prior to 3.5-4 Mya, the time when CICR appeared. The genomic variation caused by the insertion of CICR in the A (sub) genome may have played an important role in the speciation of organisms with A genomes. CONCLUSIONS: The CICR family is highly repetitive in A and B genomes of Gossypium, but not amplified in the C-G genomes. The differential amount of CICR family in At and Dt will aid in partitioning sub genome sequences for chromosome assemblies during tetraploid genome sequencing and will act as a method for assessing the accuracy of tetraploid genomes by looking at the proportion of CICR elements in resulting pseudochromosome sequences. The timeline of the expansion of CICR family provides a new reference for cotton evolutionary analysis, while the impact on gene function caused by the insertion of CICR elements will be a target for further analysis of investigating phenotypic differences between A genome and D genome species.


Assuntos
Elementos de DNA Transponíveis/genética , Gossypium/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Plantas/genética , Genoma de Planta/genética , Hibridização in Situ Fluorescente , Análise de Sequência de DNA , Tetraploidia
19.
Viruses ; 10(10)2018 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-30301244

RESUMO

The recent outbreaks of Zika virus (ZIKV), its association with Guillain⁻Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.


Assuntos
Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia , Zika virus/genética , Zika virus/patogenicidade , Células A549 , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , DNA Complementar/genética , Feminino , Humanos , Camundongos , Camundongos Knockout , RNA Viral/genética , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Genética Reversa , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Replicação Viral , Zika virus/imunologia , Infecção por Zika virus/prevenção & controle
20.
Genome Biol ; 19(1): 171, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30355328

RESUMO

BACKGROUND: The number of de novo genome sequence assemblies is increasing exponentially; however, relatively few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of conserved non-coding elements is associated with the positions of evolutionary breakpoint regions. RESULTS: We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest. CONCLUSIONS: The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80% assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements, especially interchromosomal, are fixed during evolution without deleterious effects.


Assuntos
Cromossomos/genética , Falconiformes/genética , Rearranjo Gênico/genética , Genoma , Melopsittacus/genética , Struthioniformes/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Sequência Conservada/genética , DNA Intergênico/genética , Genômica , Especificidade da Espécie
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