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1.
Arch Virol ; 164(10): 2505-2513, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377888

RESUMO

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.


Assuntos
Expressão Gênica , Vetores Genéticos , Vírus da Necrose Hematopoética Infecciosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa
2.
Gene ; 717: 144043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31400407

RESUMO

Genes involved in the repair of DNA damage are emerging as playing important roles during the disease processes caused by pathogenic fungi. However, there are potentially hundreds of genes involved in DNA repair in a fungus and some of those genes can play additional roles within the cell. One such gene is RAD23, required for virulence of the human pathogenic fungus Cryptococcus neoformans, that encodes a protein involved in the nucleotide excision repair (NER) pathway. However, Rad23 is a dual function protein, with a role in either repair of damaged DNA or protein turn over by directing proteins to the proteasome. Here, these two functions of Rad23 were tested by the creation of a series of domain deletion alleles of RAD23 and the assessment of the strains for DNA repair, proteasome functions, and virulence properties. Deletion of the different domains was able to uncouple the two functions of Rad23, and the phenotypes of strains carrying such forms indicated that the role of RAD23 in virulence is due to its function in proteasomal-mediated protein degradation rather than NER.


Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Reparo do DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/microbiologia , Mariposas/microbiologia , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética , Virulência
3.
Nat Commun ; 10(1): 2906, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266945

RESUMO

A GGGGCC hexanucleotide repeat expansion in intron 1 of chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Repeat-associated non-ATG translation of dipeptide repeat proteins (DPRs) contributes to the neuropathological features of c9FTD/ALS. Among the five DPRs, arginine-rich poly-PR are reported to be the most toxic. Here, we generate a transgenic mouse line that expresses poly-PR (GFP-PR28) specifically in neurons. GFP-PR28 homozygous mice show decreased survival time, while the heterozygous mice show motor imbalance, decreased brain weight, loss of Purkinje cells and lower motor neurons, and inflammation in the cerebellum and spinal cord. Transcriptional analysis shows that in the cerebellum, GFP-PR28 heterozygous mice show differential expression of genes related to synaptic transmission. Our findings show that GFP-PR28 transgenic mice partly model neuropathological features of c9FTD/ALS, and show a role for poly-PR in neurodegeneration.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Proteína C9orf72/genética , Dipeptídeos/genética , Modelos Animais de Doenças , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/metabolismo , Dipeptídeos/metabolismo , Dipeptídeos/toxicidade , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora
4.
Nat Commun ; 10(1): 2905, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266953

RESUMO

Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.


Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
5.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
6.
Microbiol Res ; 226: 55-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284945

RESUMO

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.


Assuntos
Agrobacterium tumefaciens/genética , Ascomicetos/genética , Transformação Genética , Ascomicetos/citologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Southern Blotting , Carbenicilina/farmacologia , Técnicas de Cocultura , DNA Bacteriano , Regulação Fúngica da Expressão Gênica , Gentamicinas/farmacologia , Proteínas de Fluorescência Verde/genética , Higromicina B/farmacologia , Canamicina/farmacologia , Reação em Cadeia da Polimerase , Análise de Sequência , Virulência/genética
7.
Adv Mater ; 31(33): e1902575, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215123

RESUMO

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Nanopartículas/química , RNA Guia/química , RNA Mensageiro/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/métodos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Oxirredução , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Guia/administração & dosagem , RNA Mensageiro/administração & dosagem
8.
Chem Commun (Camb) ; 55(56): 8170-8173, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31241120

RESUMO

Lipid-complexed small interfering RNA (siRNA) nanoparticles are promising gene regulation materials with excellent genetic, but little cellular, selectivity. Herein, we report a chemical strategy to enhance the gene silencing selectivity of these nanoparticles against cancer cells through the covalent integration of a reactive oxygen species (ROS)-degradable thioketal into the lipid nanoparticles. These lipid nanoparticles can efficiently deliver siRNA into cells, and selectively silence cancer cell gene expression in response to the high levels of intracellular ROS in cancer cells.


Assuntos
Inativação Gênica , Lipídeos/química , Nanopartículas/química , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética
9.
Virol J ; 16(1): 81, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221185

RESUMO

BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.


Assuntos
Complexo de Golgi/fisiologia , Leucina/química , Pseudorraiva , Proteínas Estruturais Virais/metabolismo , Rede trans-Golgi/fisiologia , Animais , Cercopithecus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Transfecção , Células Vero , Proteínas Estruturais Virais/genética
10.
Virol J ; 16(1): 82, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221223

RESUMO

BACKGROUND: Cucurbit chlorotic yellows virus (CCYV) is a bipartite cucurbit-infecting crinivirus within the family Closteroviridae. The crinivirus genome varies among genera. P4.9 is the first protein encoded by CCYV RNA2. P5, which is encoded by LIYV, is necessary for efficient viral infectivity in plants; however, it remains unknown whether CCYV P4.9 is involved in movement. FINDING: In this study, we used green fluorescent protein (GFP) to examine the intracellular distribution of P4.9-GFP in plant cells, and observed fluorescence in the cytoplasm and nucleus. Transient expression of P4.9 was localized to the plasmodesmata. Co-infiltration of agrobacterium carrying binary plasmids of P4.9 and GFP facilitated GFP diffusion between cells. Besides P4.9 was able to spread by itself to neighboring cells, and co-localized with a marker specific to the endoplasmic reticulum, HDEL-mCherry, but not with the Golgi marker Man49-mCherry. CONCLUSIONS: Together, these results demonstrate that CCYV P4.9 is involved in cell-cell movement.


Assuntos
Crinivirus/química , Crinivirus/genética , Proteínas do Movimento Viral em Plantas/química , Proteínas do Movimento Viral em Plantas/genética , Genoma Viral , Proteínas de Fluorescência Verde/genética , Doenças das Plantas/virologia , RNA Viral/genética
11.
Chemistry ; 25(44): 10375-10384, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31090112

RESUMO

The ester bond as a universal linker has recently been applied in gene delivery systems owing to its efficient gene release by electrostatic repulsion after its cleavage. However, the ester bond is nonlabile and is difficult to cleave in cells. This work reports a method in which a secondary amine was introduced to the ß-position of the ester bond to generate a hydrogen-bond cyclization (HBC) structure that can make the ester bond hydrolysis ultrafast. A series of molecules comprising ultrasensitive esters that can be activated by H2 O2 were synthesized, and it was found that those able to form an HBC structure showed complete ester hydrolysis within 5 h in both water and phosphate-buffered saline solution, which was several times faster than other methods reported. Then, a series of amphiphilic poly(amidoamine) dendrimers were constructed, comprising the ultrasensitive ester groups for gene delivery; it was found that they could effectively release genes under quite a low concentration of H2 O2 (<200 µm) and transport them into the nucleus within 2 h in Hela cells with high safety. Their gene transfection efficiencies were higher than that of PEI25k . The results demonstrated that the hydrogen-bond-induced ultrasensitive esters could be powerfully applied to construct gene delivery systems.


Assuntos
DNA/química , Dendrímeros/química , Ésteres/química , Técnicas de Transferência de Genes , Poliaminas/química , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclização , DNA/administração & dosagem , Proteínas de Fluorescência Verde/genética , Humanos , Ligações de Hidrogênio , Peróxido de Hidrogênio/química , Hidrólise , Cinética , Transfecção
12.
Nat Plants ; 5(5): 486-490, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036913

RESUMO

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1-3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.


Assuntos
Proteínas de Plantas/genética , Plastídeos/genética , Engenharia de Proteínas/métodos , Proteínas de Ligação a RNA/genética , Transgenes/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Fluorescência Verde/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Zea mays/genética , Zea mays/metabolismo
13.
Nanomedicine (Lond) ; 14(9): 1153-1171, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31050581

RESUMO

Aim: To develop a peptide/phospholipid hybrid system for gastrin-releasing peptide receptor (GRPR)-targeted delivery of pDNA or siRNA. Materials & methods: A multifunctional GRPR-targeted peptide R9-K(GALA)-BBN(6-14) was combined with a phospholipid oligonucleotide delivery system (1:1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleoyl-3-trimethylammonium-propane) and evaluated for pDNA and siRNA delivery in terms of complex size, toxicity, receptor-targeted delivery and gene expression or knockdown efficiency. Results: By combining peptide and phospholipid delivery systems, synergistic improvements in gene expression and knockdown were observed when compared with either system alone. The optimized formulation demonstrated high levels of EGFP expression and EGFP knockdown, GRPR-targeted delivery, enhanced endosomal release and minimal toxicity. Conclusion: The peptide/phospholipid hybrid system provides efficient GRPR-targeted DNA/siRNA delivery.


Assuntos
DNA/administração & dosagem , Ácidos Graxos Monoinsaturados/química , Peptídeos/química , Fosfatidiletanolaminas/química , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/administração & dosagem , Receptores da Bombesina/metabolismo , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Humanos , Terapia de Alvo Molecular , Células PC-3 , Plasmídeos , Transfecção
14.
Analyst ; 144(11): 3581-3589, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065636

RESUMO

The microalgal cell wall is a natural barrier that limits the efficiency of gene delivery in algae genetic engineering. Here, we report the role of hard-uptake nanoparticles (huNPs) in microalgae cell electroporation to enhance the delivery of genes in Chlamydomonas reinhardtii. This role can be divided into two categories: (i) a 'transient state' for short-term behavior under confocal visualization and (ii) a 'steady state' for long-term behavior in cell culture. First, the 'transient' role of gene-huNP complexes was investigated after washing for clear confocal imaging to observe the location of huNPs after electroporation. Second, the 'steady-state' role of the gene-huNP complexes was examined after electroporation by transferring cells to a fresh, medium-rich culture environment without washing to obtain a stable cell culture. For selection of the huNPs, we used two types of nanoparticles (NPs, 250 nm and 530 nm) larger than the threshold size of electroporation uptake to avoid unwanted endocytic uptake of NPs. In the transient state, the visualization results indicate that gene-NP (250 nm) complexes were positioned on the cells and helped to deliver more genes than did the 530 nm NPs. In the steady state, the gene-NP (530 nm) complexes helped stably deliver more genes to the cells by precipitation of NPs due to gravity. We believe that these findings illustrate how gene-NP complexes function in microalgae cell electroporation and could help set up a protocol for enhanced microalgae applications associated with NPs such as environmental waste removal and biofuel production.


Assuntos
DNA/farmacocinética , Técnicas de Transferência de Genes , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacos , Chlamydomonas reinhardtii , DNA/genética , Eletroporação/métodos , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Proteínas de Fluorescência Verde/genética , Microalgas , Nanopartículas/toxicidade , Oxazinas/química , Oxazinas/toxicidade , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/toxicidade , RNA Guia/genética
15.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067766

RESUMO

The microbial assimilation of one-carbon (C1) gases is a topic of interest, given that products developed using this pathway have the potential to act as promising substrates for the synthesis of valuable chemicals via enzymatic oxidation or C-C bonding. Despite extensive studies on C1 gas assimilation pathways, their key enzymes have yet to be subjected to high-throughput evolution studies on account of the lack of an efficient analytical tool for C1 metabolites. To address this challenging issue, we attempted to establish a fine-tuned single-cell-level biosensor system constituting a combination of transcription factors (TFs) and several C1-converting enzymes that convert target compounds to the ligand of a TF. This enzymatic conversion broadens the detection range of ligands by the genetic biosensor systems. In this study, we presented new genetic enzyme screening systems (GESSs) to detect formate, formaldehyde, and methanol from specific enzyme activities and pathways, named FA-GESS, Frm-GESS, and MeOH-GESS, respectively. All the biosensors displayed linear responses to their respective C1 molecules, namely, formate (1.0-250 mM), formaldehyde (1.0-50 µM), and methanol (5-400 mM), and they did so with high specificity. Consequently, the helper enzymes, including formaldehyde dehydrogenase and methanol dehydrogenase, were successfully combined to constitute new versatile combinations of the C1-biosensors.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Formaldeído/análise , Formiatos/análise , Metanol/análise , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fatores de Transcrição
16.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091742

RESUMO

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a 'reporter' gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.


Assuntos
Ascomicetos/genética , Técnicas de Transferência de Genes , Ascomicetos/patogenicidade , Cocos/microbiologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo
17.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
18.
Analyst ; 144(12): 3756-3764, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31070195

RESUMO

Protein phosphorylation is a very important regulatory mechanism in a majority of biological processes, and the determination of protein kinase activity plays a key role in the pathological study and drug development of kinase-related diseases. However, it is very challenging to in situ study endogenous protein kinase activity in a single living cell due to the shortage of in vivo efficient methods. Here, we propose a new strategy for direct determination of protein kinase activity in a single living cell by combining single molecule fluorescence correlation spectroscopy (FCS) with activity-based probes (ABPs). Ribosomal S6 kinase-2 (RSK2) was used as a model, and the ABPs were synthesized on the basis of RSK2 inhibitor FMK to specially label active RSK2 in living cells. Conventional FCS and MEMFCS (maximum entropy method) single molecule techniques were used to in situ determine RSK2 activity in living cells based on the difference in molecular weight between free probes and probe-RSK2 complexes. Furthermore, wild-type and mutated RSK2 were fused with enhanced green fluorescent protein (EGFP) using lentivirus infection, and fluorescence cross-correlation spectroscopy (FCCS) was used to verify the selective binding of ABPs to RSK2-EGFP fusion protein in living cells. Finally, FCS with ABPs was applied for in situ monitoring of the activation of endogenous RSK2 in the stimulation of serum, epidermal growth factor, kinase inhibitors and ultraviolet irradiation; we observed that endogenous RSK2 showed different behaviors in the cytoplasm and the nucleus in some stimulation. Our results document that FCS with ABPs is a very promising method for studying endogenous protein kinases in living cells.


Assuntos
Ensaios Enzimáticos/métodos , Proteínas Quinases S6 Ribossômicas 90-kDa/análise , Análise de Célula Única/métodos , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Compostos de Boro/química , Carbocianinas/síntese química , Carbocianinas/química , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Imagem Individual de Molécula/métodos
19.
Appl Microbiol Biotechnol ; 103(12): 4839-4857, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053916

RESUMO

The surface layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling, proteinaceous subunits non-covalently bound to the outmost bacterial cell wall envelope and is involved in the adherence of bacteria to host cells. We have previously described that the S-layer protein of L. acidophilus possesses anti-viral and anti-bacterial properties. In this work, we extracted and purified S-layer proteins from L. acidophilus ATCC 4356 cells to study their interaction with cell wall components from prokaryotic (i.e., peptidoglycan and lipoteichoic acids) and eukaryotic origin (i.e., mucin and chitin), as well as with viruses, bacteria, yeast, and blood cells. Using chimeric S-layer fused to green fluorescent protein (GFP) from different parts of the protein, we analyzed their binding capacity. Our results show that the C-terminal part of the S-layer protein presents lectin-like activity, interacting with different glycoepitopes. We further demonstrate that lipoteichoic acid (LTA) serves as an anchor for the S-layer protein. Finally, a structure for the C-terminal part of S-layer and possible binding sites were predicted by a homology-based model.


Assuntos
Proteínas de Bactérias/metabolismo , Lactobacillus acidophilus/metabolismo , Lectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Fluorescência Verde/genética , Glicoproteínas de Membrana/isolamento & purificação , Ligação Proteica
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