RESUMO
BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.
Assuntos
Sistemas CRISPR-Cas/genética , Epotilonas/biossíntese , Família Multigênica , Myxococcus xanthus/genética , Proteínas Recombinantes/genética , Transcrição Gênica , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Epotilonas/genética , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Metabolismo Secundário , Ativação TranscricionalRESUMO
In childhood the neuronal ceroid lipofuscinoses (NCL) are the most frequent lysosomal diseases and the most frequent neurodegenerative diseases but, in adulthood, they represent a small fraction among the neurodegenerative diseases. Their morphology is marked by: (i) loss of neurons, foremost in the cerebral and cerebellar cortices resulting in cerebral and cerebellar atrophy; (ii) an almost ubiquitous accumulation of lipopigments in nerve cells, but also in extracerebral tissues. Loss of cortical neurons is selective, indiscriminate depletion in early childhood forms occurring only at an advanced stage, whereas loss of neurons in subcortical grey-matter regions has not been quantitatively documented. Among the fourteen different forms of NCL described to date, CLN1 and CLN10 are marked by granular lipopigments, CLN2 by curvilinear profiles (CVPs), CLN3 by fingerprint profiles (FPPs), and other forms by a combination of these features. Among extracerebral tissues, lymphocytes, skin, rectum, skeletal muscle and, occasionally, conjunctiva are possible guiding targets for diagnostic identification, the precise type of NCL then requiring molecular analysis within the clinical and morphological context. Autosomal-recessive adult NCL has been linked molecularly to different childhood forms, i.e. CLN1, CLN5, and CLN6, whilst autosomal-dominant adult NCL, now designated as CLN4, is caused by a newly identified separate gene, DNAJC5. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.
Assuntos
Lipofuscinoses Ceroides Neuronais/patologia , Adulto , Humanos , Lipofuscinoses Ceroides Neuronais/classificação , Lipofuscinoses Ceroides Neuronais/genética , Tripeptidil-Peptidase 1RESUMO
The Hippo pathway is an essential tumor suppressor signaling network that coordinates cell proliferation, death, and differentiation in higher eukaryotes. Intriguingly, the core components of the Hippo pathway are conserved from yeast to man, with the yeast analogs of mammalian MST1/2 (fly Hippo), MOB1 (fly Mats), LATS1/2 (fly Warts), and NDR1/2 (fly Tricornered) functioning as essential components of the mitotic exit network (MEN). Here, we update our previous summary of mitotic functions of Hippo core components in Drosophila melanogaster and mammals, with particular emphasis on similarities between the yeast MEN pathway and mitotic Hippo signaling. Mitotic functions of YAP and TAZ, the two main effectors of Hippo signaling, are also discussed.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Via de Sinalização Hippo , Humanos , Pontos de Checagem da Fase M do Ciclo CelularRESUMO
We determined the protein expression of adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) and its co-repressor and co-activator complexes in adipose tissue from the obese offspring of under- and over-nourished dams. Female rats were fed either a high-fat (60% kcal) or control (10% kcal) diet before mating, and throughout pregnancy and lactation (Mat-OB). Additional dams were 50% food-restricted from pregnancy day 10 to term [intrauterine growth-restricted (IUGR)]. Adipose tissue protein expression was analyzed in newborn and adult male offspring. Normal birth weight Mat-OB and low birth weight IUGR newborns had upregulated PPARγ with variable changes in co-repressors and co-activators. As obese adults, Mat-OB and IUGR offspring had increased PPARγ with decreased co-repressor and increased co-activator expression. Nutritionally programmed increased PPARγ expression is associated with altered expression of its co-regulators in the newborn and adult offspring. Functional studies of PPARγ co-regulators are necessary to establish their role in PPARγ-mediated programmed obesity.
Assuntos
Tecido Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fenômenos Fisiológicos da Nutrição Materna , PPAR gama/genética , Animais , Peso ao Nascer , Glicemia , Tamanho Corporal , Dieta Hiperlipídica , Feminino , Insulina/sangue , PPAR gama/metabolismo , Ratos , Triglicerídeos/sangueRESUMO
The name of lysosomal storage diseases stems from the fact that in this category of disorders specific undegraded materials are stored in the lysosomes. This is usually caused by a lysosomal enzyme deficiency and leads to a cascade of pathological outcomes. Apart from deficiency of lysosomal enzymes, lysosomal storage diseases also include deficiencies in proteins necessary for enzyme functioning, proteins needed for post-translational modification of these enzymes and proteins required for export of certain compounds from the lysosomes.