RESUMO
In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn't require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.
Assuntos
Bacillus subtilis/genética , DNA Bacteriano/biossíntese , Biologia Sintética/métodos , Sequências de Repetição em Tandem/genética , Bacteriófago lambda/genética , Fragmentação do DNA , DNA Bacteriano/genética , Plasmídeos/genética , Transformação GenéticaRESUMO
Plant-specific Rac/Rop small GTPases function as molecular switches for numerous signal transduction events, including defense responses. To understand the function of each of the seven Rac/Rop family members in rice, we studied the tissue-specific expression patterns of Rac/Rop genes by semi-quantitative reverse transcription-PCR (RT-PCR), and also Rac/Rop subcellular localization using green fluorescent protein (GFP) fusion proteins in transient expression systems. We also investigated the roles of these genes in disease resistance by testing single Rac/Rop-RNAi (RNA interference) plants against the rice blast pathogen Magnaporthe grisea. Our studies show that expression of OsRac2, 6 and 7 is very low in leaf blades, and reveal a strong correlation between the number of lysine and/or arginine (KR) residues in the polybasic region of Rac/Rop GTPases and their subcellular distribution in vivo. Infection assays showed that OsRac1 is a positive regulator of blast resistance, confirming previous observations, whereas OsRac4 and OsRac5 are negative regulators of blast resistance. OsRac6 may make minor contributions to disease resistance, while OsRac3 and OsRac7 are probably not involved in defense. Therefore, our study suggests that the rice Rac/Rop family plays multiple roles in diverse cellular activities and has both positive and negative functions in disease resistance.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/fisiologia , Oryza/enzimologia , Oryza/microbiologia , Proteínas de Plantas/fisiologia , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Imunidade Inata/genética , Imunidade Inata/fisiologia , Magnaporthe/patogenicidade , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Redes e Vias Metabólicas , Biologia de Sistemas/métodos , Cromatografia Líquida , Biologia Computacional , Eletroforese Capilar , Eletroforese em Gel Bidimensional , Indução Enzimática , Repressão Enzimática , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Expressão Gênica , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.