RESUMO
Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.
Assuntos
Vírus Defeituosos/genética , Teste de Complementação Genética , Vírus da Parainfluenza 3 Bovina/genética , Animais , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Teste de Complementação Genética/instrumentação , Teste de Complementação Genética/métodos , Vetores Genéticos , Vírus da Parainfluenza 3 Bovina/crescimento & desenvolvimento , Proteínas Virais de Fusão/genética , Proteínas da Matriz Viral/genética , Replicação ViralRESUMO
Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.
Assuntos
Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Clorófitas/enzimologia , Microalgas/enzimologia , Proteínas de Plantas/metabolismo , Acetolactato Sintase/química , Sequência de Aminoácidos , Clorófitas/genética , Cloroplastos/enzimologia , Cloroplastos/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética/instrumentação , Microalgas/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ácido Pirúvico/metabolismo , Alinhamento de SequênciaRESUMO
Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein, which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further, since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.
