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Gene Cloning (gene + cloning)
Selected AbstractsCharacterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2FEMS MICROBIOLOGY LETTERS, Issue 2 2009Lei-Lei Chen Abstract Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60,65 °C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59,69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma. [source] Assessment of the rind microbial diversity in a farmhouse-produced vs a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methodsJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2004C. Feurer Abstract Aims:, The diversity of the surface flora of two French red-smear soft cheeses was examined by cultivation-dependent and cultivation-independent methods to assess their composition and to evaluate the accuracy of both approaches. Methods and Results:, Culture-independent methods used involved 16S ribosomal DNA gene cloning and sequencing and single-strand conformation polymorphism analysis (SSCP). The culture-dependent method used involved direct culture and macroscopic observation, polymerase chain reaction of the 16S rRNA gene from DNA extracted from single colonies followed by complete sequencing of the gene. Only few species were recovered by both approaches either in the pasteurized and the farmer cheese. A large diversity of isolates or 16S rDNA sequences related to marine bacteria was identified at the surface of both cheeses. Conclusions:, The results indicated that all three techniques were informative and complementary to allow a more accurate representativeness of the cheese surface biodiversity. Significance and Impact of the Study:, Cultivation and molecular methods have to be combined in order to obtain an extended view of the bacterial populations of complex ecosystems. [source] Progress in the Study of Molecular Genetic Improvements of Poplar in ChinaJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2006Shan-Zhi Lin Abstract The poplar is one of the most economically important and intensively studied tree species owing to its wide application in the timber industry and as a model material for the study of woody plants. The natural resource of poplars in China is replete. Over the past 10 years, the application of molecular biological techniques to genetic improvements in poplar species has been widely studied in China. Recent advances in molecular genetic improvements of poplar, including cDNA library construction, gene cloning and identification, genetic engineering, gene expression, genetic linkage map construction, mapping of quantitative trait loci (QTL) and molecular-assisted selection, are reviewed in the present paper. In addition, the application of modern biotechnology to molecular improvements in the genetic traits of the poplar and some unsolved problems are discussed. (Managing editor: Li-Hui Zhao) [source] Large scale purification of linear plasmid DNA for efficient high throughput cloningBIOTECHNOLOGY JOURNAL, Issue 9 2010Dr. Marjolaine Noirclerc-Savoye Abstract In this report we describe a rapid, simple, and efficient method for large-scale purification of linear plasmid DNA to answer demand from high-throughput gene cloning. The process is based on the separation of the linear vector from small DNA fragments by anion exchange chromatography. Gene cloning experiments by restriction/ligation or the In-Fusion(tm) technique confirmed the high quality of the linearized vector as 100% of the genes were successfully cloned. [source] LOC387715/ARMS2 studies , gene sequencing as a procedure of choiceACTA OPHTHALMOLOGICA, Issue 2009E WYLEGALA Purpose To search for important mutations in LOC387715/ARMS2 gene. Methods 80 patients with choroidal neovascularization subsequent to age-related macular degeneration undergoing anti-VEGF treatment were screened for LOC387715/ARMS2 mutations. PCR followed by gene sequencing was performed. If polymorphism A69S coexisted with R38STOP gene cloning with pGEM-T vector was done. Results R38STOP mutation was found in 5 patients, A69S in 59 patients, in 3 cases R38STOP coexisted with A69S on the other allele. Due to substantial shortening of the R38STOP translation product, the protein is probably not effective. In patients with A69S polymorphism coexisting with R38STOP there is only A69S effective allele , so patients should be treated as A69S homozygous. Conclusion There is increasing number of studies with A69S variant of ARMS2 but only gene sequencing provide reliable date in these cases. If R38STOP is taken into account, A69S is even more important AMD risk factor. Gene sequencing is advisable in studies with LOC387715/ARMS2. [source] | ||||||||||