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Auxotrophic Mutant (auxotrophic + mutant)
Selected AbstractsAuxotrophic mutant of the cyanobacterium Nostoc muscorum showing absolute requirement of Cs+ or Rb+ for diazotrophy and autotrophyJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2006Santosh Bhargava Dr. Caesium-resistant (Cs+ -R) mutant clones of the cyanobacterium Nostoc muscorum were characterized for diazotrophic growth in a medium devoid of Cs+ or Rb+ or both. Cs+ -R phenotype suffered severe genetic damage of a pleiotropic nature affecting diazotrophic growth, chlorophyll a content, nitrogenase activity and photosynthetic O2 evolution. Mutation leading to development of Cs+ -R phenotype could be overcome by availability of Cs+/Rb+. Parent and mutant strains were similar with respect to their Cs+/Rb+ uptake. Available data suggests operation of an efficient coupling of the two incompatible reactions viz. oxygenic photosynthesis and oxygen sensitive N2 fixation in this cyanobacterium. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Tools for the genetic manipulation of Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 8 2007Lenka Pribylova Abstract A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP,kanMX,loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase. [source] pFARs, Plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cellsTHE JOURNAL OF GENE MEDICINE, Issue 4 2010Corinne Marie Abstract Background Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system. Methods The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotransfer of LUC-encoding plasmids into various tissues. Results The introduction of pFARs into the optimized bacterial strain restored normal growth to the auxotrophic mutant and allowed an efficient production of monomeric supercoiled plasmids. The electrotransfer of LUC-encoding pFAR into muscle led to high luciferase activities, demonstrating an efficient gene delivery. In transplanted tumours, transgene expression levels were superior after electrotransfer of the pFAR derivative compared to a plasmid carrying a kanamycin resistance gene. Finally, in skin, whereas luciferase activities decreased within 3 weeks after intradermal electrotransfer of a conventional expression vector, sustained luciferase expression was observed with the pFAR plasmid. Conclusions Thus, we have designed a novel strategy for the efficient production of biosafe plasmids and demonstrated their potentiality for nonviral gene delivery and high-level transgene expression in several tissues. Copyright © 2010 John Wiley & Sons, Ltd. [source] Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzaeFEMS MICROBIOLOGY LETTERS, Issue 1 2004Feng Jie Jin Abstract We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD,sC,). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD,sC,adeA,) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD,sC,,argB adeA, or niaD,sC,,argB adeB,) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD,sC,,argB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible. [source] | ||||||||||