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Vitro Selection (vitro + selection)
Selected AbstractsIn Vitro Selection of Self-Interacting Transmembrane Segments--Membrane Proteins Approached from a Different PerspectiveIUBMB LIFE, Issue 3 2002Dieter Langosch Abstract The principles underlying the folding of integral membrane proteins are uncovered in an increasingly detailed way. Experimental determination of high-resolution structures followed by analysis of packing reveal structural similarities as well as differences to soluble globular proteins. At the same time, protein/protein interactions at the level of membrane-embedded domains have been investigated for different model proteins. More recently, self-interacting transmembrane helices have been selected from combinatorial libraries in vitro to study the mechanistic basis of protein/protein interaction in membranes in a systematic way. With an emphasis on the latter approach, this review discusses insights emerging from an integrated view on the recent advances. [source] In vitro Selection for Fusarium Wilt Resistance in GladiolusJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 5 2008Idrees Ahmad Nasir Abstract Cormels pieces of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85,95% germination after breaking dormancy of 8 weeks at 4 °C. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6 cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum. [source] An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activitiesFEBS JOURNAL, Issue 11 2010Dazhi Jiang A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. We used an in vitro -selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNAzyme with dual catalytic activities. We prepared the 46-nucleotide DNAzyme with DNA-cleaving activity (PL DNAzyme), and then grafted the deoxyribonucleotide residues from an 8,17 variant DNAzyme into the region of stem,loop I and the catalytic core of the PL DNAzyme scaffold. This deoxyribonucleotide residue grafting resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving activities (DRc DNAzyme). Drc DNAzyme has properties different from those of the original PL DNAzyme, including DNA cleavage sites and the required metal ion concentration. Interestingly, the RNA substrate and RNase A can act as effectors to mediate the DNA cleavage. Our results show that RNA-cleaving and DNA-cleaving activities simultaneously coexist in DRc DNAzyme, and the DNA cleavage activity can be reversibly regulated by a conformational transition. [source] Engineering input/output nodes in prokaryotic regulatory circuitsFEMS MICROBIOLOGY REVIEWS, Issue 5 2010Aitor De Las Heras Abstract A large number of prokaryotic regulatory elements have been interfaced artificially with biological circuits that execute specific expression programs. Engineering such circuits involves the association of input/output components that perform discrete signal-transfer steps in an autonomous fashion while connected to the rest of the network with a defined topology. Each of these nodes includes a signal-recognition component for the detection of the relevant physicochemical or biological stimulus, a molecular device able to translate the signal-sensing event into a defined output and a genetic module capable of understanding such an output as an input for the next component of the circuit. The final outcome of the process can be recorded by means of a reporter product. This review addresses three such aspects of forward engineering of signal-responding genetic parts. We first recap natural and non-natural regulatory assets for designing gene expression in response to predetermined signals , chemical or otherwise. These include transcriptional regulators developed by in vitro evolution (or designed from scratch), and synthetic riboswitches derived from in vitro selection of aptamers. Then we examine recent progress on reporter genes, whose expression allows the quantification and parametrization of signal-responding circuits in their entirety. Finally, we critically examine recent work on other reporters that confer bacteria with gross organoleptic properties (e.g. distinct odour) and the interfacing of signal-sensing devices with determinants of community behaviour. [source] Heterotachy and Functional Shift in Protein EvolutionIUBMB LIFE, Issue 4-5 2003Hervé Philippe Abstract Study of structure/function relationships constitutes an important field of research, especially for modification of protein function and drug design. However, the fact that rational design (i.e. the modification of amino acid sequences by means of directed mutagenesis, based on knowledge of the three-dimensional structure) appears to be much less efficient than irrational design (i.e. random mutagenesis followed by in vitro selection) clearly indicates that we understand little about the relationships between primary sequence, three-dimensional structure and function. The use of evolutionary approaches and concepts will bring insights to this difficult question. The increasing availability of multigene family sequences that has resulted from genome projects has inspired the creation of novel in silico evolutionary methods to predict details of protein function in duplicated (paralogous) proteins. The underlying principle of all such approaches is to compare the evolutionary properties of homologous sequence positions in paralogs. It has been proposed that the positions that show switches in substitution rate over time--i.e., 'heterotachous sites'--are good indicators of functional divergence. However, it appears that heterotachy is a much more general process, since most variable sites of homologous proteins with no evidence of functional shift are heterotachous. Similarly, it appears that switches in substitution rate are as frequent when paralogous sequences are compared as when orthologous sequences are compared. Heterotachy, instead of being indicative of functional shift, may more generally reflect a less specific process related to the many intra- and inter-molecular interactions compatible with a range of more or less equally viable protein conformations. These interactions will lead to different constraints on the nature of the primary sequences, consistently with theories suggesting the non-independence of substitutions in proteins. However, a specific type of amino acid variation might constitute a good indicator of functional divergence: substitutions occurring at positions that are generally slowly evolving. Such substitutions at constrained sites are indeed much more frequent soon after gene duplication. The identification and analysis of these sites by complementing structural information with evolutionary data may represent a promising direction to future studies dealing with the functional characterization of an ever increasing number of multi-gene families identified by complete genome analysis. IUBMB Life, 55: 257-265, 2003 [source] In vitro selection and plant regeneration of copper-tolerant plants from leaf explants of Nicotiana tabacum L. cv. ,Xanthi'PLANT BREEDING, Issue 4 2007G. R. Rout Abstract Copper tolerance of Nicotiana tabacum L. var. Xanthi in vitro was achieved through plant regeneration from leaf explants on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/l BA, 0.1,0.25 mg/l IAA and 60 ,m Cu. Tolerant organogenic calli showed more vigorous growth in medium containing 60 ,m Cu than the non-tolerant calli. Standard growth parameters such as fresh and dry weight of organogenic callus, growth tolerance index (GTI), enzyme activity (peroxidase and catalase) and copper accumulation were used as indicators of copper tolerance. The activities of peroxidase and catalase as well as estimation of protein, total amino acid and chlorophyll were greater in tolerant calli than non-tolerant ones. The GTI in the 4 weeks after the beginning of treatments yielded significant differences among the tolerant and non-tolerant organogenic callus cultures. The accumulation of copper in the tolerant calli increased significantly with an increase in copper concentration in the medium. Shoot bud regeneration was achieved in both tolerant and non-tolerant organogenic calli on MS medium containing 0.5 mg/l BA and 0.1 mg/l IAA. The tolerant regenerated shoots were rooted on half-strength basal MS medium with 60 ,m Cu for selection of tolerant clones. This study may help in the selection and characterization of Cu-tolerant lines of N. tabacum cv. ,Xanthi' for building conservation strategies and also for phytoremediation programmes. [source] Design and synthesis of functional polymers by in vitro selectionPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 1-2 2004Yoshihiro Ito Abstract Combinatorial chemistry, in which targeting molecules are selected from a random molecular library, has recently developed. Among the methods devised by combinatorial chemistry, the recent progress in functional polymers that have molecular recognition ability and catalysis ability, and in bio-device polymers based on these functionalities is reviewed. Copyright © 2004 John Wiley & Sons, Ltd. [source] Inhibition of NF-,B activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMOPROTEIN SCIENCE, Issue 9 2007Emanuel Wyler Abstract The link between the NF-,B signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the I,B kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) for NEMO, and we developed NEMO peptides inhibiting NF-,B activation at the level of the IKK complex. To improve the low-affinity inhibitors, we used ribosome display to select small and stable proteins with high affinity against the individual CC2-LZ because the entire NEMO protein is poorly soluble. Several binders with affinities in the low nanomolar range were obtained. When expressed in human cells, some of the selected molecules, despite their partial degradation, inhibited TNF-,-mediated NF-,B activation while having no effect on the basal activity. Controls with a naive library member or null plasmid had no effect. Furthermore, we could show that this NF-,B inhibition occurs through a specific interaction between the binders and the endogenous NEMO, resulting in decreased IKK activation. These results indicate that in vitro selections with the NEMO subdomain alone as a target may be sufficient to lead to interesting compounds that are able to inhibit NF-,B activation. [source] | |||||||||||||