Background: the ability to engineer zinc finger proteins binding to a dna sequence of choice is essential for targeted genome editing to be possible. Experimental techniques and molecular docking have been successful ...
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Background: the ability to engineer zinc finger proteins binding to a dna sequence of choice is essential for targeted genome editing to be possible. Experimental techniques and molecular docking have been successful in predicting protein-dna interactions, however, they are highly time and resource intensive. Here, we present a novel algorithm designed for high throughput prediction of optimal zinc finger protein for 9 bp dna sequences of choice. In accordance withthe principles of information theory, a subset identified by using K-means clustering was used as a representative for the space of all possible 9 bp dna sequences. the modeling and simulation results assuming synergistic mode of binding obtained from this subset were used to train an ensemble micro neural network. Synergistic mode of binding is the closest to the dna-protein binding seen in nature, and gives much higher quality predictions, while the time and resources increase exponentially in the trade off. Our algorithm is inspired from an ensemble machine learning approach, and incorporates the predictions made by 100 parallel neural networks, each with a different hidden layer architecture designed to pick up different features from the training dataset to predict optimal zinc finger proteins for any 9 bp target dna. Results: the model gave an accuracy of an average 83% sequence identity for the testing dataset. the BLAST e-value are well within the statistical confidence interval of E-05 for 100% of the testing samples. the geometric mean and median value for the BLAST e-values were found to be 1.70E-12 and 7.00E-12 respectively. For final validation of approach, we compared our predictions against optimal ZFPs reported in literature for a set of experimentally studied dna sequences. the accuracy, as measured by the average string identity between our predictions and the optimal zinc finger protein reported in literature for a 9 bp dna target was found to be as high as 81% for dna targets with a co
Cloning of cdnas encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cdna libraries of Bothrops jararaca (South American snake), Trimeresurus ...
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Cloning of cdnas encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cdna libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cdna library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cdna clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cdna clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (similar to 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (similar to 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa), cdna clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T, gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. the present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily. (C) 1999 Elsevier Science B.V. All nights reserved.
Non-premelanosomal melanogenic compartments and their melanogenesis-controlling functions have been further elucidated. In addition to enzymatic and nonenzymatic controlling factors, we have also been exploring the ro...
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Non-premelanosomal melanogenic compartments and their melanogenesis-controlling functions have been further elucidated. In addition to enzymatic and nonenzymatic controlling factors, we have also been exploring the role of melanogenesis-related genes. Naturally occurring intrinsic melanogenic inhibitors, MW <6,000(alpha), 6,000-30,000(beta), and >30,000(gamma), having different modes of action, have been identified within melanoma cells. One of the alpha-type melanogenic inhibitors of isolated tyrosinase(Ty) nonsuppressive types, later identified as lactic acid, induces depigmentation of cultured B-16 cells by the reduction in Ty activity level due to the inhibition of its mRNA expression. the transfection of Ty cdna, rather than nuclear dna-binding master regulatory gene, can induce, within both Ty-deficient amelanotic melanoma cells and also within fibroblasts, melanin polymer formation. this multisequential step occurs not only by the induction of Ty synthesis but also by the induction of other regulatory proteins and factors such as dopachrome tautomerase, DHICA-oxidase, catalase, Ty-glycosylation in GERL, and Ty-transfer by coated vesicles to newly assigned melanogenic vacuoles in which not only eumelanin but also rather pronounced concomitant pheomelanin formation is seen. Investigation of melanin-producing vacuoles in transfected fibroblasts and reexamination of premelanosomes in pigment cells has revealed the following: 1) Melanosomes possess phagocytic ability;2) melanosomes receive tyrosinase and hydrolases via coated vesicles from GERL;3) melanosomes possess lysosome-associated membrane protein 1 (LAMP-1);4) amelanotic melanoma contains lysosome-like vacuoles with myelin figures that acquire typical premelanosome structure after Ty-cdna transfection. thus it is proposed that melanosomes are specialized lysosomes in pigment cells. Coated vesicles synthesize melanin monomers such as DHICA and some DHI, and have a monomer-stabilizing system. thus they can tr
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