Analysis of development using reverse genetics: Studies with the UDP glucose pyrophosphorylase and a humanrac protein kinase homolog in Dictyostelium discoideum.
Item
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Title
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Analysis of development using reverse genetics: Studies with the UDP glucose pyrophosphorylase and a humanrac protein kinase homolog in Dictyostelium discoideum.
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Identifier
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AAI9432364
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identifier
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9432364
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Creator
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Moon, Byoung Chon.
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Contributor
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Adviser: Robert P. Dottin
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Genetics | Biology, Molecular
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Abstract
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Using reverse genetics, the functions of the UDP glucose pyrophosphorylase (UDPGP) and a human rac protein kinase homolog gene have been characterized in development of Dictyostelium.;The enzyme UDP glucose pyrophosphorylase plays a central role in carbohydrate metabolism in many species. To analyze its function, we have created a null mutation by homologous recombination. The mutants grow normally but with no activity and develop with no obvious morphological defect. Nevertheless, enzyme activity appears at 15 hr into development. The disruptants have a slightly lower level of cellulose during culmination but the same as wild type organisms at the end development. Therefore, a second unknown UDPGP gene is expressed in late development. We cloned part of the second gene, UDPGP2, by taking advantage of the null mutant cell lines and using a novel polymerase chain reaction (PCR) strategy. The deduced amino acid sequence of part of UDPGP2 has significant homology with the UDPGP1 of Dictyostelium (46%) and slightly greater (51%) homology with the UDPGP enzyme of potato tuber. In contrast with UDPGP1, UDPGP2 mRNA accumulates only late in development, suggesting its expression may be regulated by different signal transduction pathways.;We also isolated a serine/threonine protein kinase, DdK6 which has a pleckstrin homology (PH) domain which might be involved in signal transduction and promote protein-protein aggregation. The protein kinase is closely related to the Rac protein kinase family. A reverse genetic approach was also used to reveal the role of DdK6 gene. Null mutant cell lines were created by gene targeting through homologous recombination. The null mutants caused heterochronic development delaying formation of mounds and fruiting bodies. The expression of several stage specific genes also was retarded in the mutants. Cyclic AMP pulsing experiments suggest that the null mutant is deranged in a signalling pathway(s) via the cell surface cAMP receptor(s). Expression of the DdK6 gene in wild type cells is strictly regulated at early stages. The protein is localized in the nucleus. These data show that the DdK6 protein kinase might establish an early developmental program, through transient protein aggregation perhaps by the pleckstrin homology domain.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
