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The characterization and localization of inducible protein IP-25

University of British Columbia

An in-depth study was undertaken to biochemically characterize and localize within the chromatin the inducible protein IP-25. This protein is found in Friend-virus-transformed erythroleukemia cells which have been induced to differentiate into orthochramic erythroblasts. It was found that IP-25 is one of a very limited number of proteins which appear de novo after the induction of Friend cells with dimethyl sulfoxide as an inducer. An apparent molecular weight of approximately 20,000 daltons was obtained for IP-25 by SDS polyacrylamide gel electrophoresis. Acid-urea gel electrophoresis indicated that IP-25 is a basic protein which migrates in a manner similar to histones H1⁰ or H5 in this gel system. Migration behaviour in urea gradient - TX-100 gels also indicated that this protein was similar to histone H5. Amino acid analysis revealed a similarity in amino acid composition between IP-25 and both histones H1 and H5. However the homology between IP-25 and these histones was not enough to enable a classification into either group. Peptide mapping of IP-25 and the H1b histone of Friend cells indicated that fragments of similar molecular weights are generated when digestion is carried out using chymotrypsin or papain. Chymotrypsin did not generate enough Fragments, while digestion with papain produced too many non-specific cleavages to make a definitive comparison between these two proteins. Localization of IP-25 within the chromatin repeat unit was accomplished by micrococcal nuclease digestion of intact Friend cell nuclei. The protein composition of the nuclease generated fragments was analyzed by polyacrylamide gel electrophoresis. One dimensional analysis of nucleosomal proteins indicated that IP-25 maintained a constant quantitative relationship with H1 histones in different sized chromatin fragments. This suggests that localization of IP-25 parallels the inter-nucleosomal location of histone H1. In order to obtain more direct evidence for the localization of IP-25, two-dimensional electrophoresis was performed. These experiments verified the one-dimensional electro-phoretic evidence. The presence of IP-25 in dimethyl sulfoxide induced Friend cells did not drastically alter the nuclease generated repeat lengths. The function of IP-25 during Friend cell erythropoiesis could not be determined by the data obtained in this thesis. However, it appears that the chromosomal accumulation of IP-25 and erythropoiesis in these cell cultures are linked events.

Text

2010-03-25

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http://hdl.handle.net/2429/22492

Zoology

University of British Columbia

Graduate