The characterization and localization of inducible protein IP-25

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.Science, Faculty ofZoology, Department ofGraduat

The structure and expression of the metallothionein genes of the sea urchin Lytechinus pictus /

The structure and gene expression of a number of metallothionein (MT) genes of the sea urchin Lytechinus pictus were characterized. The MT genes in this species are members of a small gene family composed of 7 to 10 copies per haploid genome. A genomic library was constructed and 81 clones containing MT sequences were isolated of which 8 were analyzed. The complete sequence of one clone (LpMT1) and the promoter region of 7 others was determined. LpMT1 encodes a primary transcript of 3042 bp composed of 4 exons which is processed into a mRNA of 605 bases. The promoter regions of all the genes analyzed are highly conserved and contain multiple copies of the metal responsive enhancer element consensus sequence. L. pictus eggs injected with MT promoter fused to a CAT reporter gene, and cultured to various stages of development, directed transcription in a metal inducible fashion. The promoters also direct transcription in the sea urchin Strongylocentrotus purpuratus and in a human HeLa cell line. Deletion analysis indicates that the LpMT1 promoter requires regulatory elements other than the two metal responsive elements

Dicer is required for survival of differentiating neural crest cells

AbstractThe neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remain largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC-derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation