Abstract

The function of intermediate filaments composed of Endo A and B proteins will be investigated in differentiating cultures of murine embryonal carcinoma cells and mouse embryos. Plasmid DNAs designed to expresss an RNA complementary to the 5' end of Endo B mRNA will be introduced into embryonal carcinoma cells. The effect of the expression of the antisense RNA upon intermediate filament formation and visceral endoderm epithelium maturation will be assessed by immunofluorescence, immunoprecipitation, and nucleic acid hybridization techniques. The gene coding for Endo B will be cloned and characterized. DNase-hypersensitive sites of the chromatin state of the gene will be mapped and compared in embryonal carcinoma cells and their differentiated derivatives. Proteins which bind to specific sequences of the Endo B gene will be identified by DNA binding assays and chromatin footprinting experiments. Finally, the regulatory sequences of the Endo B gene will be identified by deletion analyses of recombinants designed to express the chloramphenicol acetyl transferase indicator gene. Genomic sequences will be tested for tissue-specific transcriptional enhancer activity.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA033946-06
Application #
3171693
Study Section
Molecular Biology Study Section (MBY)
Project Start
1982-06-01
Project End
1989-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
6
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
009214214
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

Kulesh, D A; Oshima, R G (1989) Complete structure of the gene for human keratin 18. Genomics 4:339-47
Kulesh, D A; Cecena, G; Darmon, Y M et al. (1989) Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8. Mol Cell Biol 9:1553-65
Oshima, R G (1988) Rapid identification of M13 phage deletions for sequence analysis. Biotechniques 6:510-1
Oshima, R G; Trevor, K; Shevinsky, L H et al. (1988) Identification of the gene coding for the Endo B murine cytokeratin and its methylated, stable inactive state in mouse nonepithelial cells. Genes Dev 2:505-16
Kulesh, D A; Oshima, R G (1988) Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells. Mol Cell Biol 8:1540-50
Trevor, K; Linney, E; Oshima, R G (1987) Suppression of endo B cytokeratin by its antisense RNA inhibits the normal coexpression of endo A cytokeratin. Proc Natl Acad Sci U S A 84:1040-4
Izhar, M; Siebert, P D; Oshima, R G et al. (1986) Trophoblastic differentiation of human teratocarcinoma cell line HT-H1. Dev Biol 116:510-8
Singer, P A; Trevor, K; Oshima, R G (1986) Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos. J Biol Chem 261:538-47
Howe, W E; Klier, F G; Oshima, R G (1986) Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament. J Histochem Cytochem 34:785-93
Oshima, R G; Millan, J L; Cecena, G (1986) Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to implantation. Differentiation 33:61-8

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