Cytoskeletal architecture is a predominant and characteristic feature of eukaryotic cell identity and function. The goal of this project is to study the activity of genes that coordinate cytoskeletal organization in cultured cells and amphibian embryos in vivo using recombinant DNA vectors including anti-sense plasmids. Anti-sense gene inhibition is a valuable molecular strategy for phenocopying gene mutations in vertebrate organisms that are not amenable to classical genetic analysis. Studies on the mechanism of DNA mediated """"""""flipped"""""""" gene anti-sense inhibition will continue. Anti-sense chicken thymidine kinase intron and anti-sense intron-exon junction plasmids will be constructed and tested by comicroinjection with the wild type gene into cultured cell nuclei and Xenopus oocyte germinal vesicles. Gene product activity and target mRNA transcript metabolism will be carefully monitored. Related strategies for gene replacement will be explored. Anti-sense and sense plasmids containing several cytoskeleton-associated protein genes (tropomyosins, yield-actinin, and a 200 kd microtubule-associated protein) will be constructed and introduced into cultured cells and Xenopus embryos. The resultant deficiencies in cellular architecture will be studied to assess the contribution that each gene makes to cellular morphology, viral transformation, and embryonic morphogenesis. Xenopus cDNA library inserts, enriched in embryo-specific sequences, will be subcloned into anti-sense expression vectors and then introduced into Xenopus embryos to identify novel genes that direct embryonic pattern formation. The objective is to use anti-sense gene inhibition as a pseudo-genetic tool to identify previously uncharacterized factors that regulated cell organization in addition to using these molecular approaches for the analysis of cloned gene function in living cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037083-05
Application #
3292056
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1986-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1992-06-30
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520