The gap junction is an ubiquitous structure which permits exchanges of ions and small molecules between neighboring cells. It is involved in many important cellular processes, synchronizing heart beat and contraction of smooth muscle, maintaining homeostasis by metabolic coupling and permitting the exchanges of messages which somehow control growth, development and differentiation. Using two separate anti-gap junction antibodies we have isolated a cDNA clone which hybridizes with synthetic oligonucleotide probes directed against known sequence of the liver gap junction protein. We plan: 1. To prove definitely that we have a gap junction coding clone by sequencing and comparing the sequences to know features of the gap junction protein. 2. To deduce an amino acid sequence based on the nucleotide sequence obtained, and use it to determine possible configuration(s) for the molecule. 3. To synthesize peptides (8-15 amino acids long) which will in turn be used to generate antibodies against potentially important and accessible portions of the molecule. The arrangement of the gap junction molecule and the structure of connexons will be examined by using gold label immunocytochemistry at the electron microscopy level, and also immunochemical analyses on proteolytically and chemically cleaved gap junction protein. 4. To use the cDNA clone and antibodies in tests of the presence of the protein and/or message in different organs, and at different stages of development. The extent of homology between the mRNAs found to encode for gap junctions in different places will be determined. We will also study the distribution of the gap junction genes in different organisms. 5. To experimentally manipulate systems where gap junction expression and function can be altered at will in order to study putative changes in amounts and kinds of message expressed. 6. To carry out in vitro translation and other experiments to test for the existence of possible junction protein precursors and analyze the mode of assembly of junctional proteins into functional structures. Eventually, we will carry out manipulations of the sequence in attempts at altering function by alteration of known portions of the protein.
|Dermietzel, R; Yancey, S B; Traub, O et al. (1987) Major loss of the 28-kD protein of gap junction in proliferating hepatocytes. J Cell Biol 105:1925-34|