Patricia A. Ormonde

Recent work has shown that the murine BRCA2 tumor suppressor protein interacts with the murine RAD51 protein. This interaction suggests that BRCA2 participates in DNA repair. Residues 3196-3232 of the murine BRCA2 protein were shown to be involved in this interaction. Here, we report the detailed mapping of additional domains that are involved in interactions between the human homologs of these two proteins. Through yeast two-hybrid and biochemical assays, we demonstrate that the RAD51 protein interacts specifically with the eight evolutionarily conserved BRC motifs encoded in exon 11 of brca2 and with a similar motif found in a Caenorhabditis elegans hypothetical protein. Deletion analysis demonstrates that residues 98-339 of human RAD51 interact with the 59-residue minimal region that is conserved in all BRC motifs. These data suggest that the BRC repeats function to bind RAD51.

Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.

To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum, both in vivo and in vitro.

Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or beta-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA "laddering" in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.