Worm Breeder's Gazette 13(4): 61 (October 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||MGH Cancer Center and Dept. of Pediatrics, Boston, MA.|
|2||MGH Cancer Center John Fleming MGH Cancer Center and Dept. of Pediatrics, Boston, MA.|
The loss of function of the human Neurofibromatosis 2 (NF2) gene predisposes for nervous system tumors. NF2 has structural similarity to the moezin-ezrin-radixin family of cytoskeletal membrane linker proteins which are thought to connect membrane receptors to the cytoskeleton. NF2 thus belongs to a new class of tumor suppressor genes directly involving members of the cytoskeleton. We are interested in studying how the cytoskeleton may contribute to growth regulation. Our hypothesis is that members of the above mentioned class of proteins are necessary to transduce negative growth regulating signals induced by cell-to-cell contact known as "contact inhibition". Using a combination of degenerate PCR and low homology screening based on conserved regions in the human NF2 -moezin-ezrin-radixingene family we have isolated and sequenced full length c-DNA clones of the C. elegans homologs of human NF2 and radixin. In spite of extensive screening we have not obtained other family members and we thus assume that these genes represent the complete gene family in C. elegans. For radixin we have also isolated genomic clones and are currently in the process of finishing the genomic sequence. The NF2 genomic sequence was kindly provided by LaDeana Hillier/Richard Wilson for the genome sequencing project. The general structure of the C. elegans proteins preserves the structure of their human counterparts with a highly conserved N-terminal half (70 to 75% homology between human/human, human/C. elegans and C. elegans/C. elegans members) and a non conserved C-terminal half. The N-terminus is predicted to be directed towards the membrane and to provide the link to transmembrane proteins whereas the C-terminal half provides the connection to actin and/or other cytoskeletal proteins. Humradixin 208 EYLKIAQDLEMYG VNY FEI KNKK 229 Ceradixin EYLKIAQDLEMYG VNY FEI RNKK C eNF2 EYLRVAQDLEMYG I L Y YPI QNKK Hu mNF2 EYLKIAQDLEMYG VNY FAI RNKK The genomic structure of NF2 preserves most of the intron/exon junctions of the human NF2 gene, but is quite distinct from that of radixin (the human genomic structure of radixin is not known). Using a peptide antibody raised against a conserved part of the human gene family and also conserved in the C. elegans homologs (kindly provided by Frank Solomon, MIT), we have obtained staining not seen with control antibodies. We are in the process of confirming these preliminary data using different fixation- and staining protocols (thanks to Ralf Baumeister and Bob Waterston). We hope that by using confocal microscopy we may also be able to get some information on the subcellular localization. Additionally, we are generating promotor-GFP constructs to obtain more expression data and to be able to differentiate between the antibody staining patterns of the two proteins. The map position of radixin (fingerprinting by Alan Coulson, Cambridge) is close to spe-11 on chromosome I, NF2 is located in proximity to daf-4 on chromosome III. Ann Rose has performed an extensive lethal screen around the location of radixin (about 120 different lethals are consistent with its location). Hoping (?!) that a radixin loss-of-function mutant will be lethal (vertebrate radixin has been shown to cap the barbed ends of actin), Colin Thacker has started to try to obtain rescue of the lethal strains generated from this area with cosmids that cover the gene. Based on the detailed protocols and generous help of Joel Rothman, Susan Mango and Ed Maryon we have generated a TC1 transposon library with the mutator strain MT 3126 and have isolated transposon insertions in NF2 as well as in radixin. We are in the process of sib selection to obtain pure strains in order to generate loss-of-function mutants. Finally, we have also constructed antisense vectors with both genes under heat shock promotor control to be able to differentially delete one or both genes at different stages of development. There is some evidence in mammalian tissue culture systems that NF2 may suppress ras induced transformation (Maruta, pers. comm.). To test this observation in C. elegans we plan to inject NF2 under control of the let-60 promotor to see if we can suppress the let-60 gain-of-function Muv phenotype.