Intermediate filament formation after transfection with modified hamster vimentin and desmin genes.
Previously we cloned and characterized the hamster intermediate filament genes coding for vimentin and desmin. It was demonstrated that the cloned desmin gene was expressed after gene transfer and that the newly synthesized protein assembles into intermediate filaments. Here we present data on the transfection of modified vimentin and desmin genes onto simian virus 40-transformed hamster lens cells and HeLa cells. Modifications included: (1) removal of exons encoding the desmin COOH-terminal domain; (2) exchange of exons encoding the COOH-terminal domain of vimentin and desmin; and (3) deletion of part of exon I of desmin, coding for the NH2-terminal amino acids 4-148. In transient transfection assays it was shown that the modifications in the COOH region had no detectable effects on the filament forming potential of the encoded proteins as demonstrated with desmin antibodies in the indirect immunofluorescence test. On the other hand, deletion of a considerable part of the first exon of the desmin gene results in a lack of bona fide intermediate filament formation. Immunoblotting with desmin antibodies of cell populations enriched for the transfected modified genes showed that the presence of the modified genes results in the synthesis of the corresponding proteins with the expected molecular weights. From our results we conclude that in vivo: (1) the presence of the COOH terminus is not essential for filament formation; (2) that an exchange of COOH-terminal parts of vimentin and desmin does not prevent assembly into intermediate filaments; and (3) that removal of the NH2 terminus of desmin affects intermediate filament formation.
van den Heuvel
R M
RM
Department of Biochemistry, University of Nijmegen, The Netherlands.
van Eys
G J
GJ
Ramaekers
F C
FC
Quax
W J
WJ
Vree Egberts
W T
WT
Schaart
G
G
Cuypers
H T
HT
Bloemendal
H
H
D000818:Animals; D006224:Cricetinae; D003599:Cytoskeleton / Q000502:physiology*; D003893:Desmin / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D007382:Intermediate Filaments / Q000502:physiology*; D014162:Transfection; D014746:Vimentin / Q000235:genetics*
D003893:Desmin; D014746:Vimentin