Molecular cloning of chick cardiac muscle tensin. Full-length cDNA sequence, expression, and characterization.
Here we describe the molecular cloning of 7.1-kilobase cDNA encoding chick cardiac muscle tensin. It contains an open reading frame of 1,744 amino acid (aa) residues. Sequence analysis reveals that, in addition to the previously noted SH2 domain (Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q., and Chen, L. B. (1991) Science 252, 712-715), tensin contains virtually all of the known sequence (362 aa) of insertin, an actin-capping protein that allows actin monomer to be "inserted" (Schroer, E., and Wegner, A. (1985) Eur. J. Biochem. 153, 515-520). Moreover, tensin shares partial homology with actin (46.7% identity in 30 aa), beta-spectrin's actin-binding consensus (40% identity in 26 aa), BCR (40% identity in 25 aa), catenin alpha (35% identity in 45 aa), synapsin Ia (25.6% identity in 156 aa), IL-3 receptor (20.2% identity in 384 aa), and IL-2/EPO receptors (14% identity in 20 aa). Recombinant full-length tensin, tagged with an influenza-derived epitope, was over-expressed by a baculovirus system and purified to apparent homogeneity. It migrates as a 200-kDa protein in SDS-polyacrylamide gel electrophoresis, similar to the native tensin. The structure of the tensin molecule has been characterized by light scattering, electron microscopy, and gel filtration. Nine monoclonal antibodies recognizing different regions of tensin have been prepared and characterized. The epitope-tagged recombinant tensin gene was subcloned into a pRcCMV vector and transfected into NIH 3T3 cells. Immunofluorescence stainings with monoclonal antibodies specific for chick tensin (not cross-reactive with mouse tensin) showed that the expressed protein is indeed localized at focal contacts, as that of native tensin.
Lo
S H
SH
Dana-Farber Cancer Institute, Boston, Massachusetts.
An
Q
Q
Bao
S
S
Wong
W K
WK
Liu
Y
Y
Janmey
P A
PA
Hartwig
J H
JH
Chen
L B
LB
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002642:Chick Embryo; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D000070876:Tensins
D018076:DNA, Complementary; D008840:Microfilament Proteins; D011994:Recombinant Proteins; D000070876:Tensins