ABSTRACT
The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.
Subject(s)
Dictyostelium/genetics , Open Reading Frames , Repetitive Sequences, Nucleic Acid , Retroelements , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Codon , DNA Primers , DNA, Complementary , Databases as Topic , Genes, gag , Genes, pol , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Restriction Mapping , Retroelements/genetics , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
In the epidermis the autoantigen BP230 is a component of the hemidesmosomal plaque. We have developed a procedure for the isolation of BP230 from bovine tongue mucosa using chromatographic means. The identity of the isolated protein was confirmed by its recognition by bullous pemphigoid autoantibodies. A monoclonal antibody (MoAb230), generated against the purified protein, localizes to the region of the plaque of the hemidesmosome with which keratin bundles interact. Furthermore, the tissue distribution of BP230, assessed using MoAb230, suggests that BP230 or an immunologically related protein is a component of all hemidesmosomes. Ultrastructural analyses of the BP230 preparation reveal that the BP230 molecules assemble into macromolecular aggregates. The few images of individual intact molecules that we have observed in platinum replicas of rotary shadowed BP230 preparations suggest that BP230 is an elongate rod-shaped molecule. This is consistent with predictions based on the primary sequence of BP230 deduced from BP230 cDNAs reported by others. We discuss our results in relation to the potential function of BP230. Isolation of BP230 should now allow more rigorous biochemical analyses of potential protein-protein interactions of BP230 in the hemidesmosome.
Subject(s)
Autoantigens/isolation & purification , Carrier Proteins , Collagen , Cytoskeletal Proteins , Nerve Tissue Proteins , Non-Fibrillar Collagens , Tongue/chemistry , Animals , Antibodies, Monoclonal , Blotting, Western , Cattle , Desmosomes/chemistry , Desmosomes/immunology , Desmosomes/ultrastructure , Dystonin , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Immunohistochemistry , Mucous Membrane/chemistry , Mucous Membrane/immunology , Pemphigoid, Bullous/immunology , Tissue Distribution , Tongue/immunology , Collagen Type XVIIABSTRACT
Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity-purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.
