Abstract
A cDNA clone of a keratin-related, intermediate filament protein, designated Endo B, was constructed from size-fractionated parietal endodermal mRNA and characterized. The 1466-nucleotide cDNA insert contains an open reading frame of 1272 nucleotides that would result in 5' and 3' noncoding sequences of 54 and 60 nucleotides, respectively. The predicted amino acid composition, molecular weight (47,400), and peptide pattern correlate well with data obtained on the isolated protein. The predicted amino acid sequence fits easily into the general domain structure suggested for all intermediate filament proteins with a unique amino-terminal head domain, a large conserved central domain of predominantly alpha-helical structure, and a relatively unique carboxyl-terminal or tail domain. Over the entire molecule, Endo B is 43% identical with human 52-kDa epidermal type I keratin. However, over two of the three regions contained in the central domain that are predicted to form coiled-coil structures, the Endo B is 54-68% identical with other type I keratin sequences. This homology, along with the presence of the completely conserved sequence DNARLAADDFR-KYE, which is found in all type I keratins, permits the unambiguous identification of Endo B as a type I keratin. Comparison of the Endo B sequence to other intermediate filament proteins reveals 22 residues which are identical in all intermediate filament proteins regardless of whether filament formation requires only one type of protein subunit (vimentin, desmin, glial fibrillar acidic protein, or a neurofilament protein) or two dissimilar types (type I and type II keratins). Endo B mRNA was detectable in RNA isolated from F9 cells treated with retinoic acid for 48 h. Approximately three to five genes homologous to Endo B were detected in the mouse genome.
Highlights
Tains an open reading frame of 1272 nucleotides that mRNA andproteinbut can be induced to synthesize the would result in 5' and 3' noncoding sequences of 54 protein along with other extraembryonic endodermal funcand 60 nucleotides, respectively
This homology, along with the Here we report the preparation and characterization of a presence of the completely conserved sequence DNAR- cDNAclone for Endo B, which includes all of the 3' noncoding
Primer extension analysis of the cDNA indicates that the cDNA extends to within a t least 10 nucleotides of the 5' end of the mRNA sequence? The digestion of Endo €3 protein with N-chlorosuccinimide was performed as described previously.' Restriction enzyme fragments of the cDNA resulting from digestion with EcoRI and HindIII or with EcoRI alone were cloned into M13 mp9, mplO,mpll, or mp19 phages
Summary
Lated from F9 cells treated with retinoicacid for 48 h. Hybridization-selection of RNA by plasmid DNA was performed essentially as described [27].Approximately 15pg ofBamHI-digested plasmid DNAwas denatured by boiling and incubation in 0.4 M NaOH. Individual DNAs representing recombinantswith insert sizes of approximately 500 bp or greater were labeled by nick translation and hybridized to replicate strips of nitrocellulose containing gel-fractionated, polyadenylated mRNA from HR9 endodermal cells and F9.22 embryonal carcinoma cells. The digestion of Endo €3 protein with N-chlorosuccinimide was performed as described previously.' Restriction enzyme fragments of the cDNA resulting from digestion with EcoRI and HindIII or with EcoRI alone were cloned into M13 mp, mplO,mpll, or mp phages. Genomic DNAwas digested with restriction enzyme (1-5 unitslpg) overnight in the buffers recommended by the manufacturer and was analyzed by the method of Southern [48] as described [22] except that theprehybridization and hybridization solutions contained 50% formamide and were performed a t 42 'C. After hybridization for 18 h, the filters were washed twice in 2 X SSC (SSC, standard saline citrate is 0.15 M NaCI, 0.015 M sodium citrate, pH 7), 0.1% SDS at room temperature, and twice in 0.1 X SSC, 0.1% SDS at 55 "C for 30 min
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