Cloning and characterization of keratin D,a murine endodermal cytoskeletal protein induced during in vitro differentiation of F9 teratocarcinoma cells

Summary We have identified a cDNA coding for the murine keratin D from a collection of clones representing F9 teratocarcinoma stem cell mRNA sequences. These sequences are synthesized specifically after the addition of retinoic acid and cAMP to the culture medium. The clone is 1,382 nucleotides long and contains the entire information for the active polypeptide, the complete 3 end and most, if not all, of the 5 non-coding region. The mRNA is found in hepatocytes, in PYS-2 cells (an endodermal cell line) and in differentiated (retinoic-acid-treated) F9 cells, but not in untreated F9 cells. The length of the mRNA is 1.4 kb, as estimated by Northern blot hybridization. Southern hybridization performed under very stringent conditions detects a single fragment hybridizing strongly with the cloned cDNA, suggesting that the mouse genome contains only one or very few copies of this gene. We present the first complete sequence of a keratin expressed in simple epithelia, i.e. keratin D, and discuss its structural features.     

Cytokeratin No. 9, an epidermal type I keratin characteristic of a special program of keratinocyte differentiation displaying body site specificity

Plantar epidermis of the bovine heel pad as well as human plantar and palmar epidermis contain large amounts of an acidic (type I) keratin polypeptide (No. 9) of Mr 64,000 which so far has not been found in epidermis of other sites of the body. We present evidence for the keratinous nature of this protein, including its ability to form cytokeratin complexes and intermediate-sized filaments in vitro. We have isolated RNA from plantar epidermis of both species and show, using translation in vitro, that these polypeptides are genuine products of distinct mRNAs. Using immunofluorescence microscopy with specific antibodies against this protein, we demonstrate its location in most cells of suprabasal layers of plantar epidermis as well as in sparse keratinocytes which occur, individually or in small clusters, in upper layers of epidermis of other body locations. We conclude that cytokeratin No. 9 is characteristic of a special program of keratinocyte differentiation which during morphogenesis is expressed in most epidermal keratinocytes of soles and palms but only in a few keratinocytes at other body sites. This example of cell type-specific expression of a member of a multigene family in relation to a body site-related program of tissue differentiation raises important biological questions concerning the regulation of keratinocyte differentiation and morphogenesis as well as the function of such topological heterogeneity within a given type of tissue.     

Identification of two types of keratin polypeptides within the acidic cytokeratin subfamily I

Cytoskeletal filaments of the α-keratin type (cytokeratins) are a characteristic of epithelial cells. In diverse mammals (man, cow and rodents) these cytokeratins consist of a family of approximately 20 polypeptides, which may be divided into the more acidic (I) and the more basic (II) subfamilies. These two subfamilies show only limited amino acid sequence homology. In contrast, nucleic acid hybridization experiments and peptide maps have been interpreted to show that polypeptides of the same subfamily share extended sequence homology.We compare two polypeptides of the acidic cytokeratin subfamily, VIb (M_r 54,000) and VII (M_r 50,000), which are co-expressed in large amounts in bovine epidermal keratinocytes. These two epidermal keratins can be distinguished by specific antibodies and show different patterns of expression among several bovine tissues and cultured cells. In addition, they differ in the stability of their complexes with basic keratin polypeptides and in their tryptic peptide maps. The amino acid sequences deduced from the nucleotide sequences of complementary DNA clones containing the 3′ ends of the messenger RNAs for these keratins are compared with each other and with available amino acid sequences of human, murine and amphibian epidermal keratins. Bovine keratins VIb and VII share considerable sequence homology in the α-helical portion (68% residues identical) but lack significant homology in the extrahelical portion. Bovine keratin VIb shows, in its α-helical region, a pronounced sequence homology (88% identity) to the murine epidermal keratin of M_r 59,000. In addition, the non-helical carboxy-terminal regions of both proteins are glycinerich and contain a canonic sequence GGG^S_GYGG, which may be repeated several times. Moreover, their mRNAs present a highly conserved stretch of 236 nucleotides containing, in the murine sequence, the end of the coding and all of the non-coding region (81% identical nucleotides). Bovine keratin VII is considerably different from the murine M_r 59,000 keratin but is almost identical to the human cytokeratin number 14 of M_r 50,000, both in the α-helical and in the non-α-helical regions of the proteins, and the mRNAs of the human and the bovine keratins also display a high homology in their 3′ non-coding ends.The results show that in the same species keratins of the same subfamily can differ considerably, whereas equivalent keratin polypeptides of different species are readily identified by characteristic sequence homologies in the α-helical and the non-helical regions as well as in the 3′ non-coding portions of their mRNAs. Among the members of the acidic subfamily I of cytokeratin polypeptides that are co-expressed in bovine epidermis, at least two types can be distinguished by their carboxy-terminal sequences. One type is characterized by its abundance of glycine residues, a consensus GGG^S_GYGG heptapeptide sequence, which may be repeated several times, and an extended stretch of high RNA sequence homology in the 3′ non-coding part. The other type shows a predominance of serine and valine residues, a subterminal GGG^S_GYGG sequence (which has been maintained in Xenopus, cow and man) and also a high level of homology in the 3′ non-coding part of the mRNA. The data indicate that individual keratin type specificity overrides species diversity, both at the protein and the mRNA level. We discuss the evolutionary conservation and the tissue distribution of these two types of acidic keratin polypeptides as well as their possible biological functions.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

Amino acid sequence diversity between bovine epidermal cytokeratin polypeptides of the basic (type II) subfamily as determined from cDNA clones

The nucleotide sequences of four cDNA clones, each representing the carboxyterminal portion of a bovine epidermal cytokeratin of the "basic" (type II) subfamily, were determined, i.e., components Ia (Mr 68,000), Ib (Mr 68,000), III (Mr 60,000), and IV (Mr 59,000). The comparison of the sequences with each other and with the human type-II cytokeratin of Mr 56,000 reported by Hanukoglu and Fuchs [24] allows the following conclusions: The four major epidermal keratins of the basic (type II) subfamily, which are co-expressed in keratinocytes of the bovine muzzle, exhibit a high homology (greater than 90%) in the alpha-helical portion, but differ considerably in their nonhelical carboxy-terminal regions. The nonhelical carboxyterminal regions of all four cytokeratins are exceptionally rich in glycine and serine. Within the extrahelical tail, three different domains can be distinguished. The consensus sequence TYR(X)LLEGE which demarcates the end of the alpha-helical rod in all intermediate filaments is followed by a relatively short (22-27 amino acids) intercept rich in hydroxy amino acids and valine (carboxyterminal tail domain C1). This is followed by a long region that is variable in size and sequence, rich in glycine di-, tri-, and tetrapeptides, and contains diverse repeated sequences (domain C2). This is followed by another short (20 residues) hydroxy-amino-acid-rich intercept (domain C3) that ends with a conspicuously basic sequence of approximately four to six carboxyterminal amino acids. The first half of domain C1 is also homologous in all four keratins, suggesting that this region also assumes a common conformation and/or serves a special common function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.