Analysis of cytokeratin domains by cloning and expression of intact and deleted polypeptides in Escherichia coli.

Using recombination of an appropriate expression vector system (pINDU) with a complete cDNA encoding a basic (type II) cytokeratin, i.e. cytokeratin 8 (1) of Xenopus laevis, we transformed Escherichia coli cells to synthesize considerable amounts of an insoluble eukaryotic cytoskeletal protein. The cytokeratin was deposited in large 'inclusion bodies' in the bacterial cytoplasm but did not form detectable filamentous structures. However, when the E. coli-expressed cytokeratin was purified and combined in vitro with an authentic cytokeratin of the complementary, i.e. acidic (type I) subfamily, it formed typical intermediate-sized filaments (IFs). Using Bal31 deletion from either the 5' or the 3' end of the cDNA, series of polypeptides progressively deleted from the amino or the carboxy terminus were produced in E. coli and identified by monoclonal antibodies. These assays allowed the mapping of epitopes. The deletion polypeptides of cytokeratin 8 were further examined to localize the region(s) involved in the heterotypic binding of alpha-helices of type I cytokeratins, using an in vitro nitrocellulose blot binding assay. We show that a region of 37 amino acids located in the central portion of coil 2 of the alpha-helical rod domain is sufficient for the specific recognition of a radiolabelled type I cytokeratin, i.e. cytokeratin 18 (D) from rat liver. In addition, deletion polypeptides containing only coil 1 of the alpha-helical rod also bind strongly the complementary cytokeratin. This indicates that the capability of heterotypic recognition and complex formation is not restricted to a single signal sequence but is located in distant and independent alpha-helical domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the carboxy-terminal part of an acidic type I cytokeratin of molecular weight 51 000 from Xenopus laevis epidermis as predicted from the cDNA sequence

The DNA sequence of a clone from a cDNA library made from Xenopus laevis skin is described. This sequence represents the 3'-terminal end of an mRNA which codes for an epidermal cytokeratin polypeptide of mol. wt. 51 000 of the acidic (type I) subfamily as identified by hybridization-selection of mRNAs, followed by gel electrophoretic identification of the polypeptides synthesized by translation in vitro. The partial amino acid sequence of the amphibian cytokeratin shows strong similarity to type I cytoskeletal keratins from human (mol. wt. 50 000) and murine (mol. wt. 59 000) epidermis. In the non alpha-helical tail region the human and the non-mammalian (Xenopus) keratins are more similar to each other than to the murine protein, indicating that the former are equivalent cytokeratin polypeptides and belonging to a special subclass of type I keratin polypeptides devoid of glycine-rich regions in the carboxy-terminal portion. The evolutionary conservativity of the genes coding for cytokeratins is discussed.     

Cytokeratin domains involved in heterotypic complex formation determined by in-vitro binding assays

Cytokeratins are constituent proteins of intermediate filaments (IFs) that form heterotypic tetrameric IF subunits containing two polypeptide chains of each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the basic (type II). To locate the molecular domains involved in the formation of these heterotypic complexes, we have developed a binding assay in which total cellular or cytoskeletal polypeptides, or proteolytically prepared cytokeratin fragments, are separated by one-, or two-dimensional gel electrophoresis, blot-transferred on to nitrocellulose paper and probed with radio-iodinated purified cytokeratin polypeptides or fragments thereof, using buffers of various ionic strengths with or without 4 M-urea. Using these polypeptides in the binding assay, specific heterotypic binding was observed between complementary cytokeratin polypeptides of the two subfamilies (but not with other IF proteins) and between the corresponding alpha-helical rod domain fragments. Both rod coils 1 and 2 of the type II cytokeratin 8 bound to the rod (coils 1 and 2) fragment of type I cytokeratins, and this binding occurred at both low and high ionic strengths. The results obtained indicate that: (1) the binding between cytokeratin polypeptides of the complementary type is stronger and more selective than interactions of cytokeratins with other IF and non-IF proteins; (2) both the head and the tail portions of the proteins are not required for heterotypic complex formation; (3) the complementarity information located in the alpha-helical portions of the rod domain, and in short sequences immediately flanking them, is sufficient to discriminate between the two types of cytokeratins and to secure the formation of heterotypic cytokeratin complexes; (4) both coils 1 and 2 of the rod can contribute to this association; and (5) the formation of the heterotypic cytokeratin complex is not critically dependent upon ionic interactions. Our results are further compatible with the concept that the heterotypic binding takes place between cytokeratin homodimer coiled-coils.     

Amino acid sequence microheterogeneities of a type I cytokeratin of Mr 51,000 from Xenopus laevis epidermis

The complete cDNA-derived sequence of a type I cytokeratin (designated no. 3) from Xenopus laevis skin is described. The deduced protein has an Mr of 51,888 and consists of a glycine-rich head domain, a well-conserved alpha-helical region and a tail rich in hydroxyamino acid residues. Various cDNA clones encoding two different mRNAs were isolated that differed by short deletions/insertions and point mutations. These microheterogeneities are mainly located in a 'hypervariable region' at the C-terminal non-alpha-helical region.     

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)     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5''-upstream sequences.

Cytokeratins are a family of approximately 20 polypeptides which form the intermediate-sized filaments (IFs) characteristic of epithelial cells. They are synthesized co-ordinately as 'pairs' consisting of one representative from each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the more basic (type II) polypeptides, in cell type-specific combinations. We have isolated and characterized the genes coding for four bovine cytokeratins of the basic (type II) subfamily, i.e. cytokeratins Ib, III, IV and 6*, by Southern blot hybridization, hybridization-selection-translation experiments, hetero-duplex mapping, and partial sequencing of the exons coding for the hypervariable carboxy-terminal 'tail' regions of the proteins and the 3'-non-translated ends of the mRNAs which are distinct for the individual cytokeratin polypeptides. Limited 'chromosomal walk' experiments demonstrated that the genes are organized into two tandems, i.e. 6*----Ib and III----IV, in which they are separated by approximately 11 kb. RNA analysis by Northern and dot blots show that both genes of the III----IV tandem are co-expressed in some bovine tissues (muzzle epidermis, hoof pad and tongue mucosa) and cultured cells (BMGE + H) but that in other tissues, cornea for example, only the gene encoding III is expressed. Unexpectedly, the genes linked in the tandem 6*----Ib are not co-expressed in any of the tissues examined. mRNA from gene 6* has been found in tongue mucosa but in none of the other cell lines and tissues examined, whereas mRNA for cytokeratin Ib is expressed in cornea and muzzle epidermis but not in, for example, tongue mucosa and in the epidermis of the heel pad.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokeratin expression in simple epithelia

Cytokeratin A (no. 8) is a cytoskeletal protein (Mr, approximately 53,000 in bovine cells) which is typical of all simple epithelia, is widespread in all cultured epithelial cells, and together with its partner cytokeratin D, is the first cytokeratin expressed during embryogenesis (synonyms for this protein are Endo A and TROMA-1 antigen). We isolated a clone (pKB8(1] from a pUC8 cDNA library prepared from poly(A)+-RNA of bovine bladder urothelium which contains the 3' nontranslated portion and the sequence coding for the carboxyterminal tail and almost the whole of the alpha-helical rod (369 amino acids). Northern-blot analysis showed that the mRNA coding for this cytokeratin is specifically synthesized in various epithelial tissues and in epithelial cell culture lines. The amino acid sequence of this cytokeratin, when compared with the sequences of other intermediate filament (IF) proteins, exhibits a high and specific homology with other cytokeratins of the basic (type II) subfamily; this homology is, however, restricted to the rod portion. The tail region, which is rich in hydroxy-amino acids (approximately 35%), is unique among the type-II cytokeratins in that it does not exhibit subdivision in three domains, specifically lacking the glycine-rich middle domain. Sequence comparison with a partial sequence of the corresponding cytokeratin of the amphibian species, Xenopus laevis, indicated high evolutionary conservation. The high sequence homology of bovine cytokeratin A with published sequences of human tissue polypeptide antigen (TPA), a soluble serum component used as tumor marker in clinical oncology, supports the view that TPA is a proteolytically solubilized fragment containing the rod portion of human cytokeratin no. 8. Our analysis of clone pKB8(1) made possible the first comparison of a simple epithelial cytokeratin with epidermal keratins and other IF proteins. This showed that, in some important molecular features, cytokeratin A (no. 8) differs drastically from the epidermal members of the same cytokeratin subfamily, probably reflecting different cellular functions of the tail region in stratified and simple epithelia.     

Cytokeratin expression in simple epithelia

We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type II) subfamily, which is highly homologous to the corresponding bovine and amphibian (Xenopus laevis) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal 'tail' domain. Comparison with the reported amino acid sequences of two fragments of human 'tissue polypeptide antigen' (TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epithelium-derived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the 'head' portion. These cDNA clones and the RNA probes derived therefrom were used to detect specifically mRNAs by Northern-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.     

Molecular characterization and expression of the stratification-related cytokeratins 4 and 15

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.     

Changes in the Pattern of Cytokeratin Polypeptides in Epidermis and Hair Follicles During Skin Development in Human Fetuses

Abstract. The cytokeratin polypeptides of microdissected epidermis and hair follicles from human fetuses (from week 10 of pregnancy until birth) have been analysed by two-dimensional gel electrophoresis. Two-layered epidermis in 10-week fetuses contains major amounts of cytokeratin polypeptides typical of simple epithelia (components Nos. 8, 18, and 19 according to Moll et al. [31]). These cytokeratins are gradually reduced in their relative amounts and eventually disappear in the multilayered epidermis of later stages. At advanced stages of development, cytokeratins characteristic of adult epidermis are detected and finally predominate. These include the large and basic epidermal cytokeratin No. 1 (apparent molecular weight 68,000) which is already present in the three-layered epidermis of 13-week fetuses. Hair follicle germ cells of 13-week fetuses differ from fetal epidermal keratinocytes and show a very simple cytokeratin pattern, dominated by only two major polypeptides (Nos. 5 and 17). More developed hair follicles of 20-week fetuses have established a cytokeratin pattern similar to, but not identical with, that of hair follicles from adult skin. Different staining patterns obtained by indirect immunofluorescence microscopy using cytokeratin antibodies with different specificities suggest that, in three-layered epidermis, different cytokeratin patterns might exist in the specific cell layers. Such a differential location might explain the high complexity of polypeptide components found in fetal skin. Possible contributions of peridermal cytokeratins to this complex pattern of fetal epidermis are discussed.     

Cytokeratin expression in simple epithelia

To study the regulation of the expression of cytokeratins characteristic of simple epithelia, i.e., human cytokeratins nos. 7, 8, 18, and 19, we prepared several cDNA clones coding for these proteins and their bovine counterparts. In the present study, we describe a cDNA clone of the mRNA coding for human cytokeratin no. 18, which was isolated from an expression library using the monoclonal antibody, KG 8.13. This clone (756 nucleotides, excluding the polyA portion), encodes approximately one-half of the mRNA (approximately 1.4 kb), identifies one mRNA band in Northern-hybridization blots, and specifically selects one mRNA species coding for cytokeratin no. 18, as demonstrated by translation in vitro. Comparison of the deduced amino acid sequence--confirmed by direct amino-acid-sequence analyses of some polypeptide fragments produced by cleavage with cyanogen bromide--indicated that cytokeratin no. 18 is a member of the acidic (type I) subfamily of cytokeratins. It has only limited sequence homologies in common with other intermediate-sized filament proteins, and these are essentially restricted to certain domains of the alpha-helical rod portion. The carboxyterminal tail sequence does not contain glycine-rich elements, thus distinguishing this cytokeratin from those acidic (type I) cytokeratins that are characterized by this feature. The similarities and differences between cytokeratin no. 18 and previously described epidermal cytokeratins are discussed in relation to the differences in the stability of the complexes which this cytokeratin forms with basic (type II) cytokeratins, as well as in relation to possible functional differences of cytokeratins in simple and stratified epithelia.     

Cytokeratin expression in simple epithelia

Abstract. We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type 11) subfamily, which is highly homologous to the corresponding bovine and amphibian ( Xenopus laevis ) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal 'tail' domain. Comparison with the reported amino acid sequences of two fragments of human 'tissue polypeptide antigen'(TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epithelium-derived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the 'head' portion. These cDNA clones and the RN A probes derived therefrom were used to detect specifically mRNAs by Northern-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.     

Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides

Cytokeratins are expressed in different types of epithelial cells in certain combinations of polypeptides of the acidic (type I) and basic (type II) subfamilies, showing "expression pairs." We have examined in vitro the ability of purified and denatured cytokeratin polypeptides of human, bovine, and rat origin to form the characteristic heterotypic subunit complexes, as determined by various electrophoretic techniques and chemical cross-linking, and, subsequently, intermediate-sized filaments (IFs), as shown by electron microscopy. We have found that all of the diverse type I cytokeratin polypeptides examined can form complexes and IFs when allowed to react with equimolar amounts of any of the type II polypeptides. Examples of successful subunit complex and IF formation in vitro include combinations of polypeptides that have never been found to occur in the same cell type in vivo, such as between epidermal cytokeratins and those from simple epithelia, and also heterologous combinations between cytokeratins from different species. The reconstituted complexes and IFs show stability properties, as determined by gradual "melting" and reassociation, that are similar to those of comparable native combinations or characteristic for the specific new pair combination. The results show that cytokeratin complex and IF formation in vitro requires the pairing of one representative of each the type I and type II subfamilies into the heterotypic tetramer but that there is no structural incompatibility between any of the members of the two subfamilies. These findings suggest that the co-expression of specific pair combinations observed in vivo has other reasons than general structural requirements for IF formation and probably rather reflects the selection of certain regulatory programs of expression during cell differentiation. Moreover, the fact that certain cytokeratin polypeptide pairs that readily form complexes in vitro and coexist in the same cells in vivo nevertheless show preferential, if not exclusive, partner relationships in the living cell points to the importance of differences of stabilities among cytokeratin complexes and/or the existence of extracytokeratinous factors involved in the specific formation of certain cytokeratin pairs.     

Amino acid sequence and gene organization of cytokeratin no. 19, an exceptional tail-less intermediate filament protein.

We have isolated a cDNA clone from a bovine bladder urothelium library which encodes the smallest intermediate filament (IF) protein known, i.e. the simple epithelial cytokeratin (equivalent to human cytokeratin 19) previously thought to have mol. wt 40,000. This clone was then used to isolate the corresponding gene from which we have determined the complete nucleotide sequence and deduced the amino acid sequence of the encoded protein. This cytokeratin of 399 amino acids (mol. wt 43,893) is identified as a typical acidic (type I) cytokeratin but differs from all other IF proteins in that it does not show the carboxyterminal, non-alpha-helical tail domain. Instead it contains a 13 amino acids extension of the alpha-helical rod. The gene encoding cytokeratin 19 is also exceptional. It contains only five introns which occur in positions corresponding to intron positions in other IF protein genes. However, an intron which in all other IF proteins demarcates the region corresponding to the transition from the alpha-helical rod into the non-alpha-helical tail is missing in the cytokeratin 19 gene. Using in vitro reconstitution of purified cytokeratin 19 we show that it reacts like other type I cytokeratins in that it does not form, in the absence of a type II cytokeratin partner, typical IF. Instead it forms 40-90 nm rods of 10-11 nm diameter which appear to represent lateral associations of a number of cytokeratin molecules. Our results demonstrate that the non-alpha-helical tail domain is not an indispensable feature of IF proteins. The gene structure of this protein provides a remarkable case of a correlation of a change in protein conformation with an exon boundary.     

Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

Synthesis of cytokeratin 13, a component characteristic of internal stratified epithelia, is not induced in human epidermal tumors

Human cytokeratin 13 is one of the most abundant intermediate filament (IF) proteins of many internal stratified epithelia and occurs, at least in certain cell cultures, in an O-glycosylated form binding the lectin, wheat germ agglutinin (WGA). As other groups have reported that, in the mouse, the synthesis of mRNA encoding the 47-kDa cytokeratin corresponding to human cytokeratin 13 is induced in epidermal keratinocytes during malignant transformation, we have examined the synthesis of cytokeratin 13 mRNA and protein in human epidermis and epidermal tumors, using specific cDNA probes and cytokeratin 13 antibodies. We isolated two different cDNA clones from the vulvar carcinoma cell line A-431, in which this protein is abundant: One clone seems to represent the entire mRNA, whereas the other is only a minor component and encodes a truncated cytokeratin 13 lacking most of the carboxy-terminal tail domain, probably a product of alternative, "incorrect" splicing. Comparison of the amino acid sequences with those of other cytokeratins revealed a high degree of conservation with respect to several other human type I cytokeratins, notably cytokeratin 15, and to the murine 47-kDa cytokeratin. When human epidermis and a series of benign and malignant epidermal tumors were examined with these cDNA probes and cytokeratin-13-specific antibodies we did not find an induction of expression in keratinocytes, normal or malignantly transformed, except for some scattered, sparse cytokeratin-13-positive cells and very low levels of cytokeratin 13 mRNA, detectable only with the highly sensitive polymerase chain reaction (PCR). We conclude that the gene(s) encoding cytokeratin 13 are not induced in human keratinocytes during epidermal carcinogenesis, in apparent contrast to reports of murine epidermal tumors, and we discuss possible explanations for this interspecies difference.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.