Differential effect of subcellular localization of communication impairing gap junction protein connexin43 on tumor cell growth in vivo.

There is a large body of evidence suggesting the connexin gap junction proteins appear to act as tumor suppressors, and their tumor inhibitory effect is usually attributed to their main function of cell coupling through gap junctions. However, some cancer cells (e.g. the rat bladder carcinoma BC31 cell line) are cell-cell communication proficient. Using specific site-directed mutagenesis in the third membrane-spanning (3M) domain of connexin43 (Cx43), we abolished the intrinsic gap junction intercellular communication (GJIC) in BC31 cells either by closing the gap junctional channels or by disruption of the transport of connexin complexes to the lateral membrane. Clones of BC31 cells transfected with a dominant negative Cx43 mutant giving rise to gap junctional channels, permeable only for a small tracer (neurobiotin), displayed accelerated growth rate in vivo, showing the critical role of selective gap junctional permeability in the regulation of cell growth in vivo. The use of other dominant-negative mutants of Cx43 also suggested that the effect of impaired communication on the tumorigenicity of cancer cells depends on the subcellular location of connexin. Inhibition of intrinsic GJIC in BC31 cells by sequestering of Cx protein inside the cytoplasm, due to expression of dominant-negative transport-deficient Cx43 mutants, did not significantly enhance the growth of transfectants in nude mice, but occasionally slightly retarded it. In contrast, augmentation of GJIC in BC31 cells by forced expression of wild-type Cx43, or a communication-silent mutant, fully suppressed tumorigenicity of these cells. Overall, these results show that cell coupling is a strong, but not the sole, mechanism by which Cx suppresses growth of tumorigenic cells in vivo; a GJIC-independent activity of Cx proteins should be considered as another strong tumor-suppressive factor.

Mechanism of cell-cell adhesion complex assembly.

Cell-cell adhesion complexes play an important role in the organization and behavior of cells in tissues. An important step in the formation of such complexes is the clustering of the adhesion receptors; this is critical for proper adhesion, for anchorage of the cytoskeleton to the plasma membrane, and for generation of different intracellular signals. Recent advances reveal that several interconnected mechanisms are responsible for clustering of the different adhesion receptors.

Adhesive but not lateral E-cadherin complexes require calcium and catenins for their formation.

We examined intercadherin interactions in epithelial A-431 cells producing endogenous E-cadherin and recombinant forms of E-cadherin tagged either by myc or by flag epitopes. Three distinct E-cadherin complexes were found. The first is a conventional E-cadherin-catenin complex consisting of one E-cadherin molecule linked either to beta-catenin/alpha-catenin or to plakoglobin/alpha-catenin dimers. The second is a lateral E-cadherin complex incorporating two E-cadherin- catenin conventional complexes combined in parallel fashion via dimerization of the NH2-terminal extracellular domain of E-cadherin. The third complex is likely to contain two E-cadherin-catenin conventional complexes derived from two opposing cells and arranged in an antiparallel fashion. Formation of the antiparallel but not lateral complex strictly depends on extracellular calcium and E-cadherin binding to catenins. Double amino acid substitution Trp156Ala/Val157Gly within the extracellular NH2-terminal E-cadherin domain completely abolished both lateral and antiparallel inter-E-cadherin association. These data support an idea that the antiparallel complex has the adhesion function. Furthermore, they allow us to suggest that antiparallel complexes derive from lateral dimers and this complex process requires catenins and calcium ions.

Molecular organization of the desmoglein-plakoglobin complex.

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1-3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell-cell adhesion.

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell-cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell-cell adhesion.

Cadherin binding sites of plakoglobin: localization, specificity and role in targeting to adhering junctions.

Plakoglobin directly interacts with cadherins and plays an essential role in the assembly of adherens junctions and desmosomes. Recently we have reported that multiple cadherin binding sites are localized along the arm repeat region of plakoglobin. To demonstrate functionally and specificity of these sites in vivo we constructed a set of chimeric proteins containing a plakoglobin sequence fused with the transmembrane vesicular protein synaptophysin. Plakoglobin fused upstream or downstream from synaptophysin (PgSy and SyPg, chimeras, respectively) is exposed on the cytoplasmic surface of synaptic-like vesicles and is able to associate with E-cadherin, and with two desmosomal cadherins, desmoglein and desmocollin. Moreover, plakoglobin targets these vesicles to cell-cell junctions. Insertion of synaptophysin within plakoglobin (PSyG chimeras) can interfere with cadherin binding of the resulting chimeric proteins, dependent on the position of the insertion. Insertion of synaptophysin in the first three arm repeats selectively inactivates plakoglobin binding to desmoglein and desmocollin. An insertion of synaptophysin within the next two repeats inactivates E-cadherin and desmocollin binding but not desmoglein binding. This localization of the desmoglein and E-cadherin binding sites was further confirmed by replacement of plakoglobin arm repeats with the corresponding sequence derived from the plakoglobin homologue, beta-catenin, and by deletion mutagenesis. Insertion of synaptophysin in most sites within arm repeats 6-13 does not change plakoglobin binding to cadherins. It does, however, strongly inhibit association of the resulting vesicles either with desmosomes and adherens junctions or with desmosomes only. Using in vitro binding assays we demonstrate that arm repeats 6-13 contain two cryptic cadherin binding sites that are masked in the intact protein. These observations suggest that the arm repeat region of plakoglobin is comprises two functionally distinct regions: the 1/5 region containing desmoglein and E-cadherin specific binding sites and the 6/13 region implicated in targeting of plakoglobin/cadherin complexes into junctional structures.

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential.

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C-domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon-gamma. Analysis of gamma-interferon activation sites.

The acid cytokeratin K17 is inducible by interferon-gamma (IFN-gamma), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-gamma in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13), cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-gamma but not IFN-alpha in transient transfection assays. Sequence analysis revealed three putative gamma-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyl-tRNA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GAS1 (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN-gamma. Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFN-gamma treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified.

Identification of amino acid sequence motifs in desmocollin, a desmosomal glycoprotein, that are required for plakoglobin binding and plaque formation.

By transfecting epithelial cells with gene constructs encoding chimeric proteins of the transmembrane part of the gap junction protein connexin 32 in combination with various segments of the cytoplasmic part of the desmosomal cadherin desmocollin 1a, we have determined that a relatively short sequence element is necessary for the formation of desmosome-like plaques and for the specific anchorage of bundles of intermediate-sized filaments (IFs). Deletion of as little as the carboxyl-terminal 37 aa resulted in a lack of IF anchorage and binding of the plaque protein plakoglobin, as shown by immunolocalization and immunoprecipitation experiments. In addition, we show that the sequence requirements for the recruitment of desmoplakin, another desmosomal plaque protein, differ and that a short (10 aa) segment of the desmocollin 1a tail, located close to the plasma membrane, is also required for the binding of plakoglobin, as well as of desmoplakin, and also for IF anchorage. The importance of the carboxyl-terminal domain, homologous in diverse types of cadherins, is emphasized, as it must harbor, in a mutually exclusive pattern, the information for assembly of the IF-anchoring desmosomal plaque in desmocollins and for formation of the alpha-/beta-catenin- and vinculin-containing, actin filament-anchoring plaque in E- and N-cadherin.

Activation of the silent human cytokeratin 17 pseudogene-promoter region by cryptic enhancer elements of the cytokeratin 17 gene.

We have previously described the three loci CK-CA, CK-CB and CK-CC in the human genome that contain clustered type-I cytokeratin genes and reported the complete nucleic acid sequences of the functional cytokeratin 17 gene located in CK-CA and two closely related pseudogenes present in CK-CB and CK-CC [Troyanovsky, S.M., Leube, R.E. & Franke, W.W. (1992) Eur. J. Cell Biol. 59, 127-137]. By nucleic acid sequence analysis, we now show that extensive similarities between the functional gene and the pseudogenes exist in the 5'-upstream region. However, despite the high degree of nucleic acid identity (94%), only the 5'-upstream region of the functional gene was able to induce significant transcriptional activity in transfected cells of epithelial origin. Using chimeric upstream regions consisting of different fragments from the pseudogene and the functional gene, we made the surprising observation that cis elements in the proximal 5'-upstream region of the pseudogene promoter can cooperate with distal enhancer elements of the functional gene to induce strong chloramphenicol-O-acetyltransferase activity in transfected HeLa cells. A major site in the proximal upstream region was identified by deoxyribonuclease protection experiments to be necessary for this cooperative effect. The structure and properties of this element were further analysed by transfection of different chloramphenicol-O-acetyltransferase gene constructs, and by nucleic acid sequence comparison to corresponding regions of the related cytokeratins 14 and 16. It is concluded that the upstream regions identified in this study contribute to the strong expression of the human cytokeratin 17 gene in a coordinated fashion.

Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage.

The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes.

The desmosome and the syndesmos: cell junctions in normal development and in malignancy.

The cells of various normal and malignantly transformed tissues are connected by "adhering junctions"-plasma membrane domains characterized by close membrane-membrane contact, a dense cytoplasmic plaque and, in most cases, the attachment of cytoskeletal filaments. On the basis of their specific ultrastructural organization and molecular composition, three major types of intercellular adhering junctions can be distinguished: 1. Adherens junctions appear in different shapes and sizes (zonula adhaerens, fascia adh., punctum adh.) and contain the transmembrane glycoprotein E-cadherin. The cytoplasmic portion of E-cadherin forms complexes with alpha-, beta-, and gamma-catenin and plakoglobin which, together with other proteins such as vinculin and radicin, constitute a plaque at which actin microfilaments insert. 2. Desmosomes (maculae adhaerentes) are mostly isodiametric (diameters up to approximately 0.5 micron) membrane domains traversed by representatives of two types of desmosomal cadherins, the desmogleins (Dsg) and desmocollins (Dsc), whose cytoplasmic tails contribute to a dense plaque containing plakoglobin and desmoplakin I (with or without an alternative splice form, desmoplakin II) which anchor IFs. The specific Dsc and Dsg subtypes can differ in different cell types and up to three different human genes have so far been identified for each desmosomal cadherin. 3. Complexus adhaerentes are junctions of variable size and shape that occur in lymphatic endothelia. They have a desmoplakin- and plakoglobin-rich plaque, whose specific transmembrane proteins have not yet been fully elucidated but can include endothelial cadherin-5. In their most elaborate subform- the "syndesmos" connecting the retothelial cells of lymph node sinus-these junctions can occupy extended portions of the cell surface. The molecular arrangements in desmosomes and complexus adhaerentes have been studied to understand the assembly and disappearance of these structures. The diagnostic potential of their constituent proteins for cell typing in tumor diagnosis is emphasized, as is the role of transient junction dissociation during invasion and metastasis of carcinomas and the general importance of tumor cell interactions with the retothelial cell system in the formation of lymph node metastases.

Cytokeratin expression in alopecia areata hair follicles.

Alopecia areata is a human hair disease of unknown etiology. Immunological mechanisms, alterations in the extracellular matrix and follicular growth abnormalities have been suggested as a possible cause. Here we compare the expression of cytokeratins in normal hair follicles to that of alopecia areata using immunohistology with monoclonal antibodies. A number of cytokeratins were specifically expressed in defined anatomical parts of the follicle; however, no gross qualitative or quantitative differences were found between normal and diseased scalp. Interestingly, the expression of cytokeratin 16, which is modulated by conditions that affect the rate of keratinocyte proliferation, was found to be unchanged in the outer root sheet of alopecia areata follicles. This is in contrast with earlier observations of a decrease in the expression of the proliferation-associated, Ki-67 nuclear antigen.

Cytoskeletal architecture and epithelial differentiation: molecular determinants of cell interaction and cytoskeletal filament anchorage.

Desmosomes are morphologically well defined junctions between epithelial cells and also some other cells such as myocardiocytes, meningeal cells and dendritic reticulum cells of lymphatic follicles. Besides their function in cell coupling, desmosomes anchor components of the cytoskeleton, i.e. intermediate-sized filaments (IFs), through their cytoplasmic plaques, thereby contributing to cytoskeletal and tissue architecture. In molecular terms, desmosomes are specific assemblies of transmembrane glycoproteins of the cadherin family, desmoglein(s) and desmocollin(s), that contribute to cell adhesion via their extracellular, aminoterminal domains and to plaque formation and IF coupling through their cytoplasmic, carboxyterminal "tails". Using transfection assays, we analyzed the function of different tail domains in plaque assembly and IF anchorage. Furthermore, we present evidence that both desmogleins and desmocollins represent multigene subfamilies showing cell type specific expression and that a desmosomal plaque protein occurring in stratified and complex epithelia, the "band 6 protein", is related to the plakoglobin family.

Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.

To examine the potential of cytoplasmic portions ("tails") of desmosomal cadherins for assembly of desmosome plaque structures and anchorage of intermediate filaments (IFs), we transfected cultured human A-431 carcinoma cells, abundant in desmosomes and cytokeratin IFs, with constructs encoding chimeric proteins in which the transmembranous region of connexin 32 had been fused with tails of desmocollin (Dsc) or desmoglein (Dsg). The results show that the tail of the long splice form a of Dsc, but not its shorter splice form b, contains sufficient information to recruit desmoplakin and plakoglobin to connexon membrane paracrystals (gap junctions) and to form a novel kind of plaque at which cytokeratin IFs attach. By contrast, chimeras containing a Dsg tail, which accumulated in the plasma membrane, showed a dominant-negative effect: they not only were unable to form gap junction structures and plaques but also led to the disappearance of all endogenous desmosomes and the detachment of IFs from the plasma membrane.

Immunohistochemical localization of cytokeratin 17 in transitional cell carcinomas of the human urinary tract.

The expression of cytokeratin (CK) 17 was studied in 28 primary transitional cell carcinomas (TCCs) of the human urinary tract using CK 17-specific monoclonal antibody E3. While CK 17 was not detectable at all or only present in some areas of basal cells in normal--appearing urothelium, a certain subpopulation of cells of all G1 and G1/G2 TCCs examined (9 cases) stained positive for CK 17. These latter cells were either restricted to the basal compartment or located also in suprabasal layers exhibiting a decreasing intensity of immunoreactivity. CK 17 was seen in practically all cells in G2 and G2/G3 tumors (7 cases). In contrast, G3 TCCs and anaplastic carcinomas showed a highly variable CK 17 staining pattern ranging from completely negative to completely positive with several intermediate phenotypes. Our results indicate that CK 17 could be a useful marker for the progression of urinary tumors.

Special program of differentiation expressed in keratinocytes of human haarscheiben: an analysis of individual cytokeratin polypeptides.

Human haarscheiben, epidermal Merkel cell-rich sensory organs of hairy skin, were studied for the expression of various cytokeratin (CK) polypeptides and other epithelial and neuronal differentiation markers by applying immunoperoxidase and immunofluorescence microscopy to frozen sections and by two-dimensional gel electrophoresis. The basal clusters of Merkel cells were specifically detected by antibodies against CK 20. Haarscheiben keratinocytes were unique mainly by the prominent expression of CK 17 in the lower and middle layers. Further differences as compared to keratinocytes of usual epidermis included the enlargement of the basal compartment, characterized by the expression of CK 5 and the absence of the maturation-associated CKs 1/10/11, and the reduction of CK 15, which is a constituent of normal basal cells. Using CK 17 as a highly sensitive Haarscheibe marker in skin tissue sections, variabilities in the spatial relationship of the haarscheibe and the corresponding hair follicle were recorded. The results show that haarscheibe keratinocytes express a special program of differentiation that may be important for optimal stimulus perception. Immunohistochemical stainings for CK 17 will facilitate further studies on the distribution and biology of haarscheibe.

Characterization of the human gene encoding cytokeratin 17 and its expression pattern.

Among the members of the cytokeratin (CK) subfamily of intermediate filament (IF) proteins, CK 17 is remarkable as it is normally expressed in the basal cells of complex epithelia but not in stratified or simple epithelia. Because of its unusual expression pattern in normal and diseased states and because of the potential importance of CK 17 in tumor diagnosis, we have characterized the gene(s) and its cDNA-derived amino acid sequence. A cDNA clone encoding CK 17 was isolated from a HeLa cDNA library and used for the determination of the amino acid sequence, for studies of expression and for the screening of human genomic libraries. A number of lambda phage clones were isolated that covered three distinct, non-contiguous gene regions. Only one of these loci contains the functional CK 17 gene which is located only approximately 5 kbp 5'-upstream of the CK 16 gene, whereas the other two contain unprocessed CK 17 pseudogenes. Each of these genes is part of a larger CK type I gene locus the arrangement of which suggests that these genes and pseudogenes have arisen during evolution by duplication events comprising whole multigene loci. The functional CK 17 gene differs from the pseudogenes by the extent of methylation of certain DNA sequences in the 5'-upstream region. The 5 kbp CK 17 gene with 8 exons and 7 introns encodes a polypeptide of 432 amino acids with a calculated molecular weight of 48,000. Using S1-nuclease protection assays and RNAs from several cell lines we identified a single transcriptional start point 26 nucleotides down-stream from a TATA box element. Northern blot hybridization experiments showed a restricted pattern of CK 17 gene expression, supporting the notion that CK 17 synthesis is essentially regulated at the transcriptional level. From these findings and from immunohistological observations, CK 17 synthesis seems to be a marker of basal cell differentiation in complex epithelia and therefore indicative of a certain type of epithelial "stem cells".

Chromosomal mapping of human cytokeratin 13 gene (KRT13).

In the present study the human cytokeratin 13 gene (KRT13), encoding a polypeptide characteristic of internal stratified epithelia, has been mapped with the help of the polymerase chain reaction and somatic cell hybrids to chromosome 17. In situ hybridization of a KRT13 cDNA probe to metaphase chromosomes allowed the assignment of the KRT13 gene within the q12-q21.2 region of chromosome 17.