Transmembrane molecular assemblies regulated by the greater cadherin family.

The cadherin family of cell-cell adhesion molecules is turning out to be much more diverse than previously thought, with members involved in several kinds of intercellular junctions. The adhesive specificity and cytoskeletal interaction of these members varies. Their cytoplasmic terminals are specialized for binding several families of 'mediator' proteins which interconnect to the actin or intermediate filament systems. These multi-molecular complexes have roles not only in cell-cell adhesion, but also in intracellular signalling of developmental information.

Antisense expression of a desmocollin gene in MDCK cells alters desmosome plaque assembly but does not affect desmoglein expression.

The desmocollins are one of two types of putative adhesive proteins present in the desmosome type of cell junctions, the other type being the desmogleins; both are members of the cadherin superfamily. Each type of desmosomal cadherin occurs as a number of isoforms which have differing tissue distribution; within stratifying epithelia some isoforms occur only suprabasally. We have sought to analyse desmocollin function by reducing the amount of protein using antisense gene expression in the widely studied Madin-Darby canine kidney (MDCK) cell line. Although this is a simple epithelial cell line, we show by Northern blot analysis that it expresses multiple isoforms of the desmosomal cadherins. Desmocollins DSC2 and DSC3 and desmogleins DSG2 and DSG3 (the pemphigus vulgaris antigen PVA) were detected, but DSC1 and DSG1, which are present exclusively in the suprabasal layers of the epidermis, were absent. The major desmocollin isoform was the type 2 (DSC2). A DSC2 clone isolated from a MDCK cDNA library had the same cell adhesion recognition sequence (Phe-Ala-Thr) as human, bovine and mouse type 2 isoforms. This sequence appears diagnostic for the three desmocollin isoforms. This cDNA clone was used to isolate a genomic DSC2 clone; antisense expression of this clone in MDCK cells resulted in a drastic reduction of desmocollin protein as judged by Western blots; Dsc3 was not upregulated to compensate for the loss of Dsc2. This antisense expression significantly altered desmosome assembly. There was a loss of punctate staining evident when using a desmosome plaque protein (desmoplakin) antibody. Electron microscopy revealed that there was a reduction in the number of desmosomes and a notable increase in the asymmetry of plaques between adjacent cells. Immunolabelling showed that similar levels of desmogleins and E-cadherin were present. Immunoelectron microscopy also showed that many vesicular structures were labelled, at intervals along the lateral membranes between cells. The distinctive loose organization of the remaining desmosomes may originate in modifications to the targeting and incorporation of proteins into fully assembled plaques. Other junctions were unaffected and the cells maintained their integrity as a confluent monolayer.

The desmocollins of human foreskin epidermis: identification and chromosomal assignment of a third gene and expression patterns of the three isoforms.

A third human desmocollin, designated DSC3, was identified in foreskin epidermis by reverse transcriptase-polymerase chain reaction (PCR) using degenerate desmocollin primers. cDNA clones covering the entire coding sequence of the longer DSC3 splice variant were isolated and sequenced. Sequence comparisons indicated that this new desmocollin showed greater homology (67% amino acid identity) with the original human desmocollin (now designated DSC2) than with DSC1 (52% amino acid identity) although it had a unique potential cell adhesion recognition site (YAS). DSC3 was assigned to chromosome 18 by PCR analysis of rodent-human somatic cell hybrids, where it appears to be closely linked to all the other desmosomal cadherin genes. The expression of the three human desmocollins was examined in foreskin epidermis by in situ hybridization with 3'-untranslated riboprobes and by immunofluorescence with isoform-specific anti-peptide antibodies. DSC1 was present in the upper spinous/granular layers but not in the basal/lower spinous layers of the tissue. DSC2 and DSC3 were present in most of the living layers of the epidermis. DSC1 was not detected in any of the nonkeratinizing human epithelia examined (buccal mucosa, cervix, esophagus), indicating that it is specific for the keratinizing epithelium of the epidermis. However, all these internal epithelia expressed DSC2 and DSC3, and both were present in most of the living layers of the tissues including the basal layers.

Expression of the "skin-type" desmosomal cadherin DSC1 is closely linked to the keratinization of epithelial tissues during mouse development.

Desmosomal junctions contain two classes of desmosomal cadherin, the desmocollins and the desmogleins, each of which occurs as three distinct isoforms. To investigate the role of the "skin-type" desmosomal cadherins (desmocollin 1 and desmoglein 1) in the formation of keratinized epithelial structures, we have now cloned full-length mouse desmocollin 1 complementary deoxyribonucleic acid and examined the expression of desmocollin 1 and desmoglein 1 and messages during murine embryonic development by in situ hybridization. In the general body epidermis, desmocollin 1 and desmoglein 1 transcripts both showed considerable upregulation at 15.5 d, which is after the onset of stratification and before the start of keratinization. Before this the epidermis expressed low levels of desmocollin 1 message, although the desmoglein 1 signal was always stronger and more extensive. In the tongue, expression of desmocollin 1 message occurred several days after desmoglein 1 and coincided with the formation of the keratinizing filiform papillae. Desmoglein 1 message was also detected in epithelial tissues in which desmocollin 1 was absent, suggesting that expression of the two "skin-type" desmosomal cadherins was not tightly coupled during embryonic development. Human desmocollin 1 monoclonal antibodies that cross-reacted with mouse skin and tongue indicated that desmocollin 1 protein was first expressed in those outermost epithelial cells destined to form the keratinized layers of the stratum corneum or the papillae. The results suggest that expression of desmocollin 1 is closely associated with the keratinization of epithelial tissues during mouse development.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Cloning and transcriptional analysis of the promoter of the human type 2 desmocollin gene (DSC2).

The desmocollins, together with the desmogleins, are members of the cadherin family and constitute the adhesive proteins of the desmosome type of cell-cell junction. Here we describe a study of the promoter of the human form of the DSC2 gene which is the equivalent of the first isoform expressed in the developing mouse embryo and that has the most widespread tissue distribution in epithelia and also in desmosome-bearing non-epithelial tissues. Analysis of the 5' upstream region by DNA sequencing and Southern blotting suggested that it contained a CpG island, and a major site of transcription initiation 201 bp upstream of the translation start site was found by RNase protection and primer extension. There were no obvious CCAAT or TATA boxes present. Analysis of 1.9 kb upstream of the translation start site revealed consensus binding sites for transcription factors including Ap-2 and Sp-1, and motifs common to the promoters of other epithelially expressed genes such as keratin 14 and the desmoglein genes DSG1 and DSG3. Deletion derivatives defined a promoter of 525 bp which was active in epithelial cells and in mouse blastocysts with an intact epithelium. This promoter showed reduced expression in non-epithelial cells.

Visualisation by electron microscopy of the unique part of the cytoplasmic domain of a desmoglein, a cadherin-like protein of the desmosome type of cell junction.

Part of the cytoplasmic domain of a human desmoglein, Dsg1, a cadherin-like protein found in desmosomes of epithelial cells, has been visualised by electron microscopy. The cloned fragment contains five repeats of a 29 +/- 4 residue sequence unique to desmogleins, followed by a glycine-rich region. In rotary shadowed preparations the molecule consists of a globular head attached to a thin tail, the latter perhaps corresponding to the glycine-rich region. This portion of the molecule is thought to span the width of the inner dense plaque. The structure and dimensions concur well to the configuration deduced from the protein sequence.

Keratinization is associated with the expression of a new protein related to the desmosomal cadherins DGII/III.

Amino acid sequencing of a 48/46 kDa glycoprotein from human plantar callus, recognised by antisera raised against the desmosomal cadherins DGII/III, has revealed N-terminal homology to the DNA-derived sequence of human and bovine DGII/III. However, a tryptic fragment has homology only with a bovine clone. We propose that there are two classes of DGII/III-like molecule, that represented by the bovine cDNA clone and the 48/46 kDa protein, a monoclonal antibody against which stains mainly the suprabasal layers of human epidermis, and that represented by the human cDNA clone, identified by a monoclonal antibody which stains uniformly the living layers of the epidermis.

Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage.

Heat-resistant derivatives of a Bacillus subtilis 168 strain carrying an xhi mutation, which causes heat-sensitive induction of the PBSX prophage, have been isolated and screened for the acquisition of auxotrophy. Two classes of auxotrophs were isolated, namely Pro- and Pro-Met-; they lacked the ability to produce PBSX, as shown by their resistance to mitomycin C-induced lysis. The proline and methionine requirements and the resistance to mitomycin C were shown to segregate together in phage PBS1-mediated transduction crosses and to be linked to thiB, which is known to be co-transducible with the PBSX prophage. It was therefore proposed that these strains had deletions which removed all or part of the PBSX prophage together with adjacent bacterial DNA encoding the pro(AB) and metC genes. The met mutation was shown to be metC in PBS1 transduction crosses; this gene is known to be co-transducible with the PBSX prophage. The proline requirement was probably due to the deletion of a pro gene which was demonstrated to lie between the PBSX prophage and metC and which was 90% co-transducible with metC. These deletions have been transduced into a strain which was cured of phage SP beta, another bacteriophage carried by B. subtilis 168. No phage particles could be seen in mitomycin C-induced cultures of such strains. The PBSX-deletion strains grew with the same generation time as the PBSX+ parent in L-broth (27 min at 35 degrees C) but they were slower in minimal medium (e.g. 72 min as against 51 min in the PBSX+ strain). Besides being resistant to mitomycin C-induced lysis, the deletion strains were also resistant to lysis induced by thymine starvation of thymine auxotrophs and the loss of viability of these strains after thymine starvation was 100-fold less than in the PBSX+ parent. The deletion strains had not, however, lost the bacterial autolytic enzymes, since they were still susceptible to lysis when placed under semi-anaerobic conditions.

A heat-sensitive lysis mutant of Bacillus subtilis 168 with a low activity of pyruvate carboxylase.

A mutant of Bacillus subtilis which grew in complex medium at 30 degrees C but lysed at 45 degrees C has been isolated. It could only grow on minimal medium at 45 degrees C with added aspartate (20 microgram ml-1) but lysed if lysine (20 microgram ml-1) was also present. The requirement for aspartate was due to a low activity of pyruvate carboxylase; the site of the mutation (pyc) was linked (16% cotransducible using phage PBSI) to the pyrD locus, and the order of markers deduced was: pyrD-cysC-pyc. This defect appeared to lead to decreased synthesis of mesodiaminopimelic acid (mesoA2pm), an amino acid unique to peptidoglycan and its precursors. At the restrictive temperature the mutant accumulated uridine-5'-diphosphate N-acetylmuramyl-L-alanyl-D-glutamate, since meso A2pm is the next amino acid to be added to the growing peptide chain of peptidoglycan. This resulted in an inhibition of peptidoglycan synthesis, determined as a reduced incorporation of N-acetyl[14C]glucosamine. Peptidoglycan synthesis was not decreased if the mutant was grown in media containing aspartate but lacking lysine. The sensitivity to lysine may arise because (i) at 45 degrees C the mutant was starved for aspartate and hence mesoA2pm even when aspartate was present, since aspartate utilization, as estimated by the incorporation of [3H]aspartate into trichloroacetic acid precipitable material, was relatively inefficient; and (ii) this diminished level of mesoA2pm synthesis from aspartate was further curtailed since lysine inhibits one of the aspartokinases in B. subtilis. Thus, addition of lysine allowed protein synthesis and hence autolysin production to proceed whilst peptidoglycan synthesis remained inhibited. When autolysis was blocked, either indirectly by stopping protein synthesis through starvation of aspartate and lysine, or directly by introducing a lyt mutation, then shifting the mutant to 45 degrees C did not result in lysis but growth still ceased.

Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX.

A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced. A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min. Heat-induced lysis of the mutant was prevented by chloramphenicol. DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis. The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage. The order of markers was argC-thiB-metA-xhi-metC. The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker. tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable. It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23. The xki mutation was separated from xhi by recombination.

Genetic analysis of thymidine-resistant and low-thymine-requiring mutants of Escherichia coli K-12 induced by bacteriophage Mu-1.

Four genes, dra, tpp, drm, and pup, that specify enzymes involved in the catabolism of nucleosides and deoxynucleosides in Escherichia coli are known to be very closely linked in the order dra-tpp-drm-pup. By infecting cells with the phage Mu-1 and isolating low-thymine-requiring derivatives of a strain lacking thymidylate synthetase and also thymidine-resistant mutants of a dra-strain, it has been possible to select for strains in which Mu-1 is inserted in this gene cluster. Making use of the polar effect of Mu-induced mutations on more distal genes in the same transcriptional unit, evidence is presented that dra and tpp are co-transcribed from a promoter to the left of dra, and drm and pup are co-transcribed from a promotor located between tpp and drm. Residual levels of purine nucleoside phosphorylase in drm- mutants induced by phage Mu seem to indicate that a weak promotor lies between drm and pup. From a strain in which Mu-1 is inserted in drm, a mutant has been isolated that has a deletion extending into tpp. Since this strain lacks thymidylate synthetase, it is unable to grow on minimal medium containing thymine. Mutants isolated from this strain that can grow on minimal medium containing thymine have been shown to have increased levels of the enzyme uridine phosphorylase.

Structure and interactions of desmosomal and other cadherins.

The cadherin superfamily of cell-cell adhesion molecules is now known to include proteins of the desmosome as well as of the adherens type of junction. The desmosomal cadherins consist of two families of proteins, the desmocollins and the desmogleins, both of which are represented by different isoforms which are differentially expressed in epidermis. The desmocollins are quite similar to the classic cadherins in overall structure, but with alternatively spliced variants; the desmogleins have extra cytoplasmic sequences added onto the basic cadherin structure. The cytoplasmic domains are specialized for binding to 'mediator' proteins, such as plakoglobin, which interconnect to the intermediate filament system rather than the actin filaments as do the classic cadherins.

Identification and sequencing of the Escherichia coli cet gene which codes for an inner membrane protein, mutation of which causes tolerance to colicin E2.

Dominant mutations of the cet gene of Escherichia coli result in tolerance to colicin E2 and increased amounts of an inner membrane protein with an Mr of 42,000. We have cloned the cet+ gene and sequenced its DNA, revealing that the gene product, coded by the longest open-reading frame, has an Mr of 49,772, with five predicted transmembrane structures towards its carboxy terminus and one at ist amino terminus. We have demonstrated that the cet locus does in fact code for the inner membrane protein that is present in increased amounts in cet mutants, and we have shown that this increased amount of Cet protein is the result of enhanced transcription. The cet gene is shown to be in the same operon as the phoM gene, which is required in a phoR background for expression of the structural gene for alkaline phosphatase, phoA. Although the Cet protein is not required for phoA expression, our experiments suggest that the Cet protein has an enhancing effect on the transcription of phoA. No effect of phosphate concentration on cet or phoM gene expression could be found and thus their primary function may not be connected to the phosphate regulon.

Cloning and insertional inactivation of the dye (sfrA) gene, mutation of which affects sex factor F expression and dye sensitivity of Escherichia coli K-12.

Deletions of the Escherichia coli K-12 chromosome between trpR and thr render the bacterium sensitive to the dye toluidine blue (Dye-), and if male (Hfr or F'), the strain is sterile (Fex-), failing to donate F' or chromosomal markers and resistant to male-specific phages as a consequence of its inability to elaborate F pili. A 6-kilobase SalI fragment of E. coli chromosomal DNA cloned into the plasmid pBR322 has been shown to complement both the Dye- and Fex- phenotypes. Insertion of the transposon gamma delta (Tn1000) into a specific part of this plasmid invariably results in both the Dye- and Fex- phenotypes, indicating that these phenotypes derive from mutation in a single gene. Complementation tests between such insertions and sfrA4, a previously isolated mutation resulting in a Fex- phenotype and reported to code for a transcriptional control factor for F (L. Beutin, P. A. Manning, M. Achtman, and N. Willetts, J. Bacteriol. 145:840-844, 1981), indicated that dye and sfrA4 were mutations in a single cistron. It is proposed that the dye (sfrA) gene product is necessary not only for efficient transcription of the F factor genes, but also for some component(s) of the bacterial envelope, loss of which results in sensitivity to toluidine blue.