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T M Magin

Publications and source records attributed to T M Magin.

32 records · Page 2Linked to original sources
PubMed1996

Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.

Animals↗
PubMed1992

A position- and orientation-dependent element in the first intron is required for expression of the mouse hprt gene in embryonic stem cells.

The gene (hprt) coding for mouse HPRT (hypoxanthine phosphoribosyltransferase) is transcribed from a promoter lacking CAAT and TATAA boxes. It is expressed ubiquitously, albeit at different levels, in all tissues and cultured cells. During investigations to characterise hprt transcription control elements required in embryonic stem (ES) cells and to develop compact hprt minigenes for gene-targeting strategies, we discovered a requirement for intron-1 sequences for expression in ES cells. The essential intron-1 element, which is 420 bp long, is located 230 bp downstream from the transcription start point and is shown to increase transcription from the hprt promoter in a position- and orientation-dependent manner. We propose that this element is an integral downstream part of the hprt promoter.

Animals↗
PubMed1992

Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene.

A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker--the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications.

Animals↗
PubMed1991

Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus.

The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.

3T3 Cells↗
PubMed1990

The hemidesmosomal plaque. I. Characterization of a major constituent protein as a differentiation marker for certain forms of epithelia.

To examine whether constituent proteins of hemidesmosomal structures can be used as markers for certain pathways of epithelial differentiation we have examined the occurrence of the major M- approximately 230,000 plaque protein, the "bullous pemphigoid" (BP) antigen. Several bovine, rat and human tissues and bovine cell culture lines were examined, using different human autoantibody preparations in immunocytochemistry and immunoblotting. We report that this protein, also unequivocally identified by cDNA cloning from expression libraries and DNA sequencing, occurs not only in different stratified epithelia but also, apparently always in hemidesmosomal structures, in urothelium of bladder and the complex epithelia of trachea, bronchus and several glands, notably myoepithelium-containing skin glands, the mammary gland and salivary glands. The protein is absent, however, in all single-layered epithelia and in several tissues reported to have subplasmalemmal densities structurally similar to hemidesmosomes, such as Purkinje fibers of heart, meninges and perineuria. A mammary-gland-derived epithelial cell line (BMGE + H) is particularly rich in hemidesmosomes. This has been used to study the endocytotic uptake of hemidesmosome-containing plasma membrane domains into cytoplasmic vesicles upon detachment of cell sheets during treatment with dispase, a proteolytic enzyme. We propose to use the Mr- approximately 230,000 plaque protein as a marker selective for certain subsets of epithelial cell types and epithelium-derived tumors in studies of fetal and tumor development, including differentiation diagnosis of carcinomas.

Amino Acid Sequence↗
PubMed1990

De novo formation of cytokeratin filaments in calf lens cells and cytoplasts after transfection with cDNAs or microinjection with mRNAs encoding human cytokeratins.

We have studied the formation of new intermediate-sized filaments (IFs) by human cytokeratins (CKs) 8 and/or 19 in cultured bovine lens cells stably transfected with the corresponding cDNAs under SV40 promoter control. In the transfected cells, polypeptides of both type I and type II CKs were synthesized to near-equimolar amounts, formed heterotypic complexes and assembled into IFs with a peculiar tendency to accumulate into variously sized, often roundish aggregates in the juxtanuclear region, usually one per cell. Electron microscopy of these large CK IF aggregates revealed typical 7 to 12-nm IFs, tightly packed together in an apparently haphazard mode. By immunoelectron microscopy, the CK IFs could be readily distinguished from the vimentin IFs which were abundant in these cells. Electron microscopy also showed that many of the CK IF aggregates were located in the vicinity of the nucleus but did not have direct contact with the nuclear envelope; moreover, their location did not regularly correspond to those of the centrosomes and the Golgi apparatus. During enucleation of transfected cells in the presence of cytochalasin B, the CK aggregates were often retained in the cytoplast. After microinjection of CK 8 and 19 mRNAs, synthesized in vitro from cDNA molecules, into enucleated cytoplasts prepared from untransfected cells, CK IFs similar to those observed in microinjected whole cells were formed but often showed a wider cytoplasmic distribution. Our observations indicate that typical CK IFs can form, in vivo, in the absence of any nuclear structures. We discuss possible reasons for the tendency of the CK IFs to accumulate, in this cell line, into a juxtanuclear aggregate, in relation to similar CK-IF aggregates formed in certain normal cell types and upon toxic damages.

Animals↗
PubMed1987

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↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1986

Cytokeratin expression in simple epithelia. I. Identification of mRNA coding for human cytokeratin no. 18 by a cDNA clone.

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.

Amino Acid Sequence↗
PubMed1986

Cytokeratin expression in simple epithelia. II. cDNA cloning and sequence characteristics of bovine cytokeratin A (no. 8).

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.

Amino Acid Sequence↗
PubMed1985

Patterns of expression and organization of cytokeratin intermediate filaments.

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.

Amino Acid Sequence↗
PubMed1984

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones.

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.

Animals↗
PubMed1983

Translational products of mRNAs coding for non-epidermal cytokeratins.

Total RNA and poly(A)+ RNA were isolated from tissues and cultured cells of various mammalian species (bovine muzzle epidermis and bladder urothelium; rat hepatoma cells; human cell lines HeLa, MCF-7 and A-431) and examined by translation in vitro using the reticulocyte lysate system. Polypeptides were separated and identified by two-dimensional electrophoresis and cytokeratins were selectively enriched from the translation assays by co-polymerization with added heterologous cytokeratins. In all three species, non-epidermal cytokeratins A, D and mol. wt. 40,000 (corresponding to numbers 8, 18 and 19 of the human cytokeratin catalog of Moll et al., 1982) were identified as translation products capable of co-polymerization with epidermal keratins. Several other basic and other acidic cytokeratins were also identified as translational products. In addition, two unidentified polypeptides (mol. wt. 52,000 and 43,000) which were minor polypeptides in cytoskeletons and translation assays were found to be specifically enriched in co-polymers with bovine epidermal keratins. The results indicate that many, perhaps all, non-epidermal cytokeratins characteristic of simple epithelia are genuine products of translation and that their diversity is not due to post-translational modification or processing. These findings, taken together with observations of in vitro translation of epidermal mRNAs, suggest that the diversity of cell type-specific expression of the different members of the cytokeratin polypeptide family is largely due to the cell type-specific synthesis of diverse mRNAs.

Animals↗