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

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

A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells

Epithelial cells contain a class of intermediate-sized filaments formed by proteins related to epidermal alpha-keratins ('cytokeratins'). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000-68 000) and isoelectric pH (5.0-8.5). We have separated, by two-dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.     

Amino acid sequence microheterogeneities of basic (type II) cytokeratins of Xenopus laevis epidermis and evolutionary conservativity of helical and non-helical domains

Three clones coding for carboxy-terminal portions of type II cytokeratins have been isolated from a cDNA library constructed from the epidermis of the frog Xenopus laevis. These clones have been identified by hybridization-selection-translation and Northern blot analysis, and contain sequences complementary to mRNAs of similar size that code for three different polypeptides of the Mr 64,000 group, Ia-c, i.e. the only major type II cytokeratins expressed in this tissue. A comparison of the corresponding nucleotide sequences and the amino acid sequences deduced therefrom shows only minor differences in these polypeptides, most of which occur as isolated point mutations. This indicates that coding sequences of the different type II cytokeratin genes in epidermis of Xenopus are very similar, in contrast to the more extended differences of type II cytokeratin genes expressed in mammalian epidermis, which probably reflects a lower degree of evolutionary divergence of members of this protein family in amphibia. A comparison of the Xenopus sequences with those of mammalian type II cytokeratins reveals the same characteristic features, i.e. an alpha-helical domain ending with the familiar consensus sequence T Y R (X Y) L E G E, followed by a non-helical domain Cl enriched in hydroxyamino acids. Both domains are remarkably conserved in sequence between Xenopus and mammals. The following glycine-rich domain (C2) displays similar oligopeptide repeats (mostly of the type G G G M in the frog keratins), and the terminal C3 domain is characterized by a region exceptionally rich in hydroxyamino acids, which immediately precedes a cluster of basic amino acids at the carboxy terminus. Our results show that the typical features of the domain of type II cytokeratins are already established in amphibia and that these homologies are not restricted to the alpha-helical rod of these proteins but, in principle, extend to the other domains located in the so-called hypervariable tail portion. This suggests that the hypervariable regions are not subject to random variability but contain functionally important domains that have been well conserved during evolution.     

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.     

Different keratin polypeptides in epidermis and other epithelia of human skin: a specific cytokeratin of molecular weight 46,000 in epithelia of the pilosebaceous tract and basal cell epitheliomas

Cytokeratin polypeptides of human epidermis, of epithelia microdissected from various zones of the pilosebaceous tract (outer root-sheath of hair follicle, sebaceous gland), and of eccrine sweat-glands have been separated by one- and two-dimensional gel electrophoresis and characterized by binding of cytokeratin antibodies and by peptide mapping. The epithelium of the pilosebaceous tract has three major keratin polypeptides in common with interfollicular epidermis (two basic components of mol wts 58,000 and 56,000 and one acidic polypeptide of mol wt 50,000); however, it lacks basic keratin polypeptides in the mol wt range of 64,000-68,000 and two acidic keratin-polypeptides of mol wts 56,000 and 56,500 and contains an additional characteristic acidic cytokeratin of mol wt 46,000. Another cytokeratin polypeptide of mol wt 48,000 that is prominent in hair-follicle epithelium is also found in nonfollicular epidermis of foot sole. Both epidermis and pilosebaceous tract are different from eccrine sweat-gland epithelium, which also contains two major cytokeratins of mol wts 52,500 and 54,000 (isoelectric at pH 5.8-6.1) and a more acidic cytokeratin of mol wt 40,000. A striking similarity between the cytokeratins of human basal-cell epitheliomas and those of the pilosebaceous tract has been found: all three major cytokeratins (mol wts 58,000; 50,000; 46,000) of the tumor cells are also expressed in hair-follicle epithelium. The cytokeratin of mol wt 46,000, which is the most prominent acidic cytokeratin in this tumor, is related, by immunological and peptide map criteria, to the acidic keratin-polypeptides of mol wts 48,000 and 50,000, but represents a distinct keratin that is also found in other human tumor cells such as in solid adamantinomas and in cultured HeLa cells. The results show that the various epithelia present in skin, albeit in physical and ontogenic continuity, can be distinguished by their specific cytokeratin-polypeptide patterns and that the cytoskeleton of basal-cell epitheliomas is related to that of cells of the pilosebaceous tract.     

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.     

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)     

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)     

Characterization of bovine keratin genes: similarities of exon patterns in genes coding for different keratins.

Four different genomic clones which contain the genes coding for epidermal keratins Ia (mol. wt. approximately 68 000), Ib (68 000), III (60 000) and VIb (54 500) have been selected using cDNA probes and identified by hybrid-selection translation. The genes vary considerably in length, primarily due to differences in intron sizes: keratin Ia, 9.3 kb (approximately 2.55 kb total exons); keratin Ib, 6.0 kb (2.25 kb exons); keratin III, 6.0 kb (2.2 kb exons); keratin VIb, 4.4 kb (1.85 kb exons). The genes for all three representatives of the basic (type II) cytokeratin subfamily, i.e., keratins Ia, Ib and III, contain eight introns of variable sizes (0.1-1.8 kb) and their exon patterns are very similar. The gene coding for keratin VIb, a representative of the acidic (type I) subfamily, contains seven introns, and the size pattern of its five innermost exons closely resembles that of the genes of the type II keratins. Most of the introns are located in regions coding for the alpha-helical cores of these proteins. Mapping of the intron positions by the S1 nuclease technique and sequencing of some exon-intron boundaries has revealed that some of the introns of all four keratin genes have similar positions to each other and to those of the hamster vimentin gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Biosynthesis and molecular nature of the T3 antigen of human T lymphocytes

Immunoprecipitates of the T3 antigen prepared from HPB-ALL cells by using the monoclonal antibody UCH-T1 were analysed by SDS-polyacrylamide gel electrophoresis. Cells which had been biosynthetically labelled for up to 4 h gave a major polypeptide of mol. wt. 19 000 plus two weaker, more diffuse bands of mol. wts. 21 000 and 23 000, whereas surface labelled cells gave a prominent band of mol. wt. 19 000, a major band of 21 000 and a weaker diffuse band of approximately 26 000. As judged from their sensitivity to proteinase-K digestion, all the above polypeptides possess a transmembrane orientation. Digestion with endoglycosidases H and F (endo-H and endo-F), and tunicamycin treatment indicate that all the polypeptides, except that of 19 000 mol. wt. are N-glycosylated. The 21 000 and 23 000 mol. wt. chains possess both immature and mature oligosaccharide units, whereas the 26 000 mol. wt. band apparently has mature units only. Pulse chase experiments combined with digestion by endo-F and endo-H suggest that the N-glycosylated polypeptides are derived from two polypeptides of mol. wts. 14 000 and 16 000. It is concluded that the T3 antigen is derived from three different non-glycosylated polypeptides two of which are subsequently N-glycosylated to give the 21 000, 23 000 and 26 000 forms. The cell surface T3 antigen most probably comprises at least two distinct, non-covalently associated polypeptides, but the number and types of polypeptides giving rise to the whole molecule and whether different complexes exist is at present unclear.     

Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.     

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.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases

With a procedure that allows the renaturation of the DNA polymerase catalytic activity in situ after SDS-polyacrylamide gel electrophoresis, we have compared the active polypeptides present in extracts from organisms covering a wide evolutionary range from prokaryotes to eukaryotes, namely: Escherichia coli, Oryza sativa, Daucus carota , Neurospora crassa, Dictyostelium discoideum, Saccharomyces cerevisiae, Ceratitis capitata, Leucophaea maderae , Xenopus laevis, rat tissues and human lymphoblastoid cells. Two main clusters of active peptides are visible in mammalian and adult insect tissues, characterized by a mol. wt. greater than 70000 and less than 50000, respectively. High mol. wt. peptides are heterogeneous in size and correspond to active fragments of DNA polymerase alpha, whereas low mol. wt. peptides show the same migration rate as purified DNA polymerase beta and are not generated by proteolysis of the high mol. wt. cluster, In the three species of fungi studied, only high mol. wt. peptides are found. The same is true in plant cells, where no DNA polymerase beta activity is detectable and the pattern of the high mol. wt. cluster is similar to that observed in E. coli extracts (which also lack low mol. wt. peptides). Also in mitochondria from higher and lower eukaryotes only high mol. wt. species are observed, and the active band(s) range from 70000 to 145000 daltons. Our results indicate that the structure of DNA polymerase has been highly conserved during evolution so that an active fragment of mol. wt. greater than or equal to 70 000 is always found in prokaryotic enzymes and in the replicative species of eukaryotic and mitochondrial DNA polymerases; at a certain stage in evolution, another species of low mol. wt. DNA polymerase (beta or beta-like) appears.     

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)     

Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of tissue-specific keratins as deduced from novel cDNA sequences of the lungfish Protopterus aethiopicus

Lungfishes are possibly the closest extant relatives of the land vertebrates (tetrapods). We report here the cDNA and predicted amino acid sequences of 13 different keratins (ten type I and three type II) of the lungfish Protopterus aethiopicus. These keratins include the orthologs of human K8 and K18. The lungfish keratins were also identified in tissue extracts using two-dimensional polyacrylamide gel electrophoresis, keratin blot binding assays and immunoblotting. The identified keratin spots were analyzed by peptide mass fingerprinting which assigned seven sequences (inclusively Protopterus K8 and K18) to their respective protein spot. The peptide mass fingerprints also revealed the fact that the major epidermal type I and type II keratins of this lungfish have not yet been sequenced. Nevertheless, phylogenetic trees constructed from multiple sequence alignments of keratins from lungfish and distantly related vertebrates such as lamprey, shark, trout, frog, and human reveal new insights into the evolution of K8 and K18, and unravel a variety of independent keratin radiation events.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.