The complete cDNA and deduced amino acid sequence of a type II mouse epidermal keratin of 60,000 Da: analysis of sequence differences between type I and type II keratins.

We present the complete nucleotide and deduced amino acid sequences of a mouse epidermal keratin subunit of 60,000 Da. The keratin possesses a central alpha-helical domain of four tracts (termed 1A, 1B, 2A, and 2B) that can form coiled-coils, interspersed by short linker sequences, and has non-alpha-helical terminal domains. This pattern of secondary structure is emerging as common to all intermediate filament subunits. The alpha-helical sequences conform to the type II class of keratins. Accordingly, this is the first type II keratin for which complete sequence information is available, and thus it facilitates elucidation of the fundamental distinctions between type I and type II keratins. It has been observed that type I keratins are acidic and type II keratins are neutral--basic in charge. We suggest that the basis for this empirical correlation between type and charge resides in the respective net charges of the 1A and 2B tracts. Calculations on interchain interactions between charged residues in the alpha-helical domains indicate that this keratin prefers to participate in dimers according to an in-register parallel arrangement. The terminal domains of this keratin possess characteristic glycine-rich sequences, and the carboxyl-terminal domain is highly homologous to that of a human epidermal keratin of 56,000 Da. According to the hypothesis that end-domains are located on the periphery of keratin filaments, we conclude that the corresponding mouse and human keratins are closely related, both structurally and functionally.     

Characterization of an almost full-length cDNA coding for human blood coagulation factor X.

A human liver cDNA library was screened by colony hybridization with a bovine factor X cDNA probe. Three of the positive plasmids contained overlapping DNA that coded for most of human factor X mRNA. DNA sequence analysis of these three clones allowed the prediction of the complete amino acid sequence of plasma factor X. From these studies, we predict that human factor X is synthesized as a single polypeptide chain precursor in which the light and heavy chains of plasma factor X are linked by the tripeptide Arg-Lys-Arg. The cDNA sequence also predicts that human factor X is synthesized as a preproprotein having an amino-terminal leader peptide of at least 28 amino acid residues. A comparison of the amino acid sequences of human and bovine factor X shows high sequence identity around the calcium-binding regions and catalytic regions but low sequence identity around the nonfunctional regions.     

Differences in keratin synthesis between normal epithelial cells and squamous cell carcinomas are mediated by vitamin A.

A number of recent studies have indicated that the expression of keratins is altered upon malignant transformation of human epithelial cells. We have shown that the altered expression of 67-kDa and 40-kDa keratins in established squamous cell carcinoma lines from tongue and epidermis stems largely from a difference in their sensitivity to vitamin A apparently acquired during tumorigenesis. When the vitamin A concentration in the medium is raised, the 40-kDa keratin is produced at increased levels. Conversely, when the amount of vitamin is reduced, the 67-kDa keratin is synthesized and the cells undergo stratification and terminal differentiation. However, even when vitamin A is quantitatively removed from the medium, the maximal degree of differentiation attained by each squamous cell carcinoma cell as judged by the synthesis of 67-kDa keratin was still less than that of the normal keratinocytes. These findings suggest that the altered patterns of keratins observed for some tissues upon malignant transformation arise from a complex mixture of intracellular changes in the differentiative pathway in addition to changes in the responsiveness of cells to extracellular regulators of keratin gene expression.     

Human 67-kDa calelectrin contains a duplication of four repeats found in 35-kDa lipocortins.

The 67-kDa calelectrin is the largest member of a family of Ca2+-binding proteins that associate with membranes and phospholipids in a Ca2+-dependent manner. Oligonucleotide probes based on peptide sequences obtained from purified bovine 67-kDa calelectrin were used to screen a human retina cDNA library, and the complete primary structure of human 67-kDa calelectrin was deduced by DNA sequence analysis. The protein consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to the human lipocortin I and II sequences, each of which contains four such repeats. The amino termini of the three proteins show no sequence similarity; however, in the repeated regions the proteins are 42-45% identical in sequence. Analysis of the 16 repeats from the three proteins provides insights into the structural basis for Ca2+-dependent phospholipid binding. These data place the calelectrins and the lipocortins into the same gene family and suggest that these proteins have similar functions and have evolved from a common ancestor.     

Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae.

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.     

Distinct forms of the beta subunit of GTP-binding regulatory proteins identified by molecular cloning.

Two distinct beta subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as beta 1 and beta 2 subunits. The bovine transducin beta subunit (beta 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the beta 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 beta 2 protein is 90% identical with beta 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine beta 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than beta 1 subunit mRNA in all tissues examined. The beta 1 and beta 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that beta 1 and beta 2 are encoded by separate genes. The amino acid sequences for the bovine and human beta 2 subunit are identical, as are the amino acid sequences for the bovine and human beta 1 subunit. This evolutionary conservation suggests that the two beta subunits have different roles in the signal transduction process.     

Monoclonal antibody analysis of keratin expression in the central nervous system

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a "neural keratin."     

cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase.

A lambda gt10 bovine brain and a lambda gt11 bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratory-chain NADH dehydrogenase. Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit. The size and amino acid composition of this protein agree with those determined for the purified 51-kDa subunit. Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur cluster. The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor. A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the alpha subunit of the soluble NAD(+)-reducing hydrogenase from Alcaligenes eutrophus. In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.     

Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units.

Large and small tumor (T)antigens of simian virus 40 were synthesized in vitro with L-cell extracts that had been treated by the method of Palmiter to prevent amino-terminal acetylation of nascent proteins. Partial amino-terminal amino acid sequences of both forms of T-antigen were determined and found to be identical. Methionine residues were located at positions 1 and 14, a lysine residue at position 3, and leucine residues at positions 5, 11, 13,16, 17, and 19. These amino acid sequence data match perfectly the amino acid sequence predicted from a sequence of nucleotides in the E strand of simian virus 40 DNA which begins near the junction between HindII/III fragments A and C at about 0.65 map units. This strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position. Furthermore, the data are consistent with a model for the synthesis of both forms of T-antigen that predicts that (i) small T-antigen is coded for by a sequence of nucleotides from the 5' end of the early region and (ii) large T-antigen is coded for by nucleotide sequences from two noncontiguous regions of simian virus 40 DNA.     

A gene encoding the tryptophan synthase beta subunit of Arabidopsis thaliana.

The DNA sequence of a tryptophan synthase gene and the flanking 5' and 3' regions has been determined for Arabidopsis thaliana. The sequence encodes only the beta subunit domain, indicating that alpha and beta subunits are specified by separate genes. The gene contains four introns and encodes 470 amino acid residues. The plant amino acid sequence is highly conserved with respect to corresponding microbial sequences. The NH2-terminal amino acid sequence is characteristic of chloroplast transit peptides. Identity of the sequences of the genomic exons and a cDNA clone and the presence of cellular RNA corresponding in size and 5' sequence to the gene indicate that the gene is expressed. Analysis of Arabidopsis genomic DNA suggests the presence of a second gene for the beta subunit.     

Deduced amino acid sequence of bovine retinal Go alpha: similarities to other guanine nucleotide-binding proteins.

A bovine retinal cDNA clone encoding the complete sequence (354 amino acids) of Go alpha, a guanine nucleotide-binding protein (G protein), was isolated by using oligonucleotide probes complementary to published sequences in two putative clones for the alpha subunit of bovine transducin (T alpha). The deduced amino acid sequence contained sequences identical to those in seven tryptic peptides (total 63 amino acids) from bovine brain Go alpha. The cDNA for bovine retinal Go alpha exhibits greater than 90% identity in both coding and 3' untranslated regions with a recently described partial cDNA clone for Go alpha from rat brain [Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K. & Kaziro, Y. (1986) Proc. Natl. Acad. Sci. USA 83, 3776-3780]. Comparison of the nucleotide and deduced amino acid sequences of the bovine Go alpha clone with those previously reported for other G proteins of bovine origin (Gs alpha, Gi alpha, and T alpha) reveals extensive regions identical to those surrounding the amino acids modified by cholera toxin and pertussis toxin. There are also marked similarities of sequence in regions of the G proteins, elongation factors, and the ras p21 gene products that are believed to be involved in guanine nucleotide binding and GTP hydrolysis.     

Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.

We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.     

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.     

Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone.

Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide found in the pancreatic amyloid deposits of type II (non-insulin-dependent) diabetic patients and insulinomas. We previously reported the nucleotide sequence of a human cDNA, which indicated that IAPP is a C-terminally amidated peptide derived by proteolytic processing of an 89-amino acid precursor. We now report the isolation of cDNA clones coding for cat, rat, mouse, and guinea pig IAPP precursors, obtained using the combination of "amplification of homologous DNA fragments" (AHF) and "rapid amplification of cDNA ends" (RACE). The predicted structure of IAPP precursors from these four mammals revealed that the IAPP moiety of each is derived from an 89- to 93-amino acid precursor by proteolytic processing and is likely to be amidated at the C terminus. The predicted amino acid sequence identities between the IAPP domains of these four mammals and human IAPP were 89% (cat), 84% (rat and mouse), and 78% (guinea pig). Within the IAPP domains, the N-terminal and C-terminal amino acid sequences are very highly conserved among the mammals, as is also the case with a structurally related neuropeptide, calcitonin-gene-related peptide (CGRP), suggesting that IAPP and CGRP interact with similar though not identical receptors. By contrast, the N- and C-terminal propeptides of the IAPP precursor show very little sequence conservation, which suggests that these regions do not represent additional biologically active molecules. Interspecies variations in the amino acid sequence of residues 20-29 of IAPP may account for the presence of amyloid deposits in the islets of humans and cats and their absence in rats and mice.