Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

GltX from Clostridium saccharobutylicum NCP262: glutamate synthase or oxidoreductase?

A full-length gene encoding a homologue of the small subunit of the glutamate synthase (GOGAT) enzyme was isolated from the anaerobic bacterium, Clostridium saccharobutylicum NCP262, which has been used extensively for the commercial production of solvents. Using a screening system designed to isolate genes involved in electron transport, plasmid pMET13C1 was isolated. Analysis of this plasmid identified a gene (1245 bp) with a predicted approximately 46-kDa product, which was associated with reductive activation of the pro-drug metronidazole. The deduced 414-amino-acid sequence was not typical of electron transport proteins, but rather shared striking homology to the small (beta) subunit of the GOGAT enzyme and other beta subunit-like polypeptides, and was thus designated gltX. Although all the functional domains typical of GOGAT beta subunits were conserved in this GltX protein, certain sequence features were not conserved. Furthermore, it was independently transcribed, did not lie adjacent to a GOGAT large subunit (alpha) domain, and its expression was not regulated by nitrogen conditions. These results provide additional support for current theories on the evolutionary relationships of GOGAT beta subunit domains in bacteria, and suggest that GltX belongs to a more general family of oxidoreductases, which is used in a context other than glutamate biosynthesis to transfer electrons to a currently unknown protein domain.     

The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression

A gene family of small membrane proteins, represented by phospholemman and the gamma subunit of Na,K-ATPase, was defined and characterized by the analysis of more than 1000 related ESTs (expressed sequence tags). In addition to new and more complete cDNA sequence for known family members (including MAT-8, CHIF, and RIC), the findings included two new family members and new splicing variants. A large number of EST replicates made it possible to derive curated DNA sequence with higher confidence and accuracy than from the sequencing of individual clones. The family has a core motif of 35 invariant and conserved amino acids centered on a single transmembrane span. Features of each predicted protein product were compared, and tissue distributions were determined. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif. The abundant proteins are involved in the control of ion transport.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum).

A pea leaf cDNA library constructed in lambda gt11 was screened with an antibody raised to the P subunit of glycine decarboxylase. One of the positive clones isolated was sequenced and shown to contain an open reading frame, which encoded the entire P subunit polypeptide. Aligning the deduced amino acid sequence with the amino acid sequence determined directly from the NH2 terminus of the mature P subunit shows the presence of a putative 86 amino acid leader sequence, presumably required for import into the mitochondria, and gives a Mr of the mature protein of 105,000. Comparison of this deduced amino acid sequence with the sequence of a pyridoxal phosphate-containing peptide isolated from the P subunit of chicken liver glycine decarboxylase shows remarkable conservation. The P subunit, however, shows little sequence homology with other published amino acid decarboxylases. Expression of the P subunit mRNA shows a pattern very similar to that of the corresponding polypeptide: it is strongly light induced and is expressed at a much higher level in leaves than in other tissues. Southern blot analysis suggests that the P subunit is encoded by a small multigene family.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.     

Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic 'B' subunit of vacuolar ATPase of B. hominis (HE87-1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87-1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.     

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.     

Structural characterization of Escherichia coli phosphatidylserine decarboxylase

Phosphatidylserine decarboxylase of Escherichia coli is one of a small group of pyruvoyl-dependent enzymes (Satre, M., and Kennedy, E.P. (1978) J. Biol. Chem. 253, 479-483). The DNA sequence of the structural gene (psd) and partial protein sequence studies demonstrate that the enzyme contains two nonidentical subunits, alpha (Mr = 7,332) and beta (Mr = 28,579), which are derived from a single proenzyme. These two subunits are blocked at their respective amino termini. Reduction of the enzyme with NaCNBH3 in the presence of radiolabeled phosphatidylserine resulted in association of the label with the alpha subunit. Similar reduction in the presence of ammonium ions exposed a new amino terminus for the alpha subunit beginning with alanine. Therefore, the pyruvate prosthetic group is in amide linkage to the amino terminus of the alpha subunit. The amino terminus of the beta subunit was determined to be formylmethionine. The carboxyl terminus of the beta subunit was determined to be glycine as predicted by the DNA sequence. Comparison of the DNA sequence and protein sequence information revealed that the decarboxylase is made as a proenzyme (Mr = 35,893), and the predicted amino acid at the position of the pyruvate within the open reading frame of the proenzyme is serine. Therefore, as with other pyruvoyl-dependent decarboxylases, the prosthetic group is derived from serine through a post-translational cleavage of a proenzyme.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

7S Nerve growth factor alpha and gamma subunits are closely related proteins

The polypeptide composition and partial amino acid sequence of the 7S nerve growth factor (NGF) alpha subunit have been determined. Residues in 76 unique positions corresponding to 35% of the molecule were identified. The sequence shows that the NGF alpha subunit is closely related to the NGF gamma subunit and thus a member of the same protein family as the serine proteases. This finding is unexpected since the NGF alpha subunit is devoid of detectable protease activity. However, the NGF alpha subunit differs in one important respect from the NGF gamma subunit and related serine proteases. The highly conserved amino-terminal activation cleavage structure, common to most serine proteases, has been deleted, and an uncleaved activation peptide remains attached to the amino terminus of the mature NGF alpha subunit. It is suggested that this feature is causally related to the apparent lack of proteolytic activity.     

A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.     

Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle [published erratum appears in J Cell Biol 1994 Feb;124(3):395]

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. A novel integrin alpha subunit partial cDNA isolated from TGF- beta stimulated guinea pig airway epithelial cells has previously been reported (Erle, D.J., D. Sheppard, J. Bruess, C. Ruegg, and R. Pytela. 1991. Am. J. Respir. Cell Mol. Biol. 5:170-177). We have now determined cDNA and amino acid sequence for the human homolog of this subunit, named alpha 9, from a human lung cDNA library, a human small intestine cDNA library, and cDNA from the cell lines U937, HL-60 and Tera-2. This sequence is predicted to encode a 1006-amino acid mature protein that shares 39% identity with the previously identified integrin subunit alpha 4. By Northern blot analysis, alpha 9 mRNA was detected in the human carcinoma cell lines Tera-2 and Caco-2. Anti-peptide antibodies against the predicted COOH-terminal sequence of alpha 9 immunoprecipitated a heterodimer (140 kD/115 kD nonreduced; 150 kD/130 kD reduced) from Tera-2 lysates. Immunodepletion of beta 1-containing integrins with Tera-2 lysates removed alpha 9 immunoreactivity, suggesting that beta 1 is the principal beta subunit partner for alpha 9 in these cells. alpha 9 was detected by immunohistochemistry in airway epithelium, in the basal layer of squamous epithelium, and in smooth muscle, skeletal muscle, and hepatocytes.     

Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA

Ribonuclease P (RNaseP) catalyses the removal of the 5'-leader sequence from pre-tRNA to produce the mature 5' terminus. The prokaryotic RNaseP holoenzyme consists of a catalytic RNA component and a protein subunit (RNaseP protein), which plays an auxiliary but essential role in vivo by binding to the 5'-leader sequence and broadening the substrate specificity of the ribozyme.We determined the three-dimensional high-resolution structure of the RNaseP protein from Staphylococcus aureus (117 amino acid residues) by nuclear magnetic resonance (NMR) spectroscopy in solution. The protein has an alphabeta-fold, similar to the ribonucleoprotein domain. We used small nucleic acid molecules as a model for the 5'-leader sequence to probe the propensity for generic single-stranded RNA binding on the protein surface. The NMR results reveal a contiguous interaction site, which is identical with the previously identified leader sequence binding site in RNaseP holoenzyme. The conserved arginine-rich motif does not bind single-stranded RNA. It is likely that this peptide segment binds selectively to double-stranded sections of P RNA, which are conformationally more rigid. Given the essentiality of RNaseP for the viability of the organism, knowledge of the S. aureus protein structure and insight into its interaction with RNA will help us to develop RNaseP and RNaseP protein as targets for novel antibiotics against this pathogen.     

The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells.

The major cytoplasmic target of various tyrosine-specific protein kinases is a 36-kd protein (p36). This protein can exist as a monomer or as a complex with a small subunit which seems to have a regulatory function. Amino acid sequence analysis of the small subunit from porcine intestine documents a unique polypeptide of 95 residues with a calculated mol. wt. close to 11 kd (p11). Since an immunologically related subunit of the same electrophoretic mobility is also found in the corresponding complex of chicken intestine p11 is well conserved across species. Unexpectedly, the sequence of p11 shows a high homology with the glia-specific protein S-100 whose biological function is not known. Although both proteins are dimers of rather small polypeptides we have not been able to detect in our preparations of p11 the moderate Ca2+ binding known for S-100. Certain implications of this sequence relation are discussed.