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PubMed1987

Complementary DNA cloning of complement C8 beta and its sequence homology to C9.

The complete amino acid sequence of mature C8 beta has been derived from the DNA sequence of a cDNA clone identified by expression screening of a human liver cDNA library. Comparison with the amino acid sequence of C9 shows an overall homology with few deletions and insertions. In particular, the cysteine-rich domains and membrane-inserting regions of C9 are well conserved. These findings are discussed in relation to a possible mechanism of membrane attack complex formation.

Amino Acid Sequence↗
PubMed1987

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA.

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Complementary DNA and derived amino acid sequence of the beta subunit of human complement protein C8: identification of a close structural and ancestral relationship to the alpha subunit and C9.

A cDNA clone encoding the beta subunit (Mr 64,000) of the eighth component of complement (C8) has been isolated from a human liver cDNA library. This clone has a cDNA insert of 1.95 kilobases (kb) and contains the entire beta sequence [1608 base pairs (bp)]. Analysis of total cellular RNA isolated from the hepatoma cell line HepG2 revealed the mRNA for beta to be approximately 2.5 kb. This is similar to the message size for the alpha subunit of C8 and confirms the existence of different mRNAs for alpha and beta. This finding supports genetic evidence that alpha and beta are encoded at different loci. Analysis of the derived amino acid sequence revealed several membrane surface seeking segments that may facilitate beta interaction with target membranes during complement-mediated cytolysis. Determination of the carbohydrate composition indicated 1 or 2 asparagine-linked but no O-linked oligosaccharide chains. Comparison of the beta sequence to that reported for alpha in the preceding paper [Rao, A. G., Howard, O. M. Z., Ng, S. C., Whitehead, A. S., Colten, H. R. & Sodetz, J. M. (1987) Biochemistry (preceding paper in this issue)] and to that of human C9 revealed a striking homology between all three proteins. For beta and alpha, the overall homology is 33% on the basis of identity and 53% when conserved substitutions are allowed. For beta and C9, the values are 26% and 47%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of a complementary DNA for the nuclear-coded precursor of the beta-subunit of bovine mitochondrial F1-ATPase.

We have isolated a cDNA clone encoding the precursor of the beta-subunit of the bovine heart mitochondrial F1-ATPase. Two probes were used to isolate this precursor from a bovine heart cDNA library. One probe was a mixed-sequence oligonucleotide directed against a portion of the amino acid sequence of the mature protein, and the other probe was the F1-ATPase beta-subunit gene from Saccharomyces cerevisiae. Determination of the nucleotide sequence of this cDNA reveals that it contains a 1584-nucleotide-long open reading frame that encodes the complete mature beta-subunit protein and a 48 amino acid long NH2-terminal extension. This amino-terminal presequence contains four basic arginine residues, one acidic glutamic acid residue, four polar uncharged serine residues, and five proline residues. Southern blot hybridization analyses suggest that the bovine F1-ATPase beta-subunit precursor is encoded by a single genetic locus. RNA blot hybridization analyses reveal a single mRNA species of approximately 1.9 kilobases from both bovine liver and heart.

Amino Acid Sequence↗
PubMed1989

Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit.

The 24-kDa subunit of mitochondrial NADH-ubiquinone reductase (complex I) is an iron-sulfur protein that is present in the flavoprotein or NADH dehydrogenase II subcomplex. It is a nuclear gene product and is imported into the organelle. A group of human patients with mitochondrial myopathy have been shown to have reduced levels of subunits of complex I in skeletal muscle mitochondria, and in one patient the 24-kDa subunit appears to be absent (Schapira et al., 1988). To investigate the genetic basis of this type of myopathy, cDNA clones have been isolated from a bovine library derived from heart and liver mRNA by hybridization with two mixtures of 48 synthetic oligonucleotides 17 bases in length that were designed on the basis of known protein sequences. The recombinant DNA sequence has been determined, and it encodes a precursor of the mature 24-kDa protein. The N terminus of the mature protein is preceded by a presequence of 32 amino acids that has properties that are characteristic of mitochondrial import sequences. The sequence of the mature protein deduced from the cDNA contains a segment of nine amino acids that was not determined in an earlier partial protein sequence analysis. The bovine clone has been employed as a hybridization probe to identify cDNA clones of the human homologue of the 24-kDa protein. Its DNA sequence has also been determined, and it codes for a protein that is closely related to the bovine protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA.

Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.

Adipose Tissue↗
PubMed1989

Mitochondrial NADH:ubiquinone reductase: complementary DNA sequence of the import precursor of the bovine 75-kDa subunit.

The 75-kDa subunit of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria is its largest subunit and is a component of the iron-sulfur (IP) fragment of the enzyme. It is encoded in nuclear DNA and is imported into the organelle. Protein sequences have been determined at the N-terminus of the intact protein and on fragments generated by partial cleavage with cyanogen bromide and with Staphylococcus aureus protease V8. Parts of these data have been used to design two mixtures of oligonucleotides 17 bases long, containing 192 and 256 different sequences, which have been synthesized and used as hybridization probes for identification of cognate cDNA clones. Two different but overlapping clones have been isolated, and the sequences of the cloned DNAs have been determined. Together they code for a precursor of the 75-kDa subunit of complex I. The mature protein is 704 amino acids in length, has a calculated molecular mass of 75,961 daltons, and contains no segments of sequence that could be folded into hydrophobic alpha-helixes of sufficient length to span the inner membrane of the mitochondrion. Its precursor has an N-terminal extension of 23 amino acids to specify its import into the mitochondrion from the cytoplasm. Seventeen cysteine residues are dispersed throughout the 75-kDa subunit; some of them are close to each other in the sequence in three separate groups and, by analogy with other iron-sulfur proteins, could be involved in iron-sulfur clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing.

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1976

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.

Animals↗
PubMedDate not supplied

Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells.

The partial amino-acid sequence of purified human transforming growth factor-beta (TGF-beta) was used to identify a series of cDNA clones encoding the protein. The cDNA sequence indicates that the 112-amino acid monomeric form of the natural TGF-beta homodimer is derived proteolytically from a much longer precursor polypeptide which may be secreted. TGF-beta messenger RNA is synthesized in various normal and transformed cells.

Amino Acid Sequence↗
PubMedDate not supplied

Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.

When stimulated with antigen, B cells are influenced by T cells to proliferate and differentiate into antibody-forming cells. Since it was reported that soluble factors could replace certain functions of helper T cells in the antibody response, several different kinds of lymphokines and monokines have been reported in B-cell growth and differentiation. Among these, human B-cell differentiation factor (BCDF or BSF-2) has been shown to induce the final maturation of B cells into immunoglobulin-secreting cells. BSF-2 was purified to homogeneity and its partial NH2-terminal amino-acid sequence was determined. These studies indicated that BSF-2 is functionally and structurally unlike other known proteins. Here, we report the molecular cloning, structural analysis and functional expression of the cDNA encoding human BSF-2. The primary sequence of BSF-2 deduced from the cDNA reveals that BSF-2 is a novel interleukin consisting of 184 amino acids.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of a complementary DNA to rat cyclophilin-like protein mRNA.

Using the technique of differential plaque filter hybridization, a rat cDNA was isolated whose corresponding gene expression in the kidney was positively modulated up to threefold by sodium depletion. This mRNA was more abundantly expressed in the kidneys of 17-week-old spontaneously hypertensive rats than those of age-matched Wistar-Kyoto rats. The putative protein encoded by this cDNA is a homologue of cyclophilin, a cytosolic binding protein for cyclosporin A. This cyclophilin-like protein mRNA was expressed in all the tissues examined, including the adrenal, atrium, brain, kidney, liver, lung, spleen, and ventricle. Sodium depletion in rats increased the expression level of this mRNA not only in the kidney but also in the liver. The administration of cyclosporin A in rats increased the expression level of this mRNA in the kidneys and livers. By virtue of its possible involvement in sodium homeostasis and its homology to cyclophilin, this molecule might have significant implications in the mechanism of cyclosporine-induced renal insufficiency and hypertension.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.

Base Sequence↗
PubMed1990

The epsilon-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence, expression in bovine tissues and evidence of homologous sequences in man and rat.

The epsilon-subunit of ATP synthase from bovine heart mitochondria is assembled into the extrinsic membrane sector, F1-ATPase. The mature protein is 50 amino acid residues in length and its function is unknown. It is a nuclear gene product that is imported into the organelle. A mixture of 64 oligonucleotides 17 bases long, designed on the basis of the known protein sequence, was synthesized and used as a hybridization probe to isolate a cognate cDNA clone from a bovine library. The DNA sequence of this clone was determined, and the protein sequence of the epsilon-subunit deduced from it agrees exactly with that determined by direct sequence analysis of the protein isolated from bovine hearts. The bovine cDNA was used as a hybridization probe to examine the expression of the epsilon-subunit in various bovine tissues. mRNAs related to the cDNA are found in all of these tissues, and no evidence was obtained of the presence of mRNAs for the epsilon-subunit with similar coding sequences and dissimilar 3' non-coding regions. By hybridization experiments with digests of DNA from cow, man and rat it has been shown that sequences related to the bovine cDNA are present in the genomes of all three species. More than one related sequence was detected in all cases, indicating the presence in all three genomes of more than one gene and/or pseudogenes.

Amino Acid Sequence↗