Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells

The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences

The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.     

Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization.

The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.     

Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III

The ubiquinone-binding protein (QP-C) is a nuclear-encoded component of ubiquinol-cytochrome c oxidoreductase in the mitochondrial respiratory chain and plays an important role in electron transfer as a ubiquinone-QP-C complex. We obtained a partial cDNA for rat liver QP-C by screening a lambda gt11 rat liver cDNA library using antiserum directed against bovine heart QP-C. Using this cDNA as a probe, a cDNA clone was isolated from a human fibroblast cDNA library by colony hybridization. The total length of the cloned cDNA was 518 base pairs with an open reading frame of 333 base pairs. The 111-amino acid sequence deduced from the nucleotide sequence of the cDNA is 85% homologous to that of bovine QP-C and contains only a single additional amino-terminal methionine. This implies that the human QP-C is synthesized without a presequence which is required for import of most nuclear-encoded mitochondrial proteins into mitochondria.     

Structural organization and chromosomal assignment of the gene encoding endothelin

Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.     

Winter flounder antifreeze proteins: a multigene family

The nucleotide sequence of a cDNA clone of winter flounder antifreeze protein was determined by the dideoxynucleotide method. The sequence would predict a protein of 91 amino acids composed of a prepropeptide of 38 amino acids and a mature protein of 53 amino acids, which includes four complete 11-amino acid repeats. This predicted sequence corresponds to an antifreeze protein of intermediate size which is one 11-amino acid repeat longer than the smallest antifreeze proteins found in the serum of winter flounder during the cold season. Southern blot hybridization analysis of winter flounder genomic DNA with radioactive cDNA probes reveals a multigene family of potential antifreeze protein genes. This conclusion is supported by amino acid sequence analysis of several serum antifreeze proteins.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein S11

A cDNA clone specific for rat ribosomal protein S11 was isolated by hybrid-selected translation from the cDNA library made for 8-9 S poly(A) RNA from regenerating rat liver. Since this cDNA had not enough length, another clone was selected by colony hybridization using a fragment of isolated cDNA as a probe. The nucleotide sequence of the cDNA was determined. The sequence contains 2 base pairs from the 5' noncoding region, the entire coding region of 477 base pairs, and the 3' noncoding region of 55 base pairs besides the poly(A) tail. The primary structure of the protein S11 was deduced from the nucleotide sequence. It consists of 157 amino acids. Its molecular weight is 18,299. The calculated amino acid composition is consistent with the reported composition of S11 determined on the protein hydrolysate. The amino acid sequence showed a marked homology with that of S16 of Halobacterium cutirubrum and an appreciable homology with that of S17 of Escherichia coli.     

Feline cytokines TNFα AND IL‐1β: PCR cloning and sequencing of cDNA

Abstract

Using oligonucleotide primers and polymerase chain reaction (PCR), cDNAs for feline cytokines TNFα and IL‐1β were amplified, cloned, and sequenced. The cDNA for PCR amplification was prepared from mRNA derived from lipopolysaccharide (LPS) stimulated feline bone marrow derived macrophages. PCR was performed using sets of oligonucleotide primers designed to specifically amplify cDNAs for IL‐1β or TNFα. PCR fragments were cloned into pGEM 3ZF(‐) or pCR 1000 vectors, sequenced and consensus nucleotide sequences reported.

The cDNA for feline TNFα had a 98.6% match with coding regions of a genomic clone for feline TNFα which was recently reported (McGraw, 1990). The two feline TNFα clones differ by 8 nucleotide base pair (bp) changes which result in 5 amino acid differences in the predicted protein sequence. A search of GenBank and EMBL determined that the feline TNFα cDNA consensus sequence had a 90, 86, 85, 82 and 83 percent overall match with human, porcine, ovine, mouse and goat TNFα cDNAs, respectively. The protein‐coding sequence for feline TNFα from start to stop codon is 702 bp in length and encodes a predicted protein of 233 amino acids with a molecular weight of approximately 25,446 daltons (precursor form of secreted form of TNFα).

The protein‐coding sequence for feline IL‐1β is 804 bp long and encodes a predicted protein of 267 amino acids with a molecular weight of 31,892 daltons (precursor form of secreted IL‐1β). The feline IL‐1β cDNA consensus sequence had an overall match of 79, 76, 77.5 and 77 percent with IL‐1β cDNA from human, bovine, rabbit and murine species, respectively.     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

Isolation and characterization of the gene and cDNA encoding human mitochondrial creatine kinase

Creatine kinase (CK; EC 2.7.3.2) isoenzymes play prominent roles in energy metabolism. Nuclear genes encode three known CK subunits: cytoplasmic muscle (MCK), cytoplasmic brain (BCK), and mitochondrial (MtCK). We have isolated the gene and cDNA encoding human placental MtCK. By using a dog heart MCK cDNA-derived probe, the 7.0-kb EcoRI fragment from one cross-hybridizing genomic clone was isolated and its complete nucleotide sequence determined. A region of this clone encoded predicted amino acid sequence identical to residues 15-26 of the human heart MtCK NH2-terminal protein sequence. The human placental MtCK cDNA was isolated by hybridization to a genomic fragment encoding this region. The human placental MtCK gene contains 9 exons encoding 416 amino acids, including a 38-amino acid transit peptide, presumably essential for mitochondrial import. Residues 1-14 of human placental MtCK cDNA-derived NH2-terminal sequence differ from the human heart MtCK protein sequence, suggesting that tissue-specific MtCK mRNAs are derived from multiple MtCK genes. RNA blot analysis demonstrated abundant MtCK mRNA in adult human ventricle and skeletal muscle, low amounts in placenta and small intestine, and a dramatic increase during in vitro differentiation induced by serum-deprivation in the non-fusing mouse smooth muscle cell line, BC3H1. These findings demonstrate coordinate regulation of MtCK and cytosolic CK gene expression and support the phosphocreatine shuttle hypothesis.     

Isolation, characterization, and mapping of a human acid beta-galactosidase cDNA

A lambda gt11 human testicular cDNA library was screened with degenerate oligonucleotide probe mixtures based on amino acid sequence data generated from cyanogen bromide fragments and tryptic fragments of purified human beta-galactosidase. Six positive clones were identified after screening 2 x 10(6) plaques. The sequences of these six clones were determined and found to be derived from two different cDNAs. The sequence of the longest of these cDNAs is nearly identical to that recently determined by Oshima et al. (1988). It codes for a 76-kD protein and all 11 peptides that were generated from the purified enzyme. The second clone is shorter by 393 bp in the central portion of the coding region. Analysis by Northern blotting revealed the presence of a single mRNA species of 2.45 kb in lymphoblasts and testicular tissue. It is deduced from the amino acid sequence data that proteolytic processing of the precursor form of beta-galactosidase must occur by cleavage in the carboxy-terminal portion of the polypeptide perhaps around amino acid 530 at a uniquely hydrophilic sequence. Using a probe generated from the 3' region of the cDNA, we have mapped the locus coding for human beta-galactosidase to chromosome 3p21-3pter.