Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA

The mRNA encoding human thyroglobulin has been cloned and sequenced. It is made up of a 8301-nucleotide segment encoding a preprotein monomer of 2767 amino acids, flanked by non-coding 5' and 3' regions of 41 and 106 nucleotides, respectively. This preprotein consists of a leader sequence of 19 amino acids, followed by the sequence of the mature monomer, corresponding to a polypeptide of 2748 amino acids (Mr = 302773). On its amino-terminal side, 70% of the monomer is characterized by the presence of three types of repetitive units. In contrast, the remaining 30% of the protein is devoid of repetitive units. This last region however shows an interesting homology (up to 64%) with the acetylcholinesterase of Torpedo californica. The sites of thyroid hormones synthesis are clustered at both ends of the thyroglobulin monomer. By contrast, the potential glycosylation sites are scattered along the polypeptide chain.     

Putative amino acid sequence of chick calcium-binding protein deduced from a complementary DNA sequence.

Two DNA fragments coding for chick CaBP have been isolated and sequenced. cDNA was prepared from enriched intestinal mRNA and cloned in pUC12. The recombinant clones were screened by differential hybridisation with 32P-cDNA probes synthesized from vitamin D replete and deficient chick intestinal mRNA. Two clones had outstanding affinity with the +D probe. Hybrid-arrested and hybrid-selected translation systems showed that both clones hybridised to mRNA coding for immunoprecipitable CaBP. The mRNA for CaBP has a 100 bp G,C rich sequence before a 786 bp coding region followed by 1250 nucleotides 3' untranslated region. Nucleotides coding for the Ca-binding sites show a high degree of homology for Ca-binding sites in chick calmodulin and rat intestinal CaBP. The amino acid sequence specified by the longest open reading frame contains five Ca-binding sites but is too large for the native CaBP; post-translational modification must therefore occur.     

Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.     

Primary structure of human proacrosin deduced from its cDNA sequence

cDNA clones encoding proacrosin, the zymogen of acrosin, were isolated from a human testis cDNA library by using a fragment of boar acrosin cDNA as a probe. Nucleotide sequencing of the longest cDNA clone has predicted that human proacrosin is synthesized with a 19-amino-acid signal peptide at the N-terminus. The cleavable signal sequence is followed by a 23-residue segment corresponding to the light chain and then by a 379-residue stretch that constitutes the heavy chain containing the catalytic site of the mature protease. The C-terminal portion of the deduced sequence for the heavy chain is very rich in proline residues, most of which are encoded by a unique repeat of CCCCCA. The active-site residues including histidine, aspartic acid, and serine are also predicted to be located at residues 69, 123, and 221, respectively.     

The sexual inducer of Volvox carteri. Primary structure deduced from cDNA sequence

The primary structure of the sexual inducer of Volvox carteri f. nagariensis has been deduced by cloning and sequence analysis of cDNA. The sexual inducer contains 208 amino acids including a signal sequence. A total of six potential N-glycosylation sites are found within the polypeptide chain. At the genomic level, the sexual inducer protein is encoded in five exons.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence

The complete amino acid sequence of acetyl-CoA carboxylase from chicken liver has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by Edman degradation of peptide fragments obtained by digestion of the enzyme polypeptide with Achromobacter proteinase I or staphylococcal serine proteinase. Chicken liver acetyl-CoA carboxylase is predicted to be composed of 2,324 amino acid residues, having a calculated molecular weight of 262,706. The biotin carboxyl carrier protein domain is located in the middle region of the enzyme polypeptide. The amino-terminal portion of the acetyl-CoA carboxylase has been found to exhibit a homologous primary structure to that of carbamyl phosphate synthetase. Localization of possible functional domains including biotin carboxylase subsite in the acetyl-CoA carboxylase polypeptide is discussed.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence

The complete amino acid sequence of prostaglandin endoperoxide synthase from sheep vesicular gland has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by digestion of the enzyme with carboxypeptidase Y and by automated Edman degradation of the intact enzyme polypeptide and peptide fragments obtained by limited proteolysis of the enzyme with Achromobacter proteinase I. Mature sheep prostaglandin endoperoxide synthase is shown to be composed of 576 amino acids with an Mr of 66,175. The precursor peptide is predicted to contain a 24-residue signal peptide. The serine residue susceptible to acetylation by aspirin is found to be located near the C-terminus of the enzyme polypeptide.     

Primary structure of Bacillus subtilis enolase gene deduced from c-DNA sequence

The nucleotide sequence for enolase gene of Bacillus subtilis was determined from recombinant clone pRE. The sequence was composed of 1570 bp which included the 1374 bp of the complete coding region, the 86 bp of the 5' noncoding region and the 110 bp of the 3' noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome binding site was located 20 nucleotides upstream of the initiation codon in the 5' noncoding region. The aminoacid sequence deduced from the nucleotide sequence was 558 aminoacids in length. The size of the mRNA was 1.5 kb by the northern transfer technique.     

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.     

Primary structure of rat liver mannan-binding protein deduced from its cDNA sequence

cDNA clones encoding rat liver mannan-binding protein (MBP), a lectin specific for mannose and N-acetylglucosamine, were isolated from a rat liver cDNA library carried in lambda gt 11, by screening with affinity purified polyclonal rabbit anti-rat liver MBP antibodies. The nucleotide sequence of the cDNA determined by the dideoxy method revealed the complete amino acid sequence of the MBP (226 residues). The NH2-terminal residue of the MBP, glutamic acid, was preceded by a predominantly hydrophobic stretch of 18 amino acids, which was assumed to be a signal peptide. Near the NH2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence Gly-X-Y. Here, X and Y were frequently proline and lysine. Three proline and lysine residues were hydroxylated, and one of the latter appeared to link to galactose. Computer analysis of several lectins for sequence homology suggested that the COOH-terminal quarter of the MBP is associated with the calcium binding as well as carbohydrate recognition.     

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence

Sweet potato (Ipomoea batatas) starch phosphorylase cDNA clones were isolated by screening an expression library prepared from the young root poly(A)⁺ RNA successively with an antiserum, a monoclonal antibody, and a specific oligonucleotide probe. One cDNA clone had 3292 nucleotide residues in which was contained an open reading frame coding for 955 amino acids. This sequence was compared with those of potato (916 residues plus 50-residue putative transit peptide) and rabbit muscle (841 residues) phosphorylases. The sweet potato phosphorylase has an overall structural feature highly homologous to that reported for potato phosphorylase, in conformity with the finding that they belong to the same class of plant phosphorylase. High divergencies of the two enzymes are found in the about 70 residue N-termini each including a putative transit peptide, and the midchain 78 residue insert typical of type I plant phosphorylase. We consider that the very high dissimilarity found in the midchain inserts is related to the difference in proteolytic lability of the two plant phosphorylases. Some structural features of the cDNA clone were also discussed.     

Primary structure of the alpha-subunit of human Na,K-ATPase deduced from cDNA sequence

Clones carrying cDNA sequences for the alpha-subunit of the Na,K-ATPase from HeLa cells have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 1,023 amino acids. The alpha-subunit of the human Na,K-ATPase exhibited 87% homology with its Torpedo counterpart and 98% homology with its sheep counterpart. The six putative transmembrane segments M1-M6 showed higher conservation than the total segments. Total genomic Southern hybridization indicated the existence of at most two copies, possibly only one, of the gene encoding the Na,K-ATPase alpha-subunit in the human genome.