Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5′ untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3′ untranslated region of 142 nucleotides. The molecular mass of the encoded polypeptide was calculated to be 96.392. Its amino acid sequence shows a high homology with that of other plant lipoxygenases identified to date.     

Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

Regulatory region of fr phage replicase cistron. II. Isolation and structure of specific fr RNA fragments

A new set of short RNA templates has been prepared for functional studies in initiation of translation in vitro. Number of individual RNA fragments which contain complete or part of the initiatory region of phage fr replicase cistron were isolated from complex fr RNA--fr coat protein. Their primary structure were determined by using standard fingerprint technique and rapid gel sequencing. Secondary structure of several RNA fragments and their binding activity with phage fr and MS2 coat proteins has been also studied.     

Primary structure of a proline-rich zein and its cDNA

Eighty-five cDNA clones for γ-zein (proline-rich zein) from a cDNA expression library were isolated using specific antibody and cDNA probes. Nucleotide sequences of seven independent clones were determined and found to be identical in regions where they overlapped. The primary structure of the mature protein, determined from the sequence of one near full-length clone, consists of 204 amino acids. It has a molecular weight of 21,824 daltons, about 5 kilodaltons less than that estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal one-half of the sequence contained eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln. The codon specifying the third proline in the hexapeptide repeating units is identical (CCG) in all of the eight repeats. The coding region has a very high G-C content (69.8%). The multiple charge components of γ-zein detected by isoelectric focusing do not seem to be encoded by members of a multigene family. Moreover, it was found that the codon preference in γ-zein is, in fact, the base preference in the wobble position. A codon usage value was devised to express this phenomenon.     

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.     

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 mouse tyrosine hydroxylase deduced from its cDNA.

The cDNAs for tyrosine hydroxylase were cloned from a mouse brain cDNA library by plaque hybridization. Since the longest cDNA clone lacked approximately 150 bp sequence of its N-terminal region, additional 5' region was obtained using polymerase chain reaction. Nucleotide sequence determination of cDNAs revealed that mouse tyrosine hydroxylase m-RNA encodes 498 amino acids with a calculated molecular mass of 55,990. The amino acid sequence of mouse tyrosine hydroxylase is highly homologous to rat (97%) and human (92%) enzymes.     

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 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 cDNA for bovine beta-casein

In this work the complete sequence of 1097 nucleotide bovine beta-casein cDNA has been determined. Sequencing of end labeled fragments was performed using the method of Maxam and Gilbert. Bovine beta-casein cDNA consist of a 672 nucleotide coding region, flanked by 60 nucleotide 5' and 358 nucleotide 3'-noncoding region. The restriction map of beta-casein cDNA was constructed. The computer analysis was done and the comparisons of the complete sequence of bovine beta-casein cDNA with other sequences, coding caseins in bovine, rat and guinea-pig was performed. It was determined, that cloned cDNA is related to genetic variant A1.     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

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.     

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 rabbit skeletal muscle glycogen synthase deduced from cDNA clones

The complete amino acid sequence of rabbit skeletal muscle glycogen synthase was deduced from cDNA clones with a composite length of 3317 bp. An mRNA of 3.6 kb was identified by Northern blot analysis of rabbit skeletal muscle RNA. The mRNA coded for a protein of 734 residues with a molecular weight of 83,480. The deduced NH2-terminal and COOH-terminal sequences corresponded to those reported for the purified protein, indicating the absence of any proteolytic processing. At the nucleotide level, the 5' untranslated and coding regions were 79 and 90% identical for rabbit and human muscle glycogen synthases, whereas the 3' untranslated regions were significantly less similar. The enzymes had 97% amino acid sequence identity. Interestingly, the NH2 and COOH termini of rabbit and human muscle glycogen synthase, the regions of phosphorylation, showed the greatest sequence variation (15 of 19 mismatches and two insertion/deletion events), which may indicate different evolutionary constraints in the regulatory and catalytic regions of the molecule.     

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 human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.