Glycinin A5A4B3 mRNA: cDNA cloning and nucleotide sequencing of a splitting storage protein subunit of soybean

The complete nucleotide sequence of a cloned cDNA, designated pGA5A4B3822, corresponding to glycinin A5A4B3 mRNA was determined. Analysis of the cDNA insert revealed that it contained 1899 nucleotides of mRNA sequence with a 5'-terminal non-translated region of 31 nucleotides, a signal peptide region corresponding to 23 amino acids, an acidic subunit region (A5) corresponding to 97 amino acids, an acidic subunit region (A4) corresponding to 257 amino acids followed by a basic subunit region (B3) corresponding to 185 amino acids, and a 3'-terminal non-translated region of 182 nucleotides. These results show that the glycinin A4 subunit, which is not found to be linked to a basic subunit via a disulfide bond, is synthesized as a full-sized precursor, i.e. the A5A4B3 subunit complex, from a single mRNA, followed by post-translational processing to generate an intermediary subunit complex (A5-B3), covalently linked by a disulfide bond, and the mature A4 subunit, which may associate with the above subunit complex by non-covalent interactions. From the results obtained by the Chou-Fasman rules we speculated that the two post-translational cleavage sites of this subunit precursor might be processed by the same proteolytic enzyme.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

Cloning and sequencing of chloroperoxidase cDNA.

An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast.     

Glycinin A4A5 subunit digesting protease in soybean seeds.

Endopeptidase was partially purified from the globulin fraction of 4-hr-imbibed soybean seeds. The protease fraction obtained had proteolytic activity on the glycinin A4A5 subunit at both pH 4 and 8. A suitable peptidic substrate for the endopeptidase was isolated from the tryptic digest of the carboxymethylated A4A5 subunit. Using the tryptic peptide of glycinin A5 subunit, a simple assay system for the soybean endopeptidase activity has been established. The activity was significantly inhibited by phenylmethylsulfonyl fluoride, indicating the endopeptidase is a serine protease.     

Cloning and sequencing of a full length cDNA corresponding to human cellular retinol-binding protein

We have isolated and sequenced a cDNA clone corresponding to the human cellular retinol-binding protein (CRBP). The deduced amino acid sequence, which encompasses 134 amino acid residues, shows significant homology with several low molecular weight proteins which bind hydrophobic ligands. No homology to the plasma retinol-binding protein was observed. Southern and Northern blot analyses suggest that the CRBP gene is present in a single copy in the haploid genome and that it is transcribed in a single mRNA species.     

Cloning and sequencing of a full length cDNA coding for human retinol-binding protein.

We have isolated and sequenced a cDNA clone coding for human Retinol Binding Protein. The sequence indicates that Retinol Binding Protein is synthesized as a single polypeptide chain precursor which is then matured to the secreted protein by removal of a leader peptide. Southern and Northern blot analysis suggest that the gene is present in one or few copies per haploid genome and is transcribed in a single mRNA species.     

Purification of cDNA complementary to sea urchin histone mRNA.

Complementary DNA (cDNA) was transcribed from a polyadenylated sea urchin histone mRNA preparation isolated by density gradient centrifugation. By hybridization, this cDNA was shown to be extensively contaminated (85% of hybridizable cDNA) with DNA complementary to RNA derived from the large ribosomal subunit. Purification of a mRNA specific cDNA fraction was achieved by hybridization of purified rRNA to cDNA followed by fractionation on hydroxyapatite. After further purification to remove nonhybridizable cDNA our purified cDNA showed only 8% hybirdization to rRNA.     

Cloning and sequencing of a cDNA encoding the rat Bcl-2 protein

A rat cDNA encoding the Bcl-2 protein was cloned and sequenced. The primary amino-acid sequence deduced from the nucleotide sequence reveals a 236-aa protein having extensive homology with the mouse (95%), human (87%) and chicken (71%) Bcl-2 proteins.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Cloning of cDNA for cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), and characterization of recombinant CBBP

Previously, we purified the cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP) from Sarcophaga and suggested its participation in the translational regulation of cathepsin B mRNA in this insect. In this study, we isolated a full length cDNA for CBBP. CBBP was an RNA-binding protein that contained four RGG boxes and four zinc finger motifs required for RNA binding. CBBP was shown to be localized in both the nuclei and cytoplasm of Sarcophaga hemocytes. Recombinant CBBP bound to the entire region of cathepsin B mRNA and repressed its translation in vitro.     

人血小板因子4基因cDNA的克隆与序列测定

为研究人血小板因子4（hPF4)的生物学性能和用于肿瘤生物治疗的可能性,运用反转录聚合酶链反应（RT－PCR）从人工白血病细胞系HEL细胞总RNA中扩增出hPF4基因cDNA序列,将其克隆至pUC18载体中。序列分析证实克隆片段与献报道的hPF4基因cDNA序列完全一致,说明已成为克隆到hPF4基因cDNA。