Molecular cloning and expression of the large subunit of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves

A cDNA clone, blpl14, corresponding to the large subunit of ADP-glucose pyrophosphorylase (AGPase), has been isolated from a cDNA library prepared from leaves of barley (Hordeum vulgare L.). An open reading frame encodes a protein of 503 aa, with a calculated molecular weight of 54 815. The derived aa sequence contains a putative transit peptide sequence, required for targeting to plastids, and has a highly conserved positioning of critical Lys residues that are believed to be involved in effector binding. The derived aa sequence shows 97% identity with the corresponding protein from wheat, but only 36% identity with AGPase from E. coli. The blpl14 gene is expressed predominantly in leaves and to a lesser degree in seed endosperm, but not roots, of barley.     

Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor

In human cells, there are three genes that encode DNA ligase polypeptides with distinct but overlapping functions. Previously small molecule inhibitors of human DNA ligases were identified using a structure-based approach. Three of these inhibitors, L82, a DNA ligase I (LigI)-selective inhibitor, and L67, an inhibitor of LigI and DNA ligases III (LigIII), and L189, an inhibitor of all three human DNA ligases, have related structures that are composed of two 6-member aromatic rings separated by different linkers. Here we have performed a structure-activity analysis to identify determinants of activity and selectivity. The majority of the LigI-selective inhibitors had a pyridazine ring whereas the LigI/III- and LigIII-selective inhibitors did not. In addition, the aromatic rings in LigI-selective inhibitors had either arylhydrazone or acylhydrazone, but not vinyl linkers. Among the LigI-selective inhibitors, L82-G17 exhibited increased activity against and selectivity for LigI compared with L82. Notably. L82-G17 is an uncompetitive inhibitor of the third step of the ligation reaction, phosphodiester bond formation. Cells expressing LigI were more sensitive to L82-G17 than isogenic LIG1 null cells. Furthermore, cells lacking nuclear LigIIIα, which can substitute for LigI in DNA replication, were also more sensitive to L82-G17 than isogenic parental cells. Together, our results demonstrate that L82-G17 is a LigI-selective inhibitor with utility as a probe of the catalytic activity and cellular functions of LigI and provide a framework for the future design of DNA ligase inhibitors.     

NTGLO: a tobacco homologue of the GLOBOSA floral homeotic gene of Antirrhinum majus: cDNA sequence and expression pattern

We report the cloning and DNA sequence of a cDNA from Nicotiana tabacum, NTGLO, as well as the pattern of expression of the NTGLO gene in wild-type tobacco plants. The NTGLO cDNA encodes a protein of 209 amino acids, which shows 73% identity with the GLO protein encoded by the GLO gene of Antirrhinum majus, a homeotic gene involved in the genetic control of flower development. Northern blot analysis shows that the NTGLO gene is expressed mainly in floral organs and, within the flower, expression is restricted to petals and stamens. The NTGLO gene most probably represents a true homologue of the GLO gene because: i) the MADS boxes, of the two genes are highly homologous (56 out of 58 amino acids are identical): ii) at the carboxyterminal a block of 19 amino acids is perfectly conserved between the NTGLO and GLO proteins and iii) their expression patterns in floral organs are identical.     

CIRP2, a major cytoplasmic RNA-binding protein in Xenopus oocytes

In an attempt to isolate mRNA-binding proteins we fractionated Xenopus oocyte lysate by oligo(dT)–cellulose chromatography. A 20 kDa protein was the major component of the eluate. cDNA cloning revealed that this protein is a Xenopus homolog of the cold-inducible RNA-binding protein (CIRP) which was originally identified in mammalian cells as a protein that is overexpressed upon a temperature downshift. This Xenopus protein, termed here xCIRP2, is highly expressed in ovary, testis and brain in adult Xenopus tissues. In oocytes it is predominantly localized in the cytoplasm. By biochemical fractionation we provide evidence that xCIRP2 is associated with ribosomes, suggesting that it participates in translational regulation in oocytes. Microinjection of labeled mRNA into oocytes followed by UV cross-linking of the oocyte lysate led to identification of two major RNA-binding activities. Immunoprecipitation of the RNA-binding proteins demonstrated that one is xCIRP2 and that the other contains FRGY2. FRGY2, which is one of the principal constituents of mRNA storage particles involved in translational masking of maternal mRNA, has an RNA-binding domain conserved to those of bacterial cold shock proteins. Possible implications of the highly abundant expression in oocytes of cold shock RNA-binding proteins of both eukaryotic and prokaryotic types are discussed.     

Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

A new member to the astasin family of metalloendopeptidases: A novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail

1,25-Dihydroxyvitamin D-3 is essential for the utilization of eggshell calcium by avian embryo through the chorioallantoic membrane (CAM). A cDNA library was constructed from poly(A)⁺ RNA extracted from vitamin D-deficient CAMs given 1,25-dihydroxyvitamin D-3. Screening this library by differential hybridization yielded a full-length (∼ 1.8 kb) cDNA, whose corresponding mRNA is increased 3-fold 2.5 h after a single injection of 1,25-(OH)₂D₃. The complete nucleotide sequence for the full-length cDNA has been determined. An open-reading frame, corresponding to a 310 amino acid, 41 kDa protein was found. Searching protein sequence data bases revealed a strong similarity to the following proteases: astacin, a crayfish digestive protease, Oryzias latipes hatching enzyme constituent protease (Orz), Xenopus laevis developmentally regulated UVS.2 protein secreted by the hatching gland of embryos, the NH₂-terminal domain of human bone morphogenetic protein (BMP-1) and Drosophila dorsal-ventral patterning tolloid. The cDNA has approximately 36% overall identity with astacin and BMP-1, and is more than 60% identical to either Orz or UVS.2. Moreover, multiple alignment analysis indicates that 37 residues, including 3 cysteine residues, are strictly conserved in the complete 200-amino acid astacin sequence. All 6 proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases. This motif is found within an extended sequence of HEXXHXXGFXHE that is unique to this subgroup of metalloendopeptidases. In addition, the 6 proteins have 50% identity (including the present cDNA) and 79% are conserved in 4 of these proteins in a 24-amino acid sequence that includes the putative active site. The level of mRNA for the new protein reaches a maximum at day 12 of embryonic life and declines thereafter. It is suggested that this clone corresponds to an mRNA encoding for a protease that may play a role in the degradation of eggshell matrix.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

On the role of Cdc2 kinase during meiotic maturation of oocyte in the Chinese mitten crab,Eriocheir sinensis

Cdc2 kinase is a catalytic subunit of maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocyte. To understand the role of Cdc2 kinase on the oocyte maturation in crustacean, a complete cDNA sequence of Cdc2 kinase was cloned from Chinese mitten crab Eriocheir sinensis and its spatial-temporal expression profiles were analyzed during oogenesis at RNA and protein levels. The crab Cdc2 cDNA (1364 bp) encodes for a 299 amino acids protein with calculated molecular weight of 34.7 kDa. The Cdc2 mRNAs level showed no significant change in the ovary during oogenesis, whereas higher protein level was found at previtellogenesis, late vitellogenesis and germinal vesicle breakdown (GVBD) stages. Two forms (35 kDa and 34 kDa) of Cdc2 proteins were simultaneously identified in ovary at all stages. Immunocytochemistry analysis revealed that Cdc2 proteins locate exclusively in ooplasm of previtellogenic oocyte, and then relocate into germinal vesicle at vitellogenesis stage and accumulate on meiotic spindle at oocyte maturation. These findings suggest that Cdc2 kinase has essential roles in inducing GVBD and generating meiotic apparatus during the crab oocyte maturation.     

Identification of the Dictyostelium discoideum homolog of the N-ethylmaleimide-sensitive fusion protein

The N-ethylmaleimide-sensitive fusion protein (NSF) is required for vesicular membrane fusion in multiple cellular functions. We have cloned a cDNA encoding the Dictyostelium discoideum homolog of the NSF protein. This cDNA hybridizes with a single fragment in Southern blots suggesting that NSF is encoded by a single gene in the amoeba. It is expressed constitutively during vegetative growth and throughout the differentiation cycle. The encoded gene product comprises 738 aa with a predicted molecular mass of 82 kDa. It shows the characteristic three-domain structure of NSF proteins. A more divergent amino-terminal part is followed by two highly conserved ATP-binding domains featuring Walker A and B signature sequences. The D. discoideum protein presents an overall aa sequence identity of 44% when compared to known NSF homologs. The monoclonal antibody 2E5 directed against Cricetellus griseus NSF recognizes a protein with a molecular weight of approx. 80 000 in a D. discoideum crude extract and the recombinant D. discoideum His₆-NSF expressed in Escherichia coli.     

Primary Structure of Cytochrome b(5) from Cauliflower (Brassica oleracea L.) Deduced from Peptide and cDNA Sequences

Cytochrome b₅ is a microsomal protein that functions as an intermediate electron donor in fatty acid desaturation and other oxidation/reduction reactions. cDNA clones were isolated from cauliflower (Brassica oleracea L.) by using oligonucleotides based on the partial amino acid sequence of the protein. The deduced amino acid sequence of the polypeptide exhibited approximately 30% sequence identity with the homologous protein from vertebrates.