Molecular cloning and characterization of RGA1 encoding a G protein α subunit from rice (Oryza sativa L. IR-36)

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library prepared from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 M [adenylate-³²P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.     

Nucleotide sequence of cDNA encoding α-luffin,a ribosome-inactivating protein from Luffa cylindrica

A cDNA library from tomato planta macho viroid (TPMV)-infected tomato was constructed. The library was screened at low stringency with a tobacco PR-R cDNA probe. An 832 bp cDNA from a mRNA present only in infected tissue was isolated. Nucleotide sequence showed high homology with the osmotin from both tobacco and tomato (NP24). This cDNA probably corresponds to the AP24 and P23 proteins previously described in tomato and induced upon fungal and viroid infection.     

Nucleotide sequence of cDNA clones encoding the β subunit of mitochondrial ATP synthase from the green alga Chlamydomonas reinhardtii: The precursor protein encoded by the cDNA contains both an N-terminal presequence and a C-terminal extension

cDNA and genomic clones encoding the subunit of mitochondrial ATP synthase from Chlamydomonas reinhardtii have been isolated using heterologous DNA probes from the photosynthetic bacterium Rhodospirillum rubrum. The protein encoded by the cDNA is 79–83% identical to corresponding proteins from higher-plant and mammalian mitochondria, and 75% identical to the R. rubrum protein. It contains both an N-terminal presequence and a unique C-terminal extension. The presequence, which is the first mitochondrial presequence determined in C. reinhardtii, is similar in structure to mitochondrial presequences from other organisms. As chloroplast presequences from C. reinhardtii also share features with mitochondrial presequences from other organisms (L.-G. Franzén et al., FEBS Lett 260 (1990) 165–168), this raises interesting questions about protein targeting to chloroplasts and mitochondria in C. reinhardtii. The possibility that the C-terminal extension is involved in targeting the protein to the mitochondrion is discussed. Southern blot analysis indicates that the protein is encoded by a single-copy gene.     

Reaction specificities of the ε-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro

Lycopene cyclases responsible for the formation of ε-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for ε-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer.     

Putative ammo-terminal presequence for β-subunit of plant mitochondrial F1ATPase deduced from the amino-terminal sequence of the mature subunit

The α- and β-subunits of sweet potato mitochondrial F₁ATPase were purified from the F₁ complex by gel filtration and ion-exchange high-performance liquid chromatography. Isoelectric focussing and N-terminal amino acid sequencing indicated that the purified β-subunit contains at least two polypeptides similar to each other. The N-terminal 18 amino acid sequence of the β-subunit showed homology to the amino acid sequence of the tobacco mitochondrial F₁ATPase β-subunit precursor deduced from the nucleotide sequence [(1985) EMBO J. 4, 2159-2165] between residues 56 and 73, suggesting that the N-terminal 55 amino acids of the tobacco precursor constitute the presequence required for mitochondrial targetting.     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

scsB,a cDNA encoding the hydrogenosomal β subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

A clone containing a Neocallimastix frontalis cDNA assumed to encode the β subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311?bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the β-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial β-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal β-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G+C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5′ (14.8%) and 3′ (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the β-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45?kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45?kDa) and Caecomyces communis (47?kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330?kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.     

Cloning and sequence of the cDNA encoding the β4 integrin subunit in rat peripheral nerve.

β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia.     

Human cDNA encoding the muscle isoform of the phosphorylase kinase γ subunit (PHKG1)

Muscle glycogenosis caused by phosphorylase kinase (Phk) deficiency may lead to exercise intolerance, weakness and musculatur atrophy. The gene encoding the muscle isoform of the Phk subunit (_M) is one of the candidate genes in which mutations responsible for this condition should be sought. Here, we report the cDNA sequence and the predicted primary structure of the human _M subunit.     

Nucleotide sequence of the gene presumably encoding the adsorption protein of filamentous phage φLf

A 1170-nucleotide fragment of φLf DNA was sequenced. This fragment contains an open reading frame, ORF367, encoding a protein of 367 amino acids (aa) (36710 Da). ORF367 is located downstream from the gene encoding the major coat protein (gVIIIp) and a Rho-independent termination signal. Sequence analysis revealed that the gene product has a Gly-rich domain (70 aa) at the center and a hydrophobic region (26 aa) at the C terminus. These structural features suggest that ORF367 may encode the adsorption protein of φLf.     

Nucleotide sequence of the LacN gene encoding thermostable β-galactosidase of a thermophilic anaerobe,strain NA10

The nucleotide sequences of the lacN gene encoding thermostable β-galactosidase of a thermophilic anaerobe, strain NA10, and its flanking regions were determined. The lacN gene was composed of 2031 base pairs coding for a polypeptide of 676 amino acids (molecular weight=79,286). Possible promoter and Shine-Dalgarno sequences were found upstream of the initiation codon, which were highly homologous to the consensus sequences in Escherichia coli.     

Identification of a β-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.)

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing β-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an ～4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS–PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K_m and V_max values of TAG were 31.7 μM and 0.25 μkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific β-glucosidase hydrolyzing TAG, which releases the physiologically active TA.