Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae

Abstract Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB ) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli . Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii . Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.     

Mutations associated with floral organ number in rice

How floral organ number is specified is an interesting subject and has been intensively studied in Arabidopsis thaliana. In rice (Oryza sativa L.), mutations associated with floral organ number have been identified. In three mutants of rice, floral organ number 1 (fon1) and the two alleles, floral organ number 2-1 (fon2-1) and floral organ number 2-2 (fon2-2), the floral organs were increased in number centripetally. Lodicules, homologous to petals, were rarely affected, and stamens were frequently increased from six to seven or eight. Of all the floral organs the number of pistils was the most frequently increased. Among the mutants, fon1 showed a different spectrum of organ number from fon2 -1 and fon2 -2. Lodicules were the most frequently affected in fon1, but pistils of more than half of fon1 flowers were unaffected; in contrast, the pistils of most flowers were increased in fon2 -1 and fon2-2. Homeotic conversion of organ identity was also detected at a low frequency in ectopically formed lodicules and stamens. Lodicules and stamens were partially converted into anthers and stigmas, respectively. Concomitant with the increased number of floral organs, each mutant had an enlarged apical meristem. Although meristem size was comparable among the three mutants and wild type in the early phase of flower development, a significant difference became apparent after the lemma primordium had differentiated. In these mutants, the size of the shoot apical meristem in the embryo and in the vegetative phase was not affected, and no phenotypic abnormalities were detected. These results do not coincide with those for Arabidopsis in which clavatal affects the sizes of both shoot and floral meristems, leading to abnormal phyllotaxis, inflorescence fasciation and increased floral organs. Accordingly, it is considered that FON1 and FON2 function exclusively in the regulation of the floral meristem, not of the vegetative meristem.Abbreviation DIC differential interference contrast This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.     

Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.     

cDNA Cloning and Expression of the Plastidic Copper/Zinc-Superoxide Dismutase from Spinach (Spinacia oleracea L.) Leaves

A cDNA clone for copper/zinc-superoxide dismutase (Cu/Zn-SOD)was isolated from spinach (Spinacia oleracea L.) leaves. Itsnucleotide sequence showed that it codes for a precursor polypeptideof 222 amino acids, including the NH₂-terminal 68-residue extensionwhich corresponds to a plastidic transit peptide. Northern hybridization,using plastidic and cytosolic Cu/Zn-SOD cDNAs as the probes,revealed that these two genes are differentially expressed inthe roots and leaves of spinach. ¹Present address: Department of Biochemistry and Microbiology,Cook College, Rutgers University New Brunswick, NJ 08903-0231,U.S.A.