Regulated spatial expression of fusion gene constructs with the 5′ upstream region of Halocynthia roretzi muscle actin gene in Ciona savignyi embryos

pHrMA4a-Z is a recombinant plasmid in which about 1.4 kb of the 5 flanking region of a gene for muscle actin HrMA4a from the ascidian Halocynthia roretzi is fused with the coding sequence of a bacterial gene for -galactosidase (lac-Z). In this study, we examined the expression of the fusion gene construct when it was introduced into eggs of another ascidian, namely Ciona savignyi. When a moderate amount of linearized pHrMA4a-Z was introduced into fertilized Ciona eggs, the expression of the reporter gene was evident in muscle cells of the larvae, suggesting that both species share a common machinery for the expression of muscle actin genes. The 5 upstream region of HrMA4a contains several consensus sequences, including a TATA box at -30, a CArG box at -116 and four E-boxes within a region of 200 bp. A deletion construct, in which only the 216-bp 5 flanking region of HrMA4a was fused with lac-Z, was expressed primarily in larval muscle cells. However, another deletion construct consisting of only the 61-bp upstream region of HrMA4a fused with lac-Z was not expressed at all. When pHrMA4a-Z or pHrMA4a-Z (–216) was injected into each of the muscle-precursor blastomeres of the 8-cell embryo, expression of the reporter gene was observed in larval muscle cells in a lineage-specific fashion. However, expression of the reporter gene was not observed when the plasmid was injected into non-muscle lineage. Therefore, the expression of the reporter gene may depend on some difference in cytoplasmic constituents between blastomeres of muscle and non-muscle lineage in the 8-cell embyo.     

Accumulation on the Cytoplasmic Membrane of the Precursor to Dimethyl Sulfoxide Reductase in Molybdenum Cofactor-Deficient Mutants of Rhodobacter sphaeroides f. sp. denitrificans

The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)     

A Nitrate-Inducible Plasma Membrane Protein of a Marine Alga, Heterosigma akashiwo

The rate of nitrate uptake by Heterosigma akashiwo cells thathad been cultured in medium with nitrate or ammonium ions asthe source of nitrogen was measured using¹⁵NO_3– The ratioof ¹⁵N/¹⁴N increased dramatically in nitrate-grown cells. Inammonium-grown cells, the ratio of ¹⁵N/¹⁴N did not increasefor 3 h but then it began to increase. Even when nitrate reductaseactivity was inhibited by tungstate, nitrate-grown cells couldtake up nitrate. Plasma membranes from nitrate-grown and ammonium-grown cellswere purified by the silica-microbead method, and polypeptidesassociated with the membranes were analyzed by SDS-PAGE andimmunostaining. A major polypeptide with a molecular mass of26 kDa appeared 3 h after the transfer of ammonium-grown cellsto nitrate-containing medium, and it disappeared 2 d after thetransfer of nitrate-grown cells to ammonium-containing medium.The 26 kDa polypeptide also appeared when cell growth shiftedfrom the logarithmic phase to the stationary phase and the ammoniumcontent of the medium decreased, even when the cells were culturedin ammonium-containing medium. (Received April 10, 1992; Accepted July 30, 1992)     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Isolation and characterization ofPleurotus ostreatus mutant strains resistant to a carboxin-derived fungicide,flutolanil

To develop a dominant genetic marker inPleurotus ostreatus, mutant strains resistant to a carboxin-derived fungicide, flutolanil, were isolated. These mutants included strains which showed resistance to 50-fold higher concentration of fluotolanil than the wild-type strain, even after successive cultivations in the absence of the drug. Dominance of the phenotype was confirmed by back-crossing between the resistant and wild-type monokaryons. The flutolanilresistance was also shown to be stably inherited by the basidiospore-derived progenies of the mutant strains.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A.

Two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural region 5A (NS5A). Recently, we found that both proteins are phosphorylated at serine residues and that p58 is a hyperphosphorylated form of p56. Furthermore, hyper-phosphorylation depends on the production of an intact form of the HCV NS4A protein. To clarify the nature of NS5A phosphorylation, pulse-chase analysis was performed with a transient protein production system in cultured cells. The study indicated that basal and hyperphosphorylation of NS5A occurred after proteolytic production of NS5A was complete. In an attempt to identify the location of the hyperphosphorylation sites in p58, proteins with sequential deletions from the C-terminal region of NS5A and with mutations of possible phosphorylated serine residues to a neutral amino acid, alanine, were constructed. The deleted or mutated proteins were then tested for hyperphosphorylation in the presence of the NS4A product. Here, we report that serine residues 2197, 2201, and/or 2204 are important for hyper-phosphorylation. Important sites for basal phosphorylation were identified in the region from residues 2200 to 2250 and in the C-terminal region of the NS5A product. A subcellular localization study showed that most of the NS5A products were localized in the nuclear periplasmic membrane fraction.     

The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A.

Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3.     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.