cDNA and deduced amino acid sequence of mouse matrix gla protein: one of five glutamic acid residues potentially modified to gla is not conserved in the mouse sequence.

A cDNA library was constructed using the mouse osteoblastic cell line MC3T3-E1 treated with 1 alpha,25-dihydroxyvitamin D3, based on the finding that the treatment increased ninefold the expression of 0.7 kb matrix gla protein (MGP) mRNA. cDNA clones encoding mouse MGP were isolated from the library. The nucleotide sequence showed an open reading frame of 312 nucleotides encoding 104 amino acids. Murine MGP shared 84-89% amino acid sequence homology with bovine, rat, and human MGP. However, there are five glutamic acid residues potentially modified to gamma-carboxyglutamic acid (gla) in those species; in murine MGP, lysine replaced glutamic acid 37. Also, an extra tyrosine was added at the carboxyl terminus. The significance of the substitution is discussed in relation to the gamma-carboxylation sites in MGP protein.

A novel human gene that is preferentially transcribed in heart muscle.

We have cloned a human cDNA fragment for a gene that is expressed primarily in the heart. To explore its biological function, we have isolated full-length cDNA clones of the gene. DNA sequencing of the 2.7-kb cDNA revealed a 2274-bp ORF. Close to the N terminus of the deduced amino acid sequence is a possible ATP-binding domain that has been assembled by a fusion of B- and A-type adenine nucleotide-binding motifs. In the middle of the sequence is a long stretch of alpha-helical residues that could form a coiled-coil. These features imply that this is a sequence encoding a new human motor protein.

Neopterin as an endogenous antioxidant.

The in vitro potency of neopterin (NP) as an antioxidant and its in vivo activity to suppress alloxan-induced diabetes were investigated. The reduced form of neopterin, 5,6,7,8-tetrahydroneopterin (NPH-4), showed an extremely high superoxide anion radical scavenging activity in two assay systems, i.e. xanthine/xanthine oxidase- and macrophage/phorbol myristate acetate (PMA)-reaction systems. NPH-4 also inhibited the oxidation of linoleic acid about as effectively as uric acid. Furthermore, NPH-4 and NP effectively suppressed alloxan-induced mouse diabetes. These results suggest that pteridines play an important role as endogenous antioxidants.

Inhibitory effect of neopterin on NADPH-dependent superoxide-generating oxidase of rat peritoneal macrophages.

The effect of the oxidized form of neopterin (NP) on the NADPH-dependent superoxide-generating oxidase (NADPH-oxidase) was investigated in both whole-cell and cell-free activation systems by using peritoneal macrophages of rats which had received an intraperitoneal injection of mineral oil. In the whole-cell system, NP remarkably inhibited the generation of superoxides in macrophages stimulated with phorbol myristate acetate (PMA). NP also showed an significant suppression of the activation of superoxide-generating NADPH-oxidase in the cell-free system using solubilized membranes and sodium dodecyl sulfate (SDS) as a stimulant. The 50%-inhibitory concentration (IC50) of NP was about 1 microM in both assay systems. In a kinetic study, competitive inhibition of the NADPH-oxidase by NP was observed in the cell-free system with a calculated inhibition constant (Ki) of 6.50 microM. These findings suggest that NP may play an important role in the suppression of superoxide generation via the inhibition of the NADPH-oxidase in phagocytes.

Protective effects of tetrahydroneopterin against free radical-induced injury.

The therapeutic potency of 5,6,7,8-tetrahydroneopterin (NPH4) was investigated in ischemic paw edema, Adriamycin (ADR)-induced cardiotoxicity, and endotoxin [lipopolysaccharide (LPS)]- and carbon tetrachloride (CCl4)-induced hepatotoxicity in mice. Ischemic paw edema was completely suppressed by pre-administration of NPH4. ADR-induced cardiotoxicity and LPS-induced hepatotoxicity were significantly decreased by post-administration of NPH4. Furthermore, NPH4 ameliorated CCl4-induced hepatotoxicity. These results suggest that NPH4 may be useful for the treatment of free radical, especially superoxide radical, -related tissue injury.

Enhancing potency of neopterin toward B-16 melanoma cell damage induced by UV-A irradiation and its possible application for skin tumor treatment.

The enhancing potency of the oxidized form of neopterin (NP) towards long-wavelength ultraviolet light (UV-A) induced cytotoxicity was examined in vitro using mouse melanoma (B-16) cells. A sufficient dose of UV-A irradiation was used to cause damage (measured in terms of reduced DNA synthesis) to about 30% of the irradiated cells. NP drastically enhanced the UV-A-induced cell damage, whereas the reduced form of neopterin (NPH4), which possesses a strong antioxidative activity, abolished the UV-A-induced cell damage in a dose-dependent manner. These data suggest that radical oxygen species (ROS) may be involved in UV-Ainduced B-16 cell damage. Among various radical scavengers examined, only catalase protected B-16 cells from UV-A-induced cell damage and also abolished the enhancing potency of NP, suggesting that hydrogen peroxide may mediate the cell damage. We obtained similar results in another experiment on B-16 cell damage induced by hydrogen peroxide instead of UV-A. In an in vitro analysis of the chemilumi-nescence induced by hydrogen peroxide, NP remarkedly enhanced the signal intensity at a high concentration, while NPH4 reduced the intensity in a dose-dependent manner. These results suggest that elevation of the hydrogen peroxide-mediated cytotoxicity by NP may be involved in its enhancing potency toward UVA-induced B-16 cell damage, and may also indicate the possible utility of the oxidized form of neopterin as an enhancer for UV-A-irradiation treatment of tumors.

Classification of stevia sweeteners in soft drinks using liquid chromatography and time-of-flight mass spectrometry.

The aim of this study was to develop a comprehensive analytical method for the characterisation of stevia sweeteners in soft drinks. By using LC and time-of-flight MS, we detected 30 steviol glycosides from nine stevia sweeteners. The mass spectral data of these compounds were applied to the analysis to determine steviol glycosides in nine soft drinks. On the basis of chromatographic data and principal-component analysis, these soft drinks were classified into three groups, and the soft drinks of each group, respectively, contained high-rebaudioside A extract, normal stevia extract or alfa-glucosyltransferase-treated stevia extract.

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpA gene.

One of the phosphatidyl glycerophosphate phosphatase genes of Escherichia coli, pgpA, was cloned, and its DNA sequence was determined. Its 507-base-pair open reading frame was consistent with the 18,000-molecular-weight product identified by a maxicell experiment. Between its possible promoter and methionine initiation codon, a repetitive extragenic palindromic sequence was found.

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product.

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.

Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi.

Four mutants of Escherichia coli that are resistant to the flagellotropic phage chi, but are motile, were isolated. When they were observed in liquid culture bylight microscopy, one mutant exhibited circular movement and another tumbled at high frequency on the surface of a glass slide. The remaining two mutants moved normally. None of these mutants adsorbed the wild-type strain of chi. P1 transduction revealed that the mutation sites of these four mutants were more than 97% contransducible with a site in hag, the structural gene for flagellin. When flagellins of these mutants were chromatographed on a diethylaminoethyl-cellulose column, two eluted slower and one eluted slightly faster than the flagellin of the parental strain. The other flagellin eluted at the same position as that of the parent. Host range mutants of phage chi, which could infect these bacterial mutants, were isolated.

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein.

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.

Metal-binding, nucleic acid-binding finger sequences in the CDC16 gene of Saccharomyces cerevisiae.

The CDC16 gene is involved in the process of chromosome segregation in mitosis and a cdc16ts mutant accumulates the predominant microtubule-associated protein at the nonpermissive temperature. We find that the CDC16 gene open reading frame (ORF) is capable of encoding a protein whose calculated molecular weight and pI are 94,967 and 6.60, respectively. This hypothetical protein contains 16 cysteine residues; five are clustered at the N-terminal, 4 are placed about 3 residues apart in the middle of the peptide, and 3 are located close to the C-terminal. Each of these could form a metal-binding, nucleic acid-binding domain, suggesting this protein acts either as a repressor of the microtubule-associated protein gene or as a component necessary for spindle elongation, possibly interacting with the DNA. The start of the CDC16 ORF is only 95 bp downstream from the end of the MAK11 ORF. In this region there are two TATA boxes in tandem, but there is no room for a UAS or other regulatory sequences. An ATG is present 5 bp upstream of the start of the large ORF. Its frame terminates after only two amino acids.

Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors.

We have devised a coupled radiochemical assay for detecting phosphatidylglycerolphosphate (PGP) phosphatase activity in Escherichia coli colonies immobilized on filter paper. There appeared to be at least two enzymes capable of dephosphorylating PGP, as judged by the characterization of mutations in two genes designated pgpA and pgpB. The former is located near min 10 and is cotransducible with proC and dnaZ. The latter is situated near min 28 and is closely linked to cysB. The available mutant alleles of pgpA reduced the specific activity of PGP phosphatase in crude extracts by about 30%, but they had no effect on phosphatidic acid (or lysophosphatidic acid) phosphatase. Mutants altered in the pgpB locus inactivated most of the residual PGP phosphatase activity present in single-step pgpA mutants, and the level of phosphatidic acid phosphatase was also reduced 20-fold. The available mutations in pgpA and pgpB elevated the cellular PGP pool by 10- to 50-fold. The maximal PGP levels never exceeded 5%, and these strains were not conditionally lethal. The simplest interpretation of our findings is that there are at least two membrane-associated phosphatases in E. coli, both distinct from alkaline phosphatase. The pgpA gene product is specific for PGP, whereas the pgpB gene product also acts on phosphatidic acid and lysophosphatidic acid.

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames.

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.

Effects of galU mutation on flagellar formation in Escherichia coli.

Two mutants of Escherichia coli strictly deficient in uridine-diphosphoglucose pyrophosphorylase activity (galU) were found to have very small numbers of flagellar filaments and hooks. In these mutants, both the rate of flagellin (flagellar protein) synthesis and the amount of messenger ribonucleic acid specific for flagellin were considerably lower than in the parental strains. Motile revertants from the galU mutants were isolated and were found to carry a suppressor mutation, which was mapped in the flaH cistron. These strains formed swarms under conditions of catabolite repression; their intracellular concentration of cyclic adenosine 5'-monophosphate was the same as that in the parental strains. These results suggest that the outer membrane affects flagellar formation through the flaH gene product.

Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli.

The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J. Biol. Chem. 260, 12084-12091). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map.

Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli.

Previous work from this laboratory had demonstrated that CDP-diglyceride hydrolase of Escherichia coli is encoded by the cdh gene that maps near minute 88 (Bulawa, C. E., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 11257-11264). We now report the construction of hybrid plasmids and the sequencing of a 1,243-base pair insert carrying cdh. The further construction of BAL31 deletions of this insert, in conjunction with maxicell experiments and in vitro enzyme assay, has led to the identification of a 756-base pair coding sequence for the cdh polypeptide. The molecular weight of the primary translation product deduced from the DNA sequence of the cdh gene is 28,450, in agreement with maxicell experiments. Parallel purification of the enzyme from extracts of wild-type and overproducing strains confirms the presence of a 27-kDa polypeptide in the overproducer, as judged by polyacrylamide gel electrophoresis of the most purified fractions. Inspection of the DNA sequence reveals a very hydrophobic N-terminal domain that may be either a signal peptide or a special region, anchoring the hydrolase to the membrane. In contrast to the CDP-diglyceride synthetase, the overall amino acid composition of the CDP-diglyceride hydrolase is not extraordinarily hydrophobic. Although both CDP-diglyceride synthetase and CDP-diglyceride hydrolase can transfer the CMP moiety of CDP-diglyceride to a suitable acceptor, the primary structures and mechanisms of action of these two enzymes are very different.

A -1 ribosomal frameshift in a double-stranded RNA virus of yeast forms a gag-pol fusion protein.

The L-A double-stranded RNA (dsRNA) virus of Saccharomyces cerevisiae has two open reading frames (ORFs). ORF1 encodes the 80-kDa major coat protein (gag). ORF2, which is expressed only as a 180-kDa fusion protein with ORF1, encodes a single-stranded RNA-binding domain and has the consensus sequence for RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses (pol). We show that the 180-kDa protein is formed by -1 ribosomal frame-shifting by a mechanism indistinguishable from that of retro-viruses. Analysis of the "slippery site" suggests that a low probability of unpairing of the aminoacyl-tRNA from the 0-frame codon at the ribosomal A site reduces the efficiency of frameshifting more than the reluctance of a given tRNA to have its wobble base mispaired. Frameshifting of L-A requires a pseudoknot structure just downstream of the shift site. The efficiency of the L-A frameshift site is 1.8%, similar to the observed molar ratio in viral particles of the 180-kDa fusion protein to the major coat protein.

Structure and nuclear localization signal of the SKI3 antiviral protein of Saccharomyces cerevisiae.

The yeast chromosomal genes SKI2, SKI3, SKI4, SKI6, SKI7 and SKI8 repress the replication of double-stranded RNA viruses, protecting the host from the otherwise lethal effects of the virus. We cloned and sequenced the SKI3 gene and found that it encodes a 163 kDa protein including a typical nuclear localization signal. Cell fractionation experiments show that the SKI3 gene product is indeed tightly associated with nuclei and that the putative nuclear localization sequence directs beta-galactosidase into the nucleus. However, fusion of a part of the SKI3 protein lacking this signal with beta-galactosidase also directs beta-galactosidase into the nucleus, suggesting the presence of a second nuclear localization signal. The SKI3 gene is only essential in the presence of an M double-stranded RNA virus.

Characterization of the gene rimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12.

Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus. The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally. Mutants deficient in this modification were isolated and characterized genetically and biochemically. The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected. The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA. The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined. Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein. An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated. The S6 protein of this mutant was apparently inaccessible to the RimK modification system. This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6.