cDNA sequence for rkST1, a novel member of the sodium ion-dependent glucose cotransporter family.

A full length cDNA for rkST1, a novel member of the Na+/glucose cotransporter family, was cloned from rabbit kidney and sequenced. The coding sequence comprised 2022 base pairs and 674 amino acids. rkST1 beared 50-60% amino acid identity to the other cotransporters and was characteristic in respect of its expression in brain in addition to kidney among the cotransporters.

Uncoupled release of protein and lipid in a protein-secreting bacterium, Bacillus brevis 47.

Bacillus brevis 47 was found to release approx. 10% of the total cellular lipids into the medium, and the protein secretion process in B. brevis 47 was studied to determine whether any relationship exists with lipid synthesis or alteration in the lipid composition. B. brevis 47 contained the usual phospholipids such as phosphatidylethanolamine, phosphatidylglycerol and cardiolipin as well as diglycerides as major neutral lipids. The extracellular lipid consisted of the same constituents as the cellular lipid but with a significantly altered composition. Divalent cations such as Ca2+, which specifically inhibit protein secretion, had no effect on the lipid release. A nonprotein-secreting mutant released lipid to the same extent as wild-type cells. Based on these results, we conclude that protein secretion occurs independently of lipid release.

Identification of acyl phosphatidylglycerol as a minor phospholipid of Pseudomonas BAL-31.

Compound X, a minor phospholipid of Pseudomonas BAL-31 and bacteriophage PM2, has been identified as X-3-phosphatidyl-1'-(3'-acyl)-glycerol, or acyl phosphatidylglycerol. The water-soluble product obtained by mild alkaline hydrolysis showed the same RF value as that of glycerophosphoryl-glycerol. The chemical analysis gave the ratio 1 : 3 : 2 for phosphate-acyl ester-glycerol. The position of the third acyl group was determined by nuclear magnetic resonance techniques.

Structurally different rat liver medium-chain acyl CoA dehydrogenases directed by complementary DNAs differing in their 5'-region.

Different forms of rat liver medium-chain acyl CoA dehydrogenase (MCAD) (EC 1.3.99.3) were produced in Escherichia coli carrying expression plasmids (pRMCADm-1 approximately 9) differing at the 5'-region of the cDNA. The proteins expressed could be readily extracted from the cells. The protein (approximately 44 kDa) directed by pRMCADm-3 showed the highest activity and was readily purified to homogeneity. The purified enzyme contained non-covalently bound FAD and was similar to rat liver mitochondrial enzyme in all respects examined. The purified protein (approximately 45 kDa) directed by pRMCADm-1 did not contain FAD and showed no enzymatic activity. Therefore, the leader peptide disturbs the binding of FAD to the apoprotein. The purified protein (approximately 40 kDa) directed by pRMCADm-6 did not contain FAD. Thus, the deletion of the NH2-terminal portion of the apoprotein to some extent results in its inability to combine with FAD.

Hexagonal surface array in a protein-secreting bacterium, Bacillus brevis 47.

Bacillus brevis 47, a protein-secreting bacterium, contained two major proteins with approximate molecular weights of 150 000 and 130 000 in the cell wall. The cell surface was covered with a hexagonally arranged array of six structural units about 4 nm in diameter with a lattice constant of 14.5 nm. The regular array structure as well as the chemical composition of cell envelopes remained the same regardless of the growth conditions. A mutant, strain 47-57, which was isolated as a phage resistant colony, contained only the 150 000 protein as a major cell wall protein. Although the mutant had hexagonally arranged arrays with the same lattice constant as that of wild-type cells, the distribution of mass in the unit cell differed considerably from that of the wild-type cells. The number of structural units in the unit cell of the mutant was reduced from six to three. Taking these results together with filtered images of the wild-type and mutant envelopes, two possible models for the surface array of B. brevis 47 are discussed.

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis.

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and beta-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 was expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.

Ascorbate-dependent expression of ubiquitin genes in guinea pigs.

In an attempt to characterize a gene(s), of which the expression is ascorbate-dependent, a cDNA fragment encoding ubiquitin was isolated from a subtracted cDNA library constructed from spleen RNAs of ascorbate-deficient or -replete guinea pigs. On Northern blot analysis, three transcripts (1.8 kb ubiX, 1.3 kb ubiY and 0.7 kb ubiZ) were detected. The ubiY encodes four direct repeats of the 76 amino acid ubiquitin sequence with seven additional amino acids, V-Y-A-S-P-I-F, at the C-terminus. The transcript ubiX appears to comprise more than five repeats of the ubiquitin-encoding unit. The ubiZ encodes a ubiquitin monomer fused to an 80 amino acid extension exhibiting 100% amino acid sequence identity to the human homolog, HUMUBA80R. The ubiX gene was expressed animal-dependently. The ubiY mRNA levels decreased under ascorbate-deficient conditions, and increased under ascorbate-replete conditions, whereas ubiZ mRNA remained unaltered at low levels under the feeding conditions used here.

Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes.

Complementary and genomic DNAs encoding Aspergillus oryzae Taka-amylase A (Taa) were cloned and sequenced. The coding sequence of the cDNA comprised the signal peptide [21 amino acids (aa)] and mature Taa (478 aa). The deduced aa sequence agrees well with the published aa sequence, except for one insertion, one deletion and ten aa substitutions. These differences might be due to the difference in the strains used. Sequence comparison of the cDNA and genomic DNA indicates the presence of eight introns ranging in size from 55 to 86 bp. Southern-blot analysis showed the presence of at least two Taa genes, and the second gene (Taa-G2) was isolated. All the intron/exon junctions follow the 'GT-AG' rule, except for intron I of the first gene (Taa-G1). The 5'-noncoding region was well conserved among the genomic genes and contained sequences similar to 'CAAT' and 'TATA' boxes at nucleotides -121 and -31, counted from the transcription start point, respectively. The 3'-noncoding regions, however, differed significantly from each other. Taa-G2 contains a sequence identical to that of several independent cDNA clones, suggesting that it may be the major transcribed gene in A. oryzae.

Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein.

A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

A quantity control mechanism regulating levels of the HapE subunit of the Hap complex in Aspergillus nidulans: no accumulation of HapE in hapC deletion mutants.

The Aspergillus nidulans CCAAT-binding complex (Hap complex) consists of at least three subunits, HapB, HapC and HapE. To investigate the quantity control mechanisms of the subunits during assembly of the Hap complex, reconstitution studies with the recombinant subunits and extracts prepared from the respective hap subunit deletion mutants were carried out. Furthermore, Western blot analysis of the Hap subunits and Northern blot analysis of the hap genes with the respective deletion mutants were also performed. From all the results together, it was suggested that the number of the HapC molecule could adjust that of the HapE molecule by forming stable heterodimers prior to assembly of the Hap complex.

Ascorbate-dependent elevation of mRNA levels for cytochrome P450s induced by polychlorinated biphenyls.

Trichlorobiphenyl induced only CYP1A2 mRNA, while pentachlorobiphenyl induced both CYP1A2 and CYP2B1 mRNAs in rat liver. The mRNA levels for these P450s were elevated when ascorbic acid-deficient ODS rats (mutant rats with a hereditary osteogenic disorder) were fed a diet supplemented with ascorbic acid. The amount of CYP2B1 mRNA increased rapidly and reached a maximum level of approximately double within 24 hr of injection of pentachlorobiphenyl. Thereafter, the amount of its mRNA decreased to a steady level. This pattern was roughly paralleled by changes in the amount of CYP1A2 mRNA.

Major proteins released by a protein-producing bacterium, Bacillus brevis 47, are derived from cell wall protein.

B. brevis 47 secretes a vast amount of protein consisting mainly of two kinds with approximate molecular weights of 130,000 and 150,000. The two major extracellular proteins were indistinguishable from those of cell wall protein by SDS-polyacrylamide gel electrophoresis. Based on the results of analysis of amino acid composition, limited proteolysis followed by electrophoresis, and the cross-reactivity of antisera, we conclude that the 130K and 150K extracellular proteins are derived from the respective cell wall proteins. Furthermore, the NH2-terminal amino acid analysis suggests that the two major extracellular proteins are released from the cell wall without any modification of the NH2-terminal portion.

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences.

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites.

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.

Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616.

A genomic gene encoding a polygalacturonase from Aspergillus oryzae, used in soy sauce production, was cloned and sequenced. The structural gene comprises 1227 bp coding for 363 amino acids with a putative prepropeptide of 28 amino acids and the open reading frame is disrupted by two short introns of 57 bp and 81 bp. The deduced amino acid sequence of the mature protein showed 63, 63, 63 and 64% homology with those of Aspergillus niger polygalacturonase I, Aspergillus niger polygalacturonase II, Aspergillus tubingensis polygalacturonase II and Cochliobolus carbonum polygalacturonase, respectively. There is, however, little homology among fungal, plant and bacterial polygalacturonases.

Structure and expression properties of the endo-beta-1,4-glucanase A gene from the filamentous fungus Aspergillus nidulans.

Endo-beta-1,4-glucanase A (EG A) of Aspergillus nidulans was purified to homogeneity, and its genomic gene (eglA) was cloned based on partial amino acid sequences of the purified enzyme and sequenced. The eglA gene comprised 1228 bp with four putative introns and encoded a polypeptide of 326 amino acids bearing high homology to the family A cellulases. The eglA promoter activity in A. nidulans was examined using the A. oryzae Taka-amylase A gene as a reporter. Expression of the reporter gene was induced by carboxymethylcellulose and cellobiose, and repressed by glucose, galactose, mannose, xylose, sorbitol, glycerol and succinate. Lactose neither induced nor repressed the expression.

A second pectin lyase gene (pel2) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene products.

A second pectin lyase gene, designated pel2, was isolated from a shoyu koji mold Aspergillus oryzae KBN616 and characterized. The structural gene comprised 1306 bp with three introns. The ORF encoded 375 amino acids with a signal peptide of 19 amino acids. The deduced amino acid sequence showed high similarity to those of A. oryzae Pel1, Aspergillus niger pectin lyases and Glomerella cingulata Pn1A. The pel2 gene was overexpressed under the control of the promoter of the A. oryzae TEF1 gene for purification and enzymatic characterization of its gene product. The gene product exhibited two molecular masses of 48 and 44 kDa due to different degrees of glycosylation. Both proteins had the same pH optimum of 6.0 and temperature optimum of 50 degrees C.

Depression of the xylanase-encoding cgxA gene of Chaetomium gracile in Aspergillus nidulans.

Regulation of the Chaetomium gracile xylanase A gene (cgxA) was investigated using Aspergillus nidulans as an intermediate host. Deletion of a 185 bp DNA fragment from its promoter region led to higher levels of the cgxA gene expression, indicating that the 185 bp DNA fragment contains an element involved in repression of the gene. A nuclear extract was assayed for proteins which bind to the 185 bp DNA fragment. A protein designated AnRP bound sequence specifically to the DNA fragment. The minimum sequence required for AnRP binding, 5'TTGACAAAT-3', was determined by means of gel mobility shift assays with various double-stranded oligonucleotides. Furthermore, this sequence repressed the expression of the cgxA gene when inserted at the 5' end of the cgxA gene on pXAH, which was deleted for the repressive element from the promoter region.

Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1.

Ascorbate-enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-gamma. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of I kappa B alpha by Western blotting, activation of the nuclear factor kappa B (NF-kappa B) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effect on the onset of either I kappa B alpha degradation or the nuclear translocation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The prolonged degradation of I kappa B alpha caused by ascorbate in LPS- and IFN-gamma-activated cells paralleled elevated NF-kappa B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascorbate exerted no effect on the expression of I kappa B alpha and ubiquitin genes in the activated cells. Ascorbate could modulate NF-kappa B DNA binding activity in response to combined LPS and IFN-gamma activation, which increases NO production in activated macrophages.