Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.     

Complete deficiency of 5'-nucleotidase activity in Escherichia coli leads to loss of growth on purine nucleotides but not of their excretion

Escherichia coli has many periplasmic phosphatase activities. To test whether it can take up and excrete purine nucleotides, we attempted to completely disrupt periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they were grown with purine nucleotides as the sole carbon source. Using DNA macroarrays to compare global gene expression in wild-type and ushA knockout mutant cells cultured with IMP or GMP as the sole carbon source, we identified two genes that were induced in the ushA knockout mutant cells and encoded signal sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase activity and was induced by IMP or inosine. An ushA aphA double knockout mutant was shown to be unable to grow on purine nucleotides as the sole carbon source. To investigate the excretion of purine nucleotides, we constructed an ushAaphA double knockout mutant of an inosine-producing strain and found that it accumulated IMP in the medium. In addition, when the guaBA operon was introduced into the ushAaphA double knockout IMP producer, GMP was released into the medium. These observations imply the existence of efflux activity for purine nucleotides in E. coli.     

乙肝病毒增强子对其基因表达的调控

本文构字线性化的含有从核心抗原启动子Ｃｐ起始的ＨＢＶ完整转录单元的基因组隆,以此为模型,通过增强子Ｉ和增强子ＩＩ的缺失或点突变分析,研究了ＥＮＩ和ＥＮＩＩ对ＨＢＶ基因组基因宾调控。结果用ＥＮＩＩ对Ｓ基因表达均有增强作用,且相互协同。     

Isolation and characterization of the human apolipoprotein A-II gene. Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.     

S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus

Using a two-plasmid system, we recently identified sigma(E)-dependent promoters directing expression of the sigma(E) regulon genes in Salmonella enterica serovar Typhimurium (S. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the sigma(E)-dependent rpoEp3 promoter directing expression of rpoE. This two-plasmid system was previously optimized to identify nucleotides critical for the rpoEp3 promoter activity. However, two highly conserved nucleotides in the sigma(E) consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the rpoEp3 promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAACtt) and -10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the sigma(E) consensus sequence. The expression of the wild-type and mutated rpoEp3 promoters was confirmed in S. Typhimurium and was found to exhibit a different pattern of sigma(E) activation compared with Escherichia coli, with a peak rpoEp3 promoter activity in early stationary phase followed by a decrease in late stationary phase.     

Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5'',5''''''-P1,P4-tetraphosphate phosphorylase.

The gene encoding diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) phosphorylase from yeast was isolated from a lambda gt11 library. The DNA sequence of the coding region was determined, and more than 90% of the deduced amino acid sequence was confirmed by peptide sequencing. The Ap4A phosphorylase gene (APA1) is unique in the yeast genome. Disruption experiments with this gene, first, supported the conclusion that, in vivo, Ap4A phosphorylase catabolizes the Ap4N nucleotides (where N is A, C, G, or U) and second, revealed the occurrence of a second Ap4A phosphorylase activity in yeast cells. Finally, evidence is provided that the APA1 gene product is responsible for most of the ADP sulfurylase activity in yeast extracts.     

Cloning, sequencing, and expression of cDNA for human beta-galactosidase

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.     

The 5' untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells

A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' untranslated region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.     

Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I

The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

Two overlapping sequence motifs within the polyomavirus enhancer are independently the targets of stimulation by both the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the Ha-ras oncogene.

A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly stimulates the activity of polyomavirus enhancer in a human erythroleukemia cell line, K562. The target of stimulation was the previously defined A element (from nucleotides 5107 to 5130) of the enhancer. We found that within the A element, two partly overlapping sequence motifs (one from nucleotides 5107 to 5117, the other from nucleotides 5113 to 5121) were independently the targets of TPA stimulation. The former is homologous to the enhancer core sequence of the adenovirus type 5 E1A gene, and the latter shares the consensus AP-1-binding site. In addition, transiently expressed Ha-ras oncogene also stimulated these two subelements in K562 cells, as we reported for NIH 3T3 cells previously.