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PubMed1991

Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is one of the two multisubunit pigment-protein complexes in the thylakoid membranes of cyanobacteria. Subunit III of photosystem I complex was isolated from a mutant of the cyanonbacterium Synechocystis sp PCC 6803, which lacks subunit II. The sequence of its NH2-terminal residues was determined and corresponding oligonucleotide probes were used to isolate the gene encoding this subunit. The gene, designated as psaF, codes for a mature protein of 15705 Da that is synthesized with a 23-amino acid extension. The deduced amino acid sequence is homologous to subunit III from spinach and Chlamydomonas reinhardtii. The presequence of subunit III shows characteristics typical of bacterial presequences and exhibits remarkable amino acid identity around the proteolytic processing site when compared to corresponding regions from the precursors of eukaryotic subunit III. There are two conserved hydrophobic regions in the mature subunit III which may cross or interact with thylakoid membrane. The gene psaF exists as a single copy in the genome and is expressed as a monocistronic RNA. A stable mutant strain in which the gene psaF was replaced by a gene conferring resistance to kanamycin was generated by targeted mutagenesis. Photoautotrophic growth of the mutant strain was comparable with that of the wild type suggesting that function of subunit III is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Addition of more MgSO4 to BG11 medium enhanced growth of the mutant strain but not of the wild type cells.

Amino Acid Sequence↗
PubMed1993

Cloning of human prourokinase cDNA without the signal peptide and expression in Escherichia coli.

Human prourokinase (pro-UK) cDNA without the signal peptide was obtained using synthetic oligonucleotide and DNA recombination techniques and was successfully expressed in E. coli. The plasmid pMMUK which contained pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with Hind III and PstI, so that the N-terminal 371-bp fragment could be recovered. A 304-bp fragment was collected from the 371-bp fragment after partial digestion with Fnu4HI in order to remove the signal peptide sequence. An intermediate plasmid was formed after this 304-bp fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestion and sequencing. By joining the PstI-PstI fragment of pro-UK to the plasmid we obtained the final plasmid which contained the entire coding sequence of pro-UK without the signal peptide. The coding sequence with correct orientation was inserted into pBV220 under the control of the temperature-induced promoter PRPL, and mature pro-UK was expressed in E. coli at 42 degrees C. Both sonicated supernatant and inclusion bodies of the bacterial host JM101 showed positive results by ELISA and FAPA assays. After renaturation, the biological activity of the expressed product was increased from 500-1000IU/L to about 60,000IU/L. The bacterial pro-UK showed a molecular weight of about 47,000 daltons by Western blot analysis. It can be completely inhibited by UK antiserum but not by t-PA antiserum nor by normal rabbit serum.

Base Sequence↗
PubMed1996

Gene recognition in cyanobacterium genomic sequence data using the hidden Markov model.

We have developed a hidden Markov model (HMM) to detect the protein coding regions within one megabase contiguous sequence data, registered in a database called GenBank in eight entries, of the genome of cyanobacterium, Synechocystis sp. strain PCC6803. Detection of the coding regions in the database entry was performed by using HMM whose parameters were determined by taking the statistics from the rests of the entries. This HMM has states modeling the di-codons and their frequencies within coding regions and those modeling its base contents in the intergenic regions. Results of the cross-validation showed that the HMM recognized 92.1% of coding regions assigned in sequence annotation. In addition, it suggested 94 potential new coding regions whose length are longer than 90 bases. The recognition accuracy calculated at the level of individual bases was 90.7% for the coding regions and 88.1% for the intergenic regions. This corresponds to a correlation coefficient for coding region recognition of 0.784. Comparison with its prediction accuracy with that by GeneMark showed that the HMM has the same level of prediction accuracy as GeneMark on average. Since we can extend the HMM to utilize information such as SD sequences, the prediction accuracy of the HMM will be enhanced. It was observed that correlation was positive between the prediction rate of the coding regions and the G + C content at the third position of the codon. This suggests the possibility that the prediction rate of coding regions in the cyanobacteria sequence can be enhanced by improving the present HMM into that reflects the classification of coding regions based on the G + C content.

Cyanobacteria↗
PubMed2006

Stem-loop structures in prokaryotic genomes.

BACKGROUND: Prediction of secondary structures in the expressed sequences of bacterial genomes allows to investigate spontaneous folding of the corresponding RNA. This is particularly relevant in untranslated mRNA regions, where base pairing is less affected by interactions with the translation machinery. Relatively large stem-loops significantly contribute to the formation of more complex secondary structures, often important for the activity of sequence elements controlling gene expression. RESULTS: Systematic analysis of the distribution of stem-loop structures (SLSs) in 40 wholly-sequenced bacterial genomes is presented. SLSs were searched as stems measuring at least 12 bp, bordering loops 5 to 100 nt in length. G-U pairing in the stems was allowed. SLSs found in natural genomes are constantly more numerous and stable than those expected to randomly form in sequences of comparable size and composition. The large majority of SLSs fall within protein-coding regions but enrichment of specific, non random, SLS sub-populations of higher stability was observed within the intergenic regions of the chromosomes of several species. In low-GC firmicutes, most higher stability intergenic SLSs resemble canonical rho-independent transcriptional terminators, but very frequently feature at the 5'-end an additional A-rich stretch complementary to the 3' uridines. In all species, a clearly biased SLS distribution was observed within the intergenic space, with most concentrating at the 3'-end side of flanking CDSs. Some intergenic SLS regions are members of novel repeated sequence families. CONCLUSION: In depth analysis of SLS features and distribution in 40 different bacterial genomes showed the presence of non random populations of such structures in all species. Many of these structures are plausibly transcribed, and might be involved in the control of transcription termination, or might serve as RNA elements which can enhance either the stability or the turnover of cotranscribed mRNAs. Three previously undescribed families of repeated sequences were found in Yersiniae, Bordetellae and Enterococci.

Base Composition↗
PubMed1994

Characterization of nickel-induced mutations.

A Chinese hamster ovary cell line (designated AS52) has been used to test the mutagenicity of nickel compounds. This line lacks the endogenous gene for hypoxanthine phosphoribosyl transferase (HPRT) but contains an inserted bacterial gene coding for the enzyme guanine-hypoxanthine phosphoribosyl transferase (gpt) which is the targeted locus for selection. Isolated mutants were clonally expanded and analysed utilizing the polymerase chain reaction (PCR) following exposure to ethyl methanesulfonate (EMS), Ni3S2, Ni(OH)2, NiSO4, and control conditions. Amplification of the gpt locus in normal AS52 cells results in the generation of two distinct bands, both of which have been characterized by restriction enzyme analysis and nucleotide sequencing. The smaller band represents the gpt gene. The larger band contains a large insert of bacterial origin and is non-functional. Analysis of mutants revealed three distinct patterns: (1) both PCR bands remain intact; (2) the smaller DNA band is deleted; (3) both bands are deleted. Mutant analysis was performed with two unique sets of DNA amplification primers with identical results. When compared to spontaneous or EMS induced mutants, those generated by exposure to nickel compounds exhibited an increase in gene deletions relative to point mutations; the extent of which was compound specific: NiSO4 > Ni(OH)2 > Ni3S2. These results clearly suggest that a variety of genotoxicological mechanisms are involved in the mutagenic activity of nickel compounds.

Animals↗
PubMed1992

The UDP-N-acetylglucosamine 1-carboxyvinyl-transferase of Enterobacter cloacae. Molecular cloning, sequencing of the gene and overexpression of the enzyme.

The UDP-N-acetylglucosamine 1-carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) which catalyses the first committed step in the biosynthesis of the bacterial cell-wall peptidoglycan was purified to near homogeneity from Enterobacter cloacae and the NH2-terminal amino-acid sequence determined. Using the polymerase chain reaction a 53-bp DNA fragment was synthesized; this fragment encodes the NH2-terminal sequence of the enzyme. A clone was then isolated which contained an open reading frame of 1257 bp coding for a protein of 419 amino acids. This protein was overexpressed 100-fold in transformed Escherichia coli cells and shown to possess the enolpyruvyltransferase activity. The overall amino-acid sequence of the enolpyruvyltransferase is significantly similar to that of the 5-enolpyruvylshikimate 3-phosphate synthase, the only other enzyme known to catalyse the transfer of the enolpyruvate moiety of phosphoenolpyruvate to a substrate.

Alkyl and Aryl Transferases↗
PubMed1990

Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion.

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (pulL, pulM, pulN and pulO) are shown to be required for pullulanase secretion, as is a fifth gene (pulK) which extends beyond the 5' end of the sequenced DNA. The products of the pulL, pulM, pulN and pulO genes (44 kD, 18 kD, 27 kD and 24 kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.

Amino Acid Sequence↗
PubMed1989

Cloning and analysis of an Escherichia coli operon containing the rpmF gene for ribosomal protein L32 and the gene for a 30-kilodalton protein.

The chromosomal DNA fragments of Escherichia coli K-12 were cloned into a mini-F cosmid, pRE435, after partial digestion with restriction endonuclease Sau3AI. The clones were first screened for PyrC+ and then for other genes, including rpmF encoding ribosomal protein L32 that had been mapped near pyrC (I. Janda, M. Kitakawa, and K. Isono, Mol. Gen. Genet. 201:443-436, 1985). Thus, we obtained a total of five rpmF-containing clones. The rpmF gene was located on the chromosomal segment in one of the clones (pAY2-5) by insertional mutagenesis with transposon gamma delta, followed by analysis of the gene products by the maxicell method. Hybridization analysis of clone pAY2-5 with the ordered clone bank (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) indicates that a gap at the 1,510-kilobase coordinates in the physical map of E. coli can be bridged by this clone. The nucleotide sequence of the region containing rpmF was accordingly established. In addition, the RNA transcripts from the chromosomal region containing rpmF were analyzed, and the transcriptional initiation sites were determined. The results suggest that rpmF forms an operon with the gene termed g30k which codes for a 30-kilodalton protein of unknown function. At least four transcripts were found to code for ribosomal protein L32.

Amino Acid Sequence↗
PubMed1999

Essential components of the Ti plasmid trb system, a type IV macromolecular transporter.

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Agrobacterium tumefaciens↗
PubMed1989

Evolution and regulation of the gene encoding superoxide dismutase from the archaebacterium Halobacterium cutirubrum.

The gene encoding the manganese-containing superoxide dismutase (SOD) of Halobacterium cutirubrum was isolated and characterized. The gene and 5'- and 3'-untranslated regions were located on a genomic DNA fragment of 1127 nucleotides. The deduced amino acid sequence is 200 residues long and has 39-42% identity with manganese-containing SODs of eubacteria and mitochondria. This homology may be due to either lateral transfer of the gene between eubacteria and archaebacteria or to high amino acid sequence conservation in the enzyme during the separate evolution of eubacteria and archaebacteria. Transcription of the gene initiates only about three nucleotides upstream of the translation initiation codon. The 5' end of the transcript does not contain a purine-rich Shine-Dalgarno sequence, and the promoter region does not contain consensus sequences found in other archaebacterial promoters. Termination of transcription occurs at 5 consecutive thymine residues that are preceded by a GC-rich region. The gene is basally expressed in anaerobically grown cells but is also inducible by paraquat, a generator of oxygen radicals. The same transcription initiation site is used in both types of expression, suggesting that one promoter is responsible for both basal and regulated expression. In addition to the single copy of the authentic SOD gene, the genome of H. cutirubrum contains a sequence that is very closely related to but does not code for the previously purified SOD of this organism.

Amino Acid Sequence↗
PubMed1992

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Amino Acid Sequence↗
PubMed1997

Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli.

The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol. there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2 stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme. The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase. The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E. coli using the T7 RNA polymerase and the Thiofusion expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

Amino Acid Sequence↗
PubMed2002

Congruent evolution of different classes of non-coding DNA in prokaryotic genomes.

Prokaryotic genomes are considered to be 'wall-to-wall' genomes, which consist largely of genes for proteins and structural RNAs, with only a small fraction of the genomic DNA allotted to intergenic regions, which are thought to typically contain regulatory signals. The majority of bacterial and archaeal genomes contain 6-14% non-coding DNA. Significant positive correlations were detected between the fraction of non-coding DNA and inter- and intra-operonic distances, suggesting that different classes of non-coding DNA evolve congruently. In contrast, no correlation was found between any of these characteristics of non-coding sequences and the number of genes or genome size. Thus, the non-coding regions and the gene sets in prokaryotes seem to evolve in different regimes. The evolution of non-coding regions appears to be determined primarily by the selective pressure to minimize the amount of non-functional DNA, while maintaining essential regulatory signals, because of which the content of non-coding DNA in different genomes is relatively uniform and intra- and inter-operonic non-coding regions evolve congruently. In contrast, the gene set is optimized for the particular environmental niche of the given microbe, which results in the lack of correlation between the gene number and the characteristics of non-coding regions.

DNA, Intergenic↗
PubMed1993

Three different types of organization of the vir regulon in group A streptococci.

The DNA of group A streptococci (GAS) encodes several important virulence factors such as the antiphagocytic M protein, the Ig-Fc-binding M-related proteins (FcrA-like and EnnX-like) and the complement factor-inactivating C5a peptidase. The corresponding genes emm, fcrA, ennX, and scpA, respectively, were assumed to be located close together in the GAS genome. Additionally, emm and scpA have been found to be under the positive, coordinate control of the virR locus, which led to the designation "vir regulon" for the corresponding genomic segment. In order to map the vir regulons of many GAS serotypes and to analyse any correlation between the organization of vir regulons and circumscribed heterogeneities within the emm, virR, and scpA genes, an approach using several distinct sets of polymerase chain reaction (PCR) experiments was chosen. By examination of the genomic DNA of 42 GAS isolates from 36 different M serotypes three patterns of vir regulon topography were found. The first, designated "large vir regulon" (LVR), consists of virR--fcrA(-like)--emm--ennX(-like)--scpA. The second, designated "small vir regulon" (SVR), contains virR--emm--scpA, and the last, designated "unusual vir regulon" (UVR), resembles SVR but contains additional heterogeneous sequences between emm and scpA. The patterns correlate with heterogeneities at the 3' ends of the virR and scpA genes, with the M classification system and the occurrence of specific non-coding intervening sequences within the vir regulons. The potential impact of these patterns on models to account for generation of vir regulons is discussed.

Amino Acid Sequence↗
PubMed1991

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.

Amino Acid Sequence↗
PubMed1996

Polar allele duplication for transcriptional analysis of consecutive essential genes: application to a cluster of Escherichia coli fatty acid biosynthetic genes.

The genes encoding acyl carrier protein and several key fatty acid biosynthetic enzymes are clustered at min 24 of the Escherichia coli chromosome. This cluster of genes is not transcribed as a classical operon, but rather multiple promoters are present and each gene is cotranscribed with at least one other gene. Transcripts specific for single genes ar also present. Transcription of acpP, the gene encoding acyl carrier protein, has been studied in detail. The acpP gene was shown to be transcribed from at least two different promoters by Northern (RNA) blot, primer extension, and deletion analyses, and the major promoter was mapped. We tested whether multiple promoters are necessary to produce acyl carrier protein by use of a new method whereby a transcriptional terminator was integrated into the chromosome upstream of the intact acpP gene. By use of this method (called polar allele duplication), we demonstrate that the promoter located immediately upstream of the coding sequence is sufficient for synthesis of this very abundant protein.

Acyl Carrier Protein↗
PubMed1998

Species-specific differences in the operational RNA code for aminoacylation of tRNAPro.

An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems. In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates. Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme. Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups. Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred. Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair. In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging. Additionally, E. coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E. coli acceptor-TpsiC stem-loop domain. Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Acylation↗