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PubMed1997

RpoN of the fish pathogen Vibrio (Listonella) anguillarum is essential for flagellum production and virulence by the water-borne but not intraperitoneal route of inoculation.

To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative sigma 54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.

Amino Acid Sequence↗

PubMed2005

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.

Adenoviridae↗

PubMed1972

Ribosomal RNA genes of Neurospora: isolation and characterization.

DNA sequences in Neurospora crassa that code for ribosomal RNA have been isolated in a highly purified state by DNA-RNA hybridization. About 100 separate ribosomal RNA genes exist in each nucleus. These genes are composed of repeated sequences that are present about 100 times per nucleus. The individual members of this group of DNA sequences are essentially identical.

Carbon Isotopes↗

PubMed1998

Analysis of the genome of Mycobacterium tuberculosis H37Rv.

The powerful combination of genomics and bioinformatics is providing a wealth of information about Mycobacterium tuberculosis, the aetiological agent of human tuberculosis, that will facilitate the conception and development of new therapies. The starting point for genome sequencing was the integrated map of the 4.4 Mb circular chromosome of the widely used, virulent reference strain, M. tuberculosis H37Rv. Cosmids and bacterial artificial chromosomes were selected from ordered libraries and subjected to systematic shotgun sequence analysis. This approach simplified sequence assembly as the genome is rich in repetitive DNA. In common with most bacteria, > 90% of the potential coding capacity is used, and probable or tentative functions could be attributed to > 70% of the genes. The potential biological roles of two of the principal driving forces in genome dynamics, insertion sequence elements and polymorphic multigene families are discussed.

DNA Transposable Elements↗

PubMedDate not supplied

Use of specific oligonucleotides for direct enumeration of Listeria monocytogenes in food samples by colony hybridization and rapid detection by PCR.

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after 48-h enrichment period. Using this technique, as little as 10(2) bacteria per ml of enrichment broth can be detected.

DNA, Bacterial↗

PubMed1995

[Analysis of amino acid sequences of Rhodobacter sphaeroides glutamine synthetase].

A comparative analysis of the amino acid sequence of glutamine synthetase (GS) of the photosynthetic purple bacterium Rhodobacter sphaeroides revealed that the enzyme is typical for first type procaryotic GSs and structurally resembles GSs of enteric bacteria. The data obtained indicate that the complex phenotype of purple bacterial mutants at the glnA gene coding for GS may be conditioned by specific regulation of nitrogen metabolism in bacterial cells rather than by structural-and-functional peculiarities of GS.

Amino Acid Sequence↗

PubMed1979

Nucleotide sequence of the intercistronic region preceding the gene for RNA polymerase subunit alpha in Escherichia coli.

The gene for RNA polymerase subunit alpha is a co-transcribed with several ribosomal protein genes (Jaskunas, S.R., Burgess, R.R., and Nomura, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5036-5040). The DNA sequence whicch codes for the COOH-terminal amino acids of S4 and the NH2-terminal amino acids of alpha, and the 25 nucleotide intercistronic sequence have been determined. This short distance supports the idea that some post-transcriptional regulation determines the differential synthesis of alpha and ribosomal proteins.

Amino Acid Sequence↗

PubMed2001

Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona.

Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.

Arizona↗

PubMed1995

Identification of an inhibitory element within the human 68-kDa (U1) ribonucleoprotein antigen.

Various nuclear proteins are the major targets of autoimmune responses in various rheumatic disorders. In particular, autoantibodies directed against a 68-kDa protein associated with the (U1) RNA-containing small nuclear ribonucleoprotein complexes typically occur in sera of patients with mixed connective tissue disease and related rheumatic disorders, such as systemic lupus erythematosus, and therefore are very useful as a serological marker. For establishing powerful immunoassays, it was necessary to generate recombinant human P68 antigen as the antigenic target. In this study we demonstrated that the cDNA coding for the full-length human P68 antigen could not be expressed by a traditional bacterial vector system due to a putative inhibitory sequence designated as inhibitory sequence X which is located between the autoreactive domains C' and D' of the human P68 antigen. The construction of corresponding hybrid plasmids carrying two functional and independent gene blocks indicated the trans-active function of the inhibitory sequence X, which could be localized by expression studies of various deletion constructs. Comparable Northern blot analysis clearly demonstrated that the inhibitory sequence X could act on the translation of the P68 mRNA. After excision of the inhibitory sequence X a dramatic increase in the production of recombinant human P68 antigen was observed.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli.

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.

Amino Acid Sequence↗

PubMed1992

Trans-acting factors and properly positioned DNA elements repress mating-type genes in fission yeast.

Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences. The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region. The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression. Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants. These mutants can de-repress the resistance gene at other positions than the one used for their selection. Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette. These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.

Alleles↗

PubMed1995

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Amino Acid Sequence↗

PubMed1984

Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions.

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

Autoradiography↗

PubMed1986

A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci.

The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25-kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements.

Amino Acid Sequence↗

PubMed1992

The expression of murine protein disulfide isomerase in Escherichia coli.

Protein disulfide isomerase (PDI), a luminal enzyme of the endoplasmic reticulum (ER), is thought to be involved in the process that assures that the correct disulfide bonds form as a newly synthesized protein folds into its appropriate three-dimensional structure (Freeman, 1984). In recent years, the ER has been shown to have at least two additional, distinct PDI-related luminal proteins (Bennett et al., 1988; Mazzarella et al., 1990). As a potential first step toward an investigation of the structure and function of PDI and of the PDI-related proteins as well, we have developed a bacterial expression system in Escherichia coli capable of synthesizing significant levels of enzymatically active PDI under the control of the inducible tac promoter. We have observed that the use of this bacterial expression system is complicated by the fact that there is a significant amount of internal initiation of protein synthesis within the PDI coding sequence and the fact that all of the PDI-related expression products are found equally distributed between the cytoplasmic and periplasmic fractions due to a single peptide-independent mechanism. Our studies with this system have demonstrated that at least some truncated PDI molecules containing the carboxy-terminal most active site have significant PDI activity.

Amino Acid Sequence↗

PubMed1995

Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.

The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.

3-Isopropylmalate Dehydrogenase↗

PubMed1994

Sequence and expression of a baculovirus protein with antigenic similarity to telokin.

A protein from baculovirus-infected cells reacted with an antibody against the smooth muscle protein telokin. Because of this unusual similarity, the protein, termed telokin-like protein-20 (TLP20), was isolated and characterized. Its M(r) on denaturing polyacrylamide gels was 28K and the protein contained a high proportion of beta structure. A cDNA for TLP20 was isolated and sequenced. The 3' non-coding sequence contained a region of high identity with the 5' end of two other baculovirus genes. The 5' non-coding region contains several baculovirus regulatory elements. Surprisingly, the derived amino acid sequence showed no homologies to telokin. The cDNA was cloned into a bacterial expression vector and the subsequently expressed protein had a slightly lower M(r) than the native protein, but cross-reacted with telokin antibody. This paper reports the characterization of a new baculovirus protein that shares some antigenic similarities to the smooth muscle protein telokin.

Amino Acid Sequence↗

PubMed1999

The insect immune protein scolexin is a novel serine proteinase homolog.

Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.

Amino Acid Sequence↗