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PubMed1981

A gene chimaera of SV40 and mouse beta-globin is transcribed and properly spliced.

A gene chimaera of the first exon from simian virus 40 (SV40) sequences coding for T antigen placed upstream from the third exon from mouse beta-globin, and separated by the resultant new chimaeric intron, was cloned into a bacterial plasmid. When transfected into monkey cells, the gene chimaera was transcribed, polyadenylated and spliced using the donor splice site from SV40 and the acceptor splice site from mouse beta-globin. This result suggests that a donor site from one gene can be spliced to an acceptor site from another gene.

Antigens, Neoplasm↗
PubMed1994

Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside.

Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro. In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C. Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene. Sequencing of the dCK coding region showed point mutations in seven patients. Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively. dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coli and analyzed for dCK activity. Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99). We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML.

Adult↗
PubMed2005

The regional rule for bacterial base composition.

If one looks at the composition of coding DNA and of three types of intergenic regions--those between convergent, parallel and divergent genes--a striking pattern is seen in a wide range of bacteria. The AT-content of coding sequence is lowest, and the AT-contents of the intergenic regions increase in the order: convergent, parallel and divergent. Why does this pattern occur? In this article, I suggest an explanation based on the structural requirements for the binding of transcription factors.

Bacteria↗
PubMed1983

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322.

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 X 10(3). We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance. The predicted Mr of the tetA protein is 43.2 X 10(3). The sequence homology between the Tn10 tetA gene and the pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1967

Fine structure of RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA.

Nucleotide sequences of 50 RNA codons recognized by amphibian and mammalian liver transfer RNA preparations were determined and compared with those recognized by Escherichia coli transfer RNA. Almost identical translations were obtained with transfer RNA from guinea pig liver, Xenopus laevis liver (South African clawed toad), and E. coli. However, guinea pig and Xenopus transfer RNA differ markedly from E. coli transfer RNA in relative response to certain trinucleotides. Transfer RNA from mammalian liver, amphibian liver, and amphibian muscle respond similarly to trinucleotide codons. Thus the genetic code is essentially universal, but transfer RNA from one organism may differ from that of another in relative response to some codons.

Amino Acid Sequence↗
PubMed1992

Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae.

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.

Animals↗
PubMed1987

Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome.

The coding of two rare lipoproteins by two genes, rlpA and rlpB, located in the leuS-dacA region (15 min) on the Escherichia coli chromosome was demonstrated by expression of subcloned genes in a maxicell system. The formation of these two proteins was inhibited by globomycin, which is an inhibitor of the signal peptidase for the known lipoproteins of E. coli. In each case, this inhibition was accompanied by formation of a new protein, which showed a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which we suppose to be a prolipoprotein with an N-terminal signal peptide sequence similar to those of the bacterial major lipoproteins and lysis proteins of some bacteriocins. The incorporation of 3H-labeled palmitate and glycerol into the two lipoproteins was also observed. Sequencing of DNA showed that the two lipoprotein genes contained sequences that could code for signal peptide sequences of 17 amino acids (rlpA lipoprotein) and 18 amino acids (rlpB lipoprotein). The deduced sequences of the mature peptides consisted of 345 amino acids (Mr 35,614, rlpA lipoprotein) and 175 amino acids (Mr 19,445, rlpB lipoprotein), with an N-terminal cysteine to which thioglyceride and N-fatty acyl residues may be attached. These two lipoproteins may be important in duplication of the cells.

Amino Acid Sequence↗
PubMed1987

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis.

The DNA sequence of a 1.77 kb region of the Streptomyces coelicolor chromosome containing the coding and regulatory regions of the extracellular agarase (dagA) gene was determined. The sequence predicts a primary translation product of 309 amino acids and Mr 35132. Comparison of the N-terminal sequence determined for the mature extracellular protein with that of the primary translation product deduced from the DNA sequence predicts the presence of a 30 amino acid signal peptide. Analysis of the transcription of the dagA gene using high resolution S1 mapping, in vitro transcription, dinucleotide-primed in vitro transcription and in vivo promoter probing identified four promoters, initiating transcription approximately 32, 77, 125 and 200 nucleotides upstream of the coding sequence.

Amino Acid Sequence↗
PubMed1995

Bacterial expression and site-directed mutagenesis of a functional recombinant apolipoprotein.

To facilitate structure-function studies of Manduca sexta apolipophorin III (apoLp-III), its nucleotide coding sequence was cloned from a fat body cDNA library by in vitro DNA amplification. The amplification product was cloned in the pET expression vector and introduced into E. coli. After induction, cultures were screened for apoLp-III protein production by immunoblotting with anti-apoLp-III serum. Data obtained indicated the presence of apoLp-III in both cell lysates and media of cell cultures harboring the apoLp-III-pET construct but not in cells containing the parent vector. The protein was isolated from the cell-free supernatant of cultures grown in minimal media 4 h after induction. Verification that the recombinant protein produced was indeed apoLp-III was obtained by electrospray mass spectrometric analysis. Circular dichroism (CD) spectroscopy of the isolated recombinant protein revealed a characteristic content of alpha-helical secondary structure with a further induction of helix upon addition of 50% trifluoroethanol. In urea denaturation studies, monitored by CD, evidence was obtained that recombinant and natural apoLp-III possess indistinguishable thermodynamic properties. In addition, lipid binding assays revealed that recombinant apoLp-III formed stable complexes with phospholipids and was capable of associating with lipoprotein surfaces. Examination of the fluorescence properties of recombinant apoLp-III revealed the presence of a noncovalently associated fluorescent contaminant that was effectively removed by reverse phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase.

The nucleotide sequence of the gene coding for xylose isomerase from Ampullariella sp. strain 3876, a gram-positive bacterium, has been determined. A clone of a fragment of strain 3876 DNA coding for a xylose isomerase activity was identified by its ability to complement a xylose isomerase-defective Escherichia coli strain. One such complementation positive fragment, 2,922 nucleotides in length, was sequenced in its entirety. There are two open reading frames 1,182 and 1,242 nucleotides in length, on opposite strands of this fragment, each of which could code for a protein the expected size of xylose isomerase. The 1,182-nucleotide open reading frame was identified as the coding sequence for the protein from the sequence analysis of the amino-terminal region and selected internal peptides. The gene initiates with GTG and has a high guanine and cytosine content (70%) and an exceptionally strong preference (97%) for guanine or cytosine in the third position of the codons. The gene codes for a 43,210-dalton polypeptide composed of 393 amino acids. The xylose isomerase from Ampullariella sp. strain 3876 is similar in size to other bacterial xylose isomerases and has limited amino acid sequence homology to the available sequences from E. coli, Bacillus subtilis, and Streptomyces violaceus-ruber. In all cases yet studied, the bacterial gene for xylulose kinase is downstream from the gene for xylose isomerase. We present evidence suggesting that in Ampullariella sp. strain 3876 these genes are similarly arranged.

Actinomycetales↗
PubMed2005

Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri.

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.

Amino Acid Sequence↗
PubMed1985

RNA polymerase-DNA interactions in Streptomyces. In vitro studies of a S. lividans plasmid promoter with S. coelicolor RNA polymerase.

DNA fragments of the Streptomyces lividans plasmid pIJ101 have been tested for their ability to bind Streptomyces coelicolor RNA polymerase in vitro or to promote transcription in Streptomyces in vivo. One DNA fragment which does both was shown to encode a transcript which was expressed at low cell-density in cultures of pIJ101-containing cells. The transcript start was located on the DNA sequence of the fragment by nucleotide-primed RNA polymerase binding experiments and by S1 nuclease mapping. The pattern of DNase I protection, the sites of enhanced DNase I cleavage and the DNA sequence of the fragment suggest that the RNA polymerase holoenzyme form, which recognizes this promoter, is similar in its interaction with DNA to the major RNA polymerase of Escherichia coli. Regions showing 3/6 nucleotide homology with each of the -35 and -10 regions of the consensus sequence of E. coli promoters are present in the same positions relative to the transcript start. Symmetrical sequences which may be involved in the regulation of expression of the promoter and a potential polypeptide coding sequence can be identified.

Autoradiography↗
PubMed1986

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control.

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.

Amino Acid Sequence↗
PubMed1982

Identification of gene products from cloned fragments of the left arm of lambda dapB2.

A set of recombinant plasmids encompassing the bacterial substitution in lambda dapB2 (Mackie, G. A. (1980) J. Biol. Chem. 255, 8928--8935) has been characterized genetically by complementation and biochemically by an analysis of the proteins encoded by individual plasmids in vitro. In addition to a cluster of four genes which includes the dapB locus, a total of seven gene products has been identified. One of these is ribosomal protein S20, whose gene (rpsT) has been localized to a 0.56-kilobase segment of DNA bounded by HindIII and HindII sites. Positive identification of this gene on plasmids pGM9 and pGP2 has been achieved by analysis of the products encoded by these plasmids in vitro and by the ability of pGM9 to complement a strain lacking S20 among its 30S subunit proteins. A second gene is that for isoleucyl tRNA synthetase (ileS). Its presence on pGM21 has been ascertained by the latter's ability to direct the synthesis of a protein in vitro with the size anticipated for this gene product. Extracts of cells harbouring this plasmid also exhibit greater isoleucyl tRNA synthetase activity than parental extracts. A third gene is at least 1.3 kilobases distant from rpsT and encodes a protein of 24 000 molecular weight, of unknown function. This locus is in turn about 6.5 kilobases from lambda gene E. The latter gene, and several others, all of which likely derive from lambda DNA are carried on a 5.2-kilobase fragment inserted in the plasmid pGM11 which includes the junction between phage and bacterial sequences on the left side of lambda dapB2. This fragment of DNA is inserted into the vector in a manner which permits the coupled transcription and translation of lambda genes D and E in vitro, despite the absence of their natural promoter. The most striking feature of the organization of the bacterial genes on lambda dapB2 is the clustering of genes for polypeptides in the right half of the bacterial substitution in contrast to the apparent minimal use of the coding potential in the left half of the substitution.

Bacterial Proteins↗
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↗