PubMed Health⌕ Search

Biomedical subjects

B E Uhlin

Publications and source records attributed to B E Uhlin.

At least 55 records · Page 3Linked to original sources
PubMed1988

Processed mRNA with differential stability in the regulation of E. coli pilin gene expression.

E. coli expressing the papA-I genes produce pili that mediate specific adhesion to mammalian cells. We show that the major pilus subunit gene, papA, is part of a polycistronic transcriptional unit subject to specific posttranscriptional processing. A primary transcript also encoding the papB regulatory gene product is endonucleolytically cleaved, resulting in the rapid decay of the papB-encoding 5' half of the mRNA, whereas the papA-encoding 3' half remains as a quite stable transcript. Processing and differential mRNA stability thereby result in accumulation of mRNAs encoding only the major pilus subunit. A sequence immediately downstream of the papA coding region may serve as a stability determinant for the papA transcript and concomitantly attenuate read-through transcription into the minor pilus subunit gene papH. This suggests that differential expression of genes within an operon may include endo- and exonucleolytic processing of the mRNA.

Bacterial Outer Membrane Proteins↗
PubMed1988

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli.

Expression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F72 and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.

Base Sequence↗
PubMed1986

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion.

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.

Antibodies, Monoclonal↗
PubMed1986

Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli.

The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.

Amino Acid Sequence↗
PubMed1985

Transcriptional activation of a pap pilus virulence operon from uropathogenic Escherichia coli.

A gene cluster mediating production of pili in uropathogenic Escherichia coli was analysed with respect to regulation of pili synthesis. Two cistrons, papB and papI, were localized upstream of the major pilus subunit gene, papA. The papI-papB-papA region was characterized by nucleotide sequencing and by transcriptional analysis. The papA gene was primarily represented by an 800 nucleotide long transcript but was also co-transcribed with papB as a less abundant 1300 nucleotide long mRNA. Both transcripts presumably terminated at the same site downstream of the papA coding sequence. The weakly expressed papI gene was transcribed in the opposite direction to that of papB and papA. Studies with lacZ operon fusions showed that the papB gene encoded a trans-active effector required for papA transcription. Similarly, the papI gene stimulated papB transcription in trans. Furthermore, full expression of papA was cis dependent upon the papI-papB region. Transcription of the papB gene was shown to be dependent upon cAMP and its receptor protein. A binding site for the cAMP-CRP complex was postulated in the DNA sequence upstream of the papB promoter.

Amino Acid Sequence↗
PubMed1985

Adhesion to human cells by Escherichia coli lacking the major subunit of a digalactoside-specific pilus-adhesin.

Pathogenic bacteria frequently possess pili with specific binding properties that allow them to attach to epithelial tissue. In Escherichia coli, the pili associated with pyelonephritis (Pap pili) bind to digalactoside-containing glycolipids on the uroepithelium. Transposon-insertion mutants and deletion mutants of the cloned genetic determinant encoding synthesis of such digalactoside-binding Pap pili have been studied in E. coli K-12. Mutants that completely lack synthesis of the major Pap pili subunit protein, the papA gene product, and thereby no longer produce pili were shown to retain the binding specificity of intact Pap pili. Reduced expression of some of the remaining pap genes, presumably due to polarity effects from papA::Tn5 insertions, was circumvented by the use of a copy-number mutant plasmid vector. Derivatives carrying the papA-D genes produced Pap pili but did not bind to human cells. The products of the genes papE-G are essential for digalactoside-specific hemagglutination and for attachment to urinary bladder cells. The papC and papD genes presumably aid in surface localization and/or polymerization of the pili-adhesin subunits and are required for expression of pili as well as of the binding properties. Serological evidence is presented that suggests that a minor pilus component(s), presumably produced by the papE, -F, or -G gene, is the actual binding moiety in the digalactoside-specific interaction of Pap pilus-adhesin.

Adhesins, Escherichia coli↗
PubMed1984

Environmental temperature regulates transcription of a virulence pili operon in E. coli.

The expression in Escherichia coli K-12 of a pilus-adhesion determinant, obtained through molecular cloning from a pyelonephritic E. coli isolate, was studied at different temperatures. Strain HB101 carrying the recombinant plasmid pRHU845 agglutinated human erythrocytes after growth at 37 degrees C but not after growth at 22 degrees C. Quantitation of pilus subunit protein by an enzyme-linked immunosorbent assay (ELISA) for pilus antigen showed that synthesis of the pilus subunits was reduced at least 20-fold at 22 degrees C as compared with 37 degrees C. The 5' end of the pilus subunit structural gene, papA, was fused to the lacZ gene such that expression could be monitored at both translational and transcriptional levels. Measurements of beta-galactosidase production by the papA-lacZ hybrids provided evidence for thermoregulation of papA gene transcription. A regulatory determinant was localized to a 2-kb EcoRI-HindIII fragment encoding the papB gene and part of papA although none of the presently known pap gene products seem to be directly involved in a thermoregulatory mechanism. Comparison with other thermoregulatory systems in E. coli suggests that pap gene expression is regulated by a novel mechanism.

Base Sequence↗
PubMed1984

Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation.

A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose-resistant hemagglutination ( MRHA ) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96 , and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 ( papB ) 19 500 ( papA , structural gene for the Pap pilus subunit), 81 000 ( papC ) and 28 500 ( papD ). Analyses of a lacZ- papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.

Antigens, Bacterial↗
PubMed1981

Physical mapping of the srl recA region of Escherichia coli: analysis of Tn10 generated insertions and deletions.

A restriction endonuclease map for the enzymes EcoRI, BamHI, SalI, and PstI covering 23.5 kilobase pairs (kb) of the srl recA region of Escherichia coli was constructed. An insertion of the transposon Tn10 in the negative regulatory gene srlR was shown to be located 5.8 kb away from the promoter proximal end of the recA gene. The extent of several Tn10 generated deletions, originating from the srlR301::Tn10 insertion, were analyzed by physical mapping. Three mutations that had removed the Tn10 encoded tetracycline resistance gene, del(srl-recA)302, del(srl-recA)304, and del(srl-recA)303, were found to be deleted for 40%, 45%, and 50% of the recA structural gene, respectively. A deletion, del(srl-recA)306, that had not affected the structure of the Tn10 in srlR301 was shown to have removed the entire recA structural gene.

Chromosome Deletion↗
PubMed1981

Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli.

We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.

Acetyltransferases↗
PubMed1981

Overproduction of the Escherichia coli recA protein without stimulation of its proteolytic activity.

Plasmid pBEU14, which carries the Escherichia coli recA+ gene and which can be amplified by manipulation of growth temperature, was constructed. When pBEU14 deoxyribonucleic acid was amplified, a high rate of synthesis and accumulation of recA protein resulted. Amplification of the recA gene and protein did not cause induction of prophage lambda, indicating that the proteolytic activity of the recA protein was not stimulated.

Bacterial Proteins↗
PubMed1980

Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop.

The replication control functions of a mutant of plasmid R1 that replicates without control at temperatures above 35 degrees C have been analyzed. Although the mutations have not been mapped precisely, the data indicate that the gene (cop) previously identified on the wild-type plasmid (S. Molin and K. Nordström, J. Bacteriol. 141:111-120, 1980) as being responsible for expressing a trans-acting replication inhibitor, as well as for incompatibility of plasmid R1, is not affected in this mutant. Thus, the conditional lack of replication control observed in this plasmid mutant presumably is not caused by the loss of inhibitor activity of the cop gene.

DNA Replication↗
PubMed1979

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products.

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.

Cloning, Molecular↗
PubMed1979

Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19.

Plasmid R1drd-19 is present in a small number of copies per cell of Escherichia coli. The plasmid was reduced in size by in vivo as well as in vitro (cloning) techniques, resulting in a series of plasmid derivatives of different molecular weight. All plasmids isolated contain a small region (about 2 x 10(6) daltons of deoxyribonucleic acid) of the resistance transfer factor part of the plasmid located close to one of the IS1 sequences that separates the resistance transfer factor part from the resistance determinant. All these derivatives were present at the same copy number, retained the incompatibility properties of plasmid R1drd-19, and were stably maintained during cell division. Genes mutated to yield copy mutations also were found to be located in the same region.

Cell Division↗