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H Bujard

Publications and source records attributed to H Bujard.

At least 73 records · Page 4Linked to original sources
PubMed1989

Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system.

The single-polypeptide RNA polymerases that are encoded by bacteriophage T7 and its relatives form the basis of highly specific and efficient transcription systems. Here, we describe the regulation of transcription from phage promoters by the lac repressor-operator system of Escherichia coli. A synthetic oligodeoxyribonucleotide that contains the core sequence of the lac operator (lacO) was cloned at various distances downstream from the transcription start point (tsp) of the T3 and T7 promoters. The ability of lac repressor to prevent transcription from the phage promoters in vitro was dependent on the position of the operator. Efficient repression was observed when the center of the operator was placed between +14 and +27 (+1 being the tsp), whereas the repressor had little effect when bound to operators centered at +64. For in vivo studies, the chloramphenicol acetyltransferase (CAT)-encoding reporter gene was placed under the control of various promoter-operator constructs, and introduced into bacterial cells containing the genes for the lac repressor and T3 or T7 RNA polymerase. As with in vitro studies, high levels of repression (greater than 4000-fold) of T3 and T7 RNA polymerase activity were achieved, and repression was reversed by the inducer isopropyl-beta-D-thiogalactopyranoside. When the T3 promoter-lacO constructs are used to regulate the expression of a target gene in combination with an inducible RNA polymerase gene under control of the lacUV5 promoter, the doubly regulated system provides extremely tight levels of repression, yet allows high levels of expression after induction. In such a system, we observed a greater than 10(5)-fold increase in CAT activity within 30 min after induction. This system should prove useful in cloning and expressing genes that are potentially toxic to the host cells.

Base Sequence↗
PubMed1989

A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection.

We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P. falciparum merozoites which is a processing product of the precursor glycoprotein gp190. This assay can be used in the diagnosis of acute malaria in individuals with primary infection. Two partial sequences of gp190 were expressed in E. coli as beta-galactosidase (beta-Gal) fusion proteins. The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots. Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity. The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.

Animals↗
PubMed1989

Regulated expression of foreign genes in mammalian cells under the control of coliphage T3 RNA polymerase and lac repressor.

Systems that stringently regulate the expression of individual genes within a complex genetic background have contributed greatly to the analysis of gene function. In this report the development of a highly regulated expression system in mammalian cells is described in which transcription of a foreign gene is mediated by the bacteriophage T3 RNA polymerase under the control of the Escherichia coli lac repressor. Rabbit kidney cell lines have been established that constitutively express the phage RNA polymerase and lac repressor. The two bacterial proteins regulate the transcription of the coding sequence of the firefly luciferase, which has been placed under the control of a T3 promoter/lac operator fusion. In the presence of the inducer isopropyl beta-D-thiogalactoside, efficient T3 polymerase-dependent transcription is observed, which is tightly repressed in the absence of inducer. Translation of the T3 transcripts can be mediated by vaccinia virus functions. The demonstration that a specific transcription activity can be regulated over a range of several orders of magnitude in higher eukaryotic cells by using a highly specific and nontoxic inducer has broad implications for a variety of studies.

Animals↗
PubMed1989

Development of the human immune response against the major surface protein (gp190) of Plasmodium falciparum.

The 190-kilodalton glycoprotein (gp190) of Plasmodium falciparum, the precursor of the major surface proteins of merozoites, is considered a promising candidate for a blood stage malaria vaccine. DNA sequences specific for the gp190 of the two isolates K1 and MAD20 were subcloned and expressed in Escherichia coli. The panel of fusion proteins obtained represents about 80% of the polymorphic sequences observed so far within various isolates of P. falciparum. Sera from individuals living in a malaria-endemic area of West Africa were tested in immunoblots against the gp190 fusion proteins, and antibody reactivity was mapped to defined regions of the gp190. Depending on the age of the individual and on the presence of parasites in the blood, distinct regions of gp190 were differentially recognized by the respective antibodies. Similarly, the analysis of sera from German patients with acute malaria revealed a distinct pattern. When grouped according to age and to parasitemia, the reactivity of the sera of people living in malaria-endemic areas may indicate a correlation between certain gp190 regions and protective immune response.

Animals↗
PubMed1988

Epitopes recognized by human T cells map within the conserved part of the GP190 of P. falciparum.

In a study aimed at developing a vaccine against the asexual blood stages of Plasmodium falciparum, two T cell epitopes were identified within a nonpolymorphic region of gp190 of Plasmodium falciparum merozoites. The two epitopes, which were revealed by deletion analysis, stimulated human T cell clones. Peptides containing sequences of the epitopes stimulated the cloned T cells and peripheral blood mononuclear cells (PBMC) from malaria-infected individuals. Moreover, the T cell clones responded to 11 different Plasmodium falciparum isolates in culture, showing that the epitopes are recognized in native parasites.

Amino Acid Sequence↗
PubMed1988

Nonpolymorphic regions of p190, a protein of the Plasmodium falciparum erythrocytic stage, contain both T and B cell epitopes.

Two conserved regions from the genetically polymorphic p190 molecule of the malaria parasite Plasmodium falciparum have previously been expressed in Escherichia coli as separate polypeptides (190.L and 190.M) or as a single fusion protein (190.N). In the present study we investigated whether human B and T lymphocytes recognize these conserved regions. The more amino-terminal region, 190.L (corresponding to residues 188-363 of the encoded protein sequence) reacted preferentially with sera from donors living in a malaria-endemic area. Also, EBV-transformed B cells, from a healthy donor living in a malaria-mesoendemic area, were fused with a human-mouse hybrid line (SPM4-0), yielding two hybridomas whose products recognized both 190.L and the fusion protein 190.N, but not the 190.M polypeptide. A large number of p190-specific T cell clones were obtained from PBMC of a noninfected donor, after in vitro stimulation with the recombinant fusion protein 190.N. The clones reacted with intact, parasite-derived p190, as well as either 190.L or 190.M. Four clones that recognized the more amino-terminal fragment also responded to infected E. According to these results the more amino-terminal conserved sequences of p190 have the requisites to be immunogenic in humans.

Adult↗
PubMed1988

PL of coliphage lambda: an alternative solution for an efficient promoter.

Promoter PL of coliphage lambda is highly active in vivo although it is recognized 15-30 times less efficiently by RNA polymerase when compared with promoters of similar strength. Moreover, it differs significantly from the consensus sequence for Escherichia coli promoters. Sequence variants of PL which are more homologous to consensus promoters bind RNA polymerase with increased efficiency. They are nevertheless significantly reduced in their in vivo strength. High activity can be restored by a downstream sequence of a typical consensus-like promoter. Evidently, such elements are required for the efficient release of a stably bound RNA polymerase into a transcriptional elongation complex. We propose that the functional programme encoded in a promoter sequence can be optimized in alternative ways.

Bacteriophage lambda↗
PubMed1988

Promoters largely determine the efficiency of repressor action.

Operator sequence and repressor protein regulate the activity of the lac promoter over a greater than 1000-fold range. Combinations of the lac operator with other promoter sequences, however, differ vastly in the level of repression. The data presented show that the extent of repression is determined largely by the rates of complex formation of the competing systems operator-repressor and promoter-RNA polymerase and by the rate at which RNA polymerase clears the promoter. Moreover, up to 70-fold differences in the level of repression were found when the operator was placed in different positions within the promoter sequence. A kinetic model is proposed that explains the observed effects and that allows predictions on promoters controlled by negatively acting elements.

Base Sequence↗
PubMed1987

Promoter recognition and promoter strength in the Escherichia coli system.

The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude. Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E. coli. The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25. Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found. This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.

Bacteriophage lambda↗
PubMed1986

Determinants for protein translocation across mammalian endoplasmic reticulum. Membrane insertion of truncated and full-length prelysozyme molecules.

The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full-length prelysozyme using transcription-coupled capping of RNA and subsequent translation in a wheat germ cell-free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal-recognition particles (SRP) to the cell-free system blocked the nascent chain synthesis. The SRP-depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP-induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum.

Animals↗
PubMed1986

Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures.

The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy. Up to 75-fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5. Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence. By contrast, a synthetic 'consensus promoter' belongs to the less efficient signals. Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that information outside of the 'classical' promoter region contributes to promoter activity.

Base Sequence↗
PubMed1986

Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process.

After binding to a promoter Escherichia coli RNA polymerase is in contact with a region of about 70 bp. Around 20 bp of this sequence are transcribed. Information encoded within this transcribed region is involved in late steps of the functional program of a promoter. By changing such 'down-stream' sequences promoter strength in vivo can be varied more than 10-fold. By contrast, information for early steps of the promoter program such as recognition by the enzyme and formation of a stable complex resides in a central core region of about 35 bp. Our data show that the strength of a promoter can be limited at different levels of the overall process. Consequently promoters of identical strength can exhibit different structures due to an alternate optimization of their program.

Base Sequence↗
PubMed1986

Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

A 240-bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors. Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N-terminally fused to E. coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively. All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus-infected individuals, but not with control sera. The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR. The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80-amino acid polypeptide which were recognized differently by the various patients.

Acquired Immunodeficiency Syndrome↗
PubMed1986

lac Repressor blocks transcribing RNA polymerase and terminates transcription.

Operator sequences are essential elements in many negatively controlled operons. By binding repressors, they prevent the formation of active complexes between RNA polymerase and promoters. Here we show that the Escherichia coli lac operator-repressor complex also efficiently interrupts ongoing transcription. This observation suggests a mechanism of action for operators located distal to promoter sequences.

DNA, Bacterial↗
PubMed1985

Polymorphism of the precursor for the major surface antigens of Plasmodium falciparum merozoites: studies at the genetic level.

The gene for the precursor of Plasmodium falciparum merozoite surface antigens has been cloned. The entire sequence of the gene from a Thai isolate of the parasite is reported. It provides evidence for a signal peptide, a region containing short repeating peptides and an anchor sequence. In addition, the 5' end of a Papua New Guinea isolate has been sequenced. Comparison of these and other sequences defines, at the genetic level, a polymorphic region in the protein, and suggests that other parts of the protein are less susceptible to variation. Furthermore it appears that several signal peptides of P. falciparum exhibit extensive sequence homologies.

Amino Acid Sequence↗
PubMed1985

Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis.

Using purified sigma 55 RNA polymerase from Bacillus subtilis in an in vitro transcription system, we have shown that both promoters and terminators of Gram negative origin are recognized by this enzyme. Furthermore, when B. subtilis is transformed with a shuttle vector containing certain of these promoters, synthesis of the Staphylococcus aureus CAT protein is achieved, and levels up to 25% of the total cellular protein can be obtained. These findings indicate a closer evolutionary relationship of the expression machinery of these two bacterial species than has been assumed so far. On the basis of these results, the construction of new expression vectors for B. subtilis is likely to be facilitated, since a variety of well-characterized signal elements from Escherichia coli are available.

Acetyltransferases↗