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H Bujard

Publications and source records attributed to H Bujard.

At least 91 records · Page 5Linked to original sources
PubMed1985

Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5.

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.

Base Sequence↗
PubMed1984

A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription-coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine-labelled proteins produced offer a number of possibilities for the study of structure-function relationships of proteins.

Cell-Free System↗
PubMed1982

Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes.

The copy number of plasmids containing the ColE1 replicon is affected by changes in the transcriptional activity within the plasmid if these changes lead to transcriptional readthrough into the replication region towards the promoter priming DNA replication. Such readthrough e.g., from the tet region in pBR322 not only causes overproduction of a peptide known to affect the copy number negatively but also appears to interfere negatively with the replication of the plasmid itself. The proper placement of efficient transcriptional terminators prevents such interference and permits the stable integration of strong promoters. Due to this termination effect, up to 9-fold differences in plasmid copy number were observed, depending upon the particular growth conditions. The higher copy number is of course reflected by higher yields of plasmid-specified gene products indicating the relevance of the above effects for studies of gene expression.

Bacteriocin Plasmids↗
PubMed1981

Promoter sites in the genome of B. subtilis phage SPP1.

Transcriptional complexes formed in vitro using DNA of B. subtilis phage SPP1 as template and E. coli and B. subtilis RNA polymerases were analyzed by electron microscopy. Both enzymes recognize the same five strong promoters in the early region of the genome. Strand selection at these sites was identical with both enzymes. These results correlate well with data obtained from in vivo transcription studies. Transcriptional activity in the late region of the genome was very low, not permitting the identification of promoter sites.

Bacillus subtilis↗
PubMed1981

Organization of transcriptional signals in plasmids pBR322 and pACYC184.

Electron microscopic analysis of in vitro transcriptional complexes of pBR322 and pACYC184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors. These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pBR322 up to 10-fold. A most interesting signal arrangement was found at the beginning of the tetracycline resistance region, where two partially overlapping promoters (P1 and P2) initiate transcription crosswise in opposite directions. Whereas P2 is known to promote tetracycline resistance and to be inactivated by HindIII cleavage, P1 is able to transcribe DNA integrated at that site and probably contributes to the expression of the beta-lactamase gene in pBR322. In pACYC184, besides P1, P2, and the cat (chloramphenicol resistance) promoter (P5), two initiation sites (P3 and P4) were mapped in a region that appears to be part of insertion sequence 1. The maps of transcription signals permit a more predictable utilization of these cloning vehicles and also allow the reinterpretation of earlier cloning results.

Chloramphenicol↗
PubMed1981

Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal.

Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and terminators, respectively. By assessing transcription within the indicator regions, the efficiency of promoters as well as termination signals can be determined in vitro and in vivo.

Base Sequence↗
PubMed1979

Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs.

The interaction of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) with restriction fragments obtained from various E. coli related DNAs was studied in vitro. The DNAs investigated included several coliphage genomes (T5, lambda, T7, fd) and plasmid DNAs (pML 21, pSC101). By using the nitrocellulose filter binding of the enzyme-DNA complexes, fragment-specific relative rates of complex formation as well as complex stabilities were determined. Promoter-specific relative rates of polymerase binding were derived from fragment-specific rates by taking into account the number of major binding sites for RNA polymerase within several DNAs. Estimates of the stability of complexes formed between some major binding sites and the enzyme were obtained by studying the rate of complex decay. Both characteristics--rate of complex formation and rate of decay--varied widely and independently of each other. The promoters reacting most efficiently with E. coli RNA polymerase were found in the early region of coliphage T5 whereas some promoters in pML 21, or for example, the lambda promoter PI, belong to signals binding the enzyme most slowly. Based on the second-order rate constant determined for the interaction of E. coli RNA polymerase with promoters of phage fd, the fastest promoters characterized so far reacted with rates in the order of 10(8) M-1s-1. The hierarchy of promoters established here is of interest from the viewpoint that promoter strength correlates with the rate of polymerase binding. Among the promoters studied here this rate spans a range of 2 orders of magnitude.

Base Sequence↗
PubMed1978

Electron microscopic analysis of in vitro transcriptional complexes: mapping of promoters of the coliphage T5 genome.

Transcriptional complexes formed in vitro with coliphage T5 DNA as template were analyzed by electron microscopy and the number and location of starting sites utilized by E. coli RNA polymerase were determined. Of the 40 promoters characterized in this way, 6 map in the two terminal "pre-early" regions, 29 in the "early" and 5 in the "late" region. The direction of transcription within the different regions determined in this study agrees with earlier findings derived from RNA synthesized in vivo.

Coliphages↗
PubMed1977

Interaction of E. coli RNA polymerase with promotors of coliphage T5: the rates of complex formation and decay and their correlation with in vitro and in vivo transcriptional activity.

The genome of virulent coliphage T5 contains about 30 sites which form stable complexes with E. coli RNA polymerase. Some of these sites bind RNA polymerase with high rates, others form extremely stable complexes as compared with promotors of other E. coli systems. The transcriptional activity of these promotors in vivo and in vitro reflects the rate of complex formation with RNA polymerase rather than the stability of the enzyme/promotor complex. The fastest, i.e. the most active promotors are found in the "early" region of gene expression followed by promotors of the "preearly" class. The few binding sites for the E. coli holoenzyme within the "late" region react more slowly with the enzyme.

Binding Sites↗
PubMed1977

Electron microscopy of DNA: determination of absolute molecular weights and linear density.

The molecular lengths of several phage DNA's and one plasmid DNA have been measured and the molecular linear density of double stranded DNA under standard conditions has been determined. This determination was possible since the absolute molecular weight for phi X 174 DNA has become known from sequence work. RF DNA of phi X 174 phage consists of 5375 +/- 20 basepairs equivalent to a molecular weight of (3.558 +/- 0.013) x 10(6) dalton and since the length of this DNA has been determined to be 1.71 +/- 0.02 micrometer the molecular linear density of double stranded DNA prepared for electron microscopy under the conditions described is (2.08 +/- 0.03) x 10(6) dalton micrometer-1. These data have been used to determine the molecular weights of several phage DNA's and of the plasmid DNA PML 21. The latter DNA exhibits a remarkable heterogeneity with respect to its size.

Coliphages↗
PubMed1976

Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA.

The DNA of bacteriophage T5+ (molecular weight 76 X 10(6) dalton) has been dissected by various specific endonucleases. The restriction enzymes HindIII and EcoRI produce 16 and 7 fragments respectively, whereas Sal and Sma produce 4 fragments each. Complete cleavage maps were established for the enzymes EcoRI, Sal and Sma and an almost complete map for HindIII. Furthermore the location and size of the deletions St 20, St 14, b3, St 0 and b1 were determined. The correlation of the genetic and functional map of the phage with the arrangement of fragments produced by the different enzymes has been established.

Binding Sites↗
PubMed1975

Structure and function of the genome of coliphage T5. Transcription in vitro of the "nicked" and "nick-free" T5+ DNA.

Purified T5+ DNA in nicked form and after repair of the specific single-strand interruptions with DNA ligase was used in transcription studies using Escherichia coli RNA polymerase (holoenzyme) in the presence and absence of E. coli termination protein rho. The transcriptional products were analyzed with respect to their size distribution and the sequences transcribed from the different templates. The results indicate that the single-strand breaks in the DNA of bacteriophage T5, though in genetically defined positions, do not have any specific effect on transcription in vitro. Furthermore, the E. coli rho protein, although it depresses net RNA synthesis and reduces the average molecular weight of the transcripts, seems to act in a non-specific way in this system.

Bacterial Proteins↗
PubMed1974

Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties.

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.

Centrifugation, Density Gradient↗
PubMed1974

Double-length, circular, single-stranded DNA from filamentous phage.

Wild-type and gene 3 mutant filamentous phage stocks, containing different relative amounts of multiple-length particles, were treated exhaustively with DNase and then were highly purified. The phage DNA was extracted and examined by electron microscopy. In all cases, about 0.03% of the molecules were circular dimers. (3)H-labeled phage DNA was separated as to size by sedimentation in a preformed CsCl density gradient. Individual fractions were then examined in the electron microscope, and the percentage of linear and circular monomer and dimer DNAs was determined. A peak of double-length, circular molecules (with the expected sedimentation constant of 38S) was found ahead of the 24S monomer peak. The double-length molecules had been purified 65-fold. As previously found for single-stranded DNA, the contour length of these molecules was strongly dependent upon ionic strength. Possible artifacts were ruled out, and it was shown that the double-length molecules arose from phage particles.

Centrifugation, Density Gradient↗