Biomedical subjects

W Messer

Publications and source records attributed to W Messer.

At least 55 records · Page 3Linked to original sources

PubMed1994

A versatile plasmid vector system for the regulated expression of genes in Escherichia coli.

A series of plasmid expression vectors, which support the regulated and efficient expression of genes in Escherichia coli, have been constructed. The vectors consist of a DNA replication origin cassette, a promoter cassette, an efficient ribosome binding site together with a polylinker region and a lacZ gene. Several types of replication origins and promoter sequences are each available on cassettes. Fusion of the 5' TG dinucleotide of the gene under consideration to the A nucleotide, present on the vector, results in an ATG start codon and allows, in combination with the plasmid-borne ribosome binding site, the efficient expression of the cloned gene. Additionally, a second fusion of the gene at its 3' end with the lacZ gene, which is available in all three reading frames relative to the polylinker region, allows rapid selection of the correctly fused genes. As an example of the cloning of a regulatory gene, this vector system was used for the expression of the dnaA gene, of Escherichia coli, the initiator protein for DNA replication.

Bacterial Proteins↗

PubMed1993

The structure of the initiation complex at the replication origin, oriC, of Escherichia coli.

Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA. The AT-rich region in the left part of oriC and the start site region in the right part of oriC. Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region. We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function. An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC. DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo. We propose a specific compact nucleoprotein structure for the initiation complex.

Bacterial Proteins↗

PubMed1992

Mutations in the DnaA binding sites of the replication origin of Escherichia coli.

Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of the Escherichia coli replication origin, oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of the oriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes in oriC.

Bacterial Proteins↗

PubMed1992

The complex for replication initiation of Escherichia coli.

We probed the complex between oriC and DnaA protein using two types of mutants in oriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivated oriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1-R2 interval. FIS protein binds to a bipartite site in oriC between DnaA boxes R2 and R3. A model for the oriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 A spacing upon which oriC is arranged.

Bacterial Proteins↗

PubMed1992

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.

Attachment Sites, Microbiological↗

PubMed1991

The FIS protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli.

The FIS protein (factor for inversion stimulation) is known to stimulate site-specific recombination processes, such as the inversion of the G segment of bacteriophage Mu, by binding to specific enhancer sequences. It has also been shown to activate transcription from rRNA promoters both in vitro and in vivo. We have identified a specific binding site for FIS in the center of the origin of chromosomal DNA replication, oriC. The DNA bends upon FIS binding. Occupation of the FIS site and binding of DnaA, the initiator protein, to its adjacent binding site (R3) are mutually exclusive. A fis mutant strain can not be efficiently transformed with plasmids which carry and replicate from oriC, suggesting that FIS is required for minichromosome replication.

Bacterial Proteins↗

PubMed1991

Localized DNA melting and structural pertubations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo.

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation.

Bacterial Proteins↗

PubMed1991

Expression of the replication protein Arp of phasyl shows dual regulation by an antisense promoter.

Phasyl is the smallest naturally occurring replicon found so far in Escherichia coli. It encodes a protein which is essential for autonomous replication (Arp). The transcriptional start of the arp gene was mapped. A strong antisense promoter was found in close proximity to the arp promoter. The inactivation of this promoter led in cis to a strong increase of the transcription of the arp gene and to the inactivation of autonomous replication of phasyl. The product of the antisense promoter is an 83 nt RNA molecule, which is not translated. The antisense RNA led in trans to the inhibition of the translation of the arp mRNA, presumably mediated by the formation of an RNA-RNA hybrid in which the Shine-Dalgarno sequence of the arp transcript is sequestered. The expression of the arp gene is thus controlled by two negatively acting mechanisms: it is subject to a transcriptional control in cis exerted by the antisense promoter and to a translational control in trans mediated by the antisense RNA. Inactivation of one mechanism of control cannot be compensated by the remaining one.

Cloning, Molecular↗

PubMed1991

Expression of the dnaA gene of Escherichia coli is inducible by DNA damage.

The DnaA protein is the key DNA initiation protein in Escherichia coli. Using transcriptional and translational fusions, comparative S1 nuclease mapping and immunoblot analysis, the regulation of dnaA in relation to inducible responses to DNA damage was studied. We found that DNA damage caused by mitomycin C (MC) and methyl methanesulfonate (MMS) led to a significant induction of the dnaA gene. These results strongly suggest that in response to DNA damage which inhibits DNA replication, an increased initiation capacity is induced at oriC and that, in addition to the known auto-repression, a new regulatory mechanism may be involved in the control of dnaA gene expression. Furthermore, this mechanism might be indirectly related to the SOS regulon, because lexA and recA mutants, which block the induction of the SOS response, prevent dnaA induction by MMS and MC.

Bacterial Proteins↗

PubMed1991

DnaA protein/DNA interaction. Modulation of the recognition sequence.

In the active orientation the DnaA protein/dnaA box complex blocks transcribing RNA polymerase. The extent of transcription termination at different dnaA boxes was used to determine in vivo their various affinities to the DnaA protein. The rate of transcription distal to the respective dnaA box was monitored by the expression of the reporter gene galK. The dnaA boxes (5'-TTATACACA and 5'-TTATCCAAA), present in oriC, showed the strongest binding affinity. The dnaA box 5'-TTTTCCACA was mutated at eight positions such that the boxes differed from the consensus sequence, 5'-TT(A/T)T(A/C)CA(A/C)A, defined so far. Based on the different properties of the dnaA boxes, a new consensus sequence was derived: 5'-(T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T(A/C).

Bacterial Proteins↗

PubMed1991

Transcription termination in the dnaA gene.

The termination of transcription in the dnaA gene of E. coli was analyzed using transcriptional fusions to the galactokinase gene, S1 nuclease mapping and quantification of translation products by Western blots. The majority of transcripts originating from dnaA promoters terminated at several positions within a 200 bp region inside the dnaA reading frame.

Bacterial Proteins↗

PubMed1991

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage.

The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 bp consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.

Bacterial Proteins↗

PubMed1991

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from Escherichia coli.

The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd). The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages. Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not. This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region. Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636. This fragment mediates replication if the Arp protein is supplied in trans. Arp is the only phage-encoded protein and is essential for plasmidlike replication. No sequence homology to other known origins was found. Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent.

Base Sequence↗

PubMed1989

[Mexiletine in terminal renal failure and various dialysis procedures].

We monitored the plasma levels of mexiletine in 20 dialysis patients with severe cardiac arrhythmias after repeated oral administration and the elimination by various dialysis procedures. The levels of mexiletine in plasma and dialysate were assayed by high-pressure liquid chromatography. After repeated administration of mexiletine 400-600 mg/day trough levels were in the range from 500-2,000 ng/ml. Treatment controlled by Holter monitoring was effective in 13/20 patients. Doses of 600 mg/day and more often were not tolerated by patients with dialysis after some weeks. There was no important removal of mexiletine from plasma during hemodialysis, hemofiltration, peritoneal dialysis, or plasmapheresis. In conclusion, we recommend a slightly reduced dosage of 400-600 mg mexiletine/day (usually 600-800 mg) for patients with end-stage renal insufficiency, irrespective of dialysis.

Adult↗

PubMed1989

Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response.

Cloning, Molecular↗

PubMed1989

Directionality of DnaA protein/DNA interaction. Active orientation of the DnaA protein/dnaA box complex in transcription termination.

The complex of DnaA protein with its 9 bp consensus binding site, the dnaA box 5'-TT(A/T)T(A/C)CA(A/C)A, blocks transcribing RNA polymerase. In a model system, the rate of transcription was monitored distal to the dnaA box 5'-TTTTCCACA by the expression of a reporter gene. DnaA-dependent transcription termination occurred irrespective of whether the dnaA box region was or was not translated. Only the dnaA box orientation 5'-TTTTCCACA on the non-coding strand, but not the reverse orientation, was active in termination. This suggests that DnaA protein contacts only one strand of the DNA duplex. Oligonucleotide-directed mutation of a dnaA box present within the dnaA coding region resulted in increased expression of dnaA. This demonstrates that DnaA protein-directed transcription termination is an element of the autoregulation of the dnaA gene.

Amino Acid Sequence↗

PubMed1988

Functions of the DnaA protein of Escherichia coli in replication and transcription.

The function of DnaA protein as a replisome organizer in the initiation of DNA replication is reviewed. A model is presented showing the construction of two basic types of DnaA-dependent replication origin. New data demonstrate that the dnaA box-DnaA protein complex is a transcription terminator. Only one orientation of the dnaA box results in termination of transcription. Mutation of the dnaA box within the dnaA reading frame shows that DnaA-mediated transcription termination has a role in the autoregulation of the dnaA gene.

Amino Acid Sequence↗

PubMed1988

Termination of the Escherichia coli asnC transcript. The DnaA protein/dnaA box complex blocks transcribing RNA polymerase.

Genes clockwise of oriC, the Escherichia coli replication origin (oriC-mioC-asnC), show anticlockwise transcription. The intergenic region between mioC and asnC contains both a terminator and a consensus DnaA-protein-binding site (dnaA box). We analysed termination in this region using galK expression to monitor for transcription. About 50% of the asnC transcripts were not terminated, and about 25% terminated at the asnC terminator. We found that the DnaA protein/dnaA box complex acts as a terminator of transcription for about 25% of the transcripts. Its efficiency could be increased by raising the level of DnaA protein, or it could be inactivated by deletion in the dnaA box or by thermal denaturation of the DnaA protein.

Bacterial Proteins↗