Biomedical subjects

R Lurz

Publications and source records attributed to R Lurz.

At least 73 records · Page 4Linked to original sources

PubMed1994

Growth-phase-dependent synthesis of histones in the archaeon Methanothermus fervidus.

Histone preparations from Methanothermus fervidus (HMf) contain two small polypeptides, HMfA and HMfB, which in solution are dimers and compact DNA to form nucleosome-like structures. These archaeal nucleosome-like structures constrain positive DNA supercoils, in contrast to the negatively supercoiled DNA in eukaryal nucleosomes. HMfA has been found to make up as much as 80% of HMf preparations synthesized by M. fervidus cells during the exponential growth phase of batch cultures but to decrease to approximately 50% as cultures enter the stationary phase. By using a nondenaturing polyacrylamide gel system at pH 6.1, we have demonstrated that HMf preparations contain HMfA homodimers, HMfB homodimers, and HMfA-HMfB heterodimers and that heating a mixture of recombinant HMfA and HMfB homodimers at 95 degrees C for 5 min generates HMfA-HMfB heterodimers. Circular dichroism spectroscopy indicates that HMfA and HMfB have very similar secondary structures, but based on agarose gel electrophoretic mobility shifts, DNA topology assays, and electron microscopy, they have different DNA binding properties. HMfA binding to DNA could be detected at lower protein/DNA ratios than HMfB, but HMfB binding resulted in more extensive DNA compaction. The increased HMfB synthesized in cells approaching the stationary phase and the highly compacted state of HMfB-bound DNA are consistent with preparations for the impending period of limited genome activity.

Archaea↗

PubMed1994

C1 repressor-mediated DNA looping is involved in C1 autoregulation of bacteriophage P1.

C1 repressor is required to repress the lytic functions of a P1 prophage in vivo. Transcription of the c1 gene is autoregulated via the C1-controlled operator Op99a,b which overlaps the promoter of the c1 gene. It is negatively affected by Lxc corepressor and the DNA region upstream of c1, which contains the additional operators Op99c, d, and e. We have explored these effects by constructing a set of lacZ reporter plasmids with Op99a,b and varying parts of the upstream DNA region. Transcription levels were measured in vivo with a two-plasmid system containing the lacZ reporter and a c1+ lxc+ or c1+ lxc- plasmid. Compared to the C1+Lxc-repressed lacZ reporter with all operators present, the basal level of beta-galactosidase activity increases successively when (i) upstream operators were deleted or inactivated, (ii) Lxc corepressor was removed, and (iii) C1 and Lxc were absent. By that means a 2 x 2 x 15-fold stepwise increase in enzyme activity was found. Using electron microscopy to visualize the interaction of C1 repressor with the operators in vitro, looped DNA molecules were observed. Although all operators can participate in C1-mediated DNA looping, loops between Op99a,b and Op99d occurred predominantly. Lxc is not required but increases drastically the frequency of loop formation. The results indicate that C1-mediated DNA looping may be a second element besides Lxc for fine-tuning the autoregulation of c1 transcription.

Bacteriophage P1↗

PubMed1994

Phage P4 DNA replication in vitro.

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.

Base Sequence↗

PubMed1993

Functional analysis of pSM19035-derived replicons in Bacillus subtilis.

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pSM19035 replication. At non-permissive temperature, plasmid replication depends on the dnaB, dnaC, dnaG and dnaI initiation replication products, but does not require the dnaA function. DNA elongation is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in dna(Ts) mutants impaired in different subunits of the enzyme. Single-stranded plasmid replication intermediates, corresponding to the leading strand, accumulate in the dnaD strain. On the basis of electron microscopic analysis of replication intermediates, we confirmed that the plasmid replicates unidirectionally by a theta mechanism.

Bacillus subtilis↗

PubMed1993

Purification and properties of the RecR protein from Bacillus subtilis 168.

Genetic evidence suggests that the Bacillus subtilis recR gene product is involved in DNA repair and recombination. To assign a biochemical function to the recR gene product, the RecR protein was labeled and purified by monitoring the radioactive label. NH2-terminal protein sequence analysis of RecR was consistent with the deduced amino acid sequence of the recR gene. The RecR protein (molecular mass of 25 kDa, isoelectric point 5.4) bound single- and double-stranded DNA in a filter binding assay. RecR-DNA complex formation is enhanced by the presence of a damage in the DNA substrate. The RecR-DNA complex formation proceeds in the absence of divalent cations and nucleotide cofactors, but is markedly stimulated by ATP and divalent cations. In our experimental conditions the apparent equilibrium constants of the optimized RecR-DNA complexes are 3 x 10(-7) M and 9 x 10(-7) M for damaged and undamaged DNA, respectively. The binding reaction is cooperative. Electron microscopy studies show that the presence of divalent cations increases the rate of RecR protein self-assembly. Addition of ATP or dATP promotes the organization of discrete series of quaternary structures on DNA, but ATP gamma S inhibits the DNA binding activity. A possible mechanism for the RecR function in DNA repair is discussed.

Bacillus subtilis↗

PubMed1993

The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry.

Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail. From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.

Biological Transport↗

PubMed1993

Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities.

alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities. First, the protein (subunit M(r) = 84,900) complexes specifically the P4 origin and the cis replication region required for replication. alpha Protein interacts with all six type I repeats (TGTTCACC) present in the origin. Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate. All common NTPs except UTP and dTTP can serve as cofactors. Strand separation of partial duplexes containing tailed ends that resemble a replication fork is preferred, although a preformed fork is not absolutely required for the enzyme to invade and unwind duplex DNA. alpha Protein catalyzes unwinding in the 3'-5' direction with respect to the strand it has bound. Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. In vitro, alpha protein generates di- to pentaribonucleotides on single-stranded phage fd DNA. The predominant product is the dimer pppApG, on which most of the longer oligoribonucleotides are based. Using DNA oligonucleotides of defined sequence as templates, synthesis of pppApG was also detectable. To date, among prokaryotic and eukaryotic replication systems, gp alpha is the only protein known that combines three activities on one single polypeptide chain.

Adenosine Triphosphate↗

PubMed1992

TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin.

Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene. By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity. traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin. The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM. Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs. The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them. Specific interactions between TraK and the DNA occur only on one side of the double helix. Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules. Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.

Amino Acid Sequence↗

PubMed1992

Penicillin-binding protein 2x of Streptococcus pneumoniae. Expression in Escherichia coli and purification of a soluble enzymatically active derivative.

A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.

Bacterial Proteins↗

PubMed1992

Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA.

The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism. Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation. Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging. The gene (gene 6 or siz) was cloned and sequenced. An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified. All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein. The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF. Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid.

Amino Acid Sequence↗

PubMed1992

The human insulin gene linked polymorphic region exhibits an altered DNA structure.

Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded.

Base Sequence↗

PubMed1992

In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins.

We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated.

Bacterial Proteins↗

PubMed1992

Circular extrachromosomal DNA codes for a surface protein in the (+) mating type of the zygomycete Absidia glauca.

A small protein with a molecular mass of 15 kDa, which is specifically found on the hyphal surface of a (+) mating-type strain of the model zygomycete Absidia glauca, was purified to electrophoretic homogeneity and partially sequenced. The corresponding gene was cloned by means of an oligonucleotide probe deduced from the protein sequence. It could be localized on an extrachromosomal circular DNA element with a total length of 1250 bp. Electron microscopic analysis of A. glauca DNA showed that small extrachromosomal DNAs with varying length are a common feature of this zygomycete. There are no indications of additional chromosomal copies of the gene for this surface protein, and the plasmid is absent from DNA preparations of the (-) mating type. The copy number ranges around three per haploid genome, and a single transcript with a length of 400 bp, coding for the surface protein, could be found by employing a hybridization probe which spans the complete fungal plasmid. This is the first report of naturally occurring extrachromosomal DNA in a Mucor-like fungus, and the only example where an integral protein of the cell wall is encoded by a plasmid.

Amino Acid Sequence↗

PubMed1992

Dissection of IncP conjugative plasmid transfer: definition of the transfer region Tra2 by mobilization of the Tra1 region in trans.

We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tra1 and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tra1 region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans. The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map. The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer. Tra2 regions of RP4 (IncP alpha) and R751 (IncP beta) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tra1 region. The transfer frequencies of both systems are similar. Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB). From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently. A second potential promoter has been located immediately upstream of trbB (kilB). Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it. This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.

Base Sequence↗

PubMed1991

Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.

Bacterial Proteins↗

PubMed1991

Physical and biochemical characterization of recombination-dependent synthesis of linear plasmid multimers in Bacillus subtilis.

The synthesis and structure of linear multimeric plasmid molecules (hmw DNA) in Bacillus subtilis were investigated. The replication of covalently-closed-circular supercoiled (form I) DNA requires the rate-limiting plasmid-encoded replication initiation protein. Unlike form I, hmw DNA synthesis is partially resistant to inhibition of cellular transcription or translation and requires the host DnaB protein. In addition, hmw DNA synthesis involves host recombination and repair functions (RecE and Poll). Analysis of hmw DNA by electron microscopy displayed linear DNA molecules up to 100 kb in size, which were either single-stranded, double-stranded or double-stranded with single-stranded ends. Structural features of hmw DNA molecules were mapped by means of heteroduplex studies using defined strand-specific probes. The results suggest that a recombination intermediate, but not plasmid-encoded replication, is involved in the initiation of hmw DNA synthesis.

Bacillus subtilis↗

PubMed1991

Biological and physicochemical characterization of the major (1.40) and minor (1.45) component of infectious avian adeno-associated virus.

Two infectious components with buoyant densities of 1.40 g/cm3 and 1.45 g/cm3, designated as major (1.40) and minor (1.45) component, were detected by banding avian adeno-associated virus (AAAV) isopycnically in CsCl. In metrizamide, however, infectious AAAV banded only as a single peak at a density of 1.32 g/cm3. Biological as well as physicochemical properties of the two AAAV components recovered from CsCl density gradient were described. Concerning the minor (1.45) component, three experimental findings may suggest that the capsid structure of this AAAV population is altered in comparison with that of the major (1.40) component: (i) the sedimentation pattern characterized by an additional peak containing slower-sedimenting noninfectious material (16 S); (ii) the specific infectivity decreased by the 3.5 fold; (iii) the ready disintegration when exposed to gently denaturing conditions.

Animals↗

PubMed1990

Identification of a seventh operon on plasmid RK2 regulated by the korA gene product.

Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon. The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncP alpha plasmids RK2 and RP4 is presented. The operon encodes a 34 kDa protein which has a net negative charge. Transcription of the operon, designated by us kfrA (korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously. The korF gene is encoded downstream from korB within the key korA/korB regulatory operon. We propose that K or F binds to a novel inverted repeat overlapping the promoter for the kfrA operon.

Amino Acid Sequence↗