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PubMed2002

Construction of pMEKm12, an expression vector for protein production in Pseudomonas syringae.

Characterization of the biological roles of proteins is essential for functional genomics of pseudomonads. Heterologous proteins overproduced in Escherichia coli frequently fail to exhibit biological function. To circumvent this problem, vector pMEKm12 was constructed and used to overexpress proteins in Pseudomonas. The vector contains the pRO1600 replication origin, the maltose-binding protein (MBP) fusion system, and an inducible tac promoter. The pMEKm12 was successfully used to overexpress the syringomycin synthetase SyrB1 protein fused to MBP in Pseudomonas syringae pv. syringae. Furthermore, expression of the MBP-SyrB1 protein in the syrB1 mutant BR132A1 resulted in the restoration of syringomycin production. This vector will facilitate confirmation of the biochemical roles of nonribosomal peptide synthetase genes in Pseudomonas syringae, and studies of gene function from a wide spectrum of pseudomonads.

Bacterial Proteins↗

PubMed2006

Eukaryotic DNA replication in a chromatin context.

There has been remarkable progress in the last 20 years in defining the molecular mechanisms that regulate initiation of DNA synthesis in eukaryotic cells. Replication origins in the DNA nucleate the ordered assembly of protein factors to form a prereplication complex (preRC) that is poised for DNA synthesis. Transition of the preRC to an active initiation complex is regulated by cyclin-dependent kinases and other signaling molecules, which promote further protein assembly and activate the mini chromosome maintenance helicase. We will review these mechanisms and describe the state of knowledge about the proteins involved. However, we will also consider an additional layer of complexity. The DNA in the cell is packaged with histone proteins into chromatin. Chromatin structure provides an additional layer of heritable information with associated epigenetic modifications. Thus, we will begin by describing chromatin structure, and how the cell generally controls access to the DNA. Access to the DNA requires active chromatin remodeling, specific histone modifications, and regulated histone deposition. Studies in transcription have revealed a variety of mechanisms that regulate DNA access, and some of these are likely to be shared with DNA replication. We will briefly describe heterochromatin as a model for an epigenetically inherited chromatin state. Next, we will describe the mechanisms of replication initiation and how these are affected by constraints of chromatin. Finally, chromatin must be reassembled with appropriate modifications following passage of the replication fork, and our third major topic will be the reassembly of chromatin and its associated epigenetic marks. Thus, in this chapter, we seek to bring together the studies of replication initiation and the studies of chromatin into a single holistic narrative.

Animals↗

PubMed2003

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b.

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

ATP-Binding Cassette Transporters↗

PubMed1997

Use of EBV-based Vector/HVJ-liposome complex vector for targeted gene therapy of EBV-associated neoplasms.

Targeted suicide gene therapy for Epstein-Barr virus (EBV)-associated neoplasms was attempted by using EBV-based plasmid vectors coupled with hemagglutinating virus of Japan (HVJ)-liposome in vitro. Expression of EBV nuclear antigen (EBNA)1 is a common feature of the neoplasms associated with EBV. When various leukemic cell lines were transduced with a vector carrying a marker gene and EBV replication origin of plasmid (oriP), the marker gene product was exclusively detected in cells expressing EBNA1. Transduction of herpes simplex virus (HSV)-1 thymidine kinase (Tk) gene resulted in a marked reduction in viable cell number by ganciclovir (GCV) specifically in EBNA1 positive cells. The results demonstrate that this virus-free system may be applicable to gene therapy of EBV-associated neoplasms.

Antiviral Agents↗

PubMed1999

Fate of unstable Bacillus subtilis subgenome: re-integration and amplification in the main genome.

The plastic Bacillus subtilis genome was dissected into two physically separate genomes, the 3.9 Mb main genome and the 0.3 Mb subgenome. DNA replication of the main genome was initiated from the normal replication origin (oriC) and that of the subgenome was from a 7.2 kb oriN-containing fragment artificially inserted. When the 7.2 kb fragment was shortened to a 1.5 kb fragment that contains oriN but lacks the segregational function, the subgenome became unstable and was rapidly lost from the cell, producing inviable cells due to the loss of essential genes carried by the subgenome. Stable survivors were isolated in which the subgenome had re-integrated and multiplied in the main genome. These results suggest that a reduced genetic stability of the subgenome induces size variation of the B. subtilis genome.

Bacillus subtilis↗

PubMed2000

Role of the mukB gene in chromosome and plasmid partition in Escherichia coli.

The intracellular locations of oriC and oriR1, the replication origins of the chromosome and plasmid R1, respectively, were visualized by fluorescence in situ hybridization (FISH) in exponentially growing populations of Escherichia coli. The locations of oriC and oriR1 (from a Par+ R1 plasmid) were unique and different in the wild-type host. In a mukB mutant, the positions were perturbed for both origins. The position of oriR1 from a plasmid with active partition (Par+) in the mukB host was as randomized as that of oriR1 from the Par- plasmid in a wild-type host. However, this mukB-induced randomization did not result in unstable inheritance of the Par+ plasmid, as measured by the conventional segregation assay. This might result from the preferential association of the Par+ plasmid with the bigger, decondensed nucleoid-containing daughters during cell division of MukB- cells, whereas the Par- plasmids were distributed at random and were lost by frequently ending up in anucleate cells.

Bacterial Proteins↗

PubMed1995

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.

Adhesins, Bacterial↗

PubMed2000

CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis.

Passage through mitosis is required to reset replication origins for the subsequent S phase. During mitosis, a series of biochemical reactions involving cyclin-dependent kinases (CDKs), the anaphase promoting complex or cyclosome (APC/C), and a mitotic exit network including Cdc5, 14, and 15 coordinates the proper separation and segregation of sister chromatids. Here we show that cyclin B/CDK inactivation can drive origin resetting in either early S phase or mitosis. This origin resetting occurs efficiently in the absence of APC/C function and mitotic exit network function. We conclude that CDK inactivation is the single essential event in mitosis required to allow pre-RC assembly for the next cell cycle.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1996

Activation of oriLyt, the lytic origin of DNA replication of Epstein-Barr virus, by BZLF1.

oriLyt, the cis-acting element of the lytic origin of DNA replication of Epstein-Barr virus, is activated by the viral transactivator BZLF1 which belongs to the extended bZIP class of transcription factors. Seven binding sites for BZLF1, so-called ZRE sites, are located within oriLyt. By mutational analysis of individual ZRE sites, we found that lytic DNA replication is dependent on only four of these sites which colocate with the promoter of the BHLF1 gene. The remaining three ZRE sites distal to the BHLF1 promoter were dispensable for DNA replication and did not contribute to long-range transcriptional activation of this promoter by BZLF1. This finding indicated that a similar set of ZRE sites is involved in DNA replication and transcriptional activation. To determine the function of BZLF1 in DNA replication, BZLF1 mutants with successive deletions in the transactivation domain were analyzed in replication assays. Unexpectedly, most BZLF1 mutants which failed to support DNA replication were found to be equally defective in transcriptional activation. Therefore, similar trans-acting domains of BZLF1 are involved both in replication and in transcription.

Binding Sites↗

PubMed2005

Genome plasticity and ori-ter rebalancing in Salmonella typhi.

Genome plasticity resulting from frequent rearrangement of the bacterial genome is a fascinating but poorly understood phenomenon. First reported in Salmonella typhi, it has been observed only in a small number of Salmonella serovars, although the over 2,500 known Salmonella serovars are all very closely related. To gain insights into this phenomenon and elucidate its roles in bacterial evolution, especially those involved in the formation of particular pathogens, we systematically analyzed the genomes of 127 wild-type S. typhi strains isolated from many places of the world and compared them with the two sequenced strains, Ty2 and CT18, attempting to find possible associations between genome rearrangement and other significant genomic features. Like other host-adapted Salmonella serovars, S. typhi contained large genome insertions, including the 134 kb Salmonella pathogenicity island, SPI7. Our analyses showed that SPI7 disrupted the physical balance of the bacterial genome between the replication origin (ori) and terminus (ter) when this DNA segment was inserted into the genome, and rearrangement in individual strains further changed the genome balance status, with a general tendency toward a better balanced genome structure. In a given S. typhi strain, genome diversification occurred and resulted in different structures among cells in the culture. Under a stressed condition, bacterial cells with better balanced genome structures were selected to greatly increase in proportion; in such cases, bacteria with better balanced genomes formed larger colonies and grew with shorter generation times. Our results support the hypothesis that genome plasticity as a result of frequent rearrangement provides the opportunity for the bacterial genome to adopt a better balanced structure and thus eventually stabilizes the genome during evolution.

Evolution, Molecular↗

PubMed2002

Promoter/origin structure of the complementary strand of hepatitis C virus genome.

Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3' termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp.

Base Sequence↗

PubMed1995

Elements of chromosome structure and function in fission yeast.

The investigation of fission yeast chromosome structure and function has moved rapidly over the past 10 years. The isolation of replication origins, telomeres and centromeres has allowed the development of minichromosomes, a yeast artificial chromosome (YAC)-like cloning system and investigations into chromosome segregation and behaviour during mitosis and meiosis. Many mutants have been isolated which are defective in chromosome segregation. The development of the fluorescent in-situ hybridization (FISH) technique for use in S. pombe has allowed the localization of centromeres and telomeres throughout mitosis and meiosis. In combination with indirect immunofluorescence to detect spindle and chromosomal proteins, the FISH technique should further advance our understanding of fission yeast chromosome structure and function. The recent discovery of a heterochromatin-like structure mediating transcriptional repression at centromeres reinforces the notion that fission yeast centromeres are similar to those of larger eukaryotes. Further characterization of such phenomena will accelerate the genetic dissection of this important chromosomal element.

Base Sequence↗

PubMedDate not supplied

Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication.

Genetic and biochemical evidence indicates that initiation of chromosomal replication in Escherichia coli occurs in a nucleoprotein complex at the replication origin (oriC) formed with DnaA protein. The frequency of initiation at oriC is tightly regulated to only once per chromosome per cell cycle. To prevent untimely, extra initiations, negative control for initiation is indispensable. Recently, we found that the function of the initiator protein, DnaA, is controlled by DNA polymerase III holoenzyme, the replicase of the chromosome. The ATP-bound form of DnaA protein, an active form for initiation, is efficiently converted to the ADP bound form, an inactive form, since a subunit of the polymerase loaded on DNA (beta subunit sliding clamp) stimulates hydrolysis of ATP bound to DnaA protein. Comparison of this system, RIDA (regulatory inactivation of DnaA), with other systems for negative regulation of initiation is included in this review, and the roles of these systems for concerted control for initiation during the cell cycle are discussed.

Adenosine Diphosphate↗

PubMed1996

Gene arrangement and organization in a approximately 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae.

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.

Amino Acid Sequence↗

PubMed2004

[Analysis of the interaction between DnaA protein which is involved in the initiation of chromosomal replication in E. coli and its specifically binding DNA].

DnaA protein binds specifically to a group of binding sites called DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. DnaA domain IV comprises 94 amino acid residues and is required for DNA binding. At first, the backbone assignments of DnaA domain IV in the complexes were determined using several nuclear magnetic resonance (NMR) spectra. Using NMR analysis, we investigated the interaction between DnaA domain IV and DnaA box R1. The 1H-15N HSQC spectrum of DnaA domain IV showed prominent chemical shift perturbations on six residues (Arg399, Ala404, Leu422, Asp433, Thr435 and Thr436). Through homology modeling, we located all of these residues on one side of the surface of the DnaA domain IV molecule. Moreover, we confirmed that these residues in DnaA domain IV bind to DnaA box R1 by mutation analysis. Finally, we compared the chemical shift perturbation of the 1H-15N HSQC spectrum in the presence of the DnaA box with that in the presence of a non-specific oligonucleotide that has a reduced affinity for DnaA, and the results suggested that Leu422 imparts specificity in binding with DnaA box R1.

Bacterial Proteins↗

PubMed2002

Padlock oligonucleotides as a tool for labeling superhelical DNA.

Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called 'padlock oligonucleotide'. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.

Base Sequence↗

PubMed1998

Nuclear matrix attachment regions of human papillomavirus type 16 point toward conservation of these genomic elements in all genital papillomaviruses.

The gene functions, transcriptional regulation, and genome replication of human papillomaviruses (HPVs) have been extensively studied. Thus far, however, there has been little research on the organization of HPV genomes in the nuclei of infected cells. As a first step to understand how chromatin and suprachromatin structures may modulate the life cycles of these viruses, we have identified and mapped interactions of HPV DNAs with the nuclear matrix. The endogenous genomes of HPV type 16 (HPV-16) which are present in SiHa, HPKI, and HPKII cells, adhere in vivo to the nuclear matrixes of these cell lines. A tight association with the nuclear matrix in vivo may be common to all genital HPV types, as the genomes of HPV-11, HPV-16, HPV-18, and HPV-33 showed high affinity in vitro to preparations of the nuclear matrix of C33A cells, as did the well-known nuclear matrix attachment region (MAR) of the cellular beta interferon gene. Affinity to the nuclear matrix is not evenly spread over the HPV-16 genome. Five genomic segments have strong MAR properties, while the other parts of the genome have low or no affinity. Some of the five MARs correlate with known cis-responsive elements: a strong MAR lies in the 5' segment of the long control region (LCR), and another one lies in the E6 gene, flanking the HPV enhancer, the replication origin, and the E6 promoter. The strongest MAR coincides with the E5 gene and the early-late intergenic region. Weak MAR activity is present in the E1 and E2 genes and in the 3' part of L2. The in vitro map of MAR activity appears to reflect MAR properties in vivo, as we found for two selected fragments with and without MAR activity. As is typical for many MARs, the two segments with highest affinity, namely, the 5' LCR and the early-late intergenic region, have an extraordinarily high A-T content (up to 85%). It is likely that these MARs have specific functions in the viral life cycle, as MARs predicted by nucleotide sequence analysis, patterns of A-T content, transcription factor YY1 binding sites, and likely topoisomerase II cleavage sites are conserved in similar positions throughout all genital HPVs.

Adenine↗

PubMed1996

Open strands adjacent to iterons promote the binding of the replication initiator protein (Rep) of pSC101 to the unit sequence of the iterons in vitro.

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.

Base Sequence↗

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