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PubMed1993

Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli.

The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sole phosphorus source. On the basis of nucleotide (nt) sequence analysis, the phn locus contains a 12.6-kb operon of seventeen genes named, in alphabetical order, phnA to phnQ [Chen et al., J. Biol. Chem. 265 (1990) 4461-4471]. New Pn+ plasmids were made which are suitable for mutational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. The construction of these plasmids showed that phnA and phnB have no role in Pn metabolism. Also, these plasmids were employed to introduce nonpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which allowed us to show that phnD probably has a role in transport. In addition, it was shown that phnP is the most distal gene required for Pn use. This was done by testing the effect of phn::uidA insertions in or near the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10.9-kb, fourteen-gene, phnCDEFGHIJKLMNOP locus.

Amino Acid Sequence↗
PubMed1994

Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida.

A shuttle cloning vector, pAKA16, and suicide derivatives pAKA19 and pAKA22 have been developed for gene transfer to Pasteurella haemolytica and P. multocida. pAKA16 was constructed by insertion of the lacZ alpha-peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from P. haemolytica serotype A1. The vector encodes ampicillin resistance, and contains at least 14 unique restriction sites and the property of phenotypic identification of recombinant clones in Escherichia coli by insertional inactivation of beta-galactosidase activity. It can be transferred by conjugation to P. haemolytica or P. multocida and is stably maintained in both species. The type-II chloramphenicol acetyltransferase-encoding gene (cat), cloned into pAKA16, was stably expressed in both P. haemolytica and P. multocida. Plasmids pAKA19 and pAKA22 were constructed by replacement of the origin of DNA replication (ori) of pAKA16 with a ColE1-type ori from pBR322 or an ori of plasmid R6K (oriR6K) from pJM703.1, respectively. These derivatives replicate in E. coli, but not in either P. haemolytica or P. multocida, and are suitable for use as suicide vectors for these Pasteurella species.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Gene organization in the dnaA-gyrA region of the Streptomyces coelicolor chromosome.

The gene organization has been determined for an 8-kb portion of the Streptomyces coelicolor chromosome close to the origin of DNA replication (oriC). Hybridization and DNA sequence analyses revealed the presence of five open reading frames (ORFs) oriented in the same direction as the proximal dnaA and dnaN genes. The deduced products of three of the ORFs have been identified as the S. coelicolor homologs of RecF, GyrB and GyrA. These genes are usually clustered in the dnaA region of bacterial chromosomes. In S. coelicolor however, the usual gene arrangement is altered. The recF gene is flanked by two ORFs, one of which encodes a protein with significant similarity to 6-phosphogluconate dehydrogenases (6PGDH), an enzyme that is not immediately linked to DNA metabolism.

Amino Acid Sequence↗
PubMed1995

Integration of an Epstein-Barr virus episome 3' into the gene encoding immunoglobulin heavy-chain alpha 1 in a lymphoblastoid cell line.

For the first time we have characterized an unoccupied site of Epstein-Barr (EBV) virus integration in a lymphoblastoid cell line, RGN1. The site of integration is about 1.5 kb downstream from the gene encoding the heavy chain constant alpha 1, specifying immunoglobulin A (IgA). Sequence and Southern analysis allowed us to hypothesize that integration occurred via a double exchange involving the viral latent origin of DNA replication (oriP) and the human DNA. The region involved in the integration is transcribed into poly(A)+ RNA in all the tested lymphoid lines, but not in the RGN1 line. We suggest a mechanism of integration primed by interactions between oriP and cell ori and its potential role in the establishment and/or evolution of EBV-carrying lines.

B-Lymphocytes↗
PubMed2006

Mutagenesis and the three R's in yeast.

Mutagenesis is a prerequisite for evolution and also is an important contributor to human diseases. Most mutations in actively dividing cells originate during DNA replication as errors introduced when copying an undamaged DNA template or during the bypass of DNA lesions. In addition, mutations can be introduced during the repair of DNA double-strand breaks by either homologous recombination or non-homologous end-joining pathways. Finally, although generally considered to be a very high-fidelity process, the excision repair of DNA damage may be an important contributor to mutagenesis in non-dividing cells. In this review, we will discuss the well-known contributions of DNA replication to mutagenesis in Saccharomyces cerevisiae, as well as the less-appreciated contributions of recombination and repair to mutagenesis in this organism.

DNA Repair↗
PubMed2006

Traffic safety for the cell: influence of cyclin-dependent kinase activity on genomic stability.

Genomic instability has long been considered a key factor in tumorigenesis. Recent evidence suggests that DNA damage may be widespread in early pre-neoplastic states, with deregulation of cyclin-dependent kinase (Cdk) activity a driving force. Increased Cdk activity may critically reduce licensing of origins of DNA replication, drive re-replication, or mediate overexpression of checkpoint proteins, inducing deleterious cell cycle delay. Conversely, inhibition of Cdk activity may compromise replication efficiency, expression of checkpoint proteins, or activation of DNA repair proteins. These vital functions point to the impact of Cdk activity on the stability of the genome. Insight into these pathways may improve our understanding of tumorigenesis and lead to more rational cancer therapies.

Animals↗
PubMed2005

Varicella vaccination of children in the United States: assessment after the first decade 1995-2005.

Live attenuated varicella vaccine (strain Oka) was approved for administration to healthy children in the United States in 1995. Over the past 10 years, varicella vaccine has been given to millions of children, usually at ages between 12 and 18 months. In states such as California, Michigan, and Texas, there has been a marked decline in the number of reported cases of varicella. Furthermore, there has been a 75% decrease in varicella-related hospitalizations across the United States, as well as a similar decrease in the number of deaths caused by complications of chickenpox. The main unanticipated result has been a growing number of outbreaks of varicella among immunized children ("breakthrough varicella"). The most cited risk factors for breakthrough varicella include the following: (1) 3-5-year interval since immunization and (2) immunization at the youngest ages, especially 12 months. Explanations for breakthrough varicella include a lessened immune response among the youngest recipients of the vaccine. Another possibility is genetic variation among circulating VZV strains. VZV strains can be separated into two geographic clades called European/North American and Asian, based on single nucleotide polymorphisms. Two mutant North American strains have been isolated from patients in the last 10 years. Several genomic differences between Oka vaccine strain and other strains have also been identified, including one site at the DNA origin of replication. Since breakthrough disease among vaccine recipients appears to be more common in the United States than in Japan, further comparisons between the varicella vaccination programs in Japan and the United States are warranted. In addition, data from varicella vaccination programs in Europe should provide further insight into the effectiveness of varicella vaccination in different geographic and ethnic populations.

Age Factors↗
PubMed2005

A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies.

Base Sequence↗
PubMed1983

Co-evolution of a filamentous bacteriophage and its defective interfering particles.

Serial passage of bacteriophage f1 at high multiplicities of infection results in the appearance of defective deletion mutants (miniphage) that harbor a tandem reiteration of regions of the f1 genome near the origin of DNA replication. These miniphage interfere with the growth of wild-type f1, and cause a sharp decrease of the viable phage titer. Upon further passage, however, the titer increases again. Viable phage variants (maxiphage) appear which harbor the same tandem reiteration of DNA as the miniphage. The maxiphage are more resistant than the wild type to interference by the miniphage. In the absence of miniphage the maxiphage grow at the same rate as the wild type. The structure of the DNA reiteration gradually changes during further passage. Miniphage and maxiphage follow, in parallel, a similar course of changes in the pattern of reiteration. In miniphage the reiterations change while the deletions are conserved. Serial passage of maxiphage quickly yields miniphage, which harbor a reiteration identical to that of the parental maxiphage. Both reiteration and deletion are relevant to the mechanism of interference by miniphage. Thus serial passage of the filamentous phage affords an experimental system to study evolution of a DNA genome in test tubes. Possible mechanisms of the interference by miniphage are discussed.

Base Sequence↗
PubMed1998

Genetic organization of the HindIII-I region of the single-nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

In order to investigate the genomic organization of the single-nucleocapsid nucleopolyhedrovirus (SNPV) of Buzura suppressaria (BusuNPV), the HindIII-I fragment located at map units (mu) 26.6-29.4 of the viral genome was sequenced. The fragment contained two partial and three complete open reading frames (ORFs) representing the 3' end of a polyhedron envelope protein gene (pep), a homologue of the AcMNPV ORF117, a conotoxin-like protein gene (ctl), an inhibitor of apoptosis gene (iap) and a superoxide dismutase gene (sod), respectively. These five genes were identified for the first time in a SNPV. Sequence analysis further revealed that these ORFs have the same conserved motifs and gene structure as those observed in their homologues from other baculoviruses. Between ctl and iap, an intergenic region of about 700 basepairs with structure similar to non-hr origins of DNA replication was observed. The genomic arrangement of the ORFs in the BusuNPV HindIII-I fragment is very different from the arrangement of their homologues in the genome of Autographa californica multiple nucleocapsid (M) NPV and other baculoviruses to date. Our data suggest that on the basis of gene arrangement, BusuNPV belongs to a distinct taxon within the Baculoviridae family, corroborating our previous conclusions derived from phylogeny analysis of several BusuNPV genes.

Amino Acid Sequence↗
PubMed1998

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant.

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage lambda gt11 called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997) 191-198) found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.

Acid Phosphatase↗
PubMed1996

Integrative vectors for constructing single-copy transcriptional fusions between Bacillus subtilis promoters and various reporter genes encoding heat-stable enzymes.

Here, we report on the construction of three integrative plasmids for Bacillus subtilis (Bs) allowing in vitro construction of transcriptional fusions. These plasmids contain a neomycin- or tetracycline-resistance cassette and one of three promoterless genes: bgaB (encoding beta-galactosidase), cat (chloramphenicol acetyltransferase), or xylE (catechol 2,3-dioxygenase). All cassettes are flanked by the 3'- and 5'-ends of the amyE gene (encoding alpha-amylase) allowing integration of these cassettes at the amyE locus of the Bs chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding bla gene. Four unique restriction sites can be used for insertion of restriction fragments carrying promoter fragments. All three reporter genes express heat-stable enzymes (stable up to at least 50 degrees C for 30 min) as shown here. We would like to point to the modular nature of these plasmids where the three reporter genes and the two resistance cassettes can be combined in any permutation. The versatility of the promoter-probe vectors was demonstrated by the integration of the promoters of the dnaK and groE operons of Bs and following their heat-inducible expression.

Bacillus subtilis↗
PubMed1993

The structure and function of yeast ARS elements.

The past year has seen significant advances in our understanding of the structure and function of yeast ARS elements. These elements, some of which function as chromosomal origins of DNA replication, are modular in structure. An essential domain, the ARS consensus sequence, binds a multiprotein complex that might be the long-sought initiator protein. The flanking domain contains a DNA unwinding element and a binding site for a multifunctional protein that acts as a replication enhancer.

Base Sequence↗
PubMed1992

Silencing: the establishment and inheritance of stable, repressed transcription states.

Silencing refers to a particular type of transcriptional repression characterized by the formation of a genetically heritable, repressed transcriptional state. Examples of silencing include position-effect variegation, X-chromosome inactivation, and the repression of the silent mating-type gene loci in yeast. Recent discoveries suggest that silencing in yeast, like silencing in larger eukaryotes, results from a particular chromatin structure that defines a chromosomal domain. In addition, a chromosomal origin of DNA replication is required for silencing in yeast, suggesting that DNA replication plays a role in forming functional chromosomal domains.

Animals↗
PubMed2000

Recent developments from the Leishmania genome project.

A first generation cosmid contig map of the Leishmania major Friedlin genome has been constructed, and genomic sequencing is well underway. Chromosome 1 (Chr1) and Chr3 have been completely sequenced, and Chr4 is virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. More than 600 completely sequenced new genes have been identified, representing approximately 8% of the total gene complement (approximately 8,600 genes) of Leishmania. Notably, a large proportion (approximately 69%) of the genes remain unclassified, with 40% of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, whereas Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. Statistical analyses of Chr1 show that the 'divergent junction' region between the two polycistronic gene clusters may be a candidate for an origin of DNA replication.

Animals↗
PubMed2000

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗
PubMed2000

Site-specific DNA binding and bending by the Borrelia burgdorferi Hbb protein.

The Borrelia burgdorferi Hbb protein shows sequence similarity to members of the Escherichia coli HU/integration host factor (IHF) family of DNA accessory factors. We have overexpressed the hbb gene product in E. coli and purified the protein to near homogeneity. Biochemical analyses have revealed that Hbb has unique properties and is neither a strict HU nor IHF analogue. Hbb was found to bind specifically to a site in the putative origin of DNA replication between dnaA and dnaN. DNA footprinting studies have shown that this site is unrelated to the consensus sequence recognized by IHF proteins. Hbb induces a dramatic bend (> 126 degrees ) at this site and was also shown to restrain negative supercoils efficiently upon DNA binding. These features of the protein suggest that Hbb may act as a DNA accessory factor that facilitates the assembly of higher order protein-DNA complexes, such as those involved in DNA replication, transcription, recombination, packaging and perhaps other DNA metabolic processes unique to Borrelia.

Bacterial Proteins↗