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PubMed2001

Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein.

Spo0J and Soj belong to the ParB/ParA family of proteins involved in chromosome and plasmid segregation in bacteria. In Bacillus subtilis, Spo0J protein binds to several specific sites, parS, located on both sides of the origin of DNA replication, oriC, and apparently self-associates to form large discrete foci visible by fluorescence microscopy. Soj protein forms large 'patches' probably associated with the nucleoid, which can undergo dynamic, co-operative jumping from nucleoid to nucleoid in the presence of Spo0J. Patches of Soj protein somehow help to bring about the condensation of Spo0J foci. Soj is also a negative regulator of transcription. In the absence of Spo0J, Soj is statically distributed on each of the nucleoids in the cell and blocks the transcription of several sporulation genes. To analyse the functional interaction between Spo0J and Soj further, we have constructed and studied a collection of spo0J mutants. Most of the mutants completely prevent Spo0J from interacting with DNA. One mutation impairs the formation of compact Spo0J foci and simultaneously results in loss of Soj movement. We also isolated one spo0J mutant, in which the frequency of Soj internucleoid oscillation is highly increased. Both mutations affecting the interaction with Soj lie in the N-terminal coding part of spo0J, whereas the substitutions affecting DNA binding lie in the mid- to C-terminal coding region.

Alleles↗
PubMed2000

UVs syndrome: establishment and characterization of fibroblastic cell lines transformed with simian virus 40 DNA.

Ultraviolet-sensitive syndrome (UVsS) is a newly established photosensitive disorder. Patients with UVsS showed mild clinical manifestations similar to classical types of xeroderma pigmentosum, and had biochemical phenotypes of Cockayne syndrome but not those of xeroderma pigmentosum. Fibroblasts from a UVsS patient were treated with simian virus 40 DNA containing the large T antigen with a defective origin of DNA replication to establish a transformed cell line. We obtained two independent transformed cell lines (Kps3SVY and Kps3SVI3) and report their initial characterization. These cells showed the same pattern in variable number of tandem repeat analyses as a primary fibroblast cell strain, Kps3, and retain the UVsS phenotype as demonstrated by increased UV sensitivity (three to four times more sensitive to UV than normal cells) and by reduced recovery of RNA synthesis after UV irradiation (20% - 30% of that of normal cells). These cells, however, showed different phenotypes as regards plating efficiency, doubling time, and transfection efficiency in spite of the fact that the same method was used to transform the cells. Kps3SVY cells were closer in phenotype to Kps3 cells than Kps3SVI3 cells. As a variable number of tandem repeat analyses also showed that Kps3SVI3 cells have lost one of the two alleles in some chromosomes, this may explain the different phenotypes between Kps3SVY and Kps3SVI3 cells. Moreover, these cells were distinct from cells with Cockayne syndrome group A or B. Thus, these cell lines provide the opportunity to conduct transfection studies on cells with the UVsS defect in DNA repair and transcription.

Cell Line, Transformed↗
PubMed2004

Gene targeting in yeast is initiated by two independent strand invasions.

To study the mechanism of gene targeting, we examined heteroduplex DNA (hDNA) formation during targeting of two separate chromosomal locations in Saccharomyces cerevisiae. We examined both replacement of the entire gene with a heterologous selectable marker and correction of a single base pair insertion mutation by gene targeting, and in all cases our results were consistent with separate strand invasion/resolution at the two ends of the targeting fragment as the dominant mechanism in wild-type cells. A small subset of transformants was consistent with assimilation of a single strand of targeting DNA encompassing both flanking homology regions and the marker into hDNA. hDNA formation during correction of a point mutation by targeted integration was conspicuously altered in a mismatch repair-deficient background and was consistent with single-strand invasion/assimilation without mismatch correction, confirming that gene targeting by this pathway is actively impeded in wild-type yeast. Finally, inversion of one targeted locus and mutation of an active origin of DNA replication at the other locus affected hDNA formation significantly, suggesting that formation of productive interactions between the targeting DNA and the targeted site in the chromosome is sensitive to local DNA dynamics.

Carrier Proteins↗
PubMed2005

Computer simulations of protein functions: searching for the molecular origin of the replication fidelity of DNA polymerases.

The use of computers to simulate the functions of complex biological macromolecules is essential to achieve a microscopic description of biological processes and to model and interpret experimental data. Here we apply theoretical computational approaches to investigate the fidelity of T7 DNA polymerase, divided into discrete steps that include contributions from substrate binding, pK(a) shifts, and rate constants for the PO bond-breaking and bond-making processes. We begin by defining the discrimination between right and wrong nucleotides in terms of the free energy landscape for the dNMP incorporation reaction. We then use the linear response approximation and the empirical valence bond methods to obtain converging results for the contribution of the binding and chemical steps to the overall fidelity. These approaches are successful in reproducing general trends in the observed polymerase incorporation fidelity. The calculations demonstrate the potential for further integration of theoretical and experimental studies to analyze high- and low-fidelity DNA polymerases.

Base Pairing↗
PubMed1978

Nucleotide sequences of the separate origins of synthesis of bacteriophage G4 viral and complementary DNA strands.

Bacteriophage G4 has physically separated origins of synthesis of its viral and complementary DNA strands. Chain termination and "plus and minus" DNA sequencing methods have been used to obtain the nucleotide sequence of these two origins. The unique origin at which the complementary DNA strand is initiated has located in the untranslated region between genes F and G. This sequence, which has considerable secondary structure, contains a stretch which is complementary to the RNA primer that is observed during synthesis in vitro of the G4 complementary DNA strand [Bouché, J.P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem., in press]. This G4 origin shows extensive sequence homology with the bacteriophage lambda origin of DNA replication [Denniston-Thompson, K., Moore, D. D., Kruger, D. E., Furth, M. E. & Blattner, F. R. (1977) Science 198, 1051-1056]. The sequence around the site in gene A at which G4 viral DNA strand synthesis is initiated by the nicking action of the cistron A protein is very similar to that of bacteriophage phiX174. An (A + T)-rich stretch flanked by (G + C)-rich sequences may be involved in the interaction between the DNA and protein.

Bacteriophages↗
PubMed1980

Ubiquitous, interspersed repeated sequences in mammalian genomes.

DNA base sequence comparisons demonstrate that the principal family of 300-nucleotide interspersed human DNA sequences, the repetitive double-strand regions of HeLa cell heterogeneous nuclear RNA, and specific RNA polymerase III in vitro transcripts of cloned human DNA sequences are all representatives of a closely related family of sequences. A segment of approximately 30 residues of these sequences is highly conserved in mammalian evolution because it is also present in the interspersed repeated DNA sequences of Chinese hamsters. Further DNA sequence comparisons demonstrate that a portion of this highly conserved segment of repetitive mamalian DNA sequence is similar to a sequence found within a low molecular weight RNA that hydrogen-bonds to poly(A)-terminated RNA molecules of Chinese hamsters and a sequence that forms half of a perfect inverted repeat near the origin of DNA replication in papovaviruses.

Animals↗
PubMed1980

Amplification of integrated viral DNA sequences in polyoma virus-transformed cells.

Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ("curing"), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA "unit" (including two 3% deletions), an 85% "unit" (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event.

Antigens, Neoplasm↗
PubMed1981

Episomal simian virus 40 genomes in human brain tumors.

Eight out of 35 human intracranial tumors were shown by restriction enzyme analysis to contain unintegrated simian virus 40 (SV40) DNA molecules. The relative amount of viral DNA was estimated to be the equivalent of one viral genome within every 10th to 20th cell. No infectious virus was detected in tissue cultures established from the tumors. From only one tumor was it possible to rescue, by cell fusion, infectious SV40 displaying wild-type properties. In those cases that permitted a more detailed analysis, the restriction enzyme cleavage patterns appeared to correspond to the wild-type patterns with one exception, in which the SV40 episomes displayed a deletion of approximately 70 base pairs close to the origin of DNA replication. From one tumor, the SV40 genomes were transferred into permissive CV-1 monkey cells by transfection with the total tumor DNA. Despite their persistence as episomes no infectious virus was produced. Furthermore, no viral antigens were detectable, although the SV40 messengers for the small and the large tumor antigens were present. These cells had, however, acquired the ability to form colonies in low concentrations of serum. Thus this report provides, by restriction enzyme analysis, direct evidence for the presence of SV40 DNA in human tumors.

Base Sequence↗
PubMed1984

DNA binding activity of polyoma virus large tumor antigen.

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.

Animals↗
PubMed1997

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Actins↗
PubMed2000

The stability of the Cdc6 protein is regulated by cyclin-dependent kinase/cyclin B complexes in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of prereplicative complexes that are a prerequisite for firing origins during DNA replication in the S phase. In budding yeast, the presence of Cdc6 protein is normally restricted to the G(1) phase of the cell cycle, at least partly because of its proteolytic degradation in the late G(1)/early S phase. Here we show that a Cdc28-dependent mechanism targets p57(CDC6) for degradation in mitotic-arrested budding yeast cells. Consistent with this observation, Cdc6-7 and Cdc6-8 proteins, mutants lacking Cdc28 phosphorylation sites, are stabilized relative to wild-type Cdc6. Our data also suggest a correlation between the absence of Cdc28/Clb kinase activity and Cdc6 protein stabilization, because a drop in Cdc28/Clb-associated kinase activity allows mitotic-arrested cells to accumulate Cdc6 protein. Finally, we also show that cdc28 temperature-sensitive G(1) mutants accumulate Cdc6 protein because of a post-transcriptional mechanism. Our data suggest that budding yeast cells target Cdc6 for degradation through a Cdc28-dependent mechanism in each cell cycle.

Base Sequence↗
PubMed2000

Evidence for class-specific factors in immunoglobulin isotype switching.

Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR.

Animals↗
PubMed1986

A method for constructing multiple tandem repeats of specific DNA fragments.

We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site.

DNA Replication↗
PubMed1993

A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection.

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

Amino Acid Sequence↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1999

Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway.

Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase. We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate. Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis. Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity. The Dbf4p phosphorylation in response to HU is RAD53 dependent. This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity. Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity. Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated. Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase.

Cell Cycle Proteins↗
PubMed1986

Evaluating quantitative variation in the genome of Zea mays.

Genomic diversity within the species Zea mays has been examined by measuring the variation in the repetitive component of the nuclear genome among North American inbred lines and varieties. This was done by preparing a set of clones of repetitive maize sequences that differ in function, molecular arrangement and multiplicity and then using these as probes for quantitative hybridization to DNA from various maize genotypes. The comparison showed that the majority of repeated sequences are markedly variable in copy number among the ten maize strains tested. The clone sample contained the rDNA and 5S genes, the major repeat of the chromosome knobs, sequences functioning as origins of DNA replication in yeast (ARS sequences) and randomly cloned sequences of unknown function and chromosomal location. The sequences ranged in reiteration frequency from 200 to greater than 10(5) copies and included both tandemly arrayed and dispersed repeats. The copy numbers were measured by hybridizing labeled cloned sequences to aliquots of high molecular weight genomic DNA that were applied to nitrocellulose filters through a slotted template (slot blotting). The hybridization signal on an autoradiogram occurred in a narrow band that could be scored reliably with a densitometer. This provided a rapid method of determining the abundance of particular repeated sequences in individual plants and plant populations. Using this technique, we found that the copy number of repeated sequences of all types generally varied among the strains by two- to threefold, although at least one sequence showed no detectable variation. In contrast to the variability found between strains, individuals within an inbred line or variety were found to be indistinguishable in terms of specific sequence multiplicity. Each genotype has a different pattern of copy numbers for the set of repeated sequence clones, and this pattern is characteristic of all individuals of a particular genotype. The data also show that the copy number of each sequence varies independently. No strains had uniformly high or low copy numbers for the entire set of probes.

DNA↗