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Bacterial DNA synthesized under phage control in a DNA-defective Salmonella-mutant and packaged into a special fraction of transducing particles of phage P22.

Lysates of P22 contain a small fraction of transducing particles with bacterial DNA replicated semiconservatively after the time of infection. It was demonstrated that the presence and relative amount of this class of transducing particles was unchanged, if infection of Salmonella occured under a condition nonpermissive for bacterial DNA replication. Analysis of particles with DNA fragments derived from different regions of the Salmonella chromosome indicated that the replication of the bacterial DNA carried by these transducing particles was not initiated specifically at the normal origin for bacterial chromosome replication.

DNA Replication↗

Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.

This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.

Alkylation↗

DNA replication analysis of FMR1, XIST, and factor 8C loci by FISH shows nontranscribed X-linked genes replicate late.

The relationship between the transcriptional state of a locus and the time when it replicates during DNA synthesis is increasingly apparent. Active autosomal genes tend to replicate early, whereas inactive ones are more permissive and frequently replicate later. Although the inactive X chromosome replicates later than its active homologue, little is known about the replication of X-linked genes. We have used FISH to examine the replication of loci on the active X chromosome that are not transcribed, either because the tissue analyzed was not the expressing tissue (F8C), because the locus is silent on all active X chromosomes (XIST), or because it has been mutated by expansion and methylation of a CpG island (FMR1). In this assay, an unreplicated locus is characterized by a single hybridization signal, and a replicated locus is characterized by a doublet hybridization signal. The percentage of doublets is used as a measure of relative time of replication in S phase. The validity of this approach has been established elsewhere, since results compare favorably with those obtained using traditional methods for studying DNA replication. Our results show that the FMR1 gene replicates relatively later in fragile X (fraX) males with the full mutation than in normal males, irrespective of the probe used. The F8C locus is late replicating in both normal and fraX males and replicates at nearly the same time on active and inactive X in females. The XIST locus replicates late in all the males studied and asynchronously in female cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

Uncoupling of S-phase and mitosis by recombinant cytotoxic necrotizing factor 2 (CNF2).

Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from virulent clinical isolates of Escherichia coli. It has been characterized in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization. The molecular mechanism of these actions is unclear. Two cellular mechanisms can be hypothesized to explain the DNA content increase (hyperploidy) induced by the toxin. The first is that the toxin interferes with cytoplasmic division without interfering with normal nuclear cycling, such that DNA is replicated in the absence of cell division. The second is that the toxin drives the nuclear machinery to replicate the DNA multiple times within one cell cycle, without interfering with cytoplasmic division. In order to investigate these phenomena, we have constructed a recombinant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag. Extracts made from E. coli that express this gene have a high multinucleating activity that colocalizes with the recombinant 115-kDa protein. To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differentiation, and stage of growth) to establish a relationship between cellular divisions and generation of hyperploidy. It was also determined that the toxin had no effect upon in vitro DNA replication using a Xenopus egg extract system. In aggregate, these data are consistent with the hypothesis that CNF2 is affecting cytoplasmic division and thereby removing the requirement for a completed mitosis before the initiation of another S-phase. These data are discussed in relation to the generation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis.

3T3 Cells↗

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster.

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with 3H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S + 18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S + 18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.

Animals↗

Heterogeneity in nuclear transport does not affect the timing of DNA synthesis in quiescent mammalian nuclei induced to replicate in Xenopus egg extracts.

Intact G0 nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in Xenopus egg extracts, despite exposure to the same concentration of replication factors. This indicates that individual nuclei differ in their ability to respond to the inducers of DNA replication. Since the induction of DNA synthesis requires the accumulation of replication factors by active nuclear transport, any variation in the rate of transport among nuclei could contribute to the variability of DNA replication. Using the naturally fluorescent protein allophycocyanin (APC) coupled with the nuclear localization sequence (NLS) of SV40 T antigen, as a marker of nuclear uptake, we show here that individual G0 nuclei differ in their rate of transport over a range of more than 20-fold. Surprisingly, this variation has no direct influence on the timing or extent of DNA synthesis. Similar results were obtained by monitoring the uptake of nucleoplasmin, a nuclear protein present at high levels in egg extracts. These experiments show that the initiation of DNA synthesis is not driven merely by the accumulation of replication factors to some threshold concentration. Instead, some other explanation is needed to account for the timing of initiation.

Amino Acid Sequence↗

Involvement of an SCFSlmb complex in timely elimination of E2F upon initiation of DNA replication in Drosophila.

BACKGROUND: Cul1 is a core component of the evolutionarily conserved SCF-type ubiquitin ligases that target specific proteins for destruction. SCF action contributes to cell cycle progression but few of the key targets of its action have been identified. RESULTS: We found that expression of the mouse Cul1 (mCul1) in the larval wing disc has a dominant negative effect. It reduces, but does not eliminate, the function of SCF complexes, promotes accumulation of Cubitus interruptus (a target of SCF action), triggers apoptosis, and causes a small wing phenotype. A screen for mutations that dominantly modify this phenotype showed effective suppression upon reduction of E2F function, suggesting that compromised downregulation of E2F contributes to the phenotype. Partial inactivation of Cul1 delayed the abrupt loss of E2F immunofluorescence beyond its normal point of downregulation at the onset of S phase. Additional screens showed that mild reduction in function of the F-box encoding gene slimb enhanced the mCul1 overexpression phenotype. Cell cycle modulation of E2F levels is virtually absent in slimb mutant cells in which slimb function is severely reduced. This implicates Slimb, a known targeting subunit of SCF, in E2F downregulation. In addition, Slimb and E2F interacted in vitro in a phosphorylation-dependent manner. CONCLUSION: We have used genetic and physical interactions to identify the G1/S transcription factor E2F as an SCFSlmb target in Drosophila. These results argue that the SCFSlmb ubiquitin ligase directs E2F destruction in S phase.

Animals↗

Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.

Polyoma gene expression is temporally regulated during productive infection of mouse cells. Early genes are expressed throughout the viral life cycle, but late mRNAs are not detected until after the onset of DNA replication. At late times, late-strand transcripts represent the great majority of viral-specific RNA in the cell. To learn more about the mechanism by which the early-late switch is regulated, we have carried out a detailed analysis of polyomavirus transcription in mouse NIH 3T6 cells. Nuclei were isolated from cells infected for 6, 12, 18, or 24 hr, and run-on assays were performed. The resulting RNAs were then hybridized to a number of immobilized early- and late-strand-specific probes, which represent the entire polyoma genome. Results indicate that the late promoter is always on, even in the absence of DNA replication. Even though the early-late switch is characterized by a greater than 300-fold difference in the ratio of steady-state early- and late-strand RNAs, there is only a 2-fold effect at the level of transcription initiation. Furthermore, the efficiency of termination for late transcripts is very high at early times during infection (greater than 90%) but drops drastically at late times (less than 40%). In other experiments, we have found an increase in splicing efficiency of late pre-mRNA molecules that parallels the decrease in termination efficiency. These results, taken together with other studies from our laboratory, have led us to propose two possible models for the temporal control of polyomavirus late gene expression.

Animals↗

Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs into mitosis.

Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cdc2 kinase undergoes partial activation before S phase, reaching in S phase a level that is sufficient for G2-M phase transition. S phase entry is not inhibited by this level of cyclin B-dependent kinase activity. Inhibition of DNA replication by aphidicolin suppresses nuclear envelope breakdown, yet it does not prevent the microtubule array from being converted from its interphasic to its mitotic state. Moreover, mitotic cytoplasmic events occur at the same time in control and aphidicolin-treated embryos. Thus unreplicated DNA only prevents mitotic nuclear, not cytoplasmic, events from occurring prematurely. These results together show that the inhibition of cyclin B-cdc2 kinase activation is probably not the only mechanism that prevents mitotic nuclear events from occurring as long as DNA replication has not been completed. In contrast, cytoplasmic mitotic events seem to be controlled by a timing mechanism independent of DNA replication, set up at fertilization, that prevents premature opening of a window for mitotic events.

Animals↗

Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection.

The relationship of two early events in the establishment of infection by avian retroviruses, the inhibition of viral DNA synthesis in stationary avian cells and the secondary infection which occurs after infection of replicating cells, was investigated. When neutralizing antibody to spleen necrosis virus was used to prevent secondary infection, the amount of unintegrated linear spleen necrosis virus DNA detected was much lower in infected stationary cells than in infected replicating cells. The amount of unintegrated linear spleen necrosis virus DNA in stationary cells was less than one copy per cell even at high multiplicities of infection. Viral DNA synthesis resumed after stimulation of the cells to replicate. The time of this viral DNA synthesis was closely correlated with renewed cellular DNA synthesis. In addition, blocking secondary infection of replicating cells prevented the rate of virus production from reaching the high levels usually associated with a normal productive infection by SNV. Virus production increased if secondary infection was allowed. However, this rise in virus production was not proportional to the amounts of viral DNA integrated after secondary infection.

Alpharetrovirus↗

Genetic dissection of a mammalian replicator in the human beta-globin locus.

The timing and localization of DNA replication initiation in mammalian cells are heritable traits, but it is not known whether initiation requires specific DNA sequences. A site-specific recombination strategy was used to show that DNA sequences previously identified as replication initiation sites could initiate replication when transferred to new chromosomal locations. An 8-kilobase DNA sequence encompassing the origin of DNA replication in the human beta-globin locus initiated replication in the simian genome. Specific deletions within the globin origin did not initiate replication in these chromosomal sites. These data suggest that initiation of DNA replication in mammalian cells requires specific sequence information and extend the replicon hypothesis to higher eukaryotes.

Animals↗

Gene-specific transactivation by herpes simplex virus type 1 alpha protein ICP27.

Herpes simplex virus type 1 (HSV-1) encodes several alpha (immediate-early) gene products that modulate gene expression during viral replication. We report here that the alpha protein ICP27 specifically stimulates expression of a later viral gene, that encoding glycoprotein B (gB). Using temperature-sensitive viral mutants, the effect of ICP27 on HSV-1 protein synthesis was examined at early times after infection or at later times when viral DNA replication was inhibited. Under these conditions, the expression of gB showed a marked dependence on the presence of functional ICP27, whereas several other beta and gamma 1 genes showed a lesser dependence. It was also noted that cells infected with ICP27 temperature sensitive mutants at the nonpermissive temperature showed a reduction in the electrophoretic mobility of the alpha protein ICP4. To examine the mechanism by which ICP27 stimulated gB expression, a plasmid was constructed in which the promoter-regulatory region of the gB gene was fused to the gene encoding chloramphenicol acetyltransferase (CAT). CAT expression from this plasmid was induced significantly by ICP27 expressed from a cotransfected plasmid. Induction of CAT activity by ICP27 correlated well with an increase in the amount of CAT transcripts initiated from the transcriptional start site of the gB gene. The transactivating activity of ICP27 was specific for the gB promoter-regulatory region, as expression from several other HSV-1 promoter-CAT chimeric genes was not stimulated by ICP27. The DNA sequences which conferred the response to ICP27 mapped within 175 base pairs upstream and 41 base pairs downstream of the gB transcriptional start site. Our results suggest that the full expression of gB and perhaps other viral genes during HSV-1 infection requires the combined action of multiple viral transactivators.

Acetyltransferases↗

Cytomegalovirus replication in primary and passaged human placental cells.

Human cytomegalovirus (HCMV) was found to replicate in passaged fibroblastic human first trimester and term placental cells. The time-course of viral DNA replication as well as virus production in these human placental fibroblasts was similar to that in human embryo fibroblast cultures. In contrast, HCMV did not replicate in primary placental epithelioid cells. Continued passage (5 or more) of primary placental epithelioid cells was necessary to convert these cells to a state of permissiveness. The permissive cells were, however, fibroblasts. HCMV DNA replication in passaged placental fibroblastic cells was not affected by treatment with insulin or human chorionic gonadotropin. Furthermore, no replication of HCMV DNA occurred in choriocarcinoma cells, the epithelioid cells derived from cancer of the placenta. These results suggest that epithelial placental trophoblasts, either normal or transformed, were nonpermissive for HCMV. The permissiveness of HCMV infection to secondary placental cells which was observed might be due to the strong selection of fibroblastic cells in vitro.

Cell Line↗

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1.

Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1/Rad53 (yeast) and ATM/CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1/Rad53, genetically and physically interacts with Sml1 in vivo. The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1Delta strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1Delta cells. Finally, Dun1 can phosphorylate Sml1 in vitro. These results suggest that Dun1 kinase function is the last step required in the Mec1/Rad53 cascade to remove Sml1 during S phase and after DNA damage.

Alleles↗

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells.

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.

Cloning, Molecular↗

Replication of deoxyribonucleic acid during the division cycle of Salmonella typhimurium.

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.

Carbon Isotopes↗

Coupling the initiation of chromosome replication to cell size in Escherichia coli.

Bacterial cells change size dramatically with change in growth rate, but the ratio between cell volume and the number of copies of the origin of chromosome replication (oriC) is roughly constant at the time of initiation of DNA replication at almost all growth rates. Recent research on the inactivation of initiator protein (DnaA) and depletion of DnaA pools by the high-affinity DnaA-binding locus datA allows us to propose a simple model to explain the long-standing question of how Escherichia coli couples DNA replication to cell size.

Adenosine Triphosphate↗

Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6.

In Saccharomyces cerevisiae, at least 17 DNA replication genes are coordinately expressed at the G1/S boundary during the cell cycle. All of these genes have the DNA sequence element ACGCGT in their 5' upstream regulatory regions. This sequence has been shown to be essential for periodic expression of the POL1, CDC9, and TMP1 genes. The cyclin (CLN1 and CLN2) and HO genes are another subset of genes that are expressed with the same timing as the DNA replication genes. Their periodic expression requires the participation of two well-characterized transcriptional activators: the SWI4 and SWI6 gene products. In this study, we present evidence that SWI6 contributes to the regulation of DNA replication genes as well. Surprisingly, a preferential requirement for SWI6 over SWI4 is observed in our studies of ACGCGT-dependent reporter gene expression in vivo. This selectivity has not been observed for the other G1/S genes. Correlating with the in vivo results, protein-DNA complexes formed in vitro on multimeric ACGCGT elements are either abolished or reduced in swi6 delta deletion mutants.

Base Sequence↗