Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone

Initiation and control of replication of the broad-host-range plasmid RK2 requires two plasmid-encoded elements, the replication origin (oriV) and the initiation protein TrfA. Purified TrfA is largely in the form of a dimer; however, only the monomeric form of the protein can bind specifically to the direct repeats (iterons) at the RK2 origin. The largely dimeric form of wild-type TrfA is inactive in the initiation of replication of RK2 in an in vitro replication system reconstituted from purified components. However, preincubation of the TrfA protein with the ClpX molecular chaperone isolated from Escherichia coli activates the initiator protein for replication in the purified system. We further observed that ClpX, in an ATP-dependent reaction, greatly increases the proportion of TrfA monomers and, therefore, the ability of this protein to bind to iterons localized within RK2 origin. Finally, a copy-up mutant of the TrfA protein which is largely in the monomer form is active in the reconstituted in vitro replication system, and its activity is not affected by ClpX.

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

A neural system for human visual working memory

Working memory is the process of actively maintaining a representation of information for a brief period of time so that it is available for use. In monkeys, visual working memory involves the concerted activity of a distributed neural system, including posterior areas in visual cortex and anterior areas in prefrontal cortex. Within visual cortex, ventral stream areas are selectively involved in object vision, whereas dorsal stream areas are selectively involved in spatial vision. This domain specificity appears to extend forward into prefrontal cortex, with ventrolateral areas involved mainly in working memory for objects and dorsolateral areas involved mainly in working memory for spatial locations. The organization of this distributed neural system for working memory in monkeys appears to be conserved in humans, though some differences between the two species exist. In humans, as compared with monkeys, areas specialized for object vision in the ventral stream have a more inferior location in temporal cortex, whereas areas specialized for spatial vision in the dorsal stream have a more superior location in parietal cortex. Displacement of both sets of visual areas away from the posterior perisylvian cortex may be related to the emergence of language over the course of brain evolution. Whereas areas specialized for object working memory in humans and monkeys are similarly located in ventrolateral prefrontal cortex, those specialized for spatial working memory occupy a more superior and posterior location within dorsal prefrontal cortex in humans than in monkeys. As in posterior cortex, this displacement in frontal cortex also may be related to the emergence of new areas to serve distinctively human cognitive abilities.

Opening of a monomer-monomer interface of the trimeric bacteriophage T4-coded GP45 sliding clamp is required for clamp loading onto DNA

The replication system of bacteriophage T4 uses a trimeric ring-shaped processivity clamp (gp45) to tether the replication polymerase (gp43) to the template-primer DNA. This ring is placed onto the DNA by an ATPase-driven clamp-loading complex (gp44/62) where it then transfers, in closed form, to the polymerase. It generally has been assumed that one of the functions of the loading machinery is to open the clamp to place it around the DNA. However, the mechanism by which this occurs has not been fully defined. In this study we design and characterize a double-mutant gp45 protein that contains pairs of cysteine residues located at each monomer-monomer interface of the trimeric clamp. This mutant protein is functionally equivalent to wild-type gp45. However, when all three monomer-monomer interfaces are tethered by covalent crosslinks formed (reversibly or irreversibly) between the cysteine pairs these closed clamps can no longer be loaded onto the DNA nor onto the polymerase, effectively eliminating processive strand-displacement DNA synthesis. Analysis of the individual steps of the clamp-loading process shows that the ATPase-dependent interactions between the clamp and the clamp loader that precede DNA binding are hyperstimulated by the covalently crosslinked ring, suggesting that binding of the closed ring induces a futile, ATP-driven, ring-opening cycle. These findings and others permit further characterization and ordering of the steps involved in the T4 clamp-loading process.

Formation of the preprimosome protects λ O from RNA transcription-dependent proteolysis by ClpP/ClpX

Using the bacteriophage λ DNA replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived λ O protein to the ClpP/ClpX protease during the various stages of λ DNA replication. We find that binding of λ O protein to its oriλ DNA sequence, leading to the so-called “O-some” formation, largely inhibits its degradation. On the contrary, under conditions permissive for transcription, the λ O protein bound to the oriλ sequence becomes largely accessible to ClpP/ClpX-mediated proteolysis. However, when the λ O protein is part of the larger oriλ:O⋅P⋅DnaB preprimosomal complex, transcription does not significantly increase ClpP/ClpX-dependent λ O degradation. These results show that transcription can stimulate proteolysis of a protein that is required for the initiation of DNA replication.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Ectopic expression of prion protein (PrP) in T lymphocytes or hepatocytes of PrP knockout mice is insufficient to sustain prion replication

The cellular form of the Prion protein (PrPC) is necessary for prion replication in mice. To determine whether it is also sufficient, we expressed PrP under the control of various cell- or tissue-specific regulatory elements in PrP knockout mice. The interferon regulatory factor-1 promoter/Eμ enhancer led to high PrP levels in the spleen and low PrP levels in the brain. Following i.p. scrapie inoculation, high prion titers were found in the spleen but not in the brain at 2 weeks and 6 months, showing that the lymphoreticular system by itself is competent to replicate prions. PrP expression directed by the Lck promoter resulted in high PrP levels on T lymphocytes only but, surprisingly, did not allow prion replication in the thymus, spleen, or brain following i.p. inoculation. A third transgenic line, which expressed PrP in the liver under the control of the albumin promoter/enhancer—albeit at low levels—also failed to replicate prions. These results show that expression of PrP alone is not sufficient to sustain prion replication and suggest that additional components are needed.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

Conditionally replicating mycobacteriophages: A system for transposon delivery to Mycobacterium tuberculosis

Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes. Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems. This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria. These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome. Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30°C but not at 37°C (TM4) or 38.5°C (D29). Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells. Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M. smegmatis whereas Tn5367 transposed apparently randomly in M. phlei, Bacille Calmette–Guérin (BCG), and M. tuberculosis. Sequence analysis of the M. tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M. tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome. Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M. phlei, BCG, and M. tuberculosis.

Efficiency of DNA replication in the polymerase chain reaction

A detailed quantitative kinetic model for the polymerase chain reaction (PCR) is developed, which allows us to predict the probability of replication of a DNA molecule in terms of the physical parameters involved in the system. The important issue of the determination of the number of PCR cycles during which this probability can be considered to be a constant is solved within the framework of the model. New phenomena of multimodality and scaling behavior in the distribution of the number of molecules after a given number of PCR cycles are presented. The relevance of the model for quantitative PCR is discussed, and a novel quantitative PCR technique is proposed.

Normal host prion protein (PrPC) is required for scrapie spread within the central nervous system

Mice devoid of PrPC (Prnpo/o) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnpo/o brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnpo/o mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnpo/o mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.

On the controllability of distributed systems

To “control” a system is to make it behave (hopefully) according to our “wishes,” in a way compatible with safety and ethics, at the least possible cost. The systems considered here are distributed—i.e., governed (modeled) by partial differential equations (PDEs) of evolution. Our “wish” is to drive the system in a given time, by an adequate choice of the controls, from a given initial state to a final given state, which is the target. If this can be achieved (respectively, if we can reach any “neighborhood” of the target) the system, with the controls at our disposal, is exactly (respectively, approximately) controllable. A very general (and fuzzy) idea is that the more a system is “unstable” (chaotic, turbulent) the “simplest,” or the “cheapest,” it is to achieve exact or approximate controllability. When the PDEs are the Navier–Stokes equations, it leads to conjectures, which are presented and explained. Recent results, reported in this expository paper, essentially prove the conjectures in two space dimensions. In three space dimensions, a large number of new questions arise, some new results support (without proving) the conjectures, such as generic controllability and cases of decrease of cost of control when the instability increases. Short comments are made on models arising in climatology, thermoelasticity, non-Newtonian fluids, and molecular chemistry. The Introduction of the paper and the first part of all sections are not technical. Many open questions are mentioned in the text.