PubMed Health⌕ Search

Biomedical subjects

Dean A Jackson

Publications and source records attributed to Dean A Jackson.

16 recordsLinked to original sources
PubMed2006

Designing nonviral vectors for efficient gene transfer and long-term gene expression.

Although the genetic therapy of human diseases has been conceptually possible for many years we still lack a vector system that allows safe and reproducible genetic modification of eukaryotic cells and ensures faithful long-term expression of transgenes. There is increasing agreement that vectors that are based exclusively on chromosomal elements, which replicate autonomously in human cells, could fulfill these criteria. The rational construction of such vectors is still hindered by our limited knowledge of the factors that regulate chromatin function in eukaryotic cells. This review sets out to summarize how our current knowledge of nuclear organization can be applied to the design of extrachromosomal gene expression vectors that can be used for human gene therapy. Within the past years a number of episomal nonviral constructs have been designed and their replication strategies, expression of transgenes, mitotic stability, and delivery strategies and the mechanisms required for their stable establishment will be discussed. To date, these nonviral vectors have not been used in clinical trials. Even so, many compelling arguments can be developed to support the view that nonviral vector systems will play a major role in future gene therapy protocols.

Animals↗
PubMed2006

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Adenosine Triphosphatases↗
PubMed2006

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase.

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1(-/-) chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1(-/-) cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3(-/-) DT40 cells. Rather, we observed an increased number of replication fibers in Chk1(-/-) cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1(-/-) cells are associated with the accumulation of aberrant replication fork structures.

Animals↗
PubMed2005

The microarchitecture of DNA replication domains.

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures--replication bodies (RB), and (2) focal replication sites with no distinct underlying structure--replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase alpha, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase alpha, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.

Antibodies, Monoclonal↗
PubMed2005

The amazing complexity of transcription factories.

Multicellular eukaryotes orchestrate immensely complex patterns of gene expression in order to define numerous specialised cell types. Patterns of expression are regulated at the level of DNA modification, chromatin structure, genome architecture and nuclear organisation. Each aspect of these features of regulation is controlled by complex nuclear systems, and each of the systems must interact to define a coherent regulatory network. In proliferating cells, and particularly during development, patterns of gene expression must be preserved in cells that have active DNA repair mechanisms and efficiently duplicate both DNA and the associated chromatin status--the histone code. The systems that regulate DNA synthesis and genome stability are integrated with those that regulate the efficacy of RNA synthesis, such that patterns of gene expression are maintained. This review analyses how different experimental approaches are revealing the amazing complexity of the transcriptionally active compartment of mammalian cells. The functional implications of this complexity will be discussed.

Animals↗
PubMed2004

The case for nuclear translation.

Although it is frequently assumed that translation does not occur in eukaryotic nuclei, recent evidence suggests that some translation can take place and that it is closely coupled to transcription. The first evidence concerns the destruction of nuclear mRNAs containing premature termination codons by nonsense-mediated decay (NMD). Only ribosomes can detect termination codons, and as some NMD occurs within the nuclear fraction, active nuclear ribosomes could perform the required detection. The second evidence is the demonstration that tagged amino acids are incorporated into nascent polypeptides in a nuclear process coupled to transcription. The third evidence is that components involved in translation, NMD and transcription colocalize, coimmunoprecipitate and co-purify. All these results are simply explained if nuclear ribosomes scan nascent transcripts for premature termination codons at the site of transcription. Alternatively, the scanning needed for NMD might take place at the nuclear membrane, and contaminating cytoplasmic ribosomes might give the appearance of some nuclear translation. We argue, however, that the balance of evidence favours bona fide nuclear translation.

Animals↗
PubMed2004

Dynamics of DNA replication: an ultrastructural study.

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.

Binding Sites↗
PubMed2004

Approaches for monitoring nuclear translation.

The nuclear membrane is the defining feature of eukaryotes. It divides the cell into two functionally specialized compartments, and it is widely assumed that translation is restricted to only one: the cytoplasm. However, recent results suggest that some translation takes place in nuclei closely coupled to transcription. Various labeling techniques are described that enable nascent peptides to be labeled and then localized wherever they might be in the cell.

Biotin↗
PubMed2003

Characterisation of the interaction between WRN, the helicase/exonuclease defective in progeroid Werner's syndrome, and an essential replication factor, PCNA.

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.

Amino Acid Sequence↗
PubMed2003

The anatomy of transcription sites.

Patterns of gene expression in multicellular eukaryotes are regulated by numerous extremely sophisticated mechanisms. Over the past year, developments in our ability to monitor the organisation and dynamic properties of the components involved in gene expression have emphasised how both global nuclear architecture and chromosome structure can influence this fundamental process.

Chromatin↗
PubMed2003

Applying microscopy to the analysis of nuclear structure and function.

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery.

Animals↗
PubMed2003

The principles of nuclear structure.

In multicellular eukaryotes, chromatin function is regulated by numerous extremely sophisticated mechanisms. Recent developments in our ability to monitor the organization and dynamic properties of the components involved in processes such as gene expression and DNA synthesis have emphasised how both global nuclear architecture and chromosome structure can influence these fundamental processes. This review sets out to evaluate our present views of the principles that dictate nuclear structure. Particular emphasis is placed on architectural themes and the concept of spatial epigenetics.

Cell Nucleus↗
PubMed2002

Asymmetry of DNA replication fork progression in Werner's syndrome.

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.

Adolescent↗
PubMed2002

Chromosome structure and nuclear architecture: implications for gene therapy.

A reductionist view of mechanisms that control chromatin function might be appealing in its simplicity. It is tempting, for example, to view the process of gene expression as a series of simple protein-DNA and protein-protein interactions that lead, ultimately, to the assembly of an engaged transcription complex. RNA synthesis would then ensue to activate the pathway of gene expression. Yet attractive as this scheme may be, it is also clear that it bears only passing resemblance to events that occur in mammalian cells where a variety of epigenetic features might profoundly affect chromatin function. This review sets out to analyze our present knowledge of epigenetic features that influences chromatin function while placing particular emphasis on those features that are likely to impact on our ability to perform stable gene expression from ectopic genes - the major requirement of gene therapy.

Cell Division↗