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PubMed2002

Production of recombinant adeno-associated type 5 (rAAV5) vectors using recombinant herpes simplex viruses containing rep and cap.

We developed a scaleable production system for adeno-associated virus type 5 (AAV5)-based vectors using a replication-defective recombinant herpes simplex type 1 virus (rHSV) containing the rep and cap genes of AAV5. Multiple rHSV isolates containing AAV5 rep and cap with normal or altered p5 promoter elements were constructed and tested in vector production. Compared with rAAV5 vector yields obtained by plasmid transfection, yields of rAAV5 using any of the rHSV isolates were low. Evidence suggests that the low vector yields are a consequence of the extensive and early cytopathology induced by the rHSV isolates. In addition, we found a correlation between the amount of Rep52 or Rep40 proteins and the amount of vector produced by each rHSV isolate, suggesting that packaging of vector DNA into virus particles is rate-limiting when using rHSV to generate rAAV5 vectors.

DNA Replication↗
PubMed2006

Nucleosome structure and function.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. How chromatin structure modulates the expression of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in nucleosome structure and dynamics.

Animals↗
PubMed1994

Drug and radiation resistance in spheroids: cell contact and kinetics.

Cells from multicellular spheroids are often more resistant than monolayers to drugs and radiation. While explanations for resistance can be based on differences in cell cycle distribution, inability of the drug to penetrate the spheroid, or the presence of hypoxic cells, these mechanisms do not adequately explain resistance to all agents. Small spheroids (containing about 25-50 cells) exposed to ionizing radiation, hyperthermia, photodynamic therapy, or topoisomerase II inhibitors, are more resistant to killing than monolayers; the close three-dimensional contact in spheroids has been implicated in this resistance. Proposed mechanisms for the 'contact effect' include gap junctional 'reciprocity', cell shape mediated changes in (repair-related) gene expression, and alterations in chromatin packaging which influence DNA repair. The consequences of the contact effect are especially important for multifraction exposures. Another form of resistance can be demonstrated during repetitive treatments; 'regrowth resistance' reflects the capacity of spheroid cells to proliferate more efficiently to compensate for cell killing.

Animals↗
PubMed1996

Chromatin structure of the MMTV promoter and its changes during hormonal induction.

1. The packaging of nuclear DNA in chromatin determines the conversion of the genetic information into a defined phenotype by influencing the availability of DNA sequences for interactions with regulatory proteins and transcription factors. 2. We have studied the influence of the first level of chromatin organization, the nucleosome, on the activity of the mouse mammary tumor virus (MMTV) promoter. The MMTV promoter is strongly transcribed in response to steroid hormones but is virtually silent in the absence of hormonal stimuli. Full hormonal induction requires binding of the hormone receptors to four hormone-responsive elements (HREs), as well as binding of nuclear factor I (NFI) and the octamer transcription factor 1 (OTF-1 or Oct-1) to sites located between the HREs and the TATA box. A full loading with transcription factors cannot be achieved on free DNA due to steric hindrance between hormone receptor and NFI and between NFI and OTF-1. 3. The low basal activity of the MMTV promoter is most likely due to its organization in a positioned nucleosome. In the intact cell, as well in reconstituted chromatin, the regulatory region of the MMTV promoter is wrapped around a histone octamer in a precise rotational orientation, which permits access of the hormone receptors to only two of the four HREs, while precluding binding of NFI and OTF-1 to their respective sites. Upon hormone induction, the nucleosome is remodeled and the path of its DNA altered in a way which makes the nucleosomal dyad axis more accessible to DNase I and enables occupancy of all relevant sites: the four HREs, as well as the binding sites for NFI and OTF-1. 4. These results suggest that the nucleosomal organization of the MMTV promoter not only is responsible for the low activity prior to hormone treatment, but also may be a prerequisite for full loading with transcription factors after hormone induction. We conclude that the DNA contains topological information which modulates the expression of the genetic program.

Animals↗
PubMed2006

Dynamic nucleosomes.

It is now widely recognized that the packaging of genomic DNA together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, repair and recombination. How chromatin structure modulates the expression and maintenance of knowledge encoded in eukaryotic genomes, and how these processes take place within the context of a highly complex and compacted genomic chromatin environment remains a major unresolved question in biology. Here we review recent advances in our understanding of how nucleosome and chromatin structure may have to adapt to promote these vital functions.

Animals↗
PubMed1986

Phage T1-mediated transduction of a plasmid containing the T1 pac site.

The T1 pac site has been cloned into a plasmid vector. This recombinant plasmid was tested for T1-mediated transduction efficiency in comparison with a plasmid containing the phage lambda T1-pac-like site esp-lambda, plasmids containing T1 sequences other than the pac site, and plasmids containing neither T1 sequences nor known pac sites. The data obtained indicate that there are at least two distinct mechanisms of T1-mediated plasmid transduction. One requires the presence of any T1 sequence on the plasmid and probably takes place via cointegrate formation with the homologous region of an infecting T1 genome. The other is specifically dependent on the presence of a pac site on the plasmid. Plasmids are packaged as head-to-tail multimers that have one heterogeneous molecular end and the other terminated at pac, and the direction of packaging with respect to the pac site is the same for plasmids as for T1. Possible roles of pac in plasmid packaging and their implications with regard to the packaging of phage DNA are discussed.

Cloning, Molecular↗
PubMed1987

Recognition and cleavage of the bacteriophage P1 packaging site (pac). II. Functional limits of pac and location of pac cleavage termini.

Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac. We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20. It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment. The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix. The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population. (Sequence: in text). Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs. While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above. The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other. Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold. Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively.

Bacteriophages↗
PubMed1983

The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element.

The adenovirus type 5 E1A transcriptional control region contains an element with enhancer properties located between -141 and -305 relative to the E1A cap site at +1. The enhancer element is located at or very close to a sequence required in cis for packaging of viral DNA. Deletion of the element reduces both the rate of transcription and steady-state levels of E1A mRNAs in virus-infected cells. Such deletions also cause a modest reduction in activity of the E1B transcription unit, which is located immediately downstream of the E1A unit. The enhancer element includes repeated core sequences. Function is retained when the element is moved to the 3' side of the E1A gene in either possible orientation, and the E1A enhancer functions in cis to enhance transformation by the herpesvirus thymidine kinase gene in both mouse and human cells.

Adenoviruses, Human↗
PubMed1994

Herpes simplex genes: the blueprint of a successful human pathogen.

The Herpes simplex virus genome encodes 75 proteins. Of these, only 37 are required for growth of the virus in culture. These essential genes encode functions related to entry of virus into cells, regulation of gene expression and replication and packaging of viral DNA into virions. The genes that are not essential for replication in culture play a key role in multiplication of the virus and its transfer from cell to cell, in complementing cellular functions lost as a consequence of viral replication, in fine-tuning viral gene expression and in overcoming the host's response to infection. No virally encoded functions are required for establishment of the latent state, but a full complement of viral genes is essential for efficient reactivation of the virus from the latent state.

Base Sequence↗
PubMed1985

High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences.

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Chromosome Deletion↗
PubMed1993

Multimeric complexes formed by DNA-binding proteins of low sequence specificity.

Some proteins bind to double-stranded DNA with low sequence specificity, forming regular multimeric complexes that extend over large regions of DNA, strongly distorting its conformation. Formation of these complexes at particular DNA sites usually depends on the structural ability of the DNA to follow the path imposed by the protein array. These complexes are found in both prokaryotic and eukaryotic organisms and participate in processes such as DNA replication, transcription and packaging.

DNA↗
PubMed2026

The opioid receptor-ligand network in human cancers: pan-cancer multi-omics profiling and translational implications.

BACKGROUND: Opioid receptor-ligand signalling has been implicated in tumour biology and perioperative outcomes; however, its pan-cancer molecular landscape and clinical relevance remain incompletely defined. METHODS: We performed a pan-cancer multi-omics analysis of eight predefined opioid receptor-ligand genes across 33 tumour types from The Cancer Genome Atlas. Analyses included gene expression analysis using the linear models for microarray data (limma) package, genomic alterations, DNA methylation, regulatory network inference, pathway activity estimation using gene set variation analysis, and survival modelling. Multivariable Cox regression models were adjusted for age, sex, and tumour stage. RESULTS: Opioid receptor-ligand genes exhibited heterogeneous and generally low-to-moderate expression across tumour types. Genomic and epigenetic alterations were tumour-specific and variably associated with gene expression. Selected genes showed associations with overall survival in a tumour-dependent manner; however, these associations were attenuated after adjustment for clinical covariates and were accompanied by wide confidence intervals in some cohorts. Pathway analyses suggested associations with broader biological programmes, including epithelial-mesenchymal transition and immune-related pathways. Regulatory analyses identified candidate transcription factors and miRNAs, although these findings are exploratory. CONCLUSIONS: This pan-cancer analysis provides a systematic overview of opioid receptor-ligand gene features across human cancers. The observed associations are context-dependent and should be interpreted as hypothesis-generating. Further mechanistic and prospective studies are required to determine the clinical relevance of opioid signalling in cancer and perioperative settings.

Humans↗
PubMed2005

Structure and dynamic properties of nucleosome core particles.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. Whereas earlier structural studies portrayed nucleosomes (the basic repeating unit of chromatin) as monolithic and static macromolecular assemblies, we now know that they are highly dynamic and capable of extensive crosstalk with the cellular machinery. Histone variants have evolved to locally alter chromatin structure, whereas histone chaperones and other cellular factors promote histone exchange and chromatin fluidity. Both of these phenomena likely facilitate interconversion between different chromatin states that show varying degrees of transcriptional activity.

Animals↗
PubMed2004

Functional characterization of a human histone gene cluster duplication.

Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin. As a result, histone gene expression is exquisitely and functionally coupled with DNA replication. Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci. Although specific mechanisms are unclear, clustering is presumed to be important for common stringent transcriptional control of these genes at the G1/S phase transition. In this study, we describe a genomic duplication of the human histone gene cluster located at chromosome 1q21, which effectively doubles the previously known size and gene number of that cluster. The duplication persists in all examined tissues and cell lines, and the duplicated genes are transcriptionally active. Levels of messenger RNAs for duplicated histone H4 genes are high relative to those for non-duplicated H4 genes. Our data suggest that transcriptionally robust histone H4 genes may have been preferentially duplicated during evolution.

Amino Acid Sequence↗
PubMed2006

The gpQ portal protein of bacteriophage P2 forms dodecameric connectors in crystals.

Double-stranded bacteriophages code for a protein called a connector or portal protein that serves as the entry and exit portal for DNA during genome packaging and ejection, as well as the connection point between heads and tails, and possibly as a nucleator for capsid assembly. The gpQ connector protein from bacteriophage P2 has been overexpressed in Escherichia coli and purified by sucrose gradient centrifugation. Negative stain electron microscopy and image analysis revealed a 135 A diameter dodecameric ring structure with a central 25 A hole. The connector showed a strong propensity to aggregate at low ionic strength and would form microcrystalline structures in solution. Consequently, the connectors were crystallized by hanging-drop vapor diffusion against low ionic strength buffer. Two crystal forms were observed: a P4(1)22 form with unit cell parameters a=b=96.33 A and c=454.42 A that diffracted X-rays to 4.5 A resolution and an I222 crystal form with a=168.86 A, b=171.88 A and c=168.68 A that diffracted to 4.1A resolution. Self-rotation functions confirmed the presence of 12-fold symmetry in the crystals.

Bacteriophage P2↗
PubMed2004

Methods for chromatin assembly and remodeling.

Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.

Adenosine Triphosphate↗
PubMed2006

Genome-wide analysis of histone modifications by ChIP-on-chip.

Post-translational modifications to histone proteins regulate the packaging of genomic DNA into chromatin, gene activity and other functions of the genome. They are understood to play key roles in embryonic development and disease pathogenesis. Recent advances in technology have made it possible to analyze chromatin structure genome-wide in mammalian cells. Global patterns of histone modifications can be observed using a technique called ChIP-on-chip, which combines the specificity of chromatin immunoprecipitation with the unbiased, high-throughput capabilities of microarrays. The resulting maps provide insight into the functions of, and relationships between, different modifications. Here, we provide validated ChIP-on-chip methods for analyzing histone modification patterns at genome-scale in mammalian cells.

Animals↗
PubMed1997

On the origin of spontaneous somatic mutations and sectored plaques detected in transgenic mice.

The use of transgenic mice with bacterial genes that can be readily recovered and analysed for mutation has made it possible to measure mutant frequencies in many tissues. The mutations are detected by packaging the murine DNA into lambda phage and then growing these phage on a bacterial lawn under conditions such that the mutants are distinguishable from nonmutants. In the lacI mouse assay, the mutant plaques are blue whereas the nonmutant plaques are clear. The mutations detected in the lacI Big Blue Mouse are, in principle, a mixture of mutations that arose in the mouse (in vivo mutations), mutations that arose in the bacterium from lesions pre-existing in the murine DNA (ex vivo mutations), and mutations that arose during growth of the phage on the bacteria (in vitro mutations). It has been suggested that plaque morphology can be used to visually distinguish in vivo mutations (which would be seen as wholly blue plaques) from ex vivo mutations (which would produce blue and white sectored plaques) and in vitro mutations (which would produce sectored or pin-point plaques). We show here that this is not the case: ex vivo mutations produce plaques that are a homogeneous blue. By superinfection of bacteria with mutant and nonmutant phage and by in vitro mutagenesis, we found that blue plaques may contain large proportions of nonmutant phage. None of the mutant plaques seen after in vitro mutagenesis were sectored but most contained nonmutant phage. In addition, we show that most spontaneous mutations from the small intestine, which has a higher than normal mutant frequency, arose in vivo, since 17 of 17 mutants were homogeneous mutants. Sectored plaques must arise in some way other than by ex vivo mutation, like perhaps by the confluence of a mutant and nonmutant plaque.

Animals↗