Epigenetics & chromatin: Interactions and processes

On 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the conference

Transcriptional Regulation of Multi-Gene Loci: Multi-Level Control.

Recent studies indicate that different levels of control operate within multigene loci. In addition to regulatory sequences immediately flanking the genes, there are also elements that act over long distances on more than one gene. Competition for these elements among genes can influence both the level and timing of gene expression during development

Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH-VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique,soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs

Fragmentation of bacteriophage S13 replicative from DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

The restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and pyrimidine isostich analysis. The Hind and HaeIII fragments were ordered relative to each other by cross digestion and a physical map of the S13 genome constructed. Comparison of the Hind cleavage patterns of S13 RF DNA and X174 RF DNA showed the majority of the fragments from the two DNAs coincided with each other except for three of the thirteen S13 fragments and three of the thirteen X174 fragments. Comparison of the HaeIII patterns of the two DNAs revealed a lack of coincidence of one S13 fragment only and two X174 fragments. From the data obtained by the cleavage of the two DNAs by Hind and HaeIII, a correlation between the physical restriction enzyme cleavage maps and the genetic map of the two phages was made. The differences in cleavage of the two DNAs by the restriction enzymes have been explained by changes in two restriction enzyme sites in the AB region and one change of site in the F region of the genetic map of the two bacteriophages

The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimerism.

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly understood. We have tested the notion that the chromosome make-up may be important in contributing both to somatic cell chimaerism and to germ line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percentage of euploid metaphases in the ES cell clones injected into the murine blastocyst. The majority of the ES cell clones that we tested, which were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in contrast, none of the ES cell clones with more than 50% of chromosomally abnormal metaphases transmitted to the germline. Euploid ES cell clones cultured in vitro for more than 20 passages rapidly became severely aneuploid, and again this correlated closely with the percentage of chimaerism and with the number of ES cell-embryo chimaeras obtained per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the proportion of euploid cells dropped below 50%. This study suggests that aneuploidy, rather than 'loss of totipotency', in ES cells, is the major cause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of germline transmission, karyotype analysis is crucial and time/cost saving in any gene-targeting experiment

Accurate Estimation of Particle Dynamics Bypassing Substrate Drift Bias: Application to Cell Nucleus Motion

In microscopic imaging, the movement of a living substrate can be caused by its own displacement (e.g., cell motion/migration) or other technical factors such as microscope stage drift. This drifting motion is one of the main biases resulting in poor estimation of particle dynamics since it seriously affects the estimation of the biophysical parameters (the diffusion constant D and anomalous exponent α), especially when performed on the basis of mean squared displacement (MSD) analysis. In this paper, we compare a few substrate drift correction/registration methods based on the use of additional fluorescent spots (landmarks). In the particular case of cell nucleus motion, we labeled telomeres spreading throughout the cell nucleus. We show that compared to the MSD analysis, the use of Gaussian processes is an effective and more accurate way to estimate the substrate drift, and major biophysical parameters of particle dynamics

DNaseI hypersensitive sites 1, 2 and 3 of the human β-globin dominant control region directs position-independent expression.

The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus