Suitability of a CMV/EGFP cassette to monitor stable expression from human artificial chromosomes but not transient transfer in the cells forming viable clones

Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4 -6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation. Copyright (C) 2004 S. Karger AG, Basel

Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner

A linkage study of candidate loci in familial Parkinson's Disease

BACKGROUND: Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Most cases are sporadic, however familial cases do exist. We examined 12 families with familial Parkinson's disease ascertained at the Movement Disorder clinic at the Oregon Health Sciences University for genetic linkage to a number of candidate loci. These loci have been implicated in familial Parkinson's disease or in syndromes with a clinical presentation that overlaps with parkinsonism, as well as potentially in the pathogenesis of the disease. METHODS: The examined loci were PARK3, Parkin, DRD (dopa-responsive dystonia), FET1 (familial essential tremor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), Ret, DAT1 (the dopamine transporter), Nurr1 and Synphilin-1. Linkage to the α-synuclein gene and the Frontotemporal dementia with parkinsonism locus on chromosome 17 had previously been excluded in the families included in this study. Using Fastlink, Genehunter and Simwalk both parametric and model-free non-parametric linkage analyses were performed. RESULTS: In the multipoint parametric linkage analysis lod scores were below -2 for all loci except FET1 and Synphilin-1 under an autosomal dominant model with incomplete penetrance. Using non-parametric linkage analysis there was no evidence for linkage, although linkage could not be excluded. A few families showed positive parametric and non-parametric lod scores indicating possible genetic heterogeneity between families, although these scores did not reach any degree of statistical significance. CONCLUSIONS: We conclude that in these families there was no evidence for linkage to any of the loci tested, although we were unable to exclude linkage with both parametric and non-parametric methods

Widespread Gene Conversion in Centromere Cores

Data from maize show that centromeres strongly suppress crossing over and instead undergo frequent genetic exchange in the form of gene conversion

Decentralized Hash Tables For Mobile Robot Teams Solving Intra-Logistics Tasks

Although a remarkably high degree of automation has been reached in production and intra-logistics nowadays, human labor is still used for transportation using handcarts and forklifts. High labor cost and risk of injury are the undesirable consequences. Alternative approaches in automated warehouses are fixed installed conveyors installed either overhead or floor-based. The drawback of such solutions is the lack of flexibility, which is necessary when the production lines of the company change. Then, such an installation has to be re-built. In this paper, we propose a novel approach of decentralized teams of autonomous robots performing intra-logistics tasks using distributed algorithms. Centralized solutions suffer from limited scalability and have a single point of failure. The task is to transport material between stations keeping the communication network structure intact and most importantly, to facilitate a fair distribution of robots among loading stations. Our approach is motivated by strategies from peer-to-peer-networks and mobile ad-hoc networks. In particular we use an adapted version of distributed heterogeneous hash tables (DHHT) for distributing the tasks and localized communication. Experimental results presented in this paper show that our method reaches a fair distribution of robots over loading stations. Categories and Subject Descriptors Autonomous vehicle

Optimal File-Distribution in Heterogeneous and Asymmetric Storage Networks

We consider an optimisation problem which is motivated from storage virtualisation in the Internet. While storage networks make use of dedicated hardware to provide homogeneous bandwidth between servers and clients, in the Internet, connections between storage servers and clients are heterogeneous and often asymmetric with respect to upload and download. Thus, for a large file, the question arises how it should be fragmented and distributed among the servers to grant “optimal ” access to the contents. We concentrate on the transfer time of a file, which is the time needed for one upload and a sequence of n downloads, using a set of m servers with heterogeneous bandwidths. We assume that fragments of the file can be transferred in parallel to and from multiple servers. This model yields a distribution problem that examines the question of how these fragments should be distributed onto those servers in order to minimise the transfer time. We present an algorithm, called FlowScaling, that finds an optimal solution within running time O(m log m). We formulate the distribution problem as a maximum flow problem, which involves a function that states whether a solution with a given transfer time bound exists. This function is then used with a scaling argument to determine an optimal solution within the claimed time complexity