Alphoid DNA from different chromosomes forms de novo minichromosomes with high efficiency.

Clones from one BAC and one PAC library carrying centromeric alphoid DNA were characterized and found to be stable but to differ according to the enzyme used to make the library. Five different clones with homogeneous alphoid DNA, derived from chromosomes 13/21, 14/22, 17 and 18, were all shown to form minichromosomes de novo after transfection into the human cell line HT1080 in greater than 29% of the cell lines analysed. Similarly sized alphoid arrays (110-160 kb) from chromosomes 17, 13/21 and 14/22 all formed minichromosomes in about 50% of the cell lines analysed while a smaller array (50 kb) of 14/22 alphoid was less efficient (29% of cell lines) and a larger array (200 kb) from chromosome 18 was more efficient (2/2 cell lines). Thus the larger arrays of alphoid DNA gave higher percentages of cell lines with minichromosomes. However, smaller arrays may be preferable for gene expression as there appeared to be more EGFP expression from these minichromosomes.

Comparison of a new pmp22 transgenic mouse line with other mouse models and human patients with CMT1A.

Charcot-Marie-Tooth disease type 1A is a dominantly inherited demyelinating disorder of the peripheral nervous system. It is most frequently caused by overexpression of peripheral myelin protein 22 (PMP22), but is also caused by point mutations in the PMP22 gene. We describe a new transgenic mouse model (My41) carrying the mouse, rather than the human, pmp22 gene. The My41 strain has a severe phenotype consisting of unstable gait and weakness of the hind limbs that becomes obvious during the first 3 weeks of life. My41 mice have a shortened life span and breed poorly. Pathologically, My41 mice have a demyelinating peripheral neuropathy in which 75% of axons do not have a measurable amount of myelin. We compare the peripheral nerve pathology seen in My41 mice, which carry the mouse pmp22 gene, with previously described transgenic mice over-expressing the human PMP22 protein and Trembler-J (TrJ) mice which have a P16L substitution. We also look at the differences between CMT1A duplication patients, patients with the P16L mutation and their appropriate mouse models.

An element in intron 1 of the CFTR gene augments intestinal expression in vivo.

The elements controlling the complex developmental and tissue-specific expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene lie outside the basal promoter region and have not been characterized. We previously identified a tissue-specific DNase I hypersensitive site (DHS) in intron 1 (185 + 10 kb) of the CFTR gene. Here we show that removal of the core element abolishes the activity of this DHS in transient transfection assays of reporter/enhancer gene constructs. We then compared expression from a 310 kb yeast artificial chromosome (YAC) that contains the entire CFTR gene with expression from the same YAC from which the DHS element had been deleted. Stable transfection of a human colon carcinoma cell line showed that transcription from the deleted YAC was reduced by approximately 60%. In transgenic mice, deletion of the intron 1 DHS had no effect on expression in the lung, but reduced expression in the intestine by approximately 60%. Thus, the regulatory element associated with the intron 1 DHS is tissue-specific and is required for normal CFTR expression levels in the intestinal epithelium in vivo.

Chromosomal mapping, gene structure and characterization of the human and murine RAB27B gene.

BACKGROUND: Rab GTPases are regulators of intracellular membrane traffic. The Rab27 subfamily consists of Rab27a and Rab27b. Rab27a has been recently implicated in Griscelli Disease, a disease combining partial albinism with severe immunodeficiency. Rab27a plays a key role in the function of lysosomal-like organelles such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Little is known about Rab27b. RESULTS: The human RAB27B gene is organised in six exons, spanning about 69 kb in the chromosome 18q21.1 region. Exon 1 is non-coding and is separated from the others by 49 kb of DNA and exon 6 contains a long 3' untranslated sequence (6.4 kb). The mouse Rab27b cDNA shows 95% identity with the human cDNA at the protein level and maps to mouse chromosome 18. The mouse mRNA was detected in stomach, large intestine, spleen and eye by RT-PCR, and in heart, brain, spleen and kidney by Northern blot. Transient over-expression of EGF-Rab27b fusion protein in cultured melanocytes revealed that Rab27b is associated with melanosomes, as observed for EGF-Rab27a. CONCLUSIONS: Our results indicate that the Rab27 subfamily of Ras-like GTPases is highly conserved in mammals. There is high degree of conservation in sequence and gene structure between RAB27A and RAB27B genes. Exogenous expression of Rab27b in melanocytes results in melanosomal association as observed for Rab27a, suggesting the two Rab27 proteins are functional homologues. As with RAB27A in Griscelli Disease, RAB27B may be also associated with human disease mapping to chromosome 18.

Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells.

Plasmids carrying the origin of plasmid replication ( oriP ) and expressing the EBNA-1 protein from the Epstein-Barr virus replicate and segregate in human cells and are thus potentially useful vectors for gene therapy. As very large circular molecules, up to 660 kb in size, can be maintained episomally using this system, it is possible to include intact human genes with all their long-range controlling elements which might give high levels of tissue-specific and controlled gene expression. We have shown previously that a 320 kb yeast artificial chromosome (YAC) carrying the intact human CFTR gene can complement the Cambridge null cystic fibrosis mice as a transgene. We have now modified this YAC to a circular molecule carrying both oriP and the EBNA-1 gene. We show that this oriP-EBNA1-YAC can be stably maintained as unrearranged episomes in mouse LA-9 cells, which do not express endogenous cftr, and in mouse CMT-93 cells, which do express endogenous cftr. The human CFTR gene is expressed in some of the cell lines, but the level of expression is very variable between cell lines and is not related to the copy number of the elements.

Stable episomal maintenance of yeast artificial chromosomes in human cells.

Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.

Replication of yeast DNA and novel chromosome formation in mouse cells.

To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.

The ecology of human performance: a framework for considering the effect of context.

In theory and in practice, context (as an area of concern to occupational therapists) has not received the same attention as performance components and performance areas. The Ecology of Human Performance serves as a framework for considering the effect of context. Context is described as a lens from which persons view their world. The interrelationship of person and context determines which tasks fall within the person's performance range. The Ecology of Human Performance framework provides guidelines for encompassing context in occupational therapy theory, practice, and research.

Insertion of a pathogenic mutation into a yeast artificial chromosome containing the human amyloid precursor protein gene.

The genetic modelling of human disease would be considerably facilitated if pathogenic mutations could be inserted into transgenes which were then expressed in an appropriate manner. Yeast artificial chromosomes (YACs), when used as transgenes appear to direct expression with the correct temporal and spatial distribution. Here we demonstrate that it is comparatively straightforward to introduce pathogenic mutations into such YACs by the use of the 'pop-in, pop-out' procedure, by inserting an Alzheimer-causing mutation (APP717Val-->lle) into an APP-containing YAC which has previously been used as a transgene. The significance of these procedures for the modelling of human disease is discussed.

IgE-mediated release of rat mast cell protease II, beta-hexosaminidase and leukotriene C4 from cultured bone marrow-derived rat mast cells.

Functional characteristics of cultured bone marrow-derived rat mast cells (BMMC) were studied. BMMC were shown to release in a time- and dose-dependent fashion the mucosal mast cell (MMC)-specific enzyme, rat mast cell protease II (RMCPII), following IgE-mediated activation in vitro. RMCPII release was temporally associated with that of the mast cell granule-derived enzyme, beta-hexosaminidase (beta-hex). Release of the pre-formed granule constituents, RMCPII and beta-hex, was associated with the generation of the membrane-derived lipid mediator, leukotriene C4 (LTC4) and, in older cultures, substantial amounts were generated (25.2 ng/10(6) BMMC). Absolute amounts of RMCPII, beta-hex and LTC4 released were dependent upon the age of the BMMC. These results extend our previous observations on the staining properties and protease content of rat BMMC and provide evidence that these cells are functionally, as well as histochemically, analogous to the MMC subset, which is so prominent during intestinal nematode infections in rats.