Evelyn W. Campbell

A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity.

Abstract The toxic trace metal Cd 2+ has been used to select a variant (designated Cd r ) of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd 2+ . Resistance of the Cd r cell to Cd 2+ -mediated cytotoxicity is not due to a decreased capability of the Cd r cell to accumulate Cd 2+ since Cd 2+ uptake in the Cd r cell is indistinguishable from that in the CHO cell at both toxic and subtoxic Cd 2+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd 2+ -binding proteins (metallothioneins) reveals that the Cd r cell has an increased capacity to induce metallothionein and to sequester intracellular Cd 2+ in metallothioneins. These results suggest that the greater competence of the Cd r cell to induce metallothionein is a major factor in the Cd 2+ -resistant phenotype of the variant.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16☆

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somatic cell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is approximately 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be approximately 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.

[19] Chromosome sorting by flow cytometry

Publisher Summary This chapter presents chromosome sorting by flow cytometry. Flow cytometers operate on the principle of rapid analysis of single cells or chromosomes. A suspension of fluorescently stained chromosomes flows at a rate between 1000 to 2000/sec through a finely focused laser beam. Individual chromosome types are resolved on the basis of DNA content and base composition as determined by the cytochemistry of the fluorescent dyes. The chromosomes flow in a small stream that is broken into uniform droplets. The droplet containing the desired chromosome type, as indicated by the fluorescence measurement, can be deflected from the mainstream. In addition to the operation of the flow cytometer, the culturing of cells and chromosome preparation techniques must be highly efficient. A chromosome preparation should provide uniformly stained chromosomes, low debris levels, a high yield of free chromosomes from mitotic cells, and for library construction, chromosomal DNA of very high molecular weight. The yield of free chromosomes is crucial because low yields decrease the chromosome number concentration while increasing the debris fraction, making the chromosome sorting rates much slower than optimum. Techniques for chromosome preparation, resolution of the normal human chromosomes for flow sorting, and the way these interact to produce sorted chromosomes of high purity at optimum rates are described in the chapter.

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2.

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, gamma-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.

Stimulation of informosomal RNA synthesis in cultured chinese hamster cells exposed to low levels of cadmium

Cadmium is a trace element that is quite toxic [l-6] . Exposure to 40 mg by inhalation or to between 0.35 and 3.5 g by ingestion can be lethal [3] . Incidents of such acute effects are infrequent [ 1,3,4] . However, chronic exposure represents a unique phenomenon in toxicology because of the ubiquity of human exposure and its continuous, cumulative nature [1,3,5] . Cadmium is widely dissipated in the environment due to (a) pollution from various industrial processes such as ore smelting and burning of coal, oil, and plastics [ 1,3] and (b) the fact that it is not recycled in most of its applications [3,4]. This environmental presence is translated to human exposure mainly through foodstuffs [ 1,3] . The average diet in the United States contains 30-180 c(g daily [3] ,3-8% of which is retained [1,3-51. Since biological turnover is very low [ 1,3,6] , cadmium accumulates in the body. An adult may have a body burden of 30 mg [I ,4,5] . Most cadmium is found in the liver and kidneys [ 1,5] , which appear to be exceptionally competent in their ability to produce a specific cadmium-binding protein called thionein [ 1,2,4,6] . Such bound cadmium is relatively nontoxic [1,4,6] . Kidney failure does not occur until the concentration of thionein-bound cadmium in the kidney cortical cells reaches 200 ppm (2 mM) [ 1,4] . Since the normal adult content is one-fourth to onesixth this level [ 1,4] , the margin of safety is low and

Mengovirus replication: III. Virus reproduction in Chinese hamster ovary cells

Abstract Following infection of Chinese hamster ovary cells with Mengovirus there is an initial, rapid reduction in the rates of synthesis of RNA and protein, followed by an increase in both synthetic rates at the time of virus maturation. The time required for release of virus is approximately 6 times as long in hamster cells as in L cells, and the yield of infective virus from hamster cells is only 20% of that from L cells.

Trans-acting factors in chromosomal instability.

The hypothesis that trans-acting factors affect chromosome stability was explored using human X Chinese hamster somatic cell hybrids. Two types of hybrids were examined. In either case, the human parent consisted of human diploid fibroblasts, the chromosomes of which tended to be lost from the hybrid cell. Comparisons were made between hybrid clones in which the hamster parent had a very stable karyotype (line CHO) and clones from a hamster parent with an unusual ongoing unstable karyotype (line CHX). Chinese hamster-human hybrid cell clones were expanded, and metaphase spreads were analyzed with an in situ hybridization procedure that uses biotin-labeled human genomic DNA as probe. Analyses of chromosome numbers and interspecies translocations were made after 20, 60, and 100 population doublings. Throughout the experiments, the generation of human-hamster-translocated chromosomes was more frequent in the hybrid cells with the CHX background. In addition, these cells also generated human acentric fragments, which were rare in cells with the CHO background. These results favor explanations for the instability of the CHX line that involve ongoing production of a diffusible clastogenic factor.