Richard L. Davidson

Chapter 18 Induction of Mammalian Somatic Cell Hybridization by Polyethylene Glycol1

Publisher Summary This chapter discusses the induction of mammalian somatic cell hybridization by polyethylene glycol (PEG). The hybridization of somatic cells is a technique for the study of the organization and regulation of the mammalian genome. PEG induces mammalian cell hybridization and procedures have been developed for treating cells with PEG that are simple, rapid, and effective. The chapter outlines experiments to optimize the conditions of PEG-induced hybridization of cells attached in monolayers. Among the parameters considered were: (1) PEG molecular weight, (2) PEG concentration, (3) cell density, (4) pH, (5) length of exposure to PEG, (6) temperature, (7) volume of residual medium at the time of PEG addition, and (8) the method of removing the PEG. The experience with PEG-induced hybridization of cells in suspension is less extensive than with cells in monolayer; however a simple procedure giving consistent success with certain cell combinations has been developed. Although only a few variables have been examined for hybridization in suspension, it seems apparent that the principles established with monolayer fusions cannot be extrapolated to suspension fusions without some modification.

Lateral asymmetry in the fluorescence of human Y chromosomes stained with 33 258 Hoechst

Abstract A difference in 33 258 Hoechst fluorescence intensity between sister chromatids is observed in the distal region of the long arm of Y chromosomes from human lymphocytes grown one replication cycle in medium containing 5-bromodeoxyuridine. This difference, which appears limited to the region exhibiting intense quinacrine-mustard fluorescence, is compatible with an unequal distribution of thymine residues between the two polynucleotide chains of the DNA in this Y chromosome region.

Regulation of gene expression in somatic cell hybrids: A review

Hybridization of somatic mammahan cells in vitro was first observed in 1960 (1), at a time when experiments on the control of enzyme synthesis in bacteria were demonstrating the importance of recombinational systems for the analysis of genetic regulatory mechanisms (2). The discovery of ~ recombinational system for mammalian cells therefore suggested a new approach to the investigation of the mechanisms of the regulation of gene expression in these cells. This approach involves the combination in a hybrid of the genomes of two cells which differ in various functions and the analysis of the mechanisms underlying the differences in terms of interactions between the genomes in the hybrids. Analyses of this type have been performed on hybrids between cells of different species, between differentiated and undifferentiated cells, and between mutant and normal cells. The results of hybridizing cells in such combinations are described in this review.

Inhibition of herpes simplex virus transformed and nontransformed cells by acycloguanosine: Mechanisms of uptake and toxicity

Abstract the toxic effects of acycloguanosine (acGuo), a potent inhibitor of herpes simplex virus (HSV) replication, were analyzed in HSV-transformed and nontransformed cells of different mammalian species. Large inter- and intraspecies variations in acGuo sensitivity were observed. In the transformed cells, the expression of the HSV thymidine kinase (TK) activity increased acGuo sensitivity by at least 10-fold, and this enhanced sensitivity was associated with a 10-fold increase in the uptake and phosphorylation of acGuo to its triphosphate (acGTP). In contrast, in the nontransformed cells, the presence or absence of the cellular TK activity did not have a significant effect on acGuo sensitivity. However, deoxyguanosine (dGuo) reversed the inhibitory effects of aeGuo on nontransformed cells, in association with decreased formation of acGTP. Thus, the major pathway for uptake and phosphorylation of acGuo in nontransformed cells does not involve TK activity, as it does in HSV transformed cells. Instead, it appears that the pathway for acGuo metabolism in nontransformed cells shares a common step with the pathway for dGuo uptake and phosphorylation. This cellular pathway for acGuo metabolism could determine the acGuo sensitivity of viruses other than HSV.

Novel phenotypes arising from selection of hamster melanoma cells for resistance to BUdR.

Abstract Pigmented clones of the Syrian hamster melanoma line RPMI 3460 were selected for resistance to high levels of the thymidine analogue 5-bromodeoxyuridine (BUdR). The resistant lines obtained were characterized as to the percentage of thymidine residues in nuclear DNA replaced by BUdR (BUdR substitution), the level of in vitro thymidine kinase (EC 2.7.1.75) (TK) activity the ability to utilize exogenous thymidine for growth, and pigmentation. In contrast to the BUdR resistant cells generally reported in the literature, most of the lines isolated in this study have large amounts of BUdR in their nuclear DNA and/or high levels of TK activity. The ability of the cells to utilize exogenous thymidine was found to correlate well with the level of BUdR substitution. However, there was a definite lack of correlation between the level of in vitro TK activity and the level of BUdR-substitution. Further, the phenotypes of the cells selected in 10−4 M BUdR appear to be to some extent clonally determined.

Deoxycytidine reverses the suppression of pigmentation caused by 5-BrdUrd without changing the amount of 5-BrdUrd in DNA.

Abstract 5-Bromodeoxyuridine (BrdUrd) suppresses pigmentation in Syrian hamster melanoma cells in vitro, and this suppression is reversed by the addition of deoxycytidine (dCyd) to the culture medium. Exogenous dCyd also decreases the incorporation of BrdUrd into nuclear DNA. Over a certain range of dCyd concentrations, however, increasing the concentration of dCyd does not lead to any significant further decrease in the level of BrdUrd incorporation. With 0.5 μM BrdUrd in the medium, increasing the dCyd concentration from 0.4 mM to 1.0 mM produces essentially no detectable change in the amount of BrdUrd incorporated. This is the range of dCyd concentrations, however, in which the addition of dCyd has the greatest effect in reversing the suppression of pigmentation caused by BrdUrd. The effects of dCyd on pigmentation are prevented by aminopterin, which blocks the conversion of dCyd to thymidine nucleotides. The results suggest that dCyd reverses the suppression of pigmentation caused by BrdUrd through a mechanism that does not involve changes in the amount of BrdUrd in nuclear DNA, and that the effect of dCyd on pigmentation depends upon the conversion of the exogenously supplied dCyd to thymidine nucleotides.

Membrane alterations associated with "transformation" by BUdR in BUdR-dependent cells. Fluorescence Polarization studies with a lipid probe.

Abstract Steady state and nanosecond fluorescence polarization studies were carried out on membranes of a “bromodeoxyuridine (BUdR) dependent” cell line (B4) derived from a malignant Syrian hamster melanoma line. When grown in the presence of BUdR B4 cells resemble transformed cells (in terms of several biological characteristics), while B4 cells grown in the absence of BUdR resemble untransformed cells. B4 cells were labelled with the lipid probe 1,6-diphenyl-1,3,5-hexatriene, which had been used previously to show that fluorescence polarization values of membrane lipids of virally transformed cells are higher than fluorescence polarization values of membrane lipids of untransformed cells. The steady state fluorescence polarization values of membrane lipids of B4 cells in BUdR were found to be larger than those of cells in the absence of BUdR, and the change in fluorescence polarization values was found to be fully reversible. Nsec rotational correlation time experiments confirmed and extended the steady state results. The results of the fluorescence polarization studies suggest that the membranes of B4 cells grown in the presence of BUdR resemble those of virally transformed cells while membranes of B4 cells grown in the absence of BUdR resemble those of untransformed cells.

Differences in the order of termination of DNA replication in human chromosomes in peripheral blood lymphocytes and skin fibroblasts from the same individual.

A comparison of the sequence of terminal replication of DNA in human chromosomes has been made between skin fibroblasts and peripheral blood lymphocytes from the same adult male individual. The chromosomes were identified by quinacrine-banding, and the order of termination of replication was determined by autoradiography. Chromosomes 4, 5, 8, and 13 were found to terminate DNA replication later in lymphocytes than in fibroblasts, wheres chromosomes 15, 19, 20, and 22 were found to terminate their replication earlier in lymphocytes than in fibroblasts.

Identification of human and mouse chromosomes in human-mouse hybrids by centromere fluorescence

Abstract Differences between the centromeric regions of mouse and human chromosomes have been revealed with a staining technique which is based on the quenching by 5-bromodeoxyuridine of the fluorescence of the dye 33258 Hoechst. The differences can be used to distinguish between human and mouse chromosomes in human-mouse hybrids.

33258 Hoechst enhances the selectivity of the bromodeoxyuridine-light method of isolating conditional lethal mutants

Summary 33258 Hoechst enhances the photosensitivity of bromodeoxyuridine-(BUdR)-substituted cells. Reconstruction experiments demonstrate that this enhanced photosensitivity can be used to improve the selectivity of the BUdR-light method of isolating conditional lethal mammalian cell mutants.