Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.     

Synergism between cysteine and alkylating agents

Contrary to expectation, l-cysteine did not protect Escherichia coli from the lethal action of two monofunctional alkylating agents (nitrosomethylurethane and methylmethane sulfonate). The antibacterial action of these compounds was actually greatly enhanced by l-cysteine. This synergistic effect was also exhibited, to some extent, by d-cysteine but not by homocysteine, S-methylcysteine, or serine. The synergistic action between methylating agents and l-cysteine was not due to the formation of S-methylcysteine. l-Cysteine had no effect on the bacteriostatic action of ethylmethane sulfonate.     

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.     

Glycidol-carbohydrate hybrids: a new family of DNA alkylating agents

Novel and chiral glycidol-carbohydrate hybrids possessing an epoxy group as a DNA alkylating moiety were designed and synthesized. These artificial hybrids selectively alkylated DNA at the N-7 sites of the guanines and cleaved DNA without any additives. The binding ability of the glycidol was significantly enhanced by the attachment of the carbohydrate.     

Unusual response to bifunctional alkylating agents in a case of Fanconi anaemia

Summary Chromosomal breakage frequencies were determined in Fanconi anaemia (FA) blood cultures treated with various concentrations of the polyfunctional alkylating agents mitomycin C, diepoxybutane, and cis-platinum(II)-diammine-dichloride, for which FA cells have a characteristic hypersensitivity. At concentrations that hardly affected control cultures, three out of four patients tested exhibited a concentration-dependent increase of cells with aberrant chromosomes, with a concomitant increase in the number of chromosomal aberrations per aberrant cell. The fourth patient, a 22-year-old male, was exceptional because with all three clastogens only 40% of his cultured cells exhibited a typical concentration-dependent response, while 60% of his cells responded like those from normal healthy controls. The possible nature and significance of this unusual response is discussed.