The objective of this proposal is the development of a mutagenesis assay system in human cell line that will provide, ultimately, the capability to analyze mutational events at the level of the DNA sequence. Prokaryotic systems measuring the fidelity of DNA replication on synthetic polymers, or single stranded plasmids/phage, containing chemically-altered nucleosides or quantitating specific locus mutagenesis in plasmid/phage/bacterial genomes have successfully predicted which chemicals will be mutagenic in mammalian cells. However, the prokaryotic systems are of unknown value for predicting the mechanisms by which mammalian cells replicate a segment of DNA containing a site of chemical, the actual mutational event. The studies proposed will validate a new mutational assay with the lambda immunoglobulin gene in a human lymphoblastoid cell as the target DNA. The lambda gene was evaluated as a potential target for a mutational assay because the gene product has a neutral survival value for the cell and because clones containing mutational events in the lambda gene can be selected by an isoelectric focusing analysis (IEF). These two criteria assure there are no inherent biases in the selection of the widest possible diversity of mutagenic events. The initial analysis of clones from chemically-exposed populations identifies three classes of mutational events; isoelectric variants (point mutations within the coding region of the gene), clones with the specific mRNA but lacking an immunologically detectable gene product (point mutations affecting translation), or clones lacking the mRNA (terminal deletion). Evaluation of over 3000 control clones has not identified a single phenotypic mutant. In contrast, a mutation rate (combination of isoelectric variants plus those not producing the lambda protein) of approximately 2% was observed in clones from populations exposed to MNNG. This assay rapidly selects a wide diversity of mutational events and provides the mutant clones for subsequent analysis at the DNA sequence level pending the development of these analytical protocols. In addition, a portion of these clones will represent a panel of mutants deficient in the individual steps of protein synthesis, a powerful group of probes for studying protein synthesis in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES003813-01
Application #
3251524
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1985-03-01
Project End
1988-02-29
Budget Start
1985-03-01
Budget End
1986-02-28
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
McFarland, R D; Smith, G J (1995) Novel electrophoretic protocol for collection of mutations in the lambda light chain immunoglobulin gene in a human B-lymphoblastoid cell strain. Teratog Carcinog Mutagen 15:43-51
Black, K A; Novicki, D L; Vincent, J L et al. (1993) Flow cytometric analysis of xenobiotic metabolism activity in isolated rat hepatocytes. Cytometry 14:334-8
McFarland, R D; Vincent, J L; Smith, G J (1992) Isolation by fluorescence-activated cell sorting of cells of a human lymphoblastoid cell strain containing mutations in the lambda immunoglobulin gene. Environ Mol Mutagen 19:297-303
Black, K A; McFarland, R D; Grisham, J W et al. (1989) S-phase block and cell death in human lymphoblasts exposed to benzo[a]pyrene diol epoxide or N-acetoxy-2-acetylaminofluorene. Toxicol Appl Pharmacol 97:463-72
Black, K A; McFarland, R D; Grisham, J W et al. (1989) Cell cycle perturbation and cell death after exposure of a human lymphoblastoid cell strain to N-methyl-N'-nitro-N-nitrosoguanidine. Am J Pathol 134:53-61
Zirker, T A; Baybick, J H; Vincent Jr, J L et al. (1988) Flow cytometric analysis of DNA ploidy in lymphomas of the thyroid. Head Neck Surg 10:324-9
Smith, G J; Grisham, J W; Bentley, K S (1988) Mutagenic potency at the Na+/K+ ATPase locus correlates with cycle-dependent killing of 10T1/2 cells. Environ Mol Mutagen 12:299-309