Single-Cell Mutation Monitoring Systems

1. Somatic-Cell Mutation Monitoring System Based on Human Hemoglobin Mutants.- 1. Introduction.- 1.1. The Approach.- 1.2. Previous Studies.- 1.3. Requirements of a Red Cell Screening System.- 2. The Hemoglobin Mutants.- 2.1. Hemoglobin Loci.- 2.2. Types of Mutations.- 3. Hemoglobin in Mutation Research: Gametal Mutation Rates.- 3.1. Indirect Estimates.- 3.2. Direct Estimates.- 4. A System for Detecting Somatic Mutations of Hemoglobin.- 4.1. Appropriate Mutants.- 4.2. Immunochemical Detection of Abnormal Hemoglobins in Single Cells.- 4.3. Detection of Rare Mutant Red Cells by Fluorescent Microscopy.- 4.4. Screening for "S Cells" or "C Cells" in Blood of Genetically A/A Subjects.- 4.5. Minimum Frequencies of Somatic Mutations at Globin-Chain Loci.- 4.6. Relationship between Somatic Mutation Frequencies and Gametal Mutation Rates.- 4.7. Relationship between Frequencies of Somatic-Cell Mutants and Compartments at Which Mutations Occur.- 5. Methodological Aspects: Monospecific Anti-Mutant-Hemoglobin Antibodies.- 5.1. Immunizations.- 5.2. Sepharose-Hb.- 5.3. Purification.- 5.4. Red Cell Labeling.- 6. Methodological Aspects: Monoclonal Anti-Globin-Chain Antibodies.- 6.1. Immunizations.- 6.2. Screening.- 6.3. Semiquantitative Assessment of Ab-Hb Binding.- 6.4. Mapping the Sites of Ab-Hb Binding.- 6.5. Possible Recognition of Mutant Hemoglobins in Animals.- References.- 2. Use of Fluorescence-Activated Cell Sorter for Screening Mutant Cells.- 1. Introduction.- 2. Immunologic Identification and Flow Detection of Erythrocytes Containing Amino Acid-Substituted Hemoglobin.- 2.1. Production of Antibodies.- 2.2. Suspension Labeling of Red Cells with Hemoglobin Antibodies.- 2.3. Flow Cytometric Processing.- 2.4. Results Using Hemoglobin S- and C-Specific Antibodies.- 3. Future of the Hemoglobin-Based Assay.- 4. Detection of Erythrocytes with Mutationally Altered Glycophorin A.- 4.1. Background.- 4.2. Gene Expression Loss Variants.- 4.3. Single-Amino Acid-Substitution Variants.- 5. Summary and Conclusions.- References.- 3. Development of a Plaque Assay for the Detection of Red Blood Cells Carrying Abnormal or Mutant Hemoglobins.- 1. Introduction.- 2. Principle of the Method.- 3. Reagents.- 3.1. Anti-Mouse RBC Ghost Sera.- 3.2. Anti-Mouse Hb Antibodies.- 3.3. Indicator Cells. Methods for Coupling Antibodies to Sheep RBC.- 3.4. Complement.- 4. Equipment.- 4.1. Plaque Chambers.- 4.2. Additional Materials.- 5. Procedure for the RBC-Antibody Plaque Assay.- 5.1. Factors Affecting the RBC Plaque Formation.- 5.2. Specificity of the RBC Plaque Assay.- 6. RBC-Protein A Plaque Assay.- 7. Conclusions.- References.- 4. Direct Assay by Autoradiography for 6-Thioguanine-Resistant Lymphocytes in Human Peripheral Blood.- 1. Introduction.- 1.1. Human Mutagenicity Monitoring.- 1.2. 6-Thioguanine-Resistant (TGr) Human Peripheral Blood T Lymphocytes (T-PBLs).- 1.3. Direct Enumeration of TGr T-PBLs by Autoradiography.- 1.4. Phenocopies.- 2. Autoradiographic TGr T-PBL Assay Method.- 2.1. Cell Preparation.- 2.2. Cryopreservation.- 2.3. Cell Culture.- 2.4. Termination, Coverslip Preparation, and Autoradiography.- 2.5. Enumeration of TGr T-PBLs and Calculation of TGr T-PBL Variant Frequency (Vf).- 3. Sample Results.- 3.1. TGr T-PBL VfAssay: Appearance of Slides.- 3.2. TGr T-PBL VfAssay: Sample Data.- 4. Statistical Analysis Methods.- 4.1. Notation and Basic Assumptions.- 4.2. Confidence Intervals for a Single Variant Frequency.- 4.3. Confidence Intervals for Ratios of Variant Frequencies.- 4.4. Sample Size Determinations.- 5. Discussion.- References.- 5. Application of Antibodies to 5-Bromodeoxyuridine for the Detection of Cells of Rare Genotype.- 1. Introduction.- 1.1. Flow Cytometry.- 1.2. The Use of 5-Bromodeoxyuridine for the Detection of Cell Proliferation.- 2. Materials and Methods.- 2.1. Cell Culture.- 2.2. Labeling with BrdUrd.- 2.3. Immunological Methods.- 2.4. Flow Cytometry.- 2.5. Hybridoma Production.- 3. Results.- 3.1. Determination of Antibody Specificity.- 3.2. Flow Cytometric Method for Immunofluorescent Detection of DNA Replication.- 3.3. Application of the Flow Immunofluorometric Anti-BrdUrd Technique to the Assessment of DNA Damage.- 3.4. Reconstruction Experiments for the Detection of Thioguanine-Resistant Variants by the Immunofluorescent Anti-BrdUrd Method.- 3.5. Preparation of Monoclonal Antibodies against 5-Bromodeoxyuridine or 5-Iododeoxyuridine.- 4. Discussion.- 5. Summary.- References.- 6. Cytogenetic Abnormalities as an Indicator of Mutagenic Exposure.- 1. Introduction.- 2. Lymphocyte Assay Methodology.- 2.1. In Vitro Cultures.- 2.2. Sampling Time.- 2.3. Fixation and Slide Preparation.- 2.4. Analysis of Cells.- 3. Chromosome Aberration Analysis Following Radiation or Chemical Exposure.- 3.1. The Sensitivity of the Lymphocyte Assay.- 3.2. Background Aberration Frequencies and "Matched" Control Groups.- 4. The Analysis of Bone Marrow Samples.- 5. The Plausibility of Estimating Genetic or Carcinogenic Risk from Aberration Frequencies in Lymphocytes.- 6. Concluding Remarks.- References.- 7. Sister Chromatid Exchange Analysis in Lymphocytes.- 1. Introduction.- 2. Methodology.- 2.1. Background.- 2.2. General Design Considerations.- 2.3. Technical.- 3. Selected Applications.- Appendix A. A Procedure for Growing and Preparing Human Lymphocytes for SCE Analysis.- Appendix B. A Procedure for Growing and Preparing Rat Lymphocytes for SCE Analysis.- References.- 8. Unscheduled DNA Synthesis as an Indication of Genotoxic Exposure.- 1. Introduction.- 2. Four Laboratory Approaches to Measuring Unscheduled DNA Synthesis.- 2.1. Liquid Scintillation Counting Measurements of UDS in Human Fibroblast DNA.- 2.2. Autoradiographic Measurements of UDS in Human Diploid Fibroblasts.- 2.3. Autoradiographic Measurements of UDS in Primary Cultures of Rat Hepatocytes.- 2.4. Measurements of UDS in Hepatocytes following in Vivo Treatment.- 3. Methods.- 3.1. Procedures for LSC UDS Assays.- 3.2. Establishment of Primary Cultures of Rat Hepatocytes from Treated or Untreated Animals.- 3.3. The in Vivo Rat Hepatocyte UDS Assay.- 3.4. Autoradiography.- 4. Reagents, Solutions, Stains, and Media.- 4.1. Reagents.- 4.2. Solutions and Stains.- 4.3. Media.- 5. Equipment and Supplies.- 5.1. Balances.- 5.2. Calculators.- 5.3. Centrifuges.- 5.4. Computer Equipment.- 5.5. Filters.- 5.6. Grain Counters.- 5.7. Incubators and Related Apparatus.- 5.8. Laminar-Flow Hoods.- 5.9. Microscopes.- 5.10. Mixer.- 5.11. Pipetting Apparatus.- 5.12. Pump Apparatus.- 5.13. Rocker Platform.- 5.14. Scintillation Counter.- 5.15. Spectrophotometer.- 5.16. Tissue Culture Supplies.- 5.17. Water Baths.- 5.18. Water System.- 5.19. Miscellaneous Equipment.- 5.20. Miscellaneous Supplies.- References.- 9. The Micronucleus Test as an Indicator of Mutagenic Exposure.- 1. Historical Background.- 2. Rationale of the Test System.- 3. Technical Procedure.- 3.1. Animals.- 3.2. Administration of the Test Substance.- 3.3. Determination of Dosage.- 3.4. Sampling Times.- 3.5. Preparation of the Bone Marrow Smears.- 3.6. Scoring of Micronuclei.- 3.7. Data Evaluation.- 3.8. Other Useful Information on the Micronucleus Test.- 3.9. Manpower and Costs.- 4. Results and Comparative Studies.- 5. Related Assay Systems.- 6. Advantages.- 7. Limitations.- 8. Application of the Micronucelus Test.- References.- 10. The Identification of Somatic Mutations in Immunoglobulin Expression and Structure.- 1. Introduction.- 2. Immunoglobulin Protein and Gene Structure.- 3. Methods for the Isolation of Mutants.- 3.1. Mutagenesis.- 3.2. Screening Techniques.- 3.3. Selective Techniques.- 4. Frequency and Phenotypes of Mutants.- 4.1. Frameshift Mutants.- 4.2. Point Mutants.- 4.3. Internal Deletion Mutants Associated with Changes in DNA or RNA.- 4.4. Class- and Subclass-Switch Mutants.- 5. Discussion.- References.- 11. Detection of Chemically Induced Y-Chromosomal Nondisjunction in Human Spermatozoa.- 1. Introduction.- 2. Background.- 3. Agents That Increase YFF Bodies in Human Sperm.- 3.1. Methodology.- 3.2. Results to Date.- 4. Discussion.- References.