The 24 human chromosome types of a normal diploid fibroblast cell strain were classified into 15 groups by high-resolution flow cytometry on the basis of 33258 Hoechst fluorescence. Chromosomes associated with each group were flow sorted onto microscope slides and identified by quinacrine banding analysis. DNA cytophotometry of metaphase chromosomes from the same cell strain supported and extended this identification. Four of the groups purified were due to chromosomes of a single type-namely, chromosomes 5,6, 13, and 17. Eight additional groups were also separated and found to contain the following chromosomes: 1 and 2; 3 and 4; 7, 8, and X; 9-12; 14 and 15; 16 and 18; 20 and Y; and 19,21, and 22. The average purity for the 12 sorted fractions was 78%.Flow cytometry of isolated chromosomes is a new approach to cytogenetics that provides rapid measurement of individual metaphase chromosomes. In this approach, chromosomes that are stained in aqueous suspension with an appropriate fluorochrome are constrained to flow at high speed through a narrow laser beam that excites the stain. The emitted fluorescence is measured photometrically and the accumulated data form a frequency distribution of chromosome fluorescence. The peaks of this frequency distribution are due to individual chromosomes or groups of chromosomes of similar fluorescence; the peak mean is proportional to chromosome fluorescence and the peak area is proportional to the chromosome frequency of occurrence. Thus, the frequency distribution serves as a karyotype (1, 2). In addition, flow sorting can be used to separate chromosomes on the basis of their staining properties (3, 4), in contrast to conventional methods for purifying metaphase chromosomes that rely upon velocity or isopycnic sedimentation, zonal centrifugation, or selective filtration (5). Purification of individual metaphase chromosomes is important for several reasons. Enriched or pure chromosome fractions have been analyzed biochemically to provide information on the structure of DNA or protein (6), to transfer genetic information to whole cells (7-9), or to map genes by in vitro hybridization (10). In general, however, conventional techniques have not been able to provide chromosomes of sufficient purity for high-resolution biological or biochemical studies.By flow sorting on the basis of ethidium bromide fluorescence, we have separated, with a purity of 90%, each chromosome of the male deer Muntiocus muntjak (2n = 7) (4) and the 14 chromosome types of the Chinese hamster M3-1 cell line into eight chromosome groups (1, 3). In our previous studies with ethidium bromide-stained human chromosomes, we resolved only eight chromosome groups from the 24 chromosome types of the male (2n = 46) (2, 3). In the present study, using the DNA fluorochrome 33258 Hoechst and improved instrumentation, we resolved 15 chromosome groups in the human male and, by sorting, purified chromosomes 5, 6, 13, and 17 and eight additional groups composed of chromosomes of more than one type. MATERIALS AND M...

The frequency of thioguanine(TG)-resistant lymphocytes in mice treated with ethylnitrosourea (ENU) was followed for a period of 51 wk using our clonogenic assay [Jones et al, 1985a,b]. The effects of dose (0-58 mg/kg), time since treatment (2-51 wk), dose rate (5 weekly X 11.7 mg/kg versus 1 X 58 mg/kg), and age at time of treatment (3 vs 15 mo) on the frequency of TG-resistant, concanavalin A-responsive spleen cells were evaluated. The frequencies of TG-resistant spleen cells were generally dose responsive for 51 wk after exposure to ENU. They also were dependent upon the time that had elapsed since treatment with ENU, increasing to maximal values at 10 wk as previously reported [Jones et al, 1985a], and holding essentially stable at values of approximately 20% of the maximum frequency from week 15 until at least week 40 for the 3-month-old mice. Fractionation of 58 mg ENU/kg into 5 weekly doses did not affect the frequency of ENU-induced TG-resistant cells detected in the spleen but did increase the rate of appearance in the spleen, and the efficiency of induction by the unit dose, of TG-resistant cells. The mice exposed to ENU at 15 mo of age appeared to have a 4-fold reduction in the rate of increase in frequency of ENU-induced TG-resistant spleen cells. One set of control mice was found to have a 10-fold elevated frequency of TG-resistant cells in both the spleen and thymus, indicating that mutations can occur in stem cells of untreated animals.

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A UV-sensitive mutant line of CHO cells, UV20, was shown to be phenotypically corrected to resistance by fusion with human lymphocytes or fibroblasts. Only human chromosome 19 correlated with the DNA repair phenotype of resistant hybrid clones and their resistant or sensitive subclones. This study demonstrates the mapping of a human repair gene by direct selection of complementing hybrids in the presence of a DNA-damaging agent (mitomycin C).

Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescenceactivated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the a-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the fl-, y-, and b-globin genes. Translocations were then used to localize further the fl-, 'y-, and b-globin genes. The first translocation t(11;22Xq25;qll), which moved nearly all of chromosome 11 to a different peak, confirmed that the ft-, y-, and 5-globin genes are on this chromosome. The second, t(4; I1Xq25;ql3), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;llXqll;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the fi-, y-, and 6-globin genes. Therefore, the ft-, 7a-, and 6-globin genes reside on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.Adult human hemoglobin molecules are principally composed of hemoglobin A (af22) with a minor hemoglobin A2 (a262) component; between 3 and 9 months' gestation, the hemoglobin is principally hemoglobin F (a°272) (1). Numerous pedigree analyses first indicated that the a-and/3-globin genes are not linked (2, 3), and more recent somatic cell hybridization experiments have described the independent chromosomal segregation of the a-and (3-globin genes (4). In contrast, the /-, y-, and 6-globin genes were first considered to be linked after the discovery of the gene fusion products designated the Lepore (5) and Kenya (6, 7) hemoglobins. Recently, the close linkage of these genes has been defined by restriction endonuclease mapping (8-10) and the isolation of cloned globin genes (11). Different laboratories have tried to localize the globin genes by in situ hybridization (12-18) but, because of the specific activity of the probe, the results are open to controversy (19,20). At the same time, somatic cell hybridization studies indicated that the human a-globin gene is on chromosome 16 and the human d-and y-globin genes are on chromosome 11 (21-23). Gene mapping using partially purified chicken chromosomes prepared by zonal centrifugation has been reported (24, 25).Herein we report the results of gene mapping by sorting normal (26) and translocated human chromosomes and analyzing the extracted DNA by restriction endonuclease digestion (27). Data obtained by this procedure indicate that the /3-, y-, and 6-globin genes are located on the distal portion of the short arm of chromosome 11. This method may be applied generally to assign other genes to chromosome segments. MATERIALS AND METHODSChromosome Preparation and Sorting. The Human Genetic Mutant Cell Reposito...