'ABSTRACT Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow.karyotyping). Inthis paper we investigate the sources and magnitudes ofvariability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the-impact ofthis variability on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocytes from 11) individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability-was sufficiently small that it did not interfere with-classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was .generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karyotypes.Recent advances in the use of flow cytometry for analyzing metaphase chromosomes suggest that this approach may be well suited for quantitative karyotyping ofhuman chromosomes (1, 2). Conventional methods of karyotyping, based on visual analysis of banded metaphase chromosomes, have provided powerful tools for classifying individual chromosome types and for identifying chromosome rearrangements or aneuploidy associated with genetic disorders (3, 4). However, quantitative interpretation of banded karyotypes can be limited by cell-to-cell variability, in chromosome condensation and staining characteristics. Thus, it may be difficult to determine whether a band is truly missing or simply-not visible in the preparation. The subjective nature of banded karyotype analysis also potentially complicates interlaboratory comparisons of the size -or staining characteristics of specific lesions or polymorphisms.In flow cytometry, isolated chromosomes suspended in a fluorescent stain solution flow one at a time through a laser beam at rates of up to LOOO chromosomes per sec. The fluorescence signals resulting from laser excitation are measured for the chromosomes yielding a frequency distribution of chromosomal fluorescence. Flow cytometry has a number of advantages over -microscopic methods for quantitative analysis of-chromosomes. Chromosomes are suspended at thermodynamic equilibrium with the stain, thereby minimizing chromosome-to-chromosome staining variability. Because of the large number of chromosomes analyzed in each experiment, flow analysis provides high-precision population averages that are insensitive to cellto-cell variations in chromosome condensation. Stain combinations can be utilized to discriminate between chromosome types based on cytochemical staining characteristics and DNA content.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that

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...

Chromosomes were isolated from a variety of human cell types using a HEPES-buffered hypotonic solution (pH 8.0) containing KCl, MgS04, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. Highquality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high-quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.

An H4-IIE-C3 hepatoma cell line derived from an ACI rat has been shown to have differentially stained regions attached to the short arms of chromosomes 3, 11 and 13 and the long arm of an unidentified small chromosome. There is cell to cell variability in the number and size of the differentially stained regions, which contain, on the average, about 5% of the total DNA. A series of secondary constrictions occur at intervals along the length of each differentially stained region. These stain with silver by the Ag-AS method, indicating that the differentially stained regions contain sites of active 45S ribosomal precursor RNA transcription. In situ hybridization to metaphase chromosomes shows that the hepatoma cells have a 10 fold increase in DNA coding for 18S and 28S ribosomal RNA, 90% of it located in the differentially stained regions, and no change in the number of genes coding for 5S RNA. These results have been confirmed by filter disc hybridization.