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DNA content and base composition of human chromosomes
DNA Content and Base Composition of Human Chromosomes *,1 MARTY F. BARTHOLDI Experimental Pathology Group, Mail Stop M888, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received October 10, 1984; accepted December 3, 1984 The relative DNA content and base composition of the 24 human chromosomes have been measured by flow cytometry. Isolated human metaphase chromosomes flowing through a finely focused laser beam were analyzed singly at a rate of 200 per s. The relative chromosomal fluorescence of the DNA specific dye propidium iodide correlated strongly (r = 0.99) with the DNA-based human karyotype determined by image cytometry. The relative fluorescence of chromosomes stained with the dye combination Hoechst 33258 (AT binding specificity) and chromomycin A3 (GC binding specificity) was compared with independent measurements of chromosomal base composition. The results correlated positively with quinacrine brightness (r = 0.88) and autoradiographic determination of base-specific radioactive DNA precursor incorporation (r = 0.55). The larger chromosomes were found to have a higher AT content than the average base composition of total human genomic DNA and the smaller chromosomes a higher GC content. Hoechst-chromomycin fluorescence depends primarily on chromosomal variation in base composition, but may also be influenced by chromosomespecific classes of repetitive DNA. © 1985AcademicPress,Inc. INTRODUCTION
Basic information on chromosome composition and structure can be obtained by flow cytometric analysis. Although chromosome analysis by flow cytometry has been used mostly as a technique to determine the flow karyotypes of individuals (1) and cell populations (2), or to purify chromosomes for molecular analysis (3), the variety of measurements possible on single chromosomes provides data on chromosome composition and structure at rates sufficient to analyze large numbers of chromosomes. Fluorescence of dyes with binding specificity to DNA has proven to be the most useful * This paper is dedicated to Dr. Milton Kerker on the occasion of his 65th birthday. The U.S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. The Los Alamos National Laboratory is operated by the University of California for the U. S. Department of Energy under Contract W-7405-ENG-36.
measurement for resolving single chromosome types. The purpose of this report is to evaluate the accuracy of fluorescence analysis by flow cytometry to determine chromosomal DNA content and base composition by correlation with independent measurements. Previous analysis of human chromosomal DNA content as measured by flow cytometry lacked precision (4, 5), and previous analysis of chromosomal base composition did not evaluate specific chromosome types (6), although similar overall correlations have been determined (7). In this study the relative fluorescence of propidium-iodide-stained human chromosomes measured by flow cytometry correlated positively (r -- 0.99) with the DNA-based human karyotype determined by image cytometry of gallocyanin-chrome-alum-stained chromosomes (8). Propidium iodide binds specifically by intercalation to DNA without base specificity (9). The strong positive correlation between the flow cytometric measurements and the DNA-based human 426
0021-9797/85 $3.00 Copyright © 1985 by Academic Press, Inc. All fights of reproduction in any form J~served.
Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
HUMAN CHROMOSOME COMPOSITION karyotype established the accuracy of propidium iodide fluorescence analysis by flow cytometry to measure DNA content. The fluorescence of Hoechst 33258- and chromomycin-A3-stained human chromosomes was analyzed by determining separately the percentage difference between the normalized Hoechst 33258 and the normalized chromomycin A3 fluorescence for each chromosome from the chromosome DNA contents tabulated in the DNA-based human karyotype (8). These "fluorescence shifts" were then reduced to deviations in chromosomal base content from the average total human genomic base composition of 60.0% AT content (11). The bisbenzimidazol derivative, Hoechst 33258, binds externally to DNA at three consecutive A- T base pairs (A = adenine, T = thymine) (10). The antitumor antibiotic chromomycin A 3 also binds externally to DNA and appears to require three G - C (G = guanine, C = cytosine) base pairs at the binding site (6). Hoechst and chromomycin fluorescence shifts from the tabulated chromosomal DNA contents (8) were found to be reciprocal for each chromosome; when Hoechst fluorescence was higher, the chromomycin fluorescence was lower than expected if these dyes depended only on DNA content. The fluorescence shifts were reduced to changes in base composition by taking into account the fact that the ratio of runs of three like base pairs will change more rapidly than the average base ratio changes (6). The fluorescence shifts are therefore enhanced for the dyes Hoechst 33258 and chromomycin A3 relative to the changes in chromosomal base composition. The percentage change in AT content from the total human genomic DNA base composition of 60% AT was estimated for each chromosome and compared with independent flow cytometric analysis of Hoechst-chromomycin-stained chromosomes (r = 0.94) (1), fluorescence microscopy of quinacrine-stained chromosomes (r = 0.88) (12), and autoradiography of base-specific
427
radioactive DNA precursor incorporation into chromosomes (r = 0.55) (13). Each method agreed in the finding that the larger chromosomes tend to be relatively high in AT content and the smaller chromosomes relatively high in GC content from the average genomic base ratio. Many factors, however, may alter the dependence of Hoechst-chromomycin fluorescence on simple variation in base composition. In particular, highly repetitive DNA may alter the distribution of short runs of like base sequences from that expected in the DNA of operationally random sequence enriched in AT content. The accuracy of base composition as determined by Hoechstchromomycin staining is therefore restricted to establishing AT or GC enrichment of the overall chromosomal base composition, but may be indicative of classes of repetitive DNA specific to one or a few chromosome types. MATERIALS AND METHODS Isolated metaphase chromosomes were prepared from four diploid human cell strains, two with the karyotype 46,XX and two with 46,XY. Cells in subconfluent cultures were blocked in the metaphase stage of mitosis with colcemid (3 X 10-7 M). Mitotic cells were harvested by shaking off, centrifuged at 150 g for 8 min, and the supernatant was removed. For staining with propidium iodide (2) the mitotic cells were resuspended in 1.0 ml of 75 m M KC1 and 50/zg/ml propidium iodide. The cells were allowed to swell for 10 rain at room temperature and then 0.5 ml of the following solution was added: 75 m M KC1, 0.1% Triton X-100, 50 #g/ml propidium iodide, and 1.0 mg/ml RNase. After 3 rain at room temperature the cells were forced through a 1½-in. 22-gauge syringe needle to disperse the chromosomes. The chromosome suspension was incubated for 30 min at 37°C to allow the RNase to degrade chromosomal RNA. For staining with Hoechst 33258 and chromomycin A3 the cells were resuspended in a dournal'ofColloid and Interface Science, Vot. 105, No. 2, June 1985
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MARTY
F. B A R T H O L D I
buffer (14) containing 15 m M Tris-HC1, 0.2 m M spermine, 0.5 m M spermidine, 2 m M EDTA, 0.5 m M EGTA, 80 mM KCI, 14 mM/3-mercaptoethanol, and 0.1% digitonin. The cell suspension was held on a Vortex mixer and agitated vigorously for 1 min to release the chromosomes. Chromomycin A3 was added to a final concentration of 10 t~M, and, after 2 h to allow chromomycin binding to reach equilibrium, Hoechst 33258 was added to a final concentration of 5 #M. The chromosome suspensions were stored at 4°C. FLOW
CYTOMETRY
Univariate analysis of propidium iodidestained chromosomes was done on an experimental flow cytometer (15, 16). Fluorescence is excited by a finely focused laser beam (1.5 W at 488 nm in a 4 X 27-tzm spot at the e-2 diameters) and collected at 90 ° relative to the direction of the laser beam. The illumination optics are a 10X, 0.4 numerical aperture objective partially filled by a toric lens of 90-mm diverging focal length and 630mm cylindrical focal length. The collection lens is a 40X, 0.75 numerical aperture objective coupled by glycerol to the square outer nozzle. The fluorescent object is brought into sharp focus at the image plane of the collection lens and then diverges before impinging on the photomultiplier tube. A colored glass filter with transmission above 560 nm blocks the scattered light. The flow chamber consists of three nozzles. The sample and inner sheath streams are injected into a second sheath contained in an outer square nozzle. The velocity of the sample stream is 7 m/s with a 2-#m diameter. The fluorescence pulse is amplified, integrated, and acquired with 10-bit resolution. A previous analysis of fluoresence measured on this flow cytometer (16) demonstrated that with high irradiance, saturation of the fluorescence transition and photobleaching occur. The coefficient of variation of fluorescence measurements from single chromosomes is between 1.5 and 1.0% (17) Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
due to an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories. Bivariate analysis of chromosomes stained with Hoechst and chromomycin was done on an EPICS V flow cytometer (Coulter Electronics, Inc., Hialeah, Fla.) equipped with two argon-ion lasers (18). A standard 76-t~mdiameter flow nozzle was used with stream in air illumination. The sample and sheath streams were cooled at 4°C. The two lasers were aligned with UV (200 mW at 351.1363.8 nm) excitation followed by excitation of 350 mW at 457.9 nm. Fluorescence was collected by a single photomultiplier tube with filters KV408 and GG495 that block light below 408 and 495 nm. A single photomultiplier tube was used for detection of the two fluorescence pulses by taking advantage of the fact that they occur separately in time by 7 #s. The signals were processed and acquired using the electronics provided with the EPICS V. Both integrated fluorescence pulses were recorded in bivariate histograms with 128 channels on each axis. The fluorescence signal measured after UV excitation consists of both the long-wavelength edge of the Hoechst fluorescence (emission maximum at 450 nm, but detected through a 495-nm longpass filter) and fluorescence from adjacently bound chromomycin A3 (emission maximum at 580 nm) excited by energy transfer from Hoechst 33258 (19, 20). Energy transfer is efficient over a distance of 2.5-3.5 nm corresponding to about 7-10 base pairs. After 457.9-nm excitation only total chromomycin A3 fluorescence is measured. The coefficients of variation of the fluorescence measurements were between 2 and 3%. RESULTS
Chromosomes from two human diploid cell strains were isolated and stained with propidium iodide. The univariate histograms
429
HUMAN CHROMOSOME COMPOSITION
of the number of chromosomes versus fluorescence intensity in Fig. 1 were measured on the experimental flow cytometer. The peaks in the histograms were identified with the autosomal chromosomes number 1-22 and the X and Y chromosomes (7). The histogram in the upper panel is from cells with the karyotype 46,XX and the peak identified with the X chromosome has the same number of counts as resolved autosome pairs. The histogram in the lower panel is 173
from cells with the karyotype 46,XY and the peak identified with X chromosomes contains only half the counts of the resolved autosomes. Most of the human chromosomes are separately resolved except for 1 and 2, 9-12, and 15 and 14. Note that certain autosomal homologs are also separately resolved, for example, chromosomes 13, 16, and 22 in each of the two cell strains analyzed here. The relative propidium iodide fluorescence
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FIG. 1. Univariate flow karyotypes of two human cell strains, 46,XX and 46,XY. Isolated metaphase chromosomes were stained with propidium iodide. More than 50,000 chromosomes were analyzed in each histogram. Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
430
M A R T Y F. B A R T H O L D I
measurements of the 24 human chromosomes from both cell strains are compared to the DNA-based human karyotype determined by image cytometry of gallocyanin-chromealum-stained chromosomes (8) in Table I. The fluorescence data (peak means in Fig. 1) were normalized so that the sum of the autosomal peak means, including homolog variants, was 100. The image cytometry data were also normalized to a sum of 100 for the autosomal measurements, but homolog variants were excluded. The DNA-based human karyotype was established from 16 unrelated donors. The correlation coefficient (r) between the two measurements was 0.99. The uncertainty
TABLE I H u m a n Chromosomal D N A Content Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
DNA-basedhuman karyotype a'b
46,XXax
46,XY~c
4.28 4.19 3.49 3.31 3.18 2.97 2.76 2.52 2.38 2.35 2.34 2.31 1.88 1.78 1.73 1.61 1.47 1.40 1.09 1.15 0.82 0.90 2.64 0.97
4.19 4.19 3.53 3.35 3.17 2.98 2.82 2.56 2.37 2.37 2.37 2.37 1.87, 1.94 1.79 1.68 1.59, 1.68 1.46 1.39 1.03 1.15 0.82 0.90, 0.75 2.68 --
4.19 4.19 3.54 3.32 3.16 3.02 2.81 2.57 2.36 2.36 2.36 2.36 1.89, 1.96 1.80 1.72 1.59, 1.69 1.48 1.32 1.08 1.17 0.82 0.95, 0.78 2.70 0.98
a Normalization to the s u m of 100 for autosomal measurements. b Measured by image cytometry ofgallocyanin--chromealum-stained chromosomes (8). c Measured by flow cytometry of propidium iodidestained chromosomes (Fig. 1). Journal of Colloid and Interface Science, Vol. 105 No. 2 June 1985
of the relative peak means in the flow cytometric determination is less than 1% and the uncertainty of the chromosome DNA content in the DNA-based human karyotype (8) is 2.5%. However, since each of the human chromosomes is not clearly resolved by flow cytometry a larger inaccuracy may occur as in, for example, chromosomes 1 and 2. Chromosomes from two additional dipoloid human cell strains were stained with Hoechst 33258 and chromomycin A3 and analyzed on the dual-laser EPICS V flow cytometer. The bivariate histograms are displayed in Fig. 2 as contour plots of the number of chromosomes with correlated Hoechst (UV-excited) and chromomycin A 3 (458-nm-excited) fluorescences. The chromosome type associated with each peak was identified by sorting onto microscope slides and classified by morphology. The normal human chromosomes, except the group 912, are clearly resolved. Again the difference between the 46,XX and 46,XY karyotype is readily detected. The UV-excited fluorescence and 458-nmexcited fluorescence were separately normalized to a sum of 100 for the autosomal measurements (peak means in Fig. 2). The percentage differences between the fluorescence values and the DNA-based human karyotype as determined by image cytometry are listed in Table II. Given that the base composition of total human genomic DNA is 60% AT and 40% GC (11), an equivalent analysis was done to normalize the UV-excited fluorescence to a sum of 60 and the 458-nm-excited fluorescence to a sum of 40. The respective percentage differences were calculated with 0.60 (the average AT content) and 0.40 (the average GC content) of the DNA-based human karyotype. The sum of both fluorescences normalized to base composition for each chromosome equaled the DNA content for each chromosome as determined by propidium iodide fluorescence. The enhancement of fluorescence shifts of base-specific dyes to changes in base corn-
431
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chromomycin fluorescence occurred for a 1% increase in AT content. The fluorescence percentage differences were adjusted by these factors in Table II to yield the corresponding change in chromosomal AT content for both Hoechst and chromomycin fluorescence shifts. The percentage change in AT content is based on a deviation from the 60.0% AT content of total genomic DNA (9). Four measurements of AT content change (UVexcited and 458-nm excited fluorescence shifts for each 46,XX and 46,XY cell strains) were averaged and added (or subtracted) from 60.0% in Table III. The standard deviations
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position (21) rests on the probability of occurrence of runs of sequential-like base pairs in DNA of a specific base ratio (6). Small changes in base composition result in larger fluorescence changes of dyes that require runs of identical base pairs for binding, for example both Hoechst and chromomycin. A detailed analysis (6) determined that a 4.7% increase in UV-excited fluorescence occurred for a 1% increase in AT content due to both binding site requirements and energy transfer, and that a 3.9% decrease in 458-nm-excited
Fluorescencepercentage difference°
Percentage AT base content change
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-2.2 -7.4 -18.6 -8.5 -6.4 -0.7 -4.1 +1.2 +2.9
+0.5 +1.3 +2.3 +0.9 +1.6 +0.8 +1.0 -0.7 -0.4
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-0.4 +11.9 -2.7 -4.8 -12.3 -21.5 +5.6 -36.2 -15.0 +5.4 -38.4 +7.9 .
+4.4 -16.0 -0.6 -0.6 +13.4 +24.6 -15.7 +33.3 +8.0 -17.1 +18.2 -11.4
-0.1 +2.4 -0.6 - 1.0 -2.5 -4.4 +1.1 -7.4 -3.1 +1.1 -7.7 +1.6 .
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a Percentage difference between normalized fluorescence for 46,XX cell strain. b UV-excited fluorescence divided by 4.7. c 458-nm-excited fluorescence divided by -3.9. Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
432
MARTY F. BARTHOLDI TABLE III Human Chromosomal Base Composition (Percentage AT Content)
Chromosome
Base ratio a
HoechstChromomyein fluorescence b'c
HoechstChromomycin fluoreseence b'a
Quinacrine fluorescence e
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
60.3 61.3 60.8 59.7 60.5 60.7 59.5 59.9 59.7 61.4 59.8 60.4 60.2 57.2 58.4 59.7 61.5 61.4 56.3 57.6 56.2 58.4 59.3 62.4
59.1 60.6 61.4 63.7 61.6 61.6 60.4 60.9 59.4 59.4 59.4 59.3 63.3 60.0 59.4 57.1 54.5 62.6 51.9 57.6 55.9 54.1 62.0 76.5
59.7 60.7 61.3 62.6 61.3 61.2 60.3 60.7 59.6 59.6 59.6 59.6 62.4 59.3 59.3 56.7 54.6 61.3 51.9 56.6 60.0 54.1 61.7 65.9
56.1 65.2 59.7 79.0 57.2 65.9 61.5 59.5 50.1 51.2 55.0 49.1 78.4 58.8 46.4 40.9 35.1 42.5 20.0 33.3 41.1 18.1 ---
Human chromosomeswere labeled with base-specific radioactive DNA precursors and examined autoradiographically (13). bCalculateddeviation fromaverage60.0% AT content. cData averaged from two cell strains (this work). d Data for independent flow cytometricanalysis of l0 individuals (1). e Relative quinacrine brightness weighted by chromosome length (12).
were approximately 30% of the percentage AT content change or +0.8 percentage unit. The standard deviations of the autoradiographic base ratio determinations were also about +1.0 percentage unit (13). The fluorescence shifts were not reciprocal for chromosome 14 in either 46,XX or 46,XY and not reciprocal for chromosome 15 in 46,XX, and these data were not used. The normalized Hoechst and chromomycin fluorescence parameters based on flow Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
karyotypes of 10 individuals (1) were analyzed in the same manner for changes in base composition (Table III). Only chromosome 21 had a nonreciprocal fluorescence shift. The correlation coefficient between the two flow cytometric determinations was 0.94. In Table III the h u m a n chromosomal base ratio as determined by autoradiography of base-specific radioactive D N A precursor incorporation (13) is listed with the chromosomal quinacrine brightness weighted by chromosome length (12). The correlation coefficients with the flow cytometric measurements were 0.55 and 0.88, respectively. DISCUSSION The accuracy of D N A content measurement of propidium iodide-stained chromosomes has been established by the strong positive correlation between the relative propidium iodide chromosomal fluorescence and the standard DNA-based h u m a n karyotype (8). This correlation has been used to confirm the chromosomal identity of the peaks in flow cytometric data (22). Variations in D N A content between homologous chromosomes are readily detected by flow cytometry, but often no detectable morphological feature is associated with D N A content variants suggesting a new class of polymorphisms (8, 23). The resolution of fluorescence analysis by flow cytometry is equivalent to 1% of a chromosome's D N A content, or about 600,000 base pairs. While this amount of D N A may contain a great deal of genetic information it is equivalent in resolution to a single band detectable by microscopic cytogenetics (24). However, shifts in fluorescence might be caused by structural differences in stain accessibility (6), or by the influence of optical and geometrical properties of the chromosomes (25). The degree of resolution of the h u m a n chromosomes by Hoechst-chromomycin fluorescence indicates that this dye combination is sensitive to chromosomal properties other than just D N A content. The fluorescence
HUMAN CHROMOSOME COMPOSITION
shifts of Hoechst and chromomycin fluorescence relative to the DNA-based human karyotype were reciprocal, but not always equivalent for each chromosome. The AT binding preference for Hoechst 33258 and GC binding preference for chromomycin A3 are well documented (6, 10). The fluorescence shifts are, therefore, primarily due to individual chromosome deviations in base composition from that of total genomic DNA. A striking area of agreement among the three methods of chromosomal base composition determination is that the larger chromosomes (2-8) tend to be relatively AT rich and the smaller chromosomes tend to be relatively GC rich in comparison to the base composition of total genomic DNA. Particular chromosomes exhibit anomalous fluorescence shifts, however. Chromosomes Y, 4, 13, and 18 have the highest relative Hoechst fluorescence and chromosomes 17, 19, 20, 21, and 22 the highest relative chromomycin fluorescence. The human Y chromosome is quinacrine bright (26) as are chromosomes 4, 13, and 18, while chromosomes 17, 19, 20, 22 are quinacrine dull (12) (Table III). The quantum yield of quinacrine is reduced in the presence of GC base pairs and its brightness is enhanced in AT-rich DNA (27). The AT content of the Hoechstand quinacine-bright chromosomes are not especially elevated in the autoradiographic analysis (except Y), however, but the chromosomes 19, 20, 21, and 22 (not 17) were depressed in AT content. The dependence of the fluorescence shifts on base composition is not expected to be strictly linear. In addition to the enhancement factor discussed above, factors such as the influence of chromosomal proteins on dye accessibility to DNA, binding site requirements, energy transfer, and local effects on dye quantum yield, may diminish or enhance dye fluorescence. The fluorescence enhancement factors used in the data reduction are valid only for DNA of operationally random sequence. The moderately positive correlation between the autoradiographic determination
433
of base ratio, quinacrine brightness, and the flow cytometric measurements indicate that the accuracy of the chromosomal base composition determination may be less than the precision of the data and suggests that other interesting chromosomal properties may influence the fluorescence shifts other than simple variation in base composition. Classes of repetitive DNA make up about 35% of the human genome (11). Repetitive DNA containing sequential-like base pairs might provide an increased number of binding sites for Hoechst or enhance the fluorescence of quinacrine and give rise to a substantial fluorescence shift even if the overall chromosomal base composition is little changed. The distribution of some human satellite sequences has been shown to be chromosome specific (28), but AT-rich satellite DNA constitutes only 5% of the human genome (29). The large variations in Hoechst and chromomycin chromosomal fluorescence measured by flow cytometry may be due to uneven distribution of repetitive DNA sequences among the chromosomes or classes of repetitive DNA specific to one of a few chromosome types. The sorting of human chromosomes by flow cytometry can potentially provide purified chromosomes for base composition and repetitive DNA analysis and thereby provide direct information on chromosome composition. ACKNOWLEDGMENTS Kevin Albright, Julie Meyne, Gayte Travis, and Scott Cram provided collaborative assistance. This work was performed under the auspices of the Los Alamos National Laboratory Flow Cytometry and Sorting Resource funded by the Division of Research Resources of the National Institutes of Health (Grant P41 RR01315) and the U. S. Department of Energy. REFERENCES 1. Langlois, R. G., Yu, L. C., Gray, J. W., and Carrano, A. V., Proc. Natl. Acad. Sci. USA 76, 7876 (1982). 2. Cram, L. S., Bartholdi, M. F., Ray, F. A., Travis, G. L., and Kraemer, P. M., Cancer Res. 43, 4828 (1983). Journal of Colloidand InterfaceScience, Vol. 105,No. 2, June 1985
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3. Lebo, R. V., Gorin, F., Fletterich, R. J., Kao, F. J., Science (Washington, D. C.) 225, 57 (1984). 4. Carrano, A. V., Gray, J. W., Langlois, R. G., Burkhart-Schultz, K. T., and Van Dilla, M. A., Proc. Natl. Acad. Sci. USA 76, 1382 (1979). 5. Young, B. D., Ferguson-Smith, M. A., SiUar, R., and Boyd, E., Proc. Natl. Acad. Sci. USA 78, 7727 (1981). 6. Langlois, R. G., Carrano, A. V., Gray, J. W., and Van Dilla, M. A., Chromosoma 77, 229 (1980). 7. Gray, J. W., Langlois, R. G., Carrano, A. V., Burkhart-Schultz, K. J., and Van Dilla, M. A., Chromosoma 73, 9 (1979). 8. Mayall, B. H., Carrano, A. V., Moore, D. H., Ashworth, L. K., Bennett, D. E., and Mendelsohn, M. L., Cytometry 5, 376 (1984). 9. Jas, J., and Westerneng, G., J. Histochem. Cytochem. 29, 929 (1981). 10. MiJller, W., and Gautier, F., Eur. J. Biochem. 54, 385 (t975). 11. Kornberg, A., "DNA Replication," p. 33. Freeman, San Francisco, 1981. 12. Kuhn, E. M., Chromosoma 57, 1 (1976). 13. Korenberg, J. R., and Engels, W. R., Proc. Natl. Acad. Sci. USA 7, 3382 (1978). 14. Sillar, R. and Young, B., J. Histochem. Cytochem. 29, 74 (1981). 15. Cram, L. S., Arndt-Jovin, D. J., Grimwade, B., and Jovin, J. M., in "Flow Cytometry IV" (O. D. Laerum, J. Lindmo, and E. Thorud, Eds.) p. 256. Universtetsforlaget, Bergen, 1980.
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16. Bartholdi, M. F., Sinclair, D. C., and Cram, L. S., Cytometry 3, 395 (1983). 17. Bartholdi, M. F., Ray, F. A., Kraemer, P. M., and Cram, L. S., Cytometry 5, 534 (1984). 18. Meyne, J., Bartholdi, M. F., Travis, G. L., and Cram, L. S., Cytometry 5, 580 (1984). 19. Sahar, E., and Latt, S. A., Proc. Natl. Acad. Sci. USA 75, 5650 (1978). 20. Langlois, R. G., and Jensen, R. A., J. Histochem. Cytochem. 27, 72 (1979). 21. Comings, P. J., and Drets, M. J., Chromosoma 56, 199 (1976). 22. Bartholdi, M. F., Travis, G. L., Cram, L. S., Porreca, P., and Leavitt, J., Ann. N. Y. Acad. Sci., in press. 23. Ray, F. A., Bartholdi, M. F., Kraemer, P. M., and Cram, L. S., Cytogenet. Cell Genet. 257 (1984). 24. Cram, L. S., Bartholdi, M. F., Ray, F. A., Travis, G. L., and Kraemer, P. M., Prog. Nucleic Acid Res. 29, 19 (1983). 25. Kerker, M., Chew, H., McNulty, P. J., Kratohvil, J. P., Cooke, D. D., Scully, M., and Lee, M. P., J. Histochem. Cytochem. 27, 250 (1979). 26. Hatfield, J. M., Pedas, K. W. C., and West, N. M., Chromosoma 52, 62 (1975). 27. Pachman, U., and Rigler, R., Exp. Cell Res. 72, 602 (1972). 28. Beauchamp, R. S., Mitchell, A., Buchland, R. A., and Bostock, C. J., Chromosoma 71, 153 (1979). 29. Frommer, M., Prosser, J., Tkachuk, D., Reisener, A. H., and Vincent, P. C., Nucleic Acids Res. 10, 547 (1982).
Basic information on chromosome composition and structure can be obtained by flow cytometric analysis. Although chromosome analysis by flow cytometry has been used mostly as a technique to determine the flow karyotypes of individuals (1) and cell populations (2), or to purify chromosomes for molecular analysis (3), the variety of measurements possible on single chromosomes provides data on chromosome composition and structure at rates sufficient to analyze large numbers of chromosomes. Fluorescence of dyes with binding specificity to DNA has proven to be the most useful * This paper is dedicated to Dr. Milton Kerker on the occasion of his 65th birthday. The U.S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. The Los Alamos National Laboratory is operated by the University of California for the U. S. Department of Energy under Contract W-7405-ENG-36.
measurement for resolving single chromosome types. The purpose of this report is to evaluate the accuracy of fluorescence analysis by flow cytometry to determine chromosomal DNA content and base composition by correlation with independent measurements. Previous analysis of human chromosomal DNA content as measured by flow cytometry lacked precision (4, 5), and previous analysis of chromosomal base composition did not evaluate specific chromosome types (6), although similar overall correlations have been determined (7). In this study the relative fluorescence of propidium-iodide-stained human chromosomes measured by flow cytometry correlated positively (r -- 0.99) with the DNA-based human karyotype determined by image cytometry of gallocyanin-chrome-alum-stained chromosomes (8). Propidium iodide binds specifically by intercalation to DNA without base specificity (9). The strong positive correlation between the flow cytometric measurements and the DNA-based human 426
0021-9797/85 $3.00 Copyright © 1985 by Academic Press, Inc. All fights of reproduction in any form J~served.
Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
HUMAN CHROMOSOME COMPOSITION karyotype established the accuracy of propidium iodide fluorescence analysis by flow cytometry to measure DNA content. The fluorescence of Hoechst 33258- and chromomycin-A3-stained human chromosomes was analyzed by determining separately the percentage difference between the normalized Hoechst 33258 and the normalized chromomycin A3 fluorescence for each chromosome from the chromosome DNA contents tabulated in the DNA-based human karyotype (8). These "fluorescence shifts" were then reduced to deviations in chromosomal base content from the average total human genomic base composition of 60.0% AT content (11). The bisbenzimidazol derivative, Hoechst 33258, binds externally to DNA at three consecutive A- T base pairs (A = adenine, T = thymine) (10). The antitumor antibiotic chromomycin A 3 also binds externally to DNA and appears to require three G - C (G = guanine, C = cytosine) base pairs at the binding site (6). Hoechst and chromomycin fluorescence shifts from the tabulated chromosomal DNA contents (8) were found to be reciprocal for each chromosome; when Hoechst fluorescence was higher, the chromomycin fluorescence was lower than expected if these dyes depended only on DNA content. The fluorescence shifts were reduced to changes in base composition by taking into account the fact that the ratio of runs of three like base pairs will change more rapidly than the average base ratio changes (6). The fluorescence shifts are therefore enhanced for the dyes Hoechst 33258 and chromomycin A3 relative to the changes in chromosomal base composition. The percentage change in AT content from the total human genomic DNA base composition of 60% AT was estimated for each chromosome and compared with independent flow cytometric analysis of Hoechst-chromomycin-stained chromosomes (r = 0.94) (1), fluorescence microscopy of quinacrine-stained chromosomes (r = 0.88) (12), and autoradiography of base-specific
427
radioactive DNA precursor incorporation into chromosomes (r = 0.55) (13). Each method agreed in the finding that the larger chromosomes tend to be relatively high in AT content and the smaller chromosomes relatively high in GC content from the average genomic base ratio. Many factors, however, may alter the dependence of Hoechst-chromomycin fluorescence on simple variation in base composition. In particular, highly repetitive DNA may alter the distribution of short runs of like base sequences from that expected in the DNA of operationally random sequence enriched in AT content. The accuracy of base composition as determined by Hoechstchromomycin staining is therefore restricted to establishing AT or GC enrichment of the overall chromosomal base composition, but may be indicative of classes of repetitive DNA specific to one or a few chromosome types. MATERIALS AND METHODS Isolated metaphase chromosomes were prepared from four diploid human cell strains, two with the karyotype 46,XX and two with 46,XY. Cells in subconfluent cultures were blocked in the metaphase stage of mitosis with colcemid (3 X 10-7 M). Mitotic cells were harvested by shaking off, centrifuged at 150 g for 8 min, and the supernatant was removed. For staining with propidium iodide (2) the mitotic cells were resuspended in 1.0 ml of 75 m M KC1 and 50/zg/ml propidium iodide. The cells were allowed to swell for 10 rain at room temperature and then 0.5 ml of the following solution was added: 75 m M KC1, 0.1% Triton X-100, 50 #g/ml propidium iodide, and 1.0 mg/ml RNase. After 3 rain at room temperature the cells were forced through a 1½-in. 22-gauge syringe needle to disperse the chromosomes. The chromosome suspension was incubated for 30 min at 37°C to allow the RNase to degrade chromosomal RNA. For staining with Hoechst 33258 and chromomycin A3 the cells were resuspended in a dournal'ofColloid and Interface Science, Vot. 105, No. 2, June 1985
428
MARTY
F. B A R T H O L D I
buffer (14) containing 15 m M Tris-HC1, 0.2 m M spermine, 0.5 m M spermidine, 2 m M EDTA, 0.5 m M EGTA, 80 mM KCI, 14 mM/3-mercaptoethanol, and 0.1% digitonin. The cell suspension was held on a Vortex mixer and agitated vigorously for 1 min to release the chromosomes. Chromomycin A3 was added to a final concentration of 10 t~M, and, after 2 h to allow chromomycin binding to reach equilibrium, Hoechst 33258 was added to a final concentration of 5 #M. The chromosome suspensions were stored at 4°C. FLOW
CYTOMETRY
Univariate analysis of propidium iodidestained chromosomes was done on an experimental flow cytometer (15, 16). Fluorescence is excited by a finely focused laser beam (1.5 W at 488 nm in a 4 X 27-tzm spot at the e-2 diameters) and collected at 90 ° relative to the direction of the laser beam. The illumination optics are a 10X, 0.4 numerical aperture objective partially filled by a toric lens of 90-mm diverging focal length and 630mm cylindrical focal length. The collection lens is a 40X, 0.75 numerical aperture objective coupled by glycerol to the square outer nozzle. The fluorescent object is brought into sharp focus at the image plane of the collection lens and then diverges before impinging on the photomultiplier tube. A colored glass filter with transmission above 560 nm blocks the scattered light. The flow chamber consists of three nozzles. The sample and inner sheath streams are injected into a second sheath contained in an outer square nozzle. The velocity of the sample stream is 7 m/s with a 2-#m diameter. The fluorescence pulse is amplified, integrated, and acquired with 10-bit resolution. A previous analysis of fluoresence measured on this flow cytometer (16) demonstrated that with high irradiance, saturation of the fluorescence transition and photobleaching occur. The coefficient of variation of fluorescence measurements from single chromosomes is between 1.5 and 1.0% (17) Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
due to an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories. Bivariate analysis of chromosomes stained with Hoechst and chromomycin was done on an EPICS V flow cytometer (Coulter Electronics, Inc., Hialeah, Fla.) equipped with two argon-ion lasers (18). A standard 76-t~mdiameter flow nozzle was used with stream in air illumination. The sample and sheath streams were cooled at 4°C. The two lasers were aligned with UV (200 mW at 351.1363.8 nm) excitation followed by excitation of 350 mW at 457.9 nm. Fluorescence was collected by a single photomultiplier tube with filters KV408 and GG495 that block light below 408 and 495 nm. A single photomultiplier tube was used for detection of the two fluorescence pulses by taking advantage of the fact that they occur separately in time by 7 #s. The signals were processed and acquired using the electronics provided with the EPICS V. Both integrated fluorescence pulses were recorded in bivariate histograms with 128 channels on each axis. The fluorescence signal measured after UV excitation consists of both the long-wavelength edge of the Hoechst fluorescence (emission maximum at 450 nm, but detected through a 495-nm longpass filter) and fluorescence from adjacently bound chromomycin A3 (emission maximum at 580 nm) excited by energy transfer from Hoechst 33258 (19, 20). Energy transfer is efficient over a distance of 2.5-3.5 nm corresponding to about 7-10 base pairs. After 457.9-nm excitation only total chromomycin A3 fluorescence is measured. The coefficients of variation of the fluorescence measurements were between 2 and 3%. RESULTS
Chromosomes from two human diploid cell strains were isolated and stained with propidium iodide. The univariate histograms
429
HUMAN CHROMOSOME COMPOSITION
of the number of chromosomes versus fluorescence intensity in Fig. 1 were measured on the experimental flow cytometer. The peaks in the histograms were identified with the autosomal chromosomes number 1-22 and the X and Y chromosomes (7). The histogram in the upper panel is from cells with the karyotype 46,XX and the peak identified with the X chromosome has the same number of counts as resolved autosome pairs. The histogram in the lower panel is 173
from cells with the karyotype 46,XY and the peak identified with X chromosomes contains only half the counts of the resolved autosomes. Most of the human chromosomes are separately resolved except for 1 and 2, 9-12, and 15 and 14. Note that certain autosomal homologs are also separately resolved, for example, chromosomes 13, 16, and 22 in each of the two cell strains analyzed here. The relative propidium iodide fluorescence
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FIG. 1. Univariate flow karyotypes of two human cell strains, 46,XX and 46,XY. Isolated metaphase chromosomes were stained with propidium iodide. More than 50,000 chromosomes were analyzed in each histogram. Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
430
M A R T Y F. B A R T H O L D I
measurements of the 24 human chromosomes from both cell strains are compared to the DNA-based human karyotype determined by image cytometry of gallocyanin-chromealum-stained chromosomes (8) in Table I. The fluorescence data (peak means in Fig. 1) were normalized so that the sum of the autosomal peak means, including homolog variants, was 100. The image cytometry data were also normalized to a sum of 100 for the autosomal measurements, but homolog variants were excluded. The DNA-based human karyotype was established from 16 unrelated donors. The correlation coefficient (r) between the two measurements was 0.99. The uncertainty
TABLE I H u m a n Chromosomal D N A Content Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
DNA-basedhuman karyotype a'b
46,XXax
46,XY~c
4.28 4.19 3.49 3.31 3.18 2.97 2.76 2.52 2.38 2.35 2.34 2.31 1.88 1.78 1.73 1.61 1.47 1.40 1.09 1.15 0.82 0.90 2.64 0.97
4.19 4.19 3.53 3.35 3.17 2.98 2.82 2.56 2.37 2.37 2.37 2.37 1.87, 1.94 1.79 1.68 1.59, 1.68 1.46 1.39 1.03 1.15 0.82 0.90, 0.75 2.68 --
4.19 4.19 3.54 3.32 3.16 3.02 2.81 2.57 2.36 2.36 2.36 2.36 1.89, 1.96 1.80 1.72 1.59, 1.69 1.48 1.32 1.08 1.17 0.82 0.95, 0.78 2.70 0.98
a Normalization to the s u m of 100 for autosomal measurements. b Measured by image cytometry ofgallocyanin--chromealum-stained chromosomes (8). c Measured by flow cytometry of propidium iodidestained chromosomes (Fig. 1). Journal of Colloid and Interface Science, Vol. 105 No. 2 June 1985
of the relative peak means in the flow cytometric determination is less than 1% and the uncertainty of the chromosome DNA content in the DNA-based human karyotype (8) is 2.5%. However, since each of the human chromosomes is not clearly resolved by flow cytometry a larger inaccuracy may occur as in, for example, chromosomes 1 and 2. Chromosomes from two additional dipoloid human cell strains were stained with Hoechst 33258 and chromomycin A3 and analyzed on the dual-laser EPICS V flow cytometer. The bivariate histograms are displayed in Fig. 2 as contour plots of the number of chromosomes with correlated Hoechst (UV-excited) and chromomycin A 3 (458-nm-excited) fluorescences. The chromosome type associated with each peak was identified by sorting onto microscope slides and classified by morphology. The normal human chromosomes, except the group 912, are clearly resolved. Again the difference between the 46,XX and 46,XY karyotype is readily detected. The UV-excited fluorescence and 458-nmexcited fluorescence were separately normalized to a sum of 100 for the autosomal measurements (peak means in Fig. 2). The percentage differences between the fluorescence values and the DNA-based human karyotype as determined by image cytometry are listed in Table II. Given that the base composition of total human genomic DNA is 60% AT and 40% GC (11), an equivalent analysis was done to normalize the UV-excited fluorescence to a sum of 60 and the 458-nm-excited fluorescence to a sum of 40. The respective percentage differences were calculated with 0.60 (the average AT content) and 0.40 (the average GC content) of the DNA-based human karyotype. The sum of both fluorescences normalized to base composition for each chromosome equaled the DNA content for each chromosome as determined by propidium iodide fluorescence. The enhancement of fluorescence shifts of base-specific dyes to changes in base corn-
431
H U M A N CHROMOSOME COMPOSITION
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46,XX
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chromomycin fluorescence occurred for a 1% increase in AT content. The fluorescence percentage differences were adjusted by these factors in Table II to yield the corresponding change in chromosomal AT content for both Hoechst and chromomycin fluorescence shifts. The percentage change in AT content is based on a deviation from the 60.0% AT content of total genomic DNA (9). Four measurements of AT content change (UVexcited and 458-nm excited fluorescence shifts for each 46,XX and 46,XY cell strains) were averaged and added (or subtracted) from 60.0% in Table III. The standard deviations
HUMAN 46,XY
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position (21) rests on the probability of occurrence of runs of sequential-like base pairs in DNA of a specific base ratio (6). Small changes in base composition result in larger fluorescence changes of dyes that require runs of identical base pairs for binding, for example both Hoechst and chromomycin. A detailed analysis (6) determined that a 4.7% increase in UV-excited fluorescence occurred for a 1% increase in AT content due to both binding site requirements and energy transfer, and that a 3.9% decrease in 458-nm-excited
Fluorescencepercentage difference°
Percentage AT base content change
Chromosome UV-excited 458-nm-excited UV-excitedb 458-nm-exeitedc 1
-1.5
+4.9
-0.3
-1.3
2 3 4 5 6 7 8 9 10
+2.6 +6.5 +11.3 +4.6 +7.7 +3.7 +4.9 -3.5 -2.2
-2.2 -7.4 -18.6 -8.5 -6.4 -0.7 -4.1 +1.2 +2.9
+0.5 +1.3 +2.3 +0.9 +1.6 +0.8 +1.0 -0.7 -0.4
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11
- 1.7
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12 13 14 15 16 17 18 19 20 21 22 X y
-0.4 +11.9 -2.7 -4.8 -12.3 -21.5 +5.6 -36.2 -15.0 +5.4 -38.4 +7.9 .
+4.4 -16.0 -0.6 -0.6 +13.4 +24.6 -15.7 +33.3 +8.0 -17.1 +18.2 -11.4
-0.1 +2.4 -0.6 - 1.0 -2.5 -4.4 +1.1 -7.4 -3.1 +1.1 -7.7 +1.6 .
-1.2 +4.3 +0.2 +0.2 -3.6 -6.6 +4.2 -8.9 -2.2 +4.4 -4.9 +3.1
.
.
a Percentage difference between normalized fluorescence for 46,XX cell strain. b UV-excited fluorescence divided by 4.7. c 458-nm-excited fluorescence divided by -3.9. Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
432
MARTY F. BARTHOLDI TABLE III Human Chromosomal Base Composition (Percentage AT Content)
Chromosome
Base ratio a
HoechstChromomyein fluorescence b'c
HoechstChromomycin fluoreseence b'a
Quinacrine fluorescence e
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
60.3 61.3 60.8 59.7 60.5 60.7 59.5 59.9 59.7 61.4 59.8 60.4 60.2 57.2 58.4 59.7 61.5 61.4 56.3 57.6 56.2 58.4 59.3 62.4
59.1 60.6 61.4 63.7 61.6 61.6 60.4 60.9 59.4 59.4 59.4 59.3 63.3 60.0 59.4 57.1 54.5 62.6 51.9 57.6 55.9 54.1 62.0 76.5
59.7 60.7 61.3 62.6 61.3 61.2 60.3 60.7 59.6 59.6 59.6 59.6 62.4 59.3 59.3 56.7 54.6 61.3 51.9 56.6 60.0 54.1 61.7 65.9
56.1 65.2 59.7 79.0 57.2 65.9 61.5 59.5 50.1 51.2 55.0 49.1 78.4 58.8 46.4 40.9 35.1 42.5 20.0 33.3 41.1 18.1 ---
Human chromosomeswere labeled with base-specific radioactive DNA precursors and examined autoradiographically (13). bCalculateddeviation fromaverage60.0% AT content. cData averaged from two cell strains (this work). d Data for independent flow cytometricanalysis of l0 individuals (1). e Relative quinacrine brightness weighted by chromosome length (12).
were approximately 30% of the percentage AT content change or +0.8 percentage unit. The standard deviations of the autoradiographic base ratio determinations were also about +1.0 percentage unit (13). The fluorescence shifts were not reciprocal for chromosome 14 in either 46,XX or 46,XY and not reciprocal for chromosome 15 in 46,XX, and these data were not used. The normalized Hoechst and chromomycin fluorescence parameters based on flow Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
karyotypes of 10 individuals (1) were analyzed in the same manner for changes in base composition (Table III). Only chromosome 21 had a nonreciprocal fluorescence shift. The correlation coefficient between the two flow cytometric determinations was 0.94. In Table III the h u m a n chromosomal base ratio as determined by autoradiography of base-specific radioactive D N A precursor incorporation (13) is listed with the chromosomal quinacrine brightness weighted by chromosome length (12). The correlation coefficients with the flow cytometric measurements were 0.55 and 0.88, respectively. DISCUSSION The accuracy of D N A content measurement of propidium iodide-stained chromosomes has been established by the strong positive correlation between the relative propidium iodide chromosomal fluorescence and the standard DNA-based h u m a n karyotype (8). This correlation has been used to confirm the chromosomal identity of the peaks in flow cytometric data (22). Variations in D N A content between homologous chromosomes are readily detected by flow cytometry, but often no detectable morphological feature is associated with D N A content variants suggesting a new class of polymorphisms (8, 23). The resolution of fluorescence analysis by flow cytometry is equivalent to 1% of a chromosome's D N A content, or about 600,000 base pairs. While this amount of D N A may contain a great deal of genetic information it is equivalent in resolution to a single band detectable by microscopic cytogenetics (24). However, shifts in fluorescence might be caused by structural differences in stain accessibility (6), or by the influence of optical and geometrical properties of the chromosomes (25). The degree of resolution of the h u m a n chromosomes by Hoechst-chromomycin fluorescence indicates that this dye combination is sensitive to chromosomal properties other than just D N A content. The fluorescence
HUMAN CHROMOSOME COMPOSITION
shifts of Hoechst and chromomycin fluorescence relative to the DNA-based human karyotype were reciprocal, but not always equivalent for each chromosome. The AT binding preference for Hoechst 33258 and GC binding preference for chromomycin A3 are well documented (6, 10). The fluorescence shifts are, therefore, primarily due to individual chromosome deviations in base composition from that of total genomic DNA. A striking area of agreement among the three methods of chromosomal base composition determination is that the larger chromosomes (2-8) tend to be relatively AT rich and the smaller chromosomes tend to be relatively GC rich in comparison to the base composition of total genomic DNA. Particular chromosomes exhibit anomalous fluorescence shifts, however. Chromosomes Y, 4, 13, and 18 have the highest relative Hoechst fluorescence and chromosomes 17, 19, 20, 21, and 22 the highest relative chromomycin fluorescence. The human Y chromosome is quinacrine bright (26) as are chromosomes 4, 13, and 18, while chromosomes 17, 19, 20, 22 are quinacrine dull (12) (Table III). The quantum yield of quinacrine is reduced in the presence of GC base pairs and its brightness is enhanced in AT-rich DNA (27). The AT content of the Hoechstand quinacine-bright chromosomes are not especially elevated in the autoradiographic analysis (except Y), however, but the chromosomes 19, 20, 21, and 22 (not 17) were depressed in AT content. The dependence of the fluorescence shifts on base composition is not expected to be strictly linear. In addition to the enhancement factor discussed above, factors such as the influence of chromosomal proteins on dye accessibility to DNA, binding site requirements, energy transfer, and local effects on dye quantum yield, may diminish or enhance dye fluorescence. The fluorescence enhancement factors used in the data reduction are valid only for DNA of operationally random sequence. The moderately positive correlation between the autoradiographic determination
433
of base ratio, quinacrine brightness, and the flow cytometric measurements indicate that the accuracy of the chromosomal base composition determination may be less than the precision of the data and suggests that other interesting chromosomal properties may influence the fluorescence shifts other than simple variation in base composition. Classes of repetitive DNA make up about 35% of the human genome (11). Repetitive DNA containing sequential-like base pairs might provide an increased number of binding sites for Hoechst or enhance the fluorescence of quinacrine and give rise to a substantial fluorescence shift even if the overall chromosomal base composition is little changed. The distribution of some human satellite sequences has been shown to be chromosome specific (28), but AT-rich satellite DNA constitutes only 5% of the human genome (29). The large variations in Hoechst and chromomycin chromosomal fluorescence measured by flow cytometry may be due to uneven distribution of repetitive DNA sequences among the chromosomes or classes of repetitive DNA specific to one of a few chromosome types. The sorting of human chromosomes by flow cytometry can potentially provide purified chromosomes for base composition and repetitive DNA analysis and thereby provide direct information on chromosome composition. ACKNOWLEDGMENTS Kevin Albright, Julie Meyne, Gayte Travis, and Scott Cram provided collaborative assistance. This work was performed under the auspices of the Los Alamos National Laboratory Flow Cytometry and Sorting Resource funded by the Division of Research Resources of the National Institutes of Health (Grant P41 RR01315) and the U. S. Department of Energy. REFERENCES 1. Langlois, R. G., Yu, L. C., Gray, J. W., and Carrano, A. V., Proc. Natl. Acad. Sci. USA 76, 7876 (1982). 2. Cram, L. S., Bartholdi, M. F., Ray, F. A., Travis, G. L., and Kraemer, P. M., Cancer Res. 43, 4828 (1983). Journal of Colloidand InterfaceScience, Vol. 105,No. 2, June 1985
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3. Lebo, R. V., Gorin, F., Fletterich, R. J., Kao, F. J., Science (Washington, D. C.) 225, 57 (1984). 4. Carrano, A. V., Gray, J. W., Langlois, R. G., Burkhart-Schultz, K. T., and Van Dilla, M. A., Proc. Natl. Acad. Sci. USA 76, 1382 (1979). 5. Young, B. D., Ferguson-Smith, M. A., SiUar, R., and Boyd, E., Proc. Natl. Acad. Sci. USA 78, 7727 (1981). 6. Langlois, R. G., Carrano, A. V., Gray, J. W., and Van Dilla, M. A., Chromosoma 77, 229 (1980). 7. Gray, J. W., Langlois, R. G., Carrano, A. V., Burkhart-Schultz, K. J., and Van Dilla, M. A., Chromosoma 73, 9 (1979). 8. Mayall, B. H., Carrano, A. V., Moore, D. H., Ashworth, L. K., Bennett, D. E., and Mendelsohn, M. L., Cytometry 5, 376 (1984). 9. Jas, J., and Westerneng, G., J. Histochem. Cytochem. 29, 929 (1981). 10. MiJller, W., and Gautier, F., Eur. J. Biochem. 54, 385 (t975). 11. Kornberg, A., "DNA Replication," p. 33. Freeman, San Francisco, 1981. 12. Kuhn, E. M., Chromosoma 57, 1 (1976). 13. Korenberg, J. R., and Engels, W. R., Proc. Natl. Acad. Sci. USA 7, 3382 (1978). 14. Sillar, R. and Young, B., J. Histochem. Cytochem. 29, 74 (1981). 15. Cram, L. S., Arndt-Jovin, D. J., Grimwade, B., and Jovin, J. M., in "Flow Cytometry IV" (O. D. Laerum, J. Lindmo, and E. Thorud, Eds.) p. 256. Universtetsforlaget, Bergen, 1980.
Journal of Colloid and Interface Science, Vol. 105, No. 2, June 1985
16. Bartholdi, M. F., Sinclair, D. C., and Cram, L. S., Cytometry 3, 395 (1983). 17. Bartholdi, M. F., Ray, F. A., Kraemer, P. M., and Cram, L. S., Cytometry 5, 534 (1984). 18. Meyne, J., Bartholdi, M. F., Travis, G. L., and Cram, L. S., Cytometry 5, 580 (1984). 19. Sahar, E., and Latt, S. A., Proc. Natl. Acad. Sci. USA 75, 5650 (1978). 20. Langlois, R. G., and Jensen, R. A., J. Histochem. Cytochem. 27, 72 (1979). 21. Comings, P. J., and Drets, M. J., Chromosoma 56, 199 (1976). 22. Bartholdi, M. F., Travis, G. L., Cram, L. S., Porreca, P., and Leavitt, J., Ann. N. Y. Acad. Sci., in press. 23. Ray, F. A., Bartholdi, M. F., Kraemer, P. M., and Cram, L. S., Cytogenet. Cell Genet. 257 (1984). 24. Cram, L. S., Bartholdi, M. F., Ray, F. A., Travis, G. L., and Kraemer, P. M., Prog. Nucleic Acid Res. 29, 19 (1983). 25. Kerker, M., Chew, H., McNulty, P. J., Kratohvil, J. P., Cooke, D. D., Scully, M., and Lee, M. P., J. Histochem. Cytochem. 27, 250 (1979). 26. Hatfield, J. M., Pedas, K. W. C., and West, N. M., Chromosoma 52, 62 (1975). 27. Pachman, U., and Rigler, R., Exp. Cell Res. 72, 602 (1972). 28. Beauchamp, R. S., Mitchell, A., Buchland, R. A., and Bostock, C. J., Chromosoma 71, 153 (1979). 29. Frommer, M., Prosser, J., Tkachuk, D., Reisener, A. H., and Vincent, P. C., Nucleic Acids Res. 10, 547 (1982).