- Email: [email protected]
Immunofluorescent labeling of chromosomes with antisera to histones and histone fractions
468
L. S. Desai et al.
Table 1. Estimation
of the proportion of cell nuclei cut and exposed at one of the surfaces of a 2 pm-thick lens section
Number of nuclei Average nuclear diameter (pm) % Nuclei detected % Labeled nuclei observeda
Epithehum
NonAnnu-degenlar erating pad fibres
Early Late pyc- pycnosis nosis
50
56
48
71
47
8.4
8.2
5.9
3.6
2.2
80.6
80.2
74.5
65.7
52.0
80.1 il.1
81.7 io.7
80.4 k1.3
64.3 +1.6
49.3 il.0
a Data derived from Modak & Bollum [5] and based on per cent labeled nuclei in sections incubated with terminal transferase and 3H-dATP for 60 min.
except for the non-degenerating fibre nuclei which are the least spherical (fig. 3~); in these the observed percentage barely falls within one-sigma limits. These results (table 1) show that the observed labellingfrequencies vary widely among different lens nuclei, probably due to the differences in their diameters. The data also show that with an increasing ratio between the nuclear diameter and section-thickness the proportion of detected nuclei increases. The good agreement between the proportion of nuclei detected and percentage labelled nuclei observed, demonstrates that all detected-nuclei necessarily fall within the tritium-autoradiographic range. These results clearly show the possible source of error inherent in comparison of labelling frequencies between cell populations with different nuclear diameters. There exists a vast quantity of published literature describing differences in the size of the replicative compartment between different cell types treated with 3H-thymidine and subsequently sectioned and autoradiographed. Most of these studies need reevaluation. We suggest that, in such studies, nuclear Exptl
Cell Res 70
diameters and section thickness be accurately determined and then, using eq. (3) the proportion of nuclei detected be estimated separately for each cell type. The observed labelling percentages can then be expressed as the proportion of detected nuclei all of which necessarily fall within the autoradiographic range. We are grateful to Professor Cecile Leuchtenberger and Dr Klaus Scherrer for enthusiastic support. We thank Prof. Leuchtenberger, Dr N. Odartchenko and Dr R. Portmann for critical discussions. Research jointly supported by CTR, New York, the Swiss National Foundation (3.164.69) and the US Atomic Energy Commission under contract with the Union Carbide Corporation.
References 1. Abercrombie, M, Acta anat 94 (1964) 239. 2. Caro. L G. Methods in cell nhvsiologv (ed D M Prescott) vol. 1, p. 327. Academic Press, New York (1964). 3. Maurer, W & Primbsch, E, Exptl cell res 33 (1964) 8. 4. Modak, S P & Bollum, F J, Ibid 62 (1970) 421. 5. - Submitted for publication. 6. Modak, S P & Perdue, S W, Exptl cell res 59 (1970) 43. 7. Perry, R P, Methods in cell physiology (ed D M Prescott) vol. 1, p. 305. Academic Press, New York (1964). 8. Modak, S P, Lever, W E & Uppuluri, V R R. In preparation. Received July 12, 1971 Revised version received November 3, 1971
Immunofluorescent labeling of chromosomes with antisera to histones and histone fractions L. S. DESAI, L. POTHIER,l G. E. FOLEY and R. A. ADAMS. The Children’s Cancer Research Foundation, and ‘Department of Pathology, Medical School, Boston, Mass. 02115, USA
Harvard
Previous studies have been concerned with the nature and biological activity of histones derived from human lymphocytic cells, particularly human leukemic lymphoblasts [6-91. Since histones appear to be antigenic [14, 151, antisera were produced against purified whole histone and histone fractions 1 Present address: Roswell Park Memorial Buffalo, N.Y. 14203, USA
Institute,
~mmu~of~~oresce~t labeling of chromosFomes with antisera to h&ones and h~stone~ract~~~s
469
Fig. I. Polytene chromosomes of second instar larvae of Drosophila melanogastev treated with antisera to f3 histone isolated from human leukemic lymphoblasts (CCRF-CEM cells): pattern of fluorescence in WV light. Orig. x 500. Fig. 2. Segment of chromosome in fig. 1. Orig. x 500. Fig. 3. Human leukemic lymphoblasts (CCRF-CEM cells) treated with antisera to the f 3 histone fraction. orig. x 500. Figs 4, 5. Metaphase chromosomes of CCRF-CEM cells treated as in fig. 2. Orig. x 1250.
derived from human leukemic lymphoblasts to examine the association of histones with chromosomes by immunofluorescence methods. Information deriving from the use of
such methods might complement thad: resulting from chromosome studies based 1qm the binding and fluorescent propertie s of quinacrine and quinacrine mustar 16-j.
470
L. S. Desai et al.
Materials
and Methods
Histones. Whole histone and histone fractions were isolated from human leukemic lymphoblasts (CCRFCEM cells) [ll, 121 maintained in log-phase growth in large-volume suspension cultures, purified on Sephadex G-100 columns, and checked for purity by gel electrophoresis and amino acid analyses, as described elsewhere [6-g]. Antisera Whole histones, the lysine-rich (f l), or the arginine-rich (f 3) fractions (5 mg) were suspended in 1 ml of distilled water, emulsified with an equal volume of complete Freund adjuvant (Difco, Detroit. Mich.). and iniected in multiole subcutaneous sites in young female New Zealand rabbits once each week for 6 weeks. Approx. 2 months later, a second course of 4 weekly injections was administered. Seven days after the final injection, the rabbits were bled, the serum was separated and inactivated (at 56°C for 30 min), Millipore-filtered, and stored at -20°C. Whole histone was similarly prepared and administered, except that in the initial phase of these studies, the course of immunization consisted of 5 subcutaneous doses of 0.8 mg each, followed by a sixth intravenous dose. Chromosomes, The giant chromosomes of the salivary gland cell of Drosophila melanogaster were used in preliminary studies because of their convenient size. The salivary glands were dissected from second instar larvae, and squashed in 25 % acetic acid on glass slides with coverslips which were later removed after brief exposure to dry ice. Metaphase chromosomes of human leukemic cells (CCRF-CEM) were prepared by a modification of the method described by Moorhead et al. [13]. Immunofluorescent reagents. Goat anti-rabbit yglobulin, fluorescein-conjugated (Hyland Laboratories, Los Angeles, Calif.), diluted 1: 10; rhodamine albumin diluted 1:20; histone antisera and preimmunization rabbit sera diluted 1:3; and phosphatebuffered saline, pH 7.0 (PBS). Indirect immunofluorescence. Chromosome preparations were flooded with preimmunization or immune rabbit serum, incubated at 37°C in a humidified chamber for l/2 h, and washed in PBS. The preparations were air-dried, flooded with fluoresceinconjugated goat anti-rabbit y-globulin, together with rhodamine albumin as a non-specific counterstain, re-incubated in a humidified chamber at 37°C for l/2 h, rinsed in PBS, air-dried and examined in a Reichert Zetopan fluorescence microscope equipped with BG-12 and OG-1 filters (figs l-3), or a Leitz Wetzlar fluorescence microscope equipped with BG-12 and 530 nm filters (figs 4, 5).
Polytene chromosomes from the salivary gland cells of second instar larvae of Drosophila treated with histone antisera fluoresced in banded regions (figs 1, 2). At dilutions of Exptl Cell Res 70
1: 3, antisera to both fractions as well as to whole histone exhibited this characteristic pattern of fluorescence; and antisera to the f 3 fraction could be diluted to 1 :24 before appreciable diminution of fluorescence was noted. Fluorescence with antisera to f 1, a fraction which has not been unequivocally demonstrated in Drosophila histones [lo], was observed with some preparations. Fluorescence was not observed when chromosomes were exposed to preimmunization serum or PBS. Absorption or cross-absorption of antisera against whole histone or the f 1 or f 3 fractions (with 5 mgjml whole histones or histone fractions, respectively) eliminated the fluorescence reaction. This presumptive evidence for the specificity of these histone antisera is of interest with respect to the presumed role of histones as regulators of gene activity, since fluorescence appears to be associated primarily with DNA-rich regions of the polytene chromosomes of Drosophila. A comparison of such banding patterns with those of metaphase chromosomes might provide information as to the association of histones with repression of specific genetic function in these very well-mapped chromosomes. As expected, treatment with antisera to the f3 fraction resulted in fluorescence of inter-phase nuclei (fig. 3) and metaphase chromosomes (figs 4, 5) of the CCRF-CEM cells from which the histones used as antigen were derived. Moreover, since the CCRFCEM cells are leukemic lymphoblasts [I, 11, 12], refinement of these immunofluorescent methods might provide an important opportunity to study human malignancy by comparison of histones on chromosomes of nonmalignant and malignant cells derived from the same individual. These studies were supported in part by research grants C-6516 from the National Cancer Insitute, and FR-05526 from the Divison of Research Facilities and
Identification Resources, National Institutes of Health. GEF holds Research Career Award K6-CA-22, 150 from the National Cancer Institute.
References 1. Adams, R A, Foley, G E, Uzman, B 6, Farber, S, Lazarus, H & Kleinman, L, Cancer res 27 (1967)
of pig chromosomes
Chromosome no. 1
2.
2
3
141.
Desai, L S & Foley, G E, Biochem j 119 (1970) 165.
Desai, L S & Foley, G E, Tenth internatl cancer congress, Houston, Texas (1970) 796. Desai, L S & Foley, G E, Exptl cell res 66 (1971) 1. Desai, L S & Foley, G E, Proc XVIII ann colloq, protides of the biological fluids, Bruge, Belgium (1970) 10.
5
Dick. C & Johns. E W. Comn biochem anhvsiol " X(1&9)529.
'
'
_
FoIey, G E, Lazarus, H, Farber, S, Uzman, B G & Adams, R A, The proliferation and spread of neoplastic cells. 21st ann symp, fundamental cancer res, University of Texas M D Anderson Hospital and Tumor Institute, Houston, Texas. The Williams & Wilkins Co., Baltimore, Md. (1968) 65. 12. Foley, G E, Lazarus, H, Farber, S, Uzman, B G, Boone, B A & McCarthy, R E, Cancer 18 (1965) 522. 13.
4
64.
IX.
Moorhead, P S, Nowell, P C, Mellman, W J, Battigis, D M & Hungerford, D A, Exptl cell res
6
7
8
20(1960)613. 14. 15.
Rumke, P & Sluyser, M, Biochem j 101 (1966) ic. Stollar, B D & Ward, M, J biol them 245 (1970)
16.
Vosa, C 6, Chromosoma 31 (1970) 446.
1261.
9
Received June 1, 1971
Identification of the pig chromosomesby the quinacrine mustard fluorescencetechnique
10
I. GUSTAVSSQN, M. HAGELTORN, C. JOHANSSON and L. ZECH, Department of Animal Genetics, Nutrition and Hygiene, Royal Veterinary College, and Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institute& 104 01 Stockholm 60, Sweden
The diploid chromosome number of 38 in the pig is today definitely established [I, 21but as in several other speciesmost pairs of the pig
4=7Y
Table I. Description of the pig k~~yQt~~e
772.
Caspersson, T, Farber, S, Foley, G E, Kudynowski, J, Modest, E J, Simonsson, E, Wagh, U & Zech, L, Exptl cell res 49 (1968) 219. 3. Caspersson, T, Zech, L, Johansson, C & Modest, E J, Chromosoma 30 (1970) 215. 4, Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128. 5. Caspersson, T, Zech, L, Modest, E J, Foley G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969)
by QM j!uorescence
11 12 13
Fluorescence characteristics
This chromosome, which is easily identified by its morphology, shows z fairly uniform fluorescence pattern. The long arm exhibits a very characteristic pattern with a distinct narrow band close to the centromere separated from a broad fluorescent region by a dark band. Nearly the whole chromosome shows an evenly distributed bright fluorescence. In preparations of good quality at least two bright bands are visible in the midpart of the long arm. The short arm exhibits a narrow bright central band. The long arm has a small band close to the centromere and a dark band in the distal half of the long arm. The proximal half of the long arm is separated from the remaining part of the arm by a narrow dark band. The proximal half of the long arm is faintly fluorescing, often with a distinct brighter band close to the centromere. The distal half of the chromosome is bright. The proximal part of the long arm is darker than the remainder of the chromosome. A bright band in the middle of the long arm can often be distinguished. The secondary constriction can most often be distinguished after staining with QM. A dark band in the proximal part of the long arm can be distinguished in fairly contracted chromosomes. A bright band is seen in the distal part of the short arm. The iong arm exhibits an evenly distributed fluorescence along the whole length. The pattern is quite similar to the pattern of the X chromosome but poorer in contrast. This chromosome is morphologically distinguishable by its secondary constriction which is also evident on the fluorescence photograph. A dark band can often be distinguished in the proximal half of the short arm. Tnis chromosome has a distinct bright band on each side-of the centromere. This chromosome shows, when compared with no. 11, a very faint overall fluorescence. The longest telocentric (t) chromoso>me shows four bright bands, separated from
L. S. Desai et al.
Table 1. Estimation
of the proportion of cell nuclei cut and exposed at one of the surfaces of a 2 pm-thick lens section
Number of nuclei Average nuclear diameter (pm) % Nuclei detected % Labeled nuclei observeda
Epithehum
NonAnnu-degenlar erating pad fibres
Early Late pyc- pycnosis nosis
50
56
48
71
47
8.4
8.2
5.9
3.6
2.2
80.6
80.2
74.5
65.7
52.0
80.1 il.1
81.7 io.7
80.4 k1.3
64.3 +1.6
49.3 il.0
a Data derived from Modak & Bollum [5] and based on per cent labeled nuclei in sections incubated with terminal transferase and 3H-dATP for 60 min.
except for the non-degenerating fibre nuclei which are the least spherical (fig. 3~); in these the observed percentage barely falls within one-sigma limits. These results (table 1) show that the observed labellingfrequencies vary widely among different lens nuclei, probably due to the differences in their diameters. The data also show that with an increasing ratio between the nuclear diameter and section-thickness the proportion of detected nuclei increases. The good agreement between the proportion of nuclei detected and percentage labelled nuclei observed, demonstrates that all detected-nuclei necessarily fall within the tritium-autoradiographic range. These results clearly show the possible source of error inherent in comparison of labelling frequencies between cell populations with different nuclear diameters. There exists a vast quantity of published literature describing differences in the size of the replicative compartment between different cell types treated with 3H-thymidine and subsequently sectioned and autoradiographed. Most of these studies need reevaluation. We suggest that, in such studies, nuclear Exptl
Cell Res 70
diameters and section thickness be accurately determined and then, using eq. (3) the proportion of nuclei detected be estimated separately for each cell type. The observed labelling percentages can then be expressed as the proportion of detected nuclei all of which necessarily fall within the autoradiographic range. We are grateful to Professor Cecile Leuchtenberger and Dr Klaus Scherrer for enthusiastic support. We thank Prof. Leuchtenberger, Dr N. Odartchenko and Dr R. Portmann for critical discussions. Research jointly supported by CTR, New York, the Swiss National Foundation (3.164.69) and the US Atomic Energy Commission under contract with the Union Carbide Corporation.
References 1. Abercrombie, M, Acta anat 94 (1964) 239. 2. Caro. L G. Methods in cell nhvsiologv (ed D M Prescott) vol. 1, p. 327. Academic Press, New York (1964). 3. Maurer, W & Primbsch, E, Exptl cell res 33 (1964) 8. 4. Modak, S P & Bollum, F J, Ibid 62 (1970) 421. 5. - Submitted for publication. 6. Modak, S P & Perdue, S W, Exptl cell res 59 (1970) 43. 7. Perry, R P, Methods in cell physiology (ed D M Prescott) vol. 1, p. 305. Academic Press, New York (1964). 8. Modak, S P, Lever, W E & Uppuluri, V R R. In preparation. Received July 12, 1971 Revised version received November 3, 1971
Immunofluorescent labeling of chromosomes with antisera to histones and histone fractions L. S. DESAI, L. POTHIER,l G. E. FOLEY and R. A. ADAMS. The Children’s Cancer Research Foundation, and ‘Department of Pathology, Medical School, Boston, Mass. 02115, USA
Harvard
Previous studies have been concerned with the nature and biological activity of histones derived from human lymphocytic cells, particularly human leukemic lymphoblasts [6-91. Since histones appear to be antigenic [14, 151, antisera were produced against purified whole histone and histone fractions 1 Present address: Roswell Park Memorial Buffalo, N.Y. 14203, USA
Institute,
~mmu~of~~oresce~t labeling of chromosFomes with antisera to h&ones and h~stone~ract~~~s
469
Fig. I. Polytene chromosomes of second instar larvae of Drosophila melanogastev treated with antisera to f3 histone isolated from human leukemic lymphoblasts (CCRF-CEM cells): pattern of fluorescence in WV light. Orig. x 500. Fig. 2. Segment of chromosome in fig. 1. Orig. x 500. Fig. 3. Human leukemic lymphoblasts (CCRF-CEM cells) treated with antisera to the f 3 histone fraction. orig. x 500. Figs 4, 5. Metaphase chromosomes of CCRF-CEM cells treated as in fig. 2. Orig. x 1250.
derived from human leukemic lymphoblasts to examine the association of histones with chromosomes by immunofluorescence methods. Information deriving from the use of
such methods might complement thad: resulting from chromosome studies based 1qm the binding and fluorescent propertie s of quinacrine and quinacrine mustar 16-j.
470
L. S. Desai et al.
Materials
and Methods
Histones. Whole histone and histone fractions were isolated from human leukemic lymphoblasts (CCRFCEM cells) [ll, 121 maintained in log-phase growth in large-volume suspension cultures, purified on Sephadex G-100 columns, and checked for purity by gel electrophoresis and amino acid analyses, as described elsewhere [6-g]. Antisera Whole histones, the lysine-rich (f l), or the arginine-rich (f 3) fractions (5 mg) were suspended in 1 ml of distilled water, emulsified with an equal volume of complete Freund adjuvant (Difco, Detroit. Mich.). and iniected in multiole subcutaneous sites in young female New Zealand rabbits once each week for 6 weeks. Approx. 2 months later, a second course of 4 weekly injections was administered. Seven days after the final injection, the rabbits were bled, the serum was separated and inactivated (at 56°C for 30 min), Millipore-filtered, and stored at -20°C. Whole histone was similarly prepared and administered, except that in the initial phase of these studies, the course of immunization consisted of 5 subcutaneous doses of 0.8 mg each, followed by a sixth intravenous dose. Chromosomes, The giant chromosomes of the salivary gland cell of Drosophila melanogaster were used in preliminary studies because of their convenient size. The salivary glands were dissected from second instar larvae, and squashed in 25 % acetic acid on glass slides with coverslips which were later removed after brief exposure to dry ice. Metaphase chromosomes of human leukemic cells (CCRF-CEM) were prepared by a modification of the method described by Moorhead et al. [13]. Immunofluorescent reagents. Goat anti-rabbit yglobulin, fluorescein-conjugated (Hyland Laboratories, Los Angeles, Calif.), diluted 1: 10; rhodamine albumin diluted 1:20; histone antisera and preimmunization rabbit sera diluted 1:3; and phosphatebuffered saline, pH 7.0 (PBS). Indirect immunofluorescence. Chromosome preparations were flooded with preimmunization or immune rabbit serum, incubated at 37°C in a humidified chamber for l/2 h, and washed in PBS. The preparations were air-dried, flooded with fluoresceinconjugated goat anti-rabbit y-globulin, together with rhodamine albumin as a non-specific counterstain, re-incubated in a humidified chamber at 37°C for l/2 h, rinsed in PBS, air-dried and examined in a Reichert Zetopan fluorescence microscope equipped with BG-12 and OG-1 filters (figs l-3), or a Leitz Wetzlar fluorescence microscope equipped with BG-12 and 530 nm filters (figs 4, 5).
Polytene chromosomes from the salivary gland cells of second instar larvae of Drosophila treated with histone antisera fluoresced in banded regions (figs 1, 2). At dilutions of Exptl Cell Res 70
1: 3, antisera to both fractions as well as to whole histone exhibited this characteristic pattern of fluorescence; and antisera to the f 3 fraction could be diluted to 1 :24 before appreciable diminution of fluorescence was noted. Fluorescence with antisera to f 1, a fraction which has not been unequivocally demonstrated in Drosophila histones [lo], was observed with some preparations. Fluorescence was not observed when chromosomes were exposed to preimmunization serum or PBS. Absorption or cross-absorption of antisera against whole histone or the f 1 or f 3 fractions (with 5 mgjml whole histones or histone fractions, respectively) eliminated the fluorescence reaction. This presumptive evidence for the specificity of these histone antisera is of interest with respect to the presumed role of histones as regulators of gene activity, since fluorescence appears to be associated primarily with DNA-rich regions of the polytene chromosomes of Drosophila. A comparison of such banding patterns with those of metaphase chromosomes might provide information as to the association of histones with repression of specific genetic function in these very well-mapped chromosomes. As expected, treatment with antisera to the f3 fraction resulted in fluorescence of inter-phase nuclei (fig. 3) and metaphase chromosomes (figs 4, 5) of the CCRF-CEM cells from which the histones used as antigen were derived. Moreover, since the CCRFCEM cells are leukemic lymphoblasts [I, 11, 12], refinement of these immunofluorescent methods might provide an important opportunity to study human malignancy by comparison of histones on chromosomes of nonmalignant and malignant cells derived from the same individual. These studies were supported in part by research grants C-6516 from the National Cancer Insitute, and FR-05526 from the Divison of Research Facilities and
Identification Resources, National Institutes of Health. GEF holds Research Career Award K6-CA-22, 150 from the National Cancer Institute.
References 1. Adams, R A, Foley, G E, Uzman, B 6, Farber, S, Lazarus, H & Kleinman, L, Cancer res 27 (1967)
of pig chromosomes
Chromosome no. 1
2.
2
3
141.
Desai, L S & Foley, G E, Biochem j 119 (1970) 165.
Desai, L S & Foley, G E, Tenth internatl cancer congress, Houston, Texas (1970) 796. Desai, L S & Foley, G E, Exptl cell res 66 (1971) 1. Desai, L S & Foley, G E, Proc XVIII ann colloq, protides of the biological fluids, Bruge, Belgium (1970) 10.
5
Dick. C & Johns. E W. Comn biochem anhvsiol " X(1&9)529.
'
'
_
FoIey, G E, Lazarus, H, Farber, S, Uzman, B G & Adams, R A, The proliferation and spread of neoplastic cells. 21st ann symp, fundamental cancer res, University of Texas M D Anderson Hospital and Tumor Institute, Houston, Texas. The Williams & Wilkins Co., Baltimore, Md. (1968) 65. 12. Foley, G E, Lazarus, H, Farber, S, Uzman, B G, Boone, B A & McCarthy, R E, Cancer 18 (1965) 522. 13.
4
64.
IX.
Moorhead, P S, Nowell, P C, Mellman, W J, Battigis, D M & Hungerford, D A, Exptl cell res
6
7
8
20(1960)613. 14. 15.
Rumke, P & Sluyser, M, Biochem j 101 (1966) ic. Stollar, B D & Ward, M, J biol them 245 (1970)
16.
Vosa, C 6, Chromosoma 31 (1970) 446.
1261.
9
Received June 1, 1971
Identification of the pig chromosomesby the quinacrine mustard fluorescencetechnique
10
I. GUSTAVSSQN, M. HAGELTORN, C. JOHANSSON and L. ZECH, Department of Animal Genetics, Nutrition and Hygiene, Royal Veterinary College, and Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institute& 104 01 Stockholm 60, Sweden
The diploid chromosome number of 38 in the pig is today definitely established [I, 21but as in several other speciesmost pairs of the pig
4=7Y
Table I. Description of the pig k~~yQt~~e
772.
Caspersson, T, Farber, S, Foley, G E, Kudynowski, J, Modest, E J, Simonsson, E, Wagh, U & Zech, L, Exptl cell res 49 (1968) 219. 3. Caspersson, T, Zech, L, Johansson, C & Modest, E J, Chromosoma 30 (1970) 215. 4, Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128. 5. Caspersson, T, Zech, L, Modest, E J, Foley G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969)
by QM j!uorescence
11 12 13
Fluorescence characteristics
This chromosome, which is easily identified by its morphology, shows z fairly uniform fluorescence pattern. The long arm exhibits a very characteristic pattern with a distinct narrow band close to the centromere separated from a broad fluorescent region by a dark band. Nearly the whole chromosome shows an evenly distributed bright fluorescence. In preparations of good quality at least two bright bands are visible in the midpart of the long arm. The short arm exhibits a narrow bright central band. The long arm has a small band close to the centromere and a dark band in the distal half of the long arm. The proximal half of the long arm is separated from the remaining part of the arm by a narrow dark band. The proximal half of the long arm is faintly fluorescing, often with a distinct brighter band close to the centromere. The distal half of the chromosome is bright. The proximal part of the long arm is darker than the remainder of the chromosome. A bright band in the middle of the long arm can often be distinguished. The secondary constriction can most often be distinguished after staining with QM. A dark band in the proximal part of the long arm can be distinguished in fairly contracted chromosomes. A bright band is seen in the distal part of the short arm. The iong arm exhibits an evenly distributed fluorescence along the whole length. The pattern is quite similar to the pattern of the X chromosome but poorer in contrast. This chromosome is morphologically distinguishable by its secondary constriction which is also evident on the fluorescence photograph. A dark band can often be distinguished in the proximal half of the short arm. Tnis chromosome has a distinct bright band on each side-of the centromere. This chromosome shows, when compared with no. 11, a very faint overall fluorescence. The longest telocentric (t) chromoso>me shows four bright bands, separated from