- Email: [email protected]
DNA content of individual chromosomes of Protemnodon bicolor
IlAVA content of chromosomes of protemnotion
217
Resulfs.--Three experiments have so far been performed. The result of one of these is seen in Table I. It appears that the frequency of sex chromatin positive cells was statistically higher among labeled than among unlabeled cells on all three days of logarithmic growth investigated. The same result was obtained in the two other experiments. These results seem to invalidate the hypothesis that cells in S-phase are sex chromatin negative, but support the hypothesis that the frequency of sex chromatin positive cells is different in different intermitotic phases. This is also supported by two preliminary experiments with cultures synchronized by treatment for 16 hr with fluorodeoxyuridine in a concentration of lo-’ M.l The results of one of these experiments are shown in Fig. I. These experiments will be published in detail elsewhere. It is shown that a steep fall in the frequency of sex chromatin positive cells occurs at the same time as the first burst of mitoses. This might be explained if all postmitotic cells (G, cells) are sex chromatin negative as recently claimed by De Mars [ 11, or possibly if all cells are negative in late S-phase and G,. Experiments to test these possibilities are in progress.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
DE MARS, R., Science 146, 424 (1964). Abstract. JAMES, J., Z. Zellforsch. 64, 178 (1964). REITALU, J., Acta Pathol. MicrobioZ. Stand. 41, 257 (1957). THERKELSEN, A. J., Lancet i, 987 (1964). __ Cytogenefics 3, 207 (1964). __ Acta Pathol. Microbial. Stand. 61, 317 (1964). THERKELSEN, A. J. and PETERSEN, G. B., Exptl Cell Res.28,588
DNA
CONTENT
OF INDIVIDUAL
PROTEMNODON J. M. Radiobiological
CHROMOSOMES
OF
BICOLOR
RADLEY
Research Unit, Cancer Melbourne. Australia Received
(1962).
September
Institute
Board,
30, 1965
5LTUDIES
on individual chromosomes of metaphase spreads of cells of mammalian origin are greatly complicated both by the large number of chromosomes which usually constitute the karyotype and by the similarity in morphology of many chromosomes. Cells derived from the Chinese hamster are often used in chromosome studies because of their comparatively simple karyotype, the diploid cell having 22 1 Kindly 1.5 - 661800
supplied
by F. Hoffman-La
Roche
& Co.,
Basle. Experimental
Cell Research
41
J. M. Radley
218
Fig.
l.-Feulgen-stained
chrotnosome
spread,
peripheral-blood
culture
of Profemnodon
bicolor.
chromosomes, although some pairs are not easily distinguished [4]. An even more suitable mammal for chromosome studies is the black-tailed Wallaby (Protemnodon Kcolor), with a diploid chromosome number of 10 in the female and 11 in the male, all of which can be identified unequivocally (Fig. I). Its usefulness in cytogenetic studies has already been reported from this laboratory by Moore et al. [6, 7, 81, and it is likely to prove of greater value following the derivation and establishment of a near diploid cell line from a culture of fibroblasts obtained from this mammal [lo]. This paper reports the application of Feulgen microdensitometry to determine the relative amounts of DNA in individual chromosomes of the diploid Wallaby cell. The cells for this study were derived from a male black-tailed Wallaby. Chromosome spreads were prepared from cultures of both leucocytes and fibroblasts. The leucocytes were obtained from peripheral blood and cultured in the presence of phytohaemagglutinin for 3 days prior to harvesting. The fibroblasts were taken from the 7th passage of a serial tissue culture of explants of buccal mucosa obtained by biopsy. The method of Moorhead et al. [9] was used to obtain the chromosome spreads, which were later stained for DNA by the Feulgen method using pararosanaline dye. The relative amounts of DNA in the various chromosomes were measured at a wavelength of 560 rnp with an integrating microdensitometer [3], manufactured by Barr & Stroud. The requirements for well spread chromosomes are more critical for microdensitometry than for morphological analysis of karyotypes. A clear area must surround the chromosome being measured in order that the “penumbra” region of the image does not overlap that of other chromosomes or the edges of the field stops. To facilitate the isolation of a particular chromosome in the field from its neighbours the Experimenfal
Cell
Research
41
BNA content of chromosomes of protemnodon
219
densitometer was modified by replacing the circular aperture in the first image plane with a rectangular slit of 0.6 mm width which could be rotated in the image plane. The aperture size was selected after examining the voltage pattern at the integrator input during scanning of chromosomes. The number of metaphase spreads on a given slide in which all chromosomes are adequately spaced for densitometry is very limited. With the leucocyte material TABLE
I.
Chromosome
1
2
3
1
x
yz
Leucocytes
9b of total DPU’A S.D.
12.4 + 0.69
11.8 20.73
7.4 20.40
6.9 kO.58
12.6 LO.54
10.4 ri:0.38
Fibroblasts
% of total DNA
12.0 L-O.76
11.7 iO.50
7.3 kO.46
7.0 kO.38
12.7 iO.64
10.9 Iko.43
12.4 LO.71
12.0 kO.53
7.4 f0.44
7.0 kO.41
12.4 k 0.69
50.21
Cells
S.D.
Leucocytes [5, S]
% of total length S.D.
10.0
five such spreads were found and measured on one slide. In the case of the fibroblasts a different approach was adopted to expedite measurements. No attempt was made to find completely satisfactory spreads, instead each spread was examined and only those chromosomes easily isolated were measured, the stage then being moved on until values had been obtained for ten chromosomes of each type. Measurements were made only if all ten chromosomes were present in a spread and were of normal appearance. The eleventh chromosome (Y,) was ignored in the study since it is very small and frequently not seen, possibly because of its loss during spreading or because its presence is masked by another chromosome. Certain optical considerations are associated with the microdensitometry of small objects [l]. The limit in size of an object below which measurement is not practicable is determined by optical laws and has been dealt with by Caspersson [a]. In addition, in a scanning instrument the size of the scanning aperture in relation to the size of the image has to be considered [3]. If the aperture is of the same size or larger than the image then the recorded extinction will be too small, the error increasing with the magnitude of the true extinction. In these measurements the width of a chromosome arm was of the order of 1 ,Uand at the magnification employed ( x 70 objective, N.A. 1.3, and x 10 projection) the image width of the arm was approximately 0.7 mm whilst the width of the scanning aperture was 0.25 mm. The maximum extinction was of the order of 0.25. Prior to each measurement the angle between the long axis of the chromosome and the direction of travel of the scanning aperture was noted, but from analysis of the final results it was concluded the recorded value was independent of this angle. The results of the measurements are summarised in Table I. The mean DNA values and standard deviations in the leucocyte preparation were derived after exExperimentnl
Cell Research
41
I. Miyoshi,
220
S. Zrino and K. Hiraki
pressing each reading as a percentage of the total DNA in the spread and then determining the mean percentage over all the spreads for each type of chromosome. With the fibroblast preparation it was necessary to first determine the mean reading for each type of chromosome and then express this as a percentage of the total of the means. The two experimental approaches adopted gave identical results for the DNA contents of the individual chromosomes, with similar standard deviations. Also presented in Table I are the relative lengths of individual chromosomes determined by Moore [5, 61 from measurements on photographic enlargements of chromosome spreads of Wallaby leucocytes. Comparison of the DNA values with these results indicates a linear relationship between the DNA contents of chromosomes and their respective lengths for chromosome spreads of this species. REFERENCES
1. CARLSON, 2. 3. 4.
5. 6.
7. 8. 9. 10.
L., CASPERSSON, T., FOLEY, G. E., KUDYNO~SKI, .J., LOMAKKA, G., SIMONSSON, E. and S~RBN, L., Ezpfl Cell Res. 31, 589 (1963). CASPERSSON,T., Skand. Arch. Physiol. 73, Suppl. 8 (1936). DEELEY, E. M., J. Sci. Inst. 32, 263 (1955). Hsu, T. C., Canad. Cancer Conf. 5, 117 (1963). MOORE, R. C., Private communication. MOORE, R. C. and GREGORY, G., Nature 200, 234 (1963). ~ Inf. .I. Rad. Biol. 7, 549 (1963). MOORE, R. C. and UREN, J., Eqtl Cell Res. 38, 341 (1965). MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. and HUXGERFORD, D. A., Exptl Cell Res. 20, 613 (1960). UREN, J., MOORE, R. C. and SAW, K., To be published.
FIBROBLAST-LIKE
TRANSFORMATION
MARROW
FAT
I. MIYOSHI, Department
of Internal
S. IRINO
October
IN
BONE
VITRO
and K. HIRAKI
Medicine, Okayama Okayama, Japan Received
DEVELOPMENT
CELLS
OF HUMAN
University
Medical
School,
4, 1965
of fibroblast-like cells in cultures of human bone marrow has been reported by a number of investigators [l-5, 7, II]. These authors, however, included no description of the fate or behavior of bone marrow fat cells in vitro. The present communication concerns a morphological transformation of fat cells into fibroblastlike cells in cultures of bone marrow obtained from patients with non-leukemic hematologic disorders. Two ml of heparinized sternal bone marrow was aspirated from one patient with hemochromatosis and three patients with iron deficiency anemia and allowed to stand in a vertical position at 4°C for 2 hr. The plasma portion containing leukocytes,
Experimental
Cell
Research
41
217
Resulfs.--Three experiments have so far been performed. The result of one of these is seen in Table I. It appears that the frequency of sex chromatin positive cells was statistically higher among labeled than among unlabeled cells on all three days of logarithmic growth investigated. The same result was obtained in the two other experiments. These results seem to invalidate the hypothesis that cells in S-phase are sex chromatin negative, but support the hypothesis that the frequency of sex chromatin positive cells is different in different intermitotic phases. This is also supported by two preliminary experiments with cultures synchronized by treatment for 16 hr with fluorodeoxyuridine in a concentration of lo-’ M.l The results of one of these experiments are shown in Fig. I. These experiments will be published in detail elsewhere. It is shown that a steep fall in the frequency of sex chromatin positive cells occurs at the same time as the first burst of mitoses. This might be explained if all postmitotic cells (G, cells) are sex chromatin negative as recently claimed by De Mars [ 11, or possibly if all cells are negative in late S-phase and G,. Experiments to test these possibilities are in progress.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
DE MARS, R., Science 146, 424 (1964). Abstract. JAMES, J., Z. Zellforsch. 64, 178 (1964). REITALU, J., Acta Pathol. MicrobioZ. Stand. 41, 257 (1957). THERKELSEN, A. J., Lancet i, 987 (1964). __ Cytogenefics 3, 207 (1964). __ Acta Pathol. Microbial. Stand. 61, 317 (1964). THERKELSEN, A. J. and PETERSEN, G. B., Exptl Cell Res.28,588
DNA
CONTENT
OF INDIVIDUAL
PROTEMNODON J. M. Radiobiological
CHROMOSOMES
OF
BICOLOR
RADLEY
Research Unit, Cancer Melbourne. Australia Received
(1962).
September
Institute
Board,
30, 1965
5LTUDIES
on individual chromosomes of metaphase spreads of cells of mammalian origin are greatly complicated both by the large number of chromosomes which usually constitute the karyotype and by the similarity in morphology of many chromosomes. Cells derived from the Chinese hamster are often used in chromosome studies because of their comparatively simple karyotype, the diploid cell having 22 1 Kindly 1.5 - 661800
supplied
by F. Hoffman-La
Roche
& Co.,
Basle. Experimental
Cell Research
41
J. M. Radley
218
Fig.
l.-Feulgen-stained
chrotnosome
spread,
peripheral-blood
culture
of Profemnodon
bicolor.
chromosomes, although some pairs are not easily distinguished [4]. An even more suitable mammal for chromosome studies is the black-tailed Wallaby (Protemnodon Kcolor), with a diploid chromosome number of 10 in the female and 11 in the male, all of which can be identified unequivocally (Fig. I). Its usefulness in cytogenetic studies has already been reported from this laboratory by Moore et al. [6, 7, 81, and it is likely to prove of greater value following the derivation and establishment of a near diploid cell line from a culture of fibroblasts obtained from this mammal [lo]. This paper reports the application of Feulgen microdensitometry to determine the relative amounts of DNA in individual chromosomes of the diploid Wallaby cell. The cells for this study were derived from a male black-tailed Wallaby. Chromosome spreads were prepared from cultures of both leucocytes and fibroblasts. The leucocytes were obtained from peripheral blood and cultured in the presence of phytohaemagglutinin for 3 days prior to harvesting. The fibroblasts were taken from the 7th passage of a serial tissue culture of explants of buccal mucosa obtained by biopsy. The method of Moorhead et al. [9] was used to obtain the chromosome spreads, which were later stained for DNA by the Feulgen method using pararosanaline dye. The relative amounts of DNA in the various chromosomes were measured at a wavelength of 560 rnp with an integrating microdensitometer [3], manufactured by Barr & Stroud. The requirements for well spread chromosomes are more critical for microdensitometry than for morphological analysis of karyotypes. A clear area must surround the chromosome being measured in order that the “penumbra” region of the image does not overlap that of other chromosomes or the edges of the field stops. To facilitate the isolation of a particular chromosome in the field from its neighbours the Experimenfal
Cell
Research
41
BNA content of chromosomes of protemnodon
219
densitometer was modified by replacing the circular aperture in the first image plane with a rectangular slit of 0.6 mm width which could be rotated in the image plane. The aperture size was selected after examining the voltage pattern at the integrator input during scanning of chromosomes. The number of metaphase spreads on a given slide in which all chromosomes are adequately spaced for densitometry is very limited. With the leucocyte material TABLE
I.
Chromosome
1
2
3
1
x
yz
Leucocytes
9b of total DPU’A S.D.
12.4 + 0.69
11.8 20.73
7.4 20.40
6.9 kO.58
12.6 LO.54
10.4 ri:0.38
Fibroblasts
% of total DNA
12.0 L-O.76
11.7 iO.50
7.3 kO.46
7.0 kO.38
12.7 iO.64
10.9 Iko.43
12.4 LO.71
12.0 kO.53
7.4 f0.44
7.0 kO.41
12.4 k 0.69
50.21
Cells
S.D.
Leucocytes [5, S]
% of total length S.D.
10.0
five such spreads were found and measured on one slide. In the case of the fibroblasts a different approach was adopted to expedite measurements. No attempt was made to find completely satisfactory spreads, instead each spread was examined and only those chromosomes easily isolated were measured, the stage then being moved on until values had been obtained for ten chromosomes of each type. Measurements were made only if all ten chromosomes were present in a spread and were of normal appearance. The eleventh chromosome (Y,) was ignored in the study since it is very small and frequently not seen, possibly because of its loss during spreading or because its presence is masked by another chromosome. Certain optical considerations are associated with the microdensitometry of small objects [l]. The limit in size of an object below which measurement is not practicable is determined by optical laws and has been dealt with by Caspersson [a]. In addition, in a scanning instrument the size of the scanning aperture in relation to the size of the image has to be considered [3]. If the aperture is of the same size or larger than the image then the recorded extinction will be too small, the error increasing with the magnitude of the true extinction. In these measurements the width of a chromosome arm was of the order of 1 ,Uand at the magnification employed ( x 70 objective, N.A. 1.3, and x 10 projection) the image width of the arm was approximately 0.7 mm whilst the width of the scanning aperture was 0.25 mm. The maximum extinction was of the order of 0.25. Prior to each measurement the angle between the long axis of the chromosome and the direction of travel of the scanning aperture was noted, but from analysis of the final results it was concluded the recorded value was independent of this angle. The results of the measurements are summarised in Table I. The mean DNA values and standard deviations in the leucocyte preparation were derived after exExperimentnl
Cell Research
41
I. Miyoshi,
220
S. Zrino and K. Hiraki
pressing each reading as a percentage of the total DNA in the spread and then determining the mean percentage over all the spreads for each type of chromosome. With the fibroblast preparation it was necessary to first determine the mean reading for each type of chromosome and then express this as a percentage of the total of the means. The two experimental approaches adopted gave identical results for the DNA contents of the individual chromosomes, with similar standard deviations. Also presented in Table I are the relative lengths of individual chromosomes determined by Moore [5, 61 from measurements on photographic enlargements of chromosome spreads of Wallaby leucocytes. Comparison of the DNA values with these results indicates a linear relationship between the DNA contents of chromosomes and their respective lengths for chromosome spreads of this species. REFERENCES
1. CARLSON, 2. 3. 4.
5. 6.
7. 8. 9. 10.
L., CASPERSSON, T., FOLEY, G. E., KUDYNO~SKI, .J., LOMAKKA, G., SIMONSSON, E. and S~RBN, L., Ezpfl Cell Res. 31, 589 (1963). CASPERSSON,T., Skand. Arch. Physiol. 73, Suppl. 8 (1936). DEELEY, E. M., J. Sci. Inst. 32, 263 (1955). Hsu, T. C., Canad. Cancer Conf. 5, 117 (1963). MOORE, R. C., Private communication. MOORE, R. C. and GREGORY, G., Nature 200, 234 (1963). ~ Inf. .I. Rad. Biol. 7, 549 (1963). MOORE, R. C. and UREN, J., Eqtl Cell Res. 38, 341 (1965). MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. and HUXGERFORD, D. A., Exptl Cell Res. 20, 613 (1960). UREN, J., MOORE, R. C. and SAW, K., To be published.
FIBROBLAST-LIKE
TRANSFORMATION
MARROW
FAT
I. MIYOSHI, Department
of Internal
S. IRINO
October
IN
BONE
VITRO
and K. HIRAKI
Medicine, Okayama Okayama, Japan Received
DEVELOPMENT
CELLS
OF HUMAN
University
Medical
School,
4, 1965
of fibroblast-like cells in cultures of human bone marrow has been reported by a number of investigators [l-5, 7, II]. These authors, however, included no description of the fate or behavior of bone marrow fat cells in vitro. The present communication concerns a morphological transformation of fat cells into fibroblastlike cells in cultures of bone marrow obtained from patients with non-leukemic hematologic disorders. Two ml of heparinized sternal bone marrow was aspirated from one patient with hemochromatosis and three patients with iron deficiency anemia and allowed to stand in a vertical position at 4°C for 2 hr. The plasma portion containing leukocytes,
Experimental
Cell
Research
41