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The pattern of DNA synthesis in short-term leucocyte cultures of Protemnodon bicolor ♂
Exprrimenfal
Cell Research
38, 341-353
341
(1965)
THE PATTERN OF DNA SYNTHESIS IN SHORT-TERM LEUCOCYTE CULTURES PROTEMNODON BICOLOR 6 RUTH Radiobiological
Research
MOORE Unit,
and
Cancer
Received
JUNE
Institute
August
OF
UREN Roard,
Melbourne,
Australia
3, 1964
Is recent years, the use of tritiated thymidine has resulted in the accumulation of information on DNA synthesis in a variety of animals and plants. In plants, the pattern of DKA synthesis in Crepis [‘Ll] appears to be relatively simple, progressing from the ends of the chromosome to the centromerc. The same may be true in Vicicr [24]. In Trrrdescantirr, synthesis apparently terminates in the ends of the chromosomes [ZZ]. The situation in animals is more complex. In general, it has been found that chromosomes label asynchronously [2, 3, 8, 15, 16, 181 but the extent to lvhich this asynchrony (especially between homologues) follows an organized pattern has not been resolved, although there appears to be marked asynchrony between the allosomes and autosomes of one or both seses in the species so far examined [l, 4-7, 9, 12, 14, 19, 201. This paper presents data on the distribution of “hot” thymidine in peripheral blood cells of Protemnodon bicolor $ exposed to tritiated thynidine during part of the S-period and examined at the first metaphase. The metaphase chromosomes of P. bicolor have been described in detail elsewhere [lo, 111. It is sufficient to reiterate here that in the male, the apparent sex chromosome constitution is XYT,Y,, and it is thought that the true X chromosome is represented by the short arm of the large apparent S, the long arm of which is the homologue of the large acrocentric Y,. The long arm of the S chromosome is significantly longer than Y2, their % values [lOI being respectively 1.027 (s.n. = 0.030) and 1.009 (s.n. = 0.030), and possibly some sexchromatin is incorporated into the long arm of this chromosome. There are four pairs of autosomes (see Fig. 1). MATERIALS
AND METHOD
Samplesof venous blood from a male black-tailed term
cultures
using
the technique
of Moorhead
wallaby were used to set up short et al. [13]. After 66 hr in culture, Experimental
Cell
Research
38
342
Ruth Moore rrnd June lrren
2 ,uCi/ml of tritiated thymidine (specific activity 14.8 curies/mM, obtained from Amersham, England) was added. The cultures were terminated 4-9 hr after the addition of thymidine and 4-6 hr after the addition of colcemid. After fixation and washing, the cells were spread on slides and air-dried. Autoradiographs mere prepared using Ilford stripping film type I<& and exposed for times varying from 3 weeks to 6 months. After development of the film, the cells were stained with Harris’s Haematoxylin diluted 6 times, for 12-15 hr, washed and dried.
RESULTS
AND
DISCUSSION
After 4 hr with tritiated thymidine, some lightly-labelled metaphases were seen, and after 9 hr, a few prophases were unlabelled, indicating a variable (;, period. In human peripheral blood cullures, the S period has also been found to be long and variable [A:. As, under the conditions of the present experiment, like that of 13ader et crl. [‘L;, the incorporation of tritiated thymidine M-ould be continuous from the time of its introduction until the termination of synthesis, the DNA of the metaphase chromosomes will be of 2 types (1) unlabelled Dr\;,1 synthcsized before the addition of tritiated thymidine and (a) labelled 1)X-A synthesized after its addition. In other words, the absence of grains on the film in contact with any part of a chromosome, after a sufficient exposure time, indicates that DNA synthesis was completed in this region before the addition of “hot” thymidine, but no information is available about the time at \vhich DNA synthesis commenced in any region. Figs. ‘L-7 are metaphases from cultures exposed to tritiated thynidine for 1 hr (Fig. 3) or 7 hr (Figs. 2, 4-S) and exposed for 6 months. It can be seen that the autosomes complete synthesis at about the same time. In this respect they differ from human cells from short-term peripheral blood cultures [a, 41, diploid cell strains [121 and the aneuploid strain HeIJa However, within each chromosome, synthesis does not follow a rigid pattern. There is a strong tendency towards synchrony bet\veen homologues, although dissimilar patterns of labelling do occur. Occasionally, the same pattern of asynchrony between homologues is seen in two cells (e.g. chromosome 2 (Fig. 6&e), chromosome 3 (Figs. 4 b and Sd), chromosome 1 (Figs. 46, 5~, d, e). This is the kind of pattern described by Bader et al. [2] in human leucocgtes, but it is not a typical pattern. The variation of the extent of labelling between different autosomes is not much more marked than that between homologues. The sex-chromosomes show marked asynchrony between them and \vith Experimental
Cell
Research
38
343
2
1
4
3
X
Yl Y2
Fig.
l.-The
normal
karyotype
of I’. hicolor
$. The
autosomes
are numbered
1 to 4.
respect to autosomes. The small ‘I’, chromosome is not always yisible as it is easily obscured or lost during spreading. However, it can be seen in 26 of the 30 metaphases illustrated and in no case does it show evidence of tritium labelling. It apparently completes synthesis at a stage when the other chromosomes still have to undergo synthesis at many or possibly all sites. In peripheral blood cells Y, is positively heteropycnotic, being fully contracted during middle and late prophase, and visible as a small spot; in the first meiotic metaphase it is visible as a short rod [ 17 1. Experimental
Cell Research
38
Ruth Moore cd
June Wren
d
e
Fig.
2.
Figs. 2-7.-Autoradiographs of metaphases from peripheral blood cultures fixed after 4 hr (Fig. 3) or ‘7 hr (Figs. 2,4-7) in contact with tritiated thymidine, and a film exposure of 6 months. These cells completed DNA synthesis in the presence of the tritiated thymidine. They are arranged in order of increased labelling, the cells illustrated in the later plates having been in contact with tritiated thymidine for a progressively longer part of DNA synthesis. x 1800. Experimental
Cell Research
38
DXA synthesis
in Protemnodon
leucocytes
345
a
b
e
Fig.
3.
The short arm of the S, on the other hand, is still synthesizing I>h’A along its entire length late in the S period, as in all cells showing any evidence of autosomal labelling, it is heavily labelled. This situation differs from that in both human male peripheral blood [a, 5, 131, long term diploid cultured cells [12] and in a hamster male near-diploid cell strain [a]. Autosomnl labelling.-The pattern of autosomal labelling shows sufficient uniformity from cell to cell, enabling a sequence to be devised in which each 23 -
651813
Experimental
Cell
Resenrch
38
Ruth Moore and June Uren
346
Fig.
4.
chromosome is divided into several regions which differ with respect to the time they complete synthesis (see Text-fig. 1). Each chromosome has been divided into several regions, which are deExperimental
Cell Research
38
DNA synthesis
in Protemnodon
347
leucocytes
b
Fig.
5.
signated by the letter L or S (indicating that they are on the Long or Short arm of the chromosome) and numbered in order from the centromere. The pattern of grain distribution in prophases (Figs. 8) show that some at least of the areas which appear at metaphase as labelled blocks are comExperimental
Cell
Research
38
4
350
Ruth Moore and June Uren
chromosomes 2 are heavily labelled, the X-chromosome cannot be identified on the basis of the constriction in its short arm and it is identified on the basis of its length. As there is some overlap with chromosome 2, its identitication is not certain. However, although the pattern of DNA synthesis in the
1
2
3
4
X
u2
Text-fig. I.-Diagrammatic representation of the chromosomes of P. hicolor 6, with the division into the main regions which vary with respect to the time at which DNA replication is completed. The regions are specified by the-letter L or S (Long or Short arm) and are nlmbered consecutively from the centromere. X and Y are the allosomic regions.
long arm of chromosome 2 and XY2 may differ in detail, it is generally similar in the three metacentrics and the acrocentric Y2, and they will be discussed together. Regions L2 and 1~4 complete synthesis first, and, in chromosome 2, Sl is synthesized before S2. Regions 1~1, L3 and L5, and S2 in chromosome 2, are the last to be synthesized. Chromosome 3.-Regions Sl, L2 and L4 complete synthesis first, followed by L3 and L5. S2 or occasionally Ll is the last to complete synthesis. Chromosome Q.-Regions L2 and L4 complete synthesis early, followed by Sl and then L5. S2, Ll and L3 complete synthesis late. It can be postulated that DNA synthesis is inaugurated at a fixed number (probably a large number) of sites, and that the position and distribution of these sites is characteristic for each chromosome. Synthesis proceeds outwards from these points, possibly in the “zipperlike” fashion found in a simpler form in Crepis [al]. It is then not necessary to assume any other general controlling mechanism than the availability of DNA precursors at any point within the nucleus. The pattern of distribution of the initiating sites would result in an overall general pattern of synthesis, and varying rates of Experimental
Cell
Research
38
DNA
synthesis
in Protemnodon
leucocytes
351
Fig. 8.-Autoradiographs of prophases from cultures fixed after 4 hr contact with tritiated thymidine, and a film exposure of 6 months. It can be seen that in some parts of the chromosomes the grains are distributed in narrow bands across two chromatids. x 1300. Experimental
Cell
Research
38
352
Ruth Moore and June Uren
synthesis within each chromosome would reflect difl’erences in the geometry of the uncoiled synthesizing strands within the nucleus, with competition for UNA precursors between strands lying more or less close together during the S-period. Under these circumstances, the fewer and larger the chromosomes, the greater bvill he the probability that they will complete synthesis at about the same time, if each chromosome has a large number of initiating sites. Differences in the number of initiating sites could also bring about different rates of synthesis between chromosomes. In any particular cell type, gene function is regulated to the specific metabolic function of that particular cell type. This regulation of gene activity could be brought about by a pattern of DNA synthesis specific for the cell type. In the framework of the pattern of DNA synthesis proposed here, the initial sites of synthesis would vary somewhat between dif’ferent types of cells, and these variations could result in a different overall pattern of 11X-4 replication. The early completion of synthesis in 1’1 may be a result of its small size, or may be causally related to its positive heteropycnosis. It is apparent, however, that synthesis in the short (allosomic) arm of the S-chromosome is subject to some form of control different from that of the autosomes.
SUMMARY
Short-term peripheral blood cultures of Protemnodon bicolor 6 were grown in contact with tritiated thpmidine for several hours and autoradiographs were prepared of the chromosomes at the first metaphase. It was found that DE;,4 synthesis was completed at different times in different regions of each chromosome and an underlying pattern of DNA synthesis could be detected, although considerable variation was found.
REFERENCES
1.
ATKINS, L., TAFT, P. D. and DALAL, K. P., J. Cell. Biol. 15, 390 (1963). BADER, S., MILLER, 0. J. and MUKHERJEE, B. B., Ezptl Cell. Res. 31, 100 (1963). FICQ, A. and PAVAN, C., Radiation Res. 9, 165 (1958). GERMAN, J. L., Trans. N. Y. Acad. Sci. Series II 24, 395 (1962). GRUMBACH, M. M., MORISHIMA, A. and TAYLOR, J. H., Proc. Nat1 Aead. Sci. 49, 581 LIMA-DE-FARIA, A., J. Biophys. Biochem. Cytol. 6, 457 (1959). ~ Hereditas 47, 674 (1961). 8. LIMA-DE-FARIA, A. and REITALU, J., J. Cell. Biol. 16, 315 (1963). 9. LIMA-DE-FARIA, A., REITALU, J. and BERGMAN, S., Hereditas 47, 695 (1961). 10. MOORE, R. and GREGORY, G., Nature 200, 234 (1963). 11. ~ Intern. J. Radialion Biol. 7, 549 (1964).
2. 3. 4. 5. 6. 7.
Experimental
Cell
Research
38
(1962).
DNA synthesis
in Pro temnodon
leucocytes
353
P. S. and DEFENDI, V., J. Cell. Biol. 15, 390 (1962). P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. and HUKGERFORD, D. A., Exptl Cell Res. 20, 613 (1960). 14. MORISHIMA, A., GRUMBACH, M. M. and TAYLOR, J. H., Proc. Natt Acad. Sci. 48, 756 (1962). 15. PAINTER, R. B., J. Biophys. Biochem. Cytol. 11, 485 (1961). 16. PAVAN, C. and FICQ, A., Radiation Res. 9, 165 (1958). 17. SHARMAN, G., Aust. J. Zool. 9, 38 (1961). 18. STUBBLEFIELD, E. and MUELLER, G. C., Cancer Res. 22, 1991 (1962). 19. TAYLOR. J. H.. Ezntl Cell Res. 15, 350 (19581. 20. ~ J.‘Bioph&. kochem. Cyfol. ?, 45< (196h). 21. TAYLOR. J. H.. WOODS. P. S. and HUGHES, \V. L., Proc. Nat1 Acad. Sci. 43. I 122 (1957). \ 22. WHITE,‘M. J. b., Am. il’aturatist 94, 301 (iSSO). 23. WIMBER, D. E., Exptl Cell Res. 23, 402 (1961). 24. W~O~ARD, J., R~SCH, E. and SWIFT, H., J. Biophys. Biochem Cytot. 9, 445 (1961). 12. 13.
MOORHEAD, MOORHEAD,
Experimental
Cell Research
38
Cell Research
38, 341-353
341
(1965)
THE PATTERN OF DNA SYNTHESIS IN SHORT-TERM LEUCOCYTE CULTURES PROTEMNODON BICOLOR 6 RUTH Radiobiological
Research
MOORE Unit,
and
Cancer
Received
JUNE
Institute
August
OF
UREN Roard,
Melbourne,
Australia
3, 1964
Is recent years, the use of tritiated thymidine has resulted in the accumulation of information on DNA synthesis in a variety of animals and plants. In plants, the pattern of DKA synthesis in Crepis [‘Ll] appears to be relatively simple, progressing from the ends of the chromosome to the centromerc. The same may be true in Vicicr [24]. In Trrrdescantirr, synthesis apparently terminates in the ends of the chromosomes [ZZ]. The situation in animals is more complex. In general, it has been found that chromosomes label asynchronously [2, 3, 8, 15, 16, 181 but the extent to lvhich this asynchrony (especially between homologues) follows an organized pattern has not been resolved, although there appears to be marked asynchrony between the allosomes and autosomes of one or both seses in the species so far examined [l, 4-7, 9, 12, 14, 19, 201. This paper presents data on the distribution of “hot” thymidine in peripheral blood cells of Protemnodon bicolor $ exposed to tritiated thynidine during part of the S-period and examined at the first metaphase. The metaphase chromosomes of P. bicolor have been described in detail elsewhere [lo, 111. It is sufficient to reiterate here that in the male, the apparent sex chromosome constitution is XYT,Y,, and it is thought that the true X chromosome is represented by the short arm of the large apparent S, the long arm of which is the homologue of the large acrocentric Y,. The long arm of the S chromosome is significantly longer than Y2, their % values [lOI being respectively 1.027 (s.n. = 0.030) and 1.009 (s.n. = 0.030), and possibly some sexchromatin is incorporated into the long arm of this chromosome. There are four pairs of autosomes (see Fig. 1). MATERIALS
AND METHOD
Samplesof venous blood from a male black-tailed term
cultures
using
the technique
of Moorhead
wallaby were used to set up short et al. [13]. After 66 hr in culture, Experimental
Cell
Research
38
342
Ruth Moore rrnd June lrren
2 ,uCi/ml of tritiated thymidine (specific activity 14.8 curies/mM, obtained from Amersham, England) was added. The cultures were terminated 4-9 hr after the addition of thymidine and 4-6 hr after the addition of colcemid. After fixation and washing, the cells were spread on slides and air-dried. Autoradiographs mere prepared using Ilford stripping film type I<& and exposed for times varying from 3 weeks to 6 months. After development of the film, the cells were stained with Harris’s Haematoxylin diluted 6 times, for 12-15 hr, washed and dried.
RESULTS
AND
DISCUSSION
After 4 hr with tritiated thymidine, some lightly-labelled metaphases were seen, and after 9 hr, a few prophases were unlabelled, indicating a variable (;, period. In human peripheral blood cullures, the S period has also been found to be long and variable [A:. As, under the conditions of the present experiment, like that of 13ader et crl. [‘L;, the incorporation of tritiated thymidine M-ould be continuous from the time of its introduction until the termination of synthesis, the DNA of the metaphase chromosomes will be of 2 types (1) unlabelled Dr\;,1 synthcsized before the addition of tritiated thymidine and (a) labelled 1)X-A synthesized after its addition. In other words, the absence of grains on the film in contact with any part of a chromosome, after a sufficient exposure time, indicates that DNA synthesis was completed in this region before the addition of “hot” thymidine, but no information is available about the time at \vhich DNA synthesis commenced in any region. Figs. ‘L-7 are metaphases from cultures exposed to tritiated thynidine for 1 hr (Fig. 3) or 7 hr (Figs. 2, 4-S) and exposed for 6 months. It can be seen that the autosomes complete synthesis at about the same time. In this respect they differ from human cells from short-term peripheral blood cultures [a, 41, diploid cell strains [121 and the aneuploid strain HeIJa However, within each chromosome, synthesis does not follow a rigid pattern. There is a strong tendency towards synchrony bet\veen homologues, although dissimilar patterns of labelling do occur. Occasionally, the same pattern of asynchrony between homologues is seen in two cells (e.g. chromosome 2 (Fig. 6&e), chromosome 3 (Figs. 4 b and Sd), chromosome 1 (Figs. 46, 5~, d, e). This is the kind of pattern described by Bader et al. [2] in human leucocgtes, but it is not a typical pattern. The variation of the extent of labelling between different autosomes is not much more marked than that between homologues. The sex-chromosomes show marked asynchrony between them and \vith Experimental
Cell
Research
38
343
2
1
4
3
X
Yl Y2
Fig.
l.-The
normal
karyotype
of I’. hicolor
$. The
autosomes
are numbered
1 to 4.
respect to autosomes. The small ‘I’, chromosome is not always yisible as it is easily obscured or lost during spreading. However, it can be seen in 26 of the 30 metaphases illustrated and in no case does it show evidence of tritium labelling. It apparently completes synthesis at a stage when the other chromosomes still have to undergo synthesis at many or possibly all sites. In peripheral blood cells Y, is positively heteropycnotic, being fully contracted during middle and late prophase, and visible as a small spot; in the first meiotic metaphase it is visible as a short rod [ 17 1. Experimental
Cell Research
38
Ruth Moore cd
June Wren
d
e
Fig.
2.
Figs. 2-7.-Autoradiographs of metaphases from peripheral blood cultures fixed after 4 hr (Fig. 3) or ‘7 hr (Figs. 2,4-7) in contact with tritiated thymidine, and a film exposure of 6 months. These cells completed DNA synthesis in the presence of the tritiated thymidine. They are arranged in order of increased labelling, the cells illustrated in the later plates having been in contact with tritiated thymidine for a progressively longer part of DNA synthesis. x 1800. Experimental
Cell Research
38
DXA synthesis
in Protemnodon
leucocytes
345
a
b
e
Fig.
3.
The short arm of the S, on the other hand, is still synthesizing I>h’A along its entire length late in the S period, as in all cells showing any evidence of autosomal labelling, it is heavily labelled. This situation differs from that in both human male peripheral blood [a, 5, 131, long term diploid cultured cells [12] and in a hamster male near-diploid cell strain [a]. Autosomnl labelling.-The pattern of autosomal labelling shows sufficient uniformity from cell to cell, enabling a sequence to be devised in which each 23 -
651813
Experimental
Cell
Resenrch
38
Ruth Moore and June Uren
346
Fig.
4.
chromosome is divided into several regions which differ with respect to the time they complete synthesis (see Text-fig. 1). Each chromosome has been divided into several regions, which are deExperimental
Cell Research
38
DNA synthesis
in Protemnodon
347
leucocytes
b
Fig.
5.
signated by the letter L or S (indicating that they are on the Long or Short arm of the chromosome) and numbered in order from the centromere. The pattern of grain distribution in prophases (Figs. 8) show that some at least of the areas which appear at metaphase as labelled blocks are comExperimental
Cell
Research
38
4
350
Ruth Moore and June Uren
chromosomes 2 are heavily labelled, the X-chromosome cannot be identified on the basis of the constriction in its short arm and it is identified on the basis of its length. As there is some overlap with chromosome 2, its identitication is not certain. However, although the pattern of DNA synthesis in the
1
2
3
4
X
u2
Text-fig. I.-Diagrammatic representation of the chromosomes of P. hicolor 6, with the division into the main regions which vary with respect to the time at which DNA replication is completed. The regions are specified by the-letter L or S (Long or Short arm) and are nlmbered consecutively from the centromere. X and Y are the allosomic regions.
long arm of chromosome 2 and XY2 may differ in detail, it is generally similar in the three metacentrics and the acrocentric Y2, and they will be discussed together. Regions L2 and 1~4 complete synthesis first, and, in chromosome 2, Sl is synthesized before S2. Regions 1~1, L3 and L5, and S2 in chromosome 2, are the last to be synthesized. Chromosome 3.-Regions Sl, L2 and L4 complete synthesis first, followed by L3 and L5. S2 or occasionally Ll is the last to complete synthesis. Chromosome Q.-Regions L2 and L4 complete synthesis early, followed by Sl and then L5. S2, Ll and L3 complete synthesis late. It can be postulated that DNA synthesis is inaugurated at a fixed number (probably a large number) of sites, and that the position and distribution of these sites is characteristic for each chromosome. Synthesis proceeds outwards from these points, possibly in the “zipperlike” fashion found in a simpler form in Crepis [al]. It is then not necessary to assume any other general controlling mechanism than the availability of DNA precursors at any point within the nucleus. The pattern of distribution of the initiating sites would result in an overall general pattern of synthesis, and varying rates of Experimental
Cell
Research
38
DNA
synthesis
in Protemnodon
leucocytes
351
Fig. 8.-Autoradiographs of prophases from cultures fixed after 4 hr contact with tritiated thymidine, and a film exposure of 6 months. It can be seen that in some parts of the chromosomes the grains are distributed in narrow bands across two chromatids. x 1300. Experimental
Cell
Research
38
352
Ruth Moore and June Uren
synthesis within each chromosome would reflect difl’erences in the geometry of the uncoiled synthesizing strands within the nucleus, with competition for UNA precursors between strands lying more or less close together during the S-period. Under these circumstances, the fewer and larger the chromosomes, the greater bvill he the probability that they will complete synthesis at about the same time, if each chromosome has a large number of initiating sites. Differences in the number of initiating sites could also bring about different rates of synthesis between chromosomes. In any particular cell type, gene function is regulated to the specific metabolic function of that particular cell type. This regulation of gene activity could be brought about by a pattern of DNA synthesis specific for the cell type. In the framework of the pattern of DNA synthesis proposed here, the initial sites of synthesis would vary somewhat between dif’ferent types of cells, and these variations could result in a different overall pattern of 11X-4 replication. The early completion of synthesis in 1’1 may be a result of its small size, or may be causally related to its positive heteropycnosis. It is apparent, however, that synthesis in the short (allosomic) arm of the S-chromosome is subject to some form of control different from that of the autosomes.
SUMMARY
Short-term peripheral blood cultures of Protemnodon bicolor 6 were grown in contact with tritiated thpmidine for several hours and autoradiographs were prepared of the chromosomes at the first metaphase. It was found that DE;,4 synthesis was completed at different times in different regions of each chromosome and an underlying pattern of DNA synthesis could be detected, although considerable variation was found.
REFERENCES
1.
ATKINS, L., TAFT, P. D. and DALAL, K. P., J. Cell. Biol. 15, 390 (1963). BADER, S., MILLER, 0. J. and MUKHERJEE, B. B., Ezptl Cell. Res. 31, 100 (1963). FICQ, A. and PAVAN, C., Radiation Res. 9, 165 (1958). GERMAN, J. L., Trans. N. Y. Acad. Sci. Series II 24, 395 (1962). GRUMBACH, M. M., MORISHIMA, A. and TAYLOR, J. H., Proc. Nat1 Aead. Sci. 49, 581 LIMA-DE-FARIA, A., J. Biophys. Biochem. Cytol. 6, 457 (1959). ~ Hereditas 47, 674 (1961). 8. LIMA-DE-FARIA, A. and REITALU, J., J. Cell. Biol. 16, 315 (1963). 9. LIMA-DE-FARIA, A., REITALU, J. and BERGMAN, S., Hereditas 47, 695 (1961). 10. MOORE, R. and GREGORY, G., Nature 200, 234 (1963). 11. ~ Intern. J. Radialion Biol. 7, 549 (1964).
2. 3. 4. 5. 6. 7.
Experimental
Cell
Research
38
(1962).
DNA synthesis
in Pro temnodon
leucocytes
353
P. S. and DEFENDI, V., J. Cell. Biol. 15, 390 (1962). P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. and HUKGERFORD, D. A., Exptl Cell Res. 20, 613 (1960). 14. MORISHIMA, A., GRUMBACH, M. M. and TAYLOR, J. H., Proc. Natt Acad. Sci. 48, 756 (1962). 15. PAINTER, R. B., J. Biophys. Biochem. Cytol. 11, 485 (1961). 16. PAVAN, C. and FICQ, A., Radiation Res. 9, 165 (1958). 17. SHARMAN, G., Aust. J. Zool. 9, 38 (1961). 18. STUBBLEFIELD, E. and MUELLER, G. C., Cancer Res. 22, 1991 (1962). 19. TAYLOR. J. H.. Ezntl Cell Res. 15, 350 (19581. 20. ~ J.‘Bioph&. kochem. Cyfol. ?, 45< (196h). 21. TAYLOR. J. H.. WOODS. P. S. and HUGHES, \V. L., Proc. Nat1 Acad. Sci. 43. I 122 (1957). \ 22. WHITE,‘M. J. b., Am. il’aturatist 94, 301 (iSSO). 23. WIMBER, D. E., Exptl Cell Res. 23, 402 (1961). 24. W~O~ARD, J., R~SCH, E. and SWIFT, H., J. Biophys. Biochem Cytot. 9, 445 (1961). 12. 13.
MOORHEAD, MOORHEAD,
Experimental
Cell Research
38