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Incorporation of 3H-arginine in chromosomes of Vicia faba
Experimental
Cell Research
34, 525-532
525
(1964)
INCORPORATION OF 3H-ARGININE IN CHROMOSOMES OF VICIA FABA1 W. PRENSKY’ Biology
Department,
Brookhaven
and
H.
National
Received
H. SMITH
Laboratory,
June
Upton,
N.Y.,
U.S.A.
7, 1963
THESEstudies were undertaken to investigate the incorporation of 3H-arginine into nuciear protein of Vicia faba root meristem cells and to follow the distribution of the label through an additional replication cycle. Tritium labeled amino acids are incorporated into cytoplasmic as well as nuclear protein of V. faba [ 131. An autoradiographic analysis of nuclear protein replication and segregation would have to contend therefore with a high background level from cytoplasmically incorporated tritium. To overcome this difficulty our study was conducted under conditions designed to take advantage of the large number of chromosomes in division stages obtainable per root tip from a partial synchronization of cell divisions. The induction of partial synchrony by 5-aminouracil (5 AU) is briefly described below, and will be reported in greater detail in forthcoming papers. Woods and Duncan [14] and Duncan and Woods [3] found that mitosis is blocked in onion root tips after 5-6 hr of treatment with 5 AU. After 24 hr in 5 AU all but a few cells are in interphase with a 2 C content of DNA per nucleus. This work was followed up by Smith, Fussel and Kugelman [lo] who observed a high proportion (30-70 per cent) of dividing cells in V. fhba root meristem cells at about 14 hr after removal of the roots from a 700 ppm solution of 5 AU. DNA synthesis for this wave of divisions is primarily restricted to the first 8 hr after removal from 5 AU [7]. MATERIALS Three 12-day-old V. used as experimental 500 ppm, then treated TRA 40, prepared by
AND METHODS
faba seedlings with a large number of secondary roots were material. They were grown for 24 hr in a 5AU solution of with 3H-arginine (2 Ccc/ml, sp. act. about 100 mc/mM, Lot 2, RCC, Amersham) for 9.5 hr. After this period, the seedlings
1 Research carried out at Brookhaven Atomic Energy Commission. 2 Postdoctoral research fellowship (GF U.S. Public Health Service.
National 12602)
Laboratory from
the
under Division
the
auspices
of General
Experimental
of the
Medical
Cell
U.S.
Studies,
Research
34
W. Prensky
and H. H. Smith
were placed for 2.5 hr in a solution of 1Om4 M unlabeled arginine followed by 5 hr in colchicine (0.035 per cent). The first group of root tips was then fixed in ethanolacetic acid (3 : l), after a total elapse of 17 hr since removal from 5 AU, in order to obtain first division stages. The remaining seedlings were transferred to 0.50 strength Hoagland’s solution then placed in the same fixative 20 hr later to obtain chromosomes in the second mitotic division after labeling. Sufficient pressure was applied to Feulgen-stained root tips to rupture the cells ensuring an abundance of free floating mitotic chromosomes. Autoradiographs were prepared using Kodak NTB liquid emulsion and slides were developed after an exposure of 116 days. RESULTS The distribution of label in the first division after tritium incorporation was observed in cells of twelve root tips. Labeling was detected over the cytoplasm and all interphase and dividing nuclei (Figs. l-3). The squashing technique used resulted in a dispersal of the material under the cover glass, so that division figures, often incomplete (Figs. 2 A, 2 B), were found in areas relatively
free
of
cytoplasm.
Some
division
figures,
or
parts
of
them,
were
imbedded in extruded cytoplasm. Individual metaphase chromosomes were also found in areas both free of, and contaminated with, extruded cytoplasm (Fig. 3). Eight root tips were prepared for cytological study of the second division after labeling. Because of the action of colchicine, the tetraploid cells appearing in these root tips are those which have undergone a second cell division. Five had very few or no tetraploid cells, one had a mixed population of some tetraploid but mostly diploid division figures, and two had a very high frequency of tetraploid cells which made them satisfactory for studying second division stages. The striking aspect of the latter two was the negligible labeling found
over
tetraploid,
i.e.
second
division,
figures.
The
cytoplasm
of these
cells was, however,
still labeled (Figs. 4A, 4B). Isolated chromosomes
showed
little
(Fig.
labeled
or no labeling
5). In the other
six of the eight
Fig. l-3.-Autoradiographs from root tip cells of Vicia removal from 9.5 hr labeling with SH-arginine.
faba
showing
root
first
Fig. I.-Cell in early prophase showing labeled cytoplasm and nucleus. density of grains over the chromatin than over the cytoplasm. Fig. 2.-Parts of mitotic cytoplasm in 2 C. Fig. Q.-Single 3 D: metacentric Experimental
figures
metaphase chromosome, Cell Research
shown
free of cytoplasm
division
There
with
figures
appears
in 2 A and 2 B, and with
3 A-C: chromosomes chromosomes. heavily labeled at one end. 34
tips both
subterminal
7 hr after
to be a higher
extruded
labeled
centromere;
Incorporation
of 3H-arginine
in chromosomes
Experimental
527
Cell Research
34
528
W. Prensky
and H. H. Smith
and unlabeled nuclei were observed and some diploid cells and interphase nuclei were still fairly heavily labeled (Fig. 6). Grains were counted over individual metaphase chromosomes in first division figures of four root tips and in division figures in the two root tips that had a high frequency of tetraploid cells. Grains directly over the chromosomes and within one grain diameter from the chromosome margin were included in the chromosomal count. The values obtained from different root tips within the same group were not significantly different from each other. The combined data are presented in Fig. 7. The mean grain count over first division chromosomes was 15.6 with a standard deviation of 4.8, based on 217 observations; for second divisions the count was 2.6 22.7 grains per chromosome, based on 230 observations. The striking aspect of these data is that 108 chromosomes from the second division group had none or only one grain per chromosome. Since all cells appeared to be labeled in the first division, the high proportion of unlabeled chromosomes in the second division indicates that there may be a particular fraction of the chromosomal protein which turns over in one division cycle. 2n division stages, which had been labeled and fixed at the same time as 4N second division nuclei, but had not yet undergone a second division, failed to show a large loss of label (Fig. 6); therefore the phenomenon observed in 4N nuclei is not dependent on the time interval per se between labeling and fixation. A reasonable working hypothesis is that the loss of a certain protein fraction Srom the nucleus occurs during metabolic activation of the chromosomal complement as a whole, such as occurs when DNA is replicated prior to cell division.
DISCUSSION
A significant question pertinent to the interpretation of these results is: At what stage in the cell cycle was 3H-arginine administered to those chromosomes which were found to be unlabeled at second division? If the evidence is to be convincing that there was a complete turnover of an arginine-rich Figs. after
4-5.-Autoradiographs from removal from 9.5 hr labeling
Fig. 4. Second chromosomes. Fig.
5 A-D,
fabn
showing
second
division colchicine figures showing labeled cytoplasm and very 4 A is a late metaphase stage and 4 B early metaphase. single
Fig. 6.-Diploid as the 4N nuclei labeled. Experimental
root tip cells of Vicia with SH-arginine.
unlabeled
chromosomes
in labeled
34
few
figures grains
over
26 hr the
cytoplasm.
first division nuclei from root tips fixed at the same time and chromosomes shown in Figs. 4 and 5. Nuclei, chromosomes,
Cell Research
division
(26 hr after labeling) and cytoplasm are
Incorporation
of 3H-arginine
in chromosomes
Experimental
529
Cell Research
34
530
W. Prensky
and H. H. Smith
fraction during the division cycle, it must be division chromosomes were in fact labeled experiments [7], by means of 3H-thymidine treatment caused appr. 80 per cent of the blocked or greatly slowed DNL4 synthesis.
60: r 9 4o[ 20.10 0
proven that the unlabeled second during the first division. In other labeling, we found that 5 AU cells to accumulate in a stage of This value is roughly the same
Fig. 7.-Frequency over individual mitotic divisions
distributions chromosomes after treatment
in
of grains counted first and second with 3H-arginine.
0 4 8 12 16 20 24 NO OF CR91NSPER IELOCENTRICCriROMOSOME
as found by Howard and Pelt [4] f or a 24-hr period of continuous labeling with 32P and is that fraction of the total cell population which is undergoing active meristematic activity. The evidence strongly suggests, therefore, that the unlabeled chromosomes in tetraploid cells actually were in the S phase of the cell cycle when the 3H-arginine was administered. Some recent reports have dealt with the fate of nuclear protein during successive cell divisions. Removal of the cytoplasm from colchicine treated hamster tissue cells permitted Prescott and Render [9] to make observations with relatively little interference from labeled cytoplasm. They concluded that after a succession of four division cycles the label from six tritiated amino acids .\ as lost from the nuclear protein. Steffensen [ 111 using WS-labeled pollen grains studied the distribution of radioactivity after fertilization in embryo and endosperm nuclei. The conclusion was reached that some 35s protein was conserved in endosperm nuclei even after two cell divisions. In V. faba, Mattingly [6] found that during each of two successive 24-hr periods t”ere is a halving in the number of grains over nuclei that had been labeled for 15 min with 3H-lysine. Superficially, her results are somewhat contradictory to ours. However, the studies are not strictly comparable, since she examined nuclei from the terminal 5 mm of the root meristem in which the fraction of nuclei in the S phase was probably less than 30 per cent. In our experiments counts were made over individual chromosomes from a population of cells about 80 per cent of which were in the S phase during the labeling period. Our work and that of Mattingly also differs in that not the same protein may Experimental
Cell
Research
34
Incorporation
of 3H-arginine
in chromosomes
531
he measured in whole nuclei as in metaphase chromosomes. Goldstein [5] and Prescott [8] found that in amoebae a large fraction of the nucleo-protein migrates to the cytoplasm during mitosis. After mitosis most of this protein returns to the reconstituting nuclei. Our own observations do not exclude the possibility that whole nuclei are more heavily labeled with 3H-arginine than mitotic chromosomes. However, if there is a migration of 3H-arginine labeled protein in V. faba, it is less pronounced than in amoebae labeled with a mixture of tritiated amino acids. The identity of the protein which was labeled by the 3H-arginine is of considerable interest. Spectrophotometric measurements of nuclear histone by the fast-green method of Alfert and Geschwind [ 11 have shown that histone synthesis occurs in the nucleus during the period of DNA synthesis [a, 121. It is quite probable, therefore, that a major fraction of the tritium label we observed came from 3H-arginine which had become incorporated into a chromosomal histone. This would be a histone which is not attacked heavily by acid fixation and Feulgen hydrolysis. The extent to which fisation procedures alter the 3H-arginine content of nuclei is presently under investigation.
SUMMARY 3H-arginine was used in an effort to label the nuclear protein of Vicia faba root meristem cells. Prior to labeling, the root tips were treated for 24 hr with 5-aminouracil in order to synchronize cell divisions and obtain a large proportion of mitotic figures. Autoradiographs of chromosomes from the first and second division after labeling were examined. Almost 100 pel cent of the chromosomes in the first division were labeled. The marked absence of label in metaphase chromosomes at second division suggests turnover of a major fraction of nuclear protein during one cell division cycle. The authors wish to thank Drs. D. M. Steffensen, P. S. Woods, and D. E. Wimber for valuable discussions during the course of the study and Mr. Robert F. Smith for preparing the photographs. REFERENCES 1. ALFERT, M. and GESCHWIND, I. I., Proc. Nafl Acnd. Sci. U.S. 39, 991 (1953). 2. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 3. DUNCAN, R. E. and WOODS, P. S., Chromosoma 6, 45 (1953). 4. HOWARD, A. and PELC, S. R., E~ptl Cell Res. 2, 178 (1951). 5. GOLDSTEIN, L., Intern. Sot. Cell Biol. Symp. Vol. 2, p. 129. R. J. C. HARRIS Press, Inc., New York, 1963.
Experimental
(ed.).
Academic
Cell Research
34
W. Prensky
and H. H. Smith
6. MATTINGLY, SISTER AUGUSTINE, Expfl Cell Res. 29, 314 (1963). 7. PRENSKY, W. and SMITH, H. H., In preparation. 8. PRESCOTT, D. M., Intern. Sot. Cell Biol. Symp. Vol. 2, p, Ill. R. J. C. HARRIS (ed.). Academic Press, Inc., New York, 1963. 9. PRESCOTT, D. M. and BENDER, M. A., Abstr. 2nd Ann. Mfg. Am. Sot. Cell Biol. 146 (1963). 10. SMITH, H. H., FUSSEL, C. and KUGELMAN, B. H., Science 142, 595 (1963). 11. STEFFENSEN, D. M., Abstr. 2nd Ann. Mfg. Am. Sot. Cell Biol. 175 (1963). 12. WOODARD, J., RASCH, E. and SWIFT, H.; J. Biophys. Biochem. Cyfol. 9,‘445 (1961). 13. WOODS, P. S., Personal communication, 1958. 14. WOODS, P. S. and DUNCAX, R. E., Genetics 37, 636 (1952).
Experimental
Cell
Research
34
Cell Research
34, 525-532
525
(1964)
INCORPORATION OF 3H-ARGININE IN CHROMOSOMES OF VICIA FABA1 W. PRENSKY’ Biology
Department,
Brookhaven
and
H.
National
Received
H. SMITH
Laboratory,
June
Upton,
N.Y.,
U.S.A.
7, 1963
THESEstudies were undertaken to investigate the incorporation of 3H-arginine into nuciear protein of Vicia faba root meristem cells and to follow the distribution of the label through an additional replication cycle. Tritium labeled amino acids are incorporated into cytoplasmic as well as nuclear protein of V. faba [ 131. An autoradiographic analysis of nuclear protein replication and segregation would have to contend therefore with a high background level from cytoplasmically incorporated tritium. To overcome this difficulty our study was conducted under conditions designed to take advantage of the large number of chromosomes in division stages obtainable per root tip from a partial synchronization of cell divisions. The induction of partial synchrony by 5-aminouracil (5 AU) is briefly described below, and will be reported in greater detail in forthcoming papers. Woods and Duncan [14] and Duncan and Woods [3] found that mitosis is blocked in onion root tips after 5-6 hr of treatment with 5 AU. After 24 hr in 5 AU all but a few cells are in interphase with a 2 C content of DNA per nucleus. This work was followed up by Smith, Fussel and Kugelman [lo] who observed a high proportion (30-70 per cent) of dividing cells in V. fhba root meristem cells at about 14 hr after removal of the roots from a 700 ppm solution of 5 AU. DNA synthesis for this wave of divisions is primarily restricted to the first 8 hr after removal from 5 AU [7]. MATERIALS Three 12-day-old V. used as experimental 500 ppm, then treated TRA 40, prepared by
AND METHODS
faba seedlings with a large number of secondary roots were material. They were grown for 24 hr in a 5AU solution of with 3H-arginine (2 Ccc/ml, sp. act. about 100 mc/mM, Lot 2, RCC, Amersham) for 9.5 hr. After this period, the seedlings
1 Research carried out at Brookhaven Atomic Energy Commission. 2 Postdoctoral research fellowship (GF U.S. Public Health Service.
National 12602)
Laboratory from
the
under Division
the
auspices
of General
Experimental
of the
Medical
Cell
U.S.
Studies,
Research
34
W. Prensky
and H. H. Smith
were placed for 2.5 hr in a solution of 1Om4 M unlabeled arginine followed by 5 hr in colchicine (0.035 per cent). The first group of root tips was then fixed in ethanolacetic acid (3 : l), after a total elapse of 17 hr since removal from 5 AU, in order to obtain first division stages. The remaining seedlings were transferred to 0.50 strength Hoagland’s solution then placed in the same fixative 20 hr later to obtain chromosomes in the second mitotic division after labeling. Sufficient pressure was applied to Feulgen-stained root tips to rupture the cells ensuring an abundance of free floating mitotic chromosomes. Autoradiographs were prepared using Kodak NTB liquid emulsion and slides were developed after an exposure of 116 days. RESULTS The distribution of label in the first division after tritium incorporation was observed in cells of twelve root tips. Labeling was detected over the cytoplasm and all interphase and dividing nuclei (Figs. l-3). The squashing technique used resulted in a dispersal of the material under the cover glass, so that division figures, often incomplete (Figs. 2 A, 2 B), were found in areas relatively
free
of
cytoplasm.
Some
division
figures,
or
parts
of
them,
were
imbedded in extruded cytoplasm. Individual metaphase chromosomes were also found in areas both free of, and contaminated with, extruded cytoplasm (Fig. 3). Eight root tips were prepared for cytological study of the second division after labeling. Because of the action of colchicine, the tetraploid cells appearing in these root tips are those which have undergone a second cell division. Five had very few or no tetraploid cells, one had a mixed population of some tetraploid but mostly diploid division figures, and two had a very high frequency of tetraploid cells which made them satisfactory for studying second division stages. The striking aspect of the latter two was the negligible labeling found
over
tetraploid,
i.e.
second
division,
figures.
The
cytoplasm
of these
cells was, however,
still labeled (Figs. 4A, 4B). Isolated chromosomes
showed
little
(Fig.
labeled
or no labeling
5). In the other
six of the eight
Fig. l-3.-Autoradiographs from root tip cells of Vicia removal from 9.5 hr labeling with SH-arginine.
faba
showing
root
first
Fig. I.-Cell in early prophase showing labeled cytoplasm and nucleus. density of grains over the chromatin than over the cytoplasm. Fig. 2.-Parts of mitotic cytoplasm in 2 C. Fig. Q.-Single 3 D: metacentric Experimental
figures
metaphase chromosome, Cell Research
shown
free of cytoplasm
division
There
with
figures
appears
in 2 A and 2 B, and with
3 A-C: chromosomes chromosomes. heavily labeled at one end. 34
tips both
subterminal
7 hr after
to be a higher
extruded
labeled
centromere;
Incorporation
of 3H-arginine
in chromosomes
Experimental
527
Cell Research
34
528
W. Prensky
and H. H. Smith
and unlabeled nuclei were observed and some diploid cells and interphase nuclei were still fairly heavily labeled (Fig. 6). Grains were counted over individual metaphase chromosomes in first division figures of four root tips and in division figures in the two root tips that had a high frequency of tetraploid cells. Grains directly over the chromosomes and within one grain diameter from the chromosome margin were included in the chromosomal count. The values obtained from different root tips within the same group were not significantly different from each other. The combined data are presented in Fig. 7. The mean grain count over first division chromosomes was 15.6 with a standard deviation of 4.8, based on 217 observations; for second divisions the count was 2.6 22.7 grains per chromosome, based on 230 observations. The striking aspect of these data is that 108 chromosomes from the second division group had none or only one grain per chromosome. Since all cells appeared to be labeled in the first division, the high proportion of unlabeled chromosomes in the second division indicates that there may be a particular fraction of the chromosomal protein which turns over in one division cycle. 2n division stages, which had been labeled and fixed at the same time as 4N second division nuclei, but had not yet undergone a second division, failed to show a large loss of label (Fig. 6); therefore the phenomenon observed in 4N nuclei is not dependent on the time interval per se between labeling and fixation. A reasonable working hypothesis is that the loss of a certain protein fraction Srom the nucleus occurs during metabolic activation of the chromosomal complement as a whole, such as occurs when DNA is replicated prior to cell division.
DISCUSSION
A significant question pertinent to the interpretation of these results is: At what stage in the cell cycle was 3H-arginine administered to those chromosomes which were found to be unlabeled at second division? If the evidence is to be convincing that there was a complete turnover of an arginine-rich Figs. after
4-5.-Autoradiographs from removal from 9.5 hr labeling
Fig. 4. Second chromosomes. Fig.
5 A-D,
fabn
showing
second
division colchicine figures showing labeled cytoplasm and very 4 A is a late metaphase stage and 4 B early metaphase. single
Fig. 6.-Diploid as the 4N nuclei labeled. Experimental
root tip cells of Vicia with SH-arginine.
unlabeled
chromosomes
in labeled
34
few
figures grains
over
26 hr the
cytoplasm.
first division nuclei from root tips fixed at the same time and chromosomes shown in Figs. 4 and 5. Nuclei, chromosomes,
Cell Research
division
(26 hr after labeling) and cytoplasm are
Incorporation
of 3H-arginine
in chromosomes
Experimental
529
Cell Research
34
530
W. Prensky
and H. H. Smith
fraction during the division cycle, it must be division chromosomes were in fact labeled experiments [7], by means of 3H-thymidine treatment caused appr. 80 per cent of the blocked or greatly slowed DNL4 synthesis.
60: r 9 4o[ 20.10 0
proven that the unlabeled second during the first division. In other labeling, we found that 5 AU cells to accumulate in a stage of This value is roughly the same
Fig. 7.-Frequency over individual mitotic divisions
distributions chromosomes after treatment
in
of grains counted first and second with 3H-arginine.
0 4 8 12 16 20 24 NO OF CR91NSPER IELOCENTRICCriROMOSOME
as found by Howard and Pelt [4] f or a 24-hr period of continuous labeling with 32P and is that fraction of the total cell population which is undergoing active meristematic activity. The evidence strongly suggests, therefore, that the unlabeled chromosomes in tetraploid cells actually were in the S phase of the cell cycle when the 3H-arginine was administered. Some recent reports have dealt with the fate of nuclear protein during successive cell divisions. Removal of the cytoplasm from colchicine treated hamster tissue cells permitted Prescott and Render [9] to make observations with relatively little interference from labeled cytoplasm. They concluded that after a succession of four division cycles the label from six tritiated amino acids .\ as lost from the nuclear protein. Steffensen [ 111 using WS-labeled pollen grains studied the distribution of radioactivity after fertilization in embryo and endosperm nuclei. The conclusion was reached that some 35s protein was conserved in endosperm nuclei even after two cell divisions. In V. faba, Mattingly [6] found that during each of two successive 24-hr periods t”ere is a halving in the number of grains over nuclei that had been labeled for 15 min with 3H-lysine. Superficially, her results are somewhat contradictory to ours. However, the studies are not strictly comparable, since she examined nuclei from the terminal 5 mm of the root meristem in which the fraction of nuclei in the S phase was probably less than 30 per cent. In our experiments counts were made over individual chromosomes from a population of cells about 80 per cent of which were in the S phase during the labeling period. Our work and that of Mattingly also differs in that not the same protein may Experimental
Cell
Research
34
Incorporation
of 3H-arginine
in chromosomes
531
he measured in whole nuclei as in metaphase chromosomes. Goldstein [5] and Prescott [8] found that in amoebae a large fraction of the nucleo-protein migrates to the cytoplasm during mitosis. After mitosis most of this protein returns to the reconstituting nuclei. Our own observations do not exclude the possibility that whole nuclei are more heavily labeled with 3H-arginine than mitotic chromosomes. However, if there is a migration of 3H-arginine labeled protein in V. faba, it is less pronounced than in amoebae labeled with a mixture of tritiated amino acids. The identity of the protein which was labeled by the 3H-arginine is of considerable interest. Spectrophotometric measurements of nuclear histone by the fast-green method of Alfert and Geschwind [ 11 have shown that histone synthesis occurs in the nucleus during the period of DNA synthesis [a, 121. It is quite probable, therefore, that a major fraction of the tritium label we observed came from 3H-arginine which had become incorporated into a chromosomal histone. This would be a histone which is not attacked heavily by acid fixation and Feulgen hydrolysis. The extent to which fisation procedures alter the 3H-arginine content of nuclei is presently under investigation.
SUMMARY 3H-arginine was used in an effort to label the nuclear protein of Vicia faba root meristem cells. Prior to labeling, the root tips were treated for 24 hr with 5-aminouracil in order to synchronize cell divisions and obtain a large proportion of mitotic figures. Autoradiographs of chromosomes from the first and second division after labeling were examined. Almost 100 pel cent of the chromosomes in the first division were labeled. The marked absence of label in metaphase chromosomes at second division suggests turnover of a major fraction of nuclear protein during one cell division cycle. The authors wish to thank Drs. D. M. Steffensen, P. S. Woods, and D. E. Wimber for valuable discussions during the course of the study and Mr. Robert F. Smith for preparing the photographs. REFERENCES 1. ALFERT, M. and GESCHWIND, I. I., Proc. Nafl Acnd. Sci. U.S. 39, 991 (1953). 2. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 3. DUNCAN, R. E. and WOODS, P. S., Chromosoma 6, 45 (1953). 4. HOWARD, A. and PELC, S. R., E~ptl Cell Res. 2, 178 (1951). 5. GOLDSTEIN, L., Intern. Sot. Cell Biol. Symp. Vol. 2, p. 129. R. J. C. HARRIS Press, Inc., New York, 1963.
Experimental
(ed.).
Academic
Cell Research
34
W. Prensky
and H. H. Smith
6. MATTINGLY, SISTER AUGUSTINE, Expfl Cell Res. 29, 314 (1963). 7. PRENSKY, W. and SMITH, H. H., In preparation. 8. PRESCOTT, D. M., Intern. Sot. Cell Biol. Symp. Vol. 2, p, Ill. R. J. C. HARRIS (ed.). Academic Press, Inc., New York, 1963. 9. PRESCOTT, D. M. and BENDER, M. A., Abstr. 2nd Ann. Mfg. Am. Sot. Cell Biol. 146 (1963). 10. SMITH, H. H., FUSSEL, C. and KUGELMAN, B. H., Science 142, 595 (1963). 11. STEFFENSEN, D. M., Abstr. 2nd Ann. Mfg. Am. Sot. Cell Biol. 175 (1963). 12. WOODARD, J., RASCH, E. and SWIFT, H.; J. Biophys. Biochem. Cyfol. 9,‘445 (1961). 13. WOODS, P. S., Personal communication, 1958. 14. WOODS, P. S. and DUNCAX, R. E., Genetics 37, 636 (1952).
Experimental
Cell
Research
34