- Email: [email protected]
Investigations of the DNA content, base composition and chromatography on ECTEOLA-cellulose of normal and tumor DNA preparations
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
87, 330-336
(1960)
Investigations of the DNA Con#ent, Base Composition and Chromatography on ECTEOLA-Cellulose of Normal and Tumor DNA Preparations’ SAUL From the Section of Nucleoprotein Texas M. D. Anderson Hospital
Metabolism, and Tumor Received
KIT
Department of Biochemistry, The University of Institute, Texas Medical Center, Houston, Texas November
2, 1959
The total deoxyribonucleic acid (DNA) content per cell, the ratios of the purine and pyrimidine bases, and the chromatographic profiles on ECTEOLA-cellulose anion exchangers of DNA preparations from normal mouse spleen, spontaneous lymphomas, diploid and tetraploid lymphomas and carcinomas, and amelanotic and melanotic melanomas have been investigated. The total DNA content was related to the chromosome numbers of the tumor cells. The DNA chromatographic profiles of the above tissues were not significantly different. The molar base ratios of the purines and pyrimidines were a&o quite iimilar.
ECTEOLA-cellulose anion exchangers into groups of molecules which d8er in their viscosity and sedimentation coefficients (l3), and, presumably, in molecular weight. The results of similar fractionations are also presented. These results show that the DNA chromatographic profiles of transplanted tumors, spontaneous leukemias of mice, and various normal tissues of the mouse and the rat are similar.
INTRODUCTION
It has been postulated that differences in the structure or composition of the deoxyribonucleic acids (DNA) distinguish normal tissues from tumors. Such differences might take one or more of several forms. There might be changes in: (a) the total amount of DNA per cell, (6) the molecular size distribution of the DNA per cell, (c) the purine and pyrimidine base composition, (d) the nucleotide sequence of bases along the polynucleotide chains, (e) the relative proportions of the various types of DNA molecules within a given cell, or (f) the organization of DNA molecules within the chromosomes. Data are as yet unavailable with regard to the latter three possibilities. The first three points will be discussed in the present paper with respect to spontan&ous and transplanted tumors of mice. It is shown that although the amounts of DNA per cell are increased in some transplanted mouse tumors, the purine and pyrimidine base compositions are very similar. DNA preparations may be fractionated on 1 Aided in part by Grants from Cancer Society (P-35), the Leukemia and the National Cancer Institute
METHODS
TUMORS AND NORMAL TISSUES DNA was prepared from the following transplanted tumors of mice : diploid ascites lymphomas 6C3HED and E9514A (C3H mice), tetraploid ascites lymphomas 6C3HED-DBA-2 (C3H or DBA-2 mice), the Lettre-Ehrlich hyperdiploid and the Ehrlich hypotetraploid ascites carcinomas (Swiss mice), the Cloudman melanotic melanoma (S91), and the Cloudman amelanotic melanoma (S91A) (DBA-2 mice). The ascites tumors were harvested after 6-13 days of growth in the intraperitoneal cavities of the mice. Melanoma-bearing animals were sacrificed aft,er 1-2 months of tumor growth. Care was taken that no grossly necrotic melanoma be included in the tissue samples of the latter tumors. Spontaneous lymphatic leukemic tissue was ob-
the American Society, Inc., (C-4238). 330
-_
1N
-----
-..-
.
---.
_”
Y Jusrlw~I’l”ND
#.-
tamed from AKr mice. The incidence of spontaneous leukemia in &la-month-old female animals of the Houston colony is about SO-85%. Leukemic nodes from the thymus, axilla, and mesentery of 2-5 animals were pooled for each DNA preparation. Normal thymusorspleens of B-weekor 4-monthold Akr mice or of adult C3H and DBA-2 mice were rapidly dissected from the animals and pooled. A maximum of 20 min. was required for the dissection. In some instances, the animals were sacrificed and the tissues excised in the cold room (4”), but, in all cases, the tissues were kept ice cold until DNA extraction was begun. All experimental animals were fed ad Zibituvn prior to sacrifice.
PRE~PARATION OF DNA AND CHROMATOGRAPHY The method of Kirby was used to prepare DNA from the above tissues (4) with the modifications described previously (5). The chromatography on ECTEOLA-caellulose (Brown Co., Berlin, N. H.; 0.39 meq./g. exhange capacity; nitrogen content, 0.55%, type 20) was as discussed in the previous paper (5). Approximately 2.5-3.5 mg. DNA (equivalent to 50-70,000 optical density units at 258 mF)Z was dissolved in 0.05 M NaC1-0.001 M phosphate buffer pH 7 and applied to 500 mg. ECTEOLAcellulose columns. A discontinuous elution schedule with eluting solutions consisting of increasing concentrations of NaCl and alkaline pH was employed to elute the DNA from the columns [see Ref. (5), Table I]. Five-milliliter fractions were collected by means of a Rinco automatic fraction collector (constant-volume operation) over a period of 3 days. The solvents passed through the column under the force of gravity at an average rate of 5-10 ml./hr. The amount of DNA eluted was measured spectrophotometrically at 258 mp by means of a Beckman DK-2 recording spectrophotometer.
ANALYSIS
OF TOTAL AND BASE
THE
DNA
331
CONTENT
VP’
DNA PER CELL RATIOS
The total DNA per cell was determined by the Burton modification of the diphenylamine reaction (6). All analyses were performed at three tissue concentrations, and standard curves were run for each experiment. Deoxyribose solutions were used as stock standards. Standard curves 2 Optical density units were defined as the optical density observed with the Beckman DK-2 spectrophotometer at 258-260 rnr for a l-cm. light path X 103 X the volume, in ml., of the solution applied to the columns.
were also constructed from analyses of highly polymerized calf-thymus DNA (Mann Assayed Biochemicals), and the conversion factors for the two curves were calculated. Tumor cell counts were routinely made at two dilutions on cells suspended in ascites fluid containing saline and heparin, using a Levy ultraplane, improved Neubauer counting chamber (C. A. Hausser and Sons, Philadelphia, Pa). The purine and pyrimidine composition of the DNA samples were determined by paper chromatography and spectrophotometry (7). From 2 to 6 mg. DNA was hydrolyzed with 12 N perchloric acid, and quadruplicate aliquots were chromatographed on Whatman B3MM filter paper, using isopropyl alcohol-HCl-water (1300:332:368, by volume) as the solvent. Standards at three connentrations were simultaneously chromatographed for each of the four bases. The purines and pyrimidines were eluted from the papers with 5 ml. of 0.1 N HCI, and the optical density was recorded between 300 and 230 rnp. Blank paper strips opposite each of the bases were cut from the papers, and the optical density of each of the blanks was substracted from the optical densities of the corresponding bases. The molecular extinction coefficients of the adenine, guanine, cytosine, and thymine standards agreed closely with those reported in the literature (8). RESULTS
AND
DNA
CONTENT
DISCUSSION
PER CELL
It is now well known that the DNA content of most cells of a given organism is approximately twice that found in sperm cells (Class I cells) and that many tissues contain cells whose content of DNA is four times (Class 11) or eight times (Class III) that of the chromatids (9, 10). Values differing from the speciesspecific constancy can generally be accounted for on the basis of: (a) polyploidy or polyteny, (b) aneuploidy or heteroploidy, and (c) the premitotic replication of DNA by dividing cells. The cellular DNA content and the modal chromosomal numbers of mouse lymphomas, carcinomas, and melanomasare presented in Table I. The DNA content of normal spleen cells is also shown for comparison. The DNA content is proportional to the chromosome number in the case of the Lettre-Ehrlich and Ehrlich carcinomas. A similar relationship obtains in the case of the E9514A diploid lymphoma and the -two be’craploid lymphomas. The
332
SAUL
TABLE DNA CONTENT NUMBERS
TlUtl0r
I
AND MODAL
agreement (25-28). The increased DNA content of tumor cells thus appears to be a manifestation of the gross remodeling of the chromosome complex which takes place during the neoplastic progression (26, 27).
CHROMOSOME
OFMOUSETUMORS DNA 10' ce i1: TYPO Y _-
E9514A (diploid) 6C3HED (diploid) 6C3HED-DBA-2 (hypotetraploid) GCSHED-DBA-2 to C3H (hypotetraploid)
Lymphoma Lymphoma Lymphoma
7.9 9.8 15.3
KIT
40 76-78
PURINE AND PYRIMIDINE
BASE
RATIOS
The molar ratios of the DNA purine and pyrimidines of diploid and tetraploid lymphomas and carcinomas, two melanomas, Lymphoma 14.2 74-79 mouse thymus, and of spleens from three mouse strains are shown in Table II. The ratio of purines to pyrimidines and the ratio of adenine plus cytosine to guanine plus Lettre-Ehrlich (hy- Carcinoma 11.1 46 thymine (6-am: 6 keto) is in each case very perdiploid) closeto 1.0. The ratios of adenine to thymine Ehrlich (hypotetraCarcinoma 18.0 78 for all of the samples vary from 0.91: 1 to ploid) 1.05: 1, whereas the ratios of guanine to Cloudman S91 (tet- Melanotic 16.6 cytosine vary from 0.89: 1 to 1.05: 1. The raploid) Melanoma theoretical ratios of all of the above paramCloudman S91A Amelanotic 17.1 eters predicted on the basis of the double(tetraploid) Melanoma stranded Watson-Crick model for the structure of DNA (29) are 1.0. Taking into Normal spleen cells 8.3 40 consideration the experimental errors of the analyses, the deviations from the theoretical / DNA content of diploid lymphoma, 6C3- values seem small. HED, is approximately 24% above the The ratio of A -I- T/G -I- C was observed diploid value (11). Both the amelanotic and to be about 1.24 for the normal tissues and the melanotic melanomas contain the all of the tumors. Diploid tumors differed but little from tetraploid tumors with respect amount of DNA characteristic of tetraploid to the purine and pyrimidine baseratios. The cells. The results here presented are in agree- observed differences between the melanomas, ment with the spectrophotometric or chemi- carcinomas, or lymphomas were also very cal determinations of the DNA of human and small. The highest observed guanine, cytoanimal tumors reported by various investisine, and thymine values differed from the gators. These have shown that primary lowest by 12, 19, and 15%, respectively. hepatomas (12, 13) and lymphomas (14,21), When analyzed for significant differences carcinomas (22), or plant tumors (23,24) and using the table of t of Fisher (40), only five some transplanted lymphomas (17-21) may of the latter values were statistically difcontain, on the average, the normal diploid ferent at the 2% level of significance. The amount of DNA or slightly above this value. guanine content of tumor 6C3HED was Other transplanted lymphomas (11, 14), significantly greater than that of DBA spleen various carcinomas, and carcinomas which or the Lettre carcinoma, but the observed have been transferred for many years (25, increaseswere only 7 and 5 %, respectively. 26), and metastatic human carcinoma cells The cytosine content of tumor S91 was 6 % may have an average DNA content in the greater than that of AKr thymus, and the polyploid range or may manifest intermedithymine content of tumor S91A was 8% ate values. When chromosome studies and greater than that of DBA spleen or tumor DNA determinations have been performed 6C3HED-DBA-2. Of the above five statison individual nuclei of the same cell popula- tically significant differences, three involve tion, the chromosome spread and DNA varitissuesin which the determinations were only ation from the modal value are in good performed two times. It is conceivable that
INVESTIGATIONS
OF
THE
TABLE DNA
BASE
RATIOS
Numbers in parentheses represent guanine, C = cytosine, T thymine, 6-keto = guanine plus thymine.
molar
base
ratios
C
CELLULOSE
A+T G+C
T
PU pyr
6 am 6 keto
f zk f f f
.038 .OlO ,025 ,090 .031
0.78 0.73 0.79 0.81 0.80
& 0.60 f .090 f .028 f ,002 f .032
0.95 0.91 0.96 0.94 1.01
i f f f f
.020 ,010 .023 .180 ,053
1.27 1.28 1.24 1.17 1.26
1.02 1.07 1.02 1.06 0.99
1.04 1.04 1.02 1.01 0.99
0.82 0.81 0.79 0.79 0.80 0.83 0.77 0.77
i i i h zk ziz f f
.038 .007 .014 3020 .026 .047 ,008 ,017
0.82 0.80 0.87 0.85 0.86 0.84 0.87 0.85
f f. f f f. z!z f f
1.05 1.00 0.98 0.91 0.98 1.04 0.96 0.96
f f f f f f f f
.044 .030 ,040 ,026 ,030 .036 .017 .027
1.25 1.24 1.19 1.16 1.19 1.22 1.27 1.21
0.97 1.01 0.97 1.02 0.98 0.97 0.97 0.98
0.97 0.99 1.06 1.09 1.04 0.99 1.08 1.07
are exnressed
ON
AND TUMORS Abbreviations: A = Adenine, G = 6 am = adenine plus cytosine,
0.76 0.76 0.79 0.85 0.80
relative
the above small differences are biologically important. However, further evidence is necessary before this conclusion can be drawn. In the meantime, interpretation as to real differences should be made with extreme caution. The purine and pyrimidine base ratios presented above agree closely with those reported by Laland and associates (30) for mouse sarcoma cells. No significant differences in DNA base composition were observed between human hepatoma and normal liver ((29) or normal and leukemic human spleen tissue (31). CHROMATOGRAPHY
TISSUES
base ratid
G
C3H spleen (4) DBA spleen (‘2) Spleen AKr (7) Thymus AKr (2) Spontaneous leukemia (AKr) (11) E9514A (4) 6C3HED (4) GCSHED-DBAS to C3H (7) , GC3HED/DBA-2 (5) s91 (10) S91A (7) Lettre (5) Ehrlich (7)
II MOUSE
Molar
333
CONTENT
the number of determinations. Pu = purines, pyr = pyrimidines,
Tissue
m The
OF NORMAL
DNA
ECTEOLA-
COLUMNS
The DNA chromatographic elution profiles on ECTEOLA-cellulose of normal AKr, C3H, and DBA-2 mouse spleen and of spontaneous AKr leukemia, diploid and tetraploid ascites lymphomas (C3H or DBA-2 mice), diploid and tetraploid strains of the Ehrlich carcinomas (Swiss mice), and melanotic or amelanotic melanomas (DBA-2 mice) are shown in Figs. l-3. Details con-
,080 .060 ,050 .016 .015 .032 .026 ,020
to adenine
= 1.00 f
the
standard
error
of the
mean
cerning the chromatographic procedures and a critical evaluation of the factors affecting the chromatography are presented in the companion paper (5). In Figs. 1-3, the cumulative per cent of DNA eluted from the columns is plotted as a functionof the solvent used for elution. The composition of the eluants is given in the footnote to Fig. 1 [see also Ref. (5), Table I]. Approximately 10 % of the DNA is eluted by 0.6 M NaC1-0.001 M phosphate buffer, pH 7 (Fraction 3). Two large peaks are eluted by 2 M NaCl, 0.2 M and 0.3 M NH3 solutions (Fractions 7 and S), and several smaller peaks in Fractions 5, 6, 9, and 10. Approximately 75 % of the DNA was eluted in Fractions 6-10. Figures l-3 show that the DNA elution profiles are rather similar in all cases. The chromatographic profile of the spontaneous AKr lymphoma does not differ from that of normal AKr mouse spleen cells (Fig. 1) nor do the transplanted tetraploid lymphomas differ greatly from C3H or DBA-2 mouse spleen cells (Fig. 2). The variation shown in Fig. 2 between the diploid and tetraploid
334
SAUL
KIT
-MOUSE SPLEEN a---- 6C3HED-TETRAPLOID a..,,a..a.,E96,4A --BCIHED-DIPLOID _
tKr SPLEEN SPONTANEOUS LEUKEMIA (AKrJ
FRACTION
NUMBER
2. DNA chromatographic elution profiles on ECTEOLA-cellulose of C3H and DBA mouse spleen, diploid lymphomas E9514A, and 6C3HED, and tetraploid lymphoma 6C3HED-2-DBA. The average recovery from the columns of four determinations with the spleen DNA, two determinations with the diploid E9514A or 6C3HED DNA, and three determinations with tetraploid 6C3NED DNA were 103, 85, and 9370, respectively. For further details, see the legend to Fig. 1 or Ref. (5)) Table I. FIG.
I
I
I 2 3 4
I
I
1
II/I
I
I
5 6 7 th 9 IO II I2 13 14 FRACTION
NUMBER
FIG. 1. DNA chromatographic elution profiles of AKr mouse spleen and AKr spontaneous leukemia. Five hundred milligrams ECTEOLAcellulose (Brown Co., Berlin, N. H.; exchange capacity, 0.39 meq./g.; nitrogen content, 0.55%.) Approximately 2.5-3.5 mg. DNA were applied to each column. The recoveries for two analyses on the AKr spleen DNA and three analyses on the AKr spontaneous leukemia were 94 and 99% respectively. Optical density was recorded at 258-260 mp. Elution rate: 5-10 ml./hr. Fivemilliliter aliquots were collected, and 30 or 60 ml. per elution fraction. The fraction numbers shown in the abcissa are: Fraction 1, 0.05 M NaCl; Fraction 2, 0.2 M NaCl; Fraction 3, 0.6 M NaCl; Fraction 4, 0.8 M NaCl. Fractions 1-4 were dissolved in 0.001 M phosphate buffer, pH 7; Fraction 5, 2.0 M NaCl: 0.01 M phosphate buffer, pH 8; Fraction 6,O.Ol M NH3 ; Fraction 7,0.1 M NH3 ; Fraction 8, 0.2 M NH, ; Fraction 9, 0.3 M NH3 ; Fraction 10, 0.4 M NH8 ; Fraction 11, 0.5 M NH, ; Fraction 12, 0.6 M NH3 ; Fraction 13, 1.0 M NH,. Fractions 6-13 contained 2.0 M NaCl; Fraction 14, 0.5 M NaOH. For further details, see Ref. (5), Table I.
lymphomas (Fractions 8 and 9) is not significant. The elution profiles of the diploid and tetraploid carcinomas are similar to those of the two melanomas (Fig. 3) and to the normal spleen cells and the lymphoma DNA. These results suggest that the molecular
size distribution of the DNA molecules of various tumors and of normal spleen cells is very similar. The chromatographic elution profiles of lung, t’hymus, kidney, and Iiver of the mouse and the brain, spleen, and kidney of the rat have also been studied. No drastic differences have been observed (32). As discussed in the companion paper (5), previous indications of chromatographic differences between the DNA of normal tissues and tumor cells could be attributed to the partial degradation or denaturation of the DNA during its preparation (32, 33). The results reported in this paper are in agreement with t,hose of Kaplan and Smith on mouse leukemic tissues (34) and of Kondo and Osawa (35) who studied the DNA of various normal rat or rabbit tissues.They are at variance, however, with the results of Polli and associates (36, 37) who reported chromatographic and physical-chemical differences between normal human leukocytes and leukemic cells, and of Bendich et al. (38) who observed very marked differences be-
INVESTIGATIONS
OF
,,,,,, LETTRE-EHRLICH (HYPEFtDIPLOID)
I2
I I I I I I I I I I I I
3
4
5 6 7 FRACTION
8 9 101112I314 NUMBER
FIG. 3. DNA chromatographic elution profiles on ECTEOLA-cellulose of the Lettre-Ehrlich hyperdiploid and the Ehrlich tetraploid carcinomas, and the Cloudman S91 melanotic and the Cloudman S91A amelanotic melanomas. The average recoveries from the columns of two determinations with the Ehrlich, one determination with the Lettre-Ehrlich, two with the S91 and two with the S91A tumors: 90,91,98, and 96%, respectively. For further details, see the legend to Fig. 1 or Ref. (5), Table I.
THE
DNA
335
CONTENT
changes in chromosome number and structural rearrangements including the presence of giant and minute chromosomes. Thus, there can be little doubt that the transplanted tumors, if not the spontaneous lymphomas, differ in their hereditary determinants from normal mouse tissues. Such differences are reflected in the amount of DNA per cell nucleus in the caseof the tetraploid tumors but not in the purine and pyrimidine base composition or the chromatographic profiles. It is of interest that unpublished results from this laboratory indicate that the purine and pyrimidine base ratios of the RNA of various mouse tumors, normal tissues, and embryonic tissues are also indistinguishable. These results do not, of course, preclude changes in the molecular size distribution, which might be detected by an exchanger of higher resolving power, or of variations in the nucleotide sequencealong the DNA chains (39) or of the other factors mentioned in the Introduction. Further studies will be required to evaluate these possibilities. ACKNOWLEDGMENTS This work was performed with the technical assistance of Frank Broo and Andrew Cox. REFERENCES
tween the DNA chromatographic profiles of rat kidney and brain. These discrepancies are probably due to differences in the procedures used for the preparation of the DNA samples, to differences in the properties of the exchangers used by the various laboratories, and to differences in the schedule of elution from the columns. Smith and Kaplan used a substituted-starch anion exchanger, whereas most of the experiments performed in this laboratory were carried out on an ECTEOL.A-cellulose exchanger of relatively high nitrogen content compared with those used by I’olli et al. (36) and Bendich el al. (38). The DNA chromatographic profiles on ECTEOLA-cellulose exchangers of varying nitrogen content are currently gated in this laboratory.
being
investi-
The present studies were carried out on a number of tumors, the chromosome sets of which differ markedly from that of normal mouse tissues. These differences include
1. 2.
ROSENKRANZ, H. S., AND BENDICH, A., J. Am. Chem. Sot., 81, 902 (1959). BENDICH, A., PAHL, H. B., ROSENKRANZ, H. S., AND ROSOFF, M., Symposia Sot. Exptl.
Biol. No. 12, 31 (1958). G. DI, AND BENDICH, 3. ROSOFF, M., MAYORCA, A., Nature 180, 1355 (1957). J. 66, 495 (1957). 4. KIRBY, K. S., Biochem. 5. KIT, S., Arch. Biochem. Biophys. 87,318 (1960). 6. BURTON, K., Biochem. J. 62, 315 (1956). R., Biochem. J. 7. SMITH, J. D., AND MARKHAM, 46, 509 (1950). 8. WYATT, G. R., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.). Vol. 1, p. 243. Academic Press, New York, 1955. 9. POLLISTER, A. W., Exptl. Cell Research, Suppl. a, 59 (1952). Rev. Cytol. 2, 1 (1953). 10. SWIFT, H., Intern. et Biophys. 11. KIT, S., AND GROSS, A., Biochim. Acta 36, 185 (1959). L., GRIFFIN, A. C., AND LUCK, 12, CUNNINGHAM, J. M., J. Gen. Physiol., 34, 59 (1950). 13. RUTMAN, R. J., CANTAROW, A., AND PASCHKIS, K. E., Cancer Research 14, 111 (1954).
336
SAUL
14. SHELTON, E., J. Natl. Cancer Inst. 16,49 (1954). 15. PETRAKIS, N. L., AND FOLSTAD, L. J., J. Natl. Cancer Inst. 16, 63 (1954). 16. PETRAKIS, N. L., BOSTICK, N, L., AND SIEGEL, B. V., J. Natl. Cancer Inst. 22, 551 (1959). 17. PETRAKIS, N. L., Blood 6, 905 (1953). 18. QUATTRONE, P. D., Exptl. Cell Research 16,505 (1959). 19. MIZEN, N., AND PETERMANN, M. L., Cancer Research 12, 723 (1952). 20. MENTEN, M. L., AND WILLMS, M., Cancer Research 13, 733 (1953). 21. MENTEN, M. L., WILLMS, M., AND WRIGHT, W. D., Cancer Research 13, 729 (1953). 22. RABOTTI, G., Nature 183, 1276 (1959). 23. PARTANEN, C. R., Cancer Research 16, 300 (1956). 24. KLEIN, R. M., RASCH, E. M., AND SWIFT, H., Cancer Research 13, 499 (1953). 25. KLEIN, G., Exptl. Cell Research 2, 518 (1951). 26. FREED, J. J., AND HUNOERFORD, D. A., Cancer Research 17, 177 (1957). 27. KIT, S., AND GRIFFIN, A. C., Cancer Research 18, 621 (1958). 28. RICHARDS, B. M., WALKER, P. M. B., AND DEELEY, E. M., Ann. N. Y. Acad. Sci. 63, 831 (1956).
KIT
29. CHARGAFF, E., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, p. 307. Academic Press, New York, 1955. 30. LALAND, S. G., OVEREND, W. G., AND WEBB, M. J. Chem. Sot. 1962, 303-10. 31. UZMAN, L. L., AND DESOER, C., Arch. Biochem. Biophys. 48, 63 (1954). 32. KIT, S., AND GROSS, A., Federation Proc. 18, 262 (1959). 33. KIT, S., GRAHAM, O., GROSS, A., RAGLAND, R. S., AND FISCUS, J., Acta Unio Intern. Contra Cancrum, in press. 34. SMITH, K. C., AND KAPLAN, H. S., Federation Proc. 18, 507 (1959). 35. KONDO, N., AND OSAWA, S., Nature 183, 1602 (1959). 36. POLLI, E. E., Abstr. Papers, Intern. Cancer Congr., 7th Congr., London, 1958, p. 38. 37. POLLI, E. E., AND SHOOTER, K. V., Biochem. J. 69, 398 (1958). 38. BENDICH, A., PAHL, H. B., AND BEISER, W. M., Cold Spring Harbor Symposia Quant. Biol. 21, 31 (1956). 39. SHAPIRO, H. S., AND CHARGAFF, E., Biochim. et Biophys. Acta 23, 451 (1967). 40. FISHER, R. A., “Statistical Methods for Research Workers,” p. 174. Hafner Publishing Co., New York, 1948.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
87, 330-336
(1960)
Investigations of the DNA Con#ent, Base Composition and Chromatography on ECTEOLA-Cellulose of Normal and Tumor DNA Preparations’ SAUL From the Section of Nucleoprotein Texas M. D. Anderson Hospital
Metabolism, and Tumor Received
KIT
Department of Biochemistry, The University of Institute, Texas Medical Center, Houston, Texas November
2, 1959
The total deoxyribonucleic acid (DNA) content per cell, the ratios of the purine and pyrimidine bases, and the chromatographic profiles on ECTEOLA-cellulose anion exchangers of DNA preparations from normal mouse spleen, spontaneous lymphomas, diploid and tetraploid lymphomas and carcinomas, and amelanotic and melanotic melanomas have been investigated. The total DNA content was related to the chromosome numbers of the tumor cells. The DNA chromatographic profiles of the above tissues were not significantly different. The molar base ratios of the purines and pyrimidines were a&o quite iimilar.
ECTEOLA-cellulose anion exchangers into groups of molecules which d8er in their viscosity and sedimentation coefficients (l3), and, presumably, in molecular weight. The results of similar fractionations are also presented. These results show that the DNA chromatographic profiles of transplanted tumors, spontaneous leukemias of mice, and various normal tissues of the mouse and the rat are similar.
INTRODUCTION
It has been postulated that differences in the structure or composition of the deoxyribonucleic acids (DNA) distinguish normal tissues from tumors. Such differences might take one or more of several forms. There might be changes in: (a) the total amount of DNA per cell, (6) the molecular size distribution of the DNA per cell, (c) the purine and pyrimidine base composition, (d) the nucleotide sequence of bases along the polynucleotide chains, (e) the relative proportions of the various types of DNA molecules within a given cell, or (f) the organization of DNA molecules within the chromosomes. Data are as yet unavailable with regard to the latter three possibilities. The first three points will be discussed in the present paper with respect to spontan&ous and transplanted tumors of mice. It is shown that although the amounts of DNA per cell are increased in some transplanted mouse tumors, the purine and pyrimidine base compositions are very similar. DNA preparations may be fractionated on 1 Aided in part by Grants from Cancer Society (P-35), the Leukemia and the National Cancer Institute
METHODS
TUMORS AND NORMAL TISSUES DNA was prepared from the following transplanted tumors of mice : diploid ascites lymphomas 6C3HED and E9514A (C3H mice), tetraploid ascites lymphomas 6C3HED-DBA-2 (C3H or DBA-2 mice), the Lettre-Ehrlich hyperdiploid and the Ehrlich hypotetraploid ascites carcinomas (Swiss mice), the Cloudman melanotic melanoma (S91), and the Cloudman amelanotic melanoma (S91A) (DBA-2 mice). The ascites tumors were harvested after 6-13 days of growth in the intraperitoneal cavities of the mice. Melanoma-bearing animals were sacrificed aft,er 1-2 months of tumor growth. Care was taken that no grossly necrotic melanoma be included in the tissue samples of the latter tumors. Spontaneous lymphatic leukemic tissue was ob-
the American Society, Inc., (C-4238). 330
-_
1N
-----
-..-
.
---.
_”
Y Jusrlw~I’l”ND
#.-
tamed from AKr mice. The incidence of spontaneous leukemia in &la-month-old female animals of the Houston colony is about SO-85%. Leukemic nodes from the thymus, axilla, and mesentery of 2-5 animals were pooled for each DNA preparation. Normal thymusorspleens of B-weekor 4-monthold Akr mice or of adult C3H and DBA-2 mice were rapidly dissected from the animals and pooled. A maximum of 20 min. was required for the dissection. In some instances, the animals were sacrificed and the tissues excised in the cold room (4”), but, in all cases, the tissues were kept ice cold until DNA extraction was begun. All experimental animals were fed ad Zibituvn prior to sacrifice.
PRE~PARATION OF DNA AND CHROMATOGRAPHY The method of Kirby was used to prepare DNA from the above tissues (4) with the modifications described previously (5). The chromatography on ECTEOLA-caellulose (Brown Co., Berlin, N. H.; 0.39 meq./g. exhange capacity; nitrogen content, 0.55%, type 20) was as discussed in the previous paper (5). Approximately 2.5-3.5 mg. DNA (equivalent to 50-70,000 optical density units at 258 mF)Z was dissolved in 0.05 M NaC1-0.001 M phosphate buffer pH 7 and applied to 500 mg. ECTEOLAcellulose columns. A discontinuous elution schedule with eluting solutions consisting of increasing concentrations of NaCl and alkaline pH was employed to elute the DNA from the columns [see Ref. (5), Table I]. Five-milliliter fractions were collected by means of a Rinco automatic fraction collector (constant-volume operation) over a period of 3 days. The solvents passed through the column under the force of gravity at an average rate of 5-10 ml./hr. The amount of DNA eluted was measured spectrophotometrically at 258 mp by means of a Beckman DK-2 recording spectrophotometer.
ANALYSIS
OF TOTAL AND BASE
THE
DNA
331
CONTENT
VP’
DNA PER CELL RATIOS
The total DNA per cell was determined by the Burton modification of the diphenylamine reaction (6). All analyses were performed at three tissue concentrations, and standard curves were run for each experiment. Deoxyribose solutions were used as stock standards. Standard curves 2 Optical density units were defined as the optical density observed with the Beckman DK-2 spectrophotometer at 258-260 rnr for a l-cm. light path X 103 X the volume, in ml., of the solution applied to the columns.
were also constructed from analyses of highly polymerized calf-thymus DNA (Mann Assayed Biochemicals), and the conversion factors for the two curves were calculated. Tumor cell counts were routinely made at two dilutions on cells suspended in ascites fluid containing saline and heparin, using a Levy ultraplane, improved Neubauer counting chamber (C. A. Hausser and Sons, Philadelphia, Pa). The purine and pyrimidine composition of the DNA samples were determined by paper chromatography and spectrophotometry (7). From 2 to 6 mg. DNA was hydrolyzed with 12 N perchloric acid, and quadruplicate aliquots were chromatographed on Whatman B3MM filter paper, using isopropyl alcohol-HCl-water (1300:332:368, by volume) as the solvent. Standards at three connentrations were simultaneously chromatographed for each of the four bases. The purines and pyrimidines were eluted from the papers with 5 ml. of 0.1 N HCI, and the optical density was recorded between 300 and 230 rnp. Blank paper strips opposite each of the bases were cut from the papers, and the optical density of each of the blanks was substracted from the optical densities of the corresponding bases. The molecular extinction coefficients of the adenine, guanine, cytosine, and thymine standards agreed closely with those reported in the literature (8). RESULTS
AND
DNA
CONTENT
DISCUSSION
PER CELL
It is now well known that the DNA content of most cells of a given organism is approximately twice that found in sperm cells (Class I cells) and that many tissues contain cells whose content of DNA is four times (Class 11) or eight times (Class III) that of the chromatids (9, 10). Values differing from the speciesspecific constancy can generally be accounted for on the basis of: (a) polyploidy or polyteny, (b) aneuploidy or heteroploidy, and (c) the premitotic replication of DNA by dividing cells. The cellular DNA content and the modal chromosomal numbers of mouse lymphomas, carcinomas, and melanomasare presented in Table I. The DNA content of normal spleen cells is also shown for comparison. The DNA content is proportional to the chromosome number in the case of the Lettre-Ehrlich and Ehrlich carcinomas. A similar relationship obtains in the case of the E9514A diploid lymphoma and the -two be’craploid lymphomas. The
332
SAUL
TABLE DNA CONTENT NUMBERS
TlUtl0r
I
AND MODAL
agreement (25-28). The increased DNA content of tumor cells thus appears to be a manifestation of the gross remodeling of the chromosome complex which takes place during the neoplastic progression (26, 27).
CHROMOSOME
OFMOUSETUMORS DNA 10' ce i1: TYPO Y _-
E9514A (diploid) 6C3HED (diploid) 6C3HED-DBA-2 (hypotetraploid) GCSHED-DBA-2 to C3H (hypotetraploid)
Lymphoma Lymphoma Lymphoma
7.9 9.8 15.3
KIT
40 76-78
PURINE AND PYRIMIDINE
BASE
RATIOS
The molar ratios of the DNA purine and pyrimidines of diploid and tetraploid lymphomas and carcinomas, two melanomas, Lymphoma 14.2 74-79 mouse thymus, and of spleens from three mouse strains are shown in Table II. The ratio of purines to pyrimidines and the ratio of adenine plus cytosine to guanine plus Lettre-Ehrlich (hy- Carcinoma 11.1 46 thymine (6-am: 6 keto) is in each case very perdiploid) closeto 1.0. The ratios of adenine to thymine Ehrlich (hypotetraCarcinoma 18.0 78 for all of the samples vary from 0.91: 1 to ploid) 1.05: 1, whereas the ratios of guanine to Cloudman S91 (tet- Melanotic 16.6 cytosine vary from 0.89: 1 to 1.05: 1. The raploid) Melanoma theoretical ratios of all of the above paramCloudman S91A Amelanotic 17.1 eters predicted on the basis of the double(tetraploid) Melanoma stranded Watson-Crick model for the structure of DNA (29) are 1.0. Taking into Normal spleen cells 8.3 40 consideration the experimental errors of the analyses, the deviations from the theoretical / DNA content of diploid lymphoma, 6C3- values seem small. HED, is approximately 24% above the The ratio of A -I- T/G -I- C was observed diploid value (11). Both the amelanotic and to be about 1.24 for the normal tissues and the melanotic melanomas contain the all of the tumors. Diploid tumors differed but little from tetraploid tumors with respect amount of DNA characteristic of tetraploid to the purine and pyrimidine baseratios. The cells. The results here presented are in agree- observed differences between the melanomas, ment with the spectrophotometric or chemi- carcinomas, or lymphomas were also very cal determinations of the DNA of human and small. The highest observed guanine, cytoanimal tumors reported by various investisine, and thymine values differed from the gators. These have shown that primary lowest by 12, 19, and 15%, respectively. hepatomas (12, 13) and lymphomas (14,21), When analyzed for significant differences carcinomas (22), or plant tumors (23,24) and using the table of t of Fisher (40), only five some transplanted lymphomas (17-21) may of the latter values were statistically difcontain, on the average, the normal diploid ferent at the 2% level of significance. The amount of DNA or slightly above this value. guanine content of tumor 6C3HED was Other transplanted lymphomas (11, 14), significantly greater than that of DBA spleen various carcinomas, and carcinomas which or the Lettre carcinoma, but the observed have been transferred for many years (25, increaseswere only 7 and 5 %, respectively. 26), and metastatic human carcinoma cells The cytosine content of tumor S91 was 6 % may have an average DNA content in the greater than that of AKr thymus, and the polyploid range or may manifest intermedithymine content of tumor S91A was 8% ate values. When chromosome studies and greater than that of DBA spleen or tumor DNA determinations have been performed 6C3HED-DBA-2. Of the above five statison individual nuclei of the same cell popula- tically significant differences, three involve tion, the chromosome spread and DNA varitissuesin which the determinations were only ation from the modal value are in good performed two times. It is conceivable that
INVESTIGATIONS
OF
THE
TABLE DNA
BASE
RATIOS
Numbers in parentheses represent guanine, C = cytosine, T thymine, 6-keto = guanine plus thymine.
molar
base
ratios
C
CELLULOSE
A+T G+C
T
PU pyr
6 am 6 keto
f zk f f f
.038 .OlO ,025 ,090 .031
0.78 0.73 0.79 0.81 0.80
& 0.60 f .090 f .028 f ,002 f .032
0.95 0.91 0.96 0.94 1.01
i f f f f
.020 ,010 .023 .180 ,053
1.27 1.28 1.24 1.17 1.26
1.02 1.07 1.02 1.06 0.99
1.04 1.04 1.02 1.01 0.99
0.82 0.81 0.79 0.79 0.80 0.83 0.77 0.77
i i i h zk ziz f f
.038 .007 .014 3020 .026 .047 ,008 ,017
0.82 0.80 0.87 0.85 0.86 0.84 0.87 0.85
f f. f f f. z!z f f
1.05 1.00 0.98 0.91 0.98 1.04 0.96 0.96
f f f f f f f f
.044 .030 ,040 ,026 ,030 .036 .017 .027
1.25 1.24 1.19 1.16 1.19 1.22 1.27 1.21
0.97 1.01 0.97 1.02 0.98 0.97 0.97 0.98
0.97 0.99 1.06 1.09 1.04 0.99 1.08 1.07
are exnressed
ON
AND TUMORS Abbreviations: A = Adenine, G = 6 am = adenine plus cytosine,
0.76 0.76 0.79 0.85 0.80
relative
the above small differences are biologically important. However, further evidence is necessary before this conclusion can be drawn. In the meantime, interpretation as to real differences should be made with extreme caution. The purine and pyrimidine base ratios presented above agree closely with those reported by Laland and associates (30) for mouse sarcoma cells. No significant differences in DNA base composition were observed between human hepatoma and normal liver ((29) or normal and leukemic human spleen tissue (31). CHROMATOGRAPHY
TISSUES
base ratid
G
C3H spleen (4) DBA spleen (‘2) Spleen AKr (7) Thymus AKr (2) Spontaneous leukemia (AKr) (11) E9514A (4) 6C3HED (4) GCSHED-DBAS to C3H (7) , GC3HED/DBA-2 (5) s91 (10) S91A (7) Lettre (5) Ehrlich (7)
II MOUSE
Molar
333
CONTENT
the number of determinations. Pu = purines, pyr = pyrimidines,
Tissue
m The
OF NORMAL
DNA
ECTEOLA-
COLUMNS
The DNA chromatographic elution profiles on ECTEOLA-cellulose of normal AKr, C3H, and DBA-2 mouse spleen and of spontaneous AKr leukemia, diploid and tetraploid ascites lymphomas (C3H or DBA-2 mice), diploid and tetraploid strains of the Ehrlich carcinomas (Swiss mice), and melanotic or amelanotic melanomas (DBA-2 mice) are shown in Figs. l-3. Details con-
,080 .060 ,050 .016 .015 .032 .026 ,020
to adenine
= 1.00 f
the
standard
error
of the
mean
cerning the chromatographic procedures and a critical evaluation of the factors affecting the chromatography are presented in the companion paper (5). In Figs. 1-3, the cumulative per cent of DNA eluted from the columns is plotted as a functionof the solvent used for elution. The composition of the eluants is given in the footnote to Fig. 1 [see also Ref. (5), Table I]. Approximately 10 % of the DNA is eluted by 0.6 M NaC1-0.001 M phosphate buffer, pH 7 (Fraction 3). Two large peaks are eluted by 2 M NaCl, 0.2 M and 0.3 M NH3 solutions (Fractions 7 and S), and several smaller peaks in Fractions 5, 6, 9, and 10. Approximately 75 % of the DNA was eluted in Fractions 6-10. Figures l-3 show that the DNA elution profiles are rather similar in all cases. The chromatographic profile of the spontaneous AKr lymphoma does not differ from that of normal AKr mouse spleen cells (Fig. 1) nor do the transplanted tetraploid lymphomas differ greatly from C3H or DBA-2 mouse spleen cells (Fig. 2). The variation shown in Fig. 2 between the diploid and tetraploid
334
SAUL
KIT
-MOUSE SPLEEN a---- 6C3HED-TETRAPLOID a..,,a..a.,E96,4A --BCIHED-DIPLOID _
tKr SPLEEN SPONTANEOUS LEUKEMIA (AKrJ
FRACTION
NUMBER
2. DNA chromatographic elution profiles on ECTEOLA-cellulose of C3H and DBA mouse spleen, diploid lymphomas E9514A, and 6C3HED, and tetraploid lymphoma 6C3HED-2-DBA. The average recovery from the columns of four determinations with the spleen DNA, two determinations with the diploid E9514A or 6C3HED DNA, and three determinations with tetraploid 6C3NED DNA were 103, 85, and 9370, respectively. For further details, see the legend to Fig. 1 or Ref. (5)) Table I. FIG.
I
I
I 2 3 4
I
I
1
II/I
I
I
5 6 7 th 9 IO II I2 13 14 FRACTION
NUMBER
FIG. 1. DNA chromatographic elution profiles of AKr mouse spleen and AKr spontaneous leukemia. Five hundred milligrams ECTEOLAcellulose (Brown Co., Berlin, N. H.; exchange capacity, 0.39 meq./g.; nitrogen content, 0.55%.) Approximately 2.5-3.5 mg. DNA were applied to each column. The recoveries for two analyses on the AKr spleen DNA and three analyses on the AKr spontaneous leukemia were 94 and 99% respectively. Optical density was recorded at 258-260 mp. Elution rate: 5-10 ml./hr. Fivemilliliter aliquots were collected, and 30 or 60 ml. per elution fraction. The fraction numbers shown in the abcissa are: Fraction 1, 0.05 M NaCl; Fraction 2, 0.2 M NaCl; Fraction 3, 0.6 M NaCl; Fraction 4, 0.8 M NaCl. Fractions 1-4 were dissolved in 0.001 M phosphate buffer, pH 7; Fraction 5, 2.0 M NaCl: 0.01 M phosphate buffer, pH 8; Fraction 6,O.Ol M NH3 ; Fraction 7,0.1 M NH3 ; Fraction 8, 0.2 M NH, ; Fraction 9, 0.3 M NH3 ; Fraction 10, 0.4 M NH8 ; Fraction 11, 0.5 M NH, ; Fraction 12, 0.6 M NH3 ; Fraction 13, 1.0 M NH,. Fractions 6-13 contained 2.0 M NaCl; Fraction 14, 0.5 M NaOH. For further details, see Ref. (5), Table I.
lymphomas (Fractions 8 and 9) is not significant. The elution profiles of the diploid and tetraploid carcinomas are similar to those of the two melanomas (Fig. 3) and to the normal spleen cells and the lymphoma DNA. These results suggest that the molecular
size distribution of the DNA molecules of various tumors and of normal spleen cells is very similar. The chromatographic elution profiles of lung, t’hymus, kidney, and Iiver of the mouse and the brain, spleen, and kidney of the rat have also been studied. No drastic differences have been observed (32). As discussed in the companion paper (5), previous indications of chromatographic differences between the DNA of normal tissues and tumor cells could be attributed to the partial degradation or denaturation of the DNA during its preparation (32, 33). The results reported in this paper are in agreement with t,hose of Kaplan and Smith on mouse leukemic tissues (34) and of Kondo and Osawa (35) who studied the DNA of various normal rat or rabbit tissues.They are at variance, however, with the results of Polli and associates (36, 37) who reported chromatographic and physical-chemical differences between normal human leukocytes and leukemic cells, and of Bendich et al. (38) who observed very marked differences be-
INVESTIGATIONS
OF
,,,,,, LETTRE-EHRLICH (HYPEFtDIPLOID)
I2
I I I I I I I I I I I I
3
4
5 6 7 FRACTION
8 9 101112I314 NUMBER
FIG. 3. DNA chromatographic elution profiles on ECTEOLA-cellulose of the Lettre-Ehrlich hyperdiploid and the Ehrlich tetraploid carcinomas, and the Cloudman S91 melanotic and the Cloudman S91A amelanotic melanomas. The average recoveries from the columns of two determinations with the Ehrlich, one determination with the Lettre-Ehrlich, two with the S91 and two with the S91A tumors: 90,91,98, and 96%, respectively. For further details, see the legend to Fig. 1 or Ref. (5), Table I.
THE
DNA
335
CONTENT
changes in chromosome number and structural rearrangements including the presence of giant and minute chromosomes. Thus, there can be little doubt that the transplanted tumors, if not the spontaneous lymphomas, differ in their hereditary determinants from normal mouse tissues. Such differences are reflected in the amount of DNA per cell nucleus in the caseof the tetraploid tumors but not in the purine and pyrimidine base composition or the chromatographic profiles. It is of interest that unpublished results from this laboratory indicate that the purine and pyrimidine base ratios of the RNA of various mouse tumors, normal tissues, and embryonic tissues are also indistinguishable. These results do not, of course, preclude changes in the molecular size distribution, which might be detected by an exchanger of higher resolving power, or of variations in the nucleotide sequencealong the DNA chains (39) or of the other factors mentioned in the Introduction. Further studies will be required to evaluate these possibilities. ACKNOWLEDGMENTS This work was performed with the technical assistance of Frank Broo and Andrew Cox. REFERENCES
tween the DNA chromatographic profiles of rat kidney and brain. These discrepancies are probably due to differences in the procedures used for the preparation of the DNA samples, to differences in the properties of the exchangers used by the various laboratories, and to differences in the schedule of elution from the columns. Smith and Kaplan used a substituted-starch anion exchanger, whereas most of the experiments performed in this laboratory were carried out on an ECTEOL.A-cellulose exchanger of relatively high nitrogen content compared with those used by I’olli et al. (36) and Bendich el al. (38). The DNA chromatographic profiles on ECTEOLA-cellulose exchangers of varying nitrogen content are currently gated in this laboratory.
being
investi-
The present studies were carried out on a number of tumors, the chromosome sets of which differ markedly from that of normal mouse tissues. These differences include
1. 2.
ROSENKRANZ, H. S., AND BENDICH, A., J. Am. Chem. Sot., 81, 902 (1959). BENDICH, A., PAHL, H. B., ROSENKRANZ, H. S., AND ROSOFF, M., Symposia Sot. Exptl.
Biol. No. 12, 31 (1958). G. DI, AND BENDICH, 3. ROSOFF, M., MAYORCA, A., Nature 180, 1355 (1957). J. 66, 495 (1957). 4. KIRBY, K. S., Biochem. 5. KIT, S., Arch. Biochem. Biophys. 87,318 (1960). 6. BURTON, K., Biochem. J. 62, 315 (1956). R., Biochem. J. 7. SMITH, J. D., AND MARKHAM, 46, 509 (1950). 8. WYATT, G. R., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.). Vol. 1, p. 243. Academic Press, New York, 1955. 9. POLLISTER, A. W., Exptl. Cell Research, Suppl. a, 59 (1952). Rev. Cytol. 2, 1 (1953). 10. SWIFT, H., Intern. et Biophys. 11. KIT, S., AND GROSS, A., Biochim. Acta 36, 185 (1959). L., GRIFFIN, A. C., AND LUCK, 12, CUNNINGHAM, J. M., J. Gen. Physiol., 34, 59 (1950). 13. RUTMAN, R. J., CANTAROW, A., AND PASCHKIS, K. E., Cancer Research 14, 111 (1954).
336
SAUL
14. SHELTON, E., J. Natl. Cancer Inst. 16,49 (1954). 15. PETRAKIS, N. L., AND FOLSTAD, L. J., J. Natl. Cancer Inst. 16, 63 (1954). 16. PETRAKIS, N. L., BOSTICK, N, L., AND SIEGEL, B. V., J. Natl. Cancer Inst. 22, 551 (1959). 17. PETRAKIS, N. L., Blood 6, 905 (1953). 18. QUATTRONE, P. D., Exptl. Cell Research 16,505 (1959). 19. MIZEN, N., AND PETERMANN, M. L., Cancer Research 12, 723 (1952). 20. MENTEN, M. L., AND WILLMS, M., Cancer Research 13, 733 (1953). 21. MENTEN, M. L., WILLMS, M., AND WRIGHT, W. D., Cancer Research 13, 729 (1953). 22. RABOTTI, G., Nature 183, 1276 (1959). 23. PARTANEN, C. R., Cancer Research 16, 300 (1956). 24. KLEIN, R. M., RASCH, E. M., AND SWIFT, H., Cancer Research 13, 499 (1953). 25. KLEIN, G., Exptl. Cell Research 2, 518 (1951). 26. FREED, J. J., AND HUNOERFORD, D. A., Cancer Research 17, 177 (1957). 27. KIT, S., AND GRIFFIN, A. C., Cancer Research 18, 621 (1958). 28. RICHARDS, B. M., WALKER, P. M. B., AND DEELEY, E. M., Ann. N. Y. Acad. Sci. 63, 831 (1956).
KIT
29. CHARGAFF, E., in “The Nucleic Acids” (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, p. 307. Academic Press, New York, 1955. 30. LALAND, S. G., OVEREND, W. G., AND WEBB, M. J. Chem. Sot. 1962, 303-10. 31. UZMAN, L. L., AND DESOER, C., Arch. Biochem. Biophys. 48, 63 (1954). 32. KIT, S., AND GROSS, A., Federation Proc. 18, 262 (1959). 33. KIT, S., GRAHAM, O., GROSS, A., RAGLAND, R. S., AND FISCUS, J., Acta Unio Intern. Contra Cancrum, in press. 34. SMITH, K. C., AND KAPLAN, H. S., Federation Proc. 18, 507 (1959). 35. KONDO, N., AND OSAWA, S., Nature 183, 1602 (1959). 36. POLLI, E. E., Abstr. Papers, Intern. Cancer Congr., 7th Congr., London, 1958, p. 38. 37. POLLI, E. E., AND SHOOTER, K. V., Biochem. J. 69, 398 (1958). 38. BENDICH, A., PAHL, H. B., AND BEISER, W. M., Cold Spring Harbor Symposia Quant. Biol. 21, 31 (1956). 39. SHAPIRO, H. S., AND CHARGAFF, E., Biochim. et Biophys. Acta 23, 451 (1967). 40. FISHER, R. A., “Statistical Methods for Research Workers,” p. 174. Hafner Publishing Co., New York, 1948.