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Patterns of nucleic acids synthesis in normal and crown gall tumor tissue cultures of tobacco
ARCHIVES
OF
BIOCHEMISTRY
Patterns
AND
of Nucleic
BIOPHYSICS
Acids
Tumor
126, 817-823
Synthesis
Tissue
Roswell
Park
Memorial Received
Institute, September
New
in Normal
Cultures
B. I. SAHAI
(1968)
and
Crown
Gall
of Tobacco’
SRIVASTAVA
York
State
18, 1967;
Department
accepted
of Health,
December
Buffalo,
New
York
4, 1967
The bacteria-free crown gall tumor tissue cultures of tobacco were 40-907, higher in nucleic acid content and up to 10-15 times higher in the capacity to incorporate 32P into RNA than the normal tissue cultures. Examination of the szP sedimentation profile of rapidly labeled nucleic acids from normal and tumor tissue cultures (12-42 days old) suggested some differences between t.he normal tissue and the tumor tissue, although the profiles also changed with the culture age. Since the s2P nucleotide composition (AMP + UMP = 54-58yo) of total RNA and of different RNA fractions, obtained by density gradient centrifugation, was similar to DNA (A + T = S170) rather than ribosomal RNA (AMP + UMP = 44yo), the RNA labeled with a2P was considered to represent principally mRNA. Both the absorbance and the 3tP nucleotide compositions of total RNA from normal tissue were similar to those from tumor tissue, and, except, for fraction “C,” consistent differences in the 32P nucleotide compositions of different RNA fractions from normal and tumor tissue were not very apparent.
Agrobacterium tumefaciens (Smith and Town, Conn.), a bacterial plant pathogen, has the ability to permanently transform the host cells into tumor cells (2) which show unrestricted growth. After transformation, the tumor cells are able to synthesize auxin, cytokinin, and other substances (2, 7) in abundance which must be supplied to the normal cells for growth in vitro. Increases in the amount of RNA ( 15), proteins (3), and the activities of certain enzymes (15) as well as the induction of new enzyme proteins (12) also occur in transformed cells. These modifications could occur by a persistent de-repression of a segment of normal cell genome which would lead to a greater synthesis of mRNA’s or by the incorporation of a self-replicating viral genome (if the actual tumorogenic agent of the crown gall organism is a virus) into h.ost genome. In this respect it is interesting to note that initiation 1 This Atomic 1)3722.
work Energy
was supported Commission
by United Contract
States AT(30817
of tumors in plants by A. tumefaciens has been shown to be inhibited both by ribonuclease(4) and histones (5). In the present work the patterns of RNA synthesis and the composition of various RNA fractions in normal and tumor tissue cultures have been examined in an attempt to find qualitative or quantitative differences. MATERIALS
AND
METHODS
Greenhouse-grown tobacco (LTicotania fabacum var. Wisconsin 38) plants (about 60 cm tall) were inoculated with Agrobacterium tumefaciens, strain 4-32 (cultured for 24 hours in nutrient broth plus 0.5$& dextrose) by needle puncture of the internodes. When the tumors were well developed the superficial layers of the tumors were discarded, and small segments of the underlying tissue after treatment with Clorox [lOy, Clorox (5.3yo NaOCl) for 10 minutes followed by repeated rinsings with sterile water] were planted on Murashige and Skoog’s (11) minus 1AA and kinetin medium (medium A). Most of the isolates contained bacteria, but a few which were bacteria-free were selected and cultured successively through several transfers on medium A to establish the independ-
818
SRIVASTAVA
ence of the tumor tissue for 1AA and cytokinin which must be supplied for the culture of the normal tissue (11). The tumor tissue has now been cultured on medium A for more than a year without any reduction of its growth. For establishing the culture of the normal tissue, the internode segments from tobacco plants, after being sterilized with 10% Clorox, were cultured on Murashige and Skoog’s (11) medium containing 0.2 mg kinetin per liter and 2 mg IAA (medium B) per liter. In both medium A and B, thiamin-HCl was at a concentration 0.4 mg/liter, and the edamin was omitted. Every 34 weeks small segments of the normal tissue and the tumor tissue were transferred to mediums (50 ml) B and A, respectively, contained in 125-ml flasks. The cultures were grown in growth chambers under continuous fluorescent light at 30”. Labeling, isolation, and analysis of nucleic acids. The normal tissue and the tumor tissue (about 10 gm fresh weight) were removed carefully under sterile conditions, divided into small segments, and transferred to weighed flasks containing 30 ml of the sterile liquid (i.e., without agar) media exactly similar (except for the omission of KHnPOl and adding of 100 rC of NazH3ZPO*) to that on which the tissues were growing. After 2 hours’ incubation on a shaker at 27” the tissue segments were quickly rinsed with 30 ml of the cold liquid media (s2P omitted) and used immediately for the
FIG. 1. The media
B and
cultures of normal A, respectively.
and
tumor
isolation of nucleic acids. Where the effect of actinomycin D on RNA synthesis was to be determined, 10 pg actinomycin D/ml was incorporated into the incubation media prior to autoclaving. The nucleic acids were isolated by a phenolsodium lauryl sulfate procedure as described by Srivastava (16) except that 0.01 M MgCI, and 5 mg bentonite/ml were added to the extracting buffer. After dialysis against phosphate buffer a part of the nucleic acid sample (l-l.2 ml out of 3 ml) was layered on 26 ml of a 5-20$$ linear sucrose gradient made in 0.05 M sodium phosphate buffer, pH 6.8, containing 0.001 M MgC12. The gradients were spun at 25,000 rpm for 16 hours in an SW25-1 rotor of a Spinco model L ultracentrifuge. Forty-drop fractions were collected by bottom puncture, diluted with 3 ml of water, and read at 260 rng. Fifty micrograms of crystalline bovine serum albumin and 1 ml of 25% trichloroacetic acid were added to each fraction. The RNA precipitates were collected on nitrocellulose membrane filters (type B6) which were washed with 5% cold trichloroacetic acid, dried, and then counted in vials containing 15 ml scintillation solution (4 gm 2,5-diphenyloxazole and 0.1 gm p-bis-[(2,5(phenyloxazolyl)] benzene per liter of toluene) in a Nuclear Chicago 720 counter. After counting, the discs containing Fractions l-10, 11-20, 21-32, and 3346 were pooled to give RNA fractions designated A, B, C, and D, respectively.
tissues
of tobacco
after
33 days
growth
on
NUCLEIC
ACID
SYNTHESIS
?T
IN
TOBACCO
-250
I’
// /
I’
,’
//
0
’
2’
Y‘a s E d 3
-100
& 0
,,jPN ,I‘
I’ N
p”
/’ ,,’ /’ ,’ /;- ‘Z
-150
1
J,’
50
I
I
I
IO
20 AGE
30 IN DAYS
I
40
s19
CULTURES
Carrier yeast RNA was added to each sample, and after the membrane filters were solubilized with acetone, the RNA was hydrolyzed with 0.3 M NaOH for 16 hours (15). The nucleotides were separated by electrophoresis at pH 3.5 and located by ultraviolet light. The bands containing the nucleotides were transferred to scintillation vials and counted as above. Where nucleotide composition of the total RNA was to be determined, the nucleic acid sample left from density gradient analysis was made free of traces of inorganic s2P by passage through a Sephadex G-25 column. The hydrolysis of RNA, separation of nucleotides, and counting of radioactivity were done as outlined above. For determining the nucleotide composition of RNA through absorbancy measurements (15), the nucleotides were eluted by 0.1 N HCl, and the absorbance of the eluates at X,,, of the nucleotides was determined against appropriate blanks. Isolation and determination of base composition of DNA. The DNA was isolated from chromatin (prepared by the procedure of Huang and Bonner) (6) of the normal tissue by Marmur’s (13) procedure. The hydrolysis of DNA by formic acid, separation of the bases by paper chromatography, and determination of the base composition of
-200
:
TISSUE
50
FIG. 2. The increase in fresh and dry weights of the normal tissue and the tumor tissue cultured on media B and A, respectively.
20-
5-
l-
--L.--L i0
A
20
30
40
! AGE
0 IN
--/-..I
IO
20
30
40
I
DAYS
FIG. 3. The changes in the percentage dry weight and the amount of nucleic acids in normal and tumor tissues growing on media B and A, respectively. The values represent means of two series of experiments. rRNA-1 and rRNA-2 represent light and heavy ribosomal RNA, respectively.
-
““092
0.0
-
Wx32
‘a.0
-
“WO92
DKl
NUCLEIC I)NA were of Markham
carried (9).
RESULTS
out
ACID according
AND
SYNTHESIS to the
TOBACCO
IN
DISCUSSION
TABLE COMPOSITIOV
OF NUCLEIC
ACIDS CULTURES
S%l
CULTURES
tumor tissue for 12-, 22-, 32-, and 42-day-old cultures presented in Fig. 4 show four absorbancy peaks corresponding to heavy ribosomal RNA, light ribosomal RNA, DNA, and soluble RNA. The 321’ radioactivity peaks do not correspond to the absorbancy peaks of the nucleic acids, indicating that labeled RNA was heterogenous in nature. The rate of 32Pincorporation into RNA of the tumor tissue was substantially higher than the normal tissue throughout the growth period invest,igated, with the incorporation being lo-15 times higher during the active growth period. Actinomycin D inhibited the incorporation of 32P into all RNA fractions of both normal tissue and the tumor tissue, indicating that RNA was being synthesized on a DNA template. However, the extent of inhibition of RNA synthesis by actinomycin D in the tumor tissue was fal greater than in the normal tissue. At this point it may be interesting to note that all plant viruses are RNA viruses and that actinomycin D does not inhibit RNA synthesis by these viruses ( 13). An examination of the 32Pprofiles of the nucleic acids in Fig. 4 suggeststhat the patterns of RNA synthesis in normal and tumor tissues were different, although the patterns also changed during the growth of both tissues. The total RNA from normal and tumor tissue was similar in nucleotide composition and had 55 % of GRIP + GRIP, whereas the DNA was rich in A + T (61%) content
procedure
The normal tissue growing on medium B produced firm, compact, slightly green callus, and the tumor tissue growing on medium A produced a massof loosely held firm green tissue (Fig. 1). Both the normal tissue and the tumor t,issue grew well on the media used for their growth but the growth of the tumor tis sue was substantially better than of the normal tissue (Fig. 2) on a fresh- or dryweight basis. The percent,age dry weight of the tumor tissue was, however, consistently lower than that of the normal tissue (Fig. 3), indicating a higher degree of hydration of the tumor tissue. The total amount of nucleic acids, heavy and light ribosomal RNA, DNA, and soluble RNA all declined with the age of the culture in the normal tissue and the tumor tissue (Fig. 3). Although the amount of total nucleic acids or the nucleic acid components was 40-90 70 higher in the tumor tissue, the rat,ios of various nucleic acid components were not significantly different. The increased amount of nucleic acid in the tumor tissue noted here has also been previously found for the tomato crown gall tissue ( 15). The sucrose density gradient profiles of the nucleic acids from normal tissue and the
B.\SIG
TISSUE
FROM
I NORM.~L
(N)
AND
(T)
TUMOR
TISSUE
OF TOBACCO Age of culture
Fraction
12 days
42 days
CMP
AMP
GMP
UMP(T)
CMP
AMP
GMP
UMP
Total
32P RNAb
N T
18.9 16.5
30.1 31.3
25.0 25.5
24.2 26.6
18.3 18.1
30.6 30.6
23.5 25.2
27.4 26.1
Total
RN&
N T
23.4 22.8
24.2 24.4
31.3 31.5
21.0 21.1
22.7 22.9
21.4 21.4
32.2 30.9
23.6 24.6
N
19.2
29.9
20.0
31.0
DNAc a Mean b Base c Base
of two separate series of experiments. composition expressed as ‘% 32P. composition determined by absorbancy
measurements
and
expressed
as mole/100
ml.
21-32)
33-46)
C (Fractions
D (Fractions
of two separate in parentheses
11-20)
B (Fractions
a Mean b Values
l-10)
Fractions
-
25.3
29.8
18.0
24.4
33.6
17.0
24.9
29.0
19.2
24.8
31.3
32.1
18.1
18.0
31.5
AMP
22.3
22.7
26.5
24.6
28.6
23.9
23.G
23.6
UMP
RNA
of actinomycin
27.8
27.6
25.6
24.8
28.0
26.8
26.1
27.7
GMP
12 days
32P) OF VARIOUS
17.1
CMP
(%
series of experiments. indicate the presence
COMPOSITIONS
A (Fractions
RNA
NUCLEOTIDE
D.
20.6 (22.5) 22.6 (21.2)
16.2 (14.1) 18.0 (17.0)
17.2 (17.2) 18.3 (17.3)
20.1 (19.0)b 17.7 (16.5)
CMP
25.2 (30.9) 27.5 (25.4) __-
30.2 (36.6) 28.2 (32.5)
28.9 (37.1) 27.9 (31.8)
24.7 (31.7) 30.1 (32.7)
II
GMP
NORMAL
29.8 (25.5) 27.5 (25.5)
25.1 (24.7) 26.8 (25.1)
27.D (22.4) 28.1 (23.4)
28.0 (23.5) 26.9 (23.4)
I-
22 days
FROM
AMP
TABLE FRACTIONS
.\ND
24.2 (21.2) 22.2 (23.0)
28.3 (24.6) 27.0 (25.3)
26.2 (23.2) 25.6 (27.4)
27.0 (25.7) 25.3 (27.4)
UMP
I
Age of culture
(N)
28.8
28.7 25.8
20.2 25.8
25.0
26.0
29.0
18.0
25.G
33.4
27.1
28.5
26.0
30.0
25.5
25.8
31.4
31.0
GMP
-
23.2
22.2
26.9
25.5
25.0
25.8
25.3
25.9
UMP
TISSUI~
AMP
32 days
(T)
15.4
19.2
17.5
18.6
16.6
CMP
TUMOR
_
26.3
20.6
25.9
28.2
31.5
34.2
17.5 17.8
32.2
31.4
21.2 21.2
30.8
33.0
AMP
OF
18.3
17.8
CMP
I-
7 I
CULTURES
24.1
27.0
23.3
21.3
19.7
19.3
25.6
24.8
GMP
42 days
TOBXCO
24.5
24.2
27.4
26.9
26.7
28.0
25.3
24.2
UMP
2 c: L-
$
g 7
NUCLEIC
ACID
SYNTHESIS
IN
(Table I). These values are in agreement with those reported (1) for RNA and DNA from tobacco. Since the 32P nucleotide composition of total RNA (AMP + UMP = 54-58%) from both normal tissue, and the tumor tissue was like DNA rather than ribosomal RNA (AMP + UMP = 44 %) (l), a significant part of the RNA labeled under experimental conditions used may represent mRNA. However, the 3ZP nucleotide composition of total RNA from normal and tumor tissue was not significantly different. Since alterations in the populations of RNA may not be so obvious if the 32Pnucleotide composition of total RNA was determined, the 32P nucleotide composition of different RNA fractions (separated by density gradient centrifugation) was determined. The 32P nucleotide composition of RNA fractions from normal and tumor tissues presented in Table II shows that fractions A, B, and C were DNA-like, whereas fraction D with an AMP + UMP content of 47-52% had a nucleotide composition between that’ of soluble RNA (AMP + UMP = 39 %) (1) and DNA (A + T = 61%). As also suggest’ed by 321’ profiles (Pig. 4), the changes in nucleotide composition of RNA fractions with age indicate changesin the populations of RNA’s during growth. The only consistent differencesin the nucleotide composition betn-een normal and tumor tissue were noted in fraction C, although lessconsistent differences in other fractions were also detected. It is interesting to note that the RNA which was synthesized in the presence of actinomycin D was more DNA-like (AMP + UMP = 60 Yo) than that which was synthesized in its absence. A similar effect of actinomycin D on the 32P-nucleotidecomposition of rat liver RNA has been reported (S). Since actinomycin D inhibits both ribosomal and messenger RNA synthesis ( 14), a more DNAlike composition of RNA synthesized in the presence of actinomycin D may be due to a stronger inhibition of any ribosomal RNA synthesis.
TOBACCO
TISSUE
CULTURES
823
A very high rate of RNA synthesis noted in the tumor tissue (compared with the normal tissue) is in line with its high enzymic activity and its ability to synthesize large numbers of essential compounds in ample amounts (2). Although some differencesin the 32Pdensity gradient pattern and the nucleotide composition of RNA from normal and tumor tissue were noted in the present studies, a more precise evaluation of the extent of similarities and differences in the populations of mRNA from normal and tumor tissue must await RNA-DNA hybridization, studies. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14.
15. 16.
BELLAMY,
A. R., Hiochim. Hiophys. Acta 123, 102 (1966). BRAUN, A. C., Ann. Rev. Plant Physiol. 13, 533 (1962). BRAWN, A. C., AND WOOD, H. N.,Advan. Cancer Res. 6, 81 (1961). BRAWN, A. C., AND WOOD, H. N., Proc. Natl. Acad. Sci. U.S. 66, 1417 (1966). FELLENDERG, G., Z. PfEanzenph@ol. 66, 446 (1967). HUANG, R. C., AND BONNER, J., Proc. Natl. Acad. Sci. U.S. 46, 1716 (1962). KLEIN, R.. M., in “Encyclopedia of Plant Physiology” (W. Ruhland, ed.), Vol. XV, p. 209. Springer-JTerlag, Berlin (1965). LAMAR, C., JR., PRIVAL, M., AND PITOT, H., Cancer Res. 26, 1909 (1966). MARKHAM, R., in “Modern Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds.) Vol. 4, p. 246. Springer-Verlag, Berlin (1955). MARMUR, J., J. Mol. Biol. 3, 208 (1961). MURASHIGE, T. and SKOOG, F., Physiol. Plant. 14, 473 (1962). REDDI, K. K., Proc. LVatl. Acad. Sci. U.S. 66, 1207 (1966). SINGER, II. L., AND KNIGHT, C. A., Biochem. Biophys. Res. Common. 13, 455 (1963). SCHERRER, K., LATHAM, H., AND DARNELL, J. E., Proc. Xafl. Acarl. Sci. U.S. 49, 240 (1963). SRIVASTAVA, B. I. S., Biochim. Biophys. Acta 103, 349 (1965). SRIVASTAVA, B. I. S., Ann. N.Y. Accd. Sci. 144, 260 (1967).
OF
BIOCHEMISTRY
Patterns
AND
of Nucleic
BIOPHYSICS
Acids
Tumor
126, 817-823
Synthesis
Tissue
Roswell
Park
Memorial Received
Institute, September
New
in Normal
Cultures
B. I. SAHAI
(1968)
and
Crown
Gall
of Tobacco’
SRIVASTAVA
York
State
18, 1967;
Department
accepted
of Health,
December
Buffalo,
New
York
4, 1967
The bacteria-free crown gall tumor tissue cultures of tobacco were 40-907, higher in nucleic acid content and up to 10-15 times higher in the capacity to incorporate 32P into RNA than the normal tissue cultures. Examination of the szP sedimentation profile of rapidly labeled nucleic acids from normal and tumor tissue cultures (12-42 days old) suggested some differences between t.he normal tissue and the tumor tissue, although the profiles also changed with the culture age. Since the s2P nucleotide composition (AMP + UMP = 54-58yo) of total RNA and of different RNA fractions, obtained by density gradient centrifugation, was similar to DNA (A + T = S170) rather than ribosomal RNA (AMP + UMP = 44yo), the RNA labeled with a2P was considered to represent principally mRNA. Both the absorbance and the 3tP nucleotide compositions of total RNA from normal tissue were similar to those from tumor tissue, and, except, for fraction “C,” consistent differences in the 32P nucleotide compositions of different RNA fractions from normal and tumor tissue were not very apparent.
Agrobacterium tumefaciens (Smith and Town, Conn.), a bacterial plant pathogen, has the ability to permanently transform the host cells into tumor cells (2) which show unrestricted growth. After transformation, the tumor cells are able to synthesize auxin, cytokinin, and other substances (2, 7) in abundance which must be supplied to the normal cells for growth in vitro. Increases in the amount of RNA ( 15), proteins (3), and the activities of certain enzymes (15) as well as the induction of new enzyme proteins (12) also occur in transformed cells. These modifications could occur by a persistent de-repression of a segment of normal cell genome which would lead to a greater synthesis of mRNA’s or by the incorporation of a self-replicating viral genome (if the actual tumorogenic agent of the crown gall organism is a virus) into h.ost genome. In this respect it is interesting to note that initiation 1 This Atomic 1)3722.
work Energy
was supported Commission
by United Contract
States AT(30817
of tumors in plants by A. tumefaciens has been shown to be inhibited both by ribonuclease(4) and histones (5). In the present work the patterns of RNA synthesis and the composition of various RNA fractions in normal and tumor tissue cultures have been examined in an attempt to find qualitative or quantitative differences. MATERIALS
AND
METHODS
Greenhouse-grown tobacco (LTicotania fabacum var. Wisconsin 38) plants (about 60 cm tall) were inoculated with Agrobacterium tumefaciens, strain 4-32 (cultured for 24 hours in nutrient broth plus 0.5$& dextrose) by needle puncture of the internodes. When the tumors were well developed the superficial layers of the tumors were discarded, and small segments of the underlying tissue after treatment with Clorox [lOy, Clorox (5.3yo NaOCl) for 10 minutes followed by repeated rinsings with sterile water] were planted on Murashige and Skoog’s (11) minus 1AA and kinetin medium (medium A). Most of the isolates contained bacteria, but a few which were bacteria-free were selected and cultured successively through several transfers on medium A to establish the independ-
818
SRIVASTAVA
ence of the tumor tissue for 1AA and cytokinin which must be supplied for the culture of the normal tissue (11). The tumor tissue has now been cultured on medium A for more than a year without any reduction of its growth. For establishing the culture of the normal tissue, the internode segments from tobacco plants, after being sterilized with 10% Clorox, were cultured on Murashige and Skoog’s (11) medium containing 0.2 mg kinetin per liter and 2 mg IAA (medium B) per liter. In both medium A and B, thiamin-HCl was at a concentration 0.4 mg/liter, and the edamin was omitted. Every 34 weeks small segments of the normal tissue and the tumor tissue were transferred to mediums (50 ml) B and A, respectively, contained in 125-ml flasks. The cultures were grown in growth chambers under continuous fluorescent light at 30”. Labeling, isolation, and analysis of nucleic acids. The normal tissue and the tumor tissue (about 10 gm fresh weight) were removed carefully under sterile conditions, divided into small segments, and transferred to weighed flasks containing 30 ml of the sterile liquid (i.e., without agar) media exactly similar (except for the omission of KHnPOl and adding of 100 rC of NazH3ZPO*) to that on which the tissues were growing. After 2 hours’ incubation on a shaker at 27” the tissue segments were quickly rinsed with 30 ml of the cold liquid media (s2P omitted) and used immediately for the
FIG. 1. The media
B and
cultures of normal A, respectively.
and
tumor
isolation of nucleic acids. Where the effect of actinomycin D on RNA synthesis was to be determined, 10 pg actinomycin D/ml was incorporated into the incubation media prior to autoclaving. The nucleic acids were isolated by a phenolsodium lauryl sulfate procedure as described by Srivastava (16) except that 0.01 M MgCI, and 5 mg bentonite/ml were added to the extracting buffer. After dialysis against phosphate buffer a part of the nucleic acid sample (l-l.2 ml out of 3 ml) was layered on 26 ml of a 5-20$$ linear sucrose gradient made in 0.05 M sodium phosphate buffer, pH 6.8, containing 0.001 M MgC12. The gradients were spun at 25,000 rpm for 16 hours in an SW25-1 rotor of a Spinco model L ultracentrifuge. Forty-drop fractions were collected by bottom puncture, diluted with 3 ml of water, and read at 260 rng. Fifty micrograms of crystalline bovine serum albumin and 1 ml of 25% trichloroacetic acid were added to each fraction. The RNA precipitates were collected on nitrocellulose membrane filters (type B6) which were washed with 5% cold trichloroacetic acid, dried, and then counted in vials containing 15 ml scintillation solution (4 gm 2,5-diphenyloxazole and 0.1 gm p-bis-[(2,5(phenyloxazolyl)] benzene per liter of toluene) in a Nuclear Chicago 720 counter. After counting, the discs containing Fractions l-10, 11-20, 21-32, and 3346 were pooled to give RNA fractions designated A, B, C, and D, respectively.
tissues
of tobacco
after
33 days
growth
on
NUCLEIC
ACID
SYNTHESIS
?T
IN
TOBACCO
-250
I’
// /
I’
,’
//
0
’
2’
Y‘a s E d 3
-100
& 0
,,jPN ,I‘
I’ N
p”
/’ ,,’ /’ ,’ /;- ‘Z
-150
1
J,’
50
I
I
I
IO
20 AGE
30 IN DAYS
I
40
s19
CULTURES
Carrier yeast RNA was added to each sample, and after the membrane filters were solubilized with acetone, the RNA was hydrolyzed with 0.3 M NaOH for 16 hours (15). The nucleotides were separated by electrophoresis at pH 3.5 and located by ultraviolet light. The bands containing the nucleotides were transferred to scintillation vials and counted as above. Where nucleotide composition of the total RNA was to be determined, the nucleic acid sample left from density gradient analysis was made free of traces of inorganic s2P by passage through a Sephadex G-25 column. The hydrolysis of RNA, separation of nucleotides, and counting of radioactivity were done as outlined above. For determining the nucleotide composition of RNA through absorbancy measurements (15), the nucleotides were eluted by 0.1 N HCl, and the absorbance of the eluates at X,,, of the nucleotides was determined against appropriate blanks. Isolation and determination of base composition of DNA. The DNA was isolated from chromatin (prepared by the procedure of Huang and Bonner) (6) of the normal tissue by Marmur’s (13) procedure. The hydrolysis of DNA by formic acid, separation of the bases by paper chromatography, and determination of the base composition of
-200
:
TISSUE
50
FIG. 2. The increase in fresh and dry weights of the normal tissue and the tumor tissue cultured on media B and A, respectively.
20-
5-
l-
--L.--L i0
A
20
30
40
! AGE
0 IN
--/-..I
IO
20
30
40
I
DAYS
FIG. 3. The changes in the percentage dry weight and the amount of nucleic acids in normal and tumor tissues growing on media B and A, respectively. The values represent means of two series of experiments. rRNA-1 and rRNA-2 represent light and heavy ribosomal RNA, respectively.
-
““092
0.0
-
Wx32
‘a.0
-
“WO92
DKl
NUCLEIC I)NA were of Markham
carried (9).
RESULTS
out
ACID according
AND
SYNTHESIS to the
TOBACCO
IN
DISCUSSION
TABLE COMPOSITIOV
OF NUCLEIC
ACIDS CULTURES
S%l
CULTURES
tumor tissue for 12-, 22-, 32-, and 42-day-old cultures presented in Fig. 4 show four absorbancy peaks corresponding to heavy ribosomal RNA, light ribosomal RNA, DNA, and soluble RNA. The 321’ radioactivity peaks do not correspond to the absorbancy peaks of the nucleic acids, indicating that labeled RNA was heterogenous in nature. The rate of 32Pincorporation into RNA of the tumor tissue was substantially higher than the normal tissue throughout the growth period invest,igated, with the incorporation being lo-15 times higher during the active growth period. Actinomycin D inhibited the incorporation of 32P into all RNA fractions of both normal tissue and the tumor tissue, indicating that RNA was being synthesized on a DNA template. However, the extent of inhibition of RNA synthesis by actinomycin D in the tumor tissue was fal greater than in the normal tissue. At this point it may be interesting to note that all plant viruses are RNA viruses and that actinomycin D does not inhibit RNA synthesis by these viruses ( 13). An examination of the 32Pprofiles of the nucleic acids in Fig. 4 suggeststhat the patterns of RNA synthesis in normal and tumor tissues were different, although the patterns also changed during the growth of both tissues. The total RNA from normal and tumor tissue was similar in nucleotide composition and had 55 % of GRIP + GRIP, whereas the DNA was rich in A + T (61%) content
procedure
The normal tissue growing on medium B produced firm, compact, slightly green callus, and the tumor tissue growing on medium A produced a massof loosely held firm green tissue (Fig. 1). Both the normal tissue and the tumor t,issue grew well on the media used for their growth but the growth of the tumor tis sue was substantially better than of the normal tissue (Fig. 2) on a fresh- or dryweight basis. The percent,age dry weight of the tumor tissue was, however, consistently lower than that of the normal tissue (Fig. 3), indicating a higher degree of hydration of the tumor tissue. The total amount of nucleic acids, heavy and light ribosomal RNA, DNA, and soluble RNA all declined with the age of the culture in the normal tissue and the tumor tissue (Fig. 3). Although the amount of total nucleic acids or the nucleic acid components was 40-90 70 higher in the tumor tissue, the rat,ios of various nucleic acid components were not significantly different. The increased amount of nucleic acid in the tumor tissue noted here has also been previously found for the tomato crown gall tissue ( 15). The sucrose density gradient profiles of the nucleic acids from normal tissue and the
B.\SIG
TISSUE
FROM
I NORM.~L
(N)
AND
(T)
TUMOR
TISSUE
OF TOBACCO Age of culture
Fraction
12 days
42 days
CMP
AMP
GMP
UMP(T)
CMP
AMP
GMP
UMP
Total
32P RNAb
N T
18.9 16.5
30.1 31.3
25.0 25.5
24.2 26.6
18.3 18.1
30.6 30.6
23.5 25.2
27.4 26.1
Total
RN&
N T
23.4 22.8
24.2 24.4
31.3 31.5
21.0 21.1
22.7 22.9
21.4 21.4
32.2 30.9
23.6 24.6
N
19.2
29.9
20.0
31.0
DNAc a Mean b Base c Base
of two separate series of experiments. composition expressed as ‘% 32P. composition determined by absorbancy
measurements
and
expressed
as mole/100
ml.
21-32)
33-46)
C (Fractions
D (Fractions
of two separate in parentheses
11-20)
B (Fractions
a Mean b Values
l-10)
Fractions
-
25.3
29.8
18.0
24.4
33.6
17.0
24.9
29.0
19.2
24.8
31.3
32.1
18.1
18.0
31.5
AMP
22.3
22.7
26.5
24.6
28.6
23.9
23.G
23.6
UMP
RNA
of actinomycin
27.8
27.6
25.6
24.8
28.0
26.8
26.1
27.7
GMP
12 days
32P) OF VARIOUS
17.1
CMP
(%
series of experiments. indicate the presence
COMPOSITIONS
A (Fractions
RNA
NUCLEOTIDE
D.
20.6 (22.5) 22.6 (21.2)
16.2 (14.1) 18.0 (17.0)
17.2 (17.2) 18.3 (17.3)
20.1 (19.0)b 17.7 (16.5)
CMP
25.2 (30.9) 27.5 (25.4) __-
30.2 (36.6) 28.2 (32.5)
28.9 (37.1) 27.9 (31.8)
24.7 (31.7) 30.1 (32.7)
II
GMP
NORMAL
29.8 (25.5) 27.5 (25.5)
25.1 (24.7) 26.8 (25.1)
27.D (22.4) 28.1 (23.4)
28.0 (23.5) 26.9 (23.4)
I-
22 days
FROM
AMP
TABLE FRACTIONS
.\ND
24.2 (21.2) 22.2 (23.0)
28.3 (24.6) 27.0 (25.3)
26.2 (23.2) 25.6 (27.4)
27.0 (25.7) 25.3 (27.4)
UMP
I
Age of culture
(N)
28.8
28.7 25.8
20.2 25.8
25.0
26.0
29.0
18.0
25.G
33.4
27.1
28.5
26.0
30.0
25.5
25.8
31.4
31.0
GMP
-
23.2
22.2
26.9
25.5
25.0
25.8
25.3
25.9
UMP
TISSUI~
AMP
32 days
(T)
15.4
19.2
17.5
18.6
16.6
CMP
TUMOR
_
26.3
20.6
25.9
28.2
31.5
34.2
17.5 17.8
32.2
31.4
21.2 21.2
30.8
33.0
AMP
OF
18.3
17.8
CMP
I-
7 I
CULTURES
24.1
27.0
23.3
21.3
19.7
19.3
25.6
24.8
GMP
42 days
TOBXCO
24.5
24.2
27.4
26.9
26.7
28.0
25.3
24.2
UMP
2 c: L-
$
g 7
NUCLEIC
ACID
SYNTHESIS
IN
(Table I). These values are in agreement with those reported (1) for RNA and DNA from tobacco. Since the 32P nucleotide composition of total RNA (AMP + UMP = 54-58%) from both normal tissue, and the tumor tissue was like DNA rather than ribosomal RNA (AMP + UMP = 44 %) (l), a significant part of the RNA labeled under experimental conditions used may represent mRNA. However, the 3ZP nucleotide composition of total RNA from normal and tumor tissue was not significantly different. Since alterations in the populations of RNA may not be so obvious if the 32Pnucleotide composition of total RNA was determined, the 32P nucleotide composition of different RNA fractions (separated by density gradient centrifugation) was determined. The 32P nucleotide composition of RNA fractions from normal and tumor tissues presented in Table II shows that fractions A, B, and C were DNA-like, whereas fraction D with an AMP + UMP content of 47-52% had a nucleotide composition between that’ of soluble RNA (AMP + UMP = 39 %) (1) and DNA (A + T = 61%). As also suggest’ed by 321’ profiles (Pig. 4), the changes in nucleotide composition of RNA fractions with age indicate changesin the populations of RNA’s during growth. The only consistent differencesin the nucleotide composition betn-een normal and tumor tissue were noted in fraction C, although lessconsistent differences in other fractions were also detected. It is interesting to note that the RNA which was synthesized in the presence of actinomycin D was more DNA-like (AMP + UMP = 60 Yo) than that which was synthesized in its absence. A similar effect of actinomycin D on the 32P-nucleotidecomposition of rat liver RNA has been reported (S). Since actinomycin D inhibits both ribosomal and messenger RNA synthesis ( 14), a more DNAlike composition of RNA synthesized in the presence of actinomycin D may be due to a stronger inhibition of any ribosomal RNA synthesis.
TOBACCO
TISSUE
CULTURES
823
A very high rate of RNA synthesis noted in the tumor tissue (compared with the normal tissue) is in line with its high enzymic activity and its ability to synthesize large numbers of essential compounds in ample amounts (2). Although some differencesin the 32Pdensity gradient pattern and the nucleotide composition of RNA from normal and tumor tissue were noted in the present studies, a more precise evaluation of the extent of similarities and differences in the populations of mRNA from normal and tumor tissue must await RNA-DNA hybridization, studies. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14.
15. 16.
BELLAMY,
A. R., Hiochim. Hiophys. Acta 123, 102 (1966). BRAUN, A. C., Ann. Rev. Plant Physiol. 13, 533 (1962). BRAWN, A. C., AND WOOD, H. N.,Advan. Cancer Res. 6, 81 (1961). BRAWN, A. C., AND WOOD, H. N., Proc. Natl. Acad. Sci. U.S. 66, 1417 (1966). FELLENDERG, G., Z. PfEanzenph@ol. 66, 446 (1967). HUANG, R. C., AND BONNER, J., Proc. Natl. Acad. Sci. U.S. 46, 1716 (1962). KLEIN, R.. M., in “Encyclopedia of Plant Physiology” (W. Ruhland, ed.), Vol. XV, p. 209. Springer-JTerlag, Berlin (1965). LAMAR, C., JR., PRIVAL, M., AND PITOT, H., Cancer Res. 26, 1909 (1966). MARKHAM, R., in “Modern Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds.) Vol. 4, p. 246. Springer-Verlag, Berlin (1955). MARMUR, J., J. Mol. Biol. 3, 208 (1961). MURASHIGE, T. and SKOOG, F., Physiol. Plant. 14, 473 (1962). REDDI, K. K., Proc. LVatl. Acad. Sci. U.S. 66, 1207 (1966). SINGER, II. L., AND KNIGHT, C. A., Biochem. Biophys. Res. Common. 13, 455 (1963). SCHERRER, K., LATHAM, H., AND DARNELL, J. E., Proc. Xafl. Acarl. Sci. U.S. 49, 240 (1963). SRIVASTAVA, B. I. S., Biochim. Biophys. Acta 103, 349 (1965). SRIVASTAVA, B. I. S., Ann. N.Y. Accd. Sci. 144, 260 (1967).