RIBOSOMAL DNA IN A NUCLEAR SATELLITE OF TOMATO
MARY-DELL CHILTON
Department of Microbiology and Immunology, University of Washington,
Seattle, Washington 98195
Manuscript received April 21, 1975
Revised copy received July 7, 1975
ABSTRACT
A satellite DNA of buoyant density 1.704 constitutes approximately 5%6% of nuclear DNA isolated from cherry tomato leaves. Isolated satellite
DNA exhibits a multi-componentmelting profile. Kinetic complexity measurements indicate that 37% of the satellite consists of repeating units of I O 5
daltons, and 48% of it consists of repeating units of 5.5 x 106 daltons. The
latter component is identified as DNA coding for ribosomal RNA on the basis
of its buoyant density, kinetic complexity, and abundance in nuclear DNA,
3.2% as determined by saturation hybridization measurements. Saturation
studies show that the more rapidly reassociating component of the satellite does
not code for 5s RNA. The question of genetic linkage between satellite
components is not resolved by this study.
ALTHOUGH dicotyledonous plants commonly possess satellite components in
their DNA (INGLE,PEARSON
and SINCLAIR
1973), few of these have been
isolated and subjected to kinetic complexity measurements. The similarity in
buoyant density profile of ribosomal DNA and satellite DNA in pumpkin led
MATSUDA
and SIEGEL (1967) to suggest that the genes for ribosomal RNA
constitute an important fraction of satellite DNA, which might be nucleolar in
origin. These investigators found further that there was a positive correlation
between high percentage of ribosomal DNA and presence of satellite DNA for
several plants. THORNBURG
and SIEGEL(1973a) found that pumpkin DNA
coding for 5s RNA is of satellite density in CsCl gradients, while 4s RNA
exhibits homology principally with main band DNA. In contrast to the view that
satellite DNA might be nucleolar in o r i g i n is the observation of INGLE,TIMMIS
and SINCLAIR
(1975) that all satellite DNAs examined (nine plants) differ
slightly in buoyant density from ribosomal DNA in the same plant. These
investigators found no correlation between high percentage of ribosomal DNA
and the occurrence of satellite DNA. MATSUDA,
SIEGEL
and LIGHTFOOT
(1970)
noted that the amount of satellite DNA did not correlate closely with the amount
of ribosomal DNA, and suggested that ribosomal DNA might constitute a part
of a complex mixture of DNAs which comprise the satellite.
BENDICHand ANDERSON
( 1974) have reported kinetic complexity measurements of a satellite DNA from muskmelon, which constitutes 30% of the total
plant DNA. Approximately one-third of the satellite reassociates extremely
rapidly (repeat size 2.5 x IO5daltons) and two-thirds slowly (repeat size 5 X 10'
Genetics 81 : 469-483 Navember, 1975
470
RI-D. CHILTON
daltons) . Ribosomal DNA cannot constitute more than one-tenth of this satellite,
for it constitutes ca. 3% of muskmelon DNA (M.-D. CHILTONand A. J. BENDICH,
unpublished data).
This paper presents a partial characterization of a small satellite component
of tomato DNA (INGLE,PEARSON
and SINCLAIR
1973) which was noted in this
and CHILTON1975). Like
laboratory in. the course of other studies (HANSON
muskmelon satellite (BENDICHand ANDERSON
1974). tomato satellite DNA
exhibits two major kinetic components. The more slowly reassociating component (48%, repeat size 5.5 x lo6 daltons) is shown to code f o r ribosoma1
RNA. Genes coding for 5 s RNA, which form a part of pumpkin satellite
(THORNBURG
and SIEGEL1973a), do not constitute the more rapidly reassociating
component of tomato satellite (37%, repeat size 10' daltons) : their abundance
is 12- to 15-fold lower. The role of this more rapidly reassociating component,
whose kinetic complexity is like that of mouse satellite DNA (WARING
and
BRITTEN1966) and the simple component of muskmelon satellite DNA (BENDICH
and ANDERSON
1974), remains obscure.
MATERIALS A N D METHODS
D N A isolation
Total leaf D N A : DNA was isolated from frozen leaves of cherry tomato plants (Lycopersicon
esculenfum Mill. var. Cerasiforme Hort.) after powdering the tissue in a coffee mill (Rollmix, R.
Bialetti and Co.) cooled with dry ice chips. Per gram of tissue was added 1 ml equilibrated
phenol (pH 7.5), 0.1 ml 1 M EDTA, pH 8, and 0.05 ml 20% sodium dodecyl sulfate. The
mixture was stirred at room temperature for 10 minutes and centrifuged (10,000 X g, 10
minutes). DNA was spooled from the aqueous phase, redissolved and dialyzed. Two cycles of
ribonuclease treatment (20 pg/ml, 37", 30 minutes), deproteinization with CHC1,-isoamyl alcohol (24:l) and spooling were followed by dialysis us. 0.1 x SSC. (SSC is 0.15 M NaC1, 0.015
M trisodium citrate.) The yield was 4.6 m g of DNA from 390 g. of tissue.
Nuclear D N A : Nuclei were isolated from small leaves of cherry t?mato plants by the
procedure of HAMILTON,
KUNSCH and T ' m w m L I (1972). The nuclear pellet was washed with
triton buffer (0.25 M sucrose, 0.05 M tris, pH 8, 2 m M MgCI,, 2 mM CaCl,, 2% triton-X-100)
until the supernate was clear and colorless ( 7 washes). The nuclear pellrt was frozen. thawed
in hot lysis buffer (0.05 M tris, 2 mM EDTA, 1 M NaCl, pH 8, 1% sodium dodecyl sulfate)
and heated at 70" for 15 minutes. DN.4 was ethanol precipitated, redissolved and treated with
predigested pronase (1 mg/ml, 4 x SSC, 37", 3 hours). After centrifugation to clarify the
solution, DNA was spooled, redissolved, treated with ribonuclease and deproteinized with phenol.
After spooling and dialysis, DNA was obtained in a yield of 4.2 mg from 140 g of tissue.
Gradient-purified nuclear D N A : More rigorously purified nuclei were isolated by the Ludox
step gradient procedure of HENDRIKS
(1972), starting with triton-washed nuclei prepared as
describzd above. Nuclei were retained in the step gradient on a shelf of 50% Ludox. Nuclei
were removed from the gradient by Pasteur pipet, dispersed in 0.1 M tris, pH 7.5, 2 m M MgCl,,
centrifuged, washed once with the same buffer and frozen. DNA was isolated as described for
nuclei above. From 53 g of small leaves and buds, 180 pg of DNA was isolated.
Purification of tomato satellite D N A
Satellite DNA was separated from main hand DNA by preparative CsCl density gradient
centrifugation. Per gradient, 300 pg of DNA was fractionated, after shearing by five passages
through a 26-gauge syringe needle to reduce viscosity. T o 4.0 ml of DNA solution in 0.1 x SSC
was added 5.1 g. of CsCl (Harshaw Chemical Co., optical grade powder). Gradients were centrifuged in the Spinco 50 rotor for 60 hours at 20" (45,000 rpm). Fractions (0.15 ml) were
TOMATO SATELLITE DNA
471
collected from the bottom of the gradient by a Hoefer fractionator A,,. was measured in
microcuvettes after suitable dilution. Appropriate fractions were pooled and rebanded twice to
remove residual main band DNA contamination (see text).
Measurement of melting profiles and reassociaiion kinetics
DNA samples were sheared by French pressure cell (single-strand molecular weight ca.
5 X IO5) and dialyzed us 0.25 x SSC, the same dialyzate being used for satellite and standard
(PS8 bacteriophage) DNA, in order to assure identity of ionic strength in the two samples. The
isolation of PS8 DNA has been described previously ( CHILTON
et al. 1974).
DNA samples overlaid with mineral oil were sealed in teff on-stoppered quartz microcuvettes.
NO evaporation was detected during the course OI denaturation and reassociation measurements,
which were performed with a GilIord recording spectrophotometer equipped with automatic
blank, offset control and temperature monitor channel. A three channel water circulating system
was employed which allowed rapid switching from denaturation conditions (95") through cold
water cooling to the reassociation condition (62") in a span of approximately three minutes. A
Haake ultrathermostat was used to maintain the reassociation temperature.
Kinetics of reassociation were analyzed after graphical subtraction of "collapse" hypochromicity, a first-order loss of A,,, observed upon cooling denatured DNA to the reassociation temperature. A standard correction of 10% of the oliginal hyperchromic shift was assigned to collapse.
This value has been arrived at through analysis of many types of DNA reassociating a t a
temperature 25" below T m (midpoint of the hyperchromic transition). Because of the unusually
heterogeneous melting profile of tomato satellite DNA, the applicability of this standard collapse
correction may be questioned. Accordingly, the amount of the rapidly reassociating component
of the satellite may be slightly in error.
Isolation of "P-labeled ribosomal RNA from iomato roots
Cherry tomato seeds were surface-sterilized by treating for 5 minutes with Roccal (0 OB%),
5 minutes with Staphene (0.5%) and 15 minutes with 1% sodium hypochlorite, with sterile
water rinses after each step. Seeds were allowed t o germinate on sterile damp filter paper in a
petri dish for one week, at which time 2 mCi "P-PO, [New England Nuclear, carrier-free,
neutralized with tris buffer, p H 8) was added. After 3 days, 1 ml of 0.1 M phosphate buffer,
p H 6.8, was added as a chase. After t w o days, the plants were harvested. Platings of the root
water on nutrient agar at 22" showed the plants to be free from viable microorganisms. Roots
were excised and ground (00) in a mortar with glass beads and 2 ml each of water-saturated
(1966). After
phenol containing bentonite and the homogenization buffer of CLICKand HACKETT
15 minutes of grinding, the preparation was centrifuged (5 minutes, 5000 x g) and the aqueous
phase extracted with phenol at 50" for 5 minutes. After centrifugation (10,000 x g, 10 minutes),
the supernate was adjusted t o 0 2 M potassium acetate and RNA precipitated by addition of 2
volumes of ethanol at -5". The RNA was redissolved in 1 ml of 0.01 M tris, p H 7.5, subjected
to gel filtration on Sephadex G25 coarse, adjusted to 0.01 M MgC1, and treated with DNase
(Worthington, DPFF, 20 pg/ml, 30 mmutes, 37"). After phenol deproteinization, precipitation
and a second gel filtration, the RNA (200 pg total yield) was fractionated by preparative sucrose
density gradient sedimentation (5%-20% sucrose, 0.01 M tris, 0.02 M EDTA, p H 8, Spinco
SW25 rotor, 22,,000 rpm, 16 hours, 5"). The ribosomal RNA had a specific activity of 7770
cpm/pg at the time of isolation.
Isolation of 32P-labeled 5s R N A from tomato roots
32P-labeled RNA was isolated as described above except that 3.5 mCi 32P-P04was added to a
smaller quantity of axenic seedlings. RNA in 0.1 M NaC1, 0.01 M tris, p H 7.5 was centrifuged in
the Spinco 40 rotor (32,000 rpm, 10 hours, 5") in order to pellet most of the ribosomal RNA. The
RNA in the supernate was precipitated and redissolved in a small volume of water. A 0.1-ml
aliquot (20 pg) was subjected to preparative electrophoresis on a 5-cm gel of 10% polyacrylamide as described by HAGEN
and YOUNG(1974). The time of emergence of marker yeast 4 6
RNA from an identical gel corresponded to that of the major peak of 32P-RNA. The smaller
472
M-D. CHILTON
peak of labeled RNA which emerged subsequently was taken as 5 s RNA. After concentration by
evaporation in an air stream and dialysis us. distilled water, this RNA was used for hybridization
to DNA filters. The concentration of 5 s RNA was too low for reliable determination of specific
activity, which was instead inferred indirectly from that of ribosomal RNA in the same extract.
Ribosomal RNA was purified from the high-speed pellet fraction by preparative sucrose gradient
sedimentation as described above. The specific activity was 52,600 cpm/pg at the time of the
hybridization measurements.
Preparation of D N A filters
DNA filters were prepared by a slight modification (FARRAND,
EDENand CHILTON1975) of
the procedure of MCCARTHY
and MCCONAUGHY
(1969). DNA bound to filters was measured by
diphenyl amine reaction performed on filters at the end of hybridization reactions, as previously
described (FARRAND,
EDENand CmLToN 1975).
RNA/DNA-filter hybridization reactions
Hybridization reactions were run in 2 x SSC at 67". All reactions were incubated for 16-18
hours, and were terminated by removal of the filter from the labeled RNA solution, washing
three times in 2 x SSC a t 67", treating with 20 pg/ml RNase in 2 x SSC at 22" f o r 30-60
minutes, and washing twice with 2 x SSC at 22". Dried filters were counted in toluene-Liquifluor
(New England Nuclear) in a Beckman CPM-I00 liquid scintillation counter.
Analytical CsCl density gradient centrifugation
Analytical gradients were centrifuged for 20 hours at 42,040 rpm in the AnF rotor of a
Spinco Model E ultracentrifuge equipped with four cell mask-timer assembly and ultraviolet
optics. Films were traced by a Joyce-Loebel densitometer. Buoyant density values were
calculated by the method of SCHILDKRAUT,
MARMUR
and DOTY(1962).
RESULTS
Isolation of tomato satellite DNA
The high-density satellite component of cherry tomato nuclear DNA was
isolated by preparative CsCl density gradient centrifugation (Figure 1A). The
dense shoulder of satellite D N A was pooled (bar) and rebanded (Figure 1B).
The indicated fractions (bar) of this gradient were subjected to a second rebanding (Figure I C ) to remove residual main band DNA. Fraction 25 (arrow) of
this gradient was found by analytical CsCl gradient analysis to be significantly
contaminated with main band DNA; thus this second rebanding is essential for
isolation of pure satellite. The indicated fractions (bar) from Figure 1C were
pooled and used for the experiments described below.
FIGURE
1.-Isolation of tomato satellite DNA by successive preparative CsCl density gradient
centrifugation.
Details of gradient preparation and centrifugation are described in MATERIALS AND METHODS.
A. One of six gradients of tomato nuclear DNA used for first stage purification of satellite.
A,,, was measured on a 1/25 dilution of each fraction. Fractions indicated by the bar
were pooled.
B. Rebanding of pooled fractions from A. A,,, was measured on the fraction (0.15 ml)
diluted with 0.1 ml distilled water. Fractions indicated by the bar were pooled.
C. Rebanding of pooled fractions from B. A,,, was measured as in B. Fractions indicated by
the bar were pooled. The fraction indicated by the arrow was 60% satellite and 40% main
band, from analytical CsCl gradient tracings.
TOMATO SATELLITE D N A
A
473
474
M-D. CHILTON
Analytical CsCl density gradient profiles of isolated satellite and total tomato
DNA preparations are shown in Figure 2. The density of isolated satellite,
1.704, (Figure 2A) was identical to that of the satellite component in the original
nuclear DNA from which it was isolated (Figure 2B), in total leaf DNA (Figure
2C) and in DNA isolated from nuclei purified in a Ludox step gradient (see
MATERIALS AND METHODS) (Figure 2D).
Melting profile and reassociation kinetics of tomato satellite DNA
Purified satellite DNA was subjected to denaturation and reassociation in a
Gilford recording spectrophotometer (see MATERIALS AND METHODS). The melting
1731
I.i
I
FIGURE
2.-Buoyant density profiles of satellite DNA i n analytical CsCl gradients.
Analytical gradients containing 0.5-3 pg DNA plus 0.5 pg Micrococcus lysodeikticus UNA as
marker ( p = 1.731) were centrifuged as described in M A T E R I ~ L S AND METHODS. The amount of
satellite in samples B, C, and D cannot be compared because the gradients are overloaded with
main band DNA, whose absorbance is beyond the linear range of film response.
A. Tomato satellite DNA (p~oledfrom Figure IC)
B. Tomato nuclear DNA
C. Tomato leaf total DNA
D. DNA from tomato nuclei purified by Ludox step gradient
475
TOMATO SATELLITE D N A
profile of this DNA proved to be very complex (Figure 3 ) with four components
clear by inspection. An approximate evaluation of these components is: 16%,
T m = 77"; 31%, Tm = 82"; 35%, T m = 86"; 18%, Tm = 89". The Tm of
standard PS8 bacteriophage DNA under these conditions (0.25 x SSC) was
83.4", and the transition was sharp (Figure 3 ) .The details of the complex melting
profile of the satellite DNA were reproducible in successive experiments. It is
surprising to observe such extreme heterogeneity of melting behavior within
what appears (Figure 2) to be a single satellite in CsC1.
The reassociation kinetics of satellite DNA at 62" in 0.25 x SSC, presented as
BRITTENand KOHNE'S(1968) Cot plot, are shown in Figure 4.From such a plot
it is difficult to estimate accurately the percentage of kinetic components, although
it is clear from inspection that at least two components are present. The
Scatchard-type plot of MARSH
and MCCARTHY
(1974) is useful for resolution of
100
80
>
t- 60
g
cc
I
150 40
0-
t
I
8
20
70
75
80
85
TEMPERATURE PC)
90
95
FIGURE
3.-Thermal dissociation profiles of native and reassociated tomato satellite DNA.
The hyperchromicity of DNA samples in 0.25 x SSC was monitored as described in
MATERIALS AND METHODS. Temperature of the cuvette chamber was raised approximately 1 per
2 minutes. Data are plotted as percent of the ultimately attained hyperchromicity reached a t each
temperature.
Native tomato satellite DNA
................ Native PS8 bacteriophage DNA
82% reassociated (Cot= 4.2) tomato satellite DNA
--_____-
476
M-D. CHILTON
IO-^
to-'
10-2
lo0
IO'
CO+
FIGURE
4.-Kinetics of reassociation of tomato satellite DNA.
Reassociation of DNA samples (sheared by French pressure cell) in 0.25 x SSC a t 62" was
measured with a Gilford recording spectrophotometer as described in MATERIALS AND METHODS.
An ideal second-order curve is drawn through the phage DNA kinetic points.
0 Tomato satellite DNA
PS8 bacteriophage DNA
n
}
0
, , , :\;
,>;,,
D.
,
O b 0
40
60
80
I( 3
% DOUBLE STRANDED
20
FIGURE
5.-Scatchard-type analysis of satellite DNA reassociation kinetics.
The method of MARSH
and MCCARTHY
(1974) is used to analyze the data of Figure 4 (see
text).
TOMATO SATELLITE D N A
477
such complex reassociation kinetics (Figure 5). The rapid component of the reaction can be described by ideal kinetics if it is assumed to constitute 37% of the total
DNA, for the linear portion of the kinetics extrapolates to 37% doublestranded
DNA at infinite Cot value (zero value of the ordinate). By similar line of
reasoning, a second kinetic component ending at approximately 85% doublestranded DNA is visible, and would constitute 85 minus 37 = 48% of the satellite
DNA.
These plateau values can now be used in analyzing the data of Figure 4 more
closely. If the fast reaction corresponds to the first 37%, then at 18.5% doubleThis value must be corrected,
stranded DNA, we find its Cotl/,value: 2.04 X
for this DNA would renature still faster if isolated: its Cot,/,value would be
0.37 X 2.04 X
= 7.5 x
By a similar line of reasoning, the Cots value
of the second component is 7.58 x
and after correction for its true concentration in the mixture, it exhibits a Cot%value of 4.17 X 10-I. Under the same
renaturation conditions, sheared standard bacteriophage PS8 DNA, whose
genome size is 4.1 X lo7 (CHILTONet al. 1974) has Cots = 3.1 (Figure 4 ) . The
kinetic complexity of the fast component of tomato satellite is therefore:
(7.5 X 10-s/3.1) X 4.1 X lo7 = 9.9 X lo4daltons. Similarly, the slow component
must have a kinetic complexity of (4.17 x 10-1/3.1) X 4.1 X IOT= 5.5 X I O 6
daltons. These estimates are only approximate because of uncertainty about the
T m of the reacting DNA component (Figure 3) compared to the T m of the
standard DNA. Exact evaluations must await physical separation of the
components.
In an effort to assess the extent of mismatching in reassociated satellite DNA,
duplexes were remelted at the end of the renaturation experiment of Figure 4.
As shown in Figure 2, the reassociated DNA exhibits a broad melting curve.
Enrichment for material melting below 80" and the occurrence of material
melting below 70" suggest that some reassociated satellite DNA is extensively
mismatched. Detailed analysis of such remelting profiles is difficult because of
the contribution of remaining single-stranded DNA to the hyperchromic shift.
In addition, the presence of multiple melting components, more than one of
which may be ascribable to a single kinetic component, make interpretation
difficult.Remelting of satellite DNA which had been allowed to reassociate only
through the rapid component of the reaction did not allow assignment of a unique
Tm value to this kinetic class (data not shown).
Quantitation of ribosomal DNA in tomato nuclear DNA
The buoyant density of tomato satellite DNA (Figure 2A) is similar to that of
known plant ribosomal DNA's (THORNBURG
and SIEGEL1973b; SCOTTand
INGLE1973; INGLE,TIMMIS
and SINCLAIR
1975). Moreover the kinetic complexity of nearly half of tomato satellite DNA (5.5 x lo6) is close to that expected
for plant ribosomal precursor genes: 4.6-5.2 x lo6 (LOENING,
JONES and BIRNSTIEL 1969) plus any spacer sequences. In order to determine whether the slower
reassociating component of tomato satellite DNA does indeed code for ribosomal
478
M-D. CHILTON
I
0
2
4
6
pg/ml RNA
8
IO
FIGURE
6.-Saturation of tomato DNA by "P-rRNA.
DNA filters contaimng 15 pg tomato nuclear DNA (6.3 mm diameter) were incubated with
the indicated concentrations of labeled rRNA, washed and treated with RNase as described in
MATERIALS ~ N D
METHODS.
Reactions were carried out in 10 x 75 mm test tubes; solutions were
overlaid with oil, and tubes were capped tightly with aluminum foil. DNA remaining on the
filter at the end of the incubation was 6.25 pg (42% of the initial value). Specific activity of
ribosomal RNA at the time the filters were counted was 5.356 cpm/pg. The saturation value is:
530/5356 x 1/6.25 = 0.0158 of tomato DNA homologous to rRNA.
RNA, the amount of ribosomal DNA in tomato nuclear DNA was estimated by
saturation hybridization measurements.
Labeled ribosomal RNA was isolated from axenic tomato seedling roots, in
order to exclude chloroplast ribosomal RNA from the preparation (MATSUDA,
SIEGELand LIGHTFOOT
(1970). Saturation data for 32P-labeledribosomal RNA
(Figure 6) show that 0.099 pg of labeled RNA is bound per 6.25 ,pg of tomato
nuclear DNA. Thus 1.6% of the DNA was hybridized, and 3.2% of tomato DNA
therefore codes for ribosomal RNA. Similar results were obtained in three experiments using two different labeled ribosomal RNA preparations.
Density profile of ribosomal DNA
In order to determine the buoyant density profile of ribosomal DNA in CsC1,
individual fractions of tomato nuclear DNA from a preparative CsCl gradient
were denatured, immobilized on nitrocellulose filters, and allowed to hybridize
with labeled ribosomal RNA. The profile of DNA sequences homologous to
of the satellite
ribosomal RNA was found to track with the profile of the AZGO
component in nuclear DNA (Figure 7). This result demonstrates that all ribosomal sequences, i.e. 3.2% of total DNA, must reside in the satellite density
stratum.
479
TOMATO SATELLITE DNA
I.4
1.2
I .o
n
0
$j 0.8
a
1600
0.6
1200
5
0
m
2
LT
L
04
800
0.2
400
9-o-o-9--p-p
I
IO
20
FRACTION NUMBER
l
l
lo
$
'o-o-o-(
1
1
30
FIGURE
7.--Hybridization of 32P-rRNA with tomato DNA fractions from a preparative CsCl
gradient.
Tomato nuclear DNA (100 fig) was subjected to preparative CsCl density gradient fractionation as described in MATERIALS A N D METHODS, except that the needle shearing step was omitted.
After A,,, measurement, fractions 17-32 were diluted with 9 ml of 0.1 N NaOH to effect
denaturation, neutralized after 5 minutes with 1 ml 1 M NaH,PO,, adjusted to 5 x SSC and
applied to prewashed 25 mm cellulose nitrate filters (Schleicher and Schuell, type B6). Filters
were washed and treated as described (FARRAND,
EDENand CHILTON1975).
For hybridization reaction, filters were mounted in serial order on a n insect pin and placed
in a 20-ml beaker containing 4 m l of labeled RNA (5 pg/ml, 2 x SSC) . The solution was overlaid with mineral oil and capped tightly with aluminum foil. After 17 hours incubation at 67",
filters were washed and treated with ribonuclease solution (25 ml) as described i n MATERIALS
A N D METHODS.
Input radioactivity was 353,000 cpm.
0 A,,, of fraction (0.15 ml) plus 0.1 ml water
D cpm 32P-rRNA bound to DNA fraction
Estimation of total amount of satellite D N A
The amount of satellite in the nuclear DNA was estimated by integration of
the A?,, profile in the preparative CsCl gradient in Figure 7. Depending upon
whether fraction 21 is considered all satellite o r only 50% satellite, the result is
5%-6% satellite DNA in the preparation. Thus the 3.2% of ribosomal DNA
situated at satellite density would constitute approximately half of the satellite
DNA sequences. The component of the reassociation kinetics of satellite DNA
corresponding to a repeat size of 5.5 x 106 can therefore be assigned to ribosomal
DNA sequences (plus, presumably, some spacer sequences).
480
M-D. CHILTON
Quantitation and density profile of genes coding for 5 s R N A
The repeat size of 5 s RNA genes, 8 x lo4 (PAYNE
and DYER1971), is close to
the repeat size of the rapidly reassociating component of tomato satellite, 9.9 X
lo4. Further, the DNA homologous to 5 s RNA in pumpkin is of satellite density
(THORNBURG
and SIEGEL1973a). In order to determine whether the more
rapidly reassociating component of tomato satellite DNA might consist of
multiple copies of 5 s RNA genes, the amount of DNA homologous to 5 s RNA
in tomato was determined.
32P-labeled 5 s RNA was isolated from tomato root RNA by preparative
electrophoresis on polyacrylamide. The gel resolved 5 s RNA cleanly from 4 s
RNA (Figure 8). Saturation measurements (Figure 9) show that the amount of
DNA coding for 5 s RNA is far lower than the amount of the more rapidly
reassociating satellite component in tomato DNA. Scatchard analysis of the saturation data (Figure 9 inset) shows that 9.7 x
pg of 5 s RNA is bound per
13.3 pg of tomato nuclear DNA; hence 0.073% of the DNA is homologous. Since
only one DNA strand is hybridized, twice this value or 0.15% of tomato DNA
codes for 5 s RNA. The more rapidly reassociating component of tomato satellite
DNA constitutes 37% of a 5%-6% satellite, or 1.8%-2.2% of tomato DNA.
Thus 5 s RNA genes are 12- to 15-fold lower in abundance than the more rapidly
reassociating satellite component, whose function remains unknown.
1000
c
FRACTION NUMBER
FIGURE
8.-Polyacrylamide gel electrophoresis of low molecular weight RNA from tomato
roots.
The low molecular weight Iracticn (see MA-TERIALS AND METHODS) of 32P-labeled tomato RNA
was subjected to preparative electrophoresis in 0.04 M tris, 0.02 M sodium acetate, 2 mM EDTA,
pH 7.0, on a 5-cm gel of 10% polyacrylamide at 25 volts (15 mA). Fractions of 2 ml were
and YOUNG1974).
collected (20-minute intervals) by elution of the base of the gel (see HAGEN
In a parallel run, marker yeast 4 s RNA emerged in fractions 30-32. Fractions 45-49 were taken
as 5 s RNA and used €or hybridization studies after concentration and dialysis.
481
TOMATO SATELLITE D N A
CPM BOUND
,
,
I
I
,
l
0.I
l
t
l
l
,
0.2
pG/ML 32P 5s RNA
FIGURE
9.-Saturation of tomato DNA bv 32P 5s RNA.
The experiment was performed as i n Figure 6, except that labeled 5 s RNA was employed.
The inset shows a Scatchard analysis of the data points, from which a saturation value of 510
cpm (0.009 p g RNA) was estimated for these filters, which contained 13.3 pg DNA at the end
of the reaction.
DISCUSSION
The evidence presented here demonstrates that a major fraction of tomato
satellite DNA codes for ribosomal RNA. The satellite, which constitutes 5%-6%
of tomato DNA, contains all osf the DNA which hybridizes with ribosomal RNA
(Figure 7). Since ribosomal DNA constitutes 3.2% of tomato nuclear DNA
(Figure 6), it must represent about half of the satellite. Consistent with this
model, 48% of isolated satellite DNA exhibits a kinetic complexity of 5.5 X lo6
(Figures 4 and 5), just sufficient to code for plant ribosomal RNA precursor
(2.3-2.6 X IO6, LOENING,JONESand BIRNSTIEL1969) plus some spacer
sequences.
Genes coding for 5s RNA constitute only 0.15% of tomato DNA (Figure 9)
and thus cannot represent a significant fraction of this 5 % 4 % satellite. The
saturation values for ribosomal and 5 s RNA suggest that there are 2-3 copies
of 5s genes for each rRNA gene; however the possibility of a 1:l ratio is not
rigorously excluded.
The kinetic complexity of the more rapidly reassociating 37% component
of tomato satellite, 9.9 x IO4,is similar to that of mouse satellite DNA, 1.8-2.4 X
1 O5 (WARING
and BRITTEN1966). More recent studies by HUTTON
and WETMUR
(1973) have shown that the kinetic behavior of mouse satellite DNA defies
conventional analysis, because of an unusual molecular weight dependence of
reassociation rate. An explanation for this novel kinetic behavior has been
proposed (CHILTON1973). Thus the true repeat size for simple, rapidly reassociating DNA's can prove to be substantially smalled than the value estimated
482
M-D. C H I L T O N
kinetically. Whether the repeat size f o r the rapid component of tomato satellite
is 9.9 x lo* or smaller yet, it is clearly present in much higher copy number, by
at least 4O-fold, than the ribosomal RNA genes. The intriguing question of
whether it is actually linked to ribosomal DNA is not settled by the present
experiments. If this should prove to be the case, tomato satellite could be designated a true ribosomal satellite. At present the alternate possibility exists that
tomato satellite is a mixture of components which exhibit by coincidence the
snme buoyant density profile in CsCl. The extreme heterogeneity in melting
behavior of tomato satellite DNA (Figure 3 ) seems to support the view that the
components are covalently linked. However muskmelon satellite contains
Components of widely different T m which are not covalently associated ( BENDICH
and ANDERSON
1974). Accordingly, the question of linkage within tomato
satellite remains open.
I thank ARNOLD
J. BBNDICH,
ALBERTSIEGELand BENJaniIN D H ~ Lfor
L discussions. I thank
FREDHAGENfor assistance in preparathe electrophoresis. This research was supported by Grant
No. CA13025 from the National Institutes of Health
LITERATURE CITED
BENEDICH.
A. J. and R. S. ANDERSON,
1974 Novel properties of satellite DNA from muskmelon.
Proc. Natl. Acad. Sci. U. S. 71: 1511-1515.
BRITTEN,R. J. and D. E. KOHNE,1968 Repeated sequences in DNA. Science 161: 529-540.
CHILTnN, M.-D., 1973 Theoretical explanation of mouse satellite DNA renaturation kinetics.
Nature New Biol. 246: 16-17.
CHILTON,M.-D., T. CURRIER,
S. K. FARRAND,
A. J. BENDICH,
M. P. GORDON
and E. W. NESTER,
1974 Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown
gall tumors. Proc. Natl. Acad. Sci. U. S. 71: 3672-3676,
CLICK,R. E. and D. P. HACKETT,1966 The isolation of ribonucleic acid from plant, bacterial
or animal cells. Biochim. Biophys. Acta 129: 74-84.
s. K., F. c. EDENand M.-D. CHILTnri, 1975 Agrobacterium tumejaciens and PS8
FARRAND,
bacteriophage DNA not detected in crown gall tumors by DNA/DNA-filter hybridization.
Biochim. Biophys. Acta. 390: 264-275.
HAGEN,
F. S. and E. T. YOUNG,1974 Preparative polyacrylamide gel electrophoresis of ribonucleic acid. Identification of multiple molecular species of bacteriophage T7 lysozynie
m-ssenger RNA. Biochemistry 13 : 3394-3400.
HAMILTON,
R. H., U. KUNSCHand A. TEMPERLI,1972 Simple rapid procedure for isolation of
tobacco leaf nuclei. Anal. Biochem. 49: 48-57.
HANSON,
R. S. and M. D. CHILTnN, 1975 On the question of integration of A . tumejaciens DNA
by tomato plants. J. Bacteriol. (in press).
HENDRICKS,,
A. W., 1972 Purification of plant nuclei using colloidal silica. FEBS Letters 24:
101-1 05.
HUTTON,J. R. and J. G. WETMUR,
1973 Length dependence of the kinetic complexity of mouse
satellite DNA. Biochem. Biophys. Res. Commun. 5 2 : 1148-1155.
J., G. G. PEARSON
and J. SINCLAIR,
1973 Species distribution and properties of nuclear
satellite DNA in higher plants. Nature New Biol. 242: 193-197.
JNGLP,
INGLE,J., J. N. TIMMIS
and J. SINCLAIR,
1975 The relationship between satellite DNA, ribosomal RNA gene redundancy and genome size in plants. Plant Physiol. 5 5 : 496-501.
TOMATO SATELLITE DNA
483
LOENING.U. E., K. W. JONES
and M. L. BIRNSTIEL,1969 Properties of the ribosomal RNA
precursor in Xenopus Ineuis: comparison to the precursor in mammals and in plants. J. Mol.
Biol. 45: 353-366.
MARSH, 5. L. and MCCARTHY,
B. J., 1974 Effect of reaction conditions on the reassociation of
divergent deoxyribonucleic acid sequences. Biochemistry 13 : 338S-3388.
K. and A. SIEGEL,1967 Hybridization of plant ribosomal RNA to DNA: the isolation
MATSUDA,
of a DNA component rich in ribosomal RNA cistrons. Proc. Natl. Acad. Sci. U.S. 58:
673-680.
MATSUDA,
K., A. SIEGELand D. LIGHTFOOT,
1370 Variability in complementarity for chloroplastic and cytoplasmic ribosomal ribonucleic acids among plant nuclear deoxyribonucleic
acids. Plant Physiol. 46:6-12.
MCCARTHY,
B. J. and B. L. MCCONAUGHY,
1968 Related base sequences in the DNA of simple
and complex organisms. I. DNA/DNA duplex formation and the incidence of partially
related base sequences in DNA. Biochem. Genet. 2: 37-53.
PAYNE,P. I. and T. A. DYER,1971 The isolation of 5-S ribosomal RNA from plants. Biochim.
Biophys. Acta. 228: 167-172.
SCHILDKRAUT,
c. L , J. MARMUR and P. DOTY,1962 Determination of the base composition of
deoxyribonucleic acid from its buoyant density i n CsC1. J. Mol. Biol. 4: 430-443.
SCOTT,N. S. and J. INGLE,1973 The genes for cytoplasmic ribosomal RNA i n higher plants.
Plant Physiol. 51 : 677-684.
THORNBURG,
W. and A. SIEGEL,1973a Characterization of the rapidly reassociating deoxyribonucleic acid of Cucurbitu pepo L. and the sequences complementary to ribosomal and
transfer ribonucleic acids. Biochemistry 12: 2759-2765. -,
197313 Rapidly renatured
DNAs of some higher plants. Biochim. Biophys. Acta 312: 211-214.
M. and R. J. BnITTEN, 1966 Nucleotide sequence repetition: a rapidly reassociating
fraction of mouse DNA. Science 154: 791-794.
Corresponding editor: B. D. HALL
WAnING,