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Synthesis of satellite DNA in Rhynchosciara hollaenderi
DEVELOPMENTAL
BIOLOGY
Synthesis
28,
649-661 (1972)
of Satellite
DNA in Rhynchosciara
hollaenderi’
JOHN PAPACONSTANTINOU, WILLIAM S. BRADSHAW, z EUGENE T. CHIN,~ AND EMILIA M. JULKU Biology Diuision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 and The Uniuersity Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Accepted April
of
26, 1972
During the latter part of the fourth instar development of Rhynchosciara larvae, DNA synthesis occurs in both germ cells and somatic cells, even though these cells do not undergo mitosis. CsCl density gradient analysis has revealed the synthesis of d(AT) satellite DNAs in the testis and in somatic tissues such as salivary gland, fat body, and gastric cecum. In these studies it has been shown that there is a tissue-specific variation in the relative proportions of synthesis of d(AT) satellite and main-band DNA in the testis during the fourth instar. The initiation of synthesis of the d(AT) satellite in the testis corresponds with the appearance and formation of the highly characteristic mitochondria which develop during the maturation of the spermatocytes. This satellite DNA has been shown to be circular and has a density of 1.681 gm/cmj in CsCl. Synthesis of a similar circular DNA cannot be detected in somatic tissues, although these tissues do synthesize a d(AT) satellite of similar density. We conclude from these studies that the specific synthesis of a unique circular DNA may be an essential part of the morphogenetic development of the highly specialized mitochondria formed during spermatocyte maturation. INTRODUCTION
Analysis of DNA by isopycnic centrifugation on cesium chloride (Meselson et al., 1957) has demonstrated, in a number of eukaryotic tissues, the existence of minor components with mean densities different from that of the bulk of the DNA. These satellite DNAs exhibit densities either higher or lower than the major band. Examples include: (1) mouse satellite DNA, comprising about 10% of the total DNA (Kit, 1961), which has been shown to be associated with centromeric heterochromatin in mouse chromosomes (Pardue and Gall, 1970; Jones, 1970); (2) the ribosomal RNA cistrons localized in the nucleoli of amphibian (Wallace and ’ Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. ’ AEC Postdoctoral Fellow under appointment by Oak Ridge Associated Universities. Present address: Department of Zoology and Entomology, Brigham Young University, Provo, Utah 84601. “Predoctoral Trainee supported by Grant No. GM 1974 from NIGMS. 649 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
Birnstiel, 1966; Brown and Dawid, 1968; Evans and Birnstiel, 1968) and insect (Gall et al., 1969) oocytes; (3) the poly d(AT) satellite of some crustaceans (Sueoka, 1961; Skinner, 1967); and (4) unique populations of extranuclear DNA originating in organelles such as mitochondria (Rabinowitz et al., 1965), kinetoplasts (Dubuy et al., 1965; Riou and Paoletti, 1967), and chloroplasts (Chun et al., 1963). In this paper we report on a d(AT)rich satellite DNA fraction extracted from larval tissues of the sciarid fly Rhynchosciaru (Diptera: Sciaridae). There is a tissue-specific variation in the relative proportions of satellite and main-band DNA synthesized in the testis during the development of fourth-instar larvae. This regulation of DNA synthesis appears to be correlated with the morphogenetic maturation of larval sperm cells. Our attempts to characterize the satellite DNA have shown that as much as 50% of the total d(AT) DNA synthesized during development of the testis is in circular form. Similar analysis of salivary gland
650
DEVELOPMENTAL BIOLOGY
DNA at corresponding stages of larval development does not reveal a circular satellite DNA. A preliminary report on these observations has appeared elsewhere (Chin et al., 1970). MATERIALS
AND
METHODS
Growth of Rhynchosciara. The procedure for maintaining Rhynchosciara larvae in the laboratory has been described (Mattingly and Ogle, 1969). The following modifications in antibiotic treatment and in preparation of food have been made: (1) The egg masses and larvae are kept in plastic petri dishes layered with Bacto agar containing terramycin (100 pg/ml) instead of streptomycin and tetracycline. (2) The food is prepared by suspending one 6-0~ block of Fleischmann’s fresh active yeast in 750 ml of distilled water at 35°C and adding to this 450 gm CSMA fly larvae medium (Ralston Purina Company, P. 0. Box 337, Richmond, Indiana 47374). The food is mixed in well and permitted to ferment 4-12 hr at 35°C. The food is prepared prior to feeding. Larvae up to the fourthinstar stage of development are fed once a week; fourth-instar larvae are fed twice a week. radioactive DNA. Frepara tion of Labeled DNA from R. hollaenderi was obtained by injecting individual larvae from synchronous cultures with 2 ~1 of either methyl[3H]thymidine (1 &i; 11.2 Ci/ mmole) or [ l’C]thymidine. Following labeling periods of 3-24 hr, partially purified DNA preparations were obtained from larval tissues which had been dissected in insect Ringer’s solution (Ephrussi and Beadle, 1936) and frozen on Dry Ice. Preparation of DNA for CsCl gradient analysis. Tissues from 50-200 larvae were homogenized in a conical ground glass homogenizer in l-2 ml of 0.14 M phosphate buffer (pH 7.0)-0.01 M EDTA-1% sodium lauryl sulfate-l M sodium perchlorate.
VOLUME 28. 1972
Homogenates were then extracted in an equal volume of chloroform-octanol (9 : 1) and centrifuged at 4°C for 30 min at 12,000 g. Two volumes of cold ethanol were then added to the aqueous phase, and the resulting precipitate was dissolved in 0.15 M NaCl-0.015 M sodium citrate (1 x SSC). The DNA samples obtained were added to 6- to 7-ml CsCl gradients in 1 x SSC and centrifuged in a Spinco No. 40 rotor at 35,000 rpm for 48-72 hr at 25°C. Following centrifugation, O.l-ml fractions were collected, and radioactivity was monitored by applying aliquots of these fractions to Whatman 3-MM filter paper disks, which were processed according to Bollum (1966) and counted in toluene-fluors at a counting efficiency for tritium of about 35%. Buoyant densities of sample fractions from each gradient were determined by measurements of their refractive index. In some instances [‘“Cl or [3H]thymidinelabeled DNA from Escherichia coli was added as a marker in the density gradients. For analyses in the analytical ultracentrifuge, the first ethanol precipitate was dissolved in 1 x SSC and treated with the following enzymes for 2 hr at 37°C: RNase A (100 pg/ml), Tl RNase (330 units/ml), and cu-amylase (100 pg/ml). The solution was again extracted with chloroform-octanol (9: l), and the DNA was precipitated from the aqueous phase with ethanol as described above. The precipitate was dissolved and dialyzed against 1 x SSC. The dialyzed solutions were adjusted to a density of 1.7 gm/cm3 by adding the appropriate amount of solid CsCl. Preparation of DNA for CsCl-ethidium bromide gradient analysis. Tissues from 50 larvae were homogenized in a Dounce homogenizer (loose-fitting No. A) with two strokes in 1-2 ml of the buffer described above. Homogenates were extracted in an equal volume of chloroformThe octanol (9: 1) by gentle mixing.
PAPACONSTANTINOU Rhynchosciara Satellite DNA
homogenate was centrifuged at 4°C for 30 min at 10,000 g. The aqueous layer (approximately 1 ml) was removed and added to 4 ml of saturated CsCl (25°C) in 1 x SSC. The interphase was reextracted with 0.5 ml of the same buffer, mixed with a pipette, and centrifuged as described above. The aqueous phase was added to the same CsCl solution and mixed gently. The precipitate formed was removed by centrifugation at 4°C for 10 min at 10,000 g. The CsCl-DNA solution was gently poured into a siliconized polyallomer tube. The density was adjusted by diluting with 1 x SSC, and ethidium bromide (in 1 x SSC) was added to a final concentration of 100 pglml. The gradients were centrifuged at 35,000 rpm for 62-68 hr at 25°C in a Ti 50 rotor. Fractions were collected on filter paper or in siliconized tubes and counted by liquid scintillation counting as described above. DNase treatment. DNA fractions (main band and satellite) from CsCl gradients were dialyzed in 0.01 M Tris (pH 7.4)0.01 M KCl-0.0015 M MgCl at 4°C for 20 hr with three l-liter changes. Half of each sample was treated with DNase (Worthington Pancreatic DNase I, final concentration 5 Kg/ml) at 25°C for 10 min. At the end of the incubation period, the DNA solutions were placed in CsCl for centrifugation. RESULTS
d(AT)-Rich Satellite DNA in Tissues of Fourth-Instar Larvae The larval development of Rhynchosciara consists of four instars, ranging over a period of approximately 55 days after hatching; the first, second and third are each approximately 6 days long, whereas the fourth lasts from day 18 to prepupal molt at day 55. At the beginning of the first instar, tellular division ceases in all tissues except the testis. DNA synthesis continues in somatic cells, resulting in the formation of giant polytene chromosomes. In the testis cellular replication ceases later, during the fourth instar, approximately 30 days after hatch-
651
ing. During this stage the testis consists of spermatocytes and a small number of polytene interstitial cells. Our previous studies have shown that DNA synthesis continues in the testis during the fourth larval instar, and we have further demonstrated that there is a satellite DNA (p = 1.681 gm/cm”) in both the testis and salivary gland of fourth-instar larvae. Although profiles of the CsCl gradients were qualitatively the same, with a main-band DNA (p = 1.695 gm/ cm”) and satellite DNA, pulse-labeling analysis indicated that a large proportion of the total [3H]thymidine was incorporated into the satellite DNA of the testis. These studies have now been extended to include a survey of DNA synthesis in other tissues from fourth-instar larvae. Figure 1 shows the buoyant density gradient profiles for thymidine-labeled DNA preparations from testis, gastric cecum, fat body, and salivary gland isolated from 43-day-old larvae from a single fourthinstar culture. It can be seen that several populations of DNA molecules are evident in each tissue: a main band with a density of 1.695 gm/cm3 (with reference to the bacterial marker at 1.710 gm/cm3) and a satellite band with a density of approximately 1.681 gm/cm J. The existence of satellite DNA species in the testis and somatic tissues can also be shown by CsCl density gradient analysis in the ultracentrifuge. The patterns shown in Fig. 2 clearly show that two satellite DNAs exist in these tissues: one having a density of 1.681 gm/cm” and the other a density of 1.675 gm/cm3. [Similar observations have been made by Eckhardt and Gall (1971), using DNA extracted from adult male Rhynchosciara. ] In order to demonstrate that both the satellite and main-band DNA are macromolecules, each species was treated with DNase. DNase-treated and control satellite and main-band DNA were analyzed on CsCl gradients, and the results shown
652
DEVELOPMENTAL BIOLOGY
VOLUME 28, 1972
b
BUOYANT
DENSITY ig/cdl
FIG. 1. CsCl density gradient centrifugation of DNA from 43-day larval tissue of R~Y&ZOSC~UFU (a) Testis, (b) gastric cecum, (c) fat body, (d) salivary gland. O--O, [“HlThymidine-labeled O--O, ‘“C-labeled Escherichia coli DNA; A--A, density in gm/cm3 at 25°C.
holluenderi. insect DNA;
minor (ll%, 11% and 17%, respectively, of the total labeled DNA), the amount of labeled satellite DNA in the testis (44% of the total labeled DNA) is unusually large.
1 681 ~1.710 1.695
BUOYANT
, 6751 1.6&
DENSITY
il.710
’
1,695
(g/cm31
FIG. 2. Analysis of DNA from the tissues of 48day Rhynchosciaru larvae. DNA samples were adjusted to a density of 1.70 gm/cm” and equilibrium density gradient centrifugation was carried out in an analytical ultracentrifuge equipped with a multiplexer and scanner. The samples were centrifuged at 40,000 rpm for 44 hr and scanned at 265 nm. Escherichia coli DNA (p = 1.710 gm/cmJ) was added as a marker. (a) Testis; (b) gastric cecum; (c) fat body; (d) salivary gland.
in Fig. 3 clearly indicate that both are degraded by DNase treatment. The results shown in Fig. 1 demonstrate that while the proportion of labeled salivary gland, satellite DNA from gastric cecum, and fat body is relatively
Developmental Kinetics of Satellite DNA Synthesis during the Latter Half of the Fourth Instar A more complete description of the developmental kinetics of the synthesis of this satellite DNA was obtained from an experiment in which larval tissues from a single culture were labeled with radioactive thymidine at regular intervals during the last half of the fourth instar. In this experiment a single culture of about 1200 larvae was divided into three equal populations. One of the groups of 400 was used as the source of animals to be sampled, and as larvae were removed from it they were replenished from the other two groups in order to keep the experimental culture at a constant size. [It has been our experience that the timing of develop-
653
PAPACONSTANTINOU Rhynchosciam Satellite DNA
b
a
> 0
=I.695
: FRACTION
NO.
FIG. 3. A CsCl density gradient analysis of DNase-treated satellite and main-band DNA. Both satellite and main-band DNA were prepared as described in Materials and Methods. (a) Testis main-band DNA; (b) testis satellite DNA; (c) salivary gland main-band DNA. e---O, Control samples; (OOOO), DNase-treated samples.
ment in the late fourth instar varies among groups of different sizes. It has also been observed that synchrony rapidly ensues when larvae of different groups but of comparable age are combined (unpublished observations).] The results of measurements of the relative proportion of labeled satellite DNA resolved by buoyant density centrifugation during development in this larval culture are shown in Fig. 4. The sequence of events during the same period in overall larval development and in larval spermatocytes, with special reference to the cessation of mitosis and subsequent onset of meiosis, is also indicated. The data can be summarized as follows: (I) At each stage during the last half of the fourth instar (after mitotic figures are no longer observable in the spermatogonia) (Mattingly and Parker, 1968), the level of satellite synthesis in testis is significantly higher than that in the other tissues examined. (2) The proportion of satellite synthesis in salivary gland, gastric cecum, and fat body is about the same (10-17’S), and this proportion is essentially constant in development. Spe-
cific radioactivity (based on the number of larval organs sampled) of total DNA from the gastric cecum and fat body decreases markedly at the end of larval life. Consequently, levels of measurable satellite DNA in the small amounts of tissue obtainable from gastric cecum were low. Fat body is more abundant, and measurements of satellite DNA in late stages were more accurate. Conversely, specific radioactivity in salivary gland DNA increases markedly in late larvae. Autoradiography studies (Mattingly and Parker, 1968) have demonstrated an increase in labeled nuclei at this time. Hence, the decrease in the ratio of satellite DNA to mainband DNA in salivary glands of older larvae may reflect a real change in their respective rates of synthesis. The resolution of these two DNA species in salivary glands from very old larvae is never as good as it is from younger larvae. This is a further indication of the differential control of main-band and satellite DNA syntheses. (3) The proportion of satellite DNA in the testis varies greatly during development. From days 34 to 46, the variations in this one culture follow a
654
DEVELOPMENTAL BIOLOGY
28, 1972
VOLUME
d 60:
-
y 402 cl u 208 0
.*
I 30
., ’
I
’
I
,
34 38 42 46 50 TIME AFTER HATCHING (days)
I
, 54
FIG. 4. Comparison of synthesis of main-band and satellite DNA in various tissues during larval development of Rhynchosciara hollanderi. Groups of 80-100 larvae were taken periodically from a single large culture (maintained at constant size by the addition of equal numbers of animals of the same age) throughout the last half of the fourth instar. The animals were injected with labeled thymidine, larval tissues were removed, and DNA was prepared and subsequently analyzed by centrifugation on cesium chloride as described in Materials and Methods. Uniform procedures were followed for every group sampled, ‘including amount and specific activity of thymidine injected, time of day of injection and dissection, and labeling period (3 hr). The percentage of labeled satellite DNA resolved by isopycnic centrifugation is plotted during larval development. The sequence of the major morphogenetic events in testis and overall larval development is also indicated on the same time scale. O-O, Testis; O-O, salivary gland; AA, fat body; A-A, gastric cecum. *, Percentages derived from other larval cultures at single stages of development.
somewhat regular, cyclical pattern, from a low of about 20% to a high near 40%. Immediately prior to cocoon formation, however, at about day 47, the percentage of the new DNA made that bands with the satellite exceeds 50%. In isolated samplings of other cultures at day 47, proportions of labeled satellite DNA of 70-80% have been observed. This relative increase in the synthesis of satellite DNA immediately precedes the maturation divisions of meiosis in the spermatogonia (Mattingly and Parker, 1968). Increases in the synthesis of satellite DNA in the testis throughout the fourth instar can also be correlated with morphological changes involving mitochondria. After about 32 days of larval development, the mitochondria of the spermatogonia begin to enlarge, continuing until they com-
pletely fill the cytoplasm of the spermatocyte. (These mitochondria will ultimately fuse to form the single large mitochondrion occupying the tail of the mature sperm cell.) In view of the temporal correlation of mitochondrial development and the large increase in synthesis of satellite DNA in the testis, it was thought that satellite DNA synthesis might be attributable to mitochondrial DNA synthesis. Resolution of Two Satellite DNA Species by Ethidium Bromide-C&l Equilibrium Density Centrifugation Since all animal mitochondrial DNAs previously described are circular, experiments were performed to determine whether circular DNA could be detected in the Rhynchosciara satellite DNA fraction. These analyses are based on the
PAPACONSTANTINOU
Rhynchosciam
differential binding of ethidium bromide to circular and linear forms of doublestranded DNA (Radloff et al., 1967; Bauer and Vinograd, 1968). A small change in buoyant density is due to the more limited amount of ethidium bromide that can be intercalated by a covalently closed circular molecule, whereas a greater change in buoyant density is due to the ability of linear forms of DNA to intercalate larger quantities of ethidium bromide. Thus, closed circular DNA can be resolved from nicked circular DNA and linear DNA by ethidium bromide-CsCl gradients. In this experiment one group of larvae was injected with [ ‘“Clthymidine and another with [3H]thymidine. The DNA was extracted and run separately on CsCl gradients to resolve the main-band and satellite DNA peaks. The results are shown in Fig. 5. The “C-labeled mainband DNA (uncorrected density = 1.687 gm/cm “) and 3H-labeled satellite DNA (uncorrected density = 1.672 gm/cm3) IOO-
Q
Satellite
655
DNA
fractions were combined and placed in a neutral CsCl gradient (Fig. 5a) and an ethidium bromide-CsCl gradient (Fig. 5b). It can be seen from these data that the satellite and main-band DNAs are resolved by the neutral CsCl gradient, and that in ethidium bromide the satellite DNA is further resolved into two peaks with significantly different densities. The species with a density of 1.596 gm/cm3 is characteristic of circular DNA, whereas the species with a density of 1.555 gml cm3 is characteristic of a linear satellite DNA which may represent either nicked circular forms or a truly linear satellite DNA. The main-band DNA does not resolve into other species in ethidium bromide and has a density of 1.564 gm/cm3. The differences in buoyant density (Ap) between the various species of testis DNA in neutral and in ethidium bromide-CsCl gradients are shown in Table 1. As is indicated by the data in this table, the satellite DNA fraction from testis can be resolved b
MAIN BAND i
100
AIN BAND
80-
0
lk37
-80
1.672
BUOYANT
DENSITY
(g/cm31
I.555
FIG. 5. CsCl-ethidium bromide density gradient analysis of testis main-band and satellite DNA. (a) Neutral CsCl density gradient analysis of main-band DNA ( p = 1.687 gm/cmJ) and satellite DNA (p = 1.672 gm/cm3), (b) CsCl-ethidium bromide density gradient centrifugation of main-band and satellite DNA. The main-band DNA bands as a single peak with a density of 1.565 gm/cm3; the satellite DNA is resolved into two peaks, one with a density of 1.596 gm/cm3 and the other with a density of-l.555 gm/cm3. Since this experiment was designed to give Ap values for DNA in neutral CsCl gradients vs CsCl-ethidium bromide gradients, Escherichia coli marker DNA was omitted. The densities reported in this experiment are uncorrected. O---a, 3H-labeled DNA; O----O, ‘“C-labeled DNA; A-A, density in gm/cm3 at 25°C.
656
DEVELOPMENTAL BIOLOGY
these experiments characterized.)
TABLE 1 CHANGESIN THE BUOYANTDENSITYOF
Rhynchc~sciaru TESTIS DNA SPECIES IN ETHIDIUMBROMIDE-CSCLDENSITY GRADIENTS DNA Main bandO Satellite’
CsCl Gradient Neutral Ethidium bromide Neutral Ethidium bromide Circular Linear
VOLUME 28, 1972
but
has
not
been
Synthesis of Circular DNA during the Development of the Testis
pa”
APO’~~
1.682 1.557 1.667
0.125 -
1.592 1.542
0.075 0.125
“Neutral minus ethidium bromide (100 @g/ml). The density values for main band and satellite DNA were not corrected. b Average of three experiments. c Average of four experiments.
into one species with Ap = 0.075 gm/cm3 and another with Ap = 0.125 gm/cm3. These significant differences can be attributed to the presence of circular and linear forms of DNA, respectively. (Note also that the Ap for main-band DNA is approximately 0.125 gm/cm3, indicating, as would be expected, that this DNA is linear.) We conclude from this experiment that circular satellite DNA can comprise as much as 40% of the total labeled satellite DNA. Through these experiments, however, we cannot determine whether the linear satellite DNA seen in the ethidium bromide gradients represents a true linear form or whether it is produced by nicked circles. Because of the excessive handling of the satellite DNA in this experiment, it is possible that much of the linear DNA in the ethidium bromide gradients may be nicked circular forms. A more accurate picture of the amount of circular DNA present in the satellite DNA fraction at a stage when this fraction represents a significant portion of the total radioactive DNA is shown in Fig. 6. The calculations indicate that 34% of the radioactive satellite DNA is circular with a density of 1.601 gm/cm3. (Note that a small peak, which represents 10% of the total counts, is seen at a density of 1.582 gm/cmS. This can be detected in all of
In view of the apparent fluctuation of satellite DNA synthesis during the development of the testis, an experiment was carried out to determine the relative amount of circular DNA during this period of development. The profiles in Fig. 7 show the relative increase in the synthesis of circular DNA throughout the latter fourth-instar period of development. It can be seen that as the larvae develop toward the stage of cocoon formation (see Fig. 4), the incorporation of radioisotope into circular and linear satellite DNA is significantly greater than that seen for the main band. MAIN BAND DNA p = 1.568
12fEtE5ITELLITE
1.610
N IO‘0 i,_ 0 iz ?? c6
1.600 \
1.590
0
1.580 8
\, _ CIRCULAR DNA /J = I.601 1
1.570
Q0
1.560
b 0
1.550 1.540
\ i
FRACTION
NO.
FIG. 6. CsCl-ethidium bromide density gradient analysis of 3H-labeled DNA from fourth-instar testis.
PAPACONSTANTINOU Rhynchosciara
FRACTION
Satellite
DISCUSSION
A survey of the DNA species in somatic tissues during fourth-instar larval development has shown that nuclear DNA (main band), which has a buoyant density in cesium chloride of 1.695 gm/cm” (Szybalski, 1968; Chin et al., 1970; Bradshaw and Papaconstantinou, 1970; Eckhardt and Gall, 1971), and a small amount of satellite DNA, which has a buoyant density of 1.681 gm/cm3 (Chin et al., 1970; Eckhardt and Gall, 1971), are synthesized. Synthesis of both species of DNA is part of the process of polytenization in these somatic tissues. Based on the empirical relationship described by Marmur and Doty (1962), the density for main-band DNA
657
NO
FIG. 7. A temporal analysis of circular DNA synthesis (arrows) larvae. (a) 34-Day larvae, (b) 40-day larvae, (c) prenet, (d) net.
In this same series of experiments, attempts were made to determine whether the circular satellite DNA is synthesized in the salivary gland. Similar analyses were carried out, therefore, with salivary gland DNA from the same larvae. It was found that, although main band DNA synthesis is significantly greater in the salivary gland, essentially no circular DNA could be detected.
DNA
during the development
of fourth-instar
represents a G+C content of 35%. The corresponding figure for the satellite DNA is 19%. It should be pointed out, however, that direct chemical analysis of base composition has indicated a much lower actual G+C content for DNAs of similar density isolated from other systems. For example, the poly d(AT) satellite of crab (density = 1.677 gm/cm3) has a G+C content of 3-5% depending on the species (Swartz et al., 1962; Smith, 1964; Skinner, 1967). Our analyses of DNA synthesis in the testis have shown a qualitatively similar CsCl profile, i.e., synthesis of both mainband and satellite DNA. However, the relative incorporation of main-band and satellite DNA in testis is significantly different from that in somatic tissues. There are stages in the development of the testis at which the relative proportions of main-band and satellite DNA synthesized are reversed-i.e., satellite DNA is the major species synthesized. This was never seen for any of the somatic tissues used to acquire the data shown in Fig. 4. In fact these data clearly show that the synthesis of satellite DNA in somatic tissues de-
658
DEVELOPMENTAL BIOLOGY
creases in relation to that of main-band DNA as polytenization and fourth-instar larval development proceed. Furthermore, we consistently find that the detection of satellite DNA synthesis in somatic tissues is easiest in early fourth-instar stages. One of the interesting questions arising from these observations is whether the satellite DNAs of the testis and of the somatic tissues are the same. Polytenization does not occur in the testis except in the interstitial cells, which make up about 1% of the total tissue and could not account for all the satellite DNA synthesis observed. On the other hand, the development of mitochondria in the testis is a morphological which unique event, might be the basis for a significant portion of the satellite DNA synthesis. In Sciaru it has been shown that the mitochondria of the spermatocytes are enlarged and contain a proteinaceous inclusion (Phillips, 1966). These enlarged mitochondria ultimately fuse to form a single large organelle which extends from the nucleus down the entire length of the tail. In Rhynchosciara we have observed that in the mid-fourth-instar stage (see Fig. 4), just after the cessation of mitosis, spermatogonial mitochondria begin to enlarge. This enlargement appears to be due to either synthesis or accumulation of a proteinaceous inclusion. Just prior to meiosis, enlargement of the mitochondria is so extensive that they occupy the entire cytoplasmic space. In view of this unique mitochondrial development, it is indeed possible that the synthesis of satellite DNA in the testis may be associated with this event. Studies of mitochondrial DNA from other systems have revealed characteristics similar to those we have observed for testis satellite DNA. For example, mitochondrial DNA from certain “petite mutants” of yeast has a density of 1.678 gm/cm3 and a G + C content of 12.6%
VOLUME 28. 1972
(Bernardi et al., 1970). All animal mitochondrial DNA previously studied has been circular (Nass, 1969a; Borst and Kroon, 1969), and our analyses of testis satellite DNA on CsCl-ethidium bromide have shown that at least 35% of the total satellite DNA synthesized is circular, providing further indirect evidence that the site of synthesis of a significant portion of satellite DNA from the testis may be associated with mitochondrial replication and development. CsCl-ethidium bromide analysis of salivary gland DNA synthesized at corresponding ages failed to show the synthesis of circular DNA. We conclude that circular DNA synthesis occurs specifically in the testis. If such a species is synthesized in the salivary gland, it is at a level below the limits of sensitivity of our analytical procedures. Satellite DNA, since it is synthesized in the salivary gland, may be nuclear in origin, linear, and may be associated with the centromeric regions of the chromosomes (Pardue and Gall, 1970; Jones, 1970; Eckhardt and Gall, 1971). Similarly, a significant amount of satellite DNA is synthesized in the testis and bands in the linear regions of the CsCl-ethidium bromide gradients. At present we cannot say whether this represents broken circular DNA or a truly linear molecule. Circular DNAs have been found to occur in animal viruses (Dulbecco and Vogt, 1963; Weil and Vinograd, 1963; Crawford and Black, 1964; Crawford, 1964, 1965), mitochondria from vertebrates (Borst and Ruttenburg, 1966; Kroon et al., 1966) and invertebrates (Pik6 et al., 1967), and other organellar structures (Riou and Delain, 1968). Most of the physicochemical characteristics of circular DNA have been established using both viral and mitochondrial DNA. Our conclusion on the existence of circular DNA is based on data obtained from the density differences between intact circular and nicked circular DNA. For
PAPACONSTANTINOU
Rhynchosciam
example, the density difference between the intact circular and linear DNA forms in SV40 has been reported to be 0.040 gm/cm3 (Bauer and Vinograd, 1968). Our data show a difference of 0.043 gm/cm3 between circular and linear satellite DNA in CsCl-ethidium bromide gradients. In addition to monomeric, dimeric, and trimeric forms of circular DNA, there are also reports of catenated forms (Riou and Delain, 1968; Hudson et al., 1968; Nass, 1969b). Catenated dimers, in which only one of the submolecules is nicked, exhibit only half the buoyant density shift observed with linear or closed circular forms in a CsCl-ethidium bromide gradient (Hudson et al., 1968). Thus, this particular species of DNA bands at a density exactly half way between the circular and linear forms. The species which is shown to band at a density of 1.582 gm/cm3 in Figs. 6 and 7 lies exactly half way between the circular DNA and linear satellite DNA, makes up approximately 10% of the total satellite DNA counts, and has the density characteristics of a catenated form. The difference we have observed in the relative syntheses of main-band and satellite DNA probably reflects a differential regulation of the rates of incorporation of nuclear and cytoplasmic DNA. The evidence described above suggests a mitochondrial origin for a significant portion of the testis satellite DNA. The fact that at day 47 in larval development the satellite comprises 80% of the newly synthesized DNA could be due either to an increase in synthesis of mitochondrial DNA or independently to a decrease in nuclear DNA synthesis. That there is a decrease in nuclear DNA synthesis has been shown by direct measurement (Mattingly and Parker, 1968). We have often observed a complete absence of main-band DNA synthesis in the testis during the late fourth-instar stage. Furthermore, in Tetruhymena (Parsons, 1965)
Satellite
DNA
659
and Physarum polycephalum (Guttes and Guttes, 1964; Evans, 1966) it has been shown that the rate of synthesis of mitochondrial DNA is relatively constant throughout the cellular replication cycle. Another indication that mitochondrial and nuclear DNA syntheses are under different temporal control mechanisms comes from the observation that maximum incorporation of thymidine into mitochondrial DNA of synchronized liver cells in culture occurs between the S phase and cytokinesis (Koch and Stokstad, 1967). It has also been shown that mitochondrial DNA synthesis in yeast can be dissociated from nuclear DNA synthesis under conditions of inhibition of protein synthesis (Grossman et al., 1969). Perhaps the most likely explanation, then, for the cyclical variations observed from days 36 to 46 is that the apparent increases in satellite DNA synthesis in the testis may be due to periodic decreases in nuclear DNA synthesis. We thank Drs. Hamilton for their We also thank Mr. Ogle for excellent
Charles G. Mead and Franklin D. critical review of this manuscript. Curtis Parker and Mrs. Shirley P. technical assistance. REFERENCES
BAUER, W., and VINOGRAD,
J. (1968). The interaction of closed circular DNA with intercalative dyes. I. The super helix density of SV40 DNA in the presence and absence of dye. J. Mol. Biol. 33, 141-171. BERNARD], G., FAURES, M., PIPERNO, G., and SLONIMSKI, P. P. (1970). Mitochondrial DNA’s from respiratory sufficient and cytoplasmic respiratory deficient mutant yeast. J. Mol. Biol. 48, 23-42. BOLLUM, F. J. (1966). Filter paper disk techniques for assaying radioactive macromolecules. In “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), p. 296. Harper and Row, New York. BORST, P., and KROON, A. M. (1969). Mitochondrial DNA; physiochemical properties, replication and genetic function. Int. Reu. Cytol. 26, 108-181. BORST, P., and RUTTENBURG, G. J. C. M. (1966). Renaturation of mitochondrial DNA. Biochim. Biophys. Acta 114, 645-647. BRADSHAW, W. S., and PAPACONSTANTINOU, J. (1970).
660
DEVELOPMENTAL BIOLOGY
VOLUME28, 1972
Differential synthesis of salivary gland DNA gradients of DNA preparations from animal tisduring development of Rhynchosciara angelae. sues. ,J. Mol. Biol. 3, 711-716. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 29, 67Oa. KOCH, J., and STOKSTAD,E. L. R. (1967). IncorpoBROWN,D. D., and DAWID,I. B. (1968). Specific gene ration of [3H]thymidine into nuclear and mitoamplification in oocytes. Science 160, 272-280. chondrial DNA in synchronized mammalian cells. CHIN, E. T., BRADSHAW, W. S., and PAPACONSTANTINOU, Eur. J. Biochem. 3, l-6. J. (1970). Specific synthesis of a unique DNA KROON,A. M., BORST,P., VANBRUGGEN,E. F. J., and’ during development of Rhynchosciara sp. Biophys. RUITENBURG,G. J. C. M. (1966). Mitochondrial Sot. Abstr. 10, 164a. DNA from sheep heart. Proc. Nut. Acad. Sci. CHUN, E. H. L., VAUGHAN,M. H., and RICH, A. U.S. 56, 1836-1843. (1963). The isolation and characterization of DNA MARMUR,J., and DOTY, P. (1962). Determination associated with chloroplast preparations. J. Mol. of the base composition of deoxyribonucleic acid Biol. ‘7, 130-141. from its thermal denaturation temperature. J. Mol. CRAWFORD, L. V. (1964). A study of Shope papilloma Biol. 5. 109-118. virus DNA. J. Mol. Biol. 8, 489-495. MATTINGLY, E., and OGLE, S. (1969). A new culture CRAWFORD, L. V. (1965). A study of human papilloma method for Rhynchosciara (Diptera). Ann. Entovirus DNA. J. Mol. Biol. 13, 362-372. mol. Sot. Amer. 62, 94-96. CRAWFORD,L. V., and BLACK, P. H. (1964). The MATUNGLY,E., and PARKER,C. (1968). Nucleic acid nucleic acid of Simian virus 40. Virology 24, 388synthesis during larval development of Rhyncho392.
sciara. J. Insect Physiol.
DUBUY,H. G., MATTERN,C. F. T., and RILEY, F. L. (1965). Isolation and characterization of DNA from kinetoplasts of Leishmania enrieti. Science 147, 754-756.
U.S. 43, 581-588.
DULBECCO,R., and VOGT, M. (1963). Evidence for a ring structure of polyoma virus DNA. Proc. Nat. Acad. Sci. U.S. 50, 236-243.
ECKHARDT,R. A., and GALL, J. G. (1971). Satellite DNA associated with heterochromatin in Rhynchosciara. Chromosoma
32, 407-427.
EPHRUSSI,B., and BEADLE,G. W. (1936). A technique of transplantation for Drosophila. Amer. Natur. 70, 218-225.
EVANS,T. E. (1966). Synthesis of a cytoplasmic DNA during the G, interphase of Physarum polycepha/urn. Biochem. 683.
Biophys.
Res.
Commun.
22, 678
EVANS,D., and BIRNSTIEL,M. (1968). Localization of amplified ribosomal DNA in the oocyte of Xenopus laevis. Biochim.
Biophys. Acta 166, 274-276.
GALL,J. G., MACGREGOR,H. C., and KIDSTON,M. E. (1969). Gene amplification in the oocytes of Dytiscid water beetles. Chromosoma 26, 169-187. GROSSMAN, L. I., GOLDRING, , E. S., and MARMUR,J. (1969). Preferential synthesis of yeast mitochondrial DNA in the absence of protein synthesis. J. Mol. Biol. 46, 367-376.
GUWES, E., and GUT~ES, F. (1964). Thymidine incorporation in mitochondria in Physarum PO&cephalum.
Science
145, 1057-1058.
HUDSON,B., CLAYTON, D. A., and VINOGRAD, J. (1968). Complex mitochondrial DNA. Cold Spring Harbor Symp.
14, 1077-1083.
MESELSON, M., STAHL, F. W., and VINOGRAD,J. (1957). Equilibrium sedimentation of macromolecules in density gradients. Proc. Nat. Acad. Sci.
Quant. Biol. 33, 435-442.
JONES, K. W. (1970). Chromosomal and nuclear location of mouse satellite DNA in individual cells. Nature (London) 225, 912-915. KIT, S. (1961). Equilibrium sedimentation in density
NASS, M. M. K. (1969a). Mitochondrial DNA: Advances, problems and goals. Science 165, 25-35. NASS, M. M. K. (1969h). Reversible generation of circular dimer and higher multiple forms of mitochondrial DNA. Nature (London) 223, 1124-1129. PARDUE,M. L., and GALL,J. G. (1970). Chromosomal localization of mouse satellite DNA. Science 168, 1356-1358.
PARSONS,J. A. (1965). Mitochondrial incorporation of tritiated thymidine in Tetrahymena pyriformis. J. Cell Biol. 25, 641-646.
PHILLIPS, D. M. (1966). Observations on spermiogenesis in the fungus gnat Sciara coprophila. J. Cell Biol. 30, 477-497.
PIKE, L., TYLER, A., and VINOGRAD,J. (1967). Amount, location, priming capacity, circularity and other properties of cytoplasmic DNA in sea urchin eggs. Biol. Bull. 132, 68-90. RABINOWITZ, M., SINCLAIR,L., DESALLE,L., HASELKORN, R., and SWIM, H. (1965). Isolation of deoxyribonucleic acid from mitochondria of chick embryo heart and liver. Proc. Nat. Acad. Sci. U.S. 53, 1126-1133.
RADLOFF,R., BAUER,W., and VINOGRAD, J. (1967). A dye-buoyant-density method for the detection and isolation of closed circular DNA in HeLa cells. Proc. Nat. Acad. Sci. U.S. 57, 1514-1521. RIOU, G., and DELAIN, E. (1968). Electron microscopy of the circular kinetoplastic DNA from Trypanosoma cruzi: Occurrence of catenated forms. Proc. Nat. Acad. Sci. U.S. 62, 210-217. RIOLJ,G., and PAOLET~I,C. (1967). Preparation and properties of nuclear and satellite deoxyribo-
PAPACONSTANTINOU
Rhynchoscimu Satellite DNA
nucleic acid of Trypanasama cruzi. J. Mol. Biol. 28, 377-382. SKINNER, D. M. (1967). Satellite DNA’s in the crab Gecarcinus lateralis and Cancer Pagurus. Proc. Nat. Acad. Sci. U.S. 58, 103-110. SMITH, M. (1964). Deoxyribonucleic acids of crustacea. J. Mol. Biol. 9, 17-23. SUEOKA, N. (1961). Variation and heterogeneity of base composition of deoxyribonucleic acids: A compilation of old and new data. J. Mol. Biol. 3, 31-40. SWARTZ, M. N., TRAUTNER, T. A., and KORNBERG, A. (1962). Enzymatic synthesis of deoxyribonucleic
661
acid. XI. Further studies on nearest neighbor base sequences in deoxyribonucleic acids. J. Biol.
Chem. 237, 1961-1967. SZYBALSKI, W. (1968). Use of cesium sulfate for equilibrium density gradient centrifugation. Methods Enzymol. 12B, 346. WALLACE, H., and BIRNSTIEL, M. L. (1966). Ribosomal cistrons and the nucleolar organizer. Biochim. Biophys. Acta 114, 296-310. WEIL, R., and VINOGRAD, J. (1963). The cyclic helix and cyclic coil forms of polyoma viral DNA.
l+oc. Nat. Acad. Sci. U.S. 50, 730-738.
BIOLOGY
Synthesis
28,
649-661 (1972)
of Satellite
DNA in Rhynchosciara
hollaenderi’
JOHN PAPACONSTANTINOU, WILLIAM S. BRADSHAW, z EUGENE T. CHIN,~ AND EMILIA M. JULKU Biology Diuision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 and The Uniuersity Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Accepted April
of
26, 1972
During the latter part of the fourth instar development of Rhynchosciara larvae, DNA synthesis occurs in both germ cells and somatic cells, even though these cells do not undergo mitosis. CsCl density gradient analysis has revealed the synthesis of d(AT) satellite DNAs in the testis and in somatic tissues such as salivary gland, fat body, and gastric cecum. In these studies it has been shown that there is a tissue-specific variation in the relative proportions of synthesis of d(AT) satellite and main-band DNA in the testis during the fourth instar. The initiation of synthesis of the d(AT) satellite in the testis corresponds with the appearance and formation of the highly characteristic mitochondria which develop during the maturation of the spermatocytes. This satellite DNA has been shown to be circular and has a density of 1.681 gm/cmj in CsCl. Synthesis of a similar circular DNA cannot be detected in somatic tissues, although these tissues do synthesize a d(AT) satellite of similar density. We conclude from these studies that the specific synthesis of a unique circular DNA may be an essential part of the morphogenetic development of the highly specialized mitochondria formed during spermatocyte maturation. INTRODUCTION
Analysis of DNA by isopycnic centrifugation on cesium chloride (Meselson et al., 1957) has demonstrated, in a number of eukaryotic tissues, the existence of minor components with mean densities different from that of the bulk of the DNA. These satellite DNAs exhibit densities either higher or lower than the major band. Examples include: (1) mouse satellite DNA, comprising about 10% of the total DNA (Kit, 1961), which has been shown to be associated with centromeric heterochromatin in mouse chromosomes (Pardue and Gall, 1970; Jones, 1970); (2) the ribosomal RNA cistrons localized in the nucleoli of amphibian (Wallace and ’ Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. ’ AEC Postdoctoral Fellow under appointment by Oak Ridge Associated Universities. Present address: Department of Zoology and Entomology, Brigham Young University, Provo, Utah 84601. “Predoctoral Trainee supported by Grant No. GM 1974 from NIGMS. 649 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
Birnstiel, 1966; Brown and Dawid, 1968; Evans and Birnstiel, 1968) and insect (Gall et al., 1969) oocytes; (3) the poly d(AT) satellite of some crustaceans (Sueoka, 1961; Skinner, 1967); and (4) unique populations of extranuclear DNA originating in organelles such as mitochondria (Rabinowitz et al., 1965), kinetoplasts (Dubuy et al., 1965; Riou and Paoletti, 1967), and chloroplasts (Chun et al., 1963). In this paper we report on a d(AT)rich satellite DNA fraction extracted from larval tissues of the sciarid fly Rhynchosciaru (Diptera: Sciaridae). There is a tissue-specific variation in the relative proportions of satellite and main-band DNA synthesized in the testis during the development of fourth-instar larvae. This regulation of DNA synthesis appears to be correlated with the morphogenetic maturation of larval sperm cells. Our attempts to characterize the satellite DNA have shown that as much as 50% of the total d(AT) DNA synthesized during development of the testis is in circular form. Similar analysis of salivary gland
650
DEVELOPMENTAL BIOLOGY
DNA at corresponding stages of larval development does not reveal a circular satellite DNA. A preliminary report on these observations has appeared elsewhere (Chin et al., 1970). MATERIALS
AND
METHODS
Growth of Rhynchosciara. The procedure for maintaining Rhynchosciara larvae in the laboratory has been described (Mattingly and Ogle, 1969). The following modifications in antibiotic treatment and in preparation of food have been made: (1) The egg masses and larvae are kept in plastic petri dishes layered with Bacto agar containing terramycin (100 pg/ml) instead of streptomycin and tetracycline. (2) The food is prepared by suspending one 6-0~ block of Fleischmann’s fresh active yeast in 750 ml of distilled water at 35°C and adding to this 450 gm CSMA fly larvae medium (Ralston Purina Company, P. 0. Box 337, Richmond, Indiana 47374). The food is mixed in well and permitted to ferment 4-12 hr at 35°C. The food is prepared prior to feeding. Larvae up to the fourthinstar stage of development are fed once a week; fourth-instar larvae are fed twice a week. radioactive DNA. Frepara tion of Labeled DNA from R. hollaenderi was obtained by injecting individual larvae from synchronous cultures with 2 ~1 of either methyl[3H]thymidine (1 &i; 11.2 Ci/ mmole) or [ l’C]thymidine. Following labeling periods of 3-24 hr, partially purified DNA preparations were obtained from larval tissues which had been dissected in insect Ringer’s solution (Ephrussi and Beadle, 1936) and frozen on Dry Ice. Preparation of DNA for CsCl gradient analysis. Tissues from 50-200 larvae were homogenized in a conical ground glass homogenizer in l-2 ml of 0.14 M phosphate buffer (pH 7.0)-0.01 M EDTA-1% sodium lauryl sulfate-l M sodium perchlorate.
VOLUME 28. 1972
Homogenates were then extracted in an equal volume of chloroform-octanol (9 : 1) and centrifuged at 4°C for 30 min at 12,000 g. Two volumes of cold ethanol were then added to the aqueous phase, and the resulting precipitate was dissolved in 0.15 M NaCl-0.015 M sodium citrate (1 x SSC). The DNA samples obtained were added to 6- to 7-ml CsCl gradients in 1 x SSC and centrifuged in a Spinco No. 40 rotor at 35,000 rpm for 48-72 hr at 25°C. Following centrifugation, O.l-ml fractions were collected, and radioactivity was monitored by applying aliquots of these fractions to Whatman 3-MM filter paper disks, which were processed according to Bollum (1966) and counted in toluene-fluors at a counting efficiency for tritium of about 35%. Buoyant densities of sample fractions from each gradient were determined by measurements of their refractive index. In some instances [‘“Cl or [3H]thymidinelabeled DNA from Escherichia coli was added as a marker in the density gradients. For analyses in the analytical ultracentrifuge, the first ethanol precipitate was dissolved in 1 x SSC and treated with the following enzymes for 2 hr at 37°C: RNase A (100 pg/ml), Tl RNase (330 units/ml), and cu-amylase (100 pg/ml). The solution was again extracted with chloroform-octanol (9: l), and the DNA was precipitated from the aqueous phase with ethanol as described above. The precipitate was dissolved and dialyzed against 1 x SSC. The dialyzed solutions were adjusted to a density of 1.7 gm/cm3 by adding the appropriate amount of solid CsCl. Preparation of DNA for CsCl-ethidium bromide gradient analysis. Tissues from 50 larvae were homogenized in a Dounce homogenizer (loose-fitting No. A) with two strokes in 1-2 ml of the buffer described above. Homogenates were extracted in an equal volume of chloroformThe octanol (9: 1) by gentle mixing.
PAPACONSTANTINOU Rhynchosciara Satellite DNA
homogenate was centrifuged at 4°C for 30 min at 10,000 g. The aqueous layer (approximately 1 ml) was removed and added to 4 ml of saturated CsCl (25°C) in 1 x SSC. The interphase was reextracted with 0.5 ml of the same buffer, mixed with a pipette, and centrifuged as described above. The aqueous phase was added to the same CsCl solution and mixed gently. The precipitate formed was removed by centrifugation at 4°C for 10 min at 10,000 g. The CsCl-DNA solution was gently poured into a siliconized polyallomer tube. The density was adjusted by diluting with 1 x SSC, and ethidium bromide (in 1 x SSC) was added to a final concentration of 100 pglml. The gradients were centrifuged at 35,000 rpm for 62-68 hr at 25°C in a Ti 50 rotor. Fractions were collected on filter paper or in siliconized tubes and counted by liquid scintillation counting as described above. DNase treatment. DNA fractions (main band and satellite) from CsCl gradients were dialyzed in 0.01 M Tris (pH 7.4)0.01 M KCl-0.0015 M MgCl at 4°C for 20 hr with three l-liter changes. Half of each sample was treated with DNase (Worthington Pancreatic DNase I, final concentration 5 Kg/ml) at 25°C for 10 min. At the end of the incubation period, the DNA solutions were placed in CsCl for centrifugation. RESULTS
d(AT)-Rich Satellite DNA in Tissues of Fourth-Instar Larvae The larval development of Rhynchosciara consists of four instars, ranging over a period of approximately 55 days after hatching; the first, second and third are each approximately 6 days long, whereas the fourth lasts from day 18 to prepupal molt at day 55. At the beginning of the first instar, tellular division ceases in all tissues except the testis. DNA synthesis continues in somatic cells, resulting in the formation of giant polytene chromosomes. In the testis cellular replication ceases later, during the fourth instar, approximately 30 days after hatch-
651
ing. During this stage the testis consists of spermatocytes and a small number of polytene interstitial cells. Our previous studies have shown that DNA synthesis continues in the testis during the fourth larval instar, and we have further demonstrated that there is a satellite DNA (p = 1.681 gm/cm”) in both the testis and salivary gland of fourth-instar larvae. Although profiles of the CsCl gradients were qualitatively the same, with a main-band DNA (p = 1.695 gm/ cm”) and satellite DNA, pulse-labeling analysis indicated that a large proportion of the total [3H]thymidine was incorporated into the satellite DNA of the testis. These studies have now been extended to include a survey of DNA synthesis in other tissues from fourth-instar larvae. Figure 1 shows the buoyant density gradient profiles for thymidine-labeled DNA preparations from testis, gastric cecum, fat body, and salivary gland isolated from 43-day-old larvae from a single fourthinstar culture. It can be seen that several populations of DNA molecules are evident in each tissue: a main band with a density of 1.695 gm/cm3 (with reference to the bacterial marker at 1.710 gm/cm3) and a satellite band with a density of approximately 1.681 gm/cm J. The existence of satellite DNA species in the testis and somatic tissues can also be shown by CsCl density gradient analysis in the ultracentrifuge. The patterns shown in Fig. 2 clearly show that two satellite DNAs exist in these tissues: one having a density of 1.681 gm/cm” and the other a density of 1.675 gm/cm3. [Similar observations have been made by Eckhardt and Gall (1971), using DNA extracted from adult male Rhynchosciara. ] In order to demonstrate that both the satellite and main-band DNA are macromolecules, each species was treated with DNase. DNase-treated and control satellite and main-band DNA were analyzed on CsCl gradients, and the results shown
652
DEVELOPMENTAL BIOLOGY
VOLUME 28, 1972
b
BUOYANT
DENSITY ig/cdl
FIG. 1. CsCl density gradient centrifugation of DNA from 43-day larval tissue of R~Y&ZOSC~UFU (a) Testis, (b) gastric cecum, (c) fat body, (d) salivary gland. O--O, [“HlThymidine-labeled O--O, ‘“C-labeled Escherichia coli DNA; A--A, density in gm/cm3 at 25°C.
holluenderi. insect DNA;
minor (ll%, 11% and 17%, respectively, of the total labeled DNA), the amount of labeled satellite DNA in the testis (44% of the total labeled DNA) is unusually large.
1 681 ~1.710 1.695
BUOYANT
, 6751 1.6&
DENSITY
il.710
’
1,695
(g/cm31
FIG. 2. Analysis of DNA from the tissues of 48day Rhynchosciaru larvae. DNA samples were adjusted to a density of 1.70 gm/cm” and equilibrium density gradient centrifugation was carried out in an analytical ultracentrifuge equipped with a multiplexer and scanner. The samples were centrifuged at 40,000 rpm for 44 hr and scanned at 265 nm. Escherichia coli DNA (p = 1.710 gm/cmJ) was added as a marker. (a) Testis; (b) gastric cecum; (c) fat body; (d) salivary gland.
in Fig. 3 clearly indicate that both are degraded by DNase treatment. The results shown in Fig. 1 demonstrate that while the proportion of labeled salivary gland, satellite DNA from gastric cecum, and fat body is relatively
Developmental Kinetics of Satellite DNA Synthesis during the Latter Half of the Fourth Instar A more complete description of the developmental kinetics of the synthesis of this satellite DNA was obtained from an experiment in which larval tissues from a single culture were labeled with radioactive thymidine at regular intervals during the last half of the fourth instar. In this experiment a single culture of about 1200 larvae was divided into three equal populations. One of the groups of 400 was used as the source of animals to be sampled, and as larvae were removed from it they were replenished from the other two groups in order to keep the experimental culture at a constant size. [It has been our experience that the timing of develop-
653
PAPACONSTANTINOU Rhynchosciam Satellite DNA
b
a
> 0
=I.695
: FRACTION
NO.
FIG. 3. A CsCl density gradient analysis of DNase-treated satellite and main-band DNA. Both satellite and main-band DNA were prepared as described in Materials and Methods. (a) Testis main-band DNA; (b) testis satellite DNA; (c) salivary gland main-band DNA. e---O, Control samples; (OOOO), DNase-treated samples.
ment in the late fourth instar varies among groups of different sizes. It has also been observed that synchrony rapidly ensues when larvae of different groups but of comparable age are combined (unpublished observations).] The results of measurements of the relative proportion of labeled satellite DNA resolved by buoyant density centrifugation during development in this larval culture are shown in Fig. 4. The sequence of events during the same period in overall larval development and in larval spermatocytes, with special reference to the cessation of mitosis and subsequent onset of meiosis, is also indicated. The data can be summarized as follows: (I) At each stage during the last half of the fourth instar (after mitotic figures are no longer observable in the spermatogonia) (Mattingly and Parker, 1968), the level of satellite synthesis in testis is significantly higher than that in the other tissues examined. (2) The proportion of satellite synthesis in salivary gland, gastric cecum, and fat body is about the same (10-17’S), and this proportion is essentially constant in development. Spe-
cific radioactivity (based on the number of larval organs sampled) of total DNA from the gastric cecum and fat body decreases markedly at the end of larval life. Consequently, levels of measurable satellite DNA in the small amounts of tissue obtainable from gastric cecum were low. Fat body is more abundant, and measurements of satellite DNA in late stages were more accurate. Conversely, specific radioactivity in salivary gland DNA increases markedly in late larvae. Autoradiography studies (Mattingly and Parker, 1968) have demonstrated an increase in labeled nuclei at this time. Hence, the decrease in the ratio of satellite DNA to mainband DNA in salivary glands of older larvae may reflect a real change in their respective rates of synthesis. The resolution of these two DNA species in salivary glands from very old larvae is never as good as it is from younger larvae. This is a further indication of the differential control of main-band and satellite DNA syntheses. (3) The proportion of satellite DNA in the testis varies greatly during development. From days 34 to 46, the variations in this one culture follow a
654
DEVELOPMENTAL BIOLOGY
28, 1972
VOLUME
d 60:
-
y 402 cl u 208 0
.*
I 30
., ’
I
’
I
,
34 38 42 46 50 TIME AFTER HATCHING (days)
I
, 54
FIG. 4. Comparison of synthesis of main-band and satellite DNA in various tissues during larval development of Rhynchosciara hollanderi. Groups of 80-100 larvae were taken periodically from a single large culture (maintained at constant size by the addition of equal numbers of animals of the same age) throughout the last half of the fourth instar. The animals were injected with labeled thymidine, larval tissues were removed, and DNA was prepared and subsequently analyzed by centrifugation on cesium chloride as described in Materials and Methods. Uniform procedures were followed for every group sampled, ‘including amount and specific activity of thymidine injected, time of day of injection and dissection, and labeling period (3 hr). The percentage of labeled satellite DNA resolved by isopycnic centrifugation is plotted during larval development. The sequence of the major morphogenetic events in testis and overall larval development is also indicated on the same time scale. O-O, Testis; O-O, salivary gland; AA, fat body; A-A, gastric cecum. *, Percentages derived from other larval cultures at single stages of development.
somewhat regular, cyclical pattern, from a low of about 20% to a high near 40%. Immediately prior to cocoon formation, however, at about day 47, the percentage of the new DNA made that bands with the satellite exceeds 50%. In isolated samplings of other cultures at day 47, proportions of labeled satellite DNA of 70-80% have been observed. This relative increase in the synthesis of satellite DNA immediately precedes the maturation divisions of meiosis in the spermatogonia (Mattingly and Parker, 1968). Increases in the synthesis of satellite DNA in the testis throughout the fourth instar can also be correlated with morphological changes involving mitochondria. After about 32 days of larval development, the mitochondria of the spermatogonia begin to enlarge, continuing until they com-
pletely fill the cytoplasm of the spermatocyte. (These mitochondria will ultimately fuse to form the single large mitochondrion occupying the tail of the mature sperm cell.) In view of the temporal correlation of mitochondrial development and the large increase in synthesis of satellite DNA in the testis, it was thought that satellite DNA synthesis might be attributable to mitochondrial DNA synthesis. Resolution of Two Satellite DNA Species by Ethidium Bromide-C&l Equilibrium Density Centrifugation Since all animal mitochondrial DNAs previously described are circular, experiments were performed to determine whether circular DNA could be detected in the Rhynchosciara satellite DNA fraction. These analyses are based on the
PAPACONSTANTINOU
Rhynchosciam
differential binding of ethidium bromide to circular and linear forms of doublestranded DNA (Radloff et al., 1967; Bauer and Vinograd, 1968). A small change in buoyant density is due to the more limited amount of ethidium bromide that can be intercalated by a covalently closed circular molecule, whereas a greater change in buoyant density is due to the ability of linear forms of DNA to intercalate larger quantities of ethidium bromide. Thus, closed circular DNA can be resolved from nicked circular DNA and linear DNA by ethidium bromide-CsCl gradients. In this experiment one group of larvae was injected with [ ‘“Clthymidine and another with [3H]thymidine. The DNA was extracted and run separately on CsCl gradients to resolve the main-band and satellite DNA peaks. The results are shown in Fig. 5. The “C-labeled mainband DNA (uncorrected density = 1.687 gm/cm “) and 3H-labeled satellite DNA (uncorrected density = 1.672 gm/cm3) IOO-
Q
Satellite
655
DNA
fractions were combined and placed in a neutral CsCl gradient (Fig. 5a) and an ethidium bromide-CsCl gradient (Fig. 5b). It can be seen from these data that the satellite and main-band DNAs are resolved by the neutral CsCl gradient, and that in ethidium bromide the satellite DNA is further resolved into two peaks with significantly different densities. The species with a density of 1.596 gm/cm3 is characteristic of circular DNA, whereas the species with a density of 1.555 gml cm3 is characteristic of a linear satellite DNA which may represent either nicked circular forms or a truly linear satellite DNA. The main-band DNA does not resolve into other species in ethidium bromide and has a density of 1.564 gm/cm3. The differences in buoyant density (Ap) between the various species of testis DNA in neutral and in ethidium bromide-CsCl gradients are shown in Table 1. As is indicated by the data in this table, the satellite DNA fraction from testis can be resolved b
MAIN BAND i
100
AIN BAND
80-
0
lk37
-80
1.672
BUOYANT
DENSITY
(g/cm31
I.555
FIG. 5. CsCl-ethidium bromide density gradient analysis of testis main-band and satellite DNA. (a) Neutral CsCl density gradient analysis of main-band DNA ( p = 1.687 gm/cmJ) and satellite DNA (p = 1.672 gm/cm3), (b) CsCl-ethidium bromide density gradient centrifugation of main-band and satellite DNA. The main-band DNA bands as a single peak with a density of 1.565 gm/cm3; the satellite DNA is resolved into two peaks, one with a density of 1.596 gm/cm3 and the other with a density of-l.555 gm/cm3. Since this experiment was designed to give Ap values for DNA in neutral CsCl gradients vs CsCl-ethidium bromide gradients, Escherichia coli marker DNA was omitted. The densities reported in this experiment are uncorrected. O---a, 3H-labeled DNA; O----O, ‘“C-labeled DNA; A-A, density in gm/cm3 at 25°C.
656
DEVELOPMENTAL BIOLOGY
these experiments characterized.)
TABLE 1 CHANGESIN THE BUOYANTDENSITYOF
Rhynchc~sciaru TESTIS DNA SPECIES IN ETHIDIUMBROMIDE-CSCLDENSITY GRADIENTS DNA Main bandO Satellite’
CsCl Gradient Neutral Ethidium bromide Neutral Ethidium bromide Circular Linear
VOLUME 28, 1972
but
has
not
been
Synthesis of Circular DNA during the Development of the Testis
pa”
APO’~~
1.682 1.557 1.667
0.125 -
1.592 1.542
0.075 0.125
“Neutral minus ethidium bromide (100 @g/ml). The density values for main band and satellite DNA were not corrected. b Average of three experiments. c Average of four experiments.
into one species with Ap = 0.075 gm/cm3 and another with Ap = 0.125 gm/cm3. These significant differences can be attributed to the presence of circular and linear forms of DNA, respectively. (Note also that the Ap for main-band DNA is approximately 0.125 gm/cm3, indicating, as would be expected, that this DNA is linear.) We conclude from this experiment that circular satellite DNA can comprise as much as 40% of the total labeled satellite DNA. Through these experiments, however, we cannot determine whether the linear satellite DNA seen in the ethidium bromide gradients represents a true linear form or whether it is produced by nicked circles. Because of the excessive handling of the satellite DNA in this experiment, it is possible that much of the linear DNA in the ethidium bromide gradients may be nicked circular forms. A more accurate picture of the amount of circular DNA present in the satellite DNA fraction at a stage when this fraction represents a significant portion of the total radioactive DNA is shown in Fig. 6. The calculations indicate that 34% of the radioactive satellite DNA is circular with a density of 1.601 gm/cm3. (Note that a small peak, which represents 10% of the total counts, is seen at a density of 1.582 gm/cmS. This can be detected in all of
In view of the apparent fluctuation of satellite DNA synthesis during the development of the testis, an experiment was carried out to determine the relative amount of circular DNA during this period of development. The profiles in Fig. 7 show the relative increase in the synthesis of circular DNA throughout the latter fourth-instar period of development. It can be seen that as the larvae develop toward the stage of cocoon formation (see Fig. 4), the incorporation of radioisotope into circular and linear satellite DNA is significantly greater than that seen for the main band. MAIN BAND DNA p = 1.568
12fEtE5ITELLITE
1.610
N IO‘0 i,_ 0 iz ?? c6
1.600 \
1.590
0
1.580 8
\, _ CIRCULAR DNA /J = I.601 1
1.570
Q0
1.560
b 0
1.550 1.540
\ i
FRACTION
NO.
FIG. 6. CsCl-ethidium bromide density gradient analysis of 3H-labeled DNA from fourth-instar testis.
PAPACONSTANTINOU Rhynchosciara
FRACTION
Satellite
DISCUSSION
A survey of the DNA species in somatic tissues during fourth-instar larval development has shown that nuclear DNA (main band), which has a buoyant density in cesium chloride of 1.695 gm/cm” (Szybalski, 1968; Chin et al., 1970; Bradshaw and Papaconstantinou, 1970; Eckhardt and Gall, 1971), and a small amount of satellite DNA, which has a buoyant density of 1.681 gm/cm3 (Chin et al., 1970; Eckhardt and Gall, 1971), are synthesized. Synthesis of both species of DNA is part of the process of polytenization in these somatic tissues. Based on the empirical relationship described by Marmur and Doty (1962), the density for main-band DNA
657
NO
FIG. 7. A temporal analysis of circular DNA synthesis (arrows) larvae. (a) 34-Day larvae, (b) 40-day larvae, (c) prenet, (d) net.
In this same series of experiments, attempts were made to determine whether the circular satellite DNA is synthesized in the salivary gland. Similar analyses were carried out, therefore, with salivary gland DNA from the same larvae. It was found that, although main band DNA synthesis is significantly greater in the salivary gland, essentially no circular DNA could be detected.
DNA
during the development
of fourth-instar
represents a G+C content of 35%. The corresponding figure for the satellite DNA is 19%. It should be pointed out, however, that direct chemical analysis of base composition has indicated a much lower actual G+C content for DNAs of similar density isolated from other systems. For example, the poly d(AT) satellite of crab (density = 1.677 gm/cm3) has a G+C content of 3-5% depending on the species (Swartz et al., 1962; Smith, 1964; Skinner, 1967). Our analyses of DNA synthesis in the testis have shown a qualitatively similar CsCl profile, i.e., synthesis of both mainband and satellite DNA. However, the relative incorporation of main-band and satellite DNA in testis is significantly different from that in somatic tissues. There are stages in the development of the testis at which the relative proportions of main-band and satellite DNA synthesized are reversed-i.e., satellite DNA is the major species synthesized. This was never seen for any of the somatic tissues used to acquire the data shown in Fig. 4. In fact these data clearly show that the synthesis of satellite DNA in somatic tissues de-
658
DEVELOPMENTAL BIOLOGY
creases in relation to that of main-band DNA as polytenization and fourth-instar larval development proceed. Furthermore, we consistently find that the detection of satellite DNA synthesis in somatic tissues is easiest in early fourth-instar stages. One of the interesting questions arising from these observations is whether the satellite DNAs of the testis and of the somatic tissues are the same. Polytenization does not occur in the testis except in the interstitial cells, which make up about 1% of the total tissue and could not account for all the satellite DNA synthesis observed. On the other hand, the development of mitochondria in the testis is a morphological which unique event, might be the basis for a significant portion of the satellite DNA synthesis. In Sciaru it has been shown that the mitochondria of the spermatocytes are enlarged and contain a proteinaceous inclusion (Phillips, 1966). These enlarged mitochondria ultimately fuse to form a single large organelle which extends from the nucleus down the entire length of the tail. In Rhynchosciara we have observed that in the mid-fourth-instar stage (see Fig. 4), just after the cessation of mitosis, spermatogonial mitochondria begin to enlarge. This enlargement appears to be due to either synthesis or accumulation of a proteinaceous inclusion. Just prior to meiosis, enlargement of the mitochondria is so extensive that they occupy the entire cytoplasmic space. In view of this unique mitochondrial development, it is indeed possible that the synthesis of satellite DNA in the testis may be associated with this event. Studies of mitochondrial DNA from other systems have revealed characteristics similar to those we have observed for testis satellite DNA. For example, mitochondrial DNA from certain “petite mutants” of yeast has a density of 1.678 gm/cm3 and a G + C content of 12.6%
VOLUME 28. 1972
(Bernardi et al., 1970). All animal mitochondrial DNA previously studied has been circular (Nass, 1969a; Borst and Kroon, 1969), and our analyses of testis satellite DNA on CsCl-ethidium bromide have shown that at least 35% of the total satellite DNA synthesized is circular, providing further indirect evidence that the site of synthesis of a significant portion of satellite DNA from the testis may be associated with mitochondrial replication and development. CsCl-ethidium bromide analysis of salivary gland DNA synthesized at corresponding ages failed to show the synthesis of circular DNA. We conclude that circular DNA synthesis occurs specifically in the testis. If such a species is synthesized in the salivary gland, it is at a level below the limits of sensitivity of our analytical procedures. Satellite DNA, since it is synthesized in the salivary gland, may be nuclear in origin, linear, and may be associated with the centromeric regions of the chromosomes (Pardue and Gall, 1970; Jones, 1970; Eckhardt and Gall, 1971). Similarly, a significant amount of satellite DNA is synthesized in the testis and bands in the linear regions of the CsCl-ethidium bromide gradients. At present we cannot say whether this represents broken circular DNA or a truly linear molecule. Circular DNAs have been found to occur in animal viruses (Dulbecco and Vogt, 1963; Weil and Vinograd, 1963; Crawford and Black, 1964; Crawford, 1964, 1965), mitochondria from vertebrates (Borst and Ruttenburg, 1966; Kroon et al., 1966) and invertebrates (Pik6 et al., 1967), and other organellar structures (Riou and Delain, 1968). Most of the physicochemical characteristics of circular DNA have been established using both viral and mitochondrial DNA. Our conclusion on the existence of circular DNA is based on data obtained from the density differences between intact circular and nicked circular DNA. For
PAPACONSTANTINOU
Rhynchosciam
example, the density difference between the intact circular and linear DNA forms in SV40 has been reported to be 0.040 gm/cm3 (Bauer and Vinograd, 1968). Our data show a difference of 0.043 gm/cm3 between circular and linear satellite DNA in CsCl-ethidium bromide gradients. In addition to monomeric, dimeric, and trimeric forms of circular DNA, there are also reports of catenated forms (Riou and Delain, 1968; Hudson et al., 1968; Nass, 1969b). Catenated dimers, in which only one of the submolecules is nicked, exhibit only half the buoyant density shift observed with linear or closed circular forms in a CsCl-ethidium bromide gradient (Hudson et al., 1968). Thus, this particular species of DNA bands at a density exactly half way between the circular and linear forms. The species which is shown to band at a density of 1.582 gm/cm3 in Figs. 6 and 7 lies exactly half way between the circular DNA and linear satellite DNA, makes up approximately 10% of the total satellite DNA counts, and has the density characteristics of a catenated form. The difference we have observed in the relative syntheses of main-band and satellite DNA probably reflects a differential regulation of the rates of incorporation of nuclear and cytoplasmic DNA. The evidence described above suggests a mitochondrial origin for a significant portion of the testis satellite DNA. The fact that at day 47 in larval development the satellite comprises 80% of the newly synthesized DNA could be due either to an increase in synthesis of mitochondrial DNA or independently to a decrease in nuclear DNA synthesis. That there is a decrease in nuclear DNA synthesis has been shown by direct measurement (Mattingly and Parker, 1968). We have often observed a complete absence of main-band DNA synthesis in the testis during the late fourth-instar stage. Furthermore, in Tetruhymena (Parsons, 1965)
Satellite
DNA
659
and Physarum polycephalum (Guttes and Guttes, 1964; Evans, 1966) it has been shown that the rate of synthesis of mitochondrial DNA is relatively constant throughout the cellular replication cycle. Another indication that mitochondrial and nuclear DNA syntheses are under different temporal control mechanisms comes from the observation that maximum incorporation of thymidine into mitochondrial DNA of synchronized liver cells in culture occurs between the S phase and cytokinesis (Koch and Stokstad, 1967). It has also been shown that mitochondrial DNA synthesis in yeast can be dissociated from nuclear DNA synthesis under conditions of inhibition of protein synthesis (Grossman et al., 1969). Perhaps the most likely explanation, then, for the cyclical variations observed from days 36 to 46 is that the apparent increases in satellite DNA synthesis in the testis may be due to periodic decreases in nuclear DNA synthesis. We thank Drs. Hamilton for their We also thank Mr. Ogle for excellent
Charles G. Mead and Franklin D. critical review of this manuscript. Curtis Parker and Mrs. Shirley P. technical assistance. REFERENCES
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