Studies on the satellite DNAs of Drosophila nasutoides: Their buoyant densities, melting temperatures, reassociation rates and localizations in polytene chromosomes

J. Mol. Biol.

(1976)

104, 1-24

Studies on the Satellite DNAs of Drosophila nasutoides: Their Buoyant Densities, Melting Temperatures, Reassociation Rates and Localizations in Polytene Chromosomes M. (?oRDEIRO-STONE~AND Department

The University

C.S.LEE$

of Zoology

of Texas at Austin,

Austin,

Tex. 78712, U.S.A.

(Received 21 October 1975, and in revised form 2 February

1976)

The karyotype of Drosophila nasutoides reveals a very large autosome pair at the metaphase plate. The application of the C-banding technique shows that this chromosome is almost entirely heterochromatic and an isochromosome (Cordeiro et al., 1975). Examination of the DNA isolated from purified nuclei of D. nasutoides in neutral CsCl gradients reveals four major satellites. As much as 60% of the total DNA appears as satellites in the DNA from larval brains. The buoyant densities of the four satellites, designated as I through IV in the order of descending density, are 1.687, 1.682, 1,669 and 1.665 g/cm3, respectively. All four satellites show strand separations in alkaline CsCl gradients with the least separation in satellite III. Thermal denaturation studies with purified native satellites show that satellites I and IV consist of repeats of identical sequences, whereas satellites II and III show a large sequence variation between repeating units. As much as 10 to 24% base-pair m&-matching is observed in the reassociated satellite II. The sequence complexities obtained from DNA reassociation kinetics data are 5, 103, 2.3 x lo6 and 46 nucleotide pairs for the satellites I, II, III and IV, respectively. The complexity of satellite III is almost as large as that of Escherichia coli, when the reassociation rate is corrected according to the amount of mismatching in this satellite. All four satellite sequences are localized in one chromosome (dot chromosome) according to in. situ hybridizations to polytene chromosomes. The large heterochromatic chromosome seen at the metaphase plate appears as the dot chromosome after polytenization. Therefore, the large heterochromatic chromosome contains all four satellite DNA components.

1. Introduction The intense search for satellite DNAs in all eukaryotes during the past decade has shown that this particular DNA class is widespread in plants and animals. Besides their different base compositions that permit their separation from the bulk of main nuclear DNA by density gradients, satellite DNAs are characterized by many repeats of very simple identical or closely related sequences, the asymmetric distribution of guanine and thymine between complementary strands, localization in the heterochromatin, probable transcriptional inactivity in vivo and apparent species specificity. The function of satellite DNAs, however, is still not clear. Although various hypotheses have been proposed, none has yet been proven conclusively (see reviews by Bostock, 1971; Walker, 1971; Flamm, 1972). t Present address: Department of Biochemistry, University $ To whom correspondence should be addressed. 1

1

of Sao Paula, Sao Paulo, Brazil.

2

M.

CORDEIRO-STONE

AND

C.

S. LEE

Because of their simple sequences, satellite DNAs in several animals have been subject to sequence analyses. Southern (1970) showed that the guinea pig cc-satellite consists of a basic repeating unit of six nucleotide pairs in length. However, some base changes bet\Treen repeating units exist according to melting temperature studies with native and reassociated satellites (Corneo et al., 1968; Flamm et al., 1969). The mouse satellite DNA seems to be similar, except that the basic repeating unit is 9 to 18 nucleotide pairs. Again, its sequence is heterogeneous, and over a long range the mouse satellite presents other periodicities of around 120 and 240 nucleotide pairs (Southern, 1975; Biro et al., 1975). The sequence simplicity of satellite DNAs is even more remarkable in Drosophila vi&is, in that each satellite consists of repeating units of an identical sequence with seven nucleotide pairs in length (Gall & Atherton, 1974). More recently it has been shown by direct sequence analyses that the Drosophila melanogaster 1.705 g/cm3 satellite has a repeating unit of only five nucleotide pairs and the 1.672 g/cm3 satellite is a mixture of two different sequences, one having a repeating unit of five nucleotide pairs long and the other seven nucleotide pairs (Brutlag & Peacock, 1975). Such sequence homogeneity in several Drosophila satellites raises some interesting questions on the origin and the maintenance of these satellite sequences. We have been interested in the satellite DNAs of D. nasutoides because of the following observations previously made in our laboratory (Cordeiro et al., 1975). (1) Karyotype analysis showed that I). nmutoides has three pairs of autosomes plus the sex chromosomes. The unusual feature is that one autosome pair is very large and shows a distinctive banding pattern along the metaphase chromosome arm according to the C-banding technique. These positive C-bands suggest a highly heterochromatic nature for this large autosome pair. (2) D. nmutoides shows four major satellites in neutral CsCl gradients amounting to 50 to 60% of the total genome. All four satellites, designated as I through IV in descending order of buoyant density, are lighter than the main nuclear DNA. All four satellites have been purified in native form by a combination of neutral CsCl, actinomycin D/C&I, and Kg2 + /C&SO, gradients. Their buoyant densities under neutral and alkaline conditions and their melting temperatures have been determined. Further, it is shown that their sequence complexities, that is, the lengths of repeating units of the four satellites as determined by reassociation kinetics, vary over a wide range. Melting studies of reassociated satellites reveal that the repeating sequences are homogeneous in satellites I and IV, but not in satellites II and III. An intriguing observation from our reassociation kinetic studies is that the sequence complexity of satellite III is almost as large as that of E. coli. Finally, their location in polytene chromosomes is described.

2. Materials and Methods of Drosophila nasutoides U. nuwtoides (UT stock 3035.2), originally collected in the Sarnoall islands by Dr M. Wheeler, was cultured using a standard cornmeal medium with sprayed yeast. (a) Culture

(b) Isolation of DNA (i) DNA from adults of D. nasutoides and D. virilis Since a significant amount of satellite DNB is selectively and irreprodllcibly lost during chloroform or phenol extraction procedures (unpublished observations in our laboratory), we avoided such conventional procedures. Instead DNA was isolated by gentle lysis of

SATELLITE

DNAs

IN

D.

NASUTOIDES

3

purified nuclei followed by 2 or more cycles of preparative ultracentrifugation in C&l gradient,s. A tota,l of 10 to 20 g of flies were homogenized in 100 ml of a. Urowphila homogenizing medium containing 0.35 M-sucrose, 0.05 M-Tris (pH S), 0.025 M-KCl, 0.005 M-MgCl, using a Virtis homogenizer. The crude homogenate was filtered through 8 layers of cheesecloth. The debris collected on cheesecloth can be homogenized further in a motor-driven Teflon homogenizer. To the combined filtrates, Triton Xl00 (New England Nuclear) was added to a final concn of 1 y0 (v/v). A second homogenization was performed in a Teflon homogenizer with 5 strokes, followed by centrifugation at 3000 to 4000 revs/min for 15 min in a Sorvall centrifuge. After washing the nuclear pellet with more Drosophila homogenizing medium, it was resuspended in 30 to 40 ml of 0.1 M-Tris-EDTA (pH 8), 0.05 M-N&Cl. Sarkosyl (Ciba-Geigy) and Pronase (Calbiochem) were added to final concentrations of 1% and 1 mg/ml, respectively. Lysis of nuclei was achieved by incubating first at 60°C for 15 min and then by gentle rocking at 37°C for 2 h or longer. Concentrated NaCl solution was then added to a final concn of 1 M, and the lysate was rocked for another 30 min. The lysate was sheared by using a hypodermic syringe to force it through either 18 or 22 gauge needles. Much of the Sarkosyl and other soluble u.v.-absorbing material were removed by dialyzing the lysate against 0.01 M-Tris, 0.001 M-EDTA. This final solution was subjected to preparative CsCl gradient centrifugation. Whenever it was necessary to reduce the volume for preparative CsCl gradients, the lysate was concentrated by dialyzing against solid sucrose. (ii) DNA

from

Micrococcus wore extracted

bacteria luteua and E. coli BB Thywere grown by the procedure of Marmur (1961). (c) Analyticul

in a Tryptone

broth.

The

DNAs

ultracentr~fugation

Buoyant densities were determined by equilibrium centrifugations in CsCl using a Spinco model E analytical ultracentrifuge. Approximately 1 to 2 pg DNA was used per cell. The initial densities of CsCl solutions were 1.69 to 1.71 g/cm3 for a neutral gradient and 1.74 to 1.76 g/cm3 for an alkaline gradient containing 0.1 M-NaOH. The ultracentrifugation was conducted at 44,770 revs/min for 20 to 22 h at 25°C using an ANF rotor. Buoyant densities were calculated according to Vinograd & Hearst (1962), using M. Zuteus DNA as a marker for neutral densities and both M. luteus DNA and E. co&i DNA for alkaline densities. (d) Preparative (i) Neutral

and alkaline

ultracentr&@ion

Cd21 gradients

Nuclear lysates or DNA preparations previously centrifuged were mixed with solid CsCl to give a density of 1.68 to l-70 g/cm3. Each centrifuge tube was filled with 4.5 ml DNA/CsCl solution with mineral oil on the top. The centrifugation was done in a Spinco model L3-50 at 38,000 revs/n& for 40 h using a fixed-angle rotor, type 65. For alkaline gradients, 100 to 200 rg DNA in 0.01 M-Tris, 0.001 M-EDTA was mixed with concentrated NaOH solution to a final concn of 0.1 M in the gradient. After at least 10 min at room temperature, solid CsCl was added to give a density of 1.74 to 1.76 g/cm3. The conditions of the ultracentrifugation were the same as described above, except that the speed was 48,000 revs/min. (ii) Actinornycin DICsCl gradient A total of 100 to 150 pg DNA in 0.01 M-Tris, 0.001 M-EDTA (pH 8.4) was mixed with a proper amount of solid CsCl. After complete solubilization of CsCl, actinomycin D was added to give 30 pg actinomycin D/l00 pg DNA (Peacock et al., 1973). The initial density was adjusted to 1.66 g/cm3. The centrifugation was done at 40,000 revs/n& for 40 to 48 h. Actinomycin D was removed by extracting the DNA/CsCl/actinomycin D solution with isopropanol equilibrated with saturated CsCl solution (Peacock et al., 1973). This extraction procedure was repeated twice or more.

4 (iii) Hg2 + /Cs&‘0~

M.

CORDEIRO-STONE

AND

C. S. LEE

gradients

The DNA (100 to 150 pg) in 0.01 ni-NasSO, was mixed with saturat,ed Cs2S0, solution and concentrated NasB,O, solution (pH 9.2) to give a final density of 1.50 g/cm3 and 0.005 M, respectively. A portion of stock HgCl, solution was added to obtain a molar ratio of Hg2+ to DNA phosphate of 0.2 (Nandi et al., 1965). The centrifugation was done at 30,000 revs/mm and 20°C for 40 h. Hg2+ was removed by dialyzing pooled fractions against 0.1 M-EDTA. (e) So&cation of DNA For melting and reassociation rate studies, DNA samples (2 to 10 ml) in 0.5 nr-NaCl, 0.01 nl-Tris, 0.001 M-EDTA were sonicated using a Bronson sonifier at power level 2 (3.5 to 4.0 A). Prior to sonication, dry nitrogen gas was bubbled through the DNA solution for 10 to 15 min. Each 10-s pulse of sonication was followed by a 1-min rest period for effective chilling. The temperature of the DNA solution was no higher than 10°C during sonication. The total sonication time was 2 min. After sonication, DNA samples were concentrated if necessary. The DNA solutions were then exhaustively dialyzed against SSC (SSC is 0.15 M-NaCl, 0.015 M-Na,-citrate, pH 7.0) of a desired concentration for melting or reassociation experiments. (f) Determination Molecular weights of sonicated sedimentation in a Spinco model E 0.9 M-NaCl, 0.1 ivr-NaOH, and DNA tion was done at 44,770 revs/mm convert sedimentation coefficients

of molecular

weight.s

DNA samples were determined by analytical ultracentrifuge. The bulk at a concn of 20 to 30 pg/ml. The and 20°C. The equation of Studier to molecular weights.

(g) Melting

temperature

boundary velocity medium contained velocity sedimenta(1965) was used to

measurements

DNA samples to be melted were simultaneously and exhaustively dialyzed against SSC of an appropriate concentration. Prior to melting, DNA solutions were degassed by suction. Optical melting curves were obtained in a Beckman Acta III spectrophotometer. The temperature was raised by a Haake circulating water bath. The rate of temperature increase was no more than 0.4 deg. C/min. (h) DNA

reassociation

rate measurement

All the measurements of satellite DNA reassociation rates were done optically. For the experiments of satellites I, II and IV, heavy and light strands were separated by alkaline CsCl gradients as described above. Equal amounts of heavy and light strands in 2 separate quartz cells were placed in the cell holder of a spectrophotometer, the temperature of which was maintained at t, - 25 deg. C. To initiate the reassociation reaction, the 2 solutions were mixed rapidly and hypochromicity was followed at 260 nm. Mixing generally took less than 10 s. There are two advantages in using separated heavy and light strands. First, monitoring of the reaction is almost instantaneous, since there should not be any reassociation until the 2 strands are mixed. This is important when dealing with very highly repetitious DNA. Secondly, there is no artifactual absorption decrease due to nonspecific intrastrand stacking interactions occurring while the temperature is shifted from above the t, to t,-25 deg. C in conventional procedures (Wetmur & Davidson, 1968; Lee & Davidson, 1970). A clean separation of heavy and light strands of satellite III is not possible by preparative alkaline CsCl gradient centrifugation (see Results). Thus, we chose an alternate route; first the DNA was maintained in low salt and at high temperature such that the DNA is dissociated; then the salt concentration was raised to initiate the reassociation. An appropriate amount of satellite III in l-2 ml was first melted in 0.1 x SSC. The t, of satellite III is 55.5”C in this salt concentration. After melting, the temperature was maintained at 62°C. In order to initiate the reaction, 0.3 ml of 20 x SSC, which was maintained at 62”C, was added to the DNA solution, thus resulting in a Na+ concn of 0.80 M. At this ionic strength, the t, is 86°C. Again by doing this, we were able to avoid the artifactual hypochromicity as mentioned above. Exactly the same procedure was followed for E. coli DNA reassociation.

SATELLITE

DNAs

The second-order reassociation of Wetmur & Davidson (1968),

IN

D.

rate constant, &-Am

5

NASUTOIDES

k,, was determined

by using the equation

hcott =-+1.

A-Am

2

In our case, A, is the sum average of the absorbancies at 260 nm (A,,,,) of heavy and light strands at the reassociation temperature (t,, i.e. t,-25 deg. C). For satellite III, A,, is simply A,,,, at t, after an addition of 20 x SSC to increase the ionic strength to 0.80 pi. Am is the absorbancy obtained by letting the reaction proceed to near completion. For the satellites I, II and IV, the initial DNA concentration, C,, in mol DNA phosphate/l was obtained from the expected absorbancy of the native DNA, A,,, by using the following formula Ad

A,=-,

1 + H,

where A, is A,,,, of DNA at a temperature 10 deg. C above t,, and H, is the hyperchromicity for an individual satellite. The use of this formula was necessary because (1) we are handling separated single strands as starting material and (2) A, and Am are not necessarily identical, especially for satellite II, due to mismatched base-pairs in reassociated DNA. (i) In situ hybridization 3H-labeled complementary RNA was prepared by in vitro transcription of purified satellite DNA using E. coli RNA polymerase according to the methods of Gall & Pardue (1971) and Davis & Hyman (1970) with some slight modifications. cRNA$ synthesis was carried out in 0.25 ml of an incubation mixture containing 5 to 10 pg DNA, 4 to 5 units E. coli RNA polymerase (Worthington; 377 units/mg), 0.15 M-KCl, 0.05 M-Tris (pH 7.9 at 25”C), 5 mM-MgCl,, 2 mM-MnCl,, 0.2 to 0.6 mM-EDTA, 0.1 Mdithiothreitol, and nucleoside triphosphates. The templates were the light strands of satellites I, II and IV, and denatured satellite III. The substrates were: 0.4 m&r-CTP, 0.2 m&r-GTP, 100 pCi each of [3H]GTP (12 C’/i mmol), [3H]UTP (15 Ci/mmol), and [3H]ATP (19 Ci/mmol). The reaction mixture was incubated at 37°C. At the end of 2 to 4 h, an equal amount of RNA polymerase was added and the incubation was continued for another 4 h. This additional RNA polymerase step helps the incorporation of radioactivity by a factor of 2 or more. The reaction was monitored by taking 5-,.~1portions and assaying for trichloroacetic acid-precipitable counts. At the end of synthesis, DNase I (Worthington, DPFF) was added to a final concn of 40 rg/ml and incubated at 37°C for 15 min. Approximately 100 rg of wheat germ RNA (Calbiochem) was then added. This mixture was deproteinized by adding sodium dodecyl sulfate to a final concn of 1% (w/v) and extracting with 2 vol. freshly distilled phenol saturated with a Tris buffer. The aqueous phase was loaded on a Sephadex G50 column (06 cm x 19 cm). The column was eluted with 2 x SSC and the fractions containing trichloroacetic acid-insoluble counts were pooled. In general, the yield of cRNA was 0.5 to 2.0 rg per incubation mixture and the spec. act. was 0.8 x 10s to 1.3 x lOa disints/min per pg RNA. The in situ hybridization to polytene chromosomes was done as described by Gall & Pardue (1971). The HCl treatment was omitted (Gall, personal communication).

3. Results (a) Satellite

DNAs

of D. nasutoides

The DNA of Drosophila naszltoides contains a large amount of satellite DNA components, constituting about 50 to 60% of the total genome. There are four major satellites observed, designated as I, II, III and IV in descending order of their t C,t,

the initial

$ Abbreviation

DNA

concn.

in mol DNA

used: cRNA, complementary

phosphate/I

RNA.

x

the time

of incubation

in s.

6

M.

CORDEIRO-STONE

AND

C.

S.

LEE

buoyant densities. Figure I(a) shows buoyant density profiles in a neutral CsCl gradient of the DNA isolated from brains and imaginal discs of D. nasutoides. The DNA from salivary glands shows no defectable satellite as seen in Figure l(b). This observation agreeswith the results from other Drosophila speciesin that the satellite DNA replicates very little if any during polytenization (Gall et al., 1971). (b) Purification

and strand

of D. nasutoides satellites

separation

All four individual satellites were purified in native form by a combination of neutral CsCl, actinomycin D/CsCl, and Hg2 +/C&SO4 gradients. Strand separations were done by alkaline CsCl gradients. The lysate of purified nuclei from D. nasutoidesadults was centrifuged in a neutral CsClgradient using a preparative ultracentrifuge. Figure 2(a) showsa typical banding profile of the total lysate. Three reasonably well separated peaksare seen,representing main DNA, satellites I plus II and III plus IV, respectively. Fractions containing

M 1702

r I 667

I

&2

m I669

i

(a) i

\

I 6%

n

(bl

FIG. 1. Buoyant density profiles of D. nasutoides DNA in neutral CsCl gradient. The isolation of DNAs from brains and imaginal discs (a), and salivary glands (b), was done according to Gall et ~2. (1971). The analytical centrifugations were done as described in Materials and Methods. The arrow indicates M. Zuteus marker DNA (p = 1.731 g/cm3). (When the buoyant densities are calculated according to Szybalski (1968), significant differences are observed. We find that the greater the separation of 2 DNA bands (one being a reference) the more serious are the discrepancies. The discrepancy is even worse in alkaline buoyant densities because of wider strand separations. Because of the uncertain universality in the empirical parameters of Szybalski (1968) for our satellites, we decided to use the method of Vinograd & Hearst (1902). However, for a direct comparison with the data published by others, the buoyant densities calculated according to Szybalski (1968) are given here: 1.706, 1.690,1~686, 1.674, and 1.671 g/cm3 for the main DNA(III), satellites I. II, III, and IV, respectively.)

SATELLITE

DNAs

IN

D.

NASUTOIDES

7

satellites I plus II and III plus IV were pooled and rebanded as described above. When necessary, rebanding in neutral CsCl gradients was repeated. The purity of satellites I plus II and III plus IV was checked routinely in the analytical centrifuge . Satellites I and II can be separated by an actinomycin D/CsCI gradient but not by Hg2+/Cs,S0, gradients. The purified mixture of satellites I plus II was subjected to centrifugation on an actinomycin D/CsCl gradient as described in Materials and Met,hods. The result is shown in Figure 2(b). Separation of the two satellites is reasonably good. Appropriate fractions containing satellites I and II were pooled and

IO

20 Fraction

30

40

no

FIG. 2. Preparative density gradient profiles. (a) Neutral C&l gradient of the total DNA isolated from purified nuclei of D. naautoides adults. (b) D. nasutoides satellites I and II separated by an actinomycin D/CsCl gradient. (c) D. neoutoides satellites III and IV separated by a Hg2 +/C&SO+ gradient. Details are described in Materials and Methods. M, main bend DNA peak.

8

M.

CORDEIRO-STONE

AND

C.

S.

LEE

actinomycin D was removed. No rebanding was necessary after checking their purities in the analytical centrifuge. Hg2 + /C&SO, gradients separate satellites III and IV, but not actinomycin D/CsCl gradients. Figure 2(c) shows the separation of satellites III and IV in a Hg2 + /Cs,SO, gradient. Heavy and light strands of all four satellites can be separated using alkaline CsCl gradients. Figure 3 shows alkaline banding profiles obtained by analytical ultracentrifugations. Buoyant densities given in the figure were calculated according to Vinograd 6 Hearst (1962) using either M. Zuteus DNA (p = 1.789 g/cm3 at pH 2 13) or E. coli DNA (p = 1.766 g/cm3 at pH 2 13) as a marker, whichever bands closer to the unknown. The separation of heavy and light strands of the satellite I is quite dramatic as can be seen in Figure 3(a). This characteristic, along with the melting

(0)

(b)

FIQ. 3. Strand separation profiles of purified D. nmzltoides using the analytical ultracentrifuge. (a) Satellite I; (b) satellite II; (c) satellite III; (d) satellite heavy; L, light.

satellites IV.

in alkaline

Arrows

indicate

C&l

meniscus.

temperature and the reassociation rate, which will be described later, reveals similarity between the D. nasutoides satellite I and the D. virilis satellite I. (c) Thermal

denaturation

pro$les

of D. nasutoides

gradients H,

a large

satellites

In order to further understand the physicochemical properties of satellites and to obtain basic information for reassociation kinetic studies described in the next section, thermal denaturation experiments were done with purified D. nmutoides

SATELLITE

DNAs

IN

D.

NASUTOIDES

9

30

20

IO

O-b 50

60

70

00

4 Temperature

o+e74 PC 1

FIG. 4. Thermal denaturation profiles of D. nasutoides satellites. Purified native satellite DNAs in proper concentrations of SSC were melted in a spectrophotometer (-O-O-). For the melting of reassociated satellites (-a-@-), the denatured satellites were incubated at t,-26 deg. C for 8 to 12 h and melted. (a) Satellite I (14.3 pg/ml) in 0.2 x SSC (0.039 M-Na+). t, value of the native = 69.3”C, t, value of the reassociated = 68,O”C. (b) Satellite II (13.8 pg/ml) in 0.2 x SSC. t, value of the native = 67.6”C, t, value of DNA sample reassociated at t,- 25’C = 51.O”C (-e-O--), t, value of DNA sample reassociated at t,40°C = 46.O”C (-A--n-), t, value of DNA sample reassociated at t,19°C = 63.4”C (-o-o-). (c) Satellite III (97.2 pg/ml) in 0.796 M-N&+. t, value of the native = 85.6’C, t, value of the reassociated = 77.6”. (d) Satellite IV (13.4 pg/ml) in 0.2 x SSC. t, value of the native = 51.5”C, t, value of the reassociated = 51.2”C.

satellites. behaviors

The results of individual

of such melting experiments are shown satellites are described below.

in Figure

4. Melting

(i) Satellite I (Fig. 4(a)) The native satellite I melts at t, = 69*3”C in O-039 M-Nit+. The difference of melting temperatures (At,) of the native and the reassociated is 1.3 deg. C indicating less than 2% of base-pair mis-matching in the reassociated satellite I. That is, the sequence of every repeating unit in the satellite I is effectively homogeneous. Approximately 5% of a residual hyperchromicity of the reassociated satellite I seen in Figure 4(a) could be probably due to a small amount of unreassociated single-strand regions at the ends of reassociated molecules. Another possibility is that some of this hyperchromicity might be due to a small portion of satellite I sequences so diverged that its reassociation was not permitted under the conditions employed. However, the observations of a small At,,, value described above suggests that the former explanation is more likely.

10

M.

(ii) Satellite II

CORDEIRO-STONE

AND

C.

S.

LEE

(Fig. 4(b))

The native satellite II has a t, of 675°C in 0.039 M-N&+. However, the reassociated satellite II melts at a much lower temperature. t, values of the satellite II reassociated at t, - 19 deg.C, t, -25 deg.C and t, -40 deg.C are 14al”C, 16.5% and 21*5”C, respectively. Such variation in At,,, values under different criteria, i.e. different incubation temperatures in our case, is typical among repetitious DNA with sequence variation. A precise estimation of the amount of mis-matched base-pairs in the reassociated satellite II is difficult to make without direct biochemical sequencing data. The studies with deaminated DNA and glyoxylated DNA as a model for various base-pair mis-matchings give a range of 0.7 deg.C to 1.6 deg.C in At,,, per 1% mismatched base-pairs (Laird et aZ., 1969; McCarthy & Farquhar, 1972; Hutton & Wetmur, 1973). In order to elucidate further the problem of mis-matching, we have attempted to study D. vi&s satellites I and III, the sequences of which are already known (Gall & Atherton, 1974). The heteroduplexes of D. vi&is satellites IL-IIIH (one A-C mismatching out of 7 base-pairs) and IH-IIIL (one G*T mismatching out of 7 base-pairs) melt at 39el”C and 47*2”C, respectively, in O-2 x SSC. Under this condition, the native virilis satellites I and III melt at 67.3”C and 61*1”C, respectively. When we use the average t, values of the native I and III and estimate At, values of the heteroduplexes IL-IIIH and IH-IIIL, we obtain 1.8 deg.C/ % AX! mis-matching and I.2 deg.C/% G*T mis-matching. These results are in general accord with Blumenfeld et al. (1973). Without knowing precise types of base-pair mis-matching, we therefore chose to use 0.7 deg.C as a lower limit and 1.8 deg.C as an upper limit per l){, mismatched base-pairs. Based on these data, the amount of mis-matching in t,he reassociated satellite II is estimated to be 10 to 24%. The residual hypochromicity seen in Figure 4(b), therefore, is largely due to the presence of mismatching besides the staggered hybrids mentioned above. In the case of a high criteria incubation, i.e. t,-19 deg.C, the reassociation of the satellite II was not complete.

(iii) Satellite

III

(Fig. 4(c))

The native satellite III melts at 85.6% in 0.80 M-Na+ . The t, value of the reassociated DNA is 775”C, showing At, value of 8 deg.C. This corresponds to 5 to 11% mis-matching in the reassociated molecules. When satellite III is melted in 0.039 M-Na+ as with other sa,tellites, the t, is 61.O”C which is almost 10 deg.C higher than that of the satellite IV (described below), although the difference in their buoyant densities is only 0.004 g/cm3.

(iv) Satellite IV (Fig. 4(d)) Satellite IV melts at 51.5% in 0.039 rvr-Na+ with a very sharp transition. The reassociated satellite IV melts essentially at the same temperature. No residual hyperchromicity was observed. Before closing this section, we wish to describe our observations on the hypochromic effects of purified light (L) and heavy (H) strands of three satellites in 0.1 x SSC. Their hyperchromicities are as follows: IL, 0.6%; IH, 0.2%; IIL, 1.6%; IIH, 2.7%; IVL, 4.9%; IVH, 0%. It is clear that there is no significant self-complementary sequence within an isolated single strand, except for perhaps satellite IVL.

SATELLITE

DNAs

(d) Effect of ionic strengths

1N

on melting

D.

NASUTOIDES

temperatures

11

of satellite

DNAs

The effects of ionic strengths on melting temperatures of complex DNAs have been made previously (Dove & Davidson, 1962 ; Schildkraut & Lifson, 1965). In order to ensure that a similar relation holds for simple sequence DNA, we have re-examined the relationship between ionic strengths and the t, value of D. nusutoides satellite II. The result is shown in Figure 5. From this figure, we obtained the following relation :

t Itll

-

t,,

=

18.7 (log p1 -

log pLz),

where p1 and p2 denote ionic strengths which produce t,, and tm2, respectively. The slope, 18.7, is in excllent agreement with that (185) obtained by Dove & Davidson (1962), but not with that (16.6) obtained by Schildkraut & Lifson (1965). This relation holds very well with other satellites, for example, satellite III described in section (c), above.

-log

FIG. 6. Effect values of the satellite The slope from this Figure examined.

t,

[No+]

of ionic strengths on t, values of D. nasutoides satellite II. II in different concentrations of SSC are plotted against - log [Na+]. gives 18.7. The same slope is obtained with other D. nmutoides satellites

(e) Reassociation

kinetics of D. nasutoides

satellites

In order to determine the degree of repetition and the sequence complexity, i.e. the length of a repeating unit, DNA reassociation kinetic studies were undertaken for D. nasutoides satellites. Separated heavy and light strands were used for satellites I, II and IV. As described in Materials and Methods, the use of separated strands has several advantages for very rapidly reassociating DNA species. The determination of second-order rate constants and the sequence complexities of satellites are described below. The results are summarized in Table 1. (i) Determination

of second-order

rate constants

The reassociation reaction was monitored by the absorption decrease at 260 nm due to DNA reassociation. Calculations for the determination of second-order rate constants, k,, were done essentially according to Wetmur & Davidson (1968) with a

470

380

III

IV

I

520

b

(0.796

(0.0195

M-Nat)

4.72

M-Ne+)

96.2+9.7 (0.0195 M-Na+) 62.7h5.4

1os* 14 (0.0196 iw-Na+) 17.310.3 (0439 M-N&+) 7.42 (0.796 M-N&+) 7.14 (0.0195 M-Nat) 104 (0.039 M-N&+ ) 350 (O@SS5 M-N~+)

kz (measured) 1 mol-1 s-1 %Na+)C

0.72

5.90 x 104

9.05 x 104

6.99 x lo3

1.13

1.20 x 103

‘Ott

2.78

3.39 x IO-5

2.21 x 10-S

2.86 x 1O-4

1.77

1.66 x 10-S

1.97 x 10-e

[“;‘~~~+‘”

and sequence complexities

1.02 x 105

kz (0.18

rate constants

1

M-N&+)~

3.15

2.58 x 105

3.96 x lo5

3.06 x lo4

4.95

5.27 x lo3

4.45 x 105

k, (0.42

of D. nasutoides

a Molecular weights of single-stranded DNA in terms of number of nucleotides are given. b Average values from 2 to 5 independent measurements. c Conversion of rate constants to higher salts was made by using the graph shown in Fig. 7. * From the sequence data of Gel1 & Atherton (1974). e A molecular weight of 2.5 x lo9 was used as the genome complexity of E. coli. * Obtained from Bolton et al. (1965).

D. vivilis satellite E. coli

(poly!A)) 360

360

II

Reference DNA8 Poly(dA).poly(dT)

480

Sat&k8 I

Molecular weight ( L)a (nucleotides)

Reassociation

TABLE

(or k’,,,) M-Na+)

0.138

1.38 x 104

7.23 x 10“

1.58 x 103

0.229

2.76 x lo2

2.03 x lo4

k,/L’ (o.42

satellites

5

(N) pairs)

3.8 x 106B

7d

1

46

2.3 x 106

103

Complexities (nucleotide

At,,

< 2’

<2

0

<0.5

8

17

<2

(deg. C)

w

SATELLITE

DNAs

IN

D.

NASUTOIDES

13

slight modificatSion. The absorption at time zero when all the DNA is dissociated was that at t,-25 deg.C, rather than that at or above the t, value. This was possible because we were using separated heavy and light DNA strands. In this way we could avoid some absorption decrease due to stacking interaction, but not due to reassociation. In fact, we fmd that the straight line in a k, plot always extrapolates to 1-O at time zero (see Fig. 6), whereas some previous observations have been above I.0 (Wetmur & Davidson, 1968; Lee $ Da#vidson, 1970). The same objective was achieved

40 u 30

a ; G rn

20

3 c 1

IO

70

50

u al f =

30

$

I-O 40

3-o

20

: 2 h

IO Time

(s

x 10s2

)

FIG. 6. Reassociation profiles of D. nasutoides satellites, D. virilis satellite I, and E. coli DNA. (a) D. nmuto-ides satellite I in 0.0196 M-Na+. -O-O-, C, = 4.06x 10e5 mol DNA phosphates/l. -a---, C, = 2.30 x 10m5 mol/l. The slopes of two straight lines due to 2 different DNA concentrations are 2.20 x 10e3 s-l and 1.23 x 10m3 s-l, resulting in rate constants of 109 1 mol- 1 s-l and 107 1 mol- ’ s-l, respectively. The last data points represent about 80 to 85% of the total reassociation. -o-n--, D. &r&s satellite I in 0.0195 M-N&+ at C, = 3.88x 10e5 mol/l. The k, value calculated from this plot was 59 1 mol- ’ s- I. The last data point of D. vuirilis satellite I represents 75% of the total reassociation. (b) D. nmutoides satellite II (-a-@-) at C, = 1.71 x lo-* mol/l and satellite IV (-O-O-) at C, = 8.06 x 10m5 mol/l, both in 0.039 M-Na+. The last data point represents 82% reassociation for satellite II and 92% for satellite IV. The insert illustrates the variation of k, values with respect to the incubation temperature for satellite II. (c) D. nasutoides satellite III (-O-O-) at C, = 2.27 x 10m4 mol/l and E. co& DNA (-e-e-) at Co = 2.12 x lo-* mol/l, both in 0.796 M-Na+. The last points represent 71% of reassociation for satellite III and 61 o/0 for E. COG.

14

M.

CORDEIRO-STONE

AND

C.

S.

LEE

with the satellite III and E. wli DNA, the strands of which have not been separated, by proper changes of salt, concentmtions as described in Mat)erials and Methods. Figure 6 illustrates second-order reassociation profiles of all four satellites of I). nasutoides, D. vi&s satellite .L and E. coli DNA. .First, as seen in this Figure, the second-order rate plots give straight lines almost until completion of the reassociation reactions, indicating that there is only one rate component in a given satellite sequence. In order to further verify that the reassociation reaction of these satellites is secondorder, two different DNA concentrations were used for the satellite I as shown in Figure 6(a). The k, values obtained were 109 and 107 1mol-l s-l,indicating the nature of a second-order reaction of the satellite DNA reassociation. Figure 6(b) shows the second-order rate plots of satellites II and IV. The insert in Figure 6(b) illustrates the effects of incubation temperature (t,) on the rate of satellite II reassociation. Even if there is a large sequence variation between repeating units of satellite II as described in the previous section, the optimal temperature of its reassociation is still t, -25 deg.C. The rate profiles of satellite III and E. coli DNA are shown in Figure 6(c). The second-order rate constants, k,, calculated from such plots are summarized in Table 1. (ii) Conversion

of the rate constants to desired ionic strengths

For fast-reassociating satellites such as I, II and IV, it was necessary to use low salt of varying ionic strengths in order to monitor the reassociation in a reasonable period of time in a conventional spectrophotometer. Under the conditions we used, the half reaction time was around 5 to 30 minutes. In order to compare these with other rate data in the literature, it was desirable to convert the rates to a common salt concentration. The rate constants of T4 DNA in varying ionic strengths from 0.06 M to 3.2 ivr-Na + are given by Wetmur & Davidson (1968). Since we have used lower ionic strengths than 0.06 M-N&+, it was necessary to extend their data to the range of ionic strengths we used for the satellites. For this purpose, we measured the rates of the satellite IV reassociation in three different ionic strengths which are given in Table 1. The data of Wetmur & Davidson (1968) and those for satellite IV are plotted in Figure 7. We find this graph very useful and reliable. For example, the rate constant of satellite II f&t obtained in 0*0195 M-Na + and converted to O-039 M-Na + using this graph agrees very well with that directly obtained in 0.039 M-N&+. Furthermore, when the rate of E. coli DNA in 0.796 M-Na + is converted to 1 M-Na + , it gives a value of 5.2 1 mol-l s-l, which isin good agreement with that of Wetmur & Davidson (1968). As shown in Table 1, all the rate constants have been converted to 0.18 M-Na+, which is the ionic strength of 0.12 M-phosphate buffer used in conventional C,t experiments, and to 0.42 M-N&+ for a direct comparison with some of the deoxyribohomopolymer studies of Lee 6 Wetmur (1972). (iii)

Determination

of sequence complexities

The second-order rate constant has been found to be inversely proportional DNA sequence complexity, or genome complexity in bacteria and viruses, following relation (Wetmur $ Davidson, 1968 ; Wetmur, 1975),

to the by the

SATELLITE

DNAs

O-2

IN

04

D.

0.6

NASUTOIDES

08

l&i

I.0

[NO+] (M) FIG. 7. Effect of ionic strengths on DNA reassociation rates. The rate data of T4 DNA from 0.06 M to 1.0 M-N&+ are taken from Table 6 of Wetmur & Davidson (1968) (open circles). The rate constants of the D. nasutoides satellite IV were determined at ionic strengths of 0.0195 M, 0.039 M and 0.0586 M-N&+ (see Table 1). Since the rate constants of T4 DNA and the satellite IV are different, the data point of satellite IV at 0.0685 M-Na+ was superimposed on that of T4 DNA at 0.06 M-Na +, based on the assumption that the relative rates with respect to ionic strengths are the same in both DNAs (c)). The other two points are plotted accordingly (a).

where L is the length of the shorter of the two reacting complementary strands, kN is a length-independent nucleation rate constant, and N is the sequence complexity. kh depends on ionic strengths, temperature, base composition, etc. N in our case is the length of a repeating unit in a given satellite sequence. In order to minimize the variable, it is best to use a reference DNA of a comparable complexity and base composition with a known sequence complexity. For the determination of the repeating length of D. nasutoides satellites, therefore, we used the D. virilis satellite I as a reference ‘DNA for our satellites I and II, poly(dA). poly(dT) for the satellite IV, and E. coli DNA for the satellite III. The length of a repeating unit of D. nasutoides satellite I is calculated to be five nucleotide pairs by using the formula above and assuming k’, is the same in both D. nasutoides satellite I and D. virilis satellite I. This value is identical to that of the D. mekznogaster 1.705 g/cm3 satellite (Brutlag & Peacock, 1975) but slightly smaller than that of D. virilis satellites (Gall & Atherton, 1974). An accurate estimation of the sequence complexity of D. wwutoides satellite II is difficult to make due to a large amount of mis-matching, the effect of which reduces the rate of DNA reassociation. According to Bonner et al. (1973), a 10 deg.C difference in At, due to mis-matching reduces the reassociation rate at an optimal temperature by a factor of two. Since t, -25 deg.C is an optimal temperature for satellite II reassociation as seen in Figure 6(b), a 3*4-fold reduction in the rate of satellite II is expected for a At, value of 17 deg.C. When this value is used for the correction of k,, and D. virilis satellite I is used as a reference DNA, the length of a basic repeating unit of D. nasutoides satellite II is calculated to be 103 nucleotide pairs. This value is given in Table 1. A considerably different value can be obtained using the data of

16

M.

CORDEIRO-STONE

AND

C.

S.

LEE

Hutton & Wetmur (1973). They observed that the reassociation rate of deaminated DNA is reduced by a factor of two for a At, value of 23 deg.C and that of glyoxylated DNA by a factor of two for a At, value of 17 deg.C. If we take the average of these two values and correct the rate for satellite II, a sequence complexity of 206 nucleotide pairs is obtained. A theoretical approach to this problem of rate reduction due to mis-matching has been made by Southern (1971). When we apply his equation to satellite II, we obtain a sequence complexity of 89 and 10 nucleotide pairs for 10 and 24% base-pair mismatching, respectively, the variation of which is given in the previous section. Reassociation of satellite III was observed to be slow, similar to that of E. coli DNA. Therefore, we have measured the reassociation of both satellite III and E. coli DNA simultaneously in a spectrophotometer. The complexity calculated using E. co& DNA as a reference is 2.3 x lo6 nucleotide pairs, which is about two-thirds as complex as the E. coli genome (Table 1). If we consider the mismatching present in the reassociated molecules (At, = 8 deg.C), the complexity is very close to that of E. coli. We believe that satellite III of D. nasutoides is the most complex among satellite DNAs of nuclear origin. A possibility of microorganism contamination has been completely eliminated (see Discussion). The length of a repeating unit of satellite IV was determined by using poly(dA). poly(dT) as a reference DNA of known sequence complexity. A value of 46 nucleotide pairs was obtained (Table 1). Before concluding this section, we would like to make a further note on the relationship between the second-order rate constant and the sequence complexity, From our rate data on the virilis satellite I and poly(dA).poly(dT), whose sequence complexities are precisely known, the following empirical relationship is obtained in 0.42 M-Nit+ : k, = 8.45 x IO4 g.

The value of 8.45 x lo4 is very close to 8.2 x lo4 of Lee & Wetmur (1972) when their k, of poly(dA)*poly(dT) at t,-25 deg.C, not at the optimal temperature, is used. Obviously, the use of this empirical equation produces sequence complexities comparable to those given in Table 1.

(f) Localization

of satellite sequences in polytene chromosomes

Details on metaphase and polytene chromosomes of D. nasutoides have already been published from our laboratory (Cordeiro et al., 1975). Therefore, only brief accounts are given here. (i) D. nasutoides shows a very large autosome pair in metaphase plates (Fig. 8(a) and (b)). (ii) This large autosome is almost entirely heterochromatic according to the Cbanding technique (Cordeiro et al., 1975 ; Lee & Collins, unpublished results). (iii) This large autosome is an isochromosome according to the C-banding patterns, the morphology of this chromosome in polytene chromosome preparations (Fig. 8(f)) and the in situ localization of satellite I to metaphase chromosomes (Arrighi, CordeiroStone, Wheeler, Lee & Hsu, manuscript in preparation).

SATELLITE

FIU. 8. Photomicrographs of (a) and (b) Metaphase chromosome by aceto-oraein stain, bars indioate salivary glands of late third instar some. The X chromosome is clearly where 2 arms are not synapsed, bar

2

DNAs

IN

D.

NASUTOIDES

metaphase and polytene chromosomes of D. nawbides. squashes prepared from brains of young third instar larvae 6 F. (c) to (e) Polyte ne chromosome squashes prepared from larvae by aceto-orcein stain. Arrows indicate the dot chromodistinguished in (e). Bars indicate 40 pm. (f) Dot chromosome indicates 10 pm.

17

Fro. 9. Photomicrographs (a) Polytene chromosomes hybridized with Same as (a) except that the dot chromosome the same slide of (b); 16 days exposure. Note II; 20 days exposure. (e) Same as (d) except hybridized with satellite II. Note the hybridization

of D. [3H]cRNA of is attached to similar silver that the slide in the

polytene chromosomes hybridized with satellites I and II. satellite I. Note the hybridization at the corner of the dot chromosome; 10 days exposure. (b) a chromosome arm; 16 days exposure. (0) Hybridization of satellite I to diploid cells present in grain densities in (b) and (0). (d) Polytene chromosomes hybridized with [3H]oRNA of satellite was exposed for 11 days. (f) The dot chromosome, which has the appearance seen in Fig. 8 (f), middle of the chromosome: 4 days exposure. Nucleoli are clearly visible in (a), (d) and (6).

nu8utoide.s

(a) Polyt.ene (c) Hybridization satellite IV;

FIG. 10. Photomicrographs of D. nnsutoides polytene chromosomes hybridized with [3H]cRNA of satellite III; of satellite III to diploid cells in a polytene chromosome 7 and 10 days exposure, respectively.

chromosomes hybridized 4 weeks exposure. (b) slide; 11 days exposure.

Same

satellites III and IV. as (a) except that the exposure (d) and (e) Polytene chromosomes

with

was hybridized

18

days. with

20

M.

CORDEIRO-STONE

AND

C. S. LEE

(iv) This large autosome appears as the dot chromosome in polytene chromosome squashes (Fig. 8(c), (d), (e) and (f); Cordeiro et al., 1975). Often the dot chromosome is attached to the tip of an autosome arm. (v) The chromocenter of polytene chromosomes is not as well-defined as that in D. vi&s or D. melarwgaster (Fig. 8(c), (d) and (e)). Our observations with regard to the presence of the large heterochromatic chromosome and the large amount of satellite components led us to postulate that all four satellites might be localized in this particular chromosome. In situ hybridizations to salivary gland polytene chromosomes, therefore, were undertaken. 3H-labeled RNA was synthesized using appropriate satellite strands as templates and hybridized to polytene chromosomes as described in Materials and Methods. The results of such cytological hybridization experiments are shown in Figures 9 and 10. As can be seen, all four satellites are localized in the dot chromosome. Further evidence that all four satellites are present in this large heterochromatic chromosome came from in, situ localization to metaphase chromosomes. We found that only the large heterochromatic chromosome is hybridized with all four satellites (Arrighi, Cordeiro-Stone, Wheeler, Lee & Hsu, manuscript in preparation). It is interesting to note that all these satellites are not localized in the centromere of other chromosomes. This is in contrast with other satellites examined. Satellites from mouse (Pardue & Gall, 1970), human (Jones & Corneo, 1971), and Drosophila (Gall d al., 1971) are all localized in the centromeric heterochromatin. As can also be seen in Figure 9(c), the density of silver grains in diploid cells is approximately identical to that of polytene chromosomes in the same slide (Fig. 9(b)). Similar observations were made with the other three satellites. This confirms an under-reduplication of satellites during polytenization as seen in the buoyant density profile of salivary gland DNA (Results, section (a)) and as shown in other Drosophila species (Gall et al., 1971; Blumenfeld & Forrest, 1972). 4. Discussion There are several notable features in D. nmutoides which have not been previously observed in other Drosophila species: (1) the presence of a very large autosome pair at the metaphase plate which is almost entirely heterochromatic and is also an isochromosome; (2) the large amount of satellites (up to 60% of the total genome) ; (3) a wide variation in their sequence complexities as well as their sequence homoor heterogeneity; and (d) the presence of a satellite with a high sequence complexity. Taxonomically, D. nmutoides belongs to a subgroup of hypocausta in the immigraas group (Wilson et al., 1969). No other speciesexamined in this subgroup hasan autosome pair as large as the one observed in D. w-sutoides (Lee & Yoon, unpublished results). The application of the C-banding technique to D. nasutoties metaphase chromosomereveals a distinctive C-band pattern along the arms of this large autosome, indicating the largely heterochromatic nature of this chromosome (Cordeiro et al., 1975). Furthermore, this banding pattern is symmetric with respect to its centromere, typical of an isochromosome. Polytene squashes prepared from D. na.sutoades salivary glands show four well-banded chromosomes,but no well-defined chromocenter. The largest chromosomeis about twice the size of the X chromosome (Fig. 8). A careful comparison between the metaphase karyotype and polytene chromosomessuggeststhat the large heterochromatic autosome appears as the dot

SATELLITE

DNAs

IN

D.

NASUTOIDES

21

chromosome after polytenization (Cordeiro et al., 1975). Sometimes this dot chromosome shows several bands (Fig. 8(f)). The band pattern is symmetric with respect to its centromere, thus confirming the isochromosomic nature of this chromosome as mentioned above. This point is further evidenced by the in situ localization of satellite I to metaphase chromosomes (Arrighi, Cordeiro-Stone, Wheeler, Lee & Hsu, manuscript in preparation) and studies of Q and G-banding patterns (Lee & Collins, unpublished results). The large amount of satellites observed in D. nasutoiaes is quite impressive. There are four major satellites amounting to about 60% of the total genome. The largest amount was seen in larval brains and brain-associated imaginal discs, which consisted largely of diploid cells. Shearing of gentle lysates to smaller molecular weights did not alter the relative amounts of satellites, ruling out the possibility that satellite sequences are interspersed within main DNA. A similar observation was made with D. vi&s satellites (Gall et al., 1971; Gall & Atherton, 1974). The asymmetric distribution of guanine plus thymine between complementary strands seems to be almost a universal characteristic of satellite DNA. Even the most complex satellite (satellite III) in D. ?zasutoid~ seems to have this characteristic by its strand separation, even if slight compared to the others. An interesting observation is that the G + T distribution of D. nasutoides satellite I is strikingly similar to that of D. vi&s satellite I. Further similarities are found in their meIting temperatures and sequence complexities. We do not think that there is any sequence similarity between these two satellites, based on the evolutionary divergence between the D. virilis group and the D. immigrans group some 3 x lo7 years ago (Throckmorton, 1975) and our observation that there is no stable heteroduplex formation between these two satellites. However, it is still possible that the two sequences might be related to each other as seen in D. virilis satellites II and III. In D. virilis, satellites II and III are related in that each is derived from the common satellite I by one base substitution in the repeating of nucleotide unit, resulting in a difference of two basepairs between II and III which does not allow the formation of stable duplex (Gall & Atherton, 1974 ; Blumenfeld et al., 1973). Only a direct sequence analysis of D. nasutoides satellite I will elucidate this point. Satellite I is the most abundant satellite species in D. nasutoides, amounting to 20 to 30% of the total genome. It consists of simple homogeneous sequence repea.ts such as those observed in all D. virilis satellites (Gall t Atherton, 1974), and in some D. me.!anoga.ster satellites (Peacock et al., 1973; Brutlag $ Peacock, 1975). The sequence complexity estimated from the second-order rate constant is five nucleotide pairs. This value coincides exactly with that of the mehnogwter l-705 satellite whose repeating sequence is A-A-G-A-G (B&lag & Peacock, 1975). However, it is slightly smaller than that of D. vi&s satellites (7 nucleotide pairs) (Gall & Atherton, 1974). An estimation for the degree of repetition per genome is diEcult to make, since we do not know the haploid genome content of D. nasutoides. The haploid genome content of D. &-ilk has a molecular weight of 2-l x 10” (data of Rasch cited in Gall & Atherton, 1974). Employing this value, even if the haploid genome content of D. nasutoides might be higher considering the presence of the large autosome pair, the number of repeats for satellite I is estimated to be about 20 x lo6 per haploid genome. Satellite II constitutes 10 to 15% of the total genome of D. wutoides. The lower melting temperature (At,,, = 17 deg.C) and the high residual hyperchromicity of

22

M.

CORDEIRO-STONE

AND

C.

S.

LEE

reassociated satellite II suggests a considerable sequence variation between repeats. A similar observation was made with the I.686 satellite of D. melanogader (Peacock et al., 1973). Furthermore, the buoyant density and the melting temperature of satellite II in D. nastio&%+s are very similar to those of the 1.686 satellite in D. mehwga-ster. It is interesting to note the similarities between the nasutoides satellite I and the virilis satellite I, and between the nasutoides satellite II and the me&nogaster l-686 satellite. From a dt, value of 17 deg.C, we estimate 10 to 24% base-pair mis-matching between repeating units in nasutoides satellite II. As described in Results, section (e), the precise sequencecomplexity of the satellite II is difficult to estimate, due to the large amount of mis-matching which reduces DNA reassociation rates. The complexity, when calculated using various data of others, ranges from 10 to 206 nucleotide pairs. When we use the value of 103 given in Table 1, there are about 4~ lo5 repeats of this sequencein satellite II of D. nasutoidesper haploid genome. There was no cross-hybridization detectable between the satellites I and II. The most unusual feature of D. ncasutoides satellites is seenin the satellite III. This satellite constitutes about 5 to 10% of the total genome. Its genomic complexity is the highest among all the satellites of nuclear origin examined thus far. The reassociation rate showsthat the sequenceof satellite III is almost as complex asthat of E. coli. Further, it is repeated only about five to ten times in the haploid genome. It could be argued that satellite III is a contaminant due to some micro-organism. We can conclusively eliminate this possibility for the following reasons. (1) Purified nuclei from adults and dechorionated embryos contain this satellite. DNA isolated from salivary glands does not show this satellite, whereas DNA from brains dissected similarly contains this satellite. (2) According to the in situ hybridization experiments, this satellite is localized in the dot chromosomein polytene chromosomes(Fig. 10(a) and (b)) and to the large heterochromatic chromosomein metaphase chromosomes (Arrighi, Cordeiro-Stone, Wheeler, Lee BEHsu, manuscript in preparation). (3) The specific activity of this satellite is comparable to other satellites when [3H]thymidine incorporation is examined with isolated larval brains of D. nasutoides(Lee, 1974). If satellite III were of microbial origin, its specific activity should be considerably higher than other DNA components including main band DNA, due to faster cell division. Satellite IV of D. nasutoides(5 to 10% of the total) is similar to the so-called dAT satellite of D. melanogasterin its buoyant density and melting temperature (Peacock et al., 1973). However, no sequencevariation within repeats has been detected in satellite IV according to melting studies on the reassociated satellite IV. A similar experiment with the D. mel&wga-ster1.672 satellite showed a 5 deg.C decreasein t, (Peacock et al., 1973). Furthermore the sequence complexity of satellite IV in D. nasutoidesis estimated to be 46 nucleotide pairs and thus this sequenceis repeated about 3 x lo5 to 7 x lo5 times per haploid genome. According to a recent study by Brutlag & Peacock (1975), the melanogaster1.672 g/cm3 satellite consists of two different sequences,one having a repeating unit, A-A-T-A-T, and the other a repeating unit, A-A-T-A-T-A-T. We have shown that all four satellites of D. nasutoidesare localized in one particular chromosomepair unlike many other satellites which are distributed in the centromerit regions of several or all chromosomes.This observation raisessomeinteresting questions on the evolution of a chromosome.Somehow, the dot chromosome, which is usually very small, such as chromosome4 of D. melarwgaster,has acquired a large

SATELLITE

DNAs

IN

D.

NASUTOIDES

23

amount of simple sequence DNA components sometime during evolution. This dot chromosome now appears as a large heterochromatic chromosome. During the process of evolution, some DNA sequence has diverged to a certain extent (satellite II). The divergence is even more extreme for satellite III to achieve a very high sequence complexity. On the other hand, certain simple sequences in the same chromosome have not diverged at all (satellites I and IV). It is curious what sort of mechanism(s) operate for the maintenance of such an extreme sequence homogeneity over a long period of evolution. We thank Dr C. Pavan for the use of his facilities, Drs H. S. Forrest and L. Puck&t for critical reading of this manuscript, and Mr D. Schulze for many discussions throughout this work. We particularly thank Dr J. G. Wetmur for many helpful suggestions. The help of Miss Linda Collins during the later part of this work was invaluable. This research has been supported by the National Science Foundation (grants GB-37253 and BMS7505377) and by the Biomedical Sciences support grant of the University of Texas at Austin. One of us (M. C.) thanks the Biochemistry Department of the University of Sao Paul0 for their generosity in providing a leave of absence and to the University of Sao Paulo and Fundecao de Amparo a Pesquisa do E&ado de Sao Paul0 for financial support.

REFERENCES Biro,

P. A., Carr-Brown, A., Southern, E. M. & Walker, P. M. B. (1976). J. Mol. Biol. 94, 71-86. Blumenfeld, M. & Forrest, H. S. (1972). Nature New Biol. 239, 170-172. Blumenfeld, M., Fox, A. S. & Forrest, H. S. (1973). Proc. Nat. Acud. Sci., U.S.A. 70, 2772-2775. Bolton, E. T., Britten, R. J., Cowie, D. B., Kohne, D. E., Roberts, R. B. & Szafranski, P. (1965). Yeurb. Carnegie In&n, 93-125. Bonner, T. I., Brenner, D. J., Neufeld, B. R. & Britten R. J. (1973). J. Mol. BioE. 81, 123-125. Bostock, C. (1971). Advan. Cell BioZ. 2, 153-223. Brutlag, D. L. & Peacock, W. J. (1975). In The Eukaqotk Chromoeomee (Peacock, W. J. & Brock, R. D., eds), pp. 35-45, Australian National University Press, Canberra. Cordeiro, M., Wheeler, L., Lee, C. S., Kastritsis, C. D. & Richardson, R. H. (1975). Chronaosoma, 51, 6&73. Corneo, G., Ginelli, E., Soave, C. & Bernardi, G. (1968). Biochemistry, 12, 4373-4379. Davis, R. W. & Hyman, R. W. (1970). Cold &nl”ing Harbor Symp. Quant. BioZ. 35, 269-281. Dove, W. F. & Davidson, N. (1962). J. Mol. BioZ. 5, 467-478. Flamm, W. G. (1972). Int. Rev. Cytol. 32, l-5. Flamm, W. G., Walker, P. M. B. & McCallum, M. (1969). J. MoZ. BioZ. 40, 423-443. Gall, J. G. & Atherton, D. D. (1974). J. MoZ. BioZ. 85, 633-664. Gall, J. G. & Pardue, M. L. (1971). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21, part D, pp. 470-480, Academic Press, New York. Gall, J. G., Cohen, E. H. & Polan, M. L. ( 1971). Chromosoma (Berlin), 33, 319-344. Hutton, J. R. & Wetmur, J. G. (1973). Biochemistry, 12, 558-563. Jones, K. W. & Corneo, G. (1971). Nature New BioZ. 233, 268-271. Laird, C. D., McConaughy, B. L. t McCarthy, B. J. (1969). Nature (London), 224, 149-154. Lee, C. H. & Wetmur, J. G. (1972). Biopolymers, 11, 1485-1497. Lee, C. S. (1974). Biochem. Gen. 12, 475-483. Lee, C. S. & Davidson, N. (1970). Biochim. Biophys. Acta, 204, 285-295. Marmur, J. (1961). J. Mol. BioZ. 3, 208-218. McCarthy, B. J. & Farquhar, M. N. (1972). In Evolution of Genetic Syateme (Smith, H. H., ed.), Brookhaven Symposium no. 23, pp. l-41, Gordon and Breach, New York. Nandi, U. S., Wang, J. C. & Davidson, N. (1965). Biochemistry, 4, 1687-1696.

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C. S. LEE

Pardue, M. L. & Gall, J. G. (1970). Science, 168, 13661358. Peacock, W. J., Brutlag, D., Goldring, E., Appels, R., Hinton, C. W. & Lindsley, D. L. (1973). Cold Spring Harbor. Symp. Qunnt. B&01. 33, 405-416. Schildkraut, C. & Lifson, S. (1966). BbpoZymers, 3, 195-208. Southern, E. M. (1970). Nature (London), 277, 794-798. Southern, E. M. (1971). Nature New Bill. 232, 82-83. Southern, E. M. (1975). J. Mol. Biol. 94, 51-09. Studier, F. W. (1965). J. Mol. Bill. 11, 373-390. Szybalski, W. (1968). In Metho& in Enzymology (Colowick, S. P. & Kaplan, N. O., eds), vol. 12, part B, pp. 33&360, Academic Press, New York. Throckmorton, L. H. (1975). In Handbook of Genetics (King, R. C., ed.), vol. 3, pp. 421-469, Plenum Press, New York. Vinograd, J. & Hearst, J. E. (1962). In Progress in the Chemistry of Organic Natural Produe& (Zechmeister, L., ed.), vol. 20, pp. 372-422, Springer Verlag, Vienna. Walker, P. M. B. (1971). Prog. Biophys. Mol. Biol. 23, 145-190. Wetmur, J. G. (1976). Annu. Rev. Biophys. Bioeng. 5, in the press. Wetmur, J. G. & Davidson, N. (1968). J. Mol. Biol. 31, 349-370. Wilson, F. D., Wheeler, M. R., Harget, M. & Kambysellis, M. (1969). In Studies in Genetics (Wheeler, M. R., ed.), University of Texas Publ. no. 6918, pp. 207-253, University of Texas Press, Austin. Note added in proof: Exhaustive digest,s of D. nasutoides satellite II with a restriction endonuclease, either EcoRI or SaZI, have recently been shown to produce multimeric patt.erns in polyacrylamide gel electrophoreses (Philippsen, Lee & Davis, unpublished results). The size of a monomeric unit is estimated to be 100 to 120 nucleotide pairs long, which agrees very well with the value of 103 for t,his sat-llite given in Table 1.