J. M ol. Biol. (1966) 17, 305-308

Renaturation of Calf Thymus Satellite DNA

Calf thymus DNA, when analysed in a caesium chloride density-gradient at equili­brium in the analytical ultracentrifuge, shows a main band at a density of 1·699 gJcm3

and satellite bands at densities of 1·713 and 1'719gJcm3 (Fig. l(a». This latter bandis very small and can scarcely be seen in the unfractionated sample. In this com­munication we report the isolation and the purification of the two satellite DNAbands, and their capacity to renature after denaturation, as shown by the densitychange in a caesium chloride density-gradient.

Calf thymus DNA was prepared and was fractionated in a preparative caesiumchloride density-gradient as previously described (Polli, Corneo, Ginelli & Bianchi

ivuc:o

.D

oon

.D-c

(0)

III

I (c )III

IJ

1'699

--Dens it y

FIG. 1. Microdensitometer tracings of calf thymus unfractionated DNA (a), and isolatesatellite DNA's (b) and (c) at equilibrium in a CsCl gradient in the Spinco analytical ultracentrifugemodel E. The peak on the left in each tracing corresponds to the density marker, StreptomycualbU8 DNA (density = 1·730 g/cm3 ) .

305

306 E. POLLI, E. GINELLI, P. BIANCHI AND G. CORNEO

1965), except that the fractions corresponding to the satellite bands collected fromthe first gradient were pooled and further purified twice in caesium chloride density­gradients.

The isolated satellite DNA's were denatured either by heating at 1000e for 10minutes in 1xsse (0·15 M-sodium chloride, 0·015 M-sodium citrate) or 0·1 sse, or bytreatment with alkali according to Vinograd, Morris, Davidson & Dove (1963).The renaturation of DNA was carried out by exposing denatured DNA samples at aconcentration of about 10 p.gfml. to a temperature of 700e in 2 X sse for five hoursand then slowly cooling to room temperature.

The satellite DNA's were analysed in a caesium chloride density-gradient in theanalytical ultracentrifuge Spinco model E, according to the technique described byMeselson, Stahl & Vinograd (1957), using Streptomyces albus DNA or Bacillus subtilisvirulent phage 2C DNA as density markers. Mter centrifuging at 44,770 rev.fmin.at 25°e for 20 hours, ultraviolet photographs were taken on Kodak commercialfilms. The ultraviolet photographs were scanned by a microdensitometer designedand provided by K. V. Shooter (Chester Beatty Research Institute, London). Thedensities of the DNA samples were determined using the method of Schildkraut,Marmur & Doty (1962) and are related to the density of Escherichia coli DNA,taken as 1·710 g/cm3 •

The thermal denaturation curve of the isolated satellite DNA was determined in1 xSSC according to Marmur & Doty (1962).

Figure 1 shows the results obtained by banding in a caesium chloride density­gradient in the analytical ultracentrifuge the satellite bands isolated from calf thymusDNA.

Upon heat denaturation, both the satellite bands increase in density by approxi­mately 0·015 g/cm3 , which is the usual increase for a double-stranded DNA. When thedenatured satellite DNA's are exposed to the conditions favouring renaturationcited above, their density returns to the native value.

Figure 2 shows the densitometer tracings of calf thymus satellite DNA (density1·713 g/cm3 ) banded in CsCl in the native, denatured and renatured forms. Theshoulder present in the renatured sample is due to some DNA which did not renatureand remained at the density of the denatured form.

Some experiments were performed using enriched fractions of satellite DNA stillcontaining some contaminant main-band DNA. Also in this case the satellite DNArenatured after denaturation, whereas the main-band DNA did not.

Some fractions yielding sharp bands in the analytical ultracentrifuge obtainedfrom the main component of calf thymus DNA by the fractionation proceduredescribed were also examined with regard to their behaviour upon renaturation.After heat denaturation, these fractions were not able to renature, as shown by theirdensity, which remained equal to the density of the denatured DNA.

In a few experiments the satellite DNA was denatured by alkali instead of byheating. After neutralizing and exposing the satellite DNA to the renaturing oondi­tions described above, its density returned to the native value.

Enough satellite DNA having a density of 1-713 gfcm3 has been isolated to deter­mine an absorbance-temperature profile, which showed a sharp transition at atemperature of 93°C (Fig. 3). This melting temperature is in agreement, withinthe limits of error of the method, with the buoyant density according to the relationsreported by Schildkraut et al. (1962) and Marmur & Doty (1962).

(0)

(b)

(el

1-742 1128

II II II II III, I

"I I

; 1 1-7131115

-e-r-r-Density

FIG. 2. Microdensitometer tracings of native (a), heat-denatured (b) and renatured (c) calfthymus satellite DNA. The peak on the left corresponds to the density marker B. aubtilis virulentphage 20 DNA (density = 1·742 g/cmS) .

1-40

...,E- 1·30

0-oN

'"Uc;0 1-20..D

6V)

..D0

'">-;:;0 1-10~a:::

1-00

60 70 80 90 100 110

Temperature (oCl

FIG. 3. Absorbance-temperature profile of calf thymus satellite DNA. The DNA suspendedin 0·15 M-NaCl-0'015 M-sodium citrate at a concentration of 20 /Lg/ml. was heated in a BeckmanDU spectrophotometer equipped with thermal spacers and glass-stoppered quartz cuvettes.

308 E. POLLI, E. GINELLI, P. BIANCHI AND G. CORNED

The results reported here show that the satellite DNA's present in the bulk ofcalf thymus DNA display, under proper conditions, a capacity to renature which isunusual for animal DNA. This has recently been shown to occur also with mitochon­drial DNA extracted from purified mitochondria of some mammalian species (Corneo,Moore, Sanadi, Grossman & Marmur, 1966).

It was also previously reported that the satellite DNA of Oancer borealis testesis able to renature after heating and quick cooling. However, in that case, renaturationoccurred spontaneously and was probably related to a very simple structure similarto that of a natural dAT copolymer (Sueoka & Cheng, 1962).

Calf thymus satellite DNA's, with regard to their property of renaturing underproper experimental conditions, behave like bacterial and viral DNA's. At present,it is not known if this behaviour is related only to the homogeneity of base composi­tion of such satellite DNA's compared to the main DNA. The function and biologicalmeaning of the satellite DNA's still remain unknown.

B. subtilis virulent phage 2C DNA was a gift from Dr J. Marmur, This investigationwas supported by U.S. Public Health Service research grant CA 05544-05 from theNational Institutes of Health, and by grant 04/57/4/6863 from the Italian ConsiglioNazionale delle Ricerche.

Laboratorio di Biologia MolecolareUniversita di MilanoVia F. Sforza 35Milano, Italy

Received 24 February 1966

ELIO POLLI

ENRICO GINELLI

PAOLO BIANCHI

GIANMARCO CORNEO

REFERENCES

Corneo, G., Moore, C., Ssnadi, D. R., Grossman, L. 1. & Marmur, J. (1966). Science, 151,687.

Mannur, J. & Doty, P. (1962). J. Mol. Biol. 5, 109.Meselson, M., Stahl, F. W. & Vinograd, J. (1957). Proc, Nat. Acad. Sci., Wash. 43, 680.Polli, E., Corneo, G., Ginelli, E. & Bianchi, P. (1965). Biochim. biophy8. Acta, 103, 672.Schildkraut, C. L., Marmur, J. & Doty, P. (1962). J. Mol. Biol. 4, 430.Sueoka, N. & Cheng, T. (1962). J. Mol. Biol. 4, 161.Vinograd, J., Morris, J., Davidson, N. & Dove, W. F., Jr. (1963). Proc. Nat. Acad.Sci.,

Wash. 49, 12.