Europ. d. Cancer Vol. 11, pp. 247-250. Pergamon Press 1975. Printed in Great Britain

Failure to Obtain Transfection wit, h XC Mitochondrial DNA*

JAN SVOBODA,'t IVO HLOZ2~NEK, J- JAN KORB:~ and OTAKAR MACH§ "~ Institute of Experimental Biology and Genetics, Czechoslovak Academy of Sciences; ~Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences,

§Institute of Haematology and Blood Transfusion, Prague, Czechoslovakia

Abstract--The transfecting activity of DNA obtained from mitochondria of XC virogenic rat cells transformed with the Prague strain of RSV was tested. Both mito- chondrial D NA isolated with EthBr, containing circular molecules, and total mitochon- drial D NA were ineffcient in transfecting sensitive chicken fibroblasts in spite of the fact that doses of mitochondrial D NA up to two orders higher than the minimum effcient dose of total XC D NA were used. These findings support the view that a D NA transcript of the RSV genome is not integrated in mitochondrial DNA.

I N T R O D U C T I O N

THE FINDINGS of Hill and Hillov~i [1, 2], showing that DNA isolated from XC virogenic rat cells transformed with the Prague strain of Rous sarcoma virus (PR RSV) [3, 4] effectively transfects chicken fibroblasts have opened the possibility of testing the subcellular localization of biologically active proviral DNA of RSV. This paper describes the results of experiments in which defined circular and total mitochon- drial XC DNA was used for transfection of chicken fibroblasts.

M A T E R I A L A N D M E T H O D S

Transfection assay The transfecting act ivi tyofXC DNA isolated

from either whole cells or nuclei or mitochon- dr ia--was tested as published previously [5].

Preparation of mitochondria by zonal centrifugation and isolation of mitochondrial DNA in the presence of ethidium bromide (EthBr) and isolation of control nuclear D NA

The cells were suspended in 3 volumes of a medium containing 0.1 M sucrose, 2 0 m M Tris-HC1, 5 m M EDTA, pH 7.4. This suspen-

Accepted 19 November 1974.

*This investigation was aided by a grant from the Jane Coffin Childs Memorial Fund for Medical Research (USA).

sion was homogenized in a Turmix using microscopic control. After disruption of 85% of the cells the suspension was diluted 5 times and sucrose was immediately added to a concentration of 0.25 M. After centrifugation for 10 min at 1000 g, the supernatant was separated and recentrifuged twice under ident- ical conditions. The mitochondrial fraction was obtained by centrifugation of the final supernatant for 30min at 20,000 g. The sediment was resuspended in the same buffer and purified by zonal centrifugation. The suspension was placed on the top of a sucrose gradient (15-50 % sucrose in 20 mM Tris-HC 1, 5 mM EDTA, pH 7.4) and centrifuged for 60 min at 30,000 rev/min in a BXIV rotor of the MSE 65 centrifuge. The fraction of purified mitochondria was diluted and pelleted. All the operations were done at 2°C.

Mitochondrial DNA was isolated by the method described previously [6]. Centrifuga- tion in the CsC1-EthBr gradient was done at 35,000 rev/min for 40 hr at 20°C in a Spinco SW 41 rotor. The bands containing circular DNA were separated and EthBr was removed by dialysis against buffer (0.15 M NaC1, 0.015 i sodium citrate, 0.001 M EDTA, pH 7"2) at 4°C for 2-3 days [7] or with a Dowex 50 resin column [8].

Control nuclear DNA was prepared from the sediment of the cell homogenate after centri- fugation at 1,000 g for 10 min (above). The

247

248 Jan Svoboda, Ivo Hlo~dnek, Jan Korb and Otakar Math

pellet was resuspended in a buffer (0.15 M NaC 1, 0.1 M EDTA, 0"02 M Tris-HC1 of pH 8.0), mixed thoroughly and sodium dodecyl sulphate was added to 1%. The mixture was incubated for 20 min at 20°C. The DNA was isolated by the s ame procedure as described for isolation of mitochondrial DNA.

EthBr treatment of total XG DNA Total XC DNA prepared according to

Marmur [9] was treated with 200 pg of EthBr per one ml of DNA solution and the solution was dialysed as described for mitochondrial DNA.

Groups of dishes, on which total or nuclear XC DNA was applied, were used as controls. In each group, some of the dishes showed positive transfection. Out of a total of eighteen control dishes, six were successfully transfected. The frequency of transfection in these experi- ments was lower than that in the experiments tabulated in Table 2. Larger doses of DNA did not produce higher transfecting activity. The efficiency of transfection was found to differ

Table 1. Attempts to transfect chicken fibroblasts with mitochondrial XC DNA prepared by EthBr gradient

centrifugation

Electron microscopy Electron microscopic control of the isolated

DNA molecules was done according to Korb [61.

Preparation of mitochondria by differential centri- fugation and isolation of mitochondrial DNA and control nuclear D NA

Mitochondria were prepared according to the method of Wilson and Cascarano [10]. Briefly, a 10 % suspension of XC tumour tissue was prepared in 0"25M sucrose--0"l mM E D T A - - 1 0 m M Tris--HCl buffer, pH 7.4, using a Potter-Elvehjem glass homogenizer and a motor driven Teflon pestle. The homo- genate was centrifuged for 10 min at 400 g and the supernatant was recentrffuged for 5 min at 400 g. The second supernatant was then centri- fuged at 7,800 g for 10 min. The mitochondrial pellet was washed twice by resuspending it in buffered sucrose solution and recentrifuging it at 7,800 g for 10 min. The resulting mitochon- drial pellet was used for the isolation of XC DNA as already described [5].

As in our previous experiments [5], nuclei were obtained by the method of Schneider [11]. The pellets obtained after centrifugation of XC homogenates at 400 g (see above) were pooled and after two washes in sucrose-EDTA- tris solution, were used for isolation of XC nuclear DNA by the same procedure as described for isolation of mitochondrial DNA.

R E S U L T S

The transfecting activity of circular DNA isolated with EthBr from XC mitochondria obtained by zonal centrifugation was tested in three experiments performed with a total of 19 dishes. No signs of transfection were observed with doses of DNA ranging from 0.17 #g to 28/~g (Table 1).

Dose of XC DNA (ug)

Number of dishes positive in transfection/

Total number of experimental dishes

Mitochondrial DNA 17-28 0/3 4-15 0/6

1.5 0/6 0.17 0[4

Total DNA 150 0/1 28 0/1 15 0/2

1-5 212

Total DNA + ethidium bromide

75 112 15 112

1.5 1/2

Nuclear DNA 150 0/2

15 112 1.5 0/2

Table 2. Attempts to transfect chicken fibroblasts with mitochondrial XC DNA prepared in the absence of EthBr

i l l

Number of dishes positive in transfection/

Dose of XC DNA Total number of experimenta (/tg) dishes

Mitochondrial DNA 150-175 0/3

15 016 1.5 0/6

Total DNA 150 4/4

15 4/6 1-5 4/6

Nuclear DNA 150 8/8 15 4/4 1.5 4/4

Failure to Obtain Transfection with X C Mitochondrial D N A 249

between different experiments. No dose depen- dence was observed with doses of XC DNA ranging from 1.5 to 150 t~g [12].

Mitochondrial DNA preparations from CsC1-EthBr gradient contained before dialysis about 85% circular molecules, the amount of which dropped to 40% after dialysis. The remaining DNA represents opened DNA mole- cules. The density equilibrium sedimentation profile of XC mitochondrial DNA in CsC1- Eth.Br gradient is shown in Fig. 1. An electron microscopic picture showing circular molecules of XC mitochondrial DNA is presented in Fig. 2.

1.2 I m l

0.8

0 10 20 30 60

Irra c t lon no.

Fig. 1. Density equilibrium sedimentation profile of mito- chondrial DNA from XC cells in CsCl-ethidium bromide gradient. Fractions 10-18 contained supercoiled DNA, fractions 19-22 contained opened circular D NA and fractions 23-32 contained linear DNA. Brackets indicate fractions which were used as mitochondrial D NA for testing the trans-

letting activity.

It should be noted that treatment of total XC DNA with EthBr under conditions used for the isolation of mitochondrial DNA does not destroy its biological activity. Similarly, nuclear DNA isolated in the presence of EthBr remains active in transfection (Table 1).

Two additional experiments were performed with doses of 1.5 to 175 #g of mitochondrial DNA isolated in the absence of EthBr from XC mitochondria obtained by differential centrifugation (Table 2). Again, all 15 dishes treated with such preparation gave negative results in transfection. Control experiments with total or nuclear XC DNA prepared from the same batch of cells used for obtaining

mitochondrial DNA gave positive results in 28 out of 32 dishes.

DISCUSSION

The lack of transfecting activity of XC mitochondrial DNA observed in our experi- ments is in agreement with the previous finding that XC nuclear DNA exhibits the same degree of transfecting activity as total cellular DNA [5, 13]. Similarly, Montagnier and Vigier [14] found no transfecting activity in the fraction containing low molecular weight DNA, which had been prepared accord- ing to Hirt [15]. This method, however, does not guarantee the isolation of circular DNA molecules. Finally, Donner et al. [16] did not obtain RSV rescue after fusion of enucleated virogenic cells with indicator chicken fibro- blasts. This is in agreement with the data discussed above, which indicate the absence of proviral RSV DNA in the cytoplasm of virogenic cells.

I t can be argued that circular mitochondrial molecules do not penetrate effectively into recipient cells. Although incorporation of such molecules was not measured in our experiments, it has clearly been shown that double- and single-stranded circular DNA of SV40 and polyoma viruses penetrate and produce a lytic infection in their respective permissive cells [17]. I t should, however, be taken into account that mitochondrial DNA is three times bigger than the viral DNAs mentioned above.

Because mitochondrial DNA represents only a small fraction of the total cell DNA about 0.1%--some activity of mitochondrial DNA could have escaped detection in the experiment in which transfection activity of total and nuclear DNA was compared. Therefore, large doses of mitochondrial DNA (up to 170/~g), two orders higher than the least active amount of total XC DNA (l'5/~g), were used in an attempt to transfect chicken fibroblasts. However, no transfection has been obtained which eliminates the possibility of the presence of rare active DNA transcripts of the RSV genome in the mitochondria. This agrees well with molecular hybridization data which indicate the absence of DNA copies of the avian myeloblastosis virus (AMV) genome in the circular DNA molecules from chicken leukaemic myeloblasts [18].

1.

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250 Jan Svoboda, Ivo Hlo2dnek, Jan Korb and Otakar Math

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Fig. 2. Electron microscopic picture of circular molecule of mitochondrial XC DNA. The bar represents 0.5 .urn.