Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA

Gene. 15 (1981) 349-359

349

Elseviez/Not~th-HollandBiomedicall~ess

Integration of foreign genome fragments into cells transformed or cotransformed with fragmented adenoviral DNA (Simian and human adenoviruses; oncogenicity; restriction endonucleases; rat kidney cells)

T.I, TJkchonenko, N,M, Chaplygina, T.I. Kalinina, A.L. Cartel, T.I. Ponomareva, B.S. Naroditsky and R.S, Dreizin The D.L Ivanovsky Institute of Virology, Academy of Medical Sciences of the USSR. Moscow, 123098 (U.S.S.R.)

(Received Match20th, 1981) (Accepted August10th, 1981)

SUMMARY The integration of DNA of highly oncogenic simian adenovirus type 7 (SAT) and non-oncogenic human adenovirus type 6 (Ad6) into the genome of newborn rat kidney cells transformed by fragmented DNA preparations was studied using reassociation kinetics and spot hybridization. Transforming DNA was fragmented with the specific endonucleases Sa/I (SA7) and B&lII (Ad6). In contrast to the cell transformation by intact viral DNA, transformation by fragmented DNA resulted in integration into the cellular genome of not only the lefthand fragment with the oncogene but also of other regions of the viral genome. Ad0itionally, integrated fragments were stable and preserved during nunwrous passages of cell lines, although they were not expressed, at least in the case of the Ad6-transformed ¢~,11line. The integration of the fragments of SA7 DNA was accompanied by loss of 25-50% of the mass of ~ach f r o n t . Adding the linear form of the pBR322 plasmid to the preparation of transforming Ad6 D N A ~ o contributed to its cointegration into the genome of the transformed cell. This technique of cell cotransformation with any foreign DNAs together with the viral oncogens may be used as an equiv. alent of.an integration vector for e ~ o t i c cells.

INTRODUCTION The analysis of DNA from cells transformed by human adenoviruses of different types shows the transformation to be most often accompanied by the integration of only the lefthand part of ~ viral Abbreviations: Ad, adenovinm; SA, simian adenovin,s; SSC, 0.15 M NaCl+ 0.015 M sodium dtmte; TK, thymidine kirtle

p

genome containing the oncogene. A similar situation develops during the transformation of ce~s with whole virus or preparations of intact Ad2 DNA (Galli. more et aL, 1974), Ad5 (Flint et al., 1976; Frolova et aL,. 1979), Ad6 (Kalinina et al., 1980), Ad7 (Sekikawa et al., 1978)and Adl2 (Lee and Mak, 1977). ~ adenovimses, in particular SA7, behav~ similarly (Chaplygina et al., 1980). . . . . . . . . . . other ~ d , we had shown earlier that transforming or tumorigenic activities of Ad6 and

0378-1119/81/0000-0000/$02.75 0 1981 Elsevier/North-HoliandBiomedicalPress

350 SA7 DNA preparations were sharply increased after their fragmentation with restriction endonucleases ,Ponomareva et at.. 1979a). In this connection, we have studied the effect of p r e ' ~ fragmentation 3f DNA on its subsequent integration into the genome of transformed cells. Simultaneously, we also ~udied the fate of heterologous DNA of the pBR322 plasmid added to the preparation of transforming DNA. A preli~nary report was published earlier (Chaplygina et al. 1981). ,, •

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MATERIALS AND METHODS

Adenoviruses SA7 and Ad6 were grown on green mc~nkey kidney cells and HeLa cells, respectively (Gavrilov et al., 1966; Dreiz~n and Zolotarskaya, 1976). 'viruses were purified with Freon-113 and CsCl-censity-gradient centrifugation (Green and Pina, 1963). Adenoviral DNAs were isolated by detergentphenol deproteinization using pro;.ase (Belle and Ginsberg, 1960). Isolation of specific endonuclease and enzymatic hydrolysis were performed according to the technique described earlier (Naroditsky et al.. 1976; 1977; 1980). DNA fragments obtained after the treatment with specific endonucleases were separated by 0.8% agarose gel electrophoresis. Fragments were isolated from the gel using the method described earlier (Mulder et al., 1974). 32P-labelled adenoviral and plasmid DNAs and their separate fragments were obtained in vitro by nick-translation (Maniatis et al., 1975). Primary cultures of kidney cells of 5-7-day-old rats of the WAG line were used for the transformation (McAllister et al., 1969). The cells were growr~ ha MEM medium supplemented with 10% calf serum. They were transformed according to Graham and Van der Eb (1973). Cell lines were derived from single transformed colonies by the standard method. For hybridization DNA was isolated from the :ells of the KC-I line after 20 passages, from those of the KC-2 line after 35 passages and from those of line 366 after 16 passages. To study the oncogenicity of the transformed cells, 5 - 6 × lOs cells wele administered to l-week-old hamsters subcutaneously in the dorsal part of the cervical area. The animals were observed for 6 months. The tumors grown were trypsinized to derive separate cell lines, which were then cloned

using the method of limit dilution. DNA was isolated from the transformed cells according to Kraiselburd et al. (1975). Viral sequences in the DNA of transformed cells were analysed by spot hybridization on nitroceilhlose falters (Fujinaga et al., 1979) and by the measurement of reassociation kinetics (Sharp et at., 1974). Spot hybridization was conducted in a solution containing 0.6 M NaCI, 0.2 M Tris- HCI pH 7.9, 0.02 M EDTA, 0.5% sodium dodecyl sulphate and 50% formamide at 37°C for 48 h. After hybridization, the filters were washed, dried and autoradiographed. Densitometry of the spots obtained was performed in a Chromoscan200 densitometer (Joyce and Loebl). The reassociation kinetics of adenovirus SA7 [32p]DNA in the presence of DNA from transformed cells was studied in a solution containing 1 M NaCI, 0.006 M Tris-HCI pH 7.5 and 0.001 M EDTA at 68°(?. The fraction of double-stranded hybrids was estimated by the stability to S~ nuclease hydrolysis (Kraiselburd et al., 1975). The number of copies of viral DNA fragments and their fraction present in the cellular genome was calculated in accordance with Sharp et al. (1974). RNA-DNA hybridization was carried out as described by Gillespie and Spiegelman (1965). The reaction was performed at 42°(? within 48 b in a buffer containing 0.9 M NaCI, 0.01 M Tris. HCI pH 7.9, 0.1% sodium dodecyl sulphate and 50% formamide. After hybridization, the filters were intensively washed with 2 X SSC and treated with RNase (50/ag/ ml) at room temperature within an hour. Then, the filters were washed with 50 ml of 2 X SSC from both sides. To obtain [3H]RNA the cells were labelled with [3H]uridine (50 mCi/ml) for 4 h. The preparation of cytoplasmic RNA was isolated using a somewhat modified technique of Flint et al. (1975). Plasmid pBR322 was prepared and purified according to Clewell and Helinski (19¢~)). Before adding the plasmid to transforming Ad6 DNA it was converted into the lbtear form with Hindlll restrictase.

RESULTS

(a) SA7 transformation SalI restriction endonuclease reaves adenovirus

SA7 DNA into six fragments (Naroditsky et al.,

351 1978) (Fig. 1). We haste shown earlier that the administration of a mixture of these fragments in primary cultures of rat kidney cells is followed by formation of transformed foci (Ponomareva et al., 1979b). In those experiments DNA was fragmented under conditions of exhaustive hydrolysis, and completeness of cleavage was checked electrophoretically. One of the cell lines transformed by fragmented SA7 DNA was named KC-I (Ponomareva et al., 1979b). The administration of 5 - 6 X 10 s KC-1 cells to hamsters resulted in tumors in 70-90% of animals after 3 0 - 5 0 days. The KC-2 line originated from the tumor caused by KC-I cells and later cloned in vitro. The viral sequences in KC-1 and KC-2 DNA were qualitatively analysed by both the method of spot hybridization on falters and the analysis of reassociation kinetics. In the first method 20 pg of denatured DNA of transformed cells, DNA of calf thymus and DNA of calf thymus + lO -4/.tg of SA7 DNA (l equivalent of viral genome per diploid cell genome) were applied in the form of small spots on a nitrocellulose falter and hybridized with 32p-labelled intact SA7 DNA or its Sall fragments ( 4 - 5 X l0 s cpm per fdter at a specific activity of 1 - 2 X l0 s cpm//ag of DNA). Densitometric tracings of the autoradiographed spots (Fig. 2) show that th,~ DNA of the KC-I and KC-2 lines contains sequences homologous to all the Sail fragments of SA7 DNA (E and F fragments were analysed in a mixture). The estimation of the area under the peaks provided an approximate evaluation of the number of equivalents of viral genome fragments present in the DNA of transformed cells (Table 1).

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Fig. 2. Spot hybridization on a nitrocellulose tilter bet,veen SA7 [32p]DNA and SA7 ~ransformed cell DNA. 20 ~lg of denatured DNA of transfoxmed cells. DNA of calf thymus and DNA of calf thymus + l0 "4 #g of SA7 DNA (1 equivalent of viral genome pet diploid cell genome) were spotted and hybridized with 32P-labelled intact SA7 DNA or its Sail fragments (4-5 × l0 s cpm/filter at a specific activity of 1-2 × l0 s cpm/~g of DNA). After hybridization filters were washed, dried and autoradiographed. Densitometric tracings of autoradiogmms obtained are shown. (l) calf thymus DNA (negative control), (2) calf thymus DNA containing SA7 DNA (positive control); (3) KC-I DNA; (4) KC-3 DNA. 32p-labelled material used in the hybridization is specified at the left margin.

Hybridization of [32p]DNA of SA7 and its Sail fragments with DNA of KC-1 and KC-2 cells on nitrocellulose filter

of SA7 D N A

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TABLE I

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Fig. 1. Physical map of the genomes of SA7 end Ad6.

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Number of equivalents of SA7 DNA fragments 13erdiploid cell genome KC-IDNA

KC~ DNA

A

4.5

4.3

B

2.9

3.2

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7.5 8.0

8.0 7.2

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intact DNA

4.8

4.5

~52 It is seen in 'Fable I that the transplantation o f KC-I cells into hamster~ and subsequent cloning of KC-2 cells did not essentially affect the content of individual fragments o~ v-~ DNA in the cellular genome. The DNAs o f KC-1 and KC-2 cells contain about 4 - 5 equivalents o f complete viral genome per diploid cell genome. To provide a more precise quantitative estimation o f the viral sequences in the DNA o f transformed ceils the reassociation kinetics of SA7 DNA and its SMI fragments were analysed. The DNA of KC-I cells caused an increase in the rate of reassociation of both ~utact SA7 DNA and all its restriction fragments (Fig. 3). Calculation according to the formula of Sharp et al. (1974) showed the sequences homolofous to Lldividual fragments o f SA7 DNA to be pres-nt in. KC-I DNA ( 7 - 8 copies per diploid cell genome) (Table II). The fraction of each o f the fragments integrated into the KC-I cell DNA was 5 0 -

A Soll

B Sall

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1.5

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TABLE I1 Viral sequences in KC-I cell DNA identified on the basis of reassociation kinetics Sa/l fragments of SA7 DNA

Number of copies of fragments per diploid cell genome

Fraction of the fragment integrated into the cell genome calculated according to Sharp et al. (1974) (% of fragment's length)

A

7.5 7.5 8.0 8.5 8.0 7.0

80 50 70 75 70 75

B

C D E+F intact DNA

80%. The amounts of equivalents of complete viral genome calculated for individual SA7 DNA fragments using the data o f Table II and taking into account the percentage of the viral genome presented by each fragment are given in Table II! . . . . The general content of viral DNA in K C I DNA, calculated on the basis of the sum of individual DNA fragments and the data on the reassociation kinetics of intact viral DNA, was 5.40 and 5.25 equivalents of viral genome per cellular genome, respectively. Repre. sented for comparison in Table Ill are also the amounts oi" equivalents of viral genome in KC-1 DNA, calculated from the data o f spot hybri6ization on

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TABLE 111 SA7 DNA sequences in KC-1 DNA

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% of SAT genome

Number of equivalents of complete viral genome per diploid cell Eenome

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Fig. 3. ReassociaUon kinetics of SA7 [32p]DNA and its Sail fragments. 10-3-10 -2 ~g/ml of [32p]DNA SA7 or its Sall fragments (sp. act. 1-2 × 10 s cpm/~g), reassociated in the presence of 1800 ag/ml of catf thymus DNA (o a); 1800 pg/ml of calf thymus DNA and 10-1 ag/ml of nonlabelled SA7 DNA (X ×); 1800/~g/ml of KC-1 cell DNA (o--------o). 30-ml samples were removed at intervals and the fraction of 32P4abelled single~trand DNA (fss) was determined by iI nuclease digestion.

A B C D E+F sum intact DNA

36.5 25 19.5 11 8 100 100

Reassociation kinetics

Hybridization on filters

2.20 0.94 1.09 0.70 0.48 5.40 5.25

1.62 0.73 1.42 0.88 0.46 5.30 4.80

353

fdters (cf. Table i). The divergence in the results obtained by these two methods does not exceed 30%. Evidently, spot hybridization on falters n~ay be recommended not only as a qualitative but also as a semiquantitative method for estimation of the content of viral sequences in the DNA of transformed cells. Thus, in contrast to what is observed during the transformation of rat kidney cells by intact SA7 I~?T?. (Chaplygina et al., 1980), the transformation of those cells by pre-fragmented DNA causes the integra*ion of 11 DNA fragments. The integrated SA7 DNA fragmen s are stably preservea in the genome of transformed :ells at least throutOout 35 passages in vitro ~nd alsc during the passage through the tumor in a hamster.

3

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I B Barn HI I I I

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(b) Ad6 transformation Restriction maps of the Ad6 genome obtained with restriction endonucleases BamHl, BgllI and HindHI (Naroditsky et al., 1980) are represented in Fig. 1. The transformation of rat kidney cells by the mixture of those fragments gave positive results in all three cases (Ponomareva et al., 1979b, c). One of the cell lines obtained after the transformation by the mixture of Bglll fragments (line 366) was studied to reveal the specific features of the integration of adenoviral DNA. To facilitate the hybridization analysis we have used larger Ad6 DNA fragments prepared with restriction endonuclease BamHI. Fig. 4 shows densitometric tracings of autoradiograms after hybridization on filters of the cell line 366 DNA with 32P.labelled intact Ad6 DNA and individual BamHI fragments of Ad6 DNA. It is seen that the level of hybrioization with intact Ad6 DNA and all four fragments significantly exceeds that of nonspecific adsorption of the label on thymus DNA used as control. By the time of the analysis, the cells of this line had undergone 16 passages. The estimation of the area under the peaks proved the presence of sequences homologous to the BamHI A, B, C, and D fragments of Ad6 DNA (Table IV) in the DNA of cell line 366.

(c) Expression of integrated viral sequences Thus, transformation with we-fragmented Ad6 and SA7 DNAs results in independent integration of

I

DNA Ad6

I

l

t T

Fig. 4. Spot hyb:idization or a nitrocellulose filte~ between Ad6 [32p]DNA and Ad6-transformed cell DNA. 20 ~g of denatured DNA of transformed cells, DNA of calf thymus and DNA of calf thymus + 10-4 ~g of Ad6 DNA (1 equiv-

alent of vital genome per diploid cell genome) wele spotted and hybridized with 32P-labelled intact Ad6 DNA or its BamHI fragments (4-.5 × l0 s cpm/filter at a specificactivity of 1-2 × l0 s cpm/~g of DNA). After hybridizationfilters were washed, dried and autoradiographed. Densitometric tzacings of autoradiogtamsobtained are shown. (1) calf thymus DNA (negative control); (2) calf thymus DNA containing Ad6 DNA (positive controD; (3) 366 cell line DNA. 32P-labelled material used in the hybridization is specifiedat the left m~gin.

separate fragments of the viral genome into the cell DNA. Therefore, it seemed of interest to study the transcription of th; fragments of viral DNA integrated into the cellular genome. To this end, we analysed the [3H]RNA preparations isolated from the cytoplasm of the KC-I and 366 cell lines using RNADNA hybridization on f'dters. As can be seen from Table V, the KC-1 cytoplasm contains somewhat more virus-specific RNA than that of 366 cells (0.078% vs. 0.036%). It should be noted that this result is consistent with the higher content of SA7 DNA sequences in the DNA of KC-1 cells (5 equivalents of viral genome per cell genome), as compared

354 T&BLE ! /

TABLE VI

Hybridiz~ t ion of intact and BamHl-fragmented Ad6 DNA with DN~ of cell line 366 a

Hybridization of cytoplasmic [3H]RNA from cells of line 366 with fragments of Ad6 genome

BmnHi fra~nents

% of genome of Ad6

Number of equivalents of Ad6 DNA and its fragments per diploid cell genome

A B C D intact DNA

37.5 31.3 17.8 13.2 100

0.25 0.45 0.50 0.15 OA0

Fragments of Ad6 genome

RNA amount in sample (dpm/min)

RNA share in hybrids (%) a

Complete genome Ad6

200 000 150 000 150 000 150 000 150 000 250 000 250 000 250 000

0.026 0.000 0.037 0.007 0.004 0.000 0.001 0.031

BamHI-A

BamHI-B

BaniHl4~ BamHI-D Hindlll-A ~

a Hybridization on nitrocellulose filters.

HindHI.C Hindlll-F

to the content of Ad6 DNA in the DNA of the 366 cell line (0.4 equivalents of viral genome per cell genome). It was of interest to find out whether or not Ad6 DNA fragments independently integrated in the cellular genome were expressed. To this end, we performed hybridization of total 3H-labelled cytoplasmic RNA from the 366 cell line with the individual /~amHl fragments of Ad6 DNA. It follows from the data given in Table VI that in the cells of the 366 line containing all four BamHl fragments of Ad6 DNA, only the leftmost oncogen-containing fragment is transcribed. The rest of the adenoviral fragments in the genome of transformed cell are silent. Since the tern,in',d BamHI-B fragment is rather large (7 Md), it seemed of L,tefest to carry out hybridization of [3H]RNA with smaller fragments of the left-hand part of the genome. The latter were represented by fragments prepared by HindlIl cleavage of the BamHI-B fragment (see !:ig. i) The last three lines in Table VI show that radioactive RNA from the transformed cells hybridizes only with the leftmost

TABLE V Oetermination of virus-specific RNA in transformed cells a Source ;~f [3HIRNA

RNA amount L~ sample (dpm/min)

RNA share in hybrids (%)

lhle 366 line KC-I

288 000 380 000

0.036 ± 0.004 0.078 4. 0.008

a Erapty filters were used as control.

a Radioactivity in RNA DNA hybrid with respect to initial radioactivity ef RNA in sample (%) after subtracting nonspecific adsorption.

HindHI-F fragment with 1.75 Md molecular weight (Naroditsky et al., 1980). The study of the expression of the adenovi~al DNA regions in KC-1 and KC-2 cells is now underway. Our results are in agreement with the data on the expression of viral DNA in the cell lines transformed I'y intact DNA of human adenoviruses (Flint et al., 1976).

(d) Ad6.pBR322 cointegration The integration of foreign DNA into eukaryotic cells may be rather nonspecific (Zhdanov and Tikchonenko, 1974; Weinberg, 1980). In one of the experimental series, an equal amount of linear pBR322 DNA was added prior to transformation to the Ad6 preparation cleaved with restriction endonuclease BamHl. The Mr Ad6 DNA being 22 X 106 (Tikchonenko et al., 1979) and that of the pBR3?2 plasmid being 2.6 X 106 (Sutcliffe, 1979), there were 8 mol of plasmid DNA per mol of adenoviral DNA. This artificial mixture efficiently transformed the newborn rat kidney cells (line 68) and the properties of the transformed cells did not differ from the cells transformed only with the mixture of Ad6 fragments (T.I. Ponomareva, to be published). Altogether we have developed in independent experiments two stable cell lines t~ansformed with the mixture of Ad6 fragments and pBR322 plaswid. The results of spot hybridization of labelled pBR322 and Ad6 DNA with DNA extracted from

355 2

3

i oO DNA p BR3~2

i 6

4

t

b) DNA Ad 0

Fig. 5. Spot hybridization on a nitrocellulose filter between: (a) pBR322 [32p]DNA and cell line 68 DNA, Co) Ad6 [32p] DNA and cell fine 68 DNA. 20 ~ug of denatured DNA of transformed cells, DNA o~" calf thymus and DNA of calf thymus containing pBR322 DNA or Ad6 DNA (1 equivalent of genome per diploid cell genome) were spotted and hybridized with 32p-labelled pBR322 DNA or Ad6 DNA (4-5 X l0 s cpm/f'flter at a specific activity of 1-2 X l08 cpm/i~g of DNA). After hybridization filters were v~ashed,dried and autotadiogtaphed. Densitometric tracing of autotadiogtams obtaL-.ed axe shown. (1, 4) calf thymus DNA (negative control); (2) calf thymus DNA containing pBR322 DNA (positive centreD; (3, 6) DNA of 68 cell fine; (5) calf thymus DNA containing Ad6 DNA (positive centred 32p-labelled material used in the hybridization is shown at the left.

transformed cells of line 68 are represented in Fig. 5. Since by the time of the analysis cell fine 68 had undergone 10 passages, the integration of foreign DNA seems quite stable. Comparison of the results

2'°o1,i

t houre Fig. 6. Reassociation kinetics of pBR322 [32p]DNA. 5 X 10-4 #g/ml of [32P]DNA pBR322 (spec. act. 1-2 X 10~ cpm//tg), reassociated in the presence of 1800 #g/ml of calf thymus DNA (a.------a); 1800 /~g/ml of calf thymus DNA and 5 × 10-3 /ag/ml of non-labelled pBR DNA (X. X); 1800/zg/ml of 68 cell line DNA (o------o).

of hybridization of [32p]pBR322 DNA with the DNA from transformed cells on the one hand, and with a known quantity of pBR322 DNA on the other, reveals the presence of about seven copies of plasmid DNA in the transformed cells. The measuremerits of the reassociation kinetics of [32p]pBR322 DNA with DNA from the cotransformed cell line (Fig. 6) confirm this conclusion and allow specifying the number of plasrnid copies present in the cell genome and the fraction of plasmid DNA lost on integration into cell chromosomes. A special report (N.M. Chaplygina, manuscript in preparation) will be devoted to the analysis of integration sites and expression of plasmid DNA in the cotransformed rat cell.

DISCUSSION

(a) Intact DNA transformation Available data on the transformation of mammalian cells with human adenoviruses indicate that during the transformation by virion or intact DNA, the transformed cell genome contains, as a rule, sequences homologous to only a part of the adenoviral genome. Thus, Gallimore et al. (1974)have shown DNA of six cell lines transformed by Ad2 to contain sequences homologous to the lefthand terminal part of adenoviral DNA (14% of genome length). When rat cells were transformed with AdS, the DNA of the four cell fines developed contained 12 to 443% of the adenoviral genome (Flint et al., 1976). Transformation of rat cells with intact DNA of human Ad7 also resulted in the development of cell lines of which the DNA contained sequences homologous to only 2/3 of the Ad7 genome (Sekikawa et al., 1978). Integration of whole viral DNA into the cell genome is an exception from the rule and was observed by Fanning and Doerfler (1976) only in the case of highly oncogenic Ad12. However, the incorporation of DNA of that virus into the cellular genome is not always complete either: DNA from five hamster tumors induced with Ad12 lacked sequences homologous to 45% of the viral genome (Lee and Mak, 1977). Adenoviruses used in the present study behaved similarly. The genome of simian highly oncogenic

3:~6 SA7 is represented in hamster tumor cells by the lefthand fragment with Mr 3.1 X 106 (Chaplygina et al., 1980). Thus, recombination of whole adenoviral DNA (Afr 20-23 X 106) with the cellular genome is most often a~compamed during the cell tranfformation by a loss of a subs*antial part of the viral genome. The variabiliW of the part o ~ the viral genome eliminated during cell transformation most likely indicates that the elimination per se is not obligatory for transformation. Naturally, since only the oncogene itself is necessary for maintainiug the transformed state, the rerrmining part of the adeno~iral genome is essentially ~edundant and may be in some part deleted dur;ng the passages of transformed cells. It is evident that such shortening due to the selection of deletion mutants is secondary with respect to the integration. The concept of deletmns in preferentially internal regions of adeno~rus DNA is supported by the well-knowt! higher probabli~ty of preservation of the righthand terminai part as compared to the internal part of the viral DNA in the transformed cell. {c) Transformation by fragmented DNA Integration, as described above, is only observed when the ceils are t~ansformed or tumors are induced witi~ intact DNA either in whole virions or in the fern, of depro*~mized preparations. The present paper shows ihat the transformation of cells with the tragmented preparations of adenoviral DNAs gives substantially different results. In this case, all or ~ea.qy all DNA fragments present in the initial sample are integrated into the cell genome. 1he integrated fragments are quite stable and are preserved throughout quite a number of generations. h~ particular, the unchanged character of the integration of all six fragments of SA7 DNA into the KC-1 Line was preserved even after the passage through the tum~3r and recloning of the cell line. Altogether, the KC-2 cells had undergone 35 passages by the time of the DNA analysis. Sharp et al. (1974-)-also reported stal'~e integration of adenoviral DNA into cells transformed with highly oncogenic Adl2. According to these authors, all eight subclones studied have the same content o¢ t~_ral sequences in the cell genome. On ~ e or_her hand, Groneberg et al. (1978) showed fl~at passage of hamster cells transformed with Adl2 resulted in the appearance of two fines of revertants that had lost practically all the adenoviral sequences.

!c) Shortening of integrated DNA We believe that our data provide some details of the mechanism of integration, in particular, a deterruination of that part of the DNA which is lost during integration. Naturally, we ~umot fully eliminate shortening of the fragments at the expense of subsequent selection of deletion mutants but the small initial amount of the integrated fragments and their stability in the cell genome during the passage of the cells make this process less probable. The findings presented in this paper indicate that the fragments are incorporated independently of each other and a part of the fragment is lost during the incorporation. In the KC-1 and KC-2 cell fines analysed, containing 75% of all the viral sequences, each fragment lost 20 to 50% of its mass. In line 366, containing 40% of all the viral sequences, each fragment lost a substantially higher percentage of its mass (50 to 80%). Greater losses of molecular mass during the integration of Ad6 DNA as compared to SA7 DNA should be regarded with certain reservations. Since the transfcrming Ad6 DNA was cut with Bglll into 11 fragments, whereas SA7 DNA was hydrolysed with Sail giving only 6 fragments. Starting from the assumption that the shortening of the integrated DNA directly accompaning the integration is more or less proportional to the degree of fragmentation (the number of fragments), the loss in molecular weight of the integrated DNA should be almost twice as large witi, Ad6 as it is wRh SA7, as observed in our experiments. To provide a more precise conclusion, an experiment in which one type of transforming virus is cleaved by a variety of restriction endonucleases is desirable. The substantial difference in the degree of shortening observed between Ad6 DNA fragments is easily exp!:cable, since the transformation was performed with Bglll fragments, and me hybridization for reasons of cor.Jenience ¢:as ~rried out with the four 8amHl fragments. Therefore, when hybridization is performed with the BamHI.A fragment, seven fragments BgllI that participated in the transformation actually correspond to this DNA region. It is evident that the loss in Mr during the integration of seven fragments would substantially exceed that during the integration of one fragment. It is quite likely that a part of the small 8glH fragments are merely lost and

357

are not incorporated into the genome of transformed cell. We are presently verifying this hypothesks. A similar situation occurs for the BamHI-B fragment to which two BgllI fragments correspond. The only exception is 8amHI-D fragment which corresponds to the A part of the BglII fragment. However, it is in this fragment that the greatest loss in Mr occurs, indicating the complexity of this phenomenon and probable influence of the primary DNA structure. Apparently, for more accurate conclusions, hybridization of DNA from transformed cels with the same fragments as were used for the transformation is required. Our findings suggest tl~at the direct act of integration of fragmented adenoviral DNA into the genome of transformed cells is accompanied, as a rule, by a decrease in the M r of the fragments by 20-50% of the i n i t ~ value. A similar loss in molecular mass is obtained during the transformation of cell cultures by individual fragments of viral DNA containing the oncogene: 20-25% for the Hsul-G fragment ofadenovirus type 5 DNA with Mr 1.7 X 106 (Van der Eb et al., 1977) and 30-40% for the EcoRI.C fragment of Adl2 DNA with Mr 3.5 X 106 (Mak et al., 1979). One should be careful when evaluating cur data on the g~'eat difference in the amount of adenoviral DNA integrated during cell transformation with highly oncogenic SA7 and non-oncogenic Ad6. Although the 366 cell line has less sequences by an order of magnitude as compared to the KC-1 and KC2 lines, this difference may be due not to the different oncogenicity of the transforming virus but to the peculiaritie,: of a particular cell line. According to preliminary data of one of us (T.I. Kalinina, unpublished results), rat kidney cell lines transformed by a mixture of the BamHI fragments of Ad6 DNA contained the same amount of viral sequences as KC-1 transformed by a mixture of Sall fragments of highly oncogenic SA7 DNA.

(d) Competence of eukaryotic cells The comparison of our own and others' data suggests that the ability to integrate foreign DNA, due first and foremost to the cellular recombination systems, is a general property of eukaryotic cells 0Veinberg, 1980). We believe that it is the right time to speak about a specific state of a eukaryotic cell which, using the analogy with bacteria, may be called

competence (Zhdanov and Tikchonenko, 1974). Competent cells have an enhanced ability to accept an~ integrate any foreign DNA, oncogenic viral DNA included. It is reasonable to assume that under usual conditions only a small quantity of such competent cell.~ are present in the cel population. Transformation with oncogenic viruses virtually creates a prerequisite for the selection of competent cells, since later in the experiment transformed cels are selected due to their ability for unlimited growth. The same selective effect results from a genetic transformation, for example, that of cells deficient in TK using intact herpes virus DNA (Bacchetti and Graham, 1977) or its isolated fragment with TK gene (Maitland and McDougall, 1977). In the latter case, in addition to herpes virus DNA, foreign DNA administered into the cel with transforming DNA was incorporated into the cell genome (Hanahan et al., 1980). These results are in agreement with our data on nonspecific integration of pBR322 plasmid administered in a mixture ~itk Ad6 DNA fragments into transformed rat kidney cells. It is evident that the technique of cel coinfection or cotransformation with transforming genes in mixture with any foreign genetic material may permit the integration of the latter into the cellular genome, if conditions for the selection of transformants may be created. Naturaly, the use of viral oncogenes in such cotransformation or cointegration techniques provides better opportunities in comparison with any other transforming genes. Obviously, this approach may be used as an equivalent of integration vectors for eukaryotic cells. It follows from our f'mdings on independent integration of DNA fragments that the cel is likely to be capable of performing numerous acts of integration, the amount of available foreign DNA being a limiting factor. In other words, when one intact adenoviral genome is available for integration it is incorporated into the host DNA mos: probably as a whole. Preliminary fragmentation of adenoviral DNA is equivalent to an increase in the amount of molecules available for integration. Ultimately, incorporation and preservation in the cellular genome of other regions of adenoviral genome besides the oncogene becomes more likely. On the other hand, it may fail to cause an increase in absolute amount of adenoviral DNA integrated into the cellular genome at the expense of losses during the processing of the integrated fragments.

358 ~ r previous data on an increase in the transforming acti~ity o f fragmented DNA as compared to intact preparations (Ponomareva et al., 1979a), together with tt~e findings presented in t-ttis paper, suggest two ideas concerning the mechanism o f DNA incorporation into transforming cells. First, a certain optimal size o f the integrated DNA is likely to exist and second, the integrated DNA undergoes processing with cellular nucleases, thus causing a certain loss in molecular rc~ass o f the fragment. Therefore, during the transformation o f cells with intact DNA the integration n ~ y be hampered due to nonoptimai sizes. P;elimina~/fragmentation o f DNA facilitates the integration, which remits in the observed 5 - 8 - f o l d increase in the transformation rate over intact DNA. This suggestior,~ is confirmed b y the fact that such increase in the SAT transformation rate was only observed when r~striction endonucleases Sall and B a m H l were applied, providing oncogen-containing DNA fragments with M r 4.2 X 106 and 6 X 106, respectively. The use o f EcoRl cleavage of the lefthand DNA fragment o f 12 X 106 failed to increase the efficiency of the transformati,~n (Ponomareva et al., 1979a).

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