Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12

37, 513-520 (1969)

VIROLOGY

Synthesis

of Deoxyribonucleic

Ceils

infected

MICHIAKI

with

Acid Human

in Human

Adenovirus

TAKAHASHI, TAKE0 AND MICHIHIKO

OGINO, ONAKA

Department of Virology,

and Types KOICHI

The Research Institute for Microbial Osaka University, &da-City, Osaka, Japan

Hamster

Kidney

5 and 12 BABA,

Diseases,

Accepted November 6, 1968

The incorporation of 3H-labeled thymidine (3H-dT) into deoxyribonucleic acid (DNA) has been studied in human embryonic kidney and hamster kidney cells infected with human adenovirus types 5 and 12 (Ad5 and Ad12). The AdSinfected human and hamster kidney cultures incorporated about 5-10 times as much 3HdT into DNA BS did uninfected cultures. The Adl2-infected human and hamster kidney cultures incorporated about 4-6 times and 3-5 times as much SH-dT into DNA as did uninfected cultures. DNA-DNA hybridization experiments using a membrane filter technique were performed to elucidate the kind of DNA that was pulse-labeled with 3H-dT. In human and hamster kidney cells infected with Ad5, there was a progressive increase in the incorporation of 3H-dT into viral DNA. Incorporation of 3H-dT into cellular DNA was not immediately shut off. An increased incorporation during the early stage of infection (16-22 hours post infection) was followed by a decreased incorporation at later stages. The shutoff of incorporation into cellular DNA was found to occur gradually in hamster kidney cultures and rather rapidly in human kidney cultures. In human embryonic kidney cells infected with Ad12, incorporation of aH-dT into viral DNA increased progressively until about 32 hours PI. Incorporation of gH-dT into cellular DNA was stimulated somewhat at the early stage of infection, and was not shut off either during the period of viral DNA synthesis or for at least 20 hours after virus maturation had begun. In abortive infection of hamster kidney cells with Ad12, incorporation of aH-dT into cellular DNA was stimulated. A small amount of incorporation of 3H-dT into viral DNA was detected at an early stage of infection. From these results, it can be concluded that in productive infection of human and hamster kidney cells by Ad5 or of human kidney cells by Ad12, cellular DNA synthesis is not immediately shut off. In abortive infection of hamster kidney cells by Ad12, cellular DNA synthesis is stimulated, whereas viral DNA is synthesized in small amounts at an early stage of infection. INTRODUCTION

Infection of human embryonic kidney cells and newborn hamster kidney cells with Ad5 results in the production of infectious virus particles, characteristic cytopathic changes, and stimulation of DNA synthesis and thymidine kinase activity (Takahashi et al., 1966a,b).

Infection by Ad12 causes similar changes in human embryonic kidney cells (Takahashi et al., 1966a,b; Ledinko, 1967). However, abortive infection occurs in hamster kidney cells infected with Ad12. That is, stimulation of DNA synthesis and thymidine kinase activity (Takahashi et al., 1966a,b) and production of tumor-specific antigen (Hog513

Copyright

0

1969 by Academic

Press,

Inc.

Printed in U.S.A.

514

TAKAHASHI

gan et al., 1965; Shimojo el al., 1966) take place whereas virus production and cytopathic changes do not. It was of interest to determine the kind of DNA synthesized in these systems, especially in cases of Ad12 infection, because it has been reported that cellular DNA synthesis is induced by polyoma and SV40 infection in productive as well as in abortive infection (Dulbecco et al., 1965; Hatanaka and Dulbecco, 1966; Kit et al., 1967). Ginsberg et al. (1967) reported that Ad5 infection of KB cells resulted in stimulation of viral DNA synthesis and, concurrently, inhibition of cellular DNA synthesis. Rapp et al. (1966) reported that in infection of green monkey kidney cells by Ad2 or Ad12, which in neither case leads to the production of infectious virus, viral DNA synthesis was observed. These authors used the MAK column (methylated-albumin-kieselguhr) and CsCl equilibrium centrifugation methods for the differentiation of the cell and virus DNA’s. However, it has not been easy to differentiate clearly DNA’s of adenovirus and of mammalian cells by these methods, because the two DNA’s are very similar with respect to both GC (guanine and cytosine) content and structure (linear structure) (Green and Pifia, 1964; Green et al., 1967). Recently, a convenient and reliable technique involving the use of membrane filters has been developed for studies of DNA-DNA hybridization (Denhardt, 1966; Warnaar and Cohen, 1966; Richards, 1967). This technique enabled us to examine, in some detail, DNA synthesis in cells infected with Ad5 or Ad12. MATERIALS

AND

METHODS

Virus. Prototype adenovirus type 5 (Takahashi et al., 1966a) and adenovirus type 12 (Huie strain, kindly provided by Dr. Y. Kanda Inoue, Kyoto University) were used in this study. These viruses were propagated in KB cells and treated with crystallized trypsin to eliminate cytotoxic substances before use as inoculum (Takahashi et al., 1966b). Cell cultures. Human embryonic kidney cells and newborn hamster kidney cells were grown in monolayer cultures as described

ET AL.

previously (Takahashi et al., 1966b). Adult hamster kidney cultures were prepared from l- to 2-month-old hamsters. Purification of adenovirus. Adenovirus was grown in KB cells and purified by CsCl equilibrium centrifugation following the procedure described by Green and Pina (1963). For the production of 3H-labeled virus, the virus was grown in the presence of culture medium containing 2.6 PCi of thymidine-3H per milliliter and was purified in the same way. Infection of cells and labeling of infected cells with thymidine-3H. Confluent monolayer cell cultures, 7-10 days old, in 7-ounce bottles were washed with PBS (phosphate-buffered saline), and 1 ml of virus suspension was added to each culture. The input multiplicity in all cases was approximately 100 TCIDso/cell for Ad5 and 10 TCIDso/cell for Ad12. The cultures were incubated for 2 hours at 37” to permit virus adsorption; during this time they were rocked every 20 min. The fluid was discarded, and 10 ml of maintenance medium was added. Infection of nearly all cells by these multiplicities was confirmed by the detection of characteristic intranuclear inclusion bodies in almost all cells after 48 hours of productive infection. For the viral growth experiments, Eagle’s MEM medium containing 5 % calf serum was added as maintenance medium. At intervals up to 60 hours PI, cells were removed from bottles by scraping, suspended in culture fluid, and disrupted by 6 cycles of freezing and thawing. The cell debris was removed by centrifugation at 800 g for 15 min. The supernatant fluid was then assayed for infectious virus in human embryonic kidney cell cultures (TCIDbo). For the labeling of infected cells, Eagle’s MEM medium was used as maintenance medium. At various times after infection, the culture medium was replaced with 5 ml of fresh Eagle’s medium containing 3H-dT (2 pCi/ml for experiments in which incorporation of 3H-dT into DNA was measured, 50 pCi/ml for the hybridization experiments in which DNA of high specific activity was required). The cultures were incubated with the radiolabel for 2 hours at 37”. Noninfected cultures were treated similarly except that

DNA

SYNTHESIS

OF ADENOVIRUS-INFECTED

Eagle’s ME&I medium was substituted for the virus preparation. After the labeling period, the cultures were washed with SSC (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) and removed from the glass with a rubber scraper. For the estimation of the total 3H-dT incorporated into DNA, the cells were washed 4 times with ice cold 5 % TCA, after which the DNA was hydrolyzed with 0.5 N perchloric acid at 90” for 15 min and assayed in a scintillation spectrometer. DNA extraction. To the suspension of cells or virus in SSC, pronase was added to a final concentration of 1 mg/ml and the mixture was incubated for 2 hours at 37”. Then SDS (sodium dodecyl sulfate) was added to a final concentration of 0.2%. The suspension was incubated at 37” overnight. It was then extracted 3 times with an equal volume of water-saturated redistilled phenol at room temperature by slow rotation (60-100 rpm) for 30 min each (Thomas et al., 1966). The aqueous phase was dialyzed against a total volume of 20 liters SSC to remove phenol. Pancreatic RNase (Worthington, heated at 80” for 10 min to inactivate contaminating DNase) and crystallized RNase Tl (purified from Takadiastase, Sankyo Co., Tokyo) were added to final concentrations of 50 pg/ml and 30 pg/ml, respectively, in order to degrade RNA completely (Saito and Miura, 1963). The mixture was incubated at room temperature for 1 hour and the RNase was then removed by one phenol extraction, The phenol was removed by dialysis as above.

515

CELLS

I2

24

36

48

I

60

(HOC’RS)

FIG.

adult

2. Growth of adenovirus types 5 and 12 in hamster kidney cell culture.

DNA-DNA hybridization. DNA-DNA hybridization experiments were carried out following essentially the procedure described by Denhardt (1966) and Warnaar and Cohen (1966). DNA dissolved in SSC was denatured by heating to 100” for 10 min and cooling quickly in ice water. After NaCl was added to give a concentration of 6 X SSC, the solution was passed through a filter (Millipore, HAWP 25 mm) prewashed with 6 X SSC. The filter was dried overnight in a vacuum desiccator, then heated at 80” by an ultrared lamp for 2 hours. The filters were placed in scintillation vials with 1 ml of preincubation medium (0.02 % each of Ficoll, polyvinylpyrrolidone, bovine serum albumin in 3 X SSC) and incubated for 4-6 hours at 60”. 3H-labeled DNA was sonicated for 1 min in a lo-kc ultrasonic disintegrator and denatured by heating to 100” for 10 min in SSC. The denatured DNA was added to the vials containing the filters, and incubation at 60” was continued for an additional 12-24 hours. The filters were then washed by suction with 100 ml of 0.003 M Tris buffer (pH 9.1) in each side, and, finally, dried and counted in a scintillation spectrometer. RESULTS

I2

21

36

I 60

48 (HOURS)

FIG.

human

1. Growth embryonic

of adenovirus types 5 and 12 in kidney cell culture.

Growth of Ad5 and Ad12 in Human Embryonic Kidney Cells and Hamster Kidney Cells

The growth curves of Ad5 and Ad12 in human embryonic kidney cells are shown in Fig. 1. In both cases,the eclipse periods were approximately 20 hours, and maximum viral

516

TAKAHASHI

El’

AL

(bl

12

24

48

36

12

60

24

36

Ill

12

60

4x

36

24

60

FIG. 3. Incorporation of 3H-dT into DNA in Ad5- or Adl2-infected cell culture. Confluent monolayer cultures were inoculated with Ad5 or Ad12. At the time indicated, 3HdT (2 &i/ml) was added and the cultures were further incubated for 2 hours at 37”. (a) Human embryonic kidney cell culture. (b) Adult hamster kidney cell culture. (c) Newborn hamster kidney cell culture.

60 L

1

I

I. 0.1

0.5

1

2

3

4 CPM

ypDNAON

5 ADDED

6

7 (X IO-')

FILTER

4. Filters carrying the indicated amount of Ad5 DNA were incubated with aH-Ad5 DNA (6300 cpm, 0.05 rg). FIG.

titers were obtained at 48-60 hours post infection (PI). The growth pattern of Ad5 in adult hamster kidney cells was almost the same as that in human embryonic kidney cells, except that the maximum viral titer was somewhat lower. No virus growth was observed in hamster kidney cells infected with Ad12 (Fig. 2). The growth curves of the two viruses in newborn hamster kidney cells were quite similar to those observed in adult hamster kidney cells. Incorporation of 3H-dT into DNA and AdId-Infected Cells

2

of A&i-

Ad5-infected human and hamster kidney cultures incorporated about 5-10 times as

FIG. 5. Filters carrying 1 pg of Ad5 DNA were incubated with various amounts of 3H-Ad5 DNA (0.005, 0.0125, 0.025, 0.05 pg).

much 3H-dT into DNA as did uninfected cultures. The Adl2-infected human and hamster kidney cultures incorporated about 4-6 times and 3-5 times as much 3H-dT into DNA as did uninfected cultures. In each case, maximum incorporation was at 24-32 hours PI. No significant difference was observed between newborn and adult hamster kidney cells with respect to the incorporation of 3H-dT into DNA (Fig. 3, a-c). From these observations, coupled with the results of the virus growth studies, it can be concluded that newborn and adult hamster kidney cells do not differ in their response to infections with Ad5 or Ad12. Therefore, adult hamster kidney cells were used for the subsequent analysis of DNA synthesized after infection by these viruses.

DNA

SYNTHESIS

OF ADENOVIRUS-INFECTED

The annealing efficiency of cell DNA was usually lower than that obtained with viral DNA. A ratio of 50-100 to 1 of immobilized hamster kidney cell DNA was needed to obtain about 30 % annealing efficiency (Fig. 6). As with the viral DNA, a linear relationship was observed between the amount of DNA annealed to filters carrying a fixed amount of denatured DNA and the amount of added DNA (Fig. 7). The presence of considerable amounts of unlabeled cell DNA in the preparations of labeled cell DNA may be a factor for the lower annealing efficiency of cell DNA. On the basis of these results, 3 pg of viral DNA and 20 pg cell DNA, respectively, were

Nature of DNA Synthesized after Injection with Ad5 or Ad12 A. Quantitative Hybridization

517

CELLS

Test

Figure 4 indicates the annealing efficiency of Ad5 DNA. As can be seen, 1 pg of denatured DNA on the filter was enough to obtain about 50 % annealing efficiency when 0.05 ,ugof 3H-labeled DNA was added to the vial (20: 1). The relation between the amount of 3H-DNA annealed to the immobilized DNA on the filter and the amount added to the vial is shown in Fig. 5. With a fixed amount of denatured DNA on the filter, a constant fraction of the input DNA binds over the range tested. Similar results were obtained with Ad12 DNA.

v

.

2

4

6

8

10

12

CPM ADDED<* 10-Y

FIG. 7. Filters carrying 20 rg of hamster kidney cell (HAK) DNA were incubated with various amounts of 3H-HAK DNA (0.02, 0.05, 0.1, 0.2 pg)

FIG. 6. Filters carrying the indicated amount of hamster kidney cell (HAK) DNA were incubated with 3H-HAK DNA (9600 cpm, 0.2 rg). TABLE HYBRIDIZATION

Time after infection (hours) 16 20 24 32 48

1

BETWEEN 3H-DNA OF ADS-INFECTED HUMAN KIDNEY KIDNEY (HEK) CELL DNA OR ADS DNA43 *

Cells

Uninfected Infected Uninfected Infected Uninfected Infected Infected Infected

CELLS

AND

HUMAN

Cpm bound to HEK Cpm bound to Ad5 Cprn bound to Jnput 3H-DNA of cell DNA immobil- DNA immobilized infected HEK cells blank filter on filter (3 pg) ized on filter (20 pg) hmAJ.2 Ecg) 1,825 4,650 1,943 1,670 1,681 985 480 133

a Background was not subtracted. * Confluent monolayer cultures were inoculated was added and the cultures were further incubated by pronase, SDS, and phenol treatment. Aliquots hybridization with immobilized human kidney cell

96 6,134 101 11,525 86 10,820 13,876 5,901

65 80 57 90 74 89 113 57

6,320 17,560 6,630 26,868 5,870 25,184 28,260 9,132

with Ad5. At the time indicated, 3H-dT (50 pCi/ml) for 2 hours at 37”. DNA was extracted from the cells of DNA (0.2 pg) from each sample were subjected to DNA (20 pg per filter) and Ad5 DNA (3 pg per filter).

518

TAKAHASHI

HYBRIDIZATION

Time after infection (hours)

~~&$!$n!,m~b~l~ ized on filter (20 rg)

Cpm bound to Ad5 virus DNA imfir$$i~~gp

3,035 4,320 3,120 11,293 3,616 6,854 5,776 2,666 2,450

142 4,705 101 33,774 112 51,807 32,222 25,032 17,150

Uninfected Infected Uninfected Infected Uninfected Infected Infected Infected Infected

20 24 28 32 43 (1Refer

TABLE 2 BETWEEN 3H-DNA OF An.5 INFECTED HAMSTER KIDNEY HAMSTER KIDNEY (HAK) CELL DNA OR ADS DNAe

Cells

16

to the footnotes

in Table

BETWEEN

Time after infection (hours) 16 20 24 28 32 39 48 a Refer

Cpm bound to HEK cell DNA immobilized on filter (20 rg)

Uninfected Infected Uninfected Infected Uninfected Infected Infected Infect,ed Infected Infected to footnotes

in Table

AND

Input 3H-DNA of Cpm bound to infected HAK cells blank filter (cpm/0.2 rg DNA) 65 106 83 129 71 140 164 131 112

9,396 22,220 9,928 63,292 11,125 75,768 73,128 62,432 33,960

3

aH-DNA OF A~12 INFECTED HUMAN KIDNEY KIDNEY (HEK) CELL DNA OR A~12 DNAa

Cells

CELLS

1. TABLE

HYBRIDIXATION

ET AL.

510 1,344 473 759 487 858 894 948 654 468

A~%nD%?n~~obilized On ‘lter (3 Pd 58 589 89 2,344 93 2,426 2,894 5,348 3,547 1,674

CELLS

AND

HUMAN

Cpm bound to ‘n;P,l;,‘~tDd\~Kof blank filter cells (cpm/0.2 pg) 52 49 65 45 73 98 109 123 70 68

2,250 4,256 2,329 5,825 2,506 6,324 7,832 12,890 9,882 6,321

1.

immobilized on the filters in the subsequent annealing experiments. Throughout these studies, 0.2 pg of 3H-labeled DNA from infected cells was employed. B. Time Course of Viral and Cellular DNA Synthesis 1. Ad5 infection. As seen in Tables 1 and 2, incorporation into viral DNA in the human and hamster kidney cells increased progressively from 16 to 32 and from 16 to 24 hours PI. Incorporation into cellular DNA was found to be stimulated somewhat at an early stage of infection (16-20 hours PI) but to decrease gradually in hamster

kidney cells and rather rapidly in human kidney cells at later times. Thus it seems clear that viral DNA synthesis in these cells is greatly stimulated by infection with Ad5, and that cellular DNA synthesis is not immediately inhibited by infection. 2. Ad12 infection. In productive infection of human embryonic kidney cells by Ad12, a progressive increase of incorporation of 3HdT into viral DNA was observed after 16 hours PI. Incorporation into cellular DNA was not shut off after infection, but increased slightly at the early stage of infection and continued during the period of viral DNA synthesis (Table 3).

DNA

SYNTHESIS

OF ADENOVIRUS-INFECTED TABLE

HYBRIDIZATION

Time after infection (hours) 1G 18 22 24 32 39 48 (1Refer

3H-DNA

BETWEEN HAMSTER

Cells

KIDNEY

in Table

4

OF A~12 INFECTED (HAK) CELL DNA

HAMSTER KIDNEY OR A~12 DNAa

CELLS

Cpm bound to HAK Cpm bound to DNA Immo- Cpm bound to cell DNA immobil- Ad12 .. blank filter ized on filter (20 pg) “llz$ o$filter

Uninfected Infected Uninfected Infected Uninfected Infected Infected Infected Infected Infected to footnote

519

CELLS

1,883 1,951 1,819 2,561 1,750 3,612 4,125 5,544 2,315 2,015

90 78 87 383 95 63 110 94 77 41

58 102 59 31 75 47 40 80 91 GO

AND

Input 3H-DNA of infected HAK cells ( cpm/ 0.2 CLg) 6,105 7,205 5,859 8,264 5,315 11,400 12,644 18,928 8,404 G,4GO

1.

In abortive infection of hamster kidney cells by Ad12, incorporation of 3H-dT into cellular DNA was stimulated. A small amount of 3H-dT was incorporated into viral DNA (estimated to be less than 10% of the total) at 18 hours PI. No incorporation into viral DNA was observed at later times (Table 4). DISCUSSION

It has been reported that the ‘Lnononcogenie” DNA viruses, namely, the vaccinia and herpes group viruses, stimulate viral DNA synthesis and concurrently rapidly inhibit cellular DNA synthesis in infected cells (Kit and Dubbs, 1963; Kit et al., 1963; Kato et al., 1964; Kaplan and Ben-Porat, 1963). In contrast, the “oncogenic” DNA viruses so far studied (polyoma and SV40 viruses) induce cellular DNA synthesis in productive as well as in abortive infection (Dulbecco et al., 1965; Hatanaka and Dulbecco, 1966; Kit et al., 1967). Thus the capacity to induce cellular DXA synthesis in infected cells seems to be a unique property of the oncogenie DNA viruses. The adenovirus group would appear to be useful agents for studies of this phenomenon, as it includes many types, having various degrees of tumorigento ranging from “nononcogenic” icity, “highly oncogenic.” Ad5 and Ad12 have been recognized as representatives of “nononcogenic” and “highly oncogenic” adenoviruses, respectively.

Our present study revealed that productive infection of cells by Ad5 does not result in immediate shut off of cellular DNA synthesis, but stimulates somewhat its synthesis at the early stage of infection. In this point, Ad5 seems to differ from the “nononcogenic” pox and herpes group viruses. Recently it has been reported that rat embryo cells are transformed by Ad2 or Ad5 under certain conditions (Freeman et al., 1967). It may be conceivable that our results have some correlation with their findings. In productive infection of human kidney cells by Ad12, cellular DNA synthesis was not shut off until a long time after infection. Even though the amount of cellular DNA synthesis induced by infection is not large, this phenomenon is similar to those observed in polyoma and SV40 infection. In abortive infection of hamster kidney cells by Ad12, cellular DNA synthesis was apparently stimulated, whereas viral DNA synthesis occurred only at a low level and at an early stage of infection. From the present study and from previous findings, it has become evident that, infection of hamster kidney cells by Ad12 results in stimulation of cellular DNA synthesis, synthesis of low levels of viral DNA at an early stage of infection, enhancement of thymidine kinase activity, and production of tumor-specific antigen. The search for the factors responsible for the discontinuation of viral DNA

520

TAKAHAS

synthesis as well as studies on other types of adenovirus msy throw some light on the mechanism of tumorigenicity of the adenovirus group. ACKNOWLEDGMENTS The authors are grateful to Professor Y. Okuno for support and encouragement to this work and to Dr. Tsuneko Tomita in our Institute for helpful suggestions in the hybridization experiments. REFERENCES DENHARDT, D. T. (1966). A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. DULBECCO, R., HARTWELL, L. H., and VOGT, M. (1965). Induction of cellular DNA synthesis by polyoma virus. Proc. Null. Acad. Sci. U.S. 53, 403410. FREEMAN, A. E., BLACK, P. H., VANDERPOOL, E. A., HENRY, P. H., AUSTIN, J. B., and HUEBNER, R. J. (1967). Transformation of primary rat embryo cells by adenovirus type 2. Proc. Null. Acad. Sci. U.S. 58, 12951212. GINSBERG, H. S., BELLO, L. J., and LEVINE, A. J. (1967). Control of biosynthesis of host macromolecules in cells infected with adenoviruses. In “The Molecular Biology of Viruses” (J. S. Colter, and W. Paranchych, eds.), pp. 547-572. Academic Press, New York. GREEN, M., and PIWA, M. (1963). Biochemical studies on adenovirus multiplication. IV. Isolation, purification, and chemical analysis of adenovirus. Virology 20, 199-207. GREEN, M., and PIRA, M. (1964). Biochemical VI. studies on adenovirus multiplication. Properties of highly purified tumorigenic human adenoviruses and their DNA’s. Proc. Natl. Acad. Sci. U.S. 51, 1251-1259. GREEN, M., PIRA, M., KIMES, R., WENSINK, P. C., MACHATTIE, L. A., and THOMAS, C. A., JR. (1967). Adenovirus DNA, I. Molecular weight and conformation. Proc. Natl. Acad. Sci. U.S. 57, 13021309. HATANSKA, M., and DULBECCO, R. (1966). Induction of DNA synthesis by SVho. Proc. Natl. Acad. Sci. U.S. 56, 736-740. HOGGAN, M. D., ROWE, W. P., BL.~cK, P. H., and HUEBNER, R. J. (1965). Production of “tumor specific” antigens by oncogenic viruses during acute cytolytic infection. Proc. Natl. Acad. Sci. U.S. 53, 12-19. KAPLAN, A. S., and BEN-PORbT, T. (1963). The pattern of viral and cellular DNA synthesis in pseudorabies virus-infected cells in the logarithmic phase of growth. Virology 19, 205-214.

IHI ET AL. KATO, S., OG~WA, M., and MIYAMOTO, H. (1964). interaction in poxvirusNucleocytoplasmic infected cells. I. Relationship between inclusion formation and DNA metabolism of the cell. Biken J. 7, 45-56. KIT, S., and DUBBS, D. R. (1963). Biochemistry of vaccinia-infected mouse fibroblasts (strain L-M). IV. 3H-Thymidine uptake into DNA of cells exposed to cold shock. Exptl. Cell Res. 31, 397406. KIT, S., DIJBBS, D. R., and Hsu, T. C. (1963). Biochemistry of vaccinia-infected mouse fibroblasts (strain L-M). III. Radioautographic and biochemical studies of thymidine-H3 uptake into DNA of L-M cells and rabbit cells in primary culture. Virology 19, 13-22. KIT, S., DE TORRES, R. A. DUBBS, D. R., and f3.4~~1, M. (1967). Induction of cellular deoxyribonucleic acid synthesis by Simian Virus 40. J. Viral. 1, 738746. LEDINKO, N. (1967). Stimulation of DNA synthesis and thymidine kinase activity in human embryonic kidney cells infected by adenovirus 2 or 12. Cancer Res. 27, 1459-1469. RAPP, F., FELDMAN, L. A., and MANDEL, M. (1966). Synthesis of virus deoxyribonucleic acid during abortive infection of simian cells by human adenoviruses. J. Bacterial. 92, 931-936. RICHARDS, 0. (1967). Hybridization of Euglena grucilis chloroplast and nuclear DNA. Proc. Natl. Acad. Sci. U.S. 57, 156163. SAITO, H., and MIURA, K. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72, 619-629. SHIMOJO, H., YAMAMOTO, H., YOSHIKAWA, E., and YAMBSHITA, T. (1966). The nature of tumor antigens of adenovirus type 12 and its formation in cultured cells after infection. Japan. J. Med. Sci. Biol. 19,9-22. TAKAHASHI, M., UEDA, S., and OGINO, T. (1966a). Enhancement of the thymidine kinase activity of human embryonic kidney cells and newborn hamster kidney cells by infection with human adenovirus types 5 and 12. Virology 30,742-743. T~KAHASHI, M., VAN HOOSIER, G., and TRENTIN, J. J. (196613). Stimulation of DNA synthesis in human and hamster cells by human adenovirus types 12 and 5. Proc. Sot. Exptl. Biol. Med. 122, 740-746. THOMAS, C. A., BERNS, K. I., and KELLY, J. J., JR. (1966). Isolation of high molecular weight DNA from bacteria and cell nuclei. In “Procedure in Nucleic Acid Research” (G. L. Cantoni and D. R. Davis, eds.), pp. 535-540. WARNAAR, S. O., and COHEN, J. A. (1966). A quantitative assay for DNA-DNA hybrids using membrane filters. Biochem. Biophys. Res. Commun. 24,554-558.