of 0ncogenic and Non-oncogenic Simian Adenovirus DNA's

Transcription

1 J. gen. Virol. 0972), I7, Printed in Great Britain 245 Chemical and Physical Relationships of 0ncogenic and Non-oncogenic Simian Adenovirus DNA's ByJ. P. BURNETT, NANCY MAYNE, LINDA K. BUTLER AND JO ANN HARRINGTON The Lilly Research Laboratories, Indianapolis, Indiana, U.S.A. (Accepted 2o July ~972) SUMMARY The DNA's of the simian adenoviruses studied belong to three groups that can be distinguished on the basis of DNA-DNA hybridization techniques: one nononcogenic group and two groups of oncogenic viruses. However, DNA base composition studies reveal at most a bimodal distribution of values, and the tool. wt. of these DNA's is randomly distributed between I7 and 23 million. INTRODUCTION Human adenoviruses have been placed into three subgroups according to similar biological and biophysical properties (Schlesinger, I969). The criteria used include DNA base composition, T-antigen production, and genetic relatedness as measured by DNA homology studies. Adenoviruses of other species might be expected to form similar subgroups of closely related types. Gilden et al. 0968) have reported that the simian adenoviruses they examined could be classified into three subgroups on the basis of cross reactions among turnout and T (infected cell) antigens. Goodheart 0970 examined the buoyant densities of the DNA's of a group of simian adenoviruses and found that they formed either a unimodal or possibly at most a bimodal distribution. We have studied the base composition, tool. wt. and genetic relatedness of a number of simian adenoviruses and find three distinct groups based on DNA homology studies, but at most a bimodal distribution of base compositions. The tool. wt. of the DNA's does not show any obvious trends. METHODS Viruses. All of the simian adenoviruses tested were obtained from Dr R. N. Hull 0968). In some cases, adeno-associated virus (AAV) particles were removed from stocks by treatment with specific antisera. The viruses were then purified by plaque or terminal dilution techniques. [s2p]-labelled and unlabelled viruses were grown in CV-I or BSC-I cells and purified by techniques previously reported (Burnett et al ~968). Virus DNA was extracted using a papain digestion and phenol extraction treatment previously described (Burnett & Harrington, I968 ). Thermal denaturation studies. Thermal transitions of the DNA's were measured in a Cary I6 Spectrophotometer fitted with a 5-position electrically heated automatic samplepositioner. The extinction and temperature of each sample was measured continuously and automatically recorded. Temperatures were measured by insertion of a thermistor probe in a blank cuvette containing buffer. Base compositions were calculated from the mid-

2 246 J.P. BURNETT AND Oq-HERS point of the thermal transition profile (T~) using the equation ~ G+ C = (T~- 53"9) 2"44 (Mandel &Marmur, 1968) since all determinations were performed in o.i SSC (SSC = o. 15 ~-NaCI + o.o t 5M-sodium citrate, ph 7). Analytical sedimentation. Sedimentation studies were performed in a Spinco Model E Analytical Ultracentrifuge equipped with u.v. optics and a photoelectric scanner. Buoyant density determinations were made in equilibrium density gradients of CsC1 containing o-oi M-tris buffer, ph 7"4. Centrifuging was at 25 C and rev/min for 24 to 36 h, and equilibrium was judged by failure to observe further changes in band shape or position. In each experiment, Micrococcus lysodeikticus or Clostridium perfringens DNA was added as a standard. The density of the adenovirus DNA was then calculated from its position relative to the marker DNA by either the method of Szybalski (I968) or Qu&ier, Guill6 & Lejus (1969). Good agreement was obtained by both methods. Sedimentation coefficients were determined by a boundary sedimentation technique in I M-NaC1 containing o-oi M-tris buffer, ph 7"4. In some cases the band technique of Vinograd et al. (I963) was also employed, and good agreement was obtained. Corrections for viscosity and density and formulas relating sedimentation coefficients to tool. wt. are those of Studier 0965). s,~0.,, values were obtained by measuring &0.~ at several concentrations and extrapolating to zero concentration. Samples were centrifuged in cells containing either Kel-F or charcoal-filled epon centrepieces (Iz or 3o ram) at 3oooo rev/min and 20 C. DNA hybrid formation. The procedure employed was a modification of that described by Denhardt (~966). Purified DNA was denatured by heating in o.i x SSC to IOO C for 15 rain followed by quick cooling in ice. The salt concentration was then adjusted to 6 x SSC by addition of a concentrated SSC solution, and the DNA solution was passed through 25 mm Millipore-type HA filters. Filters containing denatured DNA were dried and pre-incubated in ficoll, polyvinyl pyrrolidone and bovine serum albumin according to Denhardt 0966). [z~p]-labelled DNA to be tested for hybrid formation was sheared by sonication to an average tool. wt. of 4 to 5 x io 5 and denatured as above. The final salt concentration of the sheared DNA was adjusted to 3 SSC and o'5 ml aliquots added to each filter. The ratio of unlabelled membrane-bound DNA to labelled fragments was always Ioo: I. After I4 to 16 h incubation at 65 C, the filters were washed on each side with three zo ml samples of 6 SSC at 65 C. Radioactivities of the dried filters were measured in a Nuclear Chicago automatic planchet counter. Backgrounds were determined with filters containing a heterologous DNA (Escherichia coli). Triplicate sets of filters were used in each experiment. RESULTS Base composition of virus DNA's Base composition of each DNA was determined both from T~ determinations and buoyant densities (Table 0. The molar ratios were calculated from buoyant densities by the equation of Schildkraut, Marmur& Doty (I962). The agreement of determinations by both methods is good with the exception that the values for thermal denaturation data are generally somewhat lower. As shown in Table I, the guanine plus cytosine (G + C) contents of the simian adenoviruses are ali about 55 ~ with the exception of 5 viruses. The oncogenic adenoviruses SA7, SA ~7, SA 18, SV 2o and SV 30 all appear to fall in a higher G + C category. However, the other oncogenic viruses, SVI, SVII, SV25, SV33, SV34 and SV38, have approximately the same G +C contents as the rest, which have so far proved to be non-oncogenic in newborn hamsters.

3 Relationships of adenovirus DNA's 247 Table I. Base composition and mol. wt. of simian adenovirus DNA's Mol. wt. Virus Density ~ G + C Tm ~ G + C s.2o.,~, ( x t 0-6) SVI 1" "4 54"9 3o'8 22"5 SV 11 1 " "4 54" SV I "o 54"o 28"4 17"7 SV I7 SV 2o 1"714 1 " z 76"o 79" I 54"o 61 "5 28.o 'o 20-4 SV "3 54'6 27"9 16'9 SV 25 I'7I "2 54"5 30"o 20"7 SV 30 I " "7 60"5 28"9 18"7 SV 3I I' "0 54"0 28"3 I7 '6 SV 32 I " "0 54"0 29"7 2(3'2. SV33 ~ " '0 54"0 29"6 19"9 SV "4 54" I7'9 SV37 I' '6 53"o 3o' SV "8 53"2 28"3 I7.6 SA7 1' "3 57" I'7 SAI7 1' SA 18 1 "72o 6I Oncogenic i I I i 16 Non-oncogenic [] [] [] D I I I 1 I f B [] [] EB D I I i i i i I I i I I t I Million [] [] 1 I I 1 I I I I 1 I Million Mol. wt. Fig. i. Distribution of tool. wt. of simian adenovirus DNA's. Mol. wt. of virus DNA's The sedimentation coefficients and corresponding tool. wt. of the simian adenoviruses examined are listed in Table I. In all cases, a single homogenous component was observed, The tool. wt. were calculated assuming the DNA to be a linear duplex as has been reported for one of the simian adenoviruses, SA7 (Burnett & Harrington, i968 ) and the human adenoviruses (Schlesinger, I969). We have also examined other selected simian adenovirus DNA's after alkali denaturation and find results consistent with a linear structure (Burnett & Harrington, I968, unpublished observations). Oncogenic as well as non-oncogenic viruses are scattered throughout the entire tool. wt. range (Fig. I). DNA homology studies We have used the membrane filter DNA hybridization technique to attempt to estimate the genetic relatedness of the various simian adenoviruses. The data from these experiments are summarized in Tables z to 7- The data are all expressed as the percentage of hybrid found when labelled fragments were incubated with filters containing heterologous DNA as compared to filters having DNA homologous to the labelled fragments. Whereas homologous reactions are arbitrarily assigned a value of Ioo ~, the actual percentage of label

4 248 J. P. BURNETT AND OTHERS Table 2. Duplex formation* by DNA's of oncogenic simian adenoviruses Filter- Labelled DNA's bound c DNA SV I SV I ~ SV 25 SV 33 SV 34 SV 38 SVI IOO'O 89"2-+I"4 92"9_+ P5 84"8_+2"6 9o'5_+3"6 92"5-+5"3 SVII 86"o-+o'8 IOO.O 87"7+-o'9 77"8_+2"2 93'4+-3"3 89"6+3 'I SV25 82'O+_ I'O 79"3_+0'8 IOO'O 88"4_+ P9 89'6+_1"2 906_+2"3 SV33 86.o+o-I 78"9+0"7 94"o+_o! Ioo-o 93'2-+ P4 95"4+_2"4 SV34 9I'I-+ 1"o 85"8-+I'8 89"7_+09 86"9_+3"o ioo.o 94o+_1'7 SV38 86'7-+3"7 82"3-+o'7 86"3_+I2 9o'o-+3"2 86"5+o'I loo-o * Values are expressed as the ~ of the homologous reaction-+ standard error for 3 to 5 determinations. Table 3. Duplex formation by DNA's of non-oncogenic simian adenoviruses Filter- Labelled DNA's bound r "- DNA SVI5 SVI7 SV23 SV31 SV32 SV37 SVI 5 I00"o 85"7_+2"2 9I'7_+ I'O 9I'7_+ 1"6 9I'6_+ I'5 93"4+2'6 SVI7 97"9_+o'9 IOO'O 87"9+_o'9 96"6_+I'6 95"7+_2"9 92"3-+3"8 SV23 8o'2_+ I'2 8P9-+ I' 4 I00"o 9o'o-+ I'3 88"5-+ I'I 94"7+0'I SV31 96"6+_1"0 92q -+ P6 89"3_+0"9 IO0"O 94"6+-o'8 92"5+2"O SV32 87"2-+1'2 8I'5+ 2'5 92"2-+ I'3 94"2+-0'9 IOO'O 99"9+_o'1 SV37 88"o_+o'7 88"2_+5"o 88"o-+ I'5 87'6-+1"6 87"6_+I"5 Ioo'o Table 4. Duplex formation by DNA's of oncogenic simian adenoviruses Filter- Labelled DNA's bound ~ ~- DNA SA7 SV2o SV3o SA 7 IOO.O 64"z -+ z.2 65"4 +- 2"3 SV zo 74"6 _ oo.o 86" SV3o 7P "o_+ 1.6 IOO.O bound usually varied from 70 to 9. Each value in the table represents the mean of three to five determinations using triplicate sets of filters. Those viruses in Table 2 form a group of closely related DNA's as measured by this technique. All of the viruses listed in this table are oncogenic. The viruses in Table 3 are those that have so far proved to be non-oncogenic in newborn hamsters. Whether any of these agents would transform cells in tissue culture, as has been observed for some of the non-tumour inducing human adenoviruses such as type 2 (Freeman et al. I967 ), we do not know. All of these viruses appear to be closely related, with homology percentages varying from 8I to approximately IOO. The three viruses in Table 4 are less closely related from the standpoint of homology as compared to the first two groups, but as will be shown, they are more related to one another than to members of the other groups. Two of these viruses, SA7 and SV2o, are highly oncogenic; virtually IOO ~ of injected hamsters have tumours after a latent period of I to 2 months. Although SV3 o DNA hybridizes well to SV2o DNA, it is not as efficient in inducing tumours. We have found SA7 and SV2o to be most oncogenic of the simian adenoviruses we have tested. The viruses listed in Table 2 and SV3o generally have exhibited lower tumour incidence and longer latent periods when similar numbers of p.f.u.'s are injected (unpublished observations).

5 Relationships of adenovirus DNA' s 249 Table 5. Summary of heterologous duplex formation by DNA' s of simian adenoviruses Filter- Labelled DNA ~agments bound c DNA SA7 SV2o SV3o SVI 50"o±I"7 48"4±P0 5I'8±I"7 SVlI 45'8±I"3 4Z'3±I'5 45"6±0'8 SV25 54"7±o'6 52'3±I"7 53'9±P2 SV33 47"3±I"o 45"2±I'5 5o'o±I'3 SV34 4o'6±o'9 49'8±2"5 4z'7±o'9 SV38 49'2±0'4 42'6±I"4 41"5±1'2 SVI5 4t'6±I'6 39'8±P5 45"I±t'8 SVt7 39"5±0"5 38'I±o 'I 39'2±2'2 8V23 43'I±I'2 42"o±1"5 4I"O±2'4 SV3I 43"4±I"o 4I"5±o'7 39"8±I'6 SV32 43"5±P5 4o'9±z'o 46"5±P5 SV37 47'o±2'9 46"8±2"5 4o'6±I'5 Table 6. Summary of heterologous duplex formation by simian adenovirus DNA's ~lteb Labelled DNAfragments bound c ~---- DNA SVI SVIt SV25 SV33 SV34 SV38 SVI5 38q±P4 33"9±o'4 43"9±o'9 38"2±1"3 42'3±o'9 4t'2±3"o SVI7 34"6±oq 28"4±o't 38"6±o'I 37"z±o'3 37"I±P9 31'8±2.2 SV23 ~'2±I'o 35"2±1-I 46"2±1"3 41"4±1"3 43"9±0"5 38"5±I"8 SVM 43"8±o'3 32"5±I'5 48"4±I"I 46"8±o'9 4I'9±o'9 38"6±Iq SV32 37"6±~" 4 3I'8±o'6 42"6±0"4 36"6±2"I 34"5±o'3 33"6±0"6 SV37 4I'6±2"2 38'9±o'7 42"4±1"7 4Y2±I'7 42'8±2'4 37"4±I'3 Table 7. Thermal stability analysis of DNA hybrids of s#nian adenoviruses Unlabelled DNA Labelled membranefragments bound Hybrid T~ AT,.* SA7 SA7 76"8 -- SVII SVII 74"5 -- SV 17 SV 17 75"9 -- SV2o SV2o 77"I -- SV3o SV3o 76'8 -- SV3t SV3t SV 33 SV 33 75"o -- SV 34 SV 34 74'8 -- SV 11 SV 34 7t "o 3'5 SVI5 SV37 73"5 o'5 SV I7 SV31 74'3 I 'Z SA7 SV2o 65"o I I'8 SA 7 SV 30 64"5 J 2"3 SV2o SV3o 7o'o 7"I SA7 SV I I 62'5 14"3 SA7 SV 17 63"o 13'6 SV 2o SV 11 63"o 14" 1 SV2o SVI I5'6 SV3o SV 17 63'5 I3"3 SV3o SV33 61 "5 15"3 * AT m = difference between T.z of homologous and heterologcus reaction. T.]s were measured by determining the amount of labelled fragment eluted from the filter-bound hybrid in o.t SSC at increasing temperatures.

6 - 250 J. P. BURNETT AND OTHERS Table 8. Heterologous duplex formation by human and simian adenovirus DNA' s Labelled DNA's Filter- ~ bound DNA A 5 A 7 A 12 - SV t 39"0 + 3'1 43"2 _+ t'7 34" t _+ 0"7 SA 7 3o'4 _+ 3"o I '2 27"2 + o'7 SV I I 33" "5 + 3'2 28'4 + 2'7 SV I5 34'I _+ i.i 35.4_+ 2"6 28"9_+ 2-8 SV I7 38"9 +_ 2-o 37"5 -+ 2"9 24" I +_ 2'5 SV 20 30"2 _+ I " _+ 1 "4 20"4 + 5"5 SV23 32"7+3"5 33'~ -+o'i SV "2 38" "5 38"7 _+ o'6 SV3o 24-6_+o'9 24'2+o-9 2I-7+_o'I SV 33 34'3 _+ 2"3 36"2_+ t'9 31 "3_+ I '8 SV 34 39"2 3"4 42"4 + 7"8 35"4-+ 8"5 SV37 30"3_+ 3"9 32-2_+ I.o I- 5 SV38 35"9 + 1"5 39'I -+o.1 -- A5 IOO'O 43"9-+4"9 24"I _+ I'3 A7 46" IOO.O _+ I.I AI2 33"~ _+ I-O 33.8_+4-9 Ioo-o There are at least two possible explanations for the failure in some cases of homology values of reciprocal experiments to agree exactly. Some may be explainable on the basis of genome length differences (Cowie, Avery & Champe, 197I). For instance, in Table 2, SV25 fragments bind better to SVI DNA than do SVI fragments to SV25 DNA. The tool. wt. of SV I DNA is approximately 9 ~ greater than SV25, and this is about the same as the difference in binding observed. Similar reasoning holds for SV25 and SV I I. However, in cases such as SV38 and SV34, the observed tool. wt. differences are not sufficient to account for the differences in binding observed. An alternative explanation might be that certain nucleotide sequences are repeated in some of the DNA's. When the intergroup DNA-DNA interactions of Groups I to IH DNA's were measured (Tables 5, 6), binding of 3o to 55 ~ of the input label was observed. This suggests that none of these three groups of viruses are closely related to each other. Further, the data in Table 7 also supports this observation. The T,,'s of the homologous hybridization products were compared with those of the heterologous reactions (McCarthy & McConaughy, 1968). When the interactions are between closely related DNA's, the T~,,'s observed are close to those of the homologous reactions (AT~ = o'5 to 3"5 C). Although we arbitrarily grouped SA7, SV2o and SV3o together above, it is obvious that they are not as closely related as members of the other groups are to one another. However, the AT, m's observed (7 to/2. 3 C) are not as high as for lesser related viruses such as SA7 and SV 1 I, and the two viruses that appeared most related in Table 4 above (SV2o and SV3o), form the most stable hybrid of the three. In Table 8 we have compared the homologous and heterologous interactions of three human adenovirus DNA's with various simian adenoviruses and with one another. The apparent percentage homology varies from 2o to 45 ~. DISCUSSION The simian adenoviruses we have studied in these experiments were all of Rhesus origin with the exception of SA 7 which was isolated from an African green monkey (Malherbe & Harwin, 1963). These viruses can be grouped on the basis of certain biological properties.

7 Relationships of adenovirus DNA' s Of those studied, ~ I have been reported to be oncogenic in newborn hamsters. These are SVx, SA7, SV2o, SV33, SV34, SV37, SV38 (Hull et al. I965), SV~I, SV23, SV25 (Gilden et al. ~968), and SV3o (Slifkin, Merkow & Rapoza, 2968). Two of these viruses, SVz3 and SV 37, are probably not oncogenic. We have repeatedly retested SV 37 and have never observed tumours in newborn hamsters. In addition, the one tumour first reported with SV37 (Hull et al. I965) eventually regressed (H. W. Wright, personal communication). Gilden et al. 0968) reported tumour induction with high frequency by SV 23. However, a sample of this virus obtained from these authors proved identical in serum neutralization tests with our strain of SV20, and its DNA hybridized Ioo ~ with our SV2o DNA. Thus, we conclude that the strain of SV2o used in our studies is identical to the SV23 used by Gilden et al. (I968). These authors also reported that the tumour and T-antigen of SV2o and their SV23 cross-reacted Ioo ~. In addition, they were unable to group the T-antigen of SV37 with any of the known tumour viruses. The simian adenoviruses employed fall into three haemagglutination (HA) groups as determined by Rapoza & Cheever (I966). SVI5, SV17, SV23, SV31, SV32 and SV37 are members of Group II. Group III includes SV 1, SV 1 I, SV20, 8V25, SV3o, SV33, SV34 and SV38. SA7 is the one member studied of Group IV. Certain members of these groups also show relationships based on serum neutralization tests. Cross-reactions have been observed with SVI5, SV~7 and SV3~ (Group A), and with SV~, SV33, SV34 and SV38 (Group B) (Hull, I968 ). In addition, SVI, SV~I, SV25, SV33 and SV34 are the only viruses of this series that will grow in human cells (Hull, 1968 ). Gilden et al. (I968) have grouped the oncogenic simian adenoviruses on the basis of cross reactivity of turnout and T-antigen. They reported that antigens from SVI, SV 1I, SV25, SV 33, SV 34 and SV 38 cross-reacted (Group I). SV 2o and SA7 were placed in groups li and III, respectively; SV 3o was grouped with the non-oncogenic viruses, SV 15, SV 31 and SV 36. We have studied the base composition of these virus DNA's by density-gradient techniques and thermal transition analysis. Our results suggest at most a bimodal distribution, with the oncogenic adenoviruses SA7, SV 2o and SV 3o having a relatively high G + C content (58 to 62 ~). The rest of the oncogenic viruses as well as the non-oncogenic vary from 54 to 56 ~o G + C. These results agree well with those of Goodheart (I 97 I) with the exception of SV3o and SV34. Our strain of SV3o has a buoyant density in CsC1 of I'7I 9 as opposed to I'717 reported by Goodheart; our SV34 banded at 1.714, whereas Goodheart reported 1"717. In addition, Goodheart indicated that the SV39 DNA he employed had two bands, 1"715 and He suggested that this was due to the presence of a mixture of SVz3 (1.715) and SV39 (I"719). Our SV39 material showed only a single band at 1-715, and we have interpreted this to mean that this virus is identical to SV23 as reported by Hull (I968). We do not currently understand the reason for these differences. However, they do not alter the general conclusions that the simian adenovirus DNA's range in density from 1.7I 4 to I'72I in CsC1 and are distributed in at most a bimodal fashion. As noted by Goodheart (I 97 I), these results are opposite to the G + C content and oncogenicity correlations observed by Pifia & Green (I965) for the human adenoviruses. The subgroups for the viruses that we have described based on DNA homology studies correlate fairly well with other reported biological properties. Those viruses in homology Group I (Table 2) are all members of HA group II, and certain of these are members of serum neutralization cross-reacting Group A. The members of homology Group II (Table 3) belong to HA group II[ and to Gilden et al. (I968) T-antigen group II; SA7 is in Group III; and SV3o was grouped with certain non-oncogenic viruses. However, as was noted, we have grouped these agents together only because, even though the degree of relatedness of their 25 I

8 252 J.P. BURNETT AND OTHERS DNA's was not as high as for members of the other groups, they were more related to each other than to the other viruses studied. In addition, it should be noted that SA7 is an isolate of a different species of monkey than the other two. The hybridization studies we have reported for the simian and human adenovirus DNA's correlate well with the limited earlier data of Pifia & Green 0968). We believe that their interpretation of the relatively low degree of homology observed as being due to those genes that determine common features of these agents usually associated with adenoviruses is probably correct. For instance, these viruses all contain a cross-reacting group-specific complement fixation antigen. It is interesting that the degree of homology between the simian and human adenovirus DNA's is as high as that observed for reactions of some of the lesser related simian adenoviruses (homology groups I and II, Table 6). If the genetic information necessary for tumour induction is common to all oncogenic adenoviruses, our homology studies can be interpreted to suggest that it must be relatively small in size. The cross-hybridizations observed between oncogenic simian adenovirus DNA's of Groups II and III varied from 3o to 55 ~o. Cross-hybridization of these virus DNA's with human adenoviruses of low (Ad 5), intermediate (Ad 7) and high (Ad I2) oncogenicity varied from 2o to 45 ~. Thermal transition studies of these hybrids indicated that imperfect hybridization due to mismatched regions (McCarthy & McConaughy, I968) could account for as much as I5 to 25 ~ of the observed cross-hybridization. Thus, we believe any common sequence of their nucleotides must be a relatively small portion of the genome. This can be correlated with our studies (Mayne, Burnett & Butler, I97I) of the oncogenicity of SA7 DNA in which we demonstrated that separated molecular halves of SA7 DNA could induce tumours, indicating a requirement for less than the whole genome. We are indebted to Mr A. C. Dwyer for preparation of the virus pools used in these studies. We also thank Mrs Dorothy Doerflinger and Mr R. A. Voorhis for skilful technical assistance. REFERENCES BURNETT, J.V. & riarrington, J.A. (1968). Simian adenovirus SA7: chemical, physical, and biological properties. Proceedings of the National Academy of Sciences of the United States of America 60, lo23- lo29. BUR~qETT, J.V., SUMMZRS, A.O., HAR~INGTON, J. A. & DWYER, A. C. (I968). Production of highly labelled adenovirus. Applied Microbiology x6, 1245-t 250. COWlE, D. ~., AVrRY, R. J. & Cr~AMPE, S. V. (1971). DNA homology among the T-even bacteriophages. Virology 45, 3o-37. DENHARDT, O. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochemical and Biophysical Research Communications 23, FREEMAN, A. E., alack, V. r~., VANDERVOOL, E. A., r~enry, V. H., AUSTIN, J. B. & HUEBNER, R. J. (I967). Transformation of primary rat embryo cells by adenovirus type 2. Proceedings of the National Academy of Sciences of the United States of America 58, 12o5-12II. GILDEN, R. V., KERN, J., rtebe~ung, a. L. & ~VESNER, R. J. (~968). Serological studies of the T and tumor antigens of the oncogenic simian adenoviruses. Applied Microbiology r6, I 0 I5-I o 18. GOODHEARX, C. R. (1971). DNA density of oncogenic and non-oncogenic simian adenoviruses. Virology 44, riull, R. N. (I968). The simian viruses. In Virology Monographs, vol. II, p New York: Springer-Verlag. HULL, R. N., JOHNSON, I. S., CULBERTSON, C. B. & WRIGHT, H. W. (1965)- Oncogenicity of the simian adenoviruses. Science, New York 15o, lo44-1o46. MCCARTHY, B. J. & MCCONAUGHY, B. L. (I968). Related base sequences in the DNA of simple and complex organisms. I. DNA/DNA duplex formation and theincidence of partially related base sequences indna. Biochemical Genetics 2, MALRERBE, I~. & HARWIN, R. (I963). The cytopathic effects of vervet monkey viruses. South African Medical Journal 37, 4o7-4I 1. MANDEL, M. & MARMUR, J. (I968). Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. In Methods in Enzymology, vol. x11, p. I95-2o6. New York & London: Academic Press.

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