VIROLOGY
97,406-414
Alignment
(1979)
of the Restriction Map of Mouse Adenovirus with that of Human Adenovirus 2
STEVEN H. LARSEN, ROBERT F. MARGOLSKEE, Department
of Microbiology,
Johns Hopkins
University
School
AND
DANIEL
of Medicine, Baltimore,
FL
NATHANS’ Maryland
21205
Accepted May 28, 1979
A detailed restriction map of mouse adenovirus (AdFL) has been constructed based on sites of cleavage of AdFL DNA by 19 restriction endonucleases. The AdFL map has been oriented with that of human adenovirus 2 (Adz) by identifying which end of AdFL DNA is retained in virus particles with incomplete genomes compared with the end retained by Ad2 defective particles (C. Tibbetts, 1977, Cell 12, 243-249). The two maps were also oriented by cross-hybridization tests with a series of restriction fragments of each viral DNA. The latter experiments revealed two regions of homology between Ad2 and AdFL DNA, corresponding to the positions of the Ad2 hexon gene and an Ad2 hexon-associated protein gene. INTRODUCTION
Mouse adenovirus strain FL (AdFL) is a typical adenovirus, asdefined by its biological properties (Hartley and Rowe, 1960), virion architecture (Wigand et al., 1977; Larsen and Nathans, 19’77),and the structure of its DNA (Larsen and Nathans, 197’7).Although little or no sequence homology was detected by liquid hybridization tests between the DNAs of AdFL and human adenovirus (Larsen and Nathans, 1977), there is evidence for antigenic crossreactivity between virion components of mouse and human adenoviruses, probably involving the common antigenic determinants of the hexon (Hartley and Rowe, 1960;Philipson and Pettersson, 1973;Wigand et al., 1977; Larsen and Nathans, 1977). In this communication we report that AdFL and human Ad2 DNAs share nucleotide sequences that map in the Ad2 hexon gene and in a gene coding for hexon-associated protein. Based on the map positions of these homologous DNA segments and on the preferential retention of one specific end of the viral DNA in naturally arising incomplete particles (Tibbetts, 1977), the restriction map of AdFL has been aligned with that of Ad2. In addition, our previous ’ To whom reprint requests should he addressed. 0042-6822/79/120406-09$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
406
cleavage map of AdFL DNA has been extended to include sites of cleavage by other restriction endonucleases. MATERIALS
AND METHODS
Preparation of viral DNA. AdFL was grown in BALBk 3T3 or Swiss 3T6 mouse cells, and viral DNA (or DNA-protein complex) was prepared from purified virions, as previously described (Larsen and Nathans, 19’7’7).Ad2 DNA was prepared similarly by M. Stern and T. J. Kelly, Jr. AdFL DNA from incomplete particles was isolated from virions with buoyant density in CsCl between that of complete and empty particles. Restriction endonuclease digestions. Restriction endonucleases were purchased from Bethesda Research Laboratories or from New England Biolabs, and used under the following conditions. For digestions by Bali, BamHI, Boll, BgZI, BglII, BstEII, KpnI, and PstI the reaction mixture contained 20 mk! Tris-HCI (pH 7.4), 10 mM MgClz, 6 mM 2-mercaptoethanol, and 100 pg/ml bovine serum albumin. For other enzymes, additional ingredients were added to this mixture: 20 m&l KC1 for HpaI and SmaI; 90 mM NaCl for HindIII, S&I, S&II, XbaI, and XhoI; and 150 mM NaCl for PvuI and Sal I. Digestions with AccI and EcoRI were per-
RESTRICTION
MAP OF AdFL
formed in 100 mM Tris-HCl (pH 7.6), 7.5 mM MgClz, 7 mM 2-mercaptoethanol, and 50 mM NaCl. Gel electrophoresis was carried out in 0.7 or 1% agarose gels (Sharp et al., 1973), under conditions described by Brockman and Nathans (1974). Detection of DNA homology. Following electrophoresis, restriction endonuclease fragments of viral DNA were transferred from agarose gels to nitrocellulose sheets and hybridized to nick-translated probe by the method of Southern (1975), with minor modifications. The 32P-probe was prepared by nick-translation of AdFL or Ad2 DNA, as described by Man~atis (1915) and by Rigby et al. (1977).
407
endonucleases (Larsen and Nathans, 1977): EcoRI, HindHI, BamHl, SalI, HpnI, and BglII. Since that time additional restriction sites have been localized by analyzing double enzyme digests and digests of the viral DNA-protein complex to identify end fragments, as described earlier (Larsen and Nathans, 1977). Tables 1 and 2 indicate the lengths of fragments produced by each of 13 different restriction endonucleases and by digestion with pairs of enzymes. The identity of end fragments is also noted. From these data, and the restriction maps constructed earlier, we can specify the sites of cleavage to within about 2% of the genome length. (Fragments of less than about 1% would not have been detected by our analyses, however.) The resulting cleavage maps are shown in Fig. 1, as are the previously determined maps (Larsen and Nathans, 1977).
RESULTS AND DISCUSSLON
Restriction map of AdFL We have previously reported the sites of cleavage of AdFL DNA by six restriction
TABLE 1 ENWNUCLEASE
R DNA FRAGMENTS”
Endonuclease Fragment A B c D E F G H I f K L M N 0 P
Ace1
BaEI
17.3b 54b 12.6 23 11.7 15.lb 10.1 5.3 8.2 2.3 5.7 5.2 3.8 3.6 3.5’ 2.9 2.8 2.6 2.5 2.4 0.8
BclI
BglI
6P 44.5 24.5b 18 5.5 12.2c 3.3 1O.Y 8.3 4.6 1.8
B&E11
KpnI
PstI
PvuI
53 45c 2.2’
38” 35.5 11.8
16.5 15 10.5 10.0 9.9 8.2” 7.2 5.6’ 5.5 3.8 2.4 2.1 1.0
W 27.5 12” 8.2 8.1
5.9
3.9’ 3.3 2.5
SrmzI
S&I
42.5 31 31.5 27 14-l 16.5 8.5 12.5 2.7 5.0” 0.4” 4.1c o.3b 3.8
S&II
XbaI
XhoI
3@ 23.5 19 15 12.2b
19.5 18.8 19.0 16.5c 16.0’ 11.0
17 16 14 13 8.6 8.6 6.5 4.8b 4.W 3.5 2.2 1.2
o Data are expressed in percentages of AdFL DNA. Fragment lengths were estimated by electrophoretic mobility compared to fragments previously determined (Larsen and Nathans, 1977). The positions of several small fragments have not yet been determined. These include AccI N, 0, and P; HpaI G, P&I Ivl, and XhoI L. In addition, fragments of less than about 1 map unit would not have been detected. b Terminal fragments identified from double enzyme digestion patterns. c Restriction fragments identified as terminal fragments based on their failure to enter the gel when DNA-protein complex is digested or by a mobixty shift when DNA-protein complex fragments are electrophoresed in the presence of 0.5% sodium dodecyl-sulfate (Larsen and Nathana, 1977).
LARSEN, MARGOLSKEE,
408
&I ml
D , H.J. Et’ 4Bm w
so C
,
AND NATHANS
45, F,
. f? F f II RI 46
D
.K? en63
A
. a0
87.8 84 B
.I 96
MAP UNITS FIG. 1. Restriction endonuclease cleavage maps of AdFL DNA. The positions of cleavage sites were derived from the data in Tables 1 and 2. The order of P&I-E and -K fragments is unknown.
Alignment of AdFL Map with that of Ad2 The reader may have noticed that the restriction map shown in Fig. 1 has the opposite orientation to that published earlier (Larsen and Nathans, 19’7’7).This change makes the map comparable to those of human adenoviruses, as documented below. The first experiment that related the AdFL map to those of human adenoviruses involved an analysis of DNA present in AdFL particles of buoyant density intermediate between full and empty virions. Tibetts (1977) reported that in light virions of human adenovirus only the left end of the viral DNA is conserved, “left” being defined by the standard restriction endonuclease cleavage maps. Retention of the left end is thought to be related to the way adenovirus DNA is encapsidated. We therefore decided
FIG. 2. BglI digestion of DNA from incomplete particles. Incomplete viral particles were isolated by centrifugation in CsCl as described under Materials and
Methods. DNA was extracted from each ofthree buoyant density classes, digested with BglI, and electrophoresed in 0.7% agarose. The left well received a digest of DNA from “full” virions. Each successive well to the right received a digest of DNA from virions of decreasing buoyant density. Note the progressive decrease in the ratio of the right-end fragment (D) to the left-end fragment (C).
RESTRICTION
409
MAP OF AdFL
TABLE
2
ENDONUCLEASE R FRAGMENTS PRODUCED BY DOUBLE DIGESTION” Endonuclease
pairs
AccI
AccI
AccI
AccI
Ace1
AccI
AccI
AccI
PstI
PstI
PstI
+
+
+
+
+
+
+
+
+
+
+
PstI +
PstI +
PstI +
Bali
BglII
SstI
BaZI
BclI
BglI
BstEII
KpnI
BamHI
12.5 11.7 11.0 8.2 6.0 4.1 3.9 3.8 3.6 3.5 3.1 3.0 2.9 2.5 2.5 2.4 2.1 1.9 1.4 0.8
15 10.5 10.2 8.8 8.0 7.1 6.2 5.6 5.5 4.7 3.9 2.4 2.3 2.3 2.1 1.0
16 15 10.5 10.2 10.0 8.2 5.6 5.5 3.3 3.0 2.4 2.1 1.4 1.0
11.4 10.4 10.0 8.2 8.2 7.1 6.6 5.6 4.8 4.3 3.8 3.5 2.4 2.1 1.6 1.6 1.0
17 12.5 10.1 8.2 5.2 3.8 3.6 3.5 3.4 3.0 2.9 2.8 2.5 2.4 2.2 2.2 0.8
13 12.5 11.4 10 8.1 5.7 5.2 4.1 3.8 3.6 3.0 2.9 2.6 2.5 2.4 1.1 0.8
BstEII
EcoRI
14.7 12.5 11.5 10.2 8.2 5.7 5.2 3.6 3.5 3.0 2.8 2.6 2.5 2.4 2.2 0.8
17.1 11.7 10 8.3 8.3 5.7 3.7 3.5 3.4 3.0 3.0 2.9 2.7 2.6 2.5 2.1 0.8
Hind111 12.5 11.5 10.5 7.6 7.6 5.7 5.2 5.2 3.8 3.6 3.5 2.8 2.6 2.6 2.5 2.0 1.1 0.8
&a1
KpnI
13 9.6 8.2 6.6 5.7 5.7 5.3 5.3 3.8 3.8 3.6 3.5 3.0 2.9 2.6 2.5 2.4 2.1 1.5 0.8
13.3 12.7 11.7 10.1 8.2 7.0 6.4 5.6 4.4 3.8 3.6 3.5 2.8 2.6 2.5 2.4 1.1 0.8
TABLE
PstI
PstI
PstI
PstI
PAI
+ BglII
+ EcoRI
+ Hind111
+ HpaI
+ SaEI
PstI + SstI
16 15 10.0 9.9 8.2 5.6 5.5 4.2 3.8 3.6 3.5 3.1 2.4 2.3 2.2 2.1 1.0
13.7 10.4 10.1 10.0 7.2 5.6 5.1 4.2 3.8 2.7 2.7 2.5 2.1 1.4 1.3 1.1 1.0
16 10.5 10 9.8 9.0 6.0 5.6 5.5 5.3 3.8 2.4 2.4 2.1 1.1 1.0
16 13.5 10.4 10.0 9.9 7.6 7.1 5.6 5.5 3.8 2.4 2.1 1.5 1.5 1.0
16 10.5 10.4 8.2 8.0 7.2 5.5 4.9 4.9 4.8 3.8 3.5 2.4 1.9 1.9 1.5 1.0
13 10.0 10.0 7.7 6.6 6.6 5.7 5.7 5.5 3.9 3.9 3.8 3.1 3.0 2.5 2.1 1.5 1.0
16 15 10.5 9.9 8.2 8.2 7.1 5.4 3.8 3.2 2.4 2.1 2.1 1.0
16 12 10.5 10.0 8.2 7.2 5.5 3.8 3.3 3.0 2.8 2.5 2.4 1.6
15 11 10.4 9.8 7.1 5.4 5.3 5.2 4.6 3.8 2.4 2.1 2.1 1.2 1.0
XhoI + BamHI
XhoI
XhoI + EcoRI
2-Continued
XhoI + B&I 16 14.5 11.3 8.0 8.0 6.0 4.6 4.1 3.9 3.4 2.3 1.2 1.1
XhoI + BclI 16.5 15.7 12.0 8.0 8.0 4.7 3.9 3.4 3.3 3.1 2.4 2.1 1.1
XhI + BgZI
XhoI + BstEII
14.5 10.5 9.6 8.0 7.5 6.2 6.0 4.8 4.8 4.2 3.9 3.4 3.4 2.5 2.1 1.8 1.3 1.1
16.5 16 14.5 11.5 7.9 6.0 5.8 4.7 3.3 2.2 2.1 1.1
XhoI + SstI 16 15 13 12 8.3 8.3 4.8 4.0 3.5 3.1 2.7 2.2 1.3 1.1
14 13 12 8.8 8.8 6.0 3.8 3.5 3.3 2.5 2.5 2.1 1.3 1.3 1.1 1.0
+ BglII 16.5 13 12 8.6 7.9 7.9 7.2 6.0 3.9 3.3 2.5 2.2 2.1 1.6 1.2 1.1
16.5 16 14.5 12 8.0 8.0 6.0 4.8 3.9 2.5 2.2 1.1 1.0
LARSEN,
410
MARGOLSKEE, TABLE
XhoI + Hind111 15 15 8.6 8.0 7.4 7.1 6.0 5.2 4.8 3.5 3.3 2.6 2.1 1.9 1.1
XhoI + HpaI 16 12.5 8.5 8.0 6.0 4.8 3.6 2.1 2.0 1.6 1.6 1.5 1.3 1.1
BatI + BglI 38 13 12 10.5 5.5 4.6 4.2 3.1 2.4 1.8
BatI + BstEII
Bali + EcoRI
45 17.5 13 6.6 5.2 2.4 2.3
44 18.5 15 7.4 5.2 4.4 2.4
Bali + Hind111
Z-Continued Bat1 + PvuI
Bat1 BaZI + + Sat1 SmaI
26 22 15 11.5 8.0 5.2 2.4 2.1
22 19.5 14.5 12 11 5.1 3.5 2.4 2.0
44 15.5 9.5 7.4 7.4 5.1 3.5 2.4
TABLE BglI + Hind111 32 12.2 12 10.0 8.3 6.4 5.4 5.2 4.3
BgtI + Sal1
BgtI + SmaI
BgZI + S&I
20 18.5 12.0 11.0 9.4 8.4 4.6 3.5 2.2 1.8
45 18 12.2 8.3 7.0 6.0
21.5 20 8.3 6.7 6.7 6.0 6.0 6.0 5.0 4.0 4.0
BglI + XbaI 19 13.7 12.3 11.6 11.5 10.8 7.6 5.0 4.0
AND NATHANS
BetI + BstEII
BclI + EcoRI
38 18 15 12 5.4 3.0 2.4 2.4
55 24 9.5 5.5 3.0 2.3
50 19.5 16 7.4 5.5 3.0
BglI + BamHI
BglI + EcoRI
28 18 1’7 6.8 5.9 5.2 4.7 4.5 4.3 1.8
45 10.5 10.0 8.8 8.3 2.4
32 22.5 12.5 11 6.5 6.0 5.2 2.6 1.5
2-Continued
BstEII + BamHI
BstEII + KpnI
BstEII + EcoRI
BstEII + SstII
BstEII + XhaI
KpnI + EcoRI
30.5 24.5 19.5 12.6 4.6 2.9 2.1 1.2
39 35 11.8 6.0 3.3 2.5 2.1 1.6 1.2
38 36 17.8 7.6 2.3
28 21 17 13.5 10.3 2.3
19 18.3 17.8 16.4 13.5 8.2 2.5 2.2
36 30 10.5 7.6 6.0 3.9 3.3 2.5 1.4
TABLE
BclI + HpaI
KpnI + Hind111 39 24 10.4 8.0 6.0 3.5 3.3 1.8 1.5
KpnI + Sat1 29 12.3 11.8 11.5 8.8 5.9 5.5 3.9 3.5 3.3 2.5
SmaI + BamHI
SmI + EcoRi
SmaI + Hind111
SnlaI + Sal1
SstI + BgZII
SstI + EcoRI
SstI + Sat1
SstII + BamHI
sst11 + EcoRI
SstII + HpaI
SstII + Sal1
18.6 16.0 15.9 14 12 11 4.0
30 24 13.8 9.4 7.5 4.5 2.5 2.1
44 17 14 9.0 7.8 7.0
30 14.5 12 10.7 10.0 9.0 7.5 5.5
31 20 11.5 11.3 8.9 7.6 3.5 3.4 2.5
26.5 25 17 8.3 5.0 4.2 3.9 3.5 2.3 2.2
27 27 17 12.5 5.0 4.0 4.0
16.5 14 13 12.5 11.5 11.5 10.8 6.6
24 18.5 18 14 6.6 5.4 4.4 4.4
23.5 21.5 14.7 12.3 10.5 7.6 7.4
17 17 14.7 12.3 11.0 9.5 6.4 6.0 2.7
22 18 14.7 14.4 12.3 11.3 3.5 2.7
of AdFL
19 17.8 16.3 12.5 5.9 3.9
2-Continued
PWUI + X5aI
a Data are expressed in percentages
KpnI + XbaI
DNA.
See Footnote
a to Table 1.
XbaI + Hind111 19.3 17.1 14.2 11.2 7.8 7.6 7.6 6.0 4.9 3.5
RESTRICTION
to use this encapsidation polarity to define the comparable “left end” of AdFL DNA. For this purpose, 32P-AdFL virions were concentrated and centrifuged to equilibrium in CsCl of 1.30 g/cc average density. When DNA from each intermediate buoyant density fraction was analyzed by electrophoresis in agarose, short DNA molecules were observed, the size range of which correlated with the buoyant density of the particle from which the DNA was derived. A fraction of each DNA sample was then digested with BglI. As seen in Fig. 2, only one of the two BglI end fragments is conserved in the DNA from light particles, namely BglI-C. By this criterion, therefore, BgZI fragment C defines the “left” end of AdFL DNA.
411
MAP OF AdFL
The second experiment that oriented the AdFL map to those of human adenoviruses involved the location of small regions of DNA homology in the AdFL and Ad2 cleavage maps. Hartley and Rowe (1960) reported that sera made against human adenoviruses cross-reacted with lysates of AdFL-infected cells. Human adenovirus anti-T sera, however, showed no cross-reactivity with such cell lysates (Larsen and Nathans, 1977). We had earlier reported that AdFL DNA had less than about 10% sequence homology with human Ad2, Ad7, or Ad12, when tested by DNA-DNA annealing in solution (Larsen and Nathans, 1977). Nonetheless, the serologic cross-reactivity suggested possible homology in one or more genes for virion proteins, the de=
-
-
-
FIG. 3. Fragments of Ad2 DNA containing sequenice homology with AdFL DNA. Ethidium bromidestained gel of Ad2 DNA cleaved by each of four restriction enzymes (left). The DNA in this gel was transferred to a nitrocellulose filter and 3ZP-ALdFL DNA hybridized to it to detect homologous sequences (right).
412
LARSEN,
MARGOLSKEE.
tection of which should also allow orientation of restriction maps. To increase the sensitivity of detecting DNA sequence homology and at the same time to map any homologous sites, restriction fragments of Ad2 DNA were transferred to nitrocellulose sheets by the method of Southern (19’75)and then hybridized with 32P-labeled AdFL DNA. Reciprocal experiments, in which fragments of AdFL DNA were probed with 32P-Ad2DNA were also carried out. The results are presented in Figs. 3 and 4 as photographs of the ethidium bromide-stained gels and of corresponding autoradiograms that show the restriction
AND NATHANS
fragments that cross-anneal and the approximate extent of annealing. As seen in Figs. 3 and 4, cross-annealing is readily detectable and is limited to a few fragments in each digest. Generally there is one fragment in each digest that crossanneals extensively (dark bands in the autoradiograms) and one or more that crossanneals less extensively (light bands). When the homologous fragments of Ad2 or AdFL DNA were located on the cleavage maps of the respective viruses, the results shown in Fig. 5 were obtained. As seen in the figure, two discrete homologous segments are present, and the map positions of AdFL
FIG. 4. Fragments of AdFL DNA containing sequence homology with Ad2 DNA. Ethidium bromide stain of AdFL DNA cleaved by each of four restriction enzymes (left). The DNA in this gel was transferred to a nitrocellulose fdter and 5*P-Ad2 DNA hybridized to it to detect homologous sequences (right).
RESTRICTION
413
MAP OF AdFL
Ad2MAP HexanmRNA
ttFii%w k9 ’ l&d III EmRI ml Siral
Ed
.
Cc,
iF’
, I .Jf
D
. ’
A”’ G: ‘4E
B ::F::
hlO(lY Limits
:
C B
’
:‘I”:
D
:I:
D
D
B A ,HbE .S.F,6,E;C’ :
E
:
G
,F.K , F G ,N
A
:“:
Is-l8
5162
Ad FL MAP A
HiJd III ~IE;C:D: !W’ Boll mobw Limits
, B
F:
D
B
c
A
;
D:E! E
616
54-
,F,
. ’ B
C
E
,E,F
,
C D
66
FIG. 5. Maps of Ad2/AdFL homology regions. Relevant restriction maps of Ad2 and AdFL are shown with fragments corresponding to the darker bands in the autoradiogram of Figs. 3 and 4 drawn as very thick lines and the lighter bands as lines of intermediate thickness. The locations of genes coding for hexon protein and an hexon-associated protein on the Ad2 map (Chow et al., 1977) are shown at the top. The Ad2 cleavage map data is from Mulder et al., 1974 for EcoRI and HpaI and by personal communication for SmaI from C. Mulder and R. Greene, for Hind111 from R. J. Roberts and J. Sambrook, and for K*I from M. B. Mathews, R. Greene, and J. Sambrook.
fragments closely correspond to those of the homologous Ad2 fragments when the maps are oriented on the basis of the ends retained in light virions. The darker Ad2 bands all contain an overlapping region within map coordinates 51 and 62 in the standard Ad2 map, and the corresponding dark bands of AdFL DNA map within coordinates 54 and 66 map units in the AdFL map. Some of the less dense bands in each autoradiogram appear to include one or the other end of this region of homology. The other less dense bands map at a different site within coordinates 12 and 18 in the Ad2 map, and within 6 and 16 in the AdFL map. The detection of two regions of homology that correspond in position when the maps are oriented as shown in Fig. 5 thus supports this orientation of the AdFL map with respect to the Ad2 map.
coordinate 11.2 and 14.9 (Fig. 5). It is therefore likely that the AdFL genes for hexon protein and an hexon-associated protein are located in approximately corresponding positions in the viral DNA and that the cross-reactivity between AdFL and human adenoviruses is due at least in part to these gene products. We infer from the intensity of autoradiographic bands in Figs. 3 and 4, that the regions of homology between the hexon genes of AdFL and Ad2 are more extensive than between the genes for hexon-associated protein. In both eases, however, the 32P-probe DNA can be released from the filter at concentrations of salt higher than that required for disrupting homologous DNA duplexes. Therefore, even in the case of the hexon genes, homology is incomplete. ACKNOWLEDGMENTS
Nature of the Homologous Segments The two regions of Ad2 DNA that are homologous to the comparable segments of AdFL DNA can be identified by reference to the functional map of Ad2 (Fig. 5). Chow et al. (1977) found that the body of the Ad2 hexon mRNA maps between 51.6 and 62.0 map units, and that the body of an hexonassociated protein mRNA maps between
We thank Keith Peden for much helpful advice, M. J. Stem, and T. J. Kelly, Jr. for DNA samples, and J. Suthers, T. Zeller and L. K. English for able technical assistance. This research was supported by grants from the Whitehall Foundation and the USPHS, National Cancer Institute (CA16519). S.H.L. was a post-doctoral trainee of the National Cancer Institute (CA 09139), and R.F.M. is a pre-doctoral trainee under the Medical Scientist Training Program of the USPHS, National Institutes of Health (5T32 GM07399).
414
LARSEN, MARGOLSKEE, REFERENCES
AND NATHANS
virus types 2 and 5 by restriction endonuclease EcoRI and HpaI. Cold Spring Harbor Symp. BROCKMAN,W. W., and NATHANS, D. (1974). The isoQwLnt. Biol. 39, 397-400. lation of simian virus 40 variants with specifically PETTERSSON,U. (1971). Structural proteins of adenoaltered genomes. Proc. Nut. Acad. Sci. USA ‘71, viruses. Virology 43, 123-136. 942-946. CHOW, L. T., ROBERTS, J. M., LEWIS, J. B., and RIGBY, P. W., DIECKMANN, M., RHODES, C., and BERG, P. (1977). Labeling deoxyribonucleic acid to BROKER, T. R. (1977). A map of cytoplasmic RNA high specific activity in vitro by nick-translation with transcripts from lytic adenovirus type 2, determined DNA polymerase I. J. Mol. Biol. 113, 237-251. by electron microscopy of RNADNA hybrids. Cell SHARP, P. A., SUGDEN,B., and SAMBROOK,J. (1973). 11, 819-836. Detection of two restriction endonuclease activities HARTLEY, J. S., and ROWE,W. P. (1960). A new mouse in Haemophilus pamin@enzae using analytical agavirus apparently related to the adenovirus group. rose-ethidium bromide electrophoresis. BiochemVirology 11, 645-647. istry 12, 3055-3063. LARSEN, S. H., and NATHANS, D. (1977). Mouse adenovirus: Growth of plaque-purified FL virus in cell SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel lines and characterization of viral DNA. Virology electrophoresis. J. Mol. Biol. 98, 503-517. 82, 182-195. MANIATIS, T., JEFFREY, A., and KLEID, D. G. (1975). TIBBETTS, C. (1977). Viral DNA sequences from incomplete particles of human adenovirus type 7. Cell Nucleotide sequence of the rightward operator of 12, 243-249. phage h. Proc. Nat. Acad. 5%. USA 72,1184-1188. MULDER, C., ARRAND, J. R., DELIUS, H., KELLER, WIGAND, R., GELDERBLOM,H., and OZEL, M. (1977). W., PEITERSSON, Il., ROBERTS,R. J., and SHARP, Biological and biophysical characteristics of mouse P. A. (1974). Cleavage maps of DNA from adenoadenovirus, strain FL. Arch. Viral. 54, 131-142.
97,406-414
Alignment
(1979)
of the Restriction Map of Mouse Adenovirus with that of Human Adenovirus 2
STEVEN H. LARSEN, ROBERT F. MARGOLSKEE, Department
of Microbiology,
Johns Hopkins
University
School
AND
DANIEL
of Medicine, Baltimore,
FL
NATHANS’ Maryland
21205
Accepted May 28, 1979
A detailed restriction map of mouse adenovirus (AdFL) has been constructed based on sites of cleavage of AdFL DNA by 19 restriction endonucleases. The AdFL map has been oriented with that of human adenovirus 2 (Adz) by identifying which end of AdFL DNA is retained in virus particles with incomplete genomes compared with the end retained by Ad2 defective particles (C. Tibbetts, 1977, Cell 12, 243-249). The two maps were also oriented by cross-hybridization tests with a series of restriction fragments of each viral DNA. The latter experiments revealed two regions of homology between Ad2 and AdFL DNA, corresponding to the positions of the Ad2 hexon gene and an Ad2 hexon-associated protein gene. INTRODUCTION
Mouse adenovirus strain FL (AdFL) is a typical adenovirus, asdefined by its biological properties (Hartley and Rowe, 1960), virion architecture (Wigand et al., 1977; Larsen and Nathans, 19’77),and the structure of its DNA (Larsen and Nathans, 197’7).Although little or no sequence homology was detected by liquid hybridization tests between the DNAs of AdFL and human adenovirus (Larsen and Nathans, 1977), there is evidence for antigenic crossreactivity between virion components of mouse and human adenoviruses, probably involving the common antigenic determinants of the hexon (Hartley and Rowe, 1960;Philipson and Pettersson, 1973;Wigand et al., 1977; Larsen and Nathans, 1977). In this communication we report that AdFL and human Ad2 DNAs share nucleotide sequences that map in the Ad2 hexon gene and in a gene coding for hexon-associated protein. Based on the map positions of these homologous DNA segments and on the preferential retention of one specific end of the viral DNA in naturally arising incomplete particles (Tibbetts, 1977), the restriction map of AdFL has been aligned with that of Ad2. In addition, our previous ’ To whom reprint requests should he addressed. 0042-6822/79/120406-09$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
406
cleavage map of AdFL DNA has been extended to include sites of cleavage by other restriction endonucleases. MATERIALS
AND METHODS
Preparation of viral DNA. AdFL was grown in BALBk 3T3 or Swiss 3T6 mouse cells, and viral DNA (or DNA-protein complex) was prepared from purified virions, as previously described (Larsen and Nathans, 19’7’7).Ad2 DNA was prepared similarly by M. Stern and T. J. Kelly, Jr. AdFL DNA from incomplete particles was isolated from virions with buoyant density in CsCl between that of complete and empty particles. Restriction endonuclease digestions. Restriction endonucleases were purchased from Bethesda Research Laboratories or from New England Biolabs, and used under the following conditions. For digestions by Bali, BamHI, Boll, BgZI, BglII, BstEII, KpnI, and PstI the reaction mixture contained 20 mk! Tris-HCI (pH 7.4), 10 mM MgClz, 6 mM 2-mercaptoethanol, and 100 pg/ml bovine serum albumin. For other enzymes, additional ingredients were added to this mixture: 20 m&l KC1 for HpaI and SmaI; 90 mM NaCl for HindIII, S&I, S&II, XbaI, and XhoI; and 150 mM NaCl for PvuI and Sal I. Digestions with AccI and EcoRI were per-
RESTRICTION
MAP OF AdFL
formed in 100 mM Tris-HCl (pH 7.6), 7.5 mM MgClz, 7 mM 2-mercaptoethanol, and 50 mM NaCl. Gel electrophoresis was carried out in 0.7 or 1% agarose gels (Sharp et al., 1973), under conditions described by Brockman and Nathans (1974). Detection of DNA homology. Following electrophoresis, restriction endonuclease fragments of viral DNA were transferred from agarose gels to nitrocellulose sheets and hybridized to nick-translated probe by the method of Southern (1975), with minor modifications. The 32P-probe was prepared by nick-translation of AdFL or Ad2 DNA, as described by Man~atis (1915) and by Rigby et al. (1977).
407
endonucleases (Larsen and Nathans, 1977): EcoRI, HindHI, BamHl, SalI, HpnI, and BglII. Since that time additional restriction sites have been localized by analyzing double enzyme digests and digests of the viral DNA-protein complex to identify end fragments, as described earlier (Larsen and Nathans, 1977). Tables 1 and 2 indicate the lengths of fragments produced by each of 13 different restriction endonucleases and by digestion with pairs of enzymes. The identity of end fragments is also noted. From these data, and the restriction maps constructed earlier, we can specify the sites of cleavage to within about 2% of the genome length. (Fragments of less than about 1% would not have been detected by our analyses, however.) The resulting cleavage maps are shown in Fig. 1, as are the previously determined maps (Larsen and Nathans, 1977).
RESULTS AND DISCUSSLON
Restriction map of AdFL We have previously reported the sites of cleavage of AdFL DNA by six restriction
TABLE 1 ENWNUCLEASE
R DNA FRAGMENTS”
Endonuclease Fragment A B c D E F G H I f K L M N 0 P
Ace1
BaEI
17.3b 54b 12.6 23 11.7 15.lb 10.1 5.3 8.2 2.3 5.7 5.2 3.8 3.6 3.5’ 2.9 2.8 2.6 2.5 2.4 0.8
BclI
BglI
6P 44.5 24.5b 18 5.5 12.2c 3.3 1O.Y 8.3 4.6 1.8
B&E11
KpnI
PstI
PvuI
53 45c 2.2’
38” 35.5 11.8
16.5 15 10.5 10.0 9.9 8.2” 7.2 5.6’ 5.5 3.8 2.4 2.1 1.0
W 27.5 12” 8.2 8.1
5.9
3.9’ 3.3 2.5
SrmzI
S&I
42.5 31 31.5 27 14-l 16.5 8.5 12.5 2.7 5.0” 0.4” 4.1c o.3b 3.8
S&II
XbaI
XhoI
3@ 23.5 19 15 12.2b
19.5 18.8 19.0 16.5c 16.0’ 11.0
17 16 14 13 8.6 8.6 6.5 4.8b 4.W 3.5 2.2 1.2
o Data are expressed in percentages of AdFL DNA. Fragment lengths were estimated by electrophoretic mobility compared to fragments previously determined (Larsen and Nathans, 1977). The positions of several small fragments have not yet been determined. These include AccI N, 0, and P; HpaI G, P&I Ivl, and XhoI L. In addition, fragments of less than about 1 map unit would not have been detected. b Terminal fragments identified from double enzyme digestion patterns. c Restriction fragments identified as terminal fragments based on their failure to enter the gel when DNA-protein complex is digested or by a mobixty shift when DNA-protein complex fragments are electrophoresed in the presence of 0.5% sodium dodecyl-sulfate (Larsen and Nathana, 1977).
LARSEN, MARGOLSKEE,
408
&I ml
D , H.J. Et’ 4Bm w
so C
,
AND NATHANS
45, F,
. f? F f II RI 46
D
.K? en63
A
. a0
87.8 84 B
.I 96
MAP UNITS FIG. 1. Restriction endonuclease cleavage maps of AdFL DNA. The positions of cleavage sites were derived from the data in Tables 1 and 2. The order of P&I-E and -K fragments is unknown.
Alignment of AdFL Map with that of Ad2 The reader may have noticed that the restriction map shown in Fig. 1 has the opposite orientation to that published earlier (Larsen and Nathans, 19’7’7).This change makes the map comparable to those of human adenoviruses, as documented below. The first experiment that related the AdFL map to those of human adenoviruses involved an analysis of DNA present in AdFL particles of buoyant density intermediate between full and empty virions. Tibetts (1977) reported that in light virions of human adenovirus only the left end of the viral DNA is conserved, “left” being defined by the standard restriction endonuclease cleavage maps. Retention of the left end is thought to be related to the way adenovirus DNA is encapsidated. We therefore decided
FIG. 2. BglI digestion of DNA from incomplete particles. Incomplete viral particles were isolated by centrifugation in CsCl as described under Materials and
Methods. DNA was extracted from each ofthree buoyant density classes, digested with BglI, and electrophoresed in 0.7% agarose. The left well received a digest of DNA from “full” virions. Each successive well to the right received a digest of DNA from virions of decreasing buoyant density. Note the progressive decrease in the ratio of the right-end fragment (D) to the left-end fragment (C).
RESTRICTION
409
MAP OF AdFL
TABLE
2
ENDONUCLEASE R FRAGMENTS PRODUCED BY DOUBLE DIGESTION” Endonuclease
pairs
AccI
AccI
AccI
AccI
Ace1
AccI
AccI
AccI
PstI
PstI
PstI
+
+
+
+
+
+
+
+
+
+
+
PstI +
PstI +
PstI +
Bali
BglII
SstI
BaZI
BclI
BglI
BstEII
KpnI
BamHI
12.5 11.7 11.0 8.2 6.0 4.1 3.9 3.8 3.6 3.5 3.1 3.0 2.9 2.5 2.5 2.4 2.1 1.9 1.4 0.8
15 10.5 10.2 8.8 8.0 7.1 6.2 5.6 5.5 4.7 3.9 2.4 2.3 2.3 2.1 1.0
16 15 10.5 10.2 10.0 8.2 5.6 5.5 3.3 3.0 2.4 2.1 1.4 1.0
11.4 10.4 10.0 8.2 8.2 7.1 6.6 5.6 4.8 4.3 3.8 3.5 2.4 2.1 1.6 1.6 1.0
17 12.5 10.1 8.2 5.2 3.8 3.6 3.5 3.4 3.0 2.9 2.8 2.5 2.4 2.2 2.2 0.8
13 12.5 11.4 10 8.1 5.7 5.2 4.1 3.8 3.6 3.0 2.9 2.6 2.5 2.4 1.1 0.8
BstEII
EcoRI
14.7 12.5 11.5 10.2 8.2 5.7 5.2 3.6 3.5 3.0 2.8 2.6 2.5 2.4 2.2 0.8
17.1 11.7 10 8.3 8.3 5.7 3.7 3.5 3.4 3.0 3.0 2.9 2.7 2.6 2.5 2.1 0.8
Hind111 12.5 11.5 10.5 7.6 7.6 5.7 5.2 5.2 3.8 3.6 3.5 2.8 2.6 2.6 2.5 2.0 1.1 0.8
&a1
KpnI
13 9.6 8.2 6.6 5.7 5.7 5.3 5.3 3.8 3.8 3.6 3.5 3.0 2.9 2.6 2.5 2.4 2.1 1.5 0.8
13.3 12.7 11.7 10.1 8.2 7.0 6.4 5.6 4.4 3.8 3.6 3.5 2.8 2.6 2.5 2.4 1.1 0.8
TABLE
PstI
PstI
PstI
PstI
PAI
+ BglII
+ EcoRI
+ Hind111
+ HpaI
+ SaEI
PstI + SstI
16 15 10.0 9.9 8.2 5.6 5.5 4.2 3.8 3.6 3.5 3.1 2.4 2.3 2.2 2.1 1.0
13.7 10.4 10.1 10.0 7.2 5.6 5.1 4.2 3.8 2.7 2.7 2.5 2.1 1.4 1.3 1.1 1.0
16 10.5 10 9.8 9.0 6.0 5.6 5.5 5.3 3.8 2.4 2.4 2.1 1.1 1.0
16 13.5 10.4 10.0 9.9 7.6 7.1 5.6 5.5 3.8 2.4 2.1 1.5 1.5 1.0
16 10.5 10.4 8.2 8.0 7.2 5.5 4.9 4.9 4.8 3.8 3.5 2.4 1.9 1.9 1.5 1.0
13 10.0 10.0 7.7 6.6 6.6 5.7 5.7 5.5 3.9 3.9 3.8 3.1 3.0 2.5 2.1 1.5 1.0
16 15 10.5 9.9 8.2 8.2 7.1 5.4 3.8 3.2 2.4 2.1 2.1 1.0
16 12 10.5 10.0 8.2 7.2 5.5 3.8 3.3 3.0 2.8 2.5 2.4 1.6
15 11 10.4 9.8 7.1 5.4 5.3 5.2 4.6 3.8 2.4 2.1 2.1 1.2 1.0
XhoI + BamHI
XhoI
XhoI + EcoRI
2-Continued
XhoI + B&I 16 14.5 11.3 8.0 8.0 6.0 4.6 4.1 3.9 3.4 2.3 1.2 1.1
XhoI + BclI 16.5 15.7 12.0 8.0 8.0 4.7 3.9 3.4 3.3 3.1 2.4 2.1 1.1
XhI + BgZI
XhoI + BstEII
14.5 10.5 9.6 8.0 7.5 6.2 6.0 4.8 4.8 4.2 3.9 3.4 3.4 2.5 2.1 1.8 1.3 1.1
16.5 16 14.5 11.5 7.9 6.0 5.8 4.7 3.3 2.2 2.1 1.1
XhoI + SstI 16 15 13 12 8.3 8.3 4.8 4.0 3.5 3.1 2.7 2.2 1.3 1.1
14 13 12 8.8 8.8 6.0 3.8 3.5 3.3 2.5 2.5 2.1 1.3 1.3 1.1 1.0
+ BglII 16.5 13 12 8.6 7.9 7.9 7.2 6.0 3.9 3.3 2.5 2.2 2.1 1.6 1.2 1.1
16.5 16 14.5 12 8.0 8.0 6.0 4.8 3.9 2.5 2.2 1.1 1.0
LARSEN,
410
MARGOLSKEE, TABLE
XhoI + Hind111 15 15 8.6 8.0 7.4 7.1 6.0 5.2 4.8 3.5 3.3 2.6 2.1 1.9 1.1
XhoI + HpaI 16 12.5 8.5 8.0 6.0 4.8 3.6 2.1 2.0 1.6 1.6 1.5 1.3 1.1
BatI + BglI 38 13 12 10.5 5.5 4.6 4.2 3.1 2.4 1.8
BatI + BstEII
Bali + EcoRI
45 17.5 13 6.6 5.2 2.4 2.3
44 18.5 15 7.4 5.2 4.4 2.4
Bali + Hind111
Z-Continued Bat1 + PvuI
Bat1 BaZI + + Sat1 SmaI
26 22 15 11.5 8.0 5.2 2.4 2.1
22 19.5 14.5 12 11 5.1 3.5 2.4 2.0
44 15.5 9.5 7.4 7.4 5.1 3.5 2.4
TABLE BglI + Hind111 32 12.2 12 10.0 8.3 6.4 5.4 5.2 4.3
BgtI + Sal1
BgtI + SmaI
BgZI + S&I
20 18.5 12.0 11.0 9.4 8.4 4.6 3.5 2.2 1.8
45 18 12.2 8.3 7.0 6.0
21.5 20 8.3 6.7 6.7 6.0 6.0 6.0 5.0 4.0 4.0
BglI + XbaI 19 13.7 12.3 11.6 11.5 10.8 7.6 5.0 4.0
AND NATHANS
BetI + BstEII
BclI + EcoRI
38 18 15 12 5.4 3.0 2.4 2.4
55 24 9.5 5.5 3.0 2.3
50 19.5 16 7.4 5.5 3.0
BglI + BamHI
BglI + EcoRI
28 18 1’7 6.8 5.9 5.2 4.7 4.5 4.3 1.8
45 10.5 10.0 8.8 8.3 2.4
32 22.5 12.5 11 6.5 6.0 5.2 2.6 1.5
2-Continued
BstEII + BamHI
BstEII + KpnI
BstEII + EcoRI
BstEII + SstII
BstEII + XhaI
KpnI + EcoRI
30.5 24.5 19.5 12.6 4.6 2.9 2.1 1.2
39 35 11.8 6.0 3.3 2.5 2.1 1.6 1.2
38 36 17.8 7.6 2.3
28 21 17 13.5 10.3 2.3
19 18.3 17.8 16.4 13.5 8.2 2.5 2.2
36 30 10.5 7.6 6.0 3.9 3.3 2.5 1.4
TABLE
BclI + HpaI
KpnI + Hind111 39 24 10.4 8.0 6.0 3.5 3.3 1.8 1.5
KpnI + Sat1 29 12.3 11.8 11.5 8.8 5.9 5.5 3.9 3.5 3.3 2.5
SmaI + BamHI
SmI + EcoRi
SmaI + Hind111
SnlaI + Sal1
SstI + BgZII
SstI + EcoRI
SstI + Sat1
SstII + BamHI
sst11 + EcoRI
SstII + HpaI
SstII + Sal1
18.6 16.0 15.9 14 12 11 4.0
30 24 13.8 9.4 7.5 4.5 2.5 2.1
44 17 14 9.0 7.8 7.0
30 14.5 12 10.7 10.0 9.0 7.5 5.5
31 20 11.5 11.3 8.9 7.6 3.5 3.4 2.5
26.5 25 17 8.3 5.0 4.2 3.9 3.5 2.3 2.2
27 27 17 12.5 5.0 4.0 4.0
16.5 14 13 12.5 11.5 11.5 10.8 6.6
24 18.5 18 14 6.6 5.4 4.4 4.4
23.5 21.5 14.7 12.3 10.5 7.6 7.4
17 17 14.7 12.3 11.0 9.5 6.4 6.0 2.7
22 18 14.7 14.4 12.3 11.3 3.5 2.7
of AdFL
19 17.8 16.3 12.5 5.9 3.9
2-Continued
PWUI + X5aI
a Data are expressed in percentages
KpnI + XbaI
DNA.
See Footnote
a to Table 1.
XbaI + Hind111 19.3 17.1 14.2 11.2 7.8 7.6 7.6 6.0 4.9 3.5
RESTRICTION
to use this encapsidation polarity to define the comparable “left end” of AdFL DNA. For this purpose, 32P-AdFL virions were concentrated and centrifuged to equilibrium in CsCl of 1.30 g/cc average density. When DNA from each intermediate buoyant density fraction was analyzed by electrophoresis in agarose, short DNA molecules were observed, the size range of which correlated with the buoyant density of the particle from which the DNA was derived. A fraction of each DNA sample was then digested with BglI. As seen in Fig. 2, only one of the two BglI end fragments is conserved in the DNA from light particles, namely BglI-C. By this criterion, therefore, BgZI fragment C defines the “left” end of AdFL DNA.
411
MAP OF AdFL
The second experiment that oriented the AdFL map to those of human adenoviruses involved the location of small regions of DNA homology in the AdFL and Ad2 cleavage maps. Hartley and Rowe (1960) reported that sera made against human adenoviruses cross-reacted with lysates of AdFL-infected cells. Human adenovirus anti-T sera, however, showed no cross-reactivity with such cell lysates (Larsen and Nathans, 1977). We had earlier reported that AdFL DNA had less than about 10% sequence homology with human Ad2, Ad7, or Ad12, when tested by DNA-DNA annealing in solution (Larsen and Nathans, 1977). Nonetheless, the serologic cross-reactivity suggested possible homology in one or more genes for virion proteins, the de=
-
-
-
FIG. 3. Fragments of Ad2 DNA containing sequenice homology with AdFL DNA. Ethidium bromidestained gel of Ad2 DNA cleaved by each of four restriction enzymes (left). The DNA in this gel was transferred to a nitrocellulose filter and 3ZP-ALdFL DNA hybridized to it to detect homologous sequences (right).
412
LARSEN,
MARGOLSKEE.
tection of which should also allow orientation of restriction maps. To increase the sensitivity of detecting DNA sequence homology and at the same time to map any homologous sites, restriction fragments of Ad2 DNA were transferred to nitrocellulose sheets by the method of Southern (19’75)and then hybridized with 32P-labeled AdFL DNA. Reciprocal experiments, in which fragments of AdFL DNA were probed with 32P-Ad2DNA were also carried out. The results are presented in Figs. 3 and 4 as photographs of the ethidium bromide-stained gels and of corresponding autoradiograms that show the restriction
AND NATHANS
fragments that cross-anneal and the approximate extent of annealing. As seen in Figs. 3 and 4, cross-annealing is readily detectable and is limited to a few fragments in each digest. Generally there is one fragment in each digest that crossanneals extensively (dark bands in the autoradiograms) and one or more that crossanneals less extensively (light bands). When the homologous fragments of Ad2 or AdFL DNA were located on the cleavage maps of the respective viruses, the results shown in Fig. 5 were obtained. As seen in the figure, two discrete homologous segments are present, and the map positions of AdFL
FIG. 4. Fragments of AdFL DNA containing sequence homology with Ad2 DNA. Ethidium bromide stain of AdFL DNA cleaved by each of four restriction enzymes (left). The DNA in this gel was transferred to a nitrocellulose fdter and 5*P-Ad2 DNA hybridized to it to detect homologous sequences (right).
RESTRICTION
413
MAP OF AdFL
Ad2MAP HexanmRNA
ttFii%w k9 ’ l&d III EmRI ml Siral
Ed
.
Cc,
iF’
, I .Jf
D
. ’
A”’ G: ‘4E
B ::F::
hlO(lY Limits
:
C B
’
:‘I”:
D
:I:
D
D
B A ,HbE .S.F,6,E;C’ :
E
:
G
,F.K , F G ,N
A
:“:
Is-l8
5162
Ad FL MAP A
HiJd III ~IE;C:D: !W’ Boll mobw Limits
, B
F:
D
B
c
A
;
D:E! E
616
54-
,F,
. ’ B
C
E
,E,F
,
C D
66
FIG. 5. Maps of Ad2/AdFL homology regions. Relevant restriction maps of Ad2 and AdFL are shown with fragments corresponding to the darker bands in the autoradiogram of Figs. 3 and 4 drawn as very thick lines and the lighter bands as lines of intermediate thickness. The locations of genes coding for hexon protein and an hexon-associated protein on the Ad2 map (Chow et al., 1977) are shown at the top. The Ad2 cleavage map data is from Mulder et al., 1974 for EcoRI and HpaI and by personal communication for SmaI from C. Mulder and R. Greene, for Hind111 from R. J. Roberts and J. Sambrook, and for K*I from M. B. Mathews, R. Greene, and J. Sambrook.
fragments closely correspond to those of the homologous Ad2 fragments when the maps are oriented on the basis of the ends retained in light virions. The darker Ad2 bands all contain an overlapping region within map coordinates 51 and 62 in the standard Ad2 map, and the corresponding dark bands of AdFL DNA map within coordinates 54 and 66 map units in the AdFL map. Some of the less dense bands in each autoradiogram appear to include one or the other end of this region of homology. The other less dense bands map at a different site within coordinates 12 and 18 in the Ad2 map, and within 6 and 16 in the AdFL map. The detection of two regions of homology that correspond in position when the maps are oriented as shown in Fig. 5 thus supports this orientation of the AdFL map with respect to the Ad2 map.
coordinate 11.2 and 14.9 (Fig. 5). It is therefore likely that the AdFL genes for hexon protein and an hexon-associated protein are located in approximately corresponding positions in the viral DNA and that the cross-reactivity between AdFL and human adenoviruses is due at least in part to these gene products. We infer from the intensity of autoradiographic bands in Figs. 3 and 4, that the regions of homology between the hexon genes of AdFL and Ad2 are more extensive than between the genes for hexon-associated protein. In both eases, however, the 32P-probe DNA can be released from the filter at concentrations of salt higher than that required for disrupting homologous DNA duplexes. Therefore, even in the case of the hexon genes, homology is incomplete. ACKNOWLEDGMENTS
Nature of the Homologous Segments The two regions of Ad2 DNA that are homologous to the comparable segments of AdFL DNA can be identified by reference to the functional map of Ad2 (Fig. 5). Chow et al. (1977) found that the body of the Ad2 hexon mRNA maps between 51.6 and 62.0 map units, and that the body of an hexonassociated protein mRNA maps between
We thank Keith Peden for much helpful advice, M. J. Stem, and T. J. Kelly, Jr. for DNA samples, and J. Suthers, T. Zeller and L. K. English for able technical assistance. This research was supported by grants from the Whitehall Foundation and the USPHS, National Cancer Institute (CA16519). S.H.L. was a post-doctoral trainee of the National Cancer Institute (CA 09139), and R.F.M. is a pre-doctoral trainee under the Medical Scientist Training Program of the USPHS, National Institutes of Health (5T32 GM07399).
414
LARSEN, MARGOLSKEE, REFERENCES
AND NATHANS
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