Cell, Vol. 6, 223-229,
October
1975,
Copyright
Genes for VA-RNA
Michael B. Mathews Cold Spring Harbor P.O. Box 100 Cold Spring Harbor,
12 1975
by MIT
in Adenovirus
Laboratory New York
11724
2
and direction of transcription. Corresponding to the two genes are two VA-RNAs which are present in the infected cell at very disparate levels. For convenience, the major and conventional species is referred to as VA-RNA, and the novel minor RNA as VA-RNA,,. Results
VA-RNA from adenovirus 2 (Ad2) infected cells is shown to consist of two species. The gene coding for the major species maps at position 30 on the viral DNA, where it spans a site cleaved by the restriction enzyme Barn HI. The minor species, constituting a few percent of the total VA-RNA, is distantly related in oligonucleotide composition to the major species. Its template maps within 700 base pairs to the right of the gene for the major species. The direction of transcription is from left to right on the conventional Ad2 map. These results, gained with a novel method for blotting DNA from agarose gels to nitrocellulose paper (E. M. Southern, manuscript in preparation), have also led to the identification of the Barn HI recognition sequence. Introduction Late in the lytic cycle, cells infected with adenoviruses synthesize large quantities of an RNA of unusual properties (Reich et al., 1966; Ohe, Weissman, and Cooke, 1969; Ohe and Weissman, 1970, 1971; Ohe, 1972). This RNA, which was called virus-associated (VA) RNA before its adenovirus origin was established, is 156 bases long and migrates at 5%. The RNA is found in the cytoplasm, predominantly unattached to ribosomes. Its nucleotide sequence, which has been determined, allows a high degree of secondary structure and shows signs of internal duplications, but does not permit a messenger role. Its function is unknown, but several if not all adenovirus strains code for VA-RNAs, which vary in sequence from serotype to serotype. By contrast to adenovirus messenger RNA production, which is accomplished by RNA polymerase II, synthesis of VA-RNA is mediated by RNA polymerase III, the enzyme responsible for the transcription of tRNA and ribosomal 55 genes in uninfected cells (Price and Penman, 1972a, 1972b; Wallace and Kates, 1972; Weinmann, Raskas, and Roeder, 1974). Furthermore, VA-RNA is unusual in that its synthesis is initiated efficiently in vitro by nuclei isolated from infected cells, and it does not appear to be derived by cleavage from a larger precursor molecule (Price and Penman, 1972b). This paper reports the existence of two genes for VA-RNA in the adenovirus 2 (Ad2) genome, their location on the physical map, and their orientation
Existence of Two VA-RNA Species The RNA used in these studies meets all the major criteria which define VA-RNA: it migrates at 5.5S is made late in infection, and is found in the postribosomal supernatant of cells infected with adenoviruses of two different serotypes but not in a similar fraction derived from uninfected cells. Its fingerprint, obtained by digestion with Tl ribonuclease and two-dimensional paper electrophoresis, is shown in Figure 1 a. The pattern of oligonucleotides is essentially identical to that published by Ohe and Weissman (1971), with the exception of one spot (X2) which I have not observed. The key in Figure 1 b illustrates the relative positions of the spots, numbered in accordance with Ohe and Weissman. A set of minor spots, indicated in Figure 1 b by letters within broken rings, is evident in long exposures of the autoradiogram. Unexpectedly, the intensity of most of these spots is fortified, rather than attenuated, in fingerprints of VA-RNA selected by hybridization to Ad2 DNA immobilized on a nitrocellulose filter (Figure lc). Thus hybridization must enrich for a minor RNA species which embodies this subset of oligonucleotides. This RNA species must be virally coded and must share many features with VA-RNA, particularly its size as judged by electrophoretic mobility, its intracellular location, and its synthesis late in infection. The new species is called VA-RNA,,; its properties will be established below. Location of VA-RNA Genes Restriction enzymes cleave duplex DNA molecules at specific sites into a limited number of fragments. For several such enzymes, maps have been drawn showing the relative positions of these fragments on the Ad2 genome. It should be possible to define (within limits) the location of a gene on the physical map by hybridizing its transcript to a series of such fragments. This task is radically simplified by the blotting procedure (E. M. Southern, manuscript in preparation), which allows the direct transfer of DNA fragments resolved by electrophoresis in agarose gels onto nitrocellulose filters for hybridization. The results of such an analysis of Ad2 DNA with a QP-VA-RNA probe are shown in Figures 2 and 3. The DNA was digested separately with 6 restriction enzymes and the products resolved by electro-
Cell 224
b
2nd
L 1st Ad2
Ad2 Hyb
Ad5
Genes 225
for VA-RNA
in Adenovirus
2
phoresis in 1.4% and 0.7% agarose, respectively. Figures 2A and 3A show the distribution of fragments as revealed by ultraviolet illumination in the presence of ethidium bromide. Following transfer to nitrocellulose paper and hybridization to 3*P-VARNA, the greatly simplified patterns of figures 28 and 38 were visualized by autoradiography. In each digest, the probe hybridizes to only one of the Ad2 DNA fragments. These are listed together with their map positions in Table 1, and place the VA-RNA genes in the region 28.8-32.2. This stretch of DNA is 4 times longer than is required to code for two VA-RNA sequences. More precise mapping requires enzymes which cleave within this region, and the results obtained with the restriction enzymes Bal I and Barn HI, each of which generates two DNA fragments that hybridize with VA-RNA, are described below. Direction of Transcription Sharp, Gallimore, and Flint (1974) have shown that agarose gel electrophoresis can be used to separate the DNA strands of the Eco RI fragment A, and they established the chemical polarity of the strands. The slower migrating strand has its 5’ end
Table
1. Map Positions
of Fragments
Which
Hybridize
to VA-RNA
Enzyme
Fragment
Position
Figure
Bum
I
A
?
2a
Eco RI
A
O-58.5
2c, 3b
Hind
B
16.7-32.2
2e 2d. 3c
Hpa
Ill
A
20.5-57.4
Sal I
I
C
25.6-45.9
3e
Sma I
B
18-36
2b. 3d
o-1 00
3a
B M
30.3-50 28.8-30.3
4e, 4f
B D
O-30 30-43
4c, 4d
Whole Bal I Barn
HI
Ad2 DNA
The VA-RNA hybridization data are summarized from Figures 2, 3, and 4. Map positions of the Ad2 DNA fragments are from Mulder et al. (1974), and the unpublished work of these authors and R. Gelinas, R. Greene, and M. B. Mathews. Where there have been minor disparities in the placement of restriction sites, the Barn HI cleavage at position 30 has been taken as a reference point, and map lengths are related to this location.
Figure
1. Fingerprints
(a) Ad2 VA-RNA, not selected.
on the right as the map is customarily oriented, while the faster migrating species has its 5’ end on the left. Figures 4a and 4b show the results of hybridizing 32P-VA-RNA to the separated strands after transfer to nitrocellulose paper. Only the slow strand is complementary to the RNA, indicating that transcription is from left to right. This result is confirmed in the following sections by fingerprint analysis of the RNA hybridizing to the fragments generated by Bal I and Barn HI cleavage of Ad2 DNA. Fine Structure Mapping Digestion of Ad2 DNA with the Barn HI restriction enzyme yields four fragments, two of which, Barn B and Barn D, both hybridize with VA RNA (Figures 4c and 4d). These fragments occupy adjacent positions in the Ad2 genome and adjoin position 30, which must therefore lie between the two VA-RNA genes or within one of them. To investigate this, the RNA was eluted from each fragmeht after treatment with ribonuclease to remove single-stranded RNA tails, and was subjected to fingerprint analysis. Figure 5a shows that only the oligonucleotides from the 5’ half of VA-RNA, (indicated in Figure 1 b by leftward hatches) are found in the Barn B hybrid. The Barn D hybrid (Figure 5b) predominantly yields spots characteristic of the 3’ half (Figure 1 b, rightward hatches) together with all of the spots attributed to VA-RNA,,. This establishes that the Barn HI site lies somewhere between residues 70-80 of the gene coding for VA-RNA,; that the VA-RNA, gene straddles position 30 on the Ad2 DNA; and that VA-RNA,, maps to the right of VA-RNA,. These conclusions are consistent with quantitative studies which indicate that 2-3 times more radioactive VARNA hybridizes to Barn D than to Barn B. Examination of the VA-RNA, sequence (Ohe and Weissman, 1971) reveals the presence of the sequence GGAUCC running from residues 73-78. This is believed to correspond to the Barn HI recognition sequence for the following reasons: first, the frequency with which Barn HI cuts Ad2 and SV40 DNA (three times and once, respectively) indicates that its site is probably a hexanucleotide “palindrome”; second, GGATCC is the only such sequence in the vicinity of the DNA corresponding to residues 70-80 of VA-RNA,; third, the same sequence is present in SV40 DNA (Dhar et al., 1974)
of VA-RNA not selected;
(b) key to Ad2 VA-RNA
spots;
(c) Ad2 VA-RNA
selected
by hybridization
to Ad2
DNA: (d) Ad5 VA-RNA,
The key shows the major spots in complete circles, with leftward hatching for oligonucleotides from the 5’ half of the molecule (for example, spots numbered 61 and 62), and rightward hatching for oligonucleotides from the3’ half (for example, spots numbered 49 and 50). Cross-hatched spots contain oligonucleotides from both halves, and the open circles indicate one unassigned spot (Xl) and the oligonucleotide comprising part of the Barn HI cleavage site. These data are taken from Ohe and Weissman (1971). Broken circles indicate the minor spots whose prominence is increased by selection on Ad2 DNA. The positions of the xylene cyanol FF and methyl orange tracking dyes are marked XC and MO, respectively.
Cell 226
in the immediate vicinity of the Barn HI I cleavage site mapped at position 0.135 (J. Sambrook and M. B. Mathews, unpublished results); fourth, Ad5 DNA lacks a Barn HI site at position 30 (Ft. Greene and C. Mulder, unpublished results), and spot 46 (Figure 1 b) which contains the oligonucleotide AUCCG corresponding to part of the presumptive Barn H I recognition sequence is also missing from fingerprints of Ad5 VA-RNA (Figure 1 d). Finally, the Barn HI site assignment is supported by recent sequence studies (G. A. Wilson, R. J. Roberts, and F. Young, personal communication).
Fingerprint of VA-RNAII Electrophoresis in agarose gels of Ad2 DNA restricted by Bal I resolves 16 bands, two of which hybridize with VA-RNA (Figures 4e and 4f). One of these bands contains a single fragment Bal M, and the other consists of a doublet of the Bal A and B fragments which fail to separate in either 1.4% or 0.7% agarose gels. The hybridization to the doublet must be wholly attributed to Bal B DNA because the Bal A fragment maps between positions 72-93 near the right-hand end of the genome (R. Gelinas,
abcde
personal communication), and the results described above exclude the possibility of any hybridization between VA-RNA and the DNA of this region. The Bal M fragment overlaps the Barn HI site at position 30, and appears to extend about 430 base pairs to the left and about 110 base pairs to the right of the Barn HI cleavage, according to measurements of electrophoretic mobilities in gels. The Bal B fragment lies immediately to the right of this region. Fingerprints of the RNA eluted from these Bal I fragments, following trimming of the nonhybridized tails, are shown in Figures 5c and 5d. It is evident from a comparison with Figure 1 that all the spots characteristic of unselected VA-RNA, are present in the Bal M hybrid, with the exception of three, spots 3, 45, and Xl, which are missing or greatly diminished in intensity. Spots 3 and 45 come from the extreme 3’ end of VA-RNA (residues 147-148 and 151-156, respectively); Xl also appears to derive from the 3’ half of the molecule as it hybridizes to Barn D DNA, but its precise location in the molecule is unknown. Neighboring oligonucleotides, such as 18, 22, and 8, from positions slightly deeper in the molecule are recovered in good yield.
abcde abcde
Figure solved
2. Hybridization of VA-RNA in a 1.4% Agarose Gel
to Fragments
abcde
of Ad2 DNA Re-
(A) DNA fluorescence, photographed during ultraviolet illumination of the gel after soaking in ethidium bromide (0.5 fig/ml). (6) Autoradiogram of VA-RNA bound to the DNA fragments after blotting onto nitrocellulose paper and hybridization to QP-VARNA
(A) DNA fluorescence. (B) Autoradiogram of hybridized
The direction of migration in the gel is downwards. The gel slots contain 1 pg Ad2 DNA digested with the following restriction enzymes: (a) Sum I; (b) Sma I: (c) Eco RI; (d) h/pa I; (e) Hind III.
Fuller details are given in the legend to Figure 2. The gel slots contain 1 pg Ad2 DNA digested with the following restriction enzymes: (a) undigested; (b) Eco RI; (c) Hpa I; (d) Sma I; (e) Sal I.
Figure solved
3. Hybridization of VA-RNA in a 0.7% Agarose Gel
to Fragments
of Ad2 DNA Re-
“P-VA-RNA.
Genes 227
for VA-RNA
in Adenovirus
2
The fingerprint of the RNA selected on Bal B DNA (Figure 5d) contains the three spots missing from the Bal M hybrid, together with the set of oligonucleotides attributed to VA-RNA,,. Discounting spots 3, 45, and Xl, VA-RNA,, seems to possess 8 spots characteristic of VA-RNA, and to lack 13 others. In their place are 11 of the spots originally observed at low levels in unselected VA-RNA. These results place the Bal I cleavage site within the VA-RNA, gene rather than to its right, as the DNA length measurements suggest. The precise location is probably close to the region corresponding to residues 130-140 of VA-RNA,, but there are insufficient data to permit identification of the Bal I recognition sequence at present. After correcting for differential DNA recoveries, about 4 fold more labeled VA-RNA is found hybridized to the f3al M fragment than to the Bal B fragment (Figures 4e and 4f). This is presumably due to difficulties in achieving full saturation of the VA-RNA,, gene with its product because of the low relative concen-
ab
c
d
e
f
BamHI digest
Separated strands
Bal I digest
Figure 4. Hybridization of VA-RNA to Barn HI and Bal I Fragments (a) and scribed (c) and (e) and
(b): by (d): (f):
to Separated
Strands of the Eco RI A fragment, Sharp et al (1974). Ad2 DNA digested by Barn HI. Ad2 DNA digested by Sal I.
DNA Strands separated
and as de-
(a), (c), and (e) are photographs of DNA fluorescence. (b), (d), and(f) are autoradiograms after blotting onto nitrocellulose paper and hybridization to 32P-VA-RNA. The direction of migration is downward.
tration of this species. In addition, hybridization of VA-RNA,, may be hampered by cross reaction of the major species with the VA-RNA,, gene, generating hybrids which are destroyed by the subsequent stringent incubation and ribonuclease treatments. It is apparent that the sensitivity of autoradiographic procedures ensures that such effects do not obstruct detection of VA-RNA,, hybrids, although quantitative studies may be vitiated. Discussion The main conclusions of this work are summarized in Figure 6. The gene for the major VA-RNA surrounds position 30 on the Ad2 physical map and is bisected by a Barn HI cleavage. It is also cut by Bal I at a site close to its right-hand end. VA-RNA,, is a newly discovered species accounting for not more than a few percent of the total VA-RNA. Its presence in low abundance probably explains the uncertainty over whether the genome contains one (Ohe, 1972) or two (A. Jackson, personal communication) copies of the VA-RNA gene. The gene for the minor species maps to the right of the VARNA, gene on the same DNA strand, somewhere between positions 30.3 and 32.2. It is not known if the two genes are separated by a spacer region, but an upper limit of about 500 base pairs can be set upon its length. The complexity of the fingerprint of VA-RNA,, is consistent with its electrophoretic mobility and with a size of approximately 150 nucleotides. The fingerprint contains 19-22 spots of which 8-l 1 are also present in fingerprints of VA-RNA,. The latitude in these tallies is attributable to the 3 spots from the 3’ terminus of VA-RNA,, which are selected by Bal B DNA and cannot be definitely assigned to or excluded from VA-RNA,, on the basis of this study. Two of the remaining spots found in the fingerprints are trinucleotides, and two others are the dinucleotide CG and guanylic acid. Thus only 4 spots containing oligonucleotides of 4 or more bases are common to both species. The dinucleotide UG, recovered in 7 M yield from VA-RNA,, is strikingly absent from VA-RNA,,, as is the oligonucleotide AUCCG corresponding to the Barn HI site. It would seem that the differences between the two species are extensive, and almost certainly greater than the divergence between the major VA-RNA species from different serotypes (Figures la and Id; Ohe et al., 1969). This might imply that the two sequences diverged at an early stage in adenovirus evolution, but such conjectures can be substantiated only by a full sequence analysis. Similarly, further work is required to prove that oligonucleotides occupying the same spots are identical, and to determine whether the homologies are clustered or spread throughout the length of the molecule.
Cell 228
Barn B
Bal M
Bal B
Genes 229
for VA-RNA
in Adenovirus
2
The position of the genes for the two VA-RNAs on the Ad2 map is interesting: they lie at or close to the region of DNA corresponding to the 5’ end of a long tract of stable late mRNAs that extend from about 30-60 on the physical map and code for some of the major structural virion proteins (Sharp et al., 1974; Lewis et al., 1975, and personal communication). Although the role of VA-RNA is cryptic, it is tempting to speculate that this location and the juxtaposition of RNA polymerase II and III transcription units may not be insignificant, and that VA-RNA may be involved in the regulation of adenovirus gene expression late in infection. Finally, it should be mentioned that the blotting technique is a powerful and flexible tool which can be used in a variety of ways. An example of this is shown in Figure 2a, where VA-RNA is seen to hybridize to Bum A DNA, thereby locating this fragment around position 30 on the Ad2 map. Clearly such an approach can be extended to a large variety of mapping problems. Experlmental
Procedures
VA-RNA Preparation HeLa cells growing in 10 cm plates were infected with Ad2 virus at a multiplicity of lo-100 and labeled with 1-5 mCi ‘2P-phosphate in 4 ml phosphate-free medium containing 2% serum from about 16-24 hr post infection. Cells were removed by trypsin, washed, chilled, and lysed with 0.5% Nonidet P40 in 1.5 mM MgCII, IO mM NaCI. IO mM Tris-HCI (pH 7.4). RNA was isolated from the postribosomal supernatant by phenol and chloroform-isoamyl alcohol extraction, collected by alcohol precipitation, and electrophoresed in 10% acrylamide gels, The VA-RNA band was visualized by autoradiography, excised, and eluted in TNE [50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 1 mM EDTA]. Impurities were removed by passage through two successive cellulose columns (Whatman CF 11). The first column was developed as described by Franklin (1966): VA-RNA chromatographs as a single-stranded RNA. The material in this fraction was rebound to a second column, washed with 100% ethyl alcohol, eluted in distilled water, and lyophilized. Specific activities of the 32P-VA-RNA were not routinely measured, but seemed to be in the range lo*-107 cpm/yg with a yield of about 107 cpm/plate. Restriction of Ad2 DNA The preparation of Ad2 DNA and of the restriction enzymes, digestion conditions, and agarose gel electrophoresis details have been described (Sambrook et al., 1975; Sharp, Sugden, and Sambrook, 1973). The enzymes Sal I, Bum I, and Sal I were gifts from R. J. Roberts. Reaction mixtures with these enzymes were incubated at 37’C for 16 hr, 4 hr, and 4 hr, respectively, in a buffer containing 10 mM Tris-HCI (pH 7.5), 10 mM MgCb, and 1 mM dithiothreitol.
Figure
5. Fingerprints
of VA-RNA
Selected
on Barn
HI and Bal I DNA
The bacterial sources of these enzymes are: Bal I, Brevibacterium albidum; Barn HI. Bacillus amyloliquefaciens H; Bum I, Brevibacterium umbra; Eco RI, Escherichia coli RY13; Hind Ill, Haemophilus influenzae Rd; Hpa I, Haemophilus parainfluenzae; Sal I, Streptomyces albus G; Sma I, Serratia marcescens. Blotting and Hybridization Denatured DNA was transferred from agarose gels to nitrocellulose paper in a stream of 6 x SSC [l x SSC is 0.15 M NaCI, 0.015 M Na citrate (pH 7.2)] by a small modification of the blotting procedure (E. M. Southern, manuscript in preparation) to allow wide slabs to be handled. Hybridization was carried out for 6-16 hr at 65-68” in 3 ml of 6 x SSC containing 0.5% SDS, 2 mM EDTA, 20 gg/ml yeast RNA, and 0.5-3 x 106 cpm VA-RNA. The paper was washed in 2 x SSC several times at room temperature, for 1 hr at 65°C and again at room temperature after incubation at 37°C for 1 hr with 20 pg/ml RNAase A. The dried filter was autoradiographed for l-20 days. These conditions allowed detection of hybridization of both major and minor VA-RNA species. Flngerprinting Circular filters or blots were treated as above except that RNAase Tl supplanted RNAase A. RNA was eluted in the presence of 50 pg carrier RNA by heating for 90 set at 95°C in 1.5 ml 5 mM Tris-HCI (pH 8.0). After alcohol precipitation, the RNA was dissolved in a small volume of water, centrifuged to remove insoluble matter, and digested with 5 pg RNAase Tl for 45 min at 37°C in 10 mM Tris-HCI (pH 7.4), 1 mM EDTA. Oligonucleotides were fingerprinted on cellulose acetate paper at pH 3.5 in 7 M urea, followed by a second dimension on DEAE cellulose paper in 7% Eco RI
Bomz;x
,
B-M e-B
---
--It
BOl M Barn B
Figure 6. The Location RNA Genes in Ad2
II IIII
and Direction
’
B-s B-B
BOl 8
---
Barn 0
---
of Transcription
of the VA-
The top part of the figure represents the Ad2 genome. The expansion of the region surrounding position 30 indicates the map locations of the genes coding for the VA-RNAs and their direction of transcription. The distribution of restriction sites and principal fragments is illustrated. 1 map unit is equivalent to about 350 base pairs or 230,000 daltons of DNA.
Fragments
32P-VA-RNA was selected by hybridization to nitrocellulose paper bearing the following DNA fragments: (a) Barn B; (b) Barn D; (c) Bal M; (d) Sal A + B. Spots such as numbers 61 and 62 from the 5’ half of VA-RNA, are obtained from the Barn B hybrid but not from the Barn D hybrid. Conversely, spots from the 3’ half of VA-RNA,, numbers 49 and 50, for example, are present in digests of the Barn D hybrid but not of the Barn B hybrid. The Barn D hybrid also yields the spots characteristic of VA-RNA,,. designated a, c-e, g-k, m, and n in figure lb. The digest of the Bal M hybrid contains the VA-RNA, spots apart from numbers 3, 45, and Xl: these are obtained from the Sal B hybrid, together with all of the VA-RNA,, spots. Eight spots appear to be common to the two VA-RNA species. Listed with their nucleotide lengths in parentheses these are: 51 (ten); 19 (four): 46 (four): 34 and/or 35 (four); 5 (three); 10 (three); 1 (one); and 2 (two).
Cell 230
formic acid (Sanger, Brownlee, and Barrell, not selected by hybridization was digested same fashion.
1965). RNA which was and fractionated in the
Acknowledgments I thank Mark Glen for able and stimulating assistance; Mike Botchan, Joe Sambrook, and Rich Roberts for advice and discussions; Phyllis Myers for enzymes; and Ed Southern for communication of his blotting technique before publication. This work was supported by a grant from the National Cancer Institute. Received
July 14, 1975
References Dhar, R., Zain, S., Weissman, S. M., Pan, J., and Subramanian, K. (1974). Proc. Nat. Acad. Sci. USA 77, 371-375. Franklin,
R. M. (1966).
Proc.
Nat. Acad.
Sci. USA 55, 1504-1511.
Lewis, J. B., Atkins, J. F.. Anderson, C. W.. Baum, P. A., and Gesteland, R. F. (1975). Proc. Nat. Acad. Sci. USA 72, 1344-1348. Mulder, C., Arrand, J. R., Delius, H., Keller, W., Pettersson, U., Roberts, R. J., and Sharp, P. A. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 397-400. Ohe,
K. (1972).
Ohe,
K., and Weissman,
Virology
Ohe,
K., and Weissman,
Ohe. K., Weissman, 244, 5320-5332.
47, 726-733. S. (1970). S. (1971).
Science
167, 879-881.
J. Biol. Chem.
S., and Cooke,
246, 6991-7009.
N. R. (1969).
J. Biol. Chem.
Price,
R., and Penman,
S. (1972a).
J. Virol.
Price,
R., and Penman,
S. (1972b).
J. Mol. Biol. 70, 435-450.
Reich, P. R., Forget, B. G., Weissman. J. Mol. Biol. 77, 428-439. Sambrook, J.. Williams, press). J. Mol. Biol. Sanger, F., Brownlee, 13, 373-398. Sharp, Harbor
S. M., and Rose, P. A., and
G. G., and Earrell,
J. A. (1966).
Grodzicker,
B. G. (1965).
W., and Sambrook,
R. D.. and Kates,
J. (1972).
J. (1973).
J. Virol.
Weinmann, R., Raskas. H. J., and Roeder, Acad. Sci. USA 71, 3426-3430.
T. (in
J. Mol. Biol.
P. A., Gallimore, P. H., and Flint, S. J. (1974). Symp. Quant. Biol. 39, 457-474.
Sharp, P. A., Sugden, 72, 3055-3063. Wallace,
I., Sharp,
9, 621-626.
Cold Spring Biochemistry
9, 627-635.
R. G. (1974).
Proc.
Nat.
October
1975,
Copyright
Genes for VA-RNA
Michael B. Mathews Cold Spring Harbor P.O. Box 100 Cold Spring Harbor,
12 1975
by MIT
in Adenovirus
Laboratory New York
11724
2
and direction of transcription. Corresponding to the two genes are two VA-RNAs which are present in the infected cell at very disparate levels. For convenience, the major and conventional species is referred to as VA-RNA, and the novel minor RNA as VA-RNA,,. Results
VA-RNA from adenovirus 2 (Ad2) infected cells is shown to consist of two species. The gene coding for the major species maps at position 30 on the viral DNA, where it spans a site cleaved by the restriction enzyme Barn HI. The minor species, constituting a few percent of the total VA-RNA, is distantly related in oligonucleotide composition to the major species. Its template maps within 700 base pairs to the right of the gene for the major species. The direction of transcription is from left to right on the conventional Ad2 map. These results, gained with a novel method for blotting DNA from agarose gels to nitrocellulose paper (E. M. Southern, manuscript in preparation), have also led to the identification of the Barn HI recognition sequence. Introduction Late in the lytic cycle, cells infected with adenoviruses synthesize large quantities of an RNA of unusual properties (Reich et al., 1966; Ohe, Weissman, and Cooke, 1969; Ohe and Weissman, 1970, 1971; Ohe, 1972). This RNA, which was called virus-associated (VA) RNA before its adenovirus origin was established, is 156 bases long and migrates at 5%. The RNA is found in the cytoplasm, predominantly unattached to ribosomes. Its nucleotide sequence, which has been determined, allows a high degree of secondary structure and shows signs of internal duplications, but does not permit a messenger role. Its function is unknown, but several if not all adenovirus strains code for VA-RNAs, which vary in sequence from serotype to serotype. By contrast to adenovirus messenger RNA production, which is accomplished by RNA polymerase II, synthesis of VA-RNA is mediated by RNA polymerase III, the enzyme responsible for the transcription of tRNA and ribosomal 55 genes in uninfected cells (Price and Penman, 1972a, 1972b; Wallace and Kates, 1972; Weinmann, Raskas, and Roeder, 1974). Furthermore, VA-RNA is unusual in that its synthesis is initiated efficiently in vitro by nuclei isolated from infected cells, and it does not appear to be derived by cleavage from a larger precursor molecule (Price and Penman, 1972b). This paper reports the existence of two genes for VA-RNA in the adenovirus 2 (Ad2) genome, their location on the physical map, and their orientation
Existence of Two VA-RNA Species The RNA used in these studies meets all the major criteria which define VA-RNA: it migrates at 5.5S is made late in infection, and is found in the postribosomal supernatant of cells infected with adenoviruses of two different serotypes but not in a similar fraction derived from uninfected cells. Its fingerprint, obtained by digestion with Tl ribonuclease and two-dimensional paper electrophoresis, is shown in Figure 1 a. The pattern of oligonucleotides is essentially identical to that published by Ohe and Weissman (1971), with the exception of one spot (X2) which I have not observed. The key in Figure 1 b illustrates the relative positions of the spots, numbered in accordance with Ohe and Weissman. A set of minor spots, indicated in Figure 1 b by letters within broken rings, is evident in long exposures of the autoradiogram. Unexpectedly, the intensity of most of these spots is fortified, rather than attenuated, in fingerprints of VA-RNA selected by hybridization to Ad2 DNA immobilized on a nitrocellulose filter (Figure lc). Thus hybridization must enrich for a minor RNA species which embodies this subset of oligonucleotides. This RNA species must be virally coded and must share many features with VA-RNA, particularly its size as judged by electrophoretic mobility, its intracellular location, and its synthesis late in infection. The new species is called VA-RNA,,; its properties will be established below. Location of VA-RNA Genes Restriction enzymes cleave duplex DNA molecules at specific sites into a limited number of fragments. For several such enzymes, maps have been drawn showing the relative positions of these fragments on the Ad2 genome. It should be possible to define (within limits) the location of a gene on the physical map by hybridizing its transcript to a series of such fragments. This task is radically simplified by the blotting procedure (E. M. Southern, manuscript in preparation), which allows the direct transfer of DNA fragments resolved by electrophoresis in agarose gels onto nitrocellulose filters for hybridization. The results of such an analysis of Ad2 DNA with a QP-VA-RNA probe are shown in Figures 2 and 3. The DNA was digested separately with 6 restriction enzymes and the products resolved by electro-
Cell 224
b
2nd
L 1st Ad2
Ad2 Hyb
Ad5
Genes 225
for VA-RNA
in Adenovirus
2
phoresis in 1.4% and 0.7% agarose, respectively. Figures 2A and 3A show the distribution of fragments as revealed by ultraviolet illumination in the presence of ethidium bromide. Following transfer to nitrocellulose paper and hybridization to 3*P-VARNA, the greatly simplified patterns of figures 28 and 38 were visualized by autoradiography. In each digest, the probe hybridizes to only one of the Ad2 DNA fragments. These are listed together with their map positions in Table 1, and place the VA-RNA genes in the region 28.8-32.2. This stretch of DNA is 4 times longer than is required to code for two VA-RNA sequences. More precise mapping requires enzymes which cleave within this region, and the results obtained with the restriction enzymes Bal I and Barn HI, each of which generates two DNA fragments that hybridize with VA-RNA, are described below. Direction of Transcription Sharp, Gallimore, and Flint (1974) have shown that agarose gel electrophoresis can be used to separate the DNA strands of the Eco RI fragment A, and they established the chemical polarity of the strands. The slower migrating strand has its 5’ end
Table
1. Map Positions
of Fragments
Which
Hybridize
to VA-RNA
Enzyme
Fragment
Position
Figure
Bum
I
A
?
2a
Eco RI
A
O-58.5
2c, 3b
Hind
B
16.7-32.2
2e 2d. 3c
Hpa
Ill
A
20.5-57.4
Sal I
I
C
25.6-45.9
3e
Sma I
B
18-36
2b. 3d
o-1 00
3a
B M
30.3-50 28.8-30.3
4e, 4f
B D
O-30 30-43
4c, 4d
Whole Bal I Barn
HI
Ad2 DNA
The VA-RNA hybridization data are summarized from Figures 2, 3, and 4. Map positions of the Ad2 DNA fragments are from Mulder et al. (1974), and the unpublished work of these authors and R. Gelinas, R. Greene, and M. B. Mathews. Where there have been minor disparities in the placement of restriction sites, the Barn HI cleavage at position 30 has been taken as a reference point, and map lengths are related to this location.
Figure
1. Fingerprints
(a) Ad2 VA-RNA, not selected.
on the right as the map is customarily oriented, while the faster migrating species has its 5’ end on the left. Figures 4a and 4b show the results of hybridizing 32P-VA-RNA to the separated strands after transfer to nitrocellulose paper. Only the slow strand is complementary to the RNA, indicating that transcription is from left to right. This result is confirmed in the following sections by fingerprint analysis of the RNA hybridizing to the fragments generated by Bal I and Barn HI cleavage of Ad2 DNA. Fine Structure Mapping Digestion of Ad2 DNA with the Barn HI restriction enzyme yields four fragments, two of which, Barn B and Barn D, both hybridize with VA RNA (Figures 4c and 4d). These fragments occupy adjacent positions in the Ad2 genome and adjoin position 30, which must therefore lie between the two VA-RNA genes or within one of them. To investigate this, the RNA was eluted from each fragmeht after treatment with ribonuclease to remove single-stranded RNA tails, and was subjected to fingerprint analysis. Figure 5a shows that only the oligonucleotides from the 5’ half of VA-RNA, (indicated in Figure 1 b by leftward hatches) are found in the Barn B hybrid. The Barn D hybrid (Figure 5b) predominantly yields spots characteristic of the 3’ half (Figure 1 b, rightward hatches) together with all of the spots attributed to VA-RNA,,. This establishes that the Barn HI site lies somewhere between residues 70-80 of the gene coding for VA-RNA,; that the VA-RNA, gene straddles position 30 on the Ad2 DNA; and that VA-RNA,, maps to the right of VA-RNA,. These conclusions are consistent with quantitative studies which indicate that 2-3 times more radioactive VARNA hybridizes to Barn D than to Barn B. Examination of the VA-RNA, sequence (Ohe and Weissman, 1971) reveals the presence of the sequence GGAUCC running from residues 73-78. This is believed to correspond to the Barn HI recognition sequence for the following reasons: first, the frequency with which Barn HI cuts Ad2 and SV40 DNA (three times and once, respectively) indicates that its site is probably a hexanucleotide “palindrome”; second, GGATCC is the only such sequence in the vicinity of the DNA corresponding to residues 70-80 of VA-RNA,; third, the same sequence is present in SV40 DNA (Dhar et al., 1974)
of VA-RNA not selected;
(b) key to Ad2 VA-RNA
spots;
(c) Ad2 VA-RNA
selected
by hybridization
to Ad2
DNA: (d) Ad5 VA-RNA,
The key shows the major spots in complete circles, with leftward hatching for oligonucleotides from the 5’ half of the molecule (for example, spots numbered 61 and 62), and rightward hatching for oligonucleotides from the3’ half (for example, spots numbered 49 and 50). Cross-hatched spots contain oligonucleotides from both halves, and the open circles indicate one unassigned spot (Xl) and the oligonucleotide comprising part of the Barn HI cleavage site. These data are taken from Ohe and Weissman (1971). Broken circles indicate the minor spots whose prominence is increased by selection on Ad2 DNA. The positions of the xylene cyanol FF and methyl orange tracking dyes are marked XC and MO, respectively.
Cell 226
in the immediate vicinity of the Barn HI I cleavage site mapped at position 0.135 (J. Sambrook and M. B. Mathews, unpublished results); fourth, Ad5 DNA lacks a Barn HI site at position 30 (Ft. Greene and C. Mulder, unpublished results), and spot 46 (Figure 1 b) which contains the oligonucleotide AUCCG corresponding to part of the presumptive Barn H I recognition sequence is also missing from fingerprints of Ad5 VA-RNA (Figure 1 d). Finally, the Barn HI site assignment is supported by recent sequence studies (G. A. Wilson, R. J. Roberts, and F. Young, personal communication).
Fingerprint of VA-RNAII Electrophoresis in agarose gels of Ad2 DNA restricted by Bal I resolves 16 bands, two of which hybridize with VA-RNA (Figures 4e and 4f). One of these bands contains a single fragment Bal M, and the other consists of a doublet of the Bal A and B fragments which fail to separate in either 1.4% or 0.7% agarose gels. The hybridization to the doublet must be wholly attributed to Bal B DNA because the Bal A fragment maps between positions 72-93 near the right-hand end of the genome (R. Gelinas,
abcde
personal communication), and the results described above exclude the possibility of any hybridization between VA-RNA and the DNA of this region. The Bal M fragment overlaps the Barn HI site at position 30, and appears to extend about 430 base pairs to the left and about 110 base pairs to the right of the Barn HI cleavage, according to measurements of electrophoretic mobilities in gels. The Bal B fragment lies immediately to the right of this region. Fingerprints of the RNA eluted from these Bal I fragments, following trimming of the nonhybridized tails, are shown in Figures 5c and 5d. It is evident from a comparison with Figure 1 that all the spots characteristic of unselected VA-RNA, are present in the Bal M hybrid, with the exception of three, spots 3, 45, and Xl, which are missing or greatly diminished in intensity. Spots 3 and 45 come from the extreme 3’ end of VA-RNA (residues 147-148 and 151-156, respectively); Xl also appears to derive from the 3’ half of the molecule as it hybridizes to Barn D DNA, but its precise location in the molecule is unknown. Neighboring oligonucleotides, such as 18, 22, and 8, from positions slightly deeper in the molecule are recovered in good yield.
abcde abcde
Figure solved
2. Hybridization of VA-RNA in a 1.4% Agarose Gel
to Fragments
abcde
of Ad2 DNA Re-
(A) DNA fluorescence, photographed during ultraviolet illumination of the gel after soaking in ethidium bromide (0.5 fig/ml). (6) Autoradiogram of VA-RNA bound to the DNA fragments after blotting onto nitrocellulose paper and hybridization to QP-VARNA
(A) DNA fluorescence. (B) Autoradiogram of hybridized
The direction of migration in the gel is downwards. The gel slots contain 1 pg Ad2 DNA digested with the following restriction enzymes: (a) Sum I; (b) Sma I: (c) Eco RI; (d) h/pa I; (e) Hind III.
Fuller details are given in the legend to Figure 2. The gel slots contain 1 pg Ad2 DNA digested with the following restriction enzymes: (a) undigested; (b) Eco RI; (c) Hpa I; (d) Sma I; (e) Sal I.
Figure solved
3. Hybridization of VA-RNA in a 0.7% Agarose Gel
to Fragments
of Ad2 DNA Re-
“P-VA-RNA.
Genes 227
for VA-RNA
in Adenovirus
2
The fingerprint of the RNA selected on Bal B DNA (Figure 5d) contains the three spots missing from the Bal M hybrid, together with the set of oligonucleotides attributed to VA-RNA,,. Discounting spots 3, 45, and Xl, VA-RNA,, seems to possess 8 spots characteristic of VA-RNA, and to lack 13 others. In their place are 11 of the spots originally observed at low levels in unselected VA-RNA. These results place the Bal I cleavage site within the VA-RNA, gene rather than to its right, as the DNA length measurements suggest. The precise location is probably close to the region corresponding to residues 130-140 of VA-RNA,, but there are insufficient data to permit identification of the Bal I recognition sequence at present. After correcting for differential DNA recoveries, about 4 fold more labeled VA-RNA is found hybridized to the f3al M fragment than to the Bal B fragment (Figures 4e and 4f). This is presumably due to difficulties in achieving full saturation of the VA-RNA,, gene with its product because of the low relative concen-
ab
c
d
e
f
BamHI digest
Separated strands
Bal I digest
Figure 4. Hybridization of VA-RNA to Barn HI and Bal I Fragments (a) and scribed (c) and (e) and
(b): by (d): (f):
to Separated
Strands of the Eco RI A fragment, Sharp et al (1974). Ad2 DNA digested by Barn HI. Ad2 DNA digested by Sal I.
DNA Strands separated
and as de-
(a), (c), and (e) are photographs of DNA fluorescence. (b), (d), and(f) are autoradiograms after blotting onto nitrocellulose paper and hybridization to 32P-VA-RNA. The direction of migration is downward.
tration of this species. In addition, hybridization of VA-RNA,, may be hampered by cross reaction of the major species with the VA-RNA,, gene, generating hybrids which are destroyed by the subsequent stringent incubation and ribonuclease treatments. It is apparent that the sensitivity of autoradiographic procedures ensures that such effects do not obstruct detection of VA-RNA,, hybrids, although quantitative studies may be vitiated. Discussion The main conclusions of this work are summarized in Figure 6. The gene for the major VA-RNA surrounds position 30 on the Ad2 physical map and is bisected by a Barn HI cleavage. It is also cut by Bal I at a site close to its right-hand end. VA-RNA,, is a newly discovered species accounting for not more than a few percent of the total VA-RNA. Its presence in low abundance probably explains the uncertainty over whether the genome contains one (Ohe, 1972) or two (A. Jackson, personal communication) copies of the VA-RNA gene. The gene for the minor species maps to the right of the VARNA, gene on the same DNA strand, somewhere between positions 30.3 and 32.2. It is not known if the two genes are separated by a spacer region, but an upper limit of about 500 base pairs can be set upon its length. The complexity of the fingerprint of VA-RNA,, is consistent with its electrophoretic mobility and with a size of approximately 150 nucleotides. The fingerprint contains 19-22 spots of which 8-l 1 are also present in fingerprints of VA-RNA,. The latitude in these tallies is attributable to the 3 spots from the 3’ terminus of VA-RNA,, which are selected by Bal B DNA and cannot be definitely assigned to or excluded from VA-RNA,, on the basis of this study. Two of the remaining spots found in the fingerprints are trinucleotides, and two others are the dinucleotide CG and guanylic acid. Thus only 4 spots containing oligonucleotides of 4 or more bases are common to both species. The dinucleotide UG, recovered in 7 M yield from VA-RNA,, is strikingly absent from VA-RNA,,, as is the oligonucleotide AUCCG corresponding to the Barn HI site. It would seem that the differences between the two species are extensive, and almost certainly greater than the divergence between the major VA-RNA species from different serotypes (Figures la and Id; Ohe et al., 1969). This might imply that the two sequences diverged at an early stage in adenovirus evolution, but such conjectures can be substantiated only by a full sequence analysis. Similarly, further work is required to prove that oligonucleotides occupying the same spots are identical, and to determine whether the homologies are clustered or spread throughout the length of the molecule.
Cell 228
Barn B
Bal M
Bal B
Genes 229
for VA-RNA
in Adenovirus
2
The position of the genes for the two VA-RNAs on the Ad2 map is interesting: they lie at or close to the region of DNA corresponding to the 5’ end of a long tract of stable late mRNAs that extend from about 30-60 on the physical map and code for some of the major structural virion proteins (Sharp et al., 1974; Lewis et al., 1975, and personal communication). Although the role of VA-RNA is cryptic, it is tempting to speculate that this location and the juxtaposition of RNA polymerase II and III transcription units may not be insignificant, and that VA-RNA may be involved in the regulation of adenovirus gene expression late in infection. Finally, it should be mentioned that the blotting technique is a powerful and flexible tool which can be used in a variety of ways. An example of this is shown in Figure 2a, where VA-RNA is seen to hybridize to Bum A DNA, thereby locating this fragment around position 30 on the Ad2 map. Clearly such an approach can be extended to a large variety of mapping problems. Experlmental
Procedures
VA-RNA Preparation HeLa cells growing in 10 cm plates were infected with Ad2 virus at a multiplicity of lo-100 and labeled with 1-5 mCi ‘2P-phosphate in 4 ml phosphate-free medium containing 2% serum from about 16-24 hr post infection. Cells were removed by trypsin, washed, chilled, and lysed with 0.5% Nonidet P40 in 1.5 mM MgCII, IO mM NaCI. IO mM Tris-HCI (pH 7.4). RNA was isolated from the postribosomal supernatant by phenol and chloroform-isoamyl alcohol extraction, collected by alcohol precipitation, and electrophoresed in 10% acrylamide gels, The VA-RNA band was visualized by autoradiography, excised, and eluted in TNE [50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 1 mM EDTA]. Impurities were removed by passage through two successive cellulose columns (Whatman CF 11). The first column was developed as described by Franklin (1966): VA-RNA chromatographs as a single-stranded RNA. The material in this fraction was rebound to a second column, washed with 100% ethyl alcohol, eluted in distilled water, and lyophilized. Specific activities of the 32P-VA-RNA were not routinely measured, but seemed to be in the range lo*-107 cpm/yg with a yield of about 107 cpm/plate. Restriction of Ad2 DNA The preparation of Ad2 DNA and of the restriction enzymes, digestion conditions, and agarose gel electrophoresis details have been described (Sambrook et al., 1975; Sharp, Sugden, and Sambrook, 1973). The enzymes Sal I, Bum I, and Sal I were gifts from R. J. Roberts. Reaction mixtures with these enzymes were incubated at 37’C for 16 hr, 4 hr, and 4 hr, respectively, in a buffer containing 10 mM Tris-HCI (pH 7.5), 10 mM MgCb, and 1 mM dithiothreitol.
Figure
5. Fingerprints
of VA-RNA
Selected
on Barn
HI and Bal I DNA
The bacterial sources of these enzymes are: Bal I, Brevibacterium albidum; Barn HI. Bacillus amyloliquefaciens H; Bum I, Brevibacterium umbra; Eco RI, Escherichia coli RY13; Hind Ill, Haemophilus influenzae Rd; Hpa I, Haemophilus parainfluenzae; Sal I, Streptomyces albus G; Sma I, Serratia marcescens. Blotting and Hybridization Denatured DNA was transferred from agarose gels to nitrocellulose paper in a stream of 6 x SSC [l x SSC is 0.15 M NaCI, 0.015 M Na citrate (pH 7.2)] by a small modification of the blotting procedure (E. M. Southern, manuscript in preparation) to allow wide slabs to be handled. Hybridization was carried out for 6-16 hr at 65-68” in 3 ml of 6 x SSC containing 0.5% SDS, 2 mM EDTA, 20 gg/ml yeast RNA, and 0.5-3 x 106 cpm VA-RNA. The paper was washed in 2 x SSC several times at room temperature, for 1 hr at 65°C and again at room temperature after incubation at 37°C for 1 hr with 20 pg/ml RNAase A. The dried filter was autoradiographed for l-20 days. These conditions allowed detection of hybridization of both major and minor VA-RNA species. Flngerprinting Circular filters or blots were treated as above except that RNAase Tl supplanted RNAase A. RNA was eluted in the presence of 50 pg carrier RNA by heating for 90 set at 95°C in 1.5 ml 5 mM Tris-HCI (pH 8.0). After alcohol precipitation, the RNA was dissolved in a small volume of water, centrifuged to remove insoluble matter, and digested with 5 pg RNAase Tl for 45 min at 37°C in 10 mM Tris-HCI (pH 7.4), 1 mM EDTA. Oligonucleotides were fingerprinted on cellulose acetate paper at pH 3.5 in 7 M urea, followed by a second dimension on DEAE cellulose paper in 7% Eco RI
Bomz;x
,
B-M e-B
---
--It
BOl M Barn B
Figure 6. The Location RNA Genes in Ad2
II IIII
and Direction
’
B-s B-B
BOl 8
---
Barn 0
---
of Transcription
of the VA-
The top part of the figure represents the Ad2 genome. The expansion of the region surrounding position 30 indicates the map locations of the genes coding for the VA-RNAs and their direction of transcription. The distribution of restriction sites and principal fragments is illustrated. 1 map unit is equivalent to about 350 base pairs or 230,000 daltons of DNA.
Fragments
32P-VA-RNA was selected by hybridization to nitrocellulose paper bearing the following DNA fragments: (a) Barn B; (b) Barn D; (c) Bal M; (d) Sal A + B. Spots such as numbers 61 and 62 from the 5’ half of VA-RNA, are obtained from the Barn B hybrid but not from the Barn D hybrid. Conversely, spots from the 3’ half of VA-RNA,, numbers 49 and 50, for example, are present in digests of the Barn D hybrid but not of the Barn B hybrid. The Barn D hybrid also yields the spots characteristic of VA-RNA,,. designated a, c-e, g-k, m, and n in figure lb. The digest of the Bal M hybrid contains the VA-RNA, spots apart from numbers 3, 45, and Xl: these are obtained from the Sal B hybrid, together with all of the VA-RNA,, spots. Eight spots appear to be common to the two VA-RNA species. Listed with their nucleotide lengths in parentheses these are: 51 (ten); 19 (four): 46 (four): 34 and/or 35 (four); 5 (three); 10 (three); 1 (one); and 2 (two).
Cell 230
formic acid (Sanger, Brownlee, and Barrell, not selected by hybridization was digested same fashion.
1965). RNA which was and fractionated in the
Acknowledgments I thank Mark Glen for able and stimulating assistance; Mike Botchan, Joe Sambrook, and Rich Roberts for advice and discussions; Phyllis Myers for enzymes; and Ed Southern for communication of his blotting technique before publication. This work was supported by a grant from the National Cancer Institute. Received
July 14, 1975
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