Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1344-1348, April 1975

Mapping of Late Adenovirus Genes by Cell-Free Translation of RNA Selected by Hybridization to Specific DNA Fragments (urea-hydroxylapatite chromatography/endonuclease R*EcoRI/ sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

J. B. LEWIS, J. F. ATKINS, C. W. ANDERSON, P. R. BAUM, AND R. F. GESTELAND Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Communicated by J. P). Watson, January 23, 1975 Cytoplasmic RNA, isolated from cells late ABSTRACT after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides, From the known order of the R.EcoRI fragments, the following pgrtial map is deduced: (III, Hila, IVa2, V, P-VII, IX), (II, P-VI), lOOK, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

with cytoplasmic RNA, prepared from human cells late after Ad2 infection, that had first been fractionated by hybridization to the separated specific fragments of Ad2 DNA obtained after digestion with the restriction endonuclease RLEcoRI. The order of the six R- EcoRI fragments on Ad2 DNA is known (16) and so we can deduce the location of Ad2 genes. This technique provides a general method to locate genes, provided mRNA and suitable specific fragments of DNA can be obtained.

A general technique for isolation of conditional lethal mutants in conjunction with identification of the altered protein products has not been developed for eukaryotes to the degree that led to such success in the genetic analysis of prokaryotes. However, the availability of enzymes that specifically fragment DNA, and the demonstration that RNA selected by hybridization to DNA can be translated in cell-free extracts (1, 2), have opened up alternative ways to do genetic mapping by biochemical means. One system that is amenable to such an analysis is the infection of mammalian cells by adenovirus. Adenovirus 2 (Ad2) is a human virus capable of transforming certain nonpermissive cells in vitro. In the productive infection of permissive cells, two phases have been defined, an early phase which includes events occurring before the onset of DNA synthesis (8 hr) and a subsequent late phase when most of the genome is expressed (for a review see ref. 3). Approximately 18 virusspecific proteins have been found in extracts of infected cells (4), and at least 10 of these have been identified among the products of cell-free translation programmed by cytoplasmic RNA from adenovirus-infected cells (5-10). Discrete size classes of viral mRNA have been demonstrated by fractionation on a sucrose gradient and correlated with their polypeptide products (5). In addition, the mRNA homologous to Ad2 DNA has been characterized by electrophoretic mobility (11, 12) and by its hybridization with specific DNA fragments (11-15), but in neither of these latter cases have the RNA classes been correlated with their polypeptide products. Thus, the pattern of transcription of Ad2 is well characterized but the relationship of this map to the location of specific genes is unknown. We report here a partial map of the localization on the viral DNA of the genes encoding adenovirus polypeptides. This map was obtained by programming cell-free protein synthesis

Adenovirus DNA was prepared from virions (17). Batches (2 mg) of DNA were digested (18) with endonuclease R EcoRI (a gift of P. Myers and R. Roberts). Glycerol was added to 15% (v/v) and bromophenol blue to 0.04%, and the fragments were separated by electrophoresis on two 34 cm X 16 cm X 5 mm 1.4% agarose gels for 30 hr at 35 mA. The gels were then stained for 5 min in aqueous ethidium bromide (4 ,ug/ml) and examined with ultraviolet illumination. Gel slices in silane-treated tubes were dissolved with 2 volumes of 5 M sodium perchlorate (Fisher, filtered before use) at 600 (ref. 19, modified). The dissolved agarose was passed through a column at 600 containing 3-5 ml of hydroxylapatite, and the columns were then washed with 25 ml of 5 M sodium perchlorate (600) (ref. 20. modified), 15 ml of 0.05 M sodium phosphate buffer, pH 6.8. Elution was with 15 ml of 0.4 M phosphate, pH 6.8. The samples were dialyzed against 30 volumes of 10 mM Tris*HCI, pH 7.9, 1 mM EDTA, 1 mM ethyleneglycol bis(,B-aminoethyl ether)-NN'-tetraacetic acid (EGTA) for 24 hr at room temperature with two changes of dialysate. NaCl was added to 0.25 M and the samples were precipitated with 2 volumes of ethanol. The precipitate was resuspended in TSE (0.05 M Tris, pH 8.0, 0.15 M NaCl, 5mM EDTA) and extracted with an equal volume of a 1: 1 mixture of chloroform: phenol (saturated with TSE) to remove any residual agarose. After an additional ethanol precipitation, the samples were dissolved in a suitably small volume of 0.01 M Tris, pH 8.0, 1 mM EDTA, and stored at -20°. Three micrograms of recovered fragment was rerun on an analytical agarose gel, to check for lack of cross contamination. Generally the recovery was 80%. Purification of mRNA by Hybridization to DNA. DNA was denatured and fragmented to segments of about 300 base pairs by boiling in glass tubes for 25 min in 0.3 M NaOH (21). After neutralization with 0.3 M HCl in the presence of 0.20 M sodium phosphate buffer, pH 6.8, at 0°, the denatured DNA was added to a hybridization mixture containing 0.18 M sodium phosphate buffer, pH 6.8, 0.2% sodium dodecyl

MATERIALS AND METHODS -

Abbreviations: Ad2, adenovirus type 2; EGTA, ethyleneglycol bis(,8-aminoethyl ether)-N,N'-tetraacetic acid.

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Biochemical Mapping of Late Adenovirus Genes

(1975)

sulfate, and RNA at the desired concentration. A typical experiment used 100 ,ug of Ad2 DNA (or molar equivalents of the R EcoRI fragments of Ad2 DNA) and 600-1600 ,ug of total cytoplasmic RNA in a final volume of 500-800 iul. Sterile glassware was used for all manipulations. After incubation for 8 min at 650 [approximately 40 times Cotl/. for the DNA (17, 18)] the sample was cooled to room temperature and an equal volume of buffer containing 8 M urea (Schwarz/Mann, ultrapure), 0.20 M sodium phosphate, pH 6.8, and 1% dodecyl sulfate was added. Double-strand nucleic acids were separated from single strands by ureahydroxylapatite chromatography at 40° (22). Hydroxylapatite (Bio-Gel HTP) suspended in 0.14 M sodium phosphate, pH 6.8, 0.2% dodecyl sulfate, was poured into a sterile 3-ml syringe to form a 1-ml column, which was then equilibrated with the urea/sodium phosphate/dodecyl sulfate buffer at a flow rate of 30-40 ml/hr. The sample was applied to the column and the column was washed 5 times with 2 ml of buffer containing 8 M urea, 0.20 M sodium phosphate, pH 6.8, 1% dodecyl sulfate, and 20 ug/ml of Escherichia coli rRNA. The hybrids were eluted at 40° with three 2-ml portions of 0.40 M sodium phosphate, pH 6.8, 0.2% dodecyl sulfate, and 20 1ug/ml of E. coli rRNA, and dialyzed against 50 volumes of 0.15 M LiCl, 0.01 M Tris HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1% dodecyl sulfate. Dialysis was for 4 hr at room temperature followed by 10 hr at 40, followed by an additional 6-hr dialysis at 40 with fresh buffer. The nucleic acids were recovered by precipitation with 2.5 volumes of ethanol. Centrifugation was for 10 min in a clinical centrifuge followed by 15 min at 12,000 rpm in a Sorvall SS34 rotor. The pellet was well drained and then redissolved in 200 ,ul of 0.01 M TrisHC1, pH 7.4, 3 mM EDTA, 5 mM EGTA. The hybrids were denatured by heating in 6 X 50-mm glass tubes for 90 sec at 950, followed by quenching in ice water. The RNA was separated from the DNA fragments by sedimentation through a sucrose gradient (2) or by adsorption to oligo(dT)-cellulose. For the latter procedure the quenched nucleic acids were diluted as quickly as possible with 0.5 ml of 0.01 M Tris * HC1, pH 7.4, 0.5 M NaCl at 0°, and then further diluted with 3.5 ml of the same buffer at room temperature, and immediately passed through a 0.5-ml oligo(dT) column with a flow rate of approximately 25 ml/hr. The flow-through fraction was passed through the column twice more, and the column was then washed with 3 ml 0.01 M Tris HCl, 0.25 M NaCl. The RNA was eluted with 3 ml of 0.01 M Tris * HC1, pH 7.4. After addition of 80 ,g of E. coli rRNA and NaCl to 0.2 M, the RNA was precipitated with 2 volumes of ethanol. The lyophilized RNA pellets were redissolved in 50,ul of H20. KB (human) cells were grown as a suspension culture at 370 in Joklik's modified Eagle medium (cat. no. F-13, Grand Island Biological Co.) supplemented to 5% with horse serum. The preparation of virus and RNA from virus-infected KB cells has been previously described (4, 5), as has the synthesis of protein in a fractionated mammalian cell-free system (5, 27). Analysis of polypeptides by dodecyl sulfate/polyacrylamide gel electrophoresis (4) has been previously described. RESULTS Nucleic acid hybridization techniques permit fractionation of RNA by homology to specific DNA. In order to use these techniques preparatively to select RNA species for identification by subsequent translation, it is essential to minimize degradation of the RNA. To this end we chose rapid liquid phase

1345

hybridization followed by hydroxylapatite chromatography rapidly separate RNA-DNA hybrids from unhybridized RNA. The urea-phosphate procedure (22) minimized nonspecific adsorption of RNA to hydroxylapatite. To reduce degradation of the RNA during chromatography, carrier RNA to

was added to the wash and to the elution buffers (see Materials and Methods), and elution with 0.40 M phosphate at 400 replaced the elution procedure at 800 used by Goldberg et al. (22). After recovering and denaturing the hybrids we separated the mRNA from DNA fragments, which can inhibit cell-free protein synthesis, either by retention of mRNA on oligo(dT)-cellulose or by the sedimentation method of Eron and Westphal (2). At least 10 adenovirus polypeptides are synthesized in a mammalian cell-free system programmed with cytoplasmic RNA isolated from cells late after infection with Ad2 (5-10). Ad2 mRNA selected by hybridization to Ad2 DNA (Fig. if and k) programs the synthesis of the same polypeptides as are seen with the total cytoplasmic RNA from which it was purified (Fig. ld and j). Except for barely detectable amounts of hexon (II) and fiber (IV), none of these polypeptides are seen in control experiments when bacteriophage X DNA is used for the hybridization (Fig. le). The translation of RNA from mock-infected cells subjected to hybridization to Ad2 DNA (Fig. lm) is indistinguishable from the background synthesis found without addition of mRNA to the cell-free system (Fig. li). Both the sucrose gradient and the oligo(dT) method are effective in recovering mRNA from the hybrids. However, the synthesis of all the adenovirus polypeptides is programmed by the RNA recovered by the oligo(dT) method (Fig. lf), whereas the synthesis of components IX and 11.5K is programmed in greatly reduced amounts by the RNA recovered by the sucrose gradient method (Fig. lk). This result is expected, since these polypeptides are encoded by 9S mRNAs (5), and mRNAs less than about 10 S were excluded in collecting the gradients in order to minimize contamination of the mRNA by DNA fragments. At the optimum concentration, hybridization-purified late Ad2 mRNA stimulated the incorporation of [I5S]methionine into trichloroacetic-acid-insoluble material about 80% as well as did the unfractionated total cytoplasmic RNA. We estimate, from the amount of hybridization-purified RNA required to give this amount of incorporation, that about 20% of the active mRNA present in the unfractionated cytoplasmic RNA was recovered. If Ad2 mRNA is fractionated by hybridization to each of the R- EcoRI fragments of Ad2 DNA, then a subset of adenovirus polypeptides is present in the translation product of each RNA fraction (Fig. 2). RNAs homologous to the R. EcoRI A fragment encode polypeptides that correspond in electrophoretic mobility to hexon (II), penton (III), IIIa, IVa2, minor core (V), major core precursor (P-VII), P-VI, P-VIII, and hexon-associated (IX) proteins. Hexon (II) and P-VI mRNAs are also homologous to the B fragment, as is mRNA for component lOOK. The 100K mRNA is also homologous to fragments F and D, as is the mRNA for P-VIII. Fiber (IV) mRNA is homologous to fragments E and C. The order of R.EcoRI DNA fragments (16) is shown in Fig. 2. With the exception of the P-VIII mRNA, all messages that hybridize to more than one fragment do so to adjacent fragments. The apparent dual map position of P-VIII is discussed below. The hybridization of 100K to B and D as well as F is

1346

Biochemistry: Lewis et al. a *.,

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Proc. Nat. Acad. Sci. USA 72 (1975) b c d e f

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FIG. 1. Autoradiogram of a 17.5%O sodium dodecyl sulfate/polyacrylamide gel analysis of the products of cell-free protein synthesis programmed by RNA selected by hybridization to DNA. For cell-free protein synthesis, a 50-,gl reaction mixture contained 0.25 A260 units ribosomal subunits, 4pul of pH 5 enzyme (containing 68 pg of protein and 4 Ag of RNA), 0.75 pl of rabbit reticulocyte ribosome wash precipitating at 30-40% (NH4)2SO4 (16 jug of protein), and 4 ;l of the fraction of a Krebs II ascites cell ribosome wash precipitating between 40 and 65% (NH4)2SO; at 00 (58 pig of protein). The other ingredients were 1.0 mM ATP, 0.4 mM GTP, 10 mM creatine phosphate, 20 ug/ml of creatine kinase, 30 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.2, 20 mM NH4Cl, 87 mM KCl, 2.0 mM Mg(OAc)2, 0.8 mM spermidine, 0.5 mM dithiothreitol, and 30 mM each of 19 amino acids (minus methionine). Each 50-IAl reaction mix contained 10 ,Ci of ['S]methionine at greater than 100 Ci/mmol of specific activity (2 pM methionine). Incubation was for 90 min at 370. The percentage incorporation of methionine into trichloroacetic-acid-insoluble radioactivity was (b) 20%.70, (c) 27%70, (d) 75%oO (e) 17%, (f) 52%,(i) 8%, (j) 66%0, (k) 51%0, (1) 48%7O, (m) 7%70. The amount of endogenous protein synthesis was determined by incubation with no added RNA (b) or with 15 pug of E. coli rRNA (c, i) which increases the amount of endogenous protein synthesis (Atkins et al., submitted). Protein synthesis was programmed by total cytoplasmic RNA from uninfected KB cells (1) or from KB cells 24 hr after infection with Ad2 (d, j), or by fractions of these RNAs selected by hybridization to DNA. (e) is a control with Xplac DNA (gift of J. L. Manley) used for the hybridization to Ad2 mRNA. The band migrating near virion component IX, apparent in b, c, and e, is globin due to translation of globin mRNA contaminating reticulocyte initiation factors. Forf and k, Ad2 mRNA was selected by hybridization to Ad2 DNA. In m, uninfected KB cell mRNA was used for hybridization to Ad2 DNA. For k and m, the gradient variant of the hybridization procedure was used, and in e andf the oligo(dT) method was used. The exact concentration of hybridization-selected RNAs used is not known, but was 160%O of the RNA recovered from hybridization using 600 pg of cytoplasmic RNA from Ad2-infected cells or 1600 ,ug of cytoplasmic RNA from uninfected cells. Polypeptide markers were provided by disrupted virions (a, n) and by in vivo-labeled extracts of uninfected cells (g) and of cells pulse-labeled 26 hr after infection with Ad2 (h). Three microliters of each reaction mix was applied to the gel. Parts a to f, g and h, and i to n are from three different gels. The autoradiograms were exposed for: (a) 84 hr, (b-f) 8 hr, (g and h) 24 hr, (i-m) 7 hr, and (n) 68 hr. not surprising, since F can encode only 49,000 daltons of protein. In addition to these 11 components, the mRNAs for several minor polypeptides are mapped on specific DNA fragments. Some of these polypeptides probably represent incom-

plete synthesis of larger polypeptides. DISCUSSION The above results and those of Prives et al. (1) and Eron and Westphal (2) demonstrate that specific mRNAs may be selected by hybridization to DNA and translated in a cell-free system to give specific polypeptides. We find that mRNA from Ad2-infected cells stimulates cell-free synthesis of 12

Ad2-specific polypeptides-II, lOOK, III, IIIa, IV, IVa2, V, P-VI, P-VII, P-VIII, IX, and 11.5K-before and after selection by hybridization to Ad2 DNA. If the Ad2 mRNA is hybridized with bacteriophage X DNA, very little of the mRNA activity is recovered. Similarly, no detectable mRNA activity is recovered if mock-infected cell

mRNA is hybridized to Ad2 DNA. Thus, the mRNAs for these virus-specific polypeptides are homologous to Ad2 DNA, so that none of them are virus-induced host proteins. Eron and Westphal (2) have reached the same conclusion for components II, III, i11a, IV, V, and P-VII. Since most of the minor unidentified polypeptides seen among the translation products of unfractionated RNA were also seen when translation was programmed with hybridization-purified Ad2 mRNA, these polypeptides probably represent incomplete synthesis of the larger Ad2 polypeptides, or previously unidentified virus-specific components. Conceivably, some minor products result from the entrapment of nonhomologous mRNA by the hybrids. Peptides which can be assigned a unique map position as discussed below must, however, be adenovirus-specific. Approximately 20% of the mRNA activity present in unfractionated cytoplasmic RNA is recovered by the above procedure. Preparation of sufficient hybridized RNA to

Proc. Nat. Acad. Sci. USA 72

Biochemical Mapping of Late Adenovirus Genes

(1975)

1347

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FIG. 2. Autoradiogram of a 17.5%o sodium dodecyl sulfate/polyacrvlamide gel to display the product of cell-free protein syntheses. Protein synthesis occurred in the presence of the fraction of Ad2 mRNA selected by hybridization to (a) Ad2 DNA, (b) Xplac DNA, (c) R-EcoRI A fragment of Ad2 DNA, (d) B fragment, (e) F fragment, (f) D fragment, (g) E fragment, (h) C fragment. Chromatography on oligo(dT)-cellulose was used to recover the mRNA after hybridization. Cell-free protein synthesis was as described in the legend to Fig. 1, except that 8.0 mM putrescine was used instead of 0.8 mM spermidine. Of the 8 ,uCi of [36S]methionine in each 50-Ml mix, the amount incorporated into trichloroacetic acid-insoluble radioactivity was (a) 20%0, (b) 12%, (c) 22%0, (d) 14%,O (e) 12%, (f) 15%, (g) 14%, (h) 12%. Three microliters of each reaction mix was used for each sample loaded onto the gel. A comparison with viral polypeptides is provided by disrupted virions (i). The autoradiogram was exposed for 6 days. The positions of the genes for 10 viral polypeptides are shown along the R* EcoRI map of Ad2 DNA at the bottom of the figure.

saturate a standard 50-,ul reaction mixture for cell-free protein synthesis requires the equivalent of 20 ug of Ad2 DNA and 120 gg of late cytoplasmic RNA, which is the yield from 20 to 40 ml of late-infected cells. This means that it should be possible to study much less abundant mRNA species by suitLably scaling up the procedure. Although both the sucrose gradient and the oligo(dT) variants of the procedure separate DNA fragments from the mRNA, we prefer the latter because of the better recovery of small mRNAs and because it is faster than the gradient method. However, the gradient provides an alternative method for mRNAs that are not retained by oligo(dT). The results with R- EcoRI fragments (see Fig. 2) demonstrate the following order of genes along the Ad2 genome: (III, MIla, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV, where the order of the components in parentheses has not yet been determined. The genes for these components can be more precisely positioned on the Ad2 DNA molecule by the use of other specific endonucleases to cleave Ad2 DNA. Of the components listed above, only II, IV, V, P-VII, and IX have been identified with their in vivo counterparts by tryptic peptide analysis (5); the remaining components have

been identified only on the basis of migration in sodium dodecyl sulfate/polyacrylamide gels. The results give two locations for component P-VIII, and we have not included it in the map. This ambiguity is most likely due to a second polypeptide with the same apparent molecular weight as P-VIII. This additional polypeptide could be a previously unknown gene product or a fragment resulting from incomplete synthesis of one of the higher molecular weight components located in R* EcoRI A fragment. Tryptic peptide analysis should determine which of these apparent P-VIII polypeptides is equivalent to the polypeptide seen in tvo. Additional unidentified minor polypeptides have been mapped on specific R- EcoRI fragments. The polypeptide migrating slightly ahead of IV that is mapped with IV on fragments E and C most likely results from the incomplete synthesis of the larger polypeptide. On the other hand, the 38K polypeptide is most likely a legitimate viral gene product, since it is the largest polypeptide whose mRNA is selected solely by F and D. As is apparent in Fig. 2, there is a significant "background" synthesis of a few Ad2 polypeptides, notably II and IV, when mRNA is selected by hybridization to fragments that do

1348

Biochemistry: Lewis et al.

not appear to contain the structural genes for those polypeptides. Although this background is not large enough to obscure the specific selection of these mRNAs by fragments, it is larger than the barely detectable amounts of these polypeptides seen when nonhomologous DNA is used for the hybridization. This background could be a consequence of the fact that nuclear RNA contains some complementary RNA sequences transcribed from both strands of DNA (14). Nuclear leakage late in infection or during the preparation of the RNA would account for the presence of some RNA complementary to late mRNA. By annealing to Ad2 mRNA, these complementary RNA sequences could link Ad2 mRNA to DNA sequences to which the Ad2 mRNA has no homology, or to another Ad2 mRNA hybridized to DNA. Such RNA RNA hybridization superimposed on DNARNA hybridization could also account for the apparent mapping of two genes at the same position on the DNA molecule. II and P-VI are both mapped at the boundary between fragments A and B. 100K and perhaps P-VIII (see above) are both mapped at the boundary between fragments F and D. Other possible explanations include the following: (a) One mRNA molecule encodes the sequences for two unrelated polypeptides. This seems unlikely for these specific cases, since we have shown previously (5) that neither hexon and P-VI (previously designated 27K) mRNAs nor 100K and P-VIII (previously designated 26K) mRNAs cosediment through sucrose gradients containing formamide. However, this objection would not pertain if the mRNA had to be processed before it could program the synthesis of the second polypeptide. (b) The information for the two polypeptides is contained in separate mRNA molecules, but these molecules, contain some common sequences. Other studies have also suggested a map order for adenovirus genes. Williams et al. (23) have isolated temperaturesensitive mutants of adenovirus type 5 and have mapped them by three-factor crosses. Hexon and fiber have been tentatively associated with two of the complementation groups whose map positions are consistent with the results presented above. Using the ability to isolate recombinants between Ad2 and adenovirus type 5 (24, 25), T. Grodzicker, C. Anderson, and J. Sambrook (unpublished) have mapped the positions of the structural genes for several polypeptides, including 100K, hexon, and fiber. Their results are in accord with the results reported here, as are the results of serological analysis of the hexon and fiber antigens from these recombinants (28). The translation of mRNA selected by hybridization to DNA fragments should be of particular value when applied to early Ad2 mRNA. Of the nine Ad2-transformed cell lines examined, all contain DNA from the left end of the R * EcoRI A fragment but none has the complete genome (15, 26). This is also a region of the Ad2 DNA transcribed early in permissive infection (11-15). Determination of which of the Ad2 early polypeptides is located in this region of the genome would implicate that protein in the maintenance of the transformed cell phenotype by Ad2 genetic information.

Proc. Nat. Acad. Sci. USA 72

(1975)

We thank R. Solem and P. Grisafi for invaluable assistance, and

P. Myers and R. Roberts for a gift of endonuclease R -EcoRI. J.F.A. acknowledges travel funds from the European Molecular Biology Organization. This work was supported by a grant from the National Science Foundation to R. F.G. and by Public Health Service Research Grant CA 13106 from the National Cancer Institute. 1. Prives, C. L., Aviv, H., Paterson, B. M., Roberts, B. E., Rozenblatt, S., Revel, M. & Winocour, E. (1974) Proc. Nat. Acad. Sci. USA 71, 302-306. 2. Eron, L. & Westphal, H. (1974) Proc. Nat. Acad. Sci. USA 71, 3385-3389. 3. Philipson, L. & Pettersson, U. (1973) Advan. Exp. Tumor Virus Res. 18, 1-55. 4. Anderson, C. W., Baum, P. R. & Gesteland, R. F. (1973) J. Virol. 12, 241-252. 5. Anderson, C. W., Lewis, J. B., Atkins, J. F. & Gesteland, R. F. (1974) Proc. Nat. Acad. Sci. USA 71, 2756-2760. 6. Lewis, J. B., Anderson, C. W., Atkins, J. F. & Gesteland, R. F. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,

581-590. 7. Eron, L., Westphal, H. & Callahan, R. (1974) J. Virol. 14, 375-383. 8. Eron, L., Callahan, R. & Westphal, H. (1974) J. Biol. Chem. 249, 6331-6338. 9. Westphal, H., Eron, L., Ferdinand, F.-J., Callahan, R. & Lai, S.-P. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 575-579. 10. Oberg, B., Saborio, J., Persson, T., Everitt, E. & Philipson,

L. (1975) J. Virol. 15, 199-207. 11. Tal, J., Craig, E. A., Zimmer, S. & Raskas, H. J. (1974) Proc. Nat. Acad. Sci. USA 71, 4057-4061. 12. Craig, E. A., Tal, J., Nishimoto, T., Zimmer, S., McGrogan, M. & Raskas, H. J. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 483-493. 13. Tibbetts, C. & Pettersson, U. (1974) J. Mol. Biol. 88, 767784. 14. Philipson, L., Pettersson, U., Lindberg, U., Tibbetts, C., Vennstrom, B. & Persson, T. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 447-456. 15. Sharp, P. A., Gallimore, P. H. & Flint, S. J. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 457-474. 16. Mulder, C., Arrand, J. R., Delius, H., Keller, W., Pettersson, U., Roberts, R. J. & Sharp, P. A. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 397-400. 17. Pettersson, U. & Sambrook, J. (1973) J. Mol. Biol. 73, 125130. 18. Pettersson, U., Mulder, C., Delius, H. & Sharp, P. A. (1973) Proc. Nat. Acad. Sci. USA 70,200-204. 19. Fukes, M. & Thomas, C. A., Jr. (1970) J. Mol. Biol. 52,

395-397.

20. Southern, E. M. (1975) J. Mol. Biol., in press. 21. Tibbetts, C., Pettersson, U., Johansson, K. & Philipson, L. (1974) J. Virol. 13, 370-377. 22. Goldberg, R. B., Galau, G. A., Britten, R. J. & Davidson, E. H. (1973) Proc. Nat. Acad. Sci. USA 70,3516-3520. 23. Williams, J. F., Young, C. S. H. & Austin, P. E. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 427-437. 24. Grodzicker, T., Williams, J., Sharp, P. A. & Sambrook, J. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 439-446. J., Grodzicker, T., Sharp, P. A. & Sambrook, J. 25. (1975) Cell 4, 113-119. 26. Sharp, P. A., Pettersson, U. & Sambrook, J. (1974) J. Mol. Biol. 86,709-726. 27. Atkins, J. F., Lewis, J. B., Anderson, C. W. & Gesteland, R. F. (1975) J. Biol. Chem. 250, in press. 28. Mautner, V., Williams, J., Sambrook, J., Sharp, P. A. & Grodzicker, T. (1975) Cell, in press.

Willia~is,