Proc. Nati. Acad. Sci. USA

Vol. 75, No. 1, pp. 69-73, January 1978 Biochemistry

Transcription of a defective polyoma virus genome (viral transcription complexes/RNA*DNA hybridization)

RICHARD CONDIT*, ALISON COWIE, ROBERT KAMEN, AND MIKE FRIED Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, England

Communicated by Paul Berg, October 11, 1977

ABSTRACT The circular genome of the cloned defective polyoma virus D-50 consists of tandemly repeated copies of the DNA sequence between 67 and 84 units on the wild-type polyoma virus DNA map. Each repeated copy thus contains the origin of viral DNA replication, which is located at about 71 map units. Viral RNA was synthesized in vitro using viral transcription complexes extracted late (30 hr) after infection from mouse cells co-infected with D-50 and helper wild-type virus. Both wild-type and D-50 DNA molecules were active as templates for in vitro transcription. Approximately 84% of the RNA transcribed in vitro from wild-type DNA was complementary to the L DNA strand. This is normal for wild-type transcription late after infection. By contrast, at least 90% of the RNA transcribed from D-50 DNA molecules was complementary to the E DNA strand. After normalization of the data to account for the observed molar ratio of D-50 DNA repeated sequences to unit length wild-type DNA, we estimate that transcription of the E DNA strand of each D-50 repeated unit is about 1.4 times as efficient as transcription of the wild-type E DNA strand. Transcription of the D-50 L DNA strand, however, is only 0.03 times as efficient as transcription of the wild-type L DNA strand. The implications of these results concerning the nature and location of promoter sequences in polyoma DNA are discussed. The expression of polyoma virus during lytic infection is regulated, in part, at the transcriptional level. During the early phase of infection, that is, before the onset of viral DNA replication, RNA complementary to only one strand (the E strand) of the viral DNA is found. After the onset of viral DNA replication, RNA complementary to the E DNA strand continues to be synthesized, but RNA is also synthesized from the complementary L strand, in much higher relative amounts (1, 2). Thus, late during polyoma virus infection, 90-95% of the pulse-labeled viral RNA is complementary to the L DNA strand, and 5-10% is complementary to the E DNA strand (3). Study of polyoma virus transcription in vitro using an active viral DNA/RNA polymerase complex extracted from the nucleus of virus-infected cells recently showed (4) that the abundance of RNA complementary to the L strand represents preferential transcription, rather than strand-specific posttranscriptional degradation of nascent RNAs. Similar results have been obtained with the closely related papova virus, simian virus 40 (SV40) (refs. 5-9; see also ref. 10 for a comparative review of polyoma and SV40 transcription). Late nuclear polyoma virus RNA is complementary to virtually all sequences of both viral DNA strands (1, 11), and viral transcripts considerably larger than genome length are present (12-16). By contrast, the polyadenylylated cytoplasmic viral mRNAs are complementary to only one-half of the sequences of each DNA strand (17, 18). Therefore, regulation of the final sequence composition of the viral mRNA seems to be accomplished at the post-transcriptional level. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

The existence of transcriptional regulation during polyoma virus and SV40 infection implies that the viral DNA molecules contain specific signals that differentially affect transcription of the complementary DNA strands. These signals could be analogous to those well characterized in prokaryotic systems, such as promoters, terminators, or binding sites for specific viral or cellular regulatory proteins. Alternatively, unknown mechanisms unique to higher organisms may be involved. Because both E and L DNA strands are transcribed by cellular RNA polymerase 11 (4, 6), knowledge of how papova virus transcription is regulated will provide information pertinent to the mechanisms controlling cellular transcription. Moreover, because vectors containing the replication origins of polyoma virus or SV40 DNA are promising vehicles for cloning other DNA in mammalian cells, it is important to learn where transcriptional signals occur in the viral DNAs. One approach to the localization and characterization of transcriptional control sequences in polyoma virus DNA is the study of viral deletion mutants. We have chosen a naturally occurring defective polyoma virus, D-50, in which approximately 83% of the wild-type genome is deleted, and the remaining 17% exists in circular, head-to-tail tandemly repeated molecules that are 34, 51, 68, 85, and 102% the length of the wild-type genome (19-21). The 17% of the wild-type DNA sequence that is repeated in D-50 molecules, which we shall refer to as the D-50 "subunit" (see Fig. 1), includes the origin of viral DNA replication at 71 map units (22, 23). Previous experiments have suggested that the 5' ends of both early and late viral mRNAs map near the replication origin (17). MATERIALS AND METHODS Virus. Plaque-purified wild-type polyoma virus of the A-2 strain (22) was grown at low multiplicity and checked for the absence of defectives as described (17, 19). The D-50 virus has been described previously (19-21). The virus stock used for infection contained 8.5 X 108 plaque-forming units (PFU) per ml of wild-type virus, and had a PFU/hemagglutination unit (HAU) ratio of 2.7 X 105. Because highly infectious (defective-free) virus stocks have a PFU/HAU ratio of 106'(19), 73% of the virions in the D-50 stock presumably contain D-50 DNA. Purification of Transcription Complexes. Sixty 90-mm dishes of the 3T6 line of mouse fibroblasts, grown to a density of 5 X 106 cells/dish, were infected with either 50 PFU of wild-type polyoma virus per cell as described previously (4) or with an amount of D-50 virus stock that contained 10 PFU. After 30-hr incubation at 370, transcription complex was purified from the infected cells through fraction III, and stored in aliquots in liquid N2 as described previously (4). The total yield of wild-type and defective virus DNA in each of two Abbreviations: SV40, simian virus 40; PFU, plaque-forming units. * Present address: Health Sciences Center, State University of New York, Stony Brook, NY 11794.

69

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Proc. Nati. Acad. Sci. USA 75 (1978)

Biochemistry: Condit et al. Wild type A-2

6 X D-50

Hha I

\\

'Hha I Hha

I50

4.

0

17% D-50 subunit

E mRNA L mRNA .4

--

-

--

-

--

-

--

3

-

.0

5

4

84 o 75 70 DNA molecule. Reading outward virus polyoma wild-type FIG. 1. The physical maps of wild-type and D-50 polyoma DNAs. (Upper Left) A from the innermost of the concentric circles, we indicate (i) standard polyoma map units (22); (ii) the Hpa II restriction endonuclease cleavage map (22); (iii) the origin (0) of DNA replication (22, 23) and the cleavage sites of restriction endonucleases EcoRI (22), Hha I and Bam I (24). (Upper Right) A hexameric D-50 DNA molecule, comprised of a 6-fold tandem repeat of a contiguous portion of the wild-type genome that lies between 67 and 84 map units. D-50 DNA preparations also contain molecules that are 2-, 3-, 4-, and 5-fold repeats of the same portion of the genome (21). (Lower) An expanded map of the basic D-50 subunit. Reading from the top down, we indicate: (i) The origin (0) of DNA replication. (ii) The estimated map positions of the 5'-terminal portions of the polyadenylylated cytoplasmic mRNAs (solid lines). The broken line indicates further sequences of unknown function that are present in small amounts in cytoplasmic polyadenylylated RNA preparations (17). The arrows indicate the 5' to 3' polarities of the RNAs (25). (iii) Hpa II restriction fragments or portions thereof. (iv) Map units. Five map units correspond to approximately 260 base pairs. Map units 67

preparations of D-50 transcription complex was approximately 2 Mg per 107 cells. The yields of transcriptional activity per

weight of DNA were similar for complexes extracted from cells infected with wild-type virus or with D-50 virus stock. In Vitro RNA Synthesis and RNA Purification. Preparative RNA synthesis reactions (0.5-1.5 ml) contained: 90 mM TrisHCI at pH 7.5, 125 mM (NH4)2SO4, 5 mM MgCl2, 0.08 mM EDTA, 1.8 mM dithiothreitol, 0.2% Sarkosyl NL-35 (CibaGeigy), 20% (vol/vol) ethylene glycol, 800 Ml of fraction III transcription complex per ml, 0.5 mM each ATP, UTP, CTP, and GTP (P-L Biochemicals), and [3H]UTP (10-60 Ci/mmol, Amersham) at 1.0 mCi/ml. Reaction mixtures were incubated for 4 hr at 28°. RNA was purified from the reactions by phenol extraction, DNase treatment, and Sephadex G-75 chromatography as described previously (4). The specific activity of the in vitro synthesized RNA was approximately 6.5 X 106 cpm/Mg.

Preparation of Separated Strands of Polyoma DNA Restriction Fragments. 32P-Labeled polyoma viral DNA (3000 cpm/,g) was prepared as described by Hirt (26) from infected 3T6 cells (22) labeled from 24 to 48 hr after infection with [32P]orthophosphate (Radiochemical Centre, Amersham) at 2.5 MCi/ml. Superhelical DNA was purified by ethidium bromide/cesium chloride equilibrium centrifugation (27). Low levels of P2P in the viral DNA facilitated quantitation during strand separation, and did not interfere with subsequent hybridization assays. Polyoma virus DNA (160 Mg) was simultaneously digested to completion with EcoRI and Bam I restriction endonucleases and then fractionated by electrophoresis on a 15- X 20-cm, 150-ml horizontal slab gel containing 1.4% (wt/vol) agarose (SeaKem) and 0.5 Mg of ethidium bromide per ml in E buffer (28). The two EcoRI/Bam I DNA fragments were excised from the gel and the gel slices were sealed with melted agarose in E buffer into appropriately sized slots in two separate agarose slab

gels identical to the first gel. After electrophoresis through the second gels, DNA fragments visualized over a UV light were excised and eluted electrophoretically into dialysis bags. DNA strands were separated by hybridization to asymmetric polyoma virus complementary RNA (synthesized in vitro using purified polyoma virus DNA and Escherichia colt RNA polymerase) followed by hydroxyapatite chromatography (1, 3, 4). RNADNA Hybridization. Hybridizations to separated strands of polyoma virus DNA were performed as follows: Mixtures (0.08 ml) containing 0.01 Mug of 3H-labeled RNA, 0.01-0.1 Mug of DNA, 12.5 mM Tris-HCI at pH 7.5, 12.5 mM EDTA, and 0.125% sodium dodecyl sulfate were heated to 1070 for 2 min, cooled rapidly, and 20 ,ul of 5 M NaCl was added. Mixtures were then incubated at 680 for 20-24 hr. RNA-DNA hybrids were recovered by adsorbtion to nitrocellulose filters, and contaminating RNA was removed by digestion with RNase (3). RESULTS The in vitro transcription system used in our experiments measures the relative numbers of polymerase molecules active in transcription of the viral E and L DNA strands at a given time during infection (4). The in vitro system was constructed as follows: 3T6 mouse cells were infected with wild-type virus or a virus stock containing D-50 and wild-type helper virus. Late during infection (30 hr), nuclei were isolated from the infected cells, lysed with the anionic detergent Sarkosyl, and large cellular DNA was removed by centrifugation (29). The supernatant contained an active viral transcription complex that was purified further by high-speed centrifugation and agarose column chromatography. RNA synthesis directed by the transcription complex in vitro proceeds entirely through elongation of RNA chains that were initiated in vivo; no reinitiation of transcription is observed. The RNA synthesized in vitro by the viral transcription complex extracted from wild-

Biochemistry: Condit et al.

Proc. Nati. Acad. Scit. USA 75 (1978)

71

100 type polyoma-infected cells is similar to nascent viiral NA synthesized in vivo by two important criteria (4): (i) each viral 40DNA strand is transcribed completely and uniformly, and polymerase molecules can travel more than once around the circular viral genome; (ii) the ratio of L to E DNA strand 30 transcription in vitro (85:15) is approximately the same as that observed in vivo (90:10) at the time after infection when the complex is prepared. The in vitro system facilitates the analysis 20 of D-50 transcription because it readily yields large amounts of radiochemically pure unprocessed viral RNA. Moreover, the 0) fact that transcription of each DNA strand is complete and 10 uniform, as will be discussed below, is critical in the quantitative .0 N interpretation of the results obtained. Analysis of DNA in the D-50 Transcription Complex. We estimated the relative sizes and amounts of defective and wild-type viral DNA in transcription complex isolated from z cells co-infected with D-50 and wild-type virus by analyzing * 40 DNA purified from the complex by agarose gel electrophoresis before and after cleavage with various restriction endonucleases (data not shown). When, for example, the mixture of wild-type 30 and defective DNA in the complex is digested to completion with the restriction endonuclease Hhia I (see Fig. 1), four different sized DNA fragments are obtained. Three of these 20' fragments, 46%, 42%, and 12% the length of the wild-type genome, arise from wild-type DNA molecules in the mixture (24). Defective molecules yield only a 17% fragment regardless of 10 the number of tandem repeats of the basic subunit contained in a given defective (21). Quantitative microdensitometry of the gels (see Table 2) showed that the amount of DNA in the 0.02 0.06 0.04 17% fragment was consistently about 60% of the sum of the pmol polyoma DNA amount of DNA in all four fragments. FIG. 2. Hybridization of in vitro synthesized RNA to separated Analysis of RNA Synthesized In Vitro by the D-50 Transtrands of EcoRI/Bam I restriction fragments. The indicated amounts scription Complex. The RNA synthesized by the D-50 tranof polyoma virus DNA were annealed to a fixed amount of 3H-labeled scription complex could comprise transcripts from both the RNA synthesized in vitro from wild-type (A) or D-50 (B) transcripwild-type and the defective DNA molecules present in the tion complex. 0, Annealings with the E DNA strand of the 40% EcoRI/Bam I restriction fragment (40E); 0, annealings with 60E; A, preparation. To determine what proportion of the RNA was annealings with an equimolar mixture of 40E and 60E; 0, annealings transcribed from each of the complementary strands of the two with 40L;*, annealings with 60L; A, annealings with an equimolar potential template species, we hybridized the RNA to excess mixture of 40L and 60L. One pmol of polyoma virus DNA = 3.4 gg, amounts of the separated strands of two restriction fragments therefore, 1 pmol of a 40% EcoRI/Bam I single-stranded DNA reof polyoma virus DNA, one of which includes all, and the other striction fragment = 0.68 Ag. The left ordinate represents the actual none of the sequences present in D-50. The fragments used, percent of the input radioactivity that remained bound to nitrocellulose filters after RNase digestion and washing (see Materials and which are approximately 40% (41.5%) and 60% (58.5%) the Methods). The right ordinate is a normalized scale calculated from length of the wild-type genome (24), were obtained by digestion the observed 46% efficiency of the filter adsorption assay. Input raof wild-type polyoma DNA with restriction endonucleases dioactivities (per annealing): (A) 10,500 cpm, (B) 6400 cpm. A counter EcoRI and Bam I (see Fig. 1). The 40% fragment contains all background of 22 cpm was subtracted from each value. of the D-50 sequences, while the 60% contains no D-50 se(not shown) demonstrated that the efficiency of the filter adquences. Assuming that transcription of each wild-type DNA sorption method is constant within a given experiment, and that strand is uniform in vitro (see below) we can determine what the RNA-DNA hybrids that bind to nitrocellulose filters are a fractions of the total RNA anneal to the two strands of the 60% representative fraction of the total RNA-DNA hybrids formed fragment and then use these numbers to calculate the proporin solution. tions of wild-type and D-50 transcripts in the mixture of RNA Fig. 2A shows a control experiment designed to test the that hybridize to the complementary strands of the 40% fragmethods described above, and to confirm our previous finding ment. We measured RNA-DNA hybrid formation by adsorption (4) that transcription of wild-type polyoma virus DNA strands of the hybrid molecules to nitrocellulose filters followed by in vitro is uniform. We hybridized 3H-labeled RNA synthesized RNase digestion at high ionic strength (30,31). This procedure in vitro from wild-type transcription complex to unlabeled DNA from the separated strands of EcoRI/Bam I restriction has the critical advantage of discriminating between RNA-DNA hybrids and double stranded RNA molecules generated by fragments, and analyzed the RNA-DNA hybrids by the filter self-annealing of complementary transcripts. In previous exadsorption method. Averaging the results of this experiment with the results of two other similar experiments (the controls periments, we assayed hybrid formation by direct measurement to Exps. 1 and 3, Table 2), we find that of the total RNA that of RNase resistance at high ionic strength in solution and demonstrated that in sufficient excess of total polyoma DNA, hybridized to the viral L DNA strand, 61% (+2%) hybridized all of the in vitro synthesized RNA enters RNA-DNA hybrids to the 60% EcoRI/Bam I restriction fragment, and 39% (+2%) in solution (4). A disadvantage of using the nitrocellulose adhybridized to the 40% fragment. These numbers are in good sorption method is that we recover only 20-60% of these agreement with the hybridization values one would expect RNA-DNA hybrids on the filters. However, control experiments knowing the precise relative sizes of the DNA fragments, 58.5% N

._

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72

Proc. Nati. Acad. Sci. USA 75 (1978)

Table 1. Calculation of wild-type and D-50 E and L DNA strand transcripts in the D-50 transcription complex in vitro product by hybridization to the separated strands of the 60% and 40% EcoRI/Bam I DNA fragments

DNA strand L E 16 3 1 Hybridization to 60% fragment, %* 17 63 2 Hybridization to 40% fragment, %* 2 11 3 Wild-type RNA hybridized to 40% fragment, %t 27 4 Wild-type RNA in mixture, %t 5 6 61 5 D-50 RNA in mixture, %§ * Numbers shown are the average of the normalized percent RNA hybridized to the two highest amounts of DNA tested. The data are taken from the experiment shown in Fig. 2B. t (Line 1) X (length of 40% fragment/length of 60% fragment) = (line 1) X (41.5/58.5) = (line 1 X 0.71). Line 1 + line 3. § Line 2 - line 3.

and 41.5%. Of the RNA that hybridized to the E DNA strand, 54% (+1%) hybridized to the 60% fragment and 46% (+1%) hybridized to the 40% fragment. Because of the small proportion of the total radioactivity measured, the values obtained in this case are less accurate than those obtained with the more abundant RNA that hybridized to the L DNA strand of the fragments. Nevertheless, the errors are too small to affect the conclusions drawn from the experiments described below. RNA synthesized in vitro from the D-50 transcription complex mixture was hybridized to the separated strands of the EcoRI/Bam I fragments in the experiment shown in Fig. 2B. The results are strikingly different from those obtained with the wild-type complex (Fig. 2A); most of the 3H-labeled RNA synthesized in vitro from the D-50 complex hybridized to the E DNA strand of the 40% fragment. The data shown in Fig. 2B are quantitatively analyzed in Table 1, using the procedure outlined above to calculate the proportions of the in vitro product that were transcribed from the E or L strands of the defective and wild-type DNA templates. From these calculations we deduce that, of the 33% of the total in vitro synthesized RNA that hybridized to the L strand of polyoma DNA, 27% was transcribed from wild-type DNA molecules and 6% was transcribed from D-50 molecules. Similarly, of the 66% of the total RNA that hybridized to the polyoma virus DNA E strand, 5% was transcribed from wild-type DNA molecules and 61% from D-50 DNA molecules. Table 2 summarizes results obtained using three different

preparations of RNA synthesized in vitro with two different preparations of D-50 transcription complex. An analysis of RNA synthesized with an exclusively wild-type transcription complex is included for comparison. We conclude that the wild-type helper DNA molecules present in the D-50 transcription complex preparations are transcribed in the same manner as DNA molecules in a transcription complex harvested from cells infected only with wild-type virus at the same time during infection: approximately 85% of the RNA is synthesized from the L DNA strand and 15% from the E DNA strand. By contrast, only about 10% of the RNA synthesized from the D-50 DNA molecules is transcribed from the L DNA strand, while 90% is synthesized from the E DNA strand.

DISCUSSION The preferential transcription of the E DNA strand of the defective D-50 late during infection can best be understood after quantitative comparison with the transcription of the complete genome. The multiplicity of infection used in the preparation of the mixed transcription complex was such that each cell should have been infected with several defective and helper wild-type virions. Under the assumption that the distribution of replicating and transcriptionally active D-50 or wild-type DNA molecules is random among the cells 30 hr after infection, the wild-type DNA molecules provide an excellent internal standard for calculating the relative transcriptional efficiency of the defective molecules. For example, in Exp. 2 shown in Table 2, the transcription complex studied contained, by weight, 58% D-50 DNA. Because the basic D-50 subunit is only 17% the mass of the wild-type DNA molecule, for every molecule of wild-type DNA in this complex, there were effectively 8.5 molecules of the basic D-50 subunit. We can therefore calculate from Table 2 the percent of the total in vitro product synthesized from E or L DNA strand of each molar equivalent of the D-50 subunit. The results of this calculation indicate that each D-50 DNA subunit yields RNA complementary to the E DNA strand at 1.4 times the wild-type level, and RNA complementary to the L DNA strand at 0.03 times the wild-type level. Thus, relative to wild-type DNA molecules isolated from the same infected cells, the efficiency of transcription of the E DNA strand of the D-50 subunit is virtually "normal," while the efficiency of transcription of the L DNA strand is drastically impaired.

Direct comparison between transcription of D-50 and wild-type DNA molecules during mixed infection enables us to exclude any explanation for the preferential transcription of the D-50 E DNA strand that invoked trans effects. Reed et

Table 2. Calculated transcription of wild-type and D-50 E and L DNA strands in the D-50 transcription complex mixture Transcription product, % of total LRNA+ LRNA,%/ DNA, ERNA ERNA LRNA ERNA,% %oftotal D-50 wt wt D-50 D-50 wt wt D-50 D-50 wt Exp. 1*

42

58

22

5

9

64

31

69

71/29

7/93

32 66 5 9/91 61 84/16 6 27 42 58 2t 70 62 31 12/88 84/16 8 5 60 26 40 3$ 87/13 13 100 87 100 4t wt, wild type; L and E RNA, RNA complementary to L and E DNA, respectively. * Calculated from an experiment identical to that shown in Fig. 2B, using a different preparation of 3H-labeled RNA synthesized from the same preparation of transcription complex used in Exp. 3. Input radioactivity = 4600 cpm per annealing. Filter adsorption assay efficiency =

21%.

results shown for Exps. 2 and 4 were calculated from the data shown in Fig. 2B and A, respectively. Calculated from an experiment identical to that shown in Fig. 2B using RNA synthesized from a different preparation of D-50 transcription complex. Input radioactivity = 3600 cpm per annealing. Filter adsorption assay efficiency = 35%.

t The

Biochemistry:

Condit et al.

al. (8) have suggested that transcription of the E DNA -Stand of the closely related virus SV40 is negatively controlled by the A gene. In their model, the A gene product binds to the viral DNA at or near the origin of DNA replication, repressing transcription of the E DNA strand. If the same control mechanism applies to polyoma virus, one could have expected that the D-50 DNA molecules would titrate available A gene product, resulting in a generalized derepression of E DNA strand transcription in the infected cells. The present data show, however, that the helper wild-type DNA molecules in the transcription complex extracted from mixedly infected cells is transcribed with the normal strand selection ratio, and moreover, that the rate of E strand transcription of the D-50 subunit per potential transcriptional start sequence is "normal." It therefore appears that sufficient A gene product is available in the infected cells to maintain transcriptional repression. We have recently shown, in addition, that transcription of the E strand of D-50 DNA is under A gene control in vivo (R. Kamen and M. Fried, unpublished results). Experiments using transcription complexes measure only the relative numbers of active RNA polymerase molecules bound to each viral DNA strand at a particular time during infection. Therefore, one cannot determine whether the greater number of polymerases bound to the D-50 DNA E strand is the result of a bias at the point of initiation or termination. The available evidence, however, suggests that termination of polyoma virus transcription in vivo is an inefficient process. Late during infection, both viral DNA strands are transcribed in their entirety (1), and the majority of the nascent viral RNA present in the cell nucleus is larger than the size of the viral DNA; RNA polymerase molecules probably travel more than once around the circular genome (10, 13-16). Even if wild-type polyoma DNA were to contain a functional termination signal that governed the frequency of L DNA strand transcription, the likely consequence of deleting DNA from the genome would be to enhance transcription. Instead, we have found normal levels of transcription of the D-50 E DNA strand and greatly reduced transcription of the D-50 L DNA strand. We therefore favor the view that the number of RNA polymerase molecules active in transcription at a particular moment is primarily a function of the rate of initiation of RNA synthesis. The simplest interpretation of the D-50 transcription result is that polyoma virus DNA contains at least two distinct transcriptional initiation sites or promoters. The promoter for E DNA strand transcription would map between approximately 67 and 84 map units, that is, within the D-50 sequences. The promoter(s) for L DNA strand transcription would lie wholly or partially outside this region. It is interesting to note that whereas the 5' end of the 20S early mRNA almost certainly maps within the D-50 subunit, the available evidence (17) does not definitely position the 5' end of the largest late mRNA within this region of the DNA. The previous assignment of 67 map units as the 5' end of the late 19S mRNA may be incorrect if polyoma virus messengers, like those of adenovirus (32) and SV40 (33-36), have 5'-terminal "leader" sequences. There are interpretations other than the absence of initiation sites that might explain the low level of D-50 L DNA strand transcription. L DNA strand transcription may normally initiate within the sequences included in the D-50 subunit, but the rate of initiation could be influenced by adjacent DNA sequences. For example, the L DNA strand "promoter" could be a region of local DNA denaturation (perhaps the result of the initiation of DNA replication), the occurrence of which might require flanking sequences not in D-50 or might be prevented by the presence of abnormal adjacent DNA sequences. It will be in-

Proc. Natl. Acad. Sci. USA 75 (1978)

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teresting to study other defective polyoma virus genomes containing more of the late region to determine whether they contain all of the sequences sufficient for normal L strand transcription. Note Added in Proof. The capped 5' ends of the leader sequences on late polyoma virus mRNAs map within 100 nucleotides of the end of the D-50 subunit which is at 67 map units (A. J. Flavell and R. Kamen,

unpublished data).

We thank Joe Sambrook for help in the preparation of this paper. This work was supported, in part, by a U.S. Public Health Service Postdoctoral Fellowship grant to R.C. 1. Kamen, R., Lindstrom, D. M., Shure, H. & Old, R. W. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,187-198. 2. Beard, P., Acheson, N. H. & Maxwell, I. H. (1976) J. Virol. 17, 20-26. 3. Flavell, A. & Kamen, R. (1977) J. Mol. Biol. 115,237-242. 4. Condit, R. C., Cowie, A., Kamen, R. & Birg, F. (1977) J. Mol. Biol. 115,215-236. 5. Laub, 0. & Aloni, Y. (1975) J. Virol. 16, 1171-1183. 6. Laub, 0. & Aloni, Y. (1976) Virology 75,346-354. 7. Gilboa, E. & Aviv, H. (1976) Cell 7,567-573. 8. Reed, S. I., Stark, G. T. & Alwine, J. C. (1976) Proc. Natl. Acad. Sci. USA 73,3083-3087. 9. Ferdinand, F.-J., Brown, M. & Khoury, G. (1977) Virology 78, 150-161. 10. Acheson, N. H. (1976) Cell 8, 1-12. 11. Aloni, Y. & Locker, H. (1973) Virology 54,495-505. 12. Acheson, N. H., Buetti, E., Scherrer, K. & Weil, R. (1971) Proc. Natl. Acad. Sci. USA 68,2231-2235. 13. Buetti, E. (1974) J. Virol. 14, 249-260. 14. Rosenthal, L. J., Salomon, C. & Weil, R. (1976) Nucleic Acids Res. 3, 1167-1183. 15. Lev, Z. & Manor, H. (1977) J. Virol. 21, 831-842. 16. Birg, F., Favaloro, J. & Kamen, R. (1977) Proc. Natl. Acad. Sci. USA 74, 3138-3142. 17. Kamen, R. & Shure, H. (1976) Cell 7,361-371. 18. Turler, H., Salomon, C., Allet, B. & Weil, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1480-1484. 19. Fried, M. (1974) J. Virol. 13, 939-946. 20. Robberson, D. L. & Fried, M. (1974) Proc. Natl. Acad. Sci. USA 71,3497-3501. 21. Griffin, B. E. & Fried, M. (1975) Nature 256, 175-179. 22. Griffin, B. E., Fried, M. & Cowie, A. (1974) Proc. Natl. Acad. Sci. USA 71, 2077-2081. 23. Crawford, L. V., Robbins, A. K. & Nicklin, P. M. (1974) J. Gen. Virol. 25, 133-142. 24. Griffin, B. E. & Fried, M. (1976) Methods Cancer Res. 12, 49-87. 25. Kamen, R., Sedat, J. & Ziff, E. (1976) J. Virol. 17,212-218. 26. Hirt, B. (1976) J. Mol. Biol. 26,365-369. 27. Radloff, F., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. 28. Sharp, P. A., Sugden, B. & Sambrook, J. (1973) Biochemistry 12, 3055-3063. 29. Gariglio, P. & Mousset, S. (1975) FEBS Lett. 56, 149-155. 30. Nygaard, A. P. & Hall, B. D. (1964) J. Mol. Biol. 9, 125-142. 31. Gillespie, D. (1968) in Methods in Enzymology, eds. Grossmann, L. & Moldave, K. (Academic Press, New York), Vol. 12B, pp. 641-668. 32. Berget, S. M., Moore, C. & Sharp, P. A. (1977) Proc. Natl. Acad. Sci. USA 74,3171-3175. 33. Celma, M. L., Dhar, R., Pan, J. & Weissman, S. M. (1977) Nucleic Acids Res. 4, 2549-2559. 34. Lavi, S. & Groner, Y. (1977) Proc. Natl. Acad. Sci. USA 74, 5323-5327. 35. Aloni, Y., Dhar, R., Laub, O., Horowitz, M. & Khoury, G. (1977) Proc. Natl. Acad. Sci. USA 74,3686-3690. 36. Hsu, M.-T. & Ford, J. (1977) Proc. Natl. Acad. Sci. USA 74, 4982-4985.