Proc. Nati. Acad. Sci. USA
Vol. 75, No. 1, pp. 162-166, January 1978 Biochemistry
Similarity of nucleotide sequences around the origin of DNA replication in mouse polyoma virus and simian virus 40* (DNA sequencing/bacteriophage T4 DNA polymerase/DNA tumor virus)
EIICHI SOEDAt, GENKI KIMURAf, AND KIN-ICHIRO MIURAt§ t National Institute of Genetics,
Mishima 411, Japan; and t Cancer Institute, Kyushu University School of Medicine, Fukuoka 812, Japan
Communicated by Motoo Kimura, October 19, 1977
ABSTRACT The nucleotide sequence around the origin of replication in DNA of mouse polyoma virus was determined by 32p labeling of the 3' terminus of the Hap II-5/Alu I-i DNA fragment, with the use of DNA polynerase. The result coincided with our previous report on the P labeling, with the use of polynucleotide kinase, of the 5' terminus of the Hap II-5/Hha I-i DNA fragment, which corresponds to the large part of the present fragment, Hap II-5/Alu I-1. A symmetrical (A+T)rich region containing a five-A stretch (or a five-T stretch) was flanked by two small regions with a 2-fold rotational axis of symmetry. On comparison of the sequence near the replication origin of polyoma DNA with that in the corresponding region of simian virus 40 DNA, which was included in the EcoRI-G fragment sequenced by Weissman's group (Subramanian, K. N., Dahr, R. & Weissman, S. M. (1977) J. Biol. Chem. 252,355-367), a considerable similarity was detected. Several possible common sequences for important biological activities such as the starting of DNA replication and RNA synthesis were suggested.
sulting shorter fragment (Hap II-5/Alu I-1) contains Hap II5/Hha I-1. This served for the nucleotide sequencing here. Maxam and Gilbert (1-3) have developed a method for determining the nucleotide sequence in DNA by using DNA fragments labeled at the 5' terminus with [32P]phosphate by polynucleotide kinase. Because it is necessary to label the terminal part of the DNA fragment for this analysis, they labeled the 5' terminus with 32P by polynucleotide kinase. We have developed a technique for 3'-terminal labeling by the use of bacteriophage T4-induced DNA polymerase in place of polynucleotide kinase and have applied it to the polyoma DNA fragment Hap II-5/Hha I-1 to assess the fidelity of the analysis method with the DNA polymerase; the technique was found to be effective and convenient when compared with polynucleotide kinase. The sequences deduced from the 3'-termini of the strands of the fragment coincided almost with those derived from the 5'-terminal labeling with polynucleotide kinase (14). The nucleotide sequence thus obtained for the region around the replication origin of polyoma DNA was compared with the corresponding DNA fragment of the related SV40 fragment EcoRI-G (3). Some homologous regions were detected around the origins of DNA replication for polyoma virus and SV40.
Structural studies on the DNA of small tumor viruses are proceeding intensively as an initial step toward understanding the function of eukaryotic DNA as well as elucidating the process of tumorigenesis. Mouse polyoma virus has been well studied, as has simian virus 40 (SV40). For SV40, analysis of its nucleotide sequence has progressed rapidly (1-3). It is necessary to determine the nucleotide sequence of polyoma virus DNA and compare it with that of SV40 to clarify the common structure for function. The starting point of DNA replication has been located on both SV40 (4) and polyoma DNA (5). Further, both the transcription starting points of "early" and "late" viral mRNAs are located at regions close to the origin of DNA replication (6, 7). Therefore, studies on the region around the replication origin will provide much information-on the initial steps in DNA replication and RNA synthesis. The DNA replication of polyoma virus is initiated at a unique site of the viral genome, which has been mapped at 71 + 3 map units by two independent methods (8, 9). As shown in Fig. 1, a fragment, Hap 11-5/Hha I-1, which is a shorter fragment obtained by Hha I endonuclease digestion of the Hap II-5 fragment, is located from 70.8 to 72.8 map units, covering the origin of DNA replication (10). In addition, the presence of a unique segment in the fragment essential to DNA replication, which has been observed in viable and defective mutants (10-12), would narrow the range of deviation. This evidence has revealed that the origin of DNA replication falls in this fragment or is located very close to the Hap II cleavage site at 70.8 map units (Hap II-3 and -5 junction). Endonuclease Alu I cuts the Hap II-5 fragment once at 74.3 map units. The re-
MATERIALS AND METHODS Preparation of DNA. Polyoma virus strain LP 147-2 was derived in G. Kimura's laboratory by a single-plaque isolation from the large-plaque strain of Vogt and Dulbecco (15), and was passed through primary baby mouse kidney cell cultures four times consecutively at low multiplicities of infection. For preparation of viral DNA, mouse 3T6 cells infected with LP 147-2 at a multiplicity of infection of 1 plaque-forming unit per cell were incubated as described previously (14), and viral DNA was extracted from the infected cells according to Hirt (16), followed by purification by centrifuging in CsCl/ethidium bromide. Fragmentation of DNA. Restriction endonuclease Hap II, Hha I, and Alu I were used as described previously (14, 17, 18). Terminal Labeling of a DNA Fragment. Polyoma form I DNA (25 ,ug) was digested with Hap II endonuclease in 100 Al of a R buffer containing 30 mM Tris-HCI (pH 7.5), 7 mM MgCl2, and 7 mM 2-mercaptoethanol at 370 for 6 hr. To the digestion mixture, 50 pl of a solution consisting of 0.15 M Tris-HCI (pH 8.0), 0.15 M KCI, 0.02 M MgCl2, and 0.015 M dithiothreitol were added. The mixture was incubated with 100
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.
Abbreviation: SV40, simian virus 40. * Contribution no. 1176 from the National Institute of Genetics. § To whom reprint requests should be addressed. 162
Biochemistry:
Soeda et al. Alu I Hha I Hap I I
Hap II
~ -G
Proc. Natl. Acad. Sci. USA 75 (1978) B
A
C
C Hap 11-4 Map units q
|
Hap 11-5
78.5
Early mRNA
743,72.8
I
Hap 11-3
G AC T G A C T
70.8 I Origin of replication '---Late RNAIHap 11-5/Hha 1-1 |== Hap 11-5/A/u 1-1 m
DNA fragments{
FIG. 1. Cutting out of the polyoma virus DNA fragment containing the origin of replication and starting points of early and late mRNAs. The recognition site of Hap II endonuclease is the same as that of Hpa II endonuclease, and scission at such sites cuts polyoma DNA into eight discrete fragments. Hha I endonuclease cuts the DNA into four fragments (10). These fragments are designated as Hap II-1 to -8 and Hha I-1 to -4 in order of decreasing size. The origin of DNA replication is located in the Hap II-5 fragment close to the Hap II-3 and -5 junction (8, 9). Alu I and Hha I endonucleases cut Hap II-5 once on the side of this junction, and the resulting shorter fragments Hap II-5/Alu I-1 and Hap II-5/Hha I-1 are considered to contain the origin of replication. Both 5' termini of early and late mRNA are located in the vicinity of the origin (7).
163
80
A 40
3W
= _
70
-~~~T
T
In.
*
--~C XC
MCi of [a-32P]dCTP (New England Nuclear Corp., 100-130 MCi/mmol) and the T4-induced DNA polymerase at 15° for
3 hr. The T4-induced DNA polymerase was prepared by the method of Panet et al. (19) and purified further on a hydroxyapatite column. To recover the 3'-terminal labeled DNA fragments as precipitate, the mixture was shaken with an equal volume of buffer-saturated phenol twice, and the water phase was added to 2.5 volumes of ethanol and chilled with dry ice. After centrifugation and drying, the DNA fragments were dissolved in 50 ,l of R buffer, and digested with Alu I endonuclease at 370 for 4 hr. The digest was then electrophoresed in a 6% polyacrylamide slab gel (0.4 X 20 X 40 cm). The Alu I endonuclease cuts the Hap II-5 fragment at 74.3 map units. The shorter fragment, the Hap II-5/Alu I-1 contains Hap II5/Hha I-1 on the side of the Hap II-3 and -5 junction, and the fragment was labeled with the pdC moiety at the 3' terminus of the early strand, which is complementary to early mRNA. Thus, fragment Hap II-5/Alu I-1 was submitted to nucleotide sequencing after elution from the polyacrylamide gel. Determination of Nucleotide Sequence. The nucleotide sequence in the DNA fragment was determined according to the method of Maxam and Gilbert (13), except that the fragment was labeled by T4-induced DNA polymerase-catalyzed incorporation of [a-32P]dCTP into the &.-terminus as described in the preceding paragraph instead of 5'-terminal labeling by
polynucleotide kinase. RESULTS The whole Hap II digestion products of polyoma DNA were labeled with [a-32P]dCTP and the T4-induced DNA polymerase. One mole of pdC moiety was incorporated at the 3' terminus of the Hap II cleavage site. The labeling efficiency was higher than was the case with the polynucleotide kinase. The labeled fragments were digested with Alu I endonuclease. The products were separated by electrophoresis in a polyacrylamide gel. The resulting shorter fragment (Hap II-5/Alu I-1) contains Hap II-5/Hha I-i, carrying the [32P]pdC moiety at only one 3' terminus of the Hap II cleavage site as shown in Fig. 1. The nucleotide sequence in the 3'-terminal labeled fragment, Hap II-5/Aiu I-1, was analyzed according to Maxam and Gilbert (13). The autoradiogram of the modified DNA fragments
0_
*
--A
-G
60
A
G
,II
20
-
_i. _.
---A
*Am _
50
-C-
-G
40
T= _, L T-
C
~
>
~
t
0 ~~~~~~1
~
~_;
T
-
-G
FIG. 2. Sequencing of the Hap II-5/Alu I-1 fragment around the origin of DNA replication. The total Hap II digestion products labeled at the 3' termini with [a-32P]dCTP and T4-induced DNA polymerase were redigested with Alu I endonuclase. A fragment, Hap II-5/Alu I-1, was isolated from the redigestion products by electrophoresis in polyacrylamide slab gels. The fragment contains the Hap II-5/Hha I-1 fragment on the side of the Hap II-3 and -5 junction and labeled with 1 mol of pdC at the 3' end of the early strand. This fragment (6 to 9 X 105 Cerenkov counts per min) was submitted to sequencing according to the method of Maxam and Gilbert (13). BPB and XC denote the reference positions of bromphenol blue and xylene cyanol markers. Electrophoresis was carried out at 100 V for 24 hr (a) and 14 hr (b). The gels were exposed to x-ray film at -20° for 5 days.
in a slab gel is shown in Fig. 2, from which the sequence of 78 nucleotides (from positions 6 to 83 numbered from the Hap II-3 and -5 junction) can be read. The sequence is almost the same as
that
reported previously
with the kinase
labelingtechnique
(14), except that some missing nucleotides were found at this time at positions 6, 8, and near the Hha I cleavage site due to
164
Proc. Natl. Acad. Sci. USA 75 (1978)
Biobhemistry: Soeda et al. A
Hap 11-3 part
Hap 11 |
Hap 11-5 part
40
30
20
10
1
Late strand
5.. C G G G CCC CT G G CCC G C.T TACT CT G GAG A A A A A GA A GAG AG G CT T
Early strand
3'. G GC C C G G GGA C C G G G C GA AT GAG A C CT CT T T T T CT T CT CT C C GA A Hha I
Hha 1-2 part
Hha 1-1 part
' 50
60
70
4
80
CCAGAGGCAACTTGTCAAAACAGGCTGGCGCCTGGGG CC... 3'
GGTCTCCGTTGAACAGTTTTGTCCGACCGCGGACCCCGG ... 5
BB
C~~~CT CG CG
GC
T
CG
G G
60 50 40 30 GO GC 20 Late strand 5 - CC GCTTA CTCT GGAAAAAGA GAGAG GCTTCCAGAGGCAACTTGTCAAAACAGGCTG C -3 GTTTTGTCC AC G-5 Early strand 3- GGCGAATGA OCC TCTTOTTCTC CGAAGGTCTCCGTTGA
OG
OG
CG CG GC GC
C
GC C GC GC
A G FIG. 3. (A) The complete nucleotide sequence of Hap II-5/Hha I-1 around the origin of DNA replication. The early and late strands of the fragment are complementary to early and late mRNA, respectively. The numbering of nucleotides starts from the Hap II cleavage site at the Hap II-3 and -5 junction. The nucleotide sequence from positions 6 to 83 of the early strand was determined from the radioautogram shown in Fig. 2. The cleavage sites of Hap II and Hha I endonucleases are indicated within the sequence. (B) A possible transient secondary structure deduced from A. The thick box indicates a symmetrical region. The two thin boxes and both of the two projecting hairpin structures possess 2-fold rotational axes of symmetry.
G A
the intensive 3'-terminal labeling (Fig. 3A). Thus, the sequence deduced from the 3' terminus of the early strand of Hap II5/Hha I-1 was complementary to that determined from the 5' terminus of the late strand. Two bands in the G lane at or near position 13 were observed. One of the two bands is probably a ghost band, because only one band was detected at the corresponding position of the opposite strand; only one C was present as a partner (14). A long cluster of pyrimidines from positions 24 to 41 of the early strand was also seen by analyses of pyrimidine tracts of the Hap II-5 fragment that was essentially retained in the DNAsof all the variants or strains (10). In this region a characteristic true palindrome sequence of T-C-T-T-T-T-T-C-T (or A-GA-A-A-A-A-G-A) from 26 to 34 is contained. This region is sandwiched between two small symmetrical regions with a 2-fold rotational axis (20-23 and 37-40) as shown in Fig. 3B. The sequenced fragment may be characterized also by the (G+C)-rich region from 1 to 15 with a 2-fold rotational axis of symmetry close to the Hap II-3 and -5 junction, which possibly forms double hairpin loops, as well as the other (G+C)-rich region from 71 to 83.
DISCUSSION The origin of DNA replication of polyoma DNA has been localized in two earlier papers (8, 9), but the uncertainty is as long as 150 nucleotides on either side. The actual location of the origin can be further narrowed down by the presence of a short segment of nucleotides essential to DNA replication of the viable and defective mutants. Thus, one such variant contains a small deletion in the Hap II-5/Hha I-1 fragment (12) and another contains an additional deletion between 67 and 71 map units in the Hap II-3 fragment next to the Hap II-5 fragment (11); both the variants can replicate without helper viruses. A
short segment around the Hap 11-3 and -5 junction is inevitably retained in the defective mutants (20). This evidence suggests that the origin of DNA replication resides in the Hap II-5/Hha I-1 fragment or, if that is not the case, it is quite close to this fragment. As shown in Fig. 3, a long symmetrical true palindrome structure containing nine nucleotides, including five continuous A residues, is sandwiched between two symmetrical regions consisting of four nucleotides. Because it has been shown (21) that replication of polyoma DNA proceeds bidirectionally with the same rate from one origin, the replication origin would have a symmetrical structure. In regard to this, the above-mentioned long symmetrical structure would be a prominent candidate for the replication origin. Such a long definite structure with symmetry is quite rare in random distribution of nucleotides, so it would be conserved for any important function on many variations of genome for a long time. There are similarities in physical maps of genomes of polyoma virus and SV40, where several genetic markers were located. Although immunological crossreaction between the antigens induced by these two viruses was not observed, Shah et al. (22) have recently succeeded in preparing an antiserum against disrupted SV40 particles that reacts immunologically with major capsid VP-1 proteins from both the viruses, suggesting that the common determinants were hidden in the viral particles. In fact, homologous regions between the viral DNAs were detected by hybridization (23). Because mouse polyoma virus and SV40 have similar characteristics, as mentioned above, there is a hypothesis that the two viruses are descended from the same ancestor. If this is the case, a common structure would be conserved in a functionally important region of DNA. Comparing the polyoma nucleotide sequence analyzed here with the corresponding region in SV40 DNA, which was
Biochemistry: Soeda et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
1
(1)
PL SL
;-
10
AAAA
A GCTT TTT[TjAGGCCTAGGCTTTT
AA
100
(3)
2)PL SE
30
50
40
80
90
100
110
120
130
1
10
20
30
40
50
CGGCCCCT[GMCCGCTCTTCGAAAAGAAGAGAjGCTTC CG TC ATAAG(T[TTCAACT CTCCAAACCTCCG TCCG
PL SE
130
20
PL
SE
30
40
100
50
90
60
70
CTCTGA A AGAAG GCTT AGAGGAACTTGTCAAAACAGGCTG [GCTCCTC T CTTCT TAGC AGAGGCGAGGC GGCCTCGGCCTCT 20
PL SL
110
120
120
(57
70
CC GGCCCCTGGCC TCGAAAAAGAGAGG TTCAG G G T C AACTT TTTTGCAAAj GdCTCC ASAAAA C TCCTC AT TTTC
140
(4)
TCTTCAAAGATGGATAA 80
20
50
GGCTTCCAGAGG
AG
90
10
1
40
30
C GGGCCCC[CCCGCTACTC CGA 110
(2)
20
165
130
30
140
40
150 50
60
A A ACT TACAA GAGC CT CAG CTAG GG 150
120
110
160 70
AAAACAGGCTG
TTTT GCAAAAA 100
90
FIG. 4. Comparison between polyoma virus and SV40 in the nucleotide sequences around the replication origin of DNA. Common nucleotide sequences are indicated in boxes. P, polyoma; S, SV40; E, early mRNA template; L, late mRNA template.
sequenced by Subramanian et al. (3), a characteristic symmetrical region containing a five-A stretch from 28 to 32 in the polyoma late strand fits the regions in SV40 DNA as shown in Fig. 4. Other common long sequences were C-A-G-A-G-G-C, from 46 to 52 in polyoma and from 139 to 145 in SV40 and G-A-G-G-C-T-T from 38 to 44 in polyoma and from 111 to 117 in SV40; the latter is also contained in phage kX174 DNA near the replication origin (24). However, these are not symmetrical sequences. There might be similarities among the nucleic acid structures having a common function, even among very different organisms, as shown in the cloverleaf structure of tRNA. Recently Tomizawa et al. (25) showed that the replication origin of colicin El plasmid DNA contains a five-A stretch, where primer RNA switches to DNA, and both sides of this region are flanked by (G+C)-rich regions. This feature is quite similar to the structure of the polyoma DNA segment mentioned here. The
A+T cluster flanked by (G+C)-rich regions has been found also at the origin of replication in phase OX 174 DNA, although the region does not show apparent symmetry (24). Such a characteristic structure may be common for the starting of DNA synthesis in eukaryotes as well as in prokaryotes. Moreover, it has been shown that the promotor region contains a common sequence rich in the A-T pairs near the initiation point of transcription (26-29). Because the cluster of the A-T base pairing must be loosened easier than the (G-C)-rich region, it is reasonable to say that the A-T cluster is involved in the starting point of DNA replication and RNA synthesis. The starting sites of early mRNA and late mRNA are located near the replication origin of DNA in SV40 and polyoma virus (6, 7). Each mRNA is transcribed along a different strand in the opposite direction, and the initial sequences of the mRNAs overlap partially. The places, which are recognized as promotors by RNA polymerase, may be included in the present DNA
Hap 11-3 part
Polyoma late strand
5..
SV40 late strand
5'. AG A GAGGCTT C
C
A GAGG
GAG G CTTTTTT GGAGG 10~~~~
G C TG G C CC
G C TTT 80
TR G
C C . . . . . 3'
G C A A A G A T G G A T...
flHindll
+
III-Cpart
3'
70
Hindll + Ill-A part FIG. 5. Alignment of the nucleotide sequences around the replication origins of DNA from polyoma virus and SV40. Some gaps have been inserted between homologous regions. Common nucleotide sequences are indicated in boxes.
166
Biochemistry: Soeda et al.
fragment, of if this is not the case they are located very close to this fragment. The T antigens induced by both the viruses also bind preferentially to the fragment containing the origin of DNA replication (30). If the nucleotide sequence in the segment Hap II-5/Hha I-1 of polyoma DNA is aligned with the corresponding region of SV40 DNA (3) by inserting some gaps, we can observe an extensive similarity, as shown in Fig. 5. Fifty-eight nucleotides of the 82 (70%) of the polyoma segment are the same as SV40. It is known that recombination occurs most frequently in this region; many variants with addition and deletion around the replication origin have been isolated so far, including viable and defective mutants (12). If such structural changes occurred also in the course of evolution, it is likely that polyoma virus and SV40 have descended from a common ancestor. The authors are grateful to Drs. Mitsuru Takanami and Robert Young for kindly providing restriction enzymes, to Drs. Motoo Kimura and Andre von de Voorde for critical discussion, and to Miss Akiko Nakaso for preparation of the polyoma-infected cells. This work was supported partly by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan. 1. Fiers, W., Rogiers, R., Soeda, E., Van de Voorde, A., Van Heuverswyn, H., Van Herreweghe, J., Volckaert, G. & Yang, R. (1975) Fed. Eur. Biochem. Soc. 10th Meet. Proc. 39,17-33. 2. Volckaert, G., Contreras, R., Soeda, E., Van de Voorde, A. & Fiers, W. (1977) J. Mol. Biol. 110, 467-510. 3. Subramanian, K. N., Dhar, R. & Weissman, S. M. (1977) J. Biol.
Chem. 252,355-367. 4. Subramanian, K. N., Pan, J., Zain, B. S. & Weissman, S. M. (1974) Nucleic Acids Res. 1, 727-752. 5. Folk, W. R. & Wang, H. E. (1974) Virology 61, 140-155. 6. Khoury, G., Martin, M. A., Lee, T. N. H., Danna, K. J. & Nathans, D. (1973) J. Mol. Biol. 78,377-389. 7. Kamen, R. & Shure, H. (1976) Cell 7,361-371. 8. Griffin, B. E., Fried, M. & Cowei, A. (1974) Proc. Natl. Acad. Sci.
USA 71, 2077-2081.
Proc. Natl. Acad. Sci. USA 75 (1978)
9. Crawford, L. V., Robbins, A. K., Nicklin, P. M. & Osborn, K. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,219-225. 10. Griffin, B. E. & Fried, M. (1975) Nature 256, 175-179. 11. Franke, B. & Vogt, M. (1975) Cell 5,205-211. 12. Fried, M. & Griffin, B. E. (1976) in Advances in Cancer Research, eds. Klein, G. & Weinhouse, S. (Academic Press, New York), Vol. 24, pp. 67-108. 13. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,560-564. 14. Soeda, E., Miura, K., Nakaso, A. & Kimura, G. (1977) FEBS Lett.
79,383-389. 15. Vogt, M. & Dulbecco, R. (1960) Proc. Natl. Acad. Sci. USA 46, 365-370. 16. Hirt, B. (1967) J. Mol. Biol. 26,365-369. 17. Miller, L. K. & Fried, M. (1976) J. Virol. 18,824-832. 18. Yang, R. C. A., Van de Voorde, A. & Fiers, W. (1976) Eur. J. Biochem. 61, 101-117. 19. Panet, A., Van de Sande, J. H., Loewen, P. C., Khorana, H. G., Raae, A. J., Lillehaug, J. R. & Kleppe, K. (1973) Biochemistry 12, 5045-5050. 20. Fried, M., Griffin, B. E., Lund, E. & Robberson, D. L. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 45-52. 21. Crawford, L. V., Syrett, C. & Wilde, A. (1973) J. Cen. Virol. 21, 515-521. 22. Shah, K. V., Ozer, H. L., Chazey, H. N. & Kelly,. T. J. (1977) J. Virol. 21, 179-186. 23. Ferguson, J. & Davis, R. W. (1975) J. Mol. Biol. 94, 135-149. 24. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocombe, P. M. & Smith, M. (1977) Nature 265,687-695. 25. Tomizawa, J., Ohmori, H. & Bird, R. E. (1977) Proc. Natl. Acad. Sci. USA 74, 1865-1869. 26. Schaller, H., Gray, C. & Herrmann, K. (1975) Proc. Natl. Acad. Sci. USA 72,737-741. 27. Pribnow, D. (1975) Proc. Natl. Acad. Sci. USA 72,784-788. 28. Sugimoto, K., Okamoto, T., Sugisaki, H. & Takanami, M. (1975) Nature 253,410-414. 29. Dhar, R., Weissman, S. M., Zain, B. S. & Pan, J. (1974) Nucleic Acids Res. 1, 595-614. 30. Reed, S. I., Ferguson, J., Davis, R. W. & Stark, G. R. (1975) Proc. Natl. Acad. Sci. USA 72,1605-1609.
Vol. 75, No. 1, pp. 162-166, January 1978 Biochemistry
Similarity of nucleotide sequences around the origin of DNA replication in mouse polyoma virus and simian virus 40* (DNA sequencing/bacteriophage T4 DNA polymerase/DNA tumor virus)
EIICHI SOEDAt, GENKI KIMURAf, AND KIN-ICHIRO MIURAt§ t National Institute of Genetics,
Mishima 411, Japan; and t Cancer Institute, Kyushu University School of Medicine, Fukuoka 812, Japan
Communicated by Motoo Kimura, October 19, 1977
ABSTRACT The nucleotide sequence around the origin of replication in DNA of mouse polyoma virus was determined by 32p labeling of the 3' terminus of the Hap II-5/Alu I-i DNA fragment, with the use of DNA polynerase. The result coincided with our previous report on the P labeling, with the use of polynucleotide kinase, of the 5' terminus of the Hap II-5/Hha I-i DNA fragment, which corresponds to the large part of the present fragment, Hap II-5/Alu I-1. A symmetrical (A+T)rich region containing a five-A stretch (or a five-T stretch) was flanked by two small regions with a 2-fold rotational axis of symmetry. On comparison of the sequence near the replication origin of polyoma DNA with that in the corresponding region of simian virus 40 DNA, which was included in the EcoRI-G fragment sequenced by Weissman's group (Subramanian, K. N., Dahr, R. & Weissman, S. M. (1977) J. Biol. Chem. 252,355-367), a considerable similarity was detected. Several possible common sequences for important biological activities such as the starting of DNA replication and RNA synthesis were suggested.
sulting shorter fragment (Hap II-5/Alu I-1) contains Hap II5/Hha I-1. This served for the nucleotide sequencing here. Maxam and Gilbert (1-3) have developed a method for determining the nucleotide sequence in DNA by using DNA fragments labeled at the 5' terminus with [32P]phosphate by polynucleotide kinase. Because it is necessary to label the terminal part of the DNA fragment for this analysis, they labeled the 5' terminus with 32P by polynucleotide kinase. We have developed a technique for 3'-terminal labeling by the use of bacteriophage T4-induced DNA polymerase in place of polynucleotide kinase and have applied it to the polyoma DNA fragment Hap II-5/Hha I-1 to assess the fidelity of the analysis method with the DNA polymerase; the technique was found to be effective and convenient when compared with polynucleotide kinase. The sequences deduced from the 3'-termini of the strands of the fragment coincided almost with those derived from the 5'-terminal labeling with polynucleotide kinase (14). The nucleotide sequence thus obtained for the region around the replication origin of polyoma DNA was compared with the corresponding DNA fragment of the related SV40 fragment EcoRI-G (3). Some homologous regions were detected around the origins of DNA replication for polyoma virus and SV40.
Structural studies on the DNA of small tumor viruses are proceeding intensively as an initial step toward understanding the function of eukaryotic DNA as well as elucidating the process of tumorigenesis. Mouse polyoma virus has been well studied, as has simian virus 40 (SV40). For SV40, analysis of its nucleotide sequence has progressed rapidly (1-3). It is necessary to determine the nucleotide sequence of polyoma virus DNA and compare it with that of SV40 to clarify the common structure for function. The starting point of DNA replication has been located on both SV40 (4) and polyoma DNA (5). Further, both the transcription starting points of "early" and "late" viral mRNAs are located at regions close to the origin of DNA replication (6, 7). Therefore, studies on the region around the replication origin will provide much information-on the initial steps in DNA replication and RNA synthesis. The DNA replication of polyoma virus is initiated at a unique site of the viral genome, which has been mapped at 71 + 3 map units by two independent methods (8, 9). As shown in Fig. 1, a fragment, Hap 11-5/Hha I-1, which is a shorter fragment obtained by Hha I endonuclease digestion of the Hap II-5 fragment, is located from 70.8 to 72.8 map units, covering the origin of DNA replication (10). In addition, the presence of a unique segment in the fragment essential to DNA replication, which has been observed in viable and defective mutants (10-12), would narrow the range of deviation. This evidence has revealed that the origin of DNA replication falls in this fragment or is located very close to the Hap II cleavage site at 70.8 map units (Hap II-3 and -5 junction). Endonuclease Alu I cuts the Hap II-5 fragment once at 74.3 map units. The re-
MATERIALS AND METHODS Preparation of DNA. Polyoma virus strain LP 147-2 was derived in G. Kimura's laboratory by a single-plaque isolation from the large-plaque strain of Vogt and Dulbecco (15), and was passed through primary baby mouse kidney cell cultures four times consecutively at low multiplicities of infection. For preparation of viral DNA, mouse 3T6 cells infected with LP 147-2 at a multiplicity of infection of 1 plaque-forming unit per cell were incubated as described previously (14), and viral DNA was extracted from the infected cells according to Hirt (16), followed by purification by centrifuging in CsCl/ethidium bromide. Fragmentation of DNA. Restriction endonuclease Hap II, Hha I, and Alu I were used as described previously (14, 17, 18). Terminal Labeling of a DNA Fragment. Polyoma form I DNA (25 ,ug) was digested with Hap II endonuclease in 100 Al of a R buffer containing 30 mM Tris-HCI (pH 7.5), 7 mM MgCl2, and 7 mM 2-mercaptoethanol at 370 for 6 hr. To the digestion mixture, 50 pl of a solution consisting of 0.15 M Tris-HCI (pH 8.0), 0.15 M KCI, 0.02 M MgCl2, and 0.015 M dithiothreitol were added. The mixture was incubated with 100
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.
Abbreviation: SV40, simian virus 40. * Contribution no. 1176 from the National Institute of Genetics. § To whom reprint requests should be addressed. 162
Biochemistry:
Soeda et al. Alu I Hha I Hap I I
Hap II
~ -G
Proc. Natl. Acad. Sci. USA 75 (1978) B
A
C
C Hap 11-4 Map units q
|
Hap 11-5
78.5
Early mRNA
743,72.8
I
Hap 11-3
G AC T G A C T
70.8 I Origin of replication '---Late RNAIHap 11-5/Hha 1-1 |== Hap 11-5/A/u 1-1 m
DNA fragments{
FIG. 1. Cutting out of the polyoma virus DNA fragment containing the origin of replication and starting points of early and late mRNAs. The recognition site of Hap II endonuclease is the same as that of Hpa II endonuclease, and scission at such sites cuts polyoma DNA into eight discrete fragments. Hha I endonuclease cuts the DNA into four fragments (10). These fragments are designated as Hap II-1 to -8 and Hha I-1 to -4 in order of decreasing size. The origin of DNA replication is located in the Hap II-5 fragment close to the Hap II-3 and -5 junction (8, 9). Alu I and Hha I endonucleases cut Hap II-5 once on the side of this junction, and the resulting shorter fragments Hap II-5/Alu I-1 and Hap II-5/Hha I-1 are considered to contain the origin of replication. Both 5' termini of early and late mRNA are located in the vicinity of the origin (7).
163
80
A 40
3W
= _
70
-~~~T
T
In.
*
--~C XC
MCi of [a-32P]dCTP (New England Nuclear Corp., 100-130 MCi/mmol) and the T4-induced DNA polymerase at 15° for
3 hr. The T4-induced DNA polymerase was prepared by the method of Panet et al. (19) and purified further on a hydroxyapatite column. To recover the 3'-terminal labeled DNA fragments as precipitate, the mixture was shaken with an equal volume of buffer-saturated phenol twice, and the water phase was added to 2.5 volumes of ethanol and chilled with dry ice. After centrifugation and drying, the DNA fragments were dissolved in 50 ,l of R buffer, and digested with Alu I endonuclease at 370 for 4 hr. The digest was then electrophoresed in a 6% polyacrylamide slab gel (0.4 X 20 X 40 cm). The Alu I endonuclease cuts the Hap II-5 fragment at 74.3 map units. The shorter fragment, the Hap II-5/Alu I-1 contains Hap II5/Hha I-1 on the side of the Hap II-3 and -5 junction, and the fragment was labeled with the pdC moiety at the 3' terminus of the early strand, which is complementary to early mRNA. Thus, fragment Hap II-5/Alu I-1 was submitted to nucleotide sequencing after elution from the polyacrylamide gel. Determination of Nucleotide Sequence. The nucleotide sequence in the DNA fragment was determined according to the method of Maxam and Gilbert (13), except that the fragment was labeled by T4-induced DNA polymerase-catalyzed incorporation of [a-32P]dCTP into the &.-terminus as described in the preceding paragraph instead of 5'-terminal labeling by
polynucleotide kinase. RESULTS The whole Hap II digestion products of polyoma DNA were labeled with [a-32P]dCTP and the T4-induced DNA polymerase. One mole of pdC moiety was incorporated at the 3' terminus of the Hap II cleavage site. The labeling efficiency was higher than was the case with the polynucleotide kinase. The labeled fragments were digested with Alu I endonuclease. The products were separated by electrophoresis in a polyacrylamide gel. The resulting shorter fragment (Hap II-5/Alu I-1) contains Hap II-5/Hha I-i, carrying the [32P]pdC moiety at only one 3' terminus of the Hap II cleavage site as shown in Fig. 1. The nucleotide sequence in the 3'-terminal labeled fragment, Hap II-5/Aiu I-1, was analyzed according to Maxam and Gilbert (13). The autoradiogram of the modified DNA fragments
0_
*
--A
-G
60
A
G
,II
20
-
_i. _.
---A
*Am _
50
-C-
-G
40
T= _, L T-
C
~
>
~
t
0 ~~~~~~1
~
~_;
T
-
-G
FIG. 2. Sequencing of the Hap II-5/Alu I-1 fragment around the origin of DNA replication. The total Hap II digestion products labeled at the 3' termini with [a-32P]dCTP and T4-induced DNA polymerase were redigested with Alu I endonuclase. A fragment, Hap II-5/Alu I-1, was isolated from the redigestion products by electrophoresis in polyacrylamide slab gels. The fragment contains the Hap II-5/Hha I-1 fragment on the side of the Hap II-3 and -5 junction and labeled with 1 mol of pdC at the 3' end of the early strand. This fragment (6 to 9 X 105 Cerenkov counts per min) was submitted to sequencing according to the method of Maxam and Gilbert (13). BPB and XC denote the reference positions of bromphenol blue and xylene cyanol markers. Electrophoresis was carried out at 100 V for 24 hr (a) and 14 hr (b). The gels were exposed to x-ray film at -20° for 5 days.
in a slab gel is shown in Fig. 2, from which the sequence of 78 nucleotides (from positions 6 to 83 numbered from the Hap II-3 and -5 junction) can be read. The sequence is almost the same as
that
reported previously
with the kinase
labelingtechnique
(14), except that some missing nucleotides were found at this time at positions 6, 8, and near the Hha I cleavage site due to
164
Proc. Natl. Acad. Sci. USA 75 (1978)
Biobhemistry: Soeda et al. A
Hap 11-3 part
Hap 11 |
Hap 11-5 part
40
30
20
10
1
Late strand
5.. C G G G CCC CT G G CCC G C.T TACT CT G GAG A A A A A GA A GAG AG G CT T
Early strand
3'. G GC C C G G GGA C C G G G C GA AT GAG A C CT CT T T T T CT T CT CT C C GA A Hha I
Hha 1-2 part
Hha 1-1 part
' 50
60
70
4
80
CCAGAGGCAACTTGTCAAAACAGGCTGGCGCCTGGGG CC... 3'
GGTCTCCGTTGAACAGTTTTGTCCGACCGCGGACCCCGG ... 5
BB
C~~~CT CG CG
GC
T
CG
G G
60 50 40 30 GO GC 20 Late strand 5 - CC GCTTA CTCT GGAAAAAGA GAGAG GCTTCCAGAGGCAACTTGTCAAAACAGGCTG C -3 GTTTTGTCC AC G-5 Early strand 3- GGCGAATGA OCC TCTTOTTCTC CGAAGGTCTCCGTTGA
OG
OG
CG CG GC GC
C
GC C GC GC
A G FIG. 3. (A) The complete nucleotide sequence of Hap II-5/Hha I-1 around the origin of DNA replication. The early and late strands of the fragment are complementary to early and late mRNA, respectively. The numbering of nucleotides starts from the Hap II cleavage site at the Hap II-3 and -5 junction. The nucleotide sequence from positions 6 to 83 of the early strand was determined from the radioautogram shown in Fig. 2. The cleavage sites of Hap II and Hha I endonucleases are indicated within the sequence. (B) A possible transient secondary structure deduced from A. The thick box indicates a symmetrical region. The two thin boxes and both of the two projecting hairpin structures possess 2-fold rotational axes of symmetry.
G A
the intensive 3'-terminal labeling (Fig. 3A). Thus, the sequence deduced from the 3' terminus of the early strand of Hap II5/Hha I-1 was complementary to that determined from the 5' terminus of the late strand. Two bands in the G lane at or near position 13 were observed. One of the two bands is probably a ghost band, because only one band was detected at the corresponding position of the opposite strand; only one C was present as a partner (14). A long cluster of pyrimidines from positions 24 to 41 of the early strand was also seen by analyses of pyrimidine tracts of the Hap II-5 fragment that was essentially retained in the DNAsof all the variants or strains (10). In this region a characteristic true palindrome sequence of T-C-T-T-T-T-T-C-T (or A-GA-A-A-A-A-G-A) from 26 to 34 is contained. This region is sandwiched between two small symmetrical regions with a 2-fold rotational axis (20-23 and 37-40) as shown in Fig. 3B. The sequenced fragment may be characterized also by the (G+C)-rich region from 1 to 15 with a 2-fold rotational axis of symmetry close to the Hap II-3 and -5 junction, which possibly forms double hairpin loops, as well as the other (G+C)-rich region from 71 to 83.
DISCUSSION The origin of DNA replication of polyoma DNA has been localized in two earlier papers (8, 9), but the uncertainty is as long as 150 nucleotides on either side. The actual location of the origin can be further narrowed down by the presence of a short segment of nucleotides essential to DNA replication of the viable and defective mutants. Thus, one such variant contains a small deletion in the Hap II-5/Hha I-1 fragment (12) and another contains an additional deletion between 67 and 71 map units in the Hap II-3 fragment next to the Hap II-5 fragment (11); both the variants can replicate without helper viruses. A
short segment around the Hap 11-3 and -5 junction is inevitably retained in the defective mutants (20). This evidence suggests that the origin of DNA replication resides in the Hap II-5/Hha I-1 fragment or, if that is not the case, it is quite close to this fragment. As shown in Fig. 3, a long symmetrical true palindrome structure containing nine nucleotides, including five continuous A residues, is sandwiched between two symmetrical regions consisting of four nucleotides. Because it has been shown (21) that replication of polyoma DNA proceeds bidirectionally with the same rate from one origin, the replication origin would have a symmetrical structure. In regard to this, the above-mentioned long symmetrical structure would be a prominent candidate for the replication origin. Such a long definite structure with symmetry is quite rare in random distribution of nucleotides, so it would be conserved for any important function on many variations of genome for a long time. There are similarities in physical maps of genomes of polyoma virus and SV40, where several genetic markers were located. Although immunological crossreaction between the antigens induced by these two viruses was not observed, Shah et al. (22) have recently succeeded in preparing an antiserum against disrupted SV40 particles that reacts immunologically with major capsid VP-1 proteins from both the viruses, suggesting that the common determinants were hidden in the viral particles. In fact, homologous regions between the viral DNAs were detected by hybridization (23). Because mouse polyoma virus and SV40 have similar characteristics, as mentioned above, there is a hypothesis that the two viruses are descended from the same ancestor. If this is the case, a common structure would be conserved in a functionally important region of DNA. Comparing the polyoma nucleotide sequence analyzed here with the corresponding region in SV40 DNA, which was
Biochemistry: Soeda et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
1
(1)
PL SL
;-
10
AAAA
A GCTT TTT[TjAGGCCTAGGCTTTT
AA
100
(3)
2)PL SE
30
50
40
80
90
100
110
120
130
1
10
20
30
40
50
CGGCCCCT[GMCCGCTCTTCGAAAAGAAGAGAjGCTTC CG TC ATAAG(T[TTCAACT CTCCAAACCTCCG TCCG
PL SE
130
20
PL
SE
30
40
100
50
90
60
70
CTCTGA A AGAAG GCTT AGAGGAACTTGTCAAAACAGGCTG [GCTCCTC T CTTCT TAGC AGAGGCGAGGC GGCCTCGGCCTCT 20
PL SL
110
120
120
(57
70
CC GGCCCCTGGCC TCGAAAAAGAGAGG TTCAG G G T C AACTT TTTTGCAAAj GdCTCC ASAAAA C TCCTC AT TTTC
140
(4)
TCTTCAAAGATGGATAA 80
20
50
GGCTTCCAGAGG
AG
90
10
1
40
30
C GGGCCCC[CCCGCTACTC CGA 110
(2)
20
165
130
30
140
40
150 50
60
A A ACT TACAA GAGC CT CAG CTAG GG 150
120
110
160 70
AAAACAGGCTG
TTTT GCAAAAA 100
90
FIG. 4. Comparison between polyoma virus and SV40 in the nucleotide sequences around the replication origin of DNA. Common nucleotide sequences are indicated in boxes. P, polyoma; S, SV40; E, early mRNA template; L, late mRNA template.
sequenced by Subramanian et al. (3), a characteristic symmetrical region containing a five-A stretch from 28 to 32 in the polyoma late strand fits the regions in SV40 DNA as shown in Fig. 4. Other common long sequences were C-A-G-A-G-G-C, from 46 to 52 in polyoma and from 139 to 145 in SV40 and G-A-G-G-C-T-T from 38 to 44 in polyoma and from 111 to 117 in SV40; the latter is also contained in phage kX174 DNA near the replication origin (24). However, these are not symmetrical sequences. There might be similarities among the nucleic acid structures having a common function, even among very different organisms, as shown in the cloverleaf structure of tRNA. Recently Tomizawa et al. (25) showed that the replication origin of colicin El plasmid DNA contains a five-A stretch, where primer RNA switches to DNA, and both sides of this region are flanked by (G+C)-rich regions. This feature is quite similar to the structure of the polyoma DNA segment mentioned here. The
A+T cluster flanked by (G+C)-rich regions has been found also at the origin of replication in phase OX 174 DNA, although the region does not show apparent symmetry (24). Such a characteristic structure may be common for the starting of DNA synthesis in eukaryotes as well as in prokaryotes. Moreover, it has been shown that the promotor region contains a common sequence rich in the A-T pairs near the initiation point of transcription (26-29). Because the cluster of the A-T base pairing must be loosened easier than the (G-C)-rich region, it is reasonable to say that the A-T cluster is involved in the starting point of DNA replication and RNA synthesis. The starting sites of early mRNA and late mRNA are located near the replication origin of DNA in SV40 and polyoma virus (6, 7). Each mRNA is transcribed along a different strand in the opposite direction, and the initial sequences of the mRNAs overlap partially. The places, which are recognized as promotors by RNA polymerase, may be included in the present DNA
Hap 11-3 part
Polyoma late strand
5..
SV40 late strand
5'. AG A GAGGCTT C
C
A GAGG
GAG G CTTTTTT GGAGG 10~~~~
G C TG G C CC
G C TTT 80
TR G
C C . . . . . 3'
G C A A A G A T G G A T...
flHindll
+
III-Cpart
3'
70
Hindll + Ill-A part FIG. 5. Alignment of the nucleotide sequences around the replication origins of DNA from polyoma virus and SV40. Some gaps have been inserted between homologous regions. Common nucleotide sequences are indicated in boxes.
166
Biochemistry: Soeda et al.
fragment, of if this is not the case they are located very close to this fragment. The T antigens induced by both the viruses also bind preferentially to the fragment containing the origin of DNA replication (30). If the nucleotide sequence in the segment Hap II-5/Hha I-1 of polyoma DNA is aligned with the corresponding region of SV40 DNA (3) by inserting some gaps, we can observe an extensive similarity, as shown in Fig. 5. Fifty-eight nucleotides of the 82 (70%) of the polyoma segment are the same as SV40. It is known that recombination occurs most frequently in this region; many variants with addition and deletion around the replication origin have been isolated so far, including viable and defective mutants (12). If such structural changes occurred also in the course of evolution, it is likely that polyoma virus and SV40 have descended from a common ancestor. The authors are grateful to Drs. Mitsuru Takanami and Robert Young for kindly providing restriction enzymes, to Drs. Motoo Kimura and Andre von de Voorde for critical discussion, and to Miss Akiko Nakaso for preparation of the polyoma-infected cells. This work was supported partly by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan. 1. Fiers, W., Rogiers, R., Soeda, E., Van de Voorde, A., Van Heuverswyn, H., Van Herreweghe, J., Volckaert, G. & Yang, R. (1975) Fed. Eur. Biochem. Soc. 10th Meet. Proc. 39,17-33. 2. Volckaert, G., Contreras, R., Soeda, E., Van de Voorde, A. & Fiers, W. (1977) J. Mol. Biol. 110, 467-510. 3. Subramanian, K. N., Dhar, R. & Weissman, S. M. (1977) J. Biol.
Chem. 252,355-367. 4. Subramanian, K. N., Pan, J., Zain, B. S. & Weissman, S. M. (1974) Nucleic Acids Res. 1, 727-752. 5. Folk, W. R. & Wang, H. E. (1974) Virology 61, 140-155. 6. Khoury, G., Martin, M. A., Lee, T. N. H., Danna, K. J. & Nathans, D. (1973) J. Mol. Biol. 78,377-389. 7. Kamen, R. & Shure, H. (1976) Cell 7,361-371. 8. Griffin, B. E., Fried, M. & Cowei, A. (1974) Proc. Natl. Acad. Sci.
USA 71, 2077-2081.
Proc. Natl. Acad. Sci. USA 75 (1978)
9. Crawford, L. V., Robbins, A. K., Nicklin, P. M. & Osborn, K. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,219-225. 10. Griffin, B. E. & Fried, M. (1975) Nature 256, 175-179. 11. Franke, B. & Vogt, M. (1975) Cell 5,205-211. 12. Fried, M. & Griffin, B. E. (1976) in Advances in Cancer Research, eds. Klein, G. & Weinhouse, S. (Academic Press, New York), Vol. 24, pp. 67-108. 13. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74,560-564. 14. Soeda, E., Miura, K., Nakaso, A. & Kimura, G. (1977) FEBS Lett.
79,383-389. 15. Vogt, M. & Dulbecco, R. (1960) Proc. Natl. Acad. Sci. USA 46, 365-370. 16. Hirt, B. (1967) J. Mol. Biol. 26,365-369. 17. Miller, L. K. & Fried, M. (1976) J. Virol. 18,824-832. 18. Yang, R. C. A., Van de Voorde, A. & Fiers, W. (1976) Eur. J. Biochem. 61, 101-117. 19. Panet, A., Van de Sande, J. H., Loewen, P. C., Khorana, H. G., Raae, A. J., Lillehaug, J. R. & Kleppe, K. (1973) Biochemistry 12, 5045-5050. 20. Fried, M., Griffin, B. E., Lund, E. & Robberson, D. L. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 45-52. 21. Crawford, L. V., Syrett, C. & Wilde, A. (1973) J. Cen. Virol. 21, 515-521. 22. Shah, K. V., Ozer, H. L., Chazey, H. N. & Kelly,. T. J. (1977) J. Virol. 21, 179-186. 23. Ferguson, J. & Davis, R. W. (1975) J. Mol. Biol. 94, 135-149. 24. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocombe, P. M. & Smith, M. (1977) Nature 265,687-695. 25. Tomizawa, J., Ohmori, H. & Bird, R. E. (1977) Proc. Natl. Acad. Sci. USA 74, 1865-1869. 26. Schaller, H., Gray, C. & Herrmann, K. (1975) Proc. Natl. Acad. Sci. USA 72,737-741. 27. Pribnow, D. (1975) Proc. Natl. Acad. Sci. USA 72,784-788. 28. Sugimoto, K., Okamoto, T., Sugisaki, H. & Takanami, M. (1975) Nature 253,410-414. 29. Dhar, R., Weissman, S. M., Zain, B. S. & Pan, J. (1974) Nucleic Acids Res. 1, 595-614. 30. Reed, S. I., Ferguson, J., Davis, R. W. & Stark, G. R. (1975) Proc. Natl. Acad. Sci. USA 72,1605-1609.
