The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe

Volume 6 Number 8 1979

Volume 6 Number 8 1979

Research Nucleic Acids Research Nucleic Acids

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe

Antoni Rafalski*, Jurg Kohli, Paul Agrist and Dieter S611

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, and tDepartment of Biological Sciences, University of Missouri, Columbia, MO 65201, USA Received 23 April 1979

ABSTRACT The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nu4leotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac C-G-AG-D-G-G-D-D-A-A-G-G- A-m2G--U-A-G-A-N-U-U*-C-A-i 6A-A-T-C-U-A-A-U-G-G-G-C-U-U-

U-G-C-C-C-G-m5C-G-C-A-G?G-T-v-C-A-mlA-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-AOH.

The anticodon sequence U*CA is complementary to the UGA codon.

INTRODUCTION The codons UAA, UAG and UGA are chain terminators for polypeptide synthesis. Suppressor tRNAs recognizing the different termination codons have been characterized at the nucleotide sequence level in prokaryotes (1-6). To date the only eukaryotic suppressor tRNAs of known nucleotide sequence are amber (UAG) suppressors of Saccharomyces cerevisiae (7,8). In addition the nucleotide sequence of a gene coding for an ochre (UAA) suppressor tRNA is known (9). These nonsense suppressor tRNAs differ from their corresponding wild type tRNAs by a base change in the anticodon. An exception is the E. coli opal (UGA) suppressor tRNATrP which originates from a base substitution in the D-stem of the wild type tRNA (5). Recently we were able to characterize the first eukaryotic opal suppressor tRNAs which were obtained from suppressor strains of Schizosaccharomyces pombe. With the help of a new in vitro system for ochre and opal suppression we purified a suppressor tRNASer from strain sup3-e (10) and a suppressor tRNALeu from strain sup8-e (11). We then set out to sequence these tRNAs in order to determine whether an anticodon mutation gives rise to the suppressor activity of these molecules. The nucleotide sequences of the two suppressors also provide the basis for studies of the many second site mutations in these tRNA genes (12) and of antisuppressor mutations affecting the suppressor tRNAs (13). Here we report C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research the nucleotide sequence of the opal suppressor tRNASer isolated from the S. pombe strain sup3-e.

MATERIALS AND METHODS General. The S. pombe strain used was ade6-704 sup3-704/5 h (12). The experimental procedures for the growth of cells, the isolation, purification and aminoacylation of tRNA, and the methods involved in the in vitro suppression assay have been described (10). Enzyme sources were: Boehringer-Mannheim (hexokinase, Penicillium citrinum nuclease P1), PL Biochemicals (T4 polynucleotide kinase), Worthington (RNase A, BAPF alkaline phosphatase, snake venom phosphodiesterase), Calbiochem (RNase Tl, RNase U2) and Enzo Biochemicals Inc. (Physarum polycephalwn ribonuclease I). Stains-All was obtained from Eastman; Avicel and cellulose HR/DEAE 15:2 thin layer chromatographic plates were from Analtech; cellulose acetate strips from Schleicher and Schuell, and cellogel strips from Kalex Co. The Whatman DE-81 paper used was from their new process giving altered characteristics electrophoretic mobilities for nucleotides. was prepared by a modification of the High specific activity Y-( Glynn and Chappel (14) procedure described by Maxam and Gilbert (15). The stability of this ATP was dramatically increased by storage in solutions containing 2-mercaptoethanol or dithiothreitol, a phenomenon previously noted (16). Purification to Homogeneity of tRNA by Two-dimensional Gel Electrophoresis. The gel system applied was a modification of the one described by Ikemura and Dahlberg (17). A 10% acrylamide - 7M urea gel was used for the first dimension, followed by 20% acrylamide - 4M urea in the second dimension (20 x 40 cm, 3 mm gels). The tRNA loaded (1.5 A260 units) was already more than 80% pure suppressor tRNA obtained by several steps of column chromatography (10). After electrophoresis the gel was stained with Stains-All at 40C, destained with light and the single major spot excised. The tRNA was electroeluted from the gel piece (the dye remains in the gel) and used for sequencing, aminoacylation and in vitro suppression studies. RNA Sequencing Methods. Fingerprints were generated by end labelling 0.5 pg of tRNA previously digested with RNase A or Ti and two-dimensional electrophoresis, as described by Silberklang et al. (18). The resulting oligonucleotides were analyzed for their 5'-terminal nucleotide by complete snake venom phosphodiesterase or nuclease P1 digestion and thin layer

32P)-ATP

2684

Nucleic Acids Research chromatography. Sequences of oligonucleotides were deduced by two-dimensional homochromatography of partial snake venom phosphodiesterase digests ("wandering spot" method) as described (18). 5'-end labelling of intact tRNA was performed according to Silberklang et at. (18). Reaction products were purified by two-dimensional gel electrophoresis as described above. Kinasing of sup3-e tRNA gave a mixture of labelled intact tRNA and a series of smaller RNAs derived from 5'-end-labelling of tRNA fragments. These fragments (mainly with purines at their 5'ends) are products of a contaminating nuclease. They were put to use along with intact tRNA as indicated below. 3'-end labelled tRNA was prepared by partial snake venom phosphodiesterase degradation of intact sup3-e tRNA and repair of the -C-C-A terminal sequence with CTP and a-(32P)ATP by E. coZi tRNA nucleotidyltransferase (18). Partial digestion with P1 nuclease and sequencing by two-dimensional homochromatography of end labelled tRNA and fragments has been described (19) The 5'-terminal nucleotides were determined by complete P1 digestion. The fragments showed marked differences in the susceptibility to this enzyme. For this reason several concentrations were utilized on portions of the RNA. The digests were then pooled for two-dimensional homochromatography. The sequencing gels on end labelled tRNA and fragments were run according to Simoncsits et at. (19) and Donis-Keller et aZ. (20). Vlodified Nucteosides. These were investigated in several ways. (a) An and polyRNase T2 digest of sup3-e tRNA was phosphorylated with nucleotide kinase. Nuclease P1 converted the nucleoside 3',5'-diphosphates to 5'-mononucleotides which were characterized by two-dimensional thin layer chromatography and autoradiography (19). (b) Analysis of the 5'-terminal nucleotide of the oligonucleotides from the RNase Tl or A fingerprints revealed several modified nucleotides. (c) Differences in chromatographic and electrophoretic mobilities as well as reduced susceptibility to snake venom phosphodiesterase cleavage showed the presence of a modified nucleotide in an oligonucleotide fragment. (d) Nucleoside mixtures resulting from complete digests of sup3-e tRNA (by a combination of RNase P1, T2 and alkaline phosphatase) were analyzed by HPLC chromatography (21,22). In this way the modified nucleotides could be identified.

Y-(32P)ATP

RESULTS Purification of the UGA Suppressor Serine tRNA.

As described elsewhere 2685

Nucleic Acids Research (10), three successive column chromatography steps gave a preparation of approximately 85% pure suppressor tRNA from S. pombe strain sup3-e. The final purification by two-dimensional gel electrophoresis revealed one major compound in this preparation. The eluted tRNA had the opal suppressor activity and could be charged with serine to the extent of 800 pmoles/A260 unit.

1r

14

3 12

<

2

214a 1

I1

2

11

1o

10

9b

;

.7

U

6e

5

f-

4

9o

,1! ..W

'I

3

02

3

5

6*6

V Figure 1 Fingerprint of a complete RNase A digest of sup3-e serine tRNA. The unnumbered spots on the left represent an artefact of the labelling procedure ~Up ) and a contaminant of the y- ( P )ATP . .

2686

Fi gure 2. Fingerprint of

a complete RNase Tl digest of sup3-e serine tRNA. The unnumbered spot to the left of tlO arises from a contaminant of the y- (32p)ATP.

Nucleic Acids Research This low value may be due to the very low tRNA concentration used in the aminoacylation assay with a crude enzyme preparation (10). The subsequent sequencing studies performed with this material showed it to be a pure tRNA Ser species. RNA Sequencing. The sup3-e tRNA was sequenced with recently developed post-labelling procedures (18-20) that allow the determination of RNA sequences of small amounts of cold material. This involved the analysis of oligonucleotide fingerprints produced by complete RNase Tl or RNase A digestion after enzymatic phosphorylation of the 5'-hydroxyl termini of the oligonucleotides with (32P)-phosphate. The ordering of those fragments was achieved by rapid gel sequencing of partially digested tRNA. The fingerprints of 5'-(32P)-labelled fragments derived from RNase A and RNase Tl cleavage of intact tRNA are shown in Figures 1 and 2. The first step in the analysis of each eluted oligonucleotide was the determination of the 5'-terminal nucleotide after snake venom phosphodiesterase digestion. Then the oligonucleotide was sequenced by two-dimensional homochromatography of partial snake venom phosphodiesterase or nuclease P1 digests. Examples of such "wandering spot" patterns are shown in Figure 3. The assignments are summarized in Tables 1 and 2, where molar yields, based on the radioactivity of each kinased oligonucleotide, are also given. Some overdigestion occurred in the particular RNase Tl fingerprint shown in Figure 2; several of the weak (not numbered) spots were shown to be fragments of other Tl oligonucleotides. The diffuse spots appearing in corresponding

PU C ..

G t pG

o

<

E

pH 3.5

pH 3.5

(NA)

(B)

Figure 3. Autoradiogram of partial snake venom phosphodiesterase digests of the 5'-(32P)-labelled fragments p12 (a) and t9b (b). 2687

Nucleic Acids Research places in both fingerprints are not of oligonucleotide nature; they derive from a contaminant in the Y-( 32P)-ATP used. The occurrence of G-m5C in fragment p2 could not be demonTABLE I strated directly, but is deduced PANCREATIC RNase END PRODUCTS from the location of the m5C-G graent Sequence __ Number measu Nlar yeld rom sequence fragment (tl) in the tRNA se1 A-U 0.8 PI quence. Oligonucleotide p5 p2 2.6 5 G-C+G-m5C p3 A-C 0.7 1 shows considerable resistance to P4 1.1 1 U-A-C p5 0.9 1 A-G-A-N snake venom phosphodiesterase 1 p6 A-A-U 1.0 G-U 1.7 2 p7 and nuclease P1 digestion. This 1.0 A-mIA-A-U 1 P8 1 A-i6A-A-r 1.1 P9 is attributed to the nucleotide pl G-G-U+6-I-D 1.9 2 1 pll U-U-U-C 1.0 N (probably m3C) at its 3'-end, p12 2.1 2 A-U-U-T+G-A-U-0 1 1.0 p13 A-A-G-G-A-mU-y causing also an abnormal shift in the "wandering spot" pattern. Fragment p9 again was very resistant to digestion due to the presence of i6Aand T, two modiTABLE II fied bases found in the total Ti Mase END PROUCTS base analysis. The presence of Fragset Number Sequence mUSsu alr yield rom sequence both D and U, at the 3'-end of 0.7 1 tl *5C-U the oligonucleotides contained 2 2.4 t2 A-G+A- 2G 2 1.8 t3 A-C-U+C-A-G in spot 10 was suggested by the 1 t4 0.9 C-C-C-U 1 0.7 t5 C-C-A fact that these oligonucleotides t6 0.7 1 D-U t7 1.0 1 U-U could be separated by electro1 C-U-U 0.8 t8 1 t9a + t9b U-C-C-U + U-C-ac4C-G 0.3 + 0.4 phoresis on DEAE-cellulose at 2 tlO 1.7 DUD-A-A-U + -U-A-U tli 1 C-U-U-U-U 1.3 pH 3.5. Although pA and pG were 1 tl2 U-C-A-C-U-A-U-U 1.0 1 tl3 T-n-C-A-m1A-A-U-C-C-U-U 1.1 found as the 5'-termini in fragt14 + tlA4 A-N-U-U*-C-A-i6A-A-y-C-U-A-A-U-G 1 1.2 ment pl2, we were unable to separate the two oligonucleotides by the available methods. The respective sequences were deduced from the pattern shown in Figure 3a and the data obtained from the Tl oligonucleotides. The homochromatographs of the fragments t2, separated at pH 3.5 on DEAE paper, showed slight differences indicating the presence of modified G. Spot tlO could be separated by the same procedure into its components which were then readily analyzed. The yield of D-D-A-A-G varied in this spot, indicating uneven kinasing of the two fragments. The oligonucleotides A-C-G and C-A-G, contained in fragment t3 were separated in other fingerprints. Fragments t9a and t9b could be related to each other 2688

Nucleic Acids Research since it became clear that ac4C is unstable, giving rise to a double track in the "wandering spot" pattern (see Figure 3b). The long oligonucleotides t13, t14 and tl4a could only be obtained in satisfying purity by prolonged electrophoresis in the second dimension of the fingerprint. Fragment t13 displayed the characteristic mlA shift in its pattern. The 5'-half of fragment t14 was very difficult to analyze by the "wandering spot" method due to the presence of four modified bases. Fragment tl4a, always present in low amounts, seems to be an undermodified or overdigested variant of t14. The total sequence of the anticodon fragment was finally deduced by the analysis of all the data obtained from both fingerprints and from sequencing gels. Direct nucleotide sequences as well as overlap information for the fingerprint fragments were obtained from two-dimensional homochromatograms of partial nuclease P1 digests of intact tRNA labelled at the 5'- or at the 3'-end, and also from 5'-end labelled tRNA fragments. As an example the pattern obtained from the analysis of 3'-end labelled tRNA is shown in Figure 4. Furthermore gel sequencing methods (22,23) were applied to the same large Figure 4. Autoradiogram of a partial nuclease P] digest of 3'- (32P)-labelled serine tRNA. t.-, z-ev

B;@

...

E

.;...> e

pH 3.5 tRNA fragments or intact tRNA. As an example the autoradiogram of a sequencing gel of 5'-(32P)-tRNA is shown in Figure 5, which allowed to read off up to 70 nucleotides. The pronounced difference between the intensity of the bands derived from the first and second half of the tRNA is attributed to the tight secondary structure of the G-C rich extra arm and of the T loop stem (see Figure 6). The occurrence of a number of modified nucleotides is in2689

Nucleic Acids Research

40

A Ph F U2 T1 TI U2 F Ph A -E

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Sequencing gel of 5'-(32P)-labelled sup3-e serine tRNA. The 1 anes were 1 oaded with untreated tRNA (-E), or partial digests with RNase A (A), Physarum RNase (Ph), formamide (F), RNase U2 (U2) and RNase Ti (Ti). 2690

Nucleic Acids Research dicated by the absence of bands expected from the known cleavage specificity of the nucleases used. In this way more than sufficient information was gathered to order the characterized oligonucleotides (Tables I and II) into a unique sequence shown in cloverleaf form in Figure 6. S.POMBE SERINE SUP3-e Figure 6. Cloverleaf model of S. pombe AOH c sup3-e serine tRNA. N is m3C and U* is a mixture of 2S c G and mcm5U. * C

pG U C A C

D G G

G

AGac4c c u G

*A *G U * G

U G A *U C G U C C U *0

A

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0 00

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A C

MIodified Nucleotides. The determination of all modified nucleotides in small amounts of unlabelled tRNA still presents a problem. Following Silberklang et al. (18), we labelled an RNase T2 hydrolysate of sup3-e tRNA with Y-(32P)ATP and polynucleotide kinase. After treatment with nuclease P1 the resulting nucleoside 5'-phosphates were analyzed by thin layer chromatography. In this way we detected the presence of D, ', T, m1A, m2G, and of several m2G modified nucleotides not yet characterized in this chromatography system. Recent advances in High Performance Liquid Chromatography for the separation of nucleoside mixtures (21,22) made this method as sensitive a tool as sophisticated post-labelling methods (23). HPLC analysis of nucleoside hydrolysates derived from sup3-e tRNA (Figure 7) proved the occurrence of one residue each of N4-acetylcytidine (ac4C), N2-dimethylguanosine 2 N6isopentenyl.6 5 adenosine (i A), 5-methylcytidine (m C), 3-methylcytidine (m 3 C), ribothymidine (T), 1-methyladenosine (mlA) and of three pseudouridines (T) residues. In addition two different modified uridines were found in less than molar amounts.

(m22G),

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Nucleic Acids Research

Figure 7. Reversed phase HPLC separation of nucleosides from a sup3-e serine tRNA hydrolysate: The nucleoside abbreviations are given in the text: En, Enzymes; IS, internal standard (Br8G). For experimental details see ref. 21 and 22.

254 nm 0.01AUFS

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zA m~~~~~~~~ mIs ru AU Ri

o

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One of these nucleosides (mcm5U) was identified as 5-(methoxycarbonyl-methyl)uridine. The other unknown compound (s2 U) was shown to contain sulfur by mass spectroscopy and appears to be 2-thiouridine. The positions of the modified nucleosides in the tRNA sequence were deduced as follows. The dihydrouridines (D) at positions 16 and 19 were identified as 5'-ends of the Tl fragments t6 and tlO, respectively. The D at position- 20 is placed there in analogy to other serine tRNAs and could not be demonstrated directly. The split wandering spot pattern of oligonucleotide t9b (Fig.3b) led to the placement of ac4C at position 12. The m2G in position 26 gave rise to gaps in the Tl slots of all sequencing gels. Pseudouridine 27 represents one of the 5'-ends in fragment tlO. Pseudouridine in positions 39 and 55 were identified as blocks to snake venom phosphodiesterase digestion of the relevant fingerprint fragments, and due to their resistance to Physarum nuclease digestion for the sequencing gels. Both, m5C in position 48 and T in position 54 were unambiguously located as 5'-ends of fragments tl and t13. 1-Methyladenosine is easily placed in position 58 by its characteristic shift in the "wandering spot" pattern of fragment t13, and the missing band in the U2 slot of the sequencing gels. Likewise i6A (position 37) and U* (position 34) in the anticodon loop are resistant to snake venom phosphodiesterase ("wandering spot") as well as to U2 and Physarum ribonuclease (sequencing gels). In analogy to rat liver tRNASer we would like to assign m3C (found in the HPLC analysis) as nucleoside N. The modified uridine in the first anticodon position is 5-(methoxycarbonylmethyl)uridine and fractionally 2-thiouridine (to accommodate the last remaining modified nucleo2692

Nucleic Acids Research sides from the HPLC analysis).

DISCUSSION In this paper and in other work from our laboratory (10,11) we have demonstrated the occurrence of a minor tRNASer and a minor tRNALeU species in S. pombe which can be mutated to yield opal suppressor tRNAs. The nucleotide sequence of sup3-e tRNA reveals general features similar to those of other serine tRNAs (8,24). The only noticeable difference consists in the base pair at the proximal end of the D stem which is U-A instead of G.C as in all other serine tRNAs sequenced so far. As observed in other S. pombe tRNAs there is a significant difference in its sequence from the S. cerevisiae tRNASer species (11,25,26). Since the suppressor tRNA is obviously active in protein synthesis, it will be interesting to examine its physical and functional properties. For an example the determination of codon:anticodon lifetimes of the two suppressors and their corresponding wild type tRNAs should contribute to the understanding of the structural properties of the anticodon region of tRNAs and may shed light on the mechanism of codon selection (for review see 27). It is known that the presence of isopentenyladenosine (i6A) adjacent to the 3'-end of the anticodon in a number of tRNAs modulates the stability of codon:anticodon complexes (28). In parallel to earlier observations (29) with E. coZi suppressor tRNATyr the abolition of suppressor activity of sup3-e tRNASer in sini strains of S. pombe (13) was shown to be caused by the absence of this nucleotide modification (30). The anticodon sequence U*CA of both suppressor tRNAs suggests that they were derived from the corresponding wild type tRNAs by base substitution in the second anticodon position. To substantiate this result the sequences of the unmutated wild type tRNA species need to be determined. This mode of suppression contrasts markedly with the E. coli UGA suppressor tRNATrP (5) that originates by a base substitution in the D stem and retains the ability to decode UGG. It was no surprise to find a modified uridine in the first position of the anticodon. Nucleotide modification at this position has been shown to influence the decoding properties of certain tRNAs. The presence of 5-(methoxycarbonylmethyl)-2-thiouridine (31) and also of 5-(methoxycarbonylmethyl)-uridine (32) were reported to restrict the recognition to A in the third codon position. This suggests that 2-thiouridine and 5-(methoxycarbonylmethyl)-uridine found in sup3-e and sup8-e tRNA (11) fulfill a similar role. The reason for the presence of both uridines in this position of 2693

Nucleic Acids Research the suppressor tRNA is not clear. A restriction to the reading of UGA by the suppressor tRNA would prevent the insertion of serine or leucine in response to the tryptophan codon UGG. It is unlikely that this could be achieved solely by the excess of tRNA Trp in the cell. This result parallels the finding that ochre suppressors in S. cerevisiae are specific for ochre termination codons unlike bacterial ochre suppressors (12,27). By applying recombinant DNA techniques, we are currently investigating the many second site mutations described in these suppressor genes (12) and we shall also study the well characterized antisuppressor strains of S. pombe (13).

ACKNOWLEDGEMENTS We are indebted to Mr. K. Kuo and Dr. C.W. Gehrke for their help in the nucleoside analysis of the sup3-e serine tRNA by HPLC methods. This work was supported by grants from the U.S. Public Health Service and from the National Science Foundation.

*Present address: Institute of Organic Chemistry, Polish Academy of Sciences, 61 704 Poznan, Noskowskiego 12/14, Poland

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86:245-260. McClain, W.H., Barrell, B.G. and Seidman, J.G. (1975) J. Vol. BioZ. 99:71 7-732. 4. Seidman, J.G., Comer, M.M. and McClain, W.H. (1974) J. MoZ. BioZ. 90: 677-689. 5. Hirsh, D. (1971) J. MoZ. BioZ. 58:439-458. 6. Kao, S. and McClain, W.H. (1977) J. BioZ. Chem. 252:8254-8257. 7. Piper, P.W., Wasserstein, M., Engbaek, F., Kaltoft, K., Celis, J.E., Zeuthen, J., Liebman, S. and Sherman, F. (1976) Nature 262:757-761. 8. Piper, P.W. (1978) J. MoZ. BioZ. 122:217-235. 9. Goodman, H.M., Olson, M.V. and Hall, B.D. (1977) Proc. Nat. Acad. Sci. 74:5453-5457. 10. Kohli, J., Kwong, T., Altruda, F., Soll, D. andWahl, G. (1979) J. Biol. Chem. 2.54:1546-1551. 11. Wetzel, R., Kohli, J., Altruda, F. and Sbll, D. (1979) MoZec. gen. Genet., in press. 12. Hawthorne, D.C. and Leupold, U. (1974) Curr. Top. Microbiol. Irrrnunol.

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Glynn, I.M. and Chappell, J.B. (1964) Biochem. J. 90:147-149. Maxam, A.M. and Gilbert, W. (1977) Proc. Nat. Acad. Sci. 74:560-564. Schendel, P.F. andWells, R.D. (1973) J. Bio1. Chem. 248:8319-8321. Ikemura, T. and Dahlberg, J.E. (1973) J. Biot. Chem. 248:5024-5032. Silberklang, M., Gillum, A.M. and RajBhandary, U.L. (1979) Vlethods in EnzymoZogy, in press. Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269:833-836. Donis-Keller, H., Maxam, A.M. and Gilbert, W. (1977) NucZ. Acids Res. 4:2527-2538. Gehrke, C.W., Kuo, K.C., Davis, G.E., Suits, R.D., Waalkes, T.P. and Borek, E. (1978) J. Chromatog. 150:455-476. Davis, G.E., Gehrke, C.W., Kuo, K.C. and Agris, P.F. (1979) J. Chromatog. in press. Randerath, E., Yu, C.-T., and Randerath, K. (1972) AnaZ. Biochem. 48: 172-198. Gauss, D.H., GrUter, F. and Sprinzl, M. (1979) NucZ. Acids Res. 6:rl-rl9. McCutchan, T., Silverman, S., Kohli, J. and Sbll, D. (1978) Biochemistry 17:1622-1628. Wong, T-W., McCutchan, T., Kohli, J. and Soll, D. (1979) NucZ. Acids Res. 6: 2057-2068 Steege, D. and Soll, D. (1979) IN Biological Regulation and Control (Goldberger, R.F., ed.) Plenum Publishing Co., N.Y., pp.433-485. Grosjean, H.J., Soll, D. and Crothers, D.M. (1976) J. MoZ. BioZ. 103: 499-519. Gefter, M.L. and Russell, R.L. (1969) J. V1oZ. BioZ. 39:145-157. Janner, F., Vbgeli, G. and Fluri, R. (1978) Experientia 34:943 Abs. Sekiya, T., Takeishi, K. and Ukita, T. (1969) Biochim. Biophys. Acta 182:411-426. Weissenbach, J. andDirheimer, G. (1978) Biochim. Biophys. Acta 518: 530- 534.

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