Proc. Natl. Acad. Sci. USA Vol. 77, No. 7, pp. 4147-4151, July 1980 Cell Biology

Transcription of tRNA in vivo: Single-stranded compared to double-stranded templates (eukaryotic /oocyte injection/single-stranded DNA vector/DNA synthesis) RICCARDO CORTESE*t, RICHARD HARLAND, AND DOUGLAS MELTON MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England Communicated by F. Sanger, April22, 1980

ABSTRACT The expression of cloned tRNA genes has been studied by injecting single-stranded and double-stranded DNA Ceti templates into Xenopus oocyte nuclei. In both forms the genes are faithfully transcribed after injection. Some single-stranded DNA is converted into double-stranded DNA in the oocyte nu- Cut with Taq I cleus. This conversion is necessary for the expression of the in- 750-bp fragment jected tRNA gene: no tRNA transcription is observed when DNA -tNA Gap fill synthesis is inhibited. We conclude that single-stranded DNA blunt ends does not serve as a template for faithful transcription of this 6 LI p!!" gene in injected oocytes. igate EcoRI I _i1I, , linkers Isolated cellular DNA behaves as a stable duplex. However, R R R R R Cut with during transcription only one strand is copied into RNA, and a, q:I3Gq~~~~~IsltLc L2-,LI W CL,tRNAhE~] FcoRI to accomplish this the DNA helix may be made locally unstable Isolate tRNA by cellular factors. Studies of eukaryotes in vitro show that RNA gene from gel polymerase II transcribes a denatured template better than a 280-bp fragment native template, and RNA polymerase III transcribes both Insert into mp2 equally well (1, 2). Obviously, transcription by purified poly- merase and a DNA template does not necessarily reflect what happens in the cell, where initiation and termination of a transcript could be determined by additional factors. Never- Transfect and isolate clones theless, if the signals for initiation and termination are recog- nized in denatured parts of the gene, then a single strand of the Two orientations gene might be expected to contain enough information for Double strand in cell correct initiation and termination of transcription. The possibility that single strands of a tRNA gene, which is tRNA tRNA normally transcribed by RNA polymerase III (1), support faithful transcription has been tested in vvo by injection of this gene into the nucleus of Xenopus oocytes. Xenopus oocytes contain everything necessary to assemble injected DNA into chromatin (3, 4), transcribe it faithfully (5-9), process the transcripts (9-11), and translate injected mRNA (12). Single-strand virion Pure sense (coding) or antisense (noncoding) single strands of a nematode tRNA gene were obtained by using a single- stranded cloning vector derived from bacteriophage M13 (13). The gene was injected into Xenopus oocytes in either single- stranded (ss) or double-stranded (ds) form. We conclude that of &RNA gene Antisense strand of tRNA gene neither the-sense strand nor the antisense strand of the tRNA hybridizes to [3P3 tRNA does not hybridize to [32PltRNA gene alone contains enough information for correct transcrip- mCetlS mCetlA tion, but that a double-stranded template is necessary. FIG. 1. Construction of mCetlS and mCetlA clones. Conditions for enzyme reactions have been described (see ref. 9 for endonuclease and ligase reactions and ref. 14 for DNA polymerase I Klenow frag- MATERIALS AND METHODS menxt gap-filling reaction). Transfection was done as described (14). Bacterial and Bacteriophage Strains. The mp2 strain of T, Taq I cleavage site; R, EcoRI cleavage site. bacteriophage M13 was grown on Escherlchia coil K-12 71-18 performed according to Genetic Manipulation Advisory Group as described (13, 14). Ceti, a recombinant plasmid carrying a II single nematode tRNAPrO gene in ColEl, has been described guidelines: category experiment 5607/11/5. elsewhere (9). All manipulations with recombinant DNA were Abbreviations: ds, double-stranded; ss, single-stranded; araCTP, cy- tosine i6-D-arabinofuranoside 5'-triphosphate; bp, base pair. The publication costs of this article were defrayed in part by page * On leave from the Istituto di Chimica Biologica, II Facolta di Med- charge payment. This article must therefore be hereby marked "ad- icine e Chirurgia, University of Naples, Naples, Italy. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: European Molecular Biology Laboratory, 6900 this fact. Heidelberg, West Germany. 4147 Downloaded by guest on September 24, 2021 4148 Cell Biology: Cortese et al. Proc. Natl. Acad. Scd. USA 77 (1980) Preparation of ss DNA and ds DNA. ss DNA was prepared Preparation of 3P-Labeled DNA. A protocol identical to as follows: 10-ml cultures of infected cells in 1.6% Tryptone/1% the one described above was used to obtain labeled ss DNA. One yeast extract/5% (wt/vol) NaCl, pH 7.4, were grown to late millicurie (1 Ci = 3.7 X 1010 becquerels) of carrier-free [32P]- logarithmic phase. After centrifugation, the phage was pre- orthophosphate (Amersham) was added when cells reached an cipitated from the supernatant by addition of polyethylene OD6so . of 0.1. The specific activity obtained was more than glycol 6000 and NaCl, final concentrations of 4% (wt/vol) and 104 dpm/,ug of DNA. 32P-Labeled ds DNA used as marker was 0.5 M, respectively. After centrifugation at 30,000 X g for 20 prepared by nick (16). mn, the pellet was resuspended in 100 ,AI of standard saline Oocyte Microinjections. They were performed by the citrate (0.15 M NaCI/0.015 M Na citrate) and extracted with standard procedure described (17). RNA extractions and gel phenol followed by ether. The aqueous phase was brought to electrophoresis were done as before (9). DNA was extracted 0.3 M NaOAc and precipitated with ethanol. The pellet was according to Wyllie et al. (3). then resuspended in 10 Ml of H20 and used for injections. CaCI Equilibrium Density Gradient Centrifugation. ds DNA was prepared as follows: E. coil K-12 71-18 was Batches of 20 oocytes were injected with unlabeled DNA at 100 grown to an nm of 0.3 and then infected with mp2 and Mug/ml, [32P]dCTP at 2.5 mCi/ml, and BrdUTP at 7.5 mM and OD65o incubated for 4 hr at room temperature (at this concentration mp2 derivatives at a multiplicity of 10 plaque-forming units/ BrdUTP completely substitutes for dTTP in DNA; unpublished cell. After 3 hr the cells were harvested and the ds DNA (rep- observations). Oocytes were homogenized in 1.5 ml of 1% Na- licative form I) was purified as described (15). Alternatively, DodSO4/30 mM EDTA/20 mM Tris-HCl, pH 7.9/50 mM small-scale preparations of ds DNA were made by using the NaCI/500 Mg of predigested Pronase per ml, incubated at 370C bacterial pellet resulting from ss DNA preparations (see above). for 1 hr, extracted twice with phenol, and precipitated with Cells were lysed and a clear lysate was prepared as in ref. 15. ethanol. The pellet was resuspended in 100 ul of RNase A (100 The supernatant was then extracted with phenol and precipi- mg/ml) in 50 mM NaCl/10 mM TrisVHCI, pH 7.9/1 mM tated with ethanol. The resulting pellet was resuspended in 25 EDTA and incubated at 370C for 30 min. At this stage, aliquots Al of HaO. Occasionally, before injection, the DNA was filtered were electrophoresed on agarose gels to establish the purity and through glass wool. The purity and integrity of the DNA was integrity of the DNA. The solution was made up to 2.1 ml with checked by 0.8% agarose gel electrophoresis, as described 10 mM Tris-HCI, pH 7.9/1 mM EDTA and added to 2.9 g of (9). CsCl. By addition of further buffer, the refractive index was

I i i 1- --! -- 1- -1i II +- ti I F' i

-*o I- I "__ 9

L1- - --I E- FIG. 2. Analysis by electrophoresis on a 1% agarose gel of the fate of ss [32PJDNA injected into oocytes. Groups of 12 oocytes were injected with 50 ni of mCetlS [32P]DNA at 100 ,ug/ml. The injections were into either the nucleus or the cytoplasm. Incubation times were as shown. ds form I (closed circular supercoils), form II (nicked circles), and form III (linear molecules) markers and ss DNA markers are in lanes ds and ss, respectively. Downloaded by guest on September 24, 2021 Cell Biology: Cortese et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4149

( '; ': .:I 0 1.1

" 1.1 ... 40f *

i ._0

.4W 0 A* 4 x-4 *N -t * x ftt -O.* a6a

-_%F **,

10 20 30 Fraction FIG. 4. Isopycnic centrifugation of mCetlS DNA synthesized in a_*_ injected oocytes in the presence of the density label, BrdUTP. Groups of 20 oocytes were injected with mCetlS ss DNA (-) or with mCetlS ds DNA (o). Incubation was for 4 hr. HH, HL, and LL correspond to marker heavy-heavy and heavy-light strands and unsubstituted DNA, respectively, run in parallel gradients. Density in g/cm3 (A).

K-12 71-18), clones carrying inserts into the EcoRI site can be identified as white plaques, whereas the intact vector forms blue plaques (2). Phage mp2, like the parental M1S, does not kill the host cell. The (+)strand of the virus is continuously secreted into the medium as a filamentous virion containing circular ss DNA. In these same cells, however, there are always many copies of FIG. 3. Autoradiogram of 10% polyacrylamide gel electrophoresis. the supercoiled ds replicative form. Restriction enzyme digest (lanes 1 and 2 with Hpa II; lanes 3 and 4 In a previous paper (9) we described a recombinant plasmid with Taq I) ofds DNA extracted from injected oocytes (Inj.) and from control mCetlS-infected E. coli (Cont.). Reaction conditions and gel Ceti, which carries a nematode tRNA gene. We showed that electrophoresis procedufes have been'described (9). a 280-base-pair (bp) DNA fragment contains all the information necessary for the expression of this tRNA gene after injection adjusted to 1.4038 + 0.0002. The sample was overlayered with into the nucleus of the frog oocyte. Transcription of this tRNA paraffin and centrifuged in a Beckman SW 50.1 rotor at 32,000 gene in injected oocytes is accomplished by RNA polymerase rpm for 64 hr. Fractions were collected from the bottom of the III (11). This 280-bp fragment of Ceti has been inserted into tube. The refractive index of every fifth fraction (collected mp2 as shown in Fig. 1. The resulting clones, mCetlS and under paraffin) was measured. Trichloroacetic acid (10%, wt/vol-insoluble radioactivity was determined. Table 1. Inhibition of DNA synthesis by araCTP Enzymes and Chemicals. Endonuclease Taq I was a gift araCTP 32p in form I from J. L. Harris. EcoRI and Hpa II were purchased from Be- T4 DNA injected, mCetlS ds thesda Research Laboratories (Rockville, MD). ligase mM DNA, dpm was a gift from J. Karn. DNA polymerase I Klenow fragment was purchased from Boehringer Mannheim. and o 126,000 the analogues, cytosine f3-D-arabinofuranoside 5'- 0.5 62,000 triphosphate (araCTP) and 5-bromo-2'-deoxyuridine 5'-tri- 5 25,000 phosphate (BrdUTP) were purchased from P-L Biochemi- 50 0 cals. Groups of 15 oocytes were injected with 50 nl of mCetlS ss DNA per oocyte (100 Ag/ml), [32P]dATP (5 mCi/ml), and araCTP as indi- RESULTS AND DISCUSSION cated. Incubations were for 5 hr. Extraction and gel electrophoresis of DNA have been described in ref. 18. The amount of radioactivity The cloning vehicle, mp2, used in these studies has been de- comigrating with mCet1S ds DNA was determined by scanning scribed (13). It contains a unique EcoRI site in the sequence autoradiographs of the dried gels with a Joyce Loebl microdensi- coding for the a peptide of ,B-galactosidase. The a peptide tometer. Even in grossly overexposed autoradiographs, no radioac- complements some deletions of the ,B-galactosidase gene. By tivity above background was detected comigrating with mCet1S DNA use of indicator plates (13) and a suitable bacterial host (E. coil when 50 mM araCTP was injected. Downloaded by guest on September 24, 2021 4150 Cell Biology: Cortese et al. Proc. Natl. Acad. Sci. USA 77 (1980) In Fig. 2 the conversion of labeled single strands to double strands is shown as a function of time. The autoradiogram of the gel was scanned with a Joyce Loebl microdensitometer and the areas under the peaks for ss circular and ds supercoiled DNA were measured. At least 10% of the total input of ss DNA was converted to ds supercoils within 2 hr when the DNA was in- jected into the nucleus. ss DNA as such was rather unstable; that which was not converted to ds DNA was completely degraded after 10 hr [this agrees with the results of Wyllie et al. (3), though they would not have detected this level of conversion of ss to ds DNA under their experimental conditions]. The conversion of single strands to double strands occurred only in the nucleus. ss DNA injected into the cytoplasm was degraded at a rate comparable to that in the nucleus, without any being converted to double strands (Fig. 2). The change in electrophoretic mobility of ss [32P]DNA to that of ds DNA (supercoiled or nicked form) is good evidence for the conclusion that ss DNA is converted to ds DNA. Evidence for the fidelity of this DNA synthesis (shown in Fig. 3) comes from the restriction digest patterns of the newly formed ds DNA, which were identical to the ones obtained with replica- tive form ds supercoiled DNA extracted from infected E. coli cells. Finally, a third line of evidence comes from studies on DNA biosynthesis in the presence of BrdUTP. Incubation with BrdUTP led to the formation of DNA of a density corre- sponding to complete heavy-light hybrid double strands (Fig. 4). From this experiment it is also clear that, after completion of the second strand, no further DNA synthesis occurs. The synthesis of the second DNA strand is very sensitive to F- - araCTP, an inhibitor of DNA synthesis in other systems (18). Table 1 shows that, after injection of a solution of 50 mM araCTP (about 50 nl/oocyte), there was a total inhibition of FIG. 5. Effect of araCTP on transcription. An autoradiogram of DNA synthesis. These observations are similar to those made a 10%6 polyacrylamide gel is shown. ds or ss DNA was injected into oocyte nuclei with Ia-32P]GTP. Incubation was for 5 hr. RNA was on the elongation of replicating chromatin in vitro (18). extracted and subjected to electrophoresis as described (9). Groups Single-stranded mp2 DNA (without a tRNA gene insert) was of 20 oocytes were analyzed. also converted into double strands. This excludes the possibility that the structure of the tRNA gene confers some feature on the mCetlA, contain the 280-bp insert in opposite orientations, as DNA that is required to initiate synthesis of the complementary determined by sequence analysis. Nematode tRNA hybridized strand. to the ss form of mCetlS DNA, but not to mCetlA DNA. As Only ds DNA Can Act as a Template for Transcription. expected, tRNA hybridized to the ds form of the DNA of both From 5 ng of injected DNA about 10% is converted to ds DNA recombinant phages. Thus, cells infected with mCetlS secrete within 2 hr, thus'producing 1 ng of ds DNA or approximately a virion containing the sense strand of the tRNA gene-that 30 times the DNA content of the oocyte nucleus. This is suffi- is, the that is transcribed into a tRNA. Cells in- cient to account for the observed production of tRNA. An im- fected with mCetlA secrete a virion containing the noncoding portant question remains: although conversion of ss DNA to ds or antisense strand (Fig. 1). DNA normally ensues, is it necessary for transcription in the Expression of ds and ss Templates. mCetlS and mCetlA case of injection of the sense strand? There are three distinct ds DNA each contains the tRNA gene but in opposite orienta- possibilities: (i) ds DNA is the only form recognized as template tions. After injection into the oocyte nucleus, both direct the by RNA polymerase; (ii) both ss and ds can function as synthesis of the same tRNA molecule, a proline tRNA (data not template, and (Mi) only ss DNA is a good template. The third shown; data of this type are shown in Fig. 5 and ref. 9). Because possibility is compatible with the results so far obtained by us the tRNA gene is transcribed in both orientations, it is unlikely and other workers because we could not rule out the possibility that a signal external to the inserted fragment is responsible for that injected or newly synthesized ds DNA is first converted to directing the transcription of the tRNA gene. ss DNA for or during transcription (maybe only in a localized Single-stranded DNAfrom either clone directs the synthesis stretch of sequence). of tRNA. If ss DNA functions as a template for transcription, In order to examine these three possibilities, we inhibited then mCetlS ss DNA could be transcribed as such. mCetlA DNA synthesis in injected oocytes with araCTP and studied the DNA is the antisense strand and can promote synthesis of tRNA synthesis of tRNA. The results are shown in Fig. 5. tRNA is only after the complementary DNA strand is synthesized. These synthesized in the absence and presence of araCTP after the results pose the following questions: (i) can ss DNA function as injection of ds DNA. The inhibitor has only a small effect on a template or (tt) must ss DNA be converted to ds DNA, which transcription of the tRNA gene in its ds form. In contrast, sense only then can act as a template for transcription? and antisense ss DNA, which both promote tRNA synthesis, fail Injected ss DNA Is a Template for DNA Synthesis. Some to do so in the presence of araCTP. of the injected circular ss DNA was converted to a mixture of araGTP, which does not interfere with transcription when ds closed circular supercoiled, linear, and nicked circular ds DNA is injected, does not lower the half-life of ss DNA (data molecules (Figs. 2-4). not shown). Quantitation from the autoradiogram shown in Fig. Downloaded by guest on September 24, 2021 Cell Biology: Cortese et al. Proc. Nati. Acad. Sci. USA 77 (1980) 4151

2 shows that after 2.5 hr 50% of the total input of ss DNA is still 3. Wyllie, A. H., Laskey, R. A., Finch, J. & Gurdon, J. B. (1978) Dev. intact in the oocyte. Thus, if ss DNA were A physiokigcal -Biol. 64, 178-188. template for tRNA transcription, the transcripts would have 4. Laskey, R. A., Honda, B. M., Mills, A. D., Morris, N. R., Wyllie, been detected. The possibility that the presence of a large A. H., Mertz, J. E., De Robertis, E. M. & Gurdon, J. B. (1977) Cold 171-178. amount of ss DNA nonspecifically inhibits transcription from Spring Harbor Symp. Quant. Biol. 17, 5. Mertz, J. E. & Gurdon, J. B. (1977) Proc. Natl. Acad. Sci. USA the single-stranded gene can be ruled out. tRNA transcripts are 74, 1502-1506. a amount in presence observed when small of ds DNA is the of 6. Brown, D. D. & Gurdon, J. B. (1977) Proc. Natl. Acad. Sci. USA a large excess of ss DNA (data not shown). This situation occurs 74,2064-2068. in the absence of araCTP after a 2-hr incubation (Fig. 2). 7. De Robertis, E. M. & Mertz, J. E. (1977) Cell 12, 174-182. Taken together, these results (Figs. 2 and 5) show that only 8. Kressman, A., Clarkson, S. G., Pirrotta, V. & Birnstiel, M. L. (1978) ds DNA is recognized as a template for faithful transcription Proc. Natl. Acad. Sci. USA 75, 1176-1180. of a tRNA gene. These results cannot exclude the possibility that 9. Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) double strands are required on only part of the template or for Nucleic Acids Res. 5, 4593-4611. only part of the transcription process, such as recog- 10. Melton, D. A., De Robertis, E. M. & Cortese, R. (1980) Nature nition. Moreover, other genes may be able to function in sin- (London) 284, 143-148. gle-stranded form. Nevertheless, in the case of the tRNA gene 11. Melton, D. A. & Cortese, R. (1979) Cell 18, 1165-1172. tested, a double-stranded template is required for faithful 12. Gurdon, J. B., Lane, C. D., Woodland, H. R. & Marbaix, G. (1971) transcription. Nature (London) 233, 177-182. 13. Groneborn, B. & Messing, J. (1978) Nature (London) 272, We thank J. B. Gurdon, L. J. Korn, R. A. Laskey, and J. D. Smith for 275-277. critically reading the manuscript and M. Squire for technical assis- 14. Schreier, P. H. & Cortese, R. (1979) J. Mol. Biol. 129, 169- tance. 172. 1. Roeder, R. G. (1976) in RNA Polymerase, eds. Losick, R. & 15. Clewell, D. B. (1972) J. Bacteriol. 110, 667-676. Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring 16. Rigby, P. W. J., Dieckman, M., Rhodes, C. & Berg, P. (1977) J. Harbor, NY), pp. 285-329. Mol. Biol. 113,237-251. 2. Lilley, D. K. J. & Houghton, M. (1979) Nucleic Acids Res. 6, 17. Gurdon, J. B. (1976) J. Embryol. Exp. Morphol. 36,513-540. 507-523. 18. Francke, B. & Hunter, T. (1975) J. Virol. 15,759-775. Downloaded by guest on September 24, 2021