THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 9, Issue of May 10, pp. 5588-5595,1985 Printed in U. S. A.

Expression in Vivo of a Mutant Human Initiator tRNA Gene in Mammalian Cells Using a Simian Virus 40 Vector*

(Received for publication, September 7, 1984)

Harold J. Drabkin and Uttam L. RajBhandary From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

We have cloned both the wild type (A54) and mutant (T54) human initiator genes described in the preceding paper (Drabkin, H. J., and RajBhandary, U. L. (1985) J. Biol. Chem. 260,5580-5587) as 141-base pair frag- ments into the SV40-pBR322 vector pSVlGT3. These vectors were subsequently used to transfect monkey kidney CV-1 cells to obtain recombinant virus stocks carrying each of the initiator tRNA genes. Following infection of CV- 1 cells by the recombinant virus stocks, both the wild type and mutant tRNAs are produced in large quantities during a 48-h period. Fingerprint analysis of 32P-labeled tRNAs was used to characterize the tRNAs made in vivo and to show that the sequence AUCG in loop IV of the wild type tRNA is replaced by T$CG in the mutant tRNA. Modified nucleotide com- position analysis of the [“PItRNAs overproduced in vivo shows that they contain all the modified nucleo- tides found in human placenta initiator tRNA.

Both wild type and mutant initiator tRNAs can be aminoacylated by either mammalian or Escherichia coli methionyl-tRNA synthetases. Furthermore, the mutant tRNA can be easily separated from the endog- enous monkey initiator tRNA by RPC-5 column chro- matography.

All eukaryotic cytoplasmic initiator tRNAs possess the sequence AUCG’ in place of the T$CG sequence found in other tRNAs (Simsek et al., 1973; Sprinzl and Gauss, 1984). The conservation of this sequence among the eukaryotic initiator tRNAs suggests that this sequence plays an impor- tant role in the function of these tRNAs in the initiation of protein synthesis. In the preceding paper (Drabkin and RajBhandary, 1985a), we used oligonucleotide-directed site- specific mutagenesis of a human initiator tRNA gene to change the ATCG corresponding to the AUCG’ sequence to TTCG. Using HeLa cell extracts i n vitro, we showed that this mutation had no effect on either transcription or on process- ing of the transcript to produce a mature sized tRNA.

The eventual goal of generating mutations in the initiator tRNA gene is to use an appropriate expression system to produce quantities of mutant tRNA necessary for i n vitro functional studies. We report here on the use of SV40 recom-

* This work was supported by Grant GM 17151 from the National 1nstit.utes of Health and by Grant NP114 from the American Cancer

by National Research Service Award Grant 1F32 GM 07480 from the Society. During the early phase of this work, H. J. D. was supported

National Institutes of Health. 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. Section 1734 solely to indicate this fact.

In all vertebrate initiator tRNAs, the U is not modified, whereas in all plant, starfish, and some other invertebrates, the U is modified to $.

binant vectors carrying the wild type or mutant tRNA genes to obtain wild type or mutant initiator tRNA from CV-1 cells lytically infected with these virus stocks. We show that not only is the mutant tRNA overproduced in vivo but also that it can be aminoacylated with methionine and also easily separated from the endogenous CV-1 initiator tRNA.

MATERIALS AND METHODS’

RESULTS

Expression of the Initiator tRNA Genes in Mammalian Cells-Our objective in these studies is to obtain sufficient quantities of the mutant initiator tRNA for i n vitro functional studies. Elsewhere, we have reported (Drabkin and Raj- Bhandary, 1985b) on attempts to achieve expression of the human initiator tRNA gene in yeast. Although many yeast polymerase I11 genes can be transcribed and processed by mammalian cell-free systems, the human tRNA gene was not transcribed in either a yeast cell-free system or in vivo in yeast when cloned using a 2 g vector.

In view of these results and also the successful i n vivo expression of a Xenopus tyrosine tRNA gene in mammalian cells (Laski et al., 1982), we decided to attempt expression of the mutant initiator tRNA gene by cloning the gene in a SV40-pBR322 hybrid vector and isolating tRNA from cells lytically infected with the recombinant virus.

Cloning into pSVlGT3”As outlined in Fig. 1, the mutant and wild type tRNA genes were excised from the M13mp7 vector with BamHI and inserted into the BamHI site of pSVlGT3, an SV40 vector containing the sV40 origin of replication, the early region, a duplication of the region from 0.14 to 0.32 map units with the single BamHI site in place of the PvuII site at 0.71 map unit of SV40, and ~ B R 3 2 2 . ~ Recombinants (pSVA54 and pSVT54) were analyzed by BamHI and AsuII digestion followed by Southern hybridiza- tion to confirm the presence of the wild type and mutant tRNA genes. The orientation of the tRNA genes in the vector was determined by analysis of DNAs cut with HphI, which cuts the human gene asymmetrically (data not shown).

The orientation chosen for in vivo expression was such that transcription from the tRNA gene is opposite to any tran- scription from the late promoter.

Preparation of Virus Stocks-The recombinants were di-

Portions of this paper (including “Materials and Methods” and Tables 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request DOC- ument No. 84M-2809, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

R. Mulligan, personal communication.

5588

I n Vivo Expression of Mutant Human Initiator tRNA Gene 5589

FIG. 1. Scheme for cloning the hu- man initiator tRNA genes into an SV40 recombinant vector, and sub- sequent preparation of virus stocks. Abbreviations used are: B, BamHI; E, EcoRI; H , HindIII; P, PstI; Ori, SV40 origin of replication. Arrows inside circle indicate the direction of early and late transcription of SV40 DNA. The orien- tation of the tRNA gene inserted into the BamHI site of pSVlGT3 is indicated by an arrow.

Cui with hi I, -pBR322 Rdigate

c. ori

- pBR322 Cut with EcoRl , Religate

gested with PstI and religated under dilute conditions in order to free the SV40 sequences from pBR322, which is known to contain sequences inhibitory to SV40 replication in mammalian cells (Lusky and Botchan, 1981). The DNA was then mixed with similarly treated pSV1-rINS7 DNA (Fig. 1) and used to transfect monkey CV-1 cells. Virus stocks (SVA54 and SVT54) prepared from these cultures were used to lyti- cally infect CV-1 cells in the presence of [32P]04.

Analysis of viral DNA obtained from Hirt extracts of cells infected with the various virus stocks by HindIII digestion revealed that the wild type (SVA54) clone chosen for study actually contained three tandem copies of the initiator tRNA gene (Fig. 2). This is clearly seen in the Southern hybridiza- tion analysis data shown in Fig. 2, in which the HindIII fragment containing the tRNA gene is larger in the wild type (A54) than in its mutant (T54) counterpart.

Subsequent analysis of the original plasmid (pSVA54) showed that it too contained the three wild type tRNA genes in tandem, and further, that all three genes were in the same orientation against the late promoter (data not shown).

Isolation and Characterization of 32P-labeled Wild Type and Mutant tRNAs-Fig. 3 shows the results of a 15% polyacryl- amide gel analysis of the [32P]tRNA isolated from infected cells. As shown in the SVA54 lane, a very intense radioactive band not present in mock- or SV40-infected cells is seen in the SVA54-infected cells. This band co-migrates with 5'-end- labeled rabbit liver initiator tRNA (not shown) and is, there- fore, most likely the initiator tRNA which is overproduced in CV-1 cells. Cells infected with SVT54 (SV40 carrying the T54 mutant initiator tRNA gene) also overproduce an RNA. Al-

Mix , cotronsfect CV-I cells

J. Lytic Infection

Virus stock

t-

A-

3 D- E-

FIG. 2. HindIII digestion of recombinant SV40 DNAs ob- tained by Hirt extraction of infected cells. Left, ethidium bro- mide-staining pattern. Letters refer to the HindIII fragments of SV40. Right, Southern hybridization analysis using 5'-[32P]initiator tRNA as a probe.

though this RNA migrates differently from the corresponding wild type RNA, we show below that it has the same 5'- and 3'-ends as the wild type initiator tRNA and differs from the latter only in having the sequence T+CG in place of AUCG.

The radioactive bands were excised, and the isolated RNAs

5590 In Vivo Expression of M u t a n t Human Init iator tRNA Gene

32 P- RNAs ISOLATED FROM CVI CELLS INFECTED WITH SV40

. .- Mock SV40 SVA54 SVT54

I

FIG. 3. Autoradiogram of polyacrylamide gel electropho- resis of [32P]RNA obtained from Hirt extracts of CV-1 cells infected with SV4O-recombinant virus stocks. Cells were in- fected with 0.5 ml of recombinant virus stocks or 0.2 ml of SV40 virus stock. The radioactive RNAs eluted from the gel are indicated by the arrows and were further purified by hybrid selection for fingerprint analysis.

were analyzed by fingerprint analysis. Fig. 4 (A-D) shows the results of T1 RNase (A and B) and pancreatic RNase (C and D) digestions of the two RNAs. Both RNAs have the 5‘-end, pAGp, and the same 3’-end, CUACCAOH (Fig. 4, A and B). The only difference in the fingerprint pattern of these two RNAs is consistent with the expectation that the mutant RNA has the sequence T$CG in place of AUCG. Thus, (a) T1 RNase fingerprints of the mutant tRNA lack the spot corresponding to AUCG and contain instead TGCG (see be- low) and (b) pancreatic RNase fingerprints of the mutant tRNA lack the sequence GGAU and instead have the sequence GGT (Fig. 4, C and D).

Modified Nucleotide Composition Analysis of 32P-labeled Wild Type and Mutant tRNAs-Analysis of modified nucleo- tides present in the 32P-labeled tRNAs (Fig. 5) show that both the wild type and mutant tRNAs contain all of the modifica- tions previously detected in human placenta initiator tRNA

(Gillum et al., 1975). In addition, the mutant tRNA contains Tp and $p. Secondary analysis of oligonucleotides present in T1 RNase digests (Fig. 4B) show that Tp and $p are present in the sequence T+CG. One of the modified oligonucleotides, m’Ap, is not seen clearly in Fig. 5. However, other analyses (data not shown) using a different solvent system demonstrate the presence of this nucleotide.

The Mutant tRNA Is More Readily Denatured than the Wild Type tRNA-As discussed above and as seen in Fig. 3, the mutant tRNA, although it has the same size as the wild type tRNA, has an altered electrophoretic mobility compared to the wild type tRNA. In order to investigate this further, the gel electrophoretic properties of the two tRNA prepara- tions were studied under three different conditions (Fig. 6).

In Fig. 6 the left panel shows that under totally nondena- turing conditions, the two RNAs have a similar mobility. The middle panel shows that under partially denaturing condi- tions, in the presence of 7 M urea, and at room temperature, there is a large difference in the mobility of the two tRNAs, with the mutant tRNA having a slower mobility. Finally, the right panel shows that under completely denaturing condi- tions (8.3 M urea, 40 watts) the two RNAs again have similar mobilities.

Even under partially denaturing conditions, the concentra- tion of polyacrylamide in the gel has an effect on separation between the mutant and wild type tRNAs. Thus, while the analysis of in vitro transcription reactions of the wild type and mutant tRNA gene showed little difference in mobility between the corresponding transcripts in 10% polyacrylamide (see Fig. 5 in Drabkin and RajBhandary, 1985a), the same reactions run on a 15% polyacrylamide gel gave excellent separation between the mutant and wild type tRNA (data not shown).

Aminoacylation of the Mutant tRNA-Having demon- strated accurate overproduction of both mutant and wild type tRNAs, we next wished to investigate whether the wild type and mutant tRNAs could be aminoacylated and whether they could be easily separated from each other. We anticipated that the mutant tRNA would most likely be aminoacylated, since the elongator methionine tRNA species, which is ami- noacylated by the same methionyl-tRNA synthetase, has T54. However, in view of the above finding that the T54 mutant initiator tRNA denatures more readily than the A54 wild type tRNA, the possibility that the mutant tRNA may not be recognized by the corresponding aminoacyl-tRNA synthetase was not ruled out.

Low molecular weight (4 S and 5 S) RNA isolated from large scale infections was aminoacylated with either [35S] methionine using a crude rat liver aminoacyl-tRNA synthe- tase preparation (which should aminoacylate all of the me- thionine tRNA species) or with [3H]leucine using a crude yeast preparation. The time course of incorporation of labeled amino acids into trichloroacetic acid-precipitable material is shown in Fig. 7. Whereas the level of leucine acceptance of all of the tRNA preparations is about the same, the levels of methionine acceptance differ. The level of methionine ac- ceptance of the tRNA obtained from the SVA54 (wild type) infected culture is about 9 times that of a mock or SV40- infected culture. Furthermore, the tRNA obtained from the SVT54-infected culture is three times higher than the control culture. The 3-fold difference in methionine acceptance levels of tRNAs isolated from the SVA54- and the SVT54-infected cultures could be related to the fact that the wild type tRNA gene is present in three times the copy number than is the mutant gene (however, see “Discussion”). These results show that both the wild type and mutant initiator tRNA genes are

FIG. 4. Fingerprint analysis of oligonucleotides present in T1 RNase (A and B) and RNase A (C and D) digests of purified A54 ( A and C) and T54 (B and D) RNAs isolated from the polyacrylamide gel shown in Fig. 3 and further purified by hybrid selection (see "Materials and Methods"). 1 and 2 refer to electrophoresis a t pH 3.5 in the first dimension and homochromatography in the second dimension, respectively.

FIG. 5 . Modified nucleotide com- position analysis on A54 and T54 RNAs purified by hybrid selection of the RNAs eluted from the gel of Fig. 3.

I n Vivo Expression of Mutant Human Initiator tRNA Gene 5592

NATIVE

d * l n l o a +

5s-

PARTIALLY FULLY DENATURED DENATURED

x x x $ a c < I -

5 s, 5s

FIG. 6. 15% polyacrylamide gel electrophoresis of A54 and T54 RNAs under varying degrees of denaturation. All gels were run in 90 mM Tris borate, pH 8.3,2 mM EDTA until the xylene cyanole migrated about 30 cm. Nondenaturing, 500 V, no urea; partially denaturing, 7 M urea, 500 fully denaturing, 8.3 M urea, 1200 V, approximately 40 watts).

A - B

1

0 20 40 0 20 40 TIME (mm) n m (min)

FIG. 7. Kinetics of aminoacylation of tRNA isolated from SV40 recombinant virus-infected CV- 1 cells. Approximately 10 pg of DEAE-cellulose-purified RNA were used per 100-p1 assay. Each time point represents a 2 0 4 aliquot. A, aminoacylation with [3H] leucine, using a yeast synthetase. B, aminoacylation with [35S]methi- onine using a rat liver synthetase.

expressed in vivo and produce tRNAs which are aminoac- ylated.

RPC-5 Chromatography of Methionyl-tRNAs-For in vitro structure-function relationship studies of the mutant human initiator tRNA overproduced in CV-1 cells, it would be nec- essary to separate it from any endogenous CV-1 initiator tRNA, which has the same sequence as wild type human initiator tRNA (Gillum et al., 1975). Fig. 8 shows the RPC-5' chromatographic profile of [35S]methionyl-tRNA from CV-1 cells which were either mock infected (A), SV40 infected (B),

A . Mock Infected t - Meti

.

t

C. SVA54 I

8. SV40

. A f i I I I I 1 1%:' , UV! ,\- , -dl .. , I

0 20 40 60 80 0 20 40 60 80 FRACTION NUMBER

FIG. 8. RPC-5 column chromatography of [36S]methionyl- tRNAs obtained using a rat liver aminoacyl-tRNA synthetase preparation.

SVA54 infected (C), or SVT54 infected (D). Both in mock and in SV40-infected cells, three peaks of methionyl-tRNAs were seen. The first one corresponds to the initiator species, whereas the latter two correspond to the elongator species of methionine tRNAs. In SVA54-infected cells, a substantial overproduction of the initiator species is seen (C). This is consistent with an approximately 9-fold elevation of methio- nine acceptance (Fig. 7). The SVT54-infected cells also show, as expected, overproduction of a methionine tRNA (Fig. 80, middle peak) which is separated clearly from the wild type initiator tRNA (Fig. 8C, first peak), although it co-chromato- graphs with the first of the elongator methionine tRNAs.

The Mutant tRNA Is Aminoacylated by Escherichia coli Aminoacyl-tRNA Synthetase-Although the mutant initiator tRNA is clearly separated from the wild type initiator tRNA, it co-chromatographs with the elongator tRNAs. To identify the mutant tRNA separately from the elongator tRNAs, we took advantage of the fact that E. coli methionyl-tRNA syn- thetase aminoacylates only the initiator species of vertebrates (Gupta et al., 1970).

Fig. 9 shows the results of aminoacylation of the mock and virus-infected tRNAs with [35S]methionine using a crude E. coli aminoacyl-tRNA synthetase preparation. The E. coli en- zyme aminoacylates the wild type (mock, SV40, SVA54) and the mutant (SVT54) initiator tRNA. The SVA54-infected cultures have about a 9-fold elevation in the level of initiator tRNA compared to the control cultures, and the SVT54- infected cultures have about a 3-fold elevated level, in agree- ment with the results obtained with the eukaryotic aminoacyl- tRNA synthetase preparations.

Fig. 10 shows the RPC-5 chromatography of the [35S]

I n Vivo Expression of Mutant Human Initiator tRNA Gene 5593

2.5 r

SVA54

$ 0

TIME (mid

FIG. 9. Kinetics of aminoacylation with [S6S]methionine of tRNA isolated from SV40 recombinant virus-infected CV-1 cells using the E. coli aminoacyl-tRNA synthetase. Approxi- mately 10 pg of DEAE-cellulose-purified RNA were used per 100-pl assay; each time point represents a 20-4 aliquot.

FRACTION NUMBER FIG. 10. RPC-5 column chromatography of [36S]methionyl-

tRNAs obtained using the E. coli aminoacyl-tRNA synthetase.

methionyl-tRNAs obtained using the E. coli enzyme. As ex- pected, the overproduced wild type human initiator tRNA chromatographs with the endogenous CV-1 initiator tRNA and yields a single peak. The mutant initiator tRNA, however, is clearly separated from the endogenous CV-1 initiator tRNA and can be obtained essentially free of the latter.

DISCUSSION

The work presented in this study demonstrates the feasi- bility of using site-specific mutagenesis as an approach to structure-function relationship studies of initiator tRNAs. We have shown that both the wild type and the T54 mutant human initiator tRNA genes can be expressed efficiently in CV-1 cells to generate mature tRNAs. The tRNAs produced contain all the base modifications found in human placenta initiator tRNA (Gillum et al., 1975), and differ only in that the sequence AUCG in the wild type initiator tRNA is re- placed by T$CG in the mutant. Both the wild type and the mutant initiator tRNAs can be aminoacylated i n vitro (Figs. 7 and 9). The amounts of tRNAs produced are severalfold

over endogenous levels of CV-1 initiator tRNA. Additionally and most importantly, following aminoacylation i n vitro, the mutant [35S]Met-tRNA is easily separated from the corre- sponding wild type endogenous initiator tRNA (Figs. 8 and 10). Thus, the properties of the mutant initiator tRNA can be compared to those of the wild type tRNA without any complications due to its contamination by wild type initiator tRNA. The [35S]methionine attached to the initiator Met- tRNA can be used to follow its function in i n uitro protein synthesis (Housman et al., 1970), without having to purify the mutant initiator tRNA to homogeneity.

While both the A54 and T54 initiator tRNAs are overpro- duced in CV-1 cells, we have noted that the amount of A54 tRNA produced is approximately three times that of the T54 tRNA (Figs. 3, 7, and 9). This is not due to a gene dosage effect since recent experiments using an SV40 vector carrying a single A54 gene show that the amount of the A54 tRNA synthesized during the 5-h labeling period is still more than the T54 tRNA (data not shown). In control experiments, analyses of infected cells show that the amount of SVA54 and SVT54 viral DNA in infected cells is about the same. There- fore, although the A54 and T54 initiator tRNA genes are transcribed equally well i n uitro and their transcripts proc- essed at about equal rates in the HeLa cell extracts (Drabkin and RajBhandary, 1985a) i n viuo, the A54 tRNA is produced in larger amounts than the T54 tRNA. There are several possible explanations for this result. 1) I n viuo the A54 tRNA gene is actually a better template for transcription than the T54 tRNA gene; 2) the two tRNA genes are transcribed equally well, but the T54 tRNA does not accumulate to the same extent because processing of the T54 tRNA precursor is slow compared to that of the A54 tRNA precursor; 3) the T54 tRNA is transported less efficiently to the cytoplasm (Zasloff et al., 1982) and turns over in the nucleus; and 4) the T54 tRNA has a higher turnover rate in the cytoplasm, perhaps due to a defect in its function.

Since the T54 tRNA is processed as well as the A54 tRNA by HeLa cell extracts and can be aminoacylated as well as the latter by either mammalian or E. coli enzymes, under normal conditions the mutant and wild type initiator tRNAs have basically the same overall three-dimensional structure (Kim et al., 1972; Robertus et al., 1974). However, although the T54 and A54 tRNAs have the same 5'- and 3'-ends (Fig. 4), and hence the same size, they separate quite well during electro- phoresis in 15% polyacrylamide gels under partially denatur- ing conditions (Fig. 3). Under nondenaturing or completely denaturing conditions, the two tRNAs behave similarly (Fig. 6). Thus, the T54 initiator tRNA denatures somewhat more readily than the A54 tRNA. It is possible that the potential for alternate base pairing within the T$CG loop between T54 and A60 on one hand (Jank et al., 1977) and T54 and m'A58 on the other (Kim et al., 1972; Robertus et al., 1974) accounts partly for this phenomenon. This denaturation could involve just small structural changes in the T$CG loop itself or a more complete destruction of the D loop-T$CG loop interac- tions (Westhof et al., 1983). In addition, the presence of $55 in mutant tRNA instead of U55 in the wild type tRNA may also contribute toward this ease of denaturation.

The only naturally occurring tRNA which contains U54 and A60 in the T$CG loop is a species of valine tRNA from several mammalian sources (Sprinzl and Gauss, 1984). In this tRNA, U54 is not methylated to T, as it is in virtually all other tRNAs (Sprinzl and Gauss, 1984). This lack of meth- ylation has been attributed to inaccessibility of U54 toward methylation, possibly because of the involvement of U54 in a Watson-Crick base pair with A60 (Jank et al., 1977). However,

5594 In Vivo Expression of Mutant Human Initiator tRNA Gene

the T54 mutant initiator tRNA which also has U54 and A60 A. (1970) J . Mol. Bid. 54, 145-154 is quantitatively methylated at U54 to form T54 (Fig. 5). ~ ~ ; ~ ; t ~ ~ ~ ~ , ) ~ ~ ~ ~ ~ ~ , , 2 ~ ; ~ ~ ~ ~ ~ p, A, (1983) J . Mol. Appl, Thus, sequences outside of the TqCG loop region must be Genet. 2, 147-159 involved in recognition of a tRNA by the tRNA (uracil-5) Housman, D., Jacobs-Lorena, M., RajBhandary, U. L., and Lodish, methyltransferase. Presumably these nucleotide residues are H. F. (1970) Nature 227,913-918 absent in the mammalian valine tRNA. Jank, P., Riesner, D., and Gross, H. J . (1977) Nucleic Acids Res. 4,

2009-2020

Acknowledgments-We wish to thank Drs. Richard Mulligan and Philip Sharp for their advice and for providing the pSVlGT3 and the pSVlrINS vectors. We also thank Dr. Philip Sharp for access to the P-3 factilities at the Center for Cancer Research used in this work. In addition, we acknowledge Dr. Frank Laski for advice and Mary Esteve of Dr. Sharp's laboratory for supplying the CV-1 cells.

REFERENCES Beikirch, H., von der Haar, F., and Cramer, F. (1972) Eur. J. Biochem.

Birhboim, H. C., and Doly, J. (1980) Nucleic Acids Res. 7, 1513- 1523 DasSarma, S., RajBhandary, U. L., and Khorana, H. G. (1984) Proc.

Drabkin. H. J.. and RaiBhandarv, U. L. (1985a) J. Bid. Chem. 260,

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Drabkin, H. J., and RajBhandary, U. L. (1985b) J. Biol. Chem. 260, 5596-5602

Gillum, A. M., Roe, B. A., Anandaraj, M. P. J . S., and RajBhandary, U. L. (1975) Cell 6, 407-413

Gupta, N. K.,'Chatterjee, N. K., Bose, K. K., Bhaduri, S., and Chung,

K&S. H., Suddath, F. L., Quigley, G. J., McPherson, A., Sussman, J. S., Wang, A. H., Seeman, N. C., and Rich, A. (1972) Science 185,435-440

Laski, F. A,, Alzner-DeWeerd, B., RajBhandary, U. L., and Sharp, P. A. (1982) Nucleic Acids Res. 10,4609-4626

Lusky, M., and Botchan, M. (1981) Nature 293, 79-81 Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S.,

Silberklang, M., Gillum, A. M., and RajBhandary, U. L. (1979)

Simsek, M., Ziegenmeyer, J., Heckman, J. E., and RajBhandary, U.

Sompayrac, L. M., and Danna, K. J. (1981) Proc. Natl. Acad. Sci. U.

Southern, E. M. (1975) J. Mol. Bid. 98, 503-517 Sprinzl, M., and Gauss, D. H. (1984) Nucleic Acids Res. 12, rl-r58 Stanley, W. M. (1974) Methods Enzymol. 29,530-547 Tollervey, D., Wise, J. A,, and Guthrie, C. (1983) Cell 35, 753-762 Westhof, E., Duman, P., and Moras, D. (1983) J. Biomol. Struct.

Yang, W.-K., and Novelli, G. D. (1971) Methods Enzymol. 20,44-55 Zasloff, M., Rosenberg, M., and Santos, T. (1982) Nature 300,81-84

Clark, B. F. C., and Klug, A. (1974) Nature 250, 546-551

Methods Enzymol. 59, 58-109

L. (1973) Proc. Natl. Acad. Sci. U. S. A . 70, 1041-1045

S. A. 78, 7575-7578

Dynamics 1,337-355

I n Vivo Expression of Mutant Human Initiator tRNA Gene 5595

svpp1ernentary Material to

Expresslan In Vivo of a Muranr Human lnltlator tRNA gene I" M a m a l l a n Cells Uslnq a SV40 Vector "

Harold J. Drabkln and Utrarn L . RaIBhandary

Haferlals and Methods

E n l y m e I : All restrlctlon enzymes w e r e purchased from elther Betheida Re- search Laboratories or New Enqland B101abs. T1-RNase and T2-RNase were pur- chased from Calbiochem. Pancreatic RNase was obtalned from warthlnqton. A

crvde rat liver aminoacyl-tRNA synthetase preparatlan was a kind g1ft of M .

Deutscher. E.coli amlnaacyl-tRNA synthetase was prepared by T.Y. Hal of thls laboratory, and a yeast mlnoacyl- tRNA synthetase preparatlon was obtalned from L. Gehrke.

-: The SVIO-pBR322 hybrid vector pSVlGT3. contalnlng a Single Bad1 nlte and almost no SV40 late region. was a klnd q1ft of Dr. Richard Hulllqan. Olgesrron of thls vector vlth PStI releases the pBR322 Sequences. Th15 vec- tor provldes the early functions necessary for SVIO replication. The SV40

hybrid vector pSY1-rINS7. contalnlng d rabblt insulin gene fragrnent inserted lntO the SV40 early reqlon, was used as a helper virus IHorowltz. et al.,

Thrs vector provides the late functions necessary for SV40 replicdtlon. 19831. Dlgestion of thls vector wlth EcoRI releases the pBR322 sequences.

lnitlator tRNA genes have been descrlbed ~n the preceding paper IDrabkln and RaiBhandary, 1985al.

The N13mp7 clones contalninq the wlld type 11\54) and mutant IT541 hwan

The l4lbp BamHI fragment containing the human lnltiator tRNA gene was

cloned lnto the slngle B a r n 1 Slte of the pSVlGT3. Recombinants (pSVA54 or pSW541 obtalned from transformation of HBlOl were rdentlfied by analysis Of plasmld DNA obtalned from minl-plasnld preparations IBlrnboim and Doly, 1980) Using restrlctlon enzyme dlqestlon and Southern hybrldizarion (Southern, 19751

DNA Transfection Into Mamallan Cells: Prior to use ~n CV-1 ce l l transfec- t lon. the PSVA54 and pSVT54 vectors, and the helper vector were diqested wlth PStI and E C O R I , respectively. and rellqated under conditions favoring CITCY-

larllatlan of the fragments Over religatlon. as deacrlbed ~n lanki, et al. (19821 (see flgYre 1 . ) .

CY-1 cells grown ~n Dulbecco's Modlfled Eagles wlth 2% calf serum were transfected with a mnlxture of vectors contalnlng either the mutant or wild type l n i t l a t o r tRNA qene and the helper by either the calcium phosphate method ILaskl. et al., 1982) or DEAE-dextran method Isornpayrac and Danna. 1981).

medla and cells by scraplnq, and treatmg the mixtures wlth three freeze- Vlrus stocks were made Of cultures showing cytopathic effects by harvesting

thawing cycles lwlth vortexlnql. Transfection of the CV-1 cells. as well as lnfectlons wlth recomhlnant Virus stocks 1SVR54 or S V T 5 0 were performed under P-3 contamment.

AnalySLS of RNA and DNA: RNA and DNA were isolated from l o r n dishes of SV-1 cells Infected vlth 0.5 to 1.Oml Of vlrus stocks by the extraction method Of Hlrt 119671.

The Hlrt supernatant was extracted w t h an equal vel-e of water-satura- ed phenol, followed by extractlon wlth chloroform The total nucleic acids were recovered by ethanol precipitation. In some cases, addrtlonai ethanol precipltatlons from 2.51 NH Acetate were carried out.

Unlabeled RNA from 15cm dishes to be used for amlnoacyldrlon a s s a y s vas

further purified by absorption Onto 0.5ml DEAE-cellulose beds. washed w l C h

lOml of O . 3 M NaC1, l O m H TrlI HC1 pH 7.4, ImM EDTR, and e l u t e d wlth 1.0ml of 1.OM NaCl l n the same buffer. The RNA w a s recovered by ethanol p r r c l p r -

t a t i o n . deacylated as descrlbed In Yang and Novellr 119111. reprrclpltarcd and dissolved in lorn NaCl. lorn Tris HC1. pH 7.4, ImM EDTR. One 15cm dlsh yields about 100-200 g of DEAE-~ellulose purlfied materlai.

Labeled RNA was prepared fror .Ocm dishes incubated for 5 hours vlth 500

YU Of 32P04 (New England Nuclear) between 4 8 to 56 hours after Infection, as descrlbed I" Laskl. et al. 119821.

Labeled RNA was analyzed on 7M urea-158 l1:20, bls to monoacrylanlde)

wild type and mutant tRNAB were vlsualxzed by autoradlography, exclsed, and polyacrylamide gels l90m Trls borate pH 8 . 3 , 4 m EDTA). The overproduced

purlfied 6 5 described (Drabkin and Ra~Bhandary, 1 9 8 5 a i .

Analysis of 32P-labeled t R N A by fingerprint analysls was performed e s -

senrlally as descrlbed by Sllberklanq, et al. 119191. Due t~ 1tS dlfferent mabllity. the mutant t R N A w a s found to be lmpure as exclsed dlrectly from

and mutant rRNAs were further purlfled by one round of hybrld-selectlo" on the onedlmensional gel. Therefore. for flnqerprlnt analysls. both wlld type

nrtrocellulose filters carrylng M13rnp7 vlrlon DNA wlth the codlng Strand of the human lnltrator tRNA qene ITollevervey. et al., 1983; DaSSarrna. et a l . .

19841.

allqonvcleotldes was done a5 descrlbed I" the prevlous paper IDrabkln and RalBhandary, 1985ai.

Hodlfled base compasltlon analysls of either lntact t R N A S Or indlvldual

Analysls of Amlnoacylated tRNAs: Total DEI\E-cellulose purlfied RNA was

arnlnoacylated wlth erther [35Sl-rnethlonlne (New England Nuclear] using

elfher the rat 1lver synthetase as described by Yang and Novel11 119711,

wlLh 1'Rl-leucine (hersham) using the yeast synthetase preparation, as de- or the E.call enzyme as described by Stanley (1974). RNA was amlnoacylated

scclbed ~n Beiklrsch, et a l . 11972). The amlnoacylated tRNA was prepared f o r RPC-5 chromatography by phenol-extraction and ethanol preclpltatron IDrabkln and Ra~Bhandary. 1985bi.

~ m l e 1 Sequence of oliganucleotldes present I" flnqerprlnts of TI -RNase

dlqeste l f l g . 4 a and b).

Spot ldentlty C O m e " t *

1 G

2 CG 3 AG 4 CAG 5 CAAG 6 CUG UCG found ~n ~n vitro transcripts 1s absent

slnce the G preceding UCG is modlfled to m7G (see spot 13 below].

-~

7 UmlG 8 Um2G

10 AUCG 9 RUG

11 CCCAU~~AACCCAG 12 ~'AAACCAUCCUCUG

present ~n A54 only

1 3 rn7Ghn5CG not present ~ _ _ ln vitro; m7G resistant to T1-RNase 5"end of mature tRNA 1 4 pAGp

1 5 TLCG

1 6 Um1Gm2G 17 CUACCAOH 3"end of mature rRNA

present 1n T 5 4 mutant only

Table 2 Sequence of oligonucleotides present I" flnqerprints of pancreatic RNase digests IFlq. 4 c and dl

Spot Identity Comments

1 u 2 AC 3 G c 4 AU 5 GU

6 AGC 7 Gm'AAAC 8 ~'G~'Gc 9 t6-C

10 GAU

I l a AGAGm7GD llb pAGCp 12 GGGC 13 GGAU present in 1154 only 14 AGRGU 15 GGAAGC 16 GGT present I" T54 mYtant only

5"end of mature tRNA