©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure-Function Relationship of Yeast S-II in Terms of Stimulation of RNA Polymerase II, Arrest Relief, and Suppression of 6-Azauracil Sensitivity (*)

Toshiyuki Nakanishi , Makoto Shimoaraiso , Takeo Kubo , Shunji Natori (§)

From the (1) Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.


INTRODUCTION

Transcription factor S-II, originally isolated from Ehrlich ascites tumor cells as a specific stimulator of RNA polymerase II, has been studied extensively (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . Results have shown that S-II is one of the transcription elongation factors that promote read-through by RNA polymerase II of pausing sites within genes in vitro (16, 17) . S-II has been proposed to release pausing by inducing cleavage of the 3`-end of the nascent transcript in the ternary elongation complex (Refs. 18-24; for review, see Ref. 25).

Recently, we purified S-II from yeast ( Saccharomyces cerevisiae) and determined its complete amino acid sequence by isolating its gene (26) . Significant sequence similarity was found between yeast S-II and other S-IIs so far sequenced (10, 27, 28) . Yeast S-II specifically stimulated yeast RNA polymerase II (26) and was shown to promote cleavage and elongation of nascent RNA in the elongation complex of transcription in vitro (29, 30) . Therefore, like the S-IIs of higher eukaryotes, yeast S-II is a transcription elongation factor.

The nucleotide sequence of the yeast S-II gene indicated that it is the same protein as STP and the product of the PPR2/DST1 gene. The PPR2/DST1 gene was originally identified as the gene responsible for 6-azauracil sensitivity of yeast (31) . STP catalyzes the transfer of a strand from a duplex linear molecule of DNA to a complementary circular single strand (32, 33) , indicating that this protein has pleiotropic functions. On the other hand, gene disruption experiments revealed that the S-II null mutant is viable (26, 31, 33) but that the mutation induces sensitivity to 6-azauracil (31) . It is not known why the S-II null mutant is sensitive to 6-azauracil. However, 6-azauracil sensitivity is the only prominent phenotype of the S-II null mutant, and the function of S-II as a transcription factor may be indispensable for suppressing 6-azauracil sensitivity. To gain more insight into the structure-function relationship of S-II, we created various deletion mutants of S-II and examined their abilities to stimulate RNA polymerase II, relieve arrest, and suppress the 6-azauracil sensitivity of the S-II null mutant. Results indicated that all of these activities are due to the same motif in the S-II molecule, suggesting that 6-azauracil sensitivity is caused by loss of function of S-II as a transcription factor.


EXPERIMENTAL PROCEDURES

S-II Null Mutant TNY14

The S-II null mutant was established previously from ANY102 ( Mata/Mat, leu2/leu2, his/his, trp1/trp1, ura3/ura3) by inserting the LEU2 gene into the S-II gene (26) . Therefore, the null mutant required uracil and could not be used for assay of suppression of 6-azauracil sensitivity. So, we established a new S-II null mutant by inserting the URA3 gene. Gene disruption method used was essentially as reported before (26) . Briefly, pBSM13 carrying a 4.0-kilobase fragment of YSII::LEU2 was digested with XbaI and ClaI to remove the LEU2 gene, and the URA3 gene was ligated instead of the LEU2 gene. The insert was purified and used for transformation of TNY04 obtained by tetrad dissection of ANY102 (34) . One of the URAtransformants thus obtained was named TNY14. Disruption of the S-II gene in TNY14 was confirmed by Southern blot analysis as described before (26) .

Yeast Transformation

Yeast cells were transformed essentially as described by Gietz et al. (34) . They were grown in 5 ml of YPD medium (1% polypeptone, 1% yeast extract, 2% glucose) at 30 °C to an optical density at 600 nm ( A) of 2.0 and then collected and suspended in 50 µl of TELiAc (10 m M Tris/HCl buffer, pH 7.5, 1 m M EDTA, 100 m M CHCOOLi). This suspension was incubated with 100 ng of plasmids, 50 µg of heat-denatured calf thymus DNA, and 300 µl of TELiAc containing 40% polyethylene glycol 4000 for 30 min at 30 °C and then for 20 min at 42 °C. The cells were collected, suspended in sterilized water, and spread on YNBD plates (0.67% yeast nitrogen base without amino acids, 2% glucose), supplemented with 20 µg/ml each of adenine sulfate, tryptophan, histidine, and 30 µg/ml of leucine, and incubated for 3 days at 30 °C.

Plasmids containing S-II deletion mutants were constructed as follows. S-II deletion mutants were amplified by polymerase chain reaction using the S-II gene (pYSII-2) as a template, and the resulting DNA fragments were ligated to the GAL1 promoter in pYO324. Mutant S-II proteins were induced by galactose.

Assay of Suppression of 6-Azauracil Sensitivity

Transformed cells were cultured in EMD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acids technical, 2% glucose) with an appropriate supplement(s) at 30 °C until Areached about 2.0. Then 2.5 10cells were transferred to 0.5 ml of fresh medium and incubated at 30 °C for 2 h. The cell suspension was diluted 1000-fold with sterilized water, and 120 µl of the diluted cell suspension was spread on YNBD or YNBGS (0.67% yeast nitrogen base without amino acids, 5% galactose, and 0.2% sucrose) plates containing an appropriate supplement(s) with or without 100 µg/ml 6-azauracil. Colonies on YNBGS plates were examined after incubation at 30 °C for 5 days.

Assay of Stimulation of RNA Polymerase II by S-II Deletion Mutants

This assay was done essentially as described before in the presence and absence of each recombinant S-II deletion mutant protein (26) . The reaction mixture (60 µl) contained 50 m M Tris/HCl (pH 7.9), 1.6 m M MnCl, 0.5 m M each of ATP, GTP, and CTP, 0.01 m M UTP, 18.5 kBq of [H]UTP, 10 m M 2-mercaptoethanol, 2 µg of calf thymus DNA, and 8 units of partially purified S. cerevisiae RNA polymerase II. The reaction mixture was incubated for 20 min at 30 °C, and then the radioactivity incorporated into the acid-insoluble fraction was counted. 1 unit of RNA polymerase II was defined as the amount incorporating 1 pmol of UMP into the acid-insoluble fraction.

Assay of Arrest-relief Activity of S-II Deletion Mutants

This assay was done essentially as described by Christie et al. (29) , using a 3`-deoxycytidine-extended template (35) of the TaqI fragment containing the human histone H3.3 gene (36) , RNA polymerase II, and S-II mutant proteins. The dC-tailed template used was kindly provided by Dr. C. M. Kane (University of California, Berkeley Dept. of Molecular and Cell Biology).

Recombinant S-II Deletion Mutant Proteins

Recombinant S-II mutant proteins were expressed in Escherichia coli using the T7 expression system, as described before (37) . Plasmids containing S-II deletion mutants were constructed as follows. S-II deletion mutants were amplified by polymerase chain reaction using pYSII-2 as a template, and the resulting DNA fragments were ligated into an expression vector, pET-3d. The resulting plasmids were transfected into E. coli BL21(DE3)/pLysE, and induction of S-II mutant proteins was performed by treating the E. coli cells with isopropyl-1-thio-- D-galactopyranoside.

Recombinant S-II mutant proteins were purified as follows. Freshly harvested E. coli cells were lysed by treatment with lysozyme-EDTA and deoxycholate, and the lysate was centrifuged at 100,000 g for 1 h. The resulting supernatant was diluted with buffer 1 (50 m M Tris/HCl, pH 7.9, 5 m M 2-mercaptoethanol, 0.1% Triton X-100) and then subjected to fast protein liquid chromatography on a Mono-S HR 5/5 column equilibrated with buffer 1. Recombinant S-II deletion mutant proteins were eluted with buffer 1 containing 0.05-0.2 M NaCl. The fraction of 3-151 protein from the Mono-S column was further purified on a column of Superose 12 with buffer 1 containing 1 M NaCl. Recombinant S-II mutant proteins were detected by immunoblotting with antibody against yeast S-II.

Other Methods

S-II and partially purified RNA polymerase II from S. cerevisiae were prepared as described before (26) . Antibody against S-II was raised by injecting purified S-II into male albino rabbits. On immunoblotting, this antibody detected S-II in the crude extract of yeast as a single band. DNA manipulations including restriction enzyme digestion, gel electrophoresis, DNA ligation, plasmid isolation, and E. coli transformation were carried out by standard methods. SDS-polyacrylamide gel electrophoresis and immunoblot analysis were performed as described before (38, 39) . Protein was determined by the method of Bradford (40) .


RESULTS

Suppression of 6-Azauracil Sensitivity of the S-II Null Mutant by S-II Deletion Mutant Proteins in Vivo

Previous studies showed that S-II, the PPR2/DST1 gene product, and STP are almost certainly the same protein (26, 31, 33) . We are interested in the functional motifs in the S-II molecule. To examine whether the region needed for stimulation of RNA polymerase II and that needed for 6-azauracil sensitivity in the S-II molecule could be separated, we constructed plasmids carrying the genes for various S-II deletion mutant proteins and used these plasmids for in vivo and in vitro experiments. We examined the ability of S-II deletion mutant proteins to suppress 6-azauracil sensitivity by introducing these plasmids into the yeast S-II null mutant and expressing the mutant proteins in vivo. We also expressed these plasmids in E. coli, isolated mutant S-II proteins, and examined their ability to stimulate yeast RNA polymerase II in vitro.

The S-II null mutants that we established previously required uracil (26) , so they could not be used for testing suppression of 6-azauracil sensitivity. Therefore, we constructed a new S-II null mutant. Southern blot analysis of genomic DNA derived from TNY14 showed that its S-II gene was disrupted. We further confirmed that S-II is not present in TNY14 by an immunofluorescence study with affinity-purified anti-S-II antibody. Immunofluorescence was exclusively detected in the nuclei of wild type yeast, whereas no immunofluorescence was detected in TNY14 (data not shown). Thus, we concluded that S-II is a nuclear protein and that TNY14 lacks this protein.

TNY14 was sensitive to 6-azauracil and did not form colonies on MVD agar plates containing 100 µg/ml of 6-azauracil (Fig. 1, A and B). When pYSG6 (an expression vector containing the GAL1 promoter-yeast S-II gene coding region) was introduced into TNY14, the resulting transformant could form colonies on MVGS agar plates in the presence of 6-azauracil (Fig. 1, C and D), indicating that introduction of full-length S-II cDNA suppressed the 6-azauracil sensitivity of TNY14. However, it did not form colonies on MVD plates as expected. Using this system, we examined the suppressions of 6-azauracil sensitivity by various S-II deletion mutants.


Figure 1: 6-Azauracil sensitivity of TNY14 and its suppression by S-II. To detect suppression of the 6-azauracil sensitivity of TNY14, pYSG6 carrying the full-length S-II gene was introduced, and its expression was induced by galactose in the presence of 6-azauracil. A and B, YNBD agar plates; C and D, YNBGS agar plates containing galactose; A and C contained no drug; B and D contained 100 µg/ml 6-azauracil.



The amino acid sequence of S-II is known to be conserved in various eukaryotes. In particular, about 50 residues in the carboxyl-terminal region are highly conserved (about 70% similarity), and about 80 residues in the amino-terminal region are also relatively well conserved (about 38% similarity) (10, 13, 15, 26, 27, 28) . Christie et al. (29) demonstrated that in yeast S-II, lacking the first 113 residues has the same activity as the full-length form to read-through the pausing site.

On the bases of these results, we constructed seven clones of S-II deletion mutants. Of these, five were deletion mutants of the amino-terminal side, and two were those of the carboxyl-terminal side. We found that the amino-terminal mutants 2-123 (in which residues 2-123 were deleted), 2-141, and 3-147 suppressed the 6-azauracil sensitivity of TNY14 but that 2-149 and 2-151 did not. These results indicated that residues 1-147, including the relatively well conserved sequence in the amino-terminal region, are not essential for suppression of 6-azauracil sensitivity. The carboxyl-terminal mutant, 260-309, did not suppress 6-azauracil sensitivity, indicating that the 49 carboxyl-terminal residues are necessary for suppressing 6-azauracil sensitivity. We confirmed the expression of each deletion mutant protein in the corresponding transformant by immunoblot analysis, as shown in Fig. 2.

Stimulation of RNA Polymerase II and Relief of Transcription Arrest by S-II Deletion Mutant Proteins in Vitro

We then expressed these S-II deletion mutants in E. coli, purified each protein to near homogeneity, and examined its ability to stimulate RNA polymerase II and relieve arrest in vitro. As shown in Fig. 3 A, each protein gave essentially a single band on SDS-polyacrylamide gel electrophoresis. Immunoblotting again showed that all the mutant proteins reacted with antibody against S-II (Fig. 3 B). As is evident from Fig. 4, wild type S-II and the 2-123 protein had almost the same stimulatory activity, but the 2-141 protein had less, and the 2-151 protein had none. Therefore, it is clear that the first 141 residues are not essential for stimulation of RNA polymerase II. As the 266-309 protein had no stimulatory activity, the last 44 residues in the carboxyl-terminal region are essential for stimulation of RNA polymerase II.


Figure 3: Purification of various S-II deletion mutant proteins and their immunoblotting. Recombinant S-II deletion mutants were expressed in E. coli, and each recombinant protein was purified. A, SDS-polyacrylamide gel electrophoresis of recombinant S-II deletion mutant proteins stained with Coomassie Brilliant Blue (47). B, Immunoblotting of recombinant S-II deletion mutant proteins by affinity-purified anti-S-II antibody. Lane 1, intact S-II; lane 2, 2-123 protein; lane 3, 2-141 protein; lane 4, 3-151 protein; lane 5, 266-309 protein.



We also examined arrest-relief activity of these mutant proteins. As shown in Fig. 5, wild type S-II, 2-123, and 2-141 proteins promoted read-through by RNA polymerase II at specific blocks to elongation in the human histone H3.3 gene, sites designated TIa, TIb, and TII (36, 41) . However, 3-151 and 266-309 proteins had no appreciable activity to relieve transcription arrest at these sites. Summary of two independent arrest-relief experiments is shown in Table I. These results strongly suggested that the ability to stimulate RNA polymerase II and to relieve arrest at specific pausing sites are the same activity, and that this activity is essential for suppression of 6-azauracil sensitivity of S-II null mutants, as summarized in Fig. 6.


DISCUSSION

Using various S-II deletion mutants of yeast, we demonstrated that at least 1-147 residues from the amino-terminal are not essential, and the last 162 residues are sufficient for suppression of the 6-azauracil sensitivity of an S-II null mutant. We also showed that for activity to stimulate RNA polymerase II and relieve arrest, there is a critical point in S-II between residues 142 and 151, and that 168 but not 158 of the carboxyl-terminal residues were necessary for stimulation of RNA polymerase II and relief of transcription arrest in vitro. Therefore, this region should contain binding regions for both RNA polymerase II and nucleic acid (6, 42, 43) . For technical reasons, we could not purify the 2-147 and 2-149 proteins in an E. coli lysate and thus could not examine their abilities to stimulate RNA polymerase II and relieve arrest. Nonetheless, it is quite clear that there is a good correlation between the activity for suppression of 6-azauracil sensitivity in vivo and that for stimulation of RNA polymerase II (and thus for arrest relief) in vitro, and that nearly half of the 309 residues of S-II are not directly related to these activities. Christie et al. (29) also reported that there is essentially no difference in the cleavage or read-through activities between complete yeast S-II and a mutant S-II lacking the first 113 residues. Probably, the functional motif of S-II is in the carboxyl-terminal half, and the amino-terminal half is the regulatory motif, since this region of S-II of Ehrlich cell has been shown to contain phosphorylation sites (7) . However, read-through mechanism of S-II may not be so simple because Cipres-Palacin and Kane (44) recently reported S-II mutants that are inactive for promoting read-through, although they stimulated cleavage of the nascent transcript in stalled elongation complexes (44) .

Unlike mutants lacking regions of the amino-terminal half, those lacking about 50 residues from the carboxyl-terminal were shown to have lost all the stimulatory, arrest-relief, and suppressive activities. Again, we could not examine these activities with a single mutant for technical reasons, but we deduced that the 260-309 protein had no stimulatory and arrest-relief activity from the fact that the 6-residue-longer 266-309 protein had no activity.

These genetical and biochemical results strongly suggest that 6-azauracil sensitivity is due to loss of function of S-II as a transcription factor. There seem to be two possible reasons why the S-II null mutant has acquired the phenotype of 6-azauracil sensitivity. One is that S-II is essential for the transcription of another gene that causes 6-azauracil sensitivity, and the S-II null mutant lacks this gene product. This gene product may not be essential for the growth of yeast, since the S-II null mutant is not lethal (26, 31, 33) . The other possibility is that many pausing sites are introduced into the yeast genome in the presence of 6-azauracil, and the S-II null mutant becomes lethal in the presence of 6-azauracil, as these pausing sites cannot be read-through by RNA polymerase II in the absence of S-II. With respect to the latter possibility, Exinger and Lacroute (45) reported that 6-azauracil inhibits GTP synthesis in S. cerevisiae and also causes significant decrease in the UTP content. In vitro transcription experiments showed that reduction of at least one of four nucleoside triphosphates in the reaction mixture induced pause of transcription elongation and that addition of S-II released this cessation of transcription elongation (18, 20, 21, 29) . Therefore, it is possible that many pausing sites are created in various class II genes and that their transcriptions are inhibited in the presence of 6-azauracil, interfering with the growth of the S-II null mutant (46) . If the former possibility is the case, there should be a specific gene(s) responsible for 6-azauracil sensitivity that is transcribed only in the presence of S-II. Identification and isolation of this gene(s) may give a clue to the function of S-II in vivo. If the latter possibility is correct, this S-II null mutant should become lethal when the nucleoside triphospate pool in the cells is reduced by other methods.

It is unlikely that S-II itself has activity to detoxify 6-azauracil or to disturb the uptake of 6-azauracil, thus making the S-II null mutant sensitive to 6-azauracil, because, in general, metabolic enzymes are present in the cytoplasm and barrier proteins are present in the membrane, whereas S-II was found almost exclusively in the nuclei.

  
Table: Summary of activities of S-II mutant proteins to promote read-through of RNA polymerase II

Data were obtained from the quantitation of the read-through experiments described in the legend to Fig. 5. Percentage radioactivity of run-off transcript (RO) or RNA stalled at TIa/total radioactivity (RO + TIa) was determined by counting the radioactivity in each band in Fig. 5. Each value represents the average of two experiments with S.D.



FOOTNOTES

*
This work was supported by a grant-in-aid for specifically promoted research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-5684-2973; Fax: 81-3-3813-5099.


ACKNOWLEDGEMENTS

We thank Dr. Caroline M. Kane for supplying the template and protocol for arrest-relief assay.


REFERENCES
  1. Sekimizu, K., Kobayashi, N., Mizuno, D., and Natori, S. (1976) Biochemistry 15, 5064-5070 [Medline] [Order article via Infotrieve]
  2. Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588 [Medline] [Order article via Infotrieve]
  3. Sekimizu, K., Yokoi, H., and Natori, S. (1982) J. Biol. Chem. 257, 2719-2721 [Abstract/Free Full Text]
  4. Ueno, K., Sekimizu, K., Mizuno, D., and Natori, S. (1979) Nature 277, 145-146 [Medline] [Order article via Infotrieve]
  5. Natori, S. (1982) Mol. Cell. Biochem. 46, 173-187 [Medline] [Order article via Infotrieve]
  6. Horikoshi, M., Sekimizu, K., and Natori, S. (1984) J. Biol. Chem. 259, 608-611 [Abstract/Free Full Text]
  7. Horikoshi, M., Sekimizu, K., Hirashima, S., Mitsuhashi, Y., and Natori, S. (1985) J. Biol. Chem. 260, 5739-5744 [Abstract]
  8. Rappaport, J., Reinberg, D., Zandomein, R., and Weinmann, R. (1987) J. Biol. Chem. 262, 5227-5232 [Abstract/Free Full Text]
  9. Reinberg, D., and Roeder, R. G. (1987) J. Biol. Chem. 262, 3331-3337 [Abstract/Free Full Text]
  10. Hirashima, S., Hirai, H., Nakanishi, Y., and Natori, S. (1988) J Biol. Chem. 263, 3858-3863 [Abstract/Free Full Text]
  11. Sluder, A. E., Greenleaf, A. L., and Price, D. H. (1989) J. Biol. Chem. 264, 8963-8969 [Abstract/Free Full Text]
  12. Bengal, E., Flores, O., Krauskopf, A., Reinberg, D., and Aloni, Y. (1991) Mol. Cell. Biol. 11, 1195-1206 [Medline] [Order article via Infotrieve]
  13. Kanai, A., Kuzuhara, T., Sekimizu, K., and Natori, S. (1991) J. Biochem. 109, 674-677 [Abstract]
  14. Wiest, D. K., Wang, D., and Hawley, D. K. (1992) J. Biol. Chem. 267, 7733-7744 [Abstract/Free Full Text]
  15. Xu, Q., Nakanishi, T., Sekimizu, K., and Natori, S. (1994) J. Biol. Chem. 269, 3100-3103 [Abstract/Free Full Text]
  16. Reines, D., Chamberlin, M. J., and Kane, C. M. (1989) J. Biol. Chem. 264, 10799-10809 [Abstract/Free Full Text]
  17. SivaRaman, L., Reines, D., and Kane, C. M. (1990) J. Biol. Chem. 265, 14554-14560 [Abstract/Free Full Text]
  18. Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6, 1342-1356
  19. Izban, M. G., and Luse, D. S. (1993) J. Biol. Chem. 268, 12864-12873 [Abstract/Free Full Text]
  20. Reines, D. (1992) J. Biol. Chem. 267, 3795-3800 [Abstract/Free Full Text]
  21. Reines, D., Ghanouni, P., Li, Q.-q, and Mote, J., Jr. (1992) J. Biol. Chem. 267, 15516-15522 [Abstract/Free Full Text]
  22. Reines, D., and Mote, J., Jr. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1917-1921 [Abstract]
  23. Gu, W., Powell, W., Mote, J., Jr., and Reines, D. (1993) J. Biol. Chem 268, 25604-25616 [Abstract/Free Full Text]
  24. Guo, H., and Price, D. H. (1993) J. Biol. Chem. 268, 18762-18770 [Abstract/Free Full Text]
  25. Kassavatis, G. A., and Geiduschek, E. P. (1993) Science 259, 944-945 [Medline] [Order article via Infotrieve]
  26. Nakanishi, T., Nakano, A., Nomura, K., Sekimizu, K., and Natori, S. (1992) J. Biol. Chem. 267, 13200-13204 [Abstract/Free Full Text]
  27. Marshall, T. K., Guo, H., and Price, D. H. (1990) Nucleic Acids Res. 18, 6293-6298 [Abstract]
  28. Yoo, O.-J., Yoon, H.-S., Beak, K.-H., Jeon, C.-J., Miyamoto, K., Ueno, A., and Agarwal, K. (1991) Nucleic Acids Res. 19, 1073-1079 [Abstract]
  29. Christie, K. R., Awrey, D. E., Edwards, A. M., and Kane, C. M. (1994) J. Biol. Chem. 269, 936-943 [Abstract/Free Full Text]
  30. Johnson, T. L., and Chamberlin, M. J. (1994) Cell 77, 217-224 [Medline] [Order article via Infotrieve]
  31. Hubert, J.-C., Guyonvarch, A., Kammerer, B., Exinger, F., Liljelund, P., and Lacroute, F. (1983) EMBO J. 2, 2071-2073 [Medline] [Order article via Infotrieve]
  32. Sugino, A., Nitiss, J., and Pesnick, M. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3683-3687 [Abstract]
  33. Clark, A. B., Dykstra, C. C., and Sugino, A. (1991) Mol. Cell. Biol. 11, 2576-2582 [Medline] [Order article via Infotrieve]
  34. Gietz, D., Jean, A. S., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425 [Medline] [Order article via Infotrieve]
  35. Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295 [Abstract/Free Full Text]
  36. Reines, D., Wells, D., Chamberlin, M. J., and Kane, C. M. (1987) Mol. Cell. Biol. 196, 299-312
  37. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline] [Order article via Infotrieve]
  38. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  39. Homma, K., Kurata, S., and Natori, S. (1994) J. Biol. Chem. 269, 15258-15264 [Abstract/Free Full Text]
  40. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  41. Kerpolla, T. K., and Kane, C. M. (1990) Biochemistry 29, 269-278 [Medline] [Order article via Infotrieve]
  42. Agarwal, K., Beak, K.-H., Jeon, C.-J., Miyamoto, K., Ueno, A., and Yoon, H.-S. (1991) Biochemistry 30, 7842-7851 [Medline] [Order article via Infotrieve]
  43. Quian, X., Joen, C.-J., Yoon, H.-S., Agarwal, K., and Weiss, M. A. (1993) Nature 365, 277-279 [CrossRef][Medline] [Order article via Infotrieve]
  44. Cipres-Palacin, G., and Kane, C. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8087-8091 [Abstract]
  45. Exinger, F., and Lacroute, F. (1992) Curr. Genet. 22, 9-11 [Medline] [Order article via Infotrieve]
  46. Archambault, J., Lacroute, F., Ruet, A., and Friesen, J. D. (1992) Mol. Cell. Biol. 12, 4142-4152 [Abstract]
  47. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617 [Medline] [Order article via Infotrieve]

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