©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural and Functional Analysis of Yeast Putative Adaptors
EVIDENCE FOR AN ADAPTOR COMPLEX IN VIVO(*)

(Received for publication, September 12, 1995; and in revised form, December 15, 1995)

Reyes Candau Shelley L. Berger (§)

From the Wistar Institute, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identified regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for interaction with ADA3. Deletions of the region that was required for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex.


INTRODUCTION

Transcriptional activation of RNA polymerase II (RNAPII) (^1)genes requires the coordinated function of several classes of proteins (Tjian and Maniatis, 1994). Transcriptional activators, or activator complexes, bind to specific DNA sequences generally upstream of the core promoter. A second class of more general factors together constitute the basal transcription apparatus, which includes RNAPII and other factors required for initiation and elongation (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH; for review, see Buratowski(1994) and Zawel and Reinberg (1993)). Activators induce high levels of transcription by stimulating the activity of the basal complex, either increasing the rate of initiation or elongation by RNAPII.

Functional and physical interactions between activators and basal factors require a third class of cofactors, which are not well characterized, but may physically bridge or functionally modify interactions between activators and basal factors (Gill and Tjian, 1992; Lewin, 1990; Ptashne and Gann, 1990; Roeder, 1991). In general these cofactors are required for optimal responses to activators, but their particular mechanisms may be quite varied. These cofactors include coactivators (Gill and Tjian, 1992), adaptors (Berger et al., 1990; Berger et al., 1992), mediators/SRBs (suppressors of RNA polymerase B) (Koleske and Young, 1995), and SWI/SNFs switch/sucrose non-fermenting) (Carlson and Laurent, 1994; Winston and Carlson, 1992). Interestingly, most of these proteins are highly conserved in eukaryotes, since putative homologues have been found from yeast to humans (for example, see Candau et al. (1996), Kwon et al.(1994), and Reese et al. (1994)), suggesting that their roles are fundamentally important in transcriptional activation.

One commonality among these factors is that they may all constitute large multicomponent complexes, perhaps to provide a high degree of flexibility in transcriptional regulation. Thus, the TFIID complex consists of the TATA-binding protein (TBP), associated with accessory factors (TBP-associated factors and coactivators; Dynlacht et al.(1991)), which are necessary for activated transcription (Chen et al., 1994). Mediators (Kim et al., 1994) and SRBs (Koleske and Young, 1994; Koleske et al., 1992) are associated with the carboxyl-terminal tail of the largest subunit of RNAPII and may form a RNAPII holoenzyme complex, which is recruited en masse to promoters during activation. The SWI/SNF complex (Peterson et al., 1994) may be required for activators to overcome chromatin-mediated repression, perhaps by associations with activators (Cote et al., 1994; Kwon et al., 1994) and TBP (Imbalzano et al., 1994).

Finally, putative transcriptional adaptors are required for full function of certain acidic activators. In previous work we identified these cofactors by their biochemical (Berger et al., 1990) and genetic interaction (Berger et al., 1992) with the activation domain of herpes simplex virus VP16. Several genes were cloned, termed ADA2 (Berger et al., 1992), ADA3 (Piña et al., 1993), and GCN5 (Marcus et al., 1994), and mutations in them affected transcription in a manner suggestive of adaptor function. For example, mutations in ADA2 affected transcriptional activation in vivo and in vitro by certain acidic activators including VP16 and yeast GCN4, but did not affect activation by the acidic activator HAP4 (Berger et al., 1992). This functional specificity was reflected in physical interactions between ADA2 and activation domains derived from VP16 (Silverman et al., 1994) and GCN4, but not HAP4 (Barlev et al., 1995). In yeast nuclear extracts, ADA2 both interacted with the TBP component of TFIID and was required for VP16 activation domain interaction with TBP (Barlev et al., 1995). These data suggested that adaptors may be required for physical interaction between activation domains and the basal transcription apparatus.

ADA2, ADA3, and GCN5 interacted in vitro (Horiuchi et al., 1995; Marcus et al., 1994), and activation in vivo by ADA2 fused to the bacterial lexA DNA binding domain (lexA-ADA2) was dependent on GCN5 and ADA3 (Marcus et al., 1994; Silverman et al., 1994). Here we report a primary structure and functional study of the adaptors. We mapped regions of GCN5 necessary for interaction with ADA2. We also defined regions of ADA2 required for interaction with GCN5 and ADA3 in vitro and then analyzed the dependence of these ADA2 mutants on ADA3 for in vivo function. We found parallels in the behavior of these mutants in vitro and in vivo. Moreover, we detected physical interaction between ADA2, ADA3, and GCN5 in yeast extracts. These data indicate that the adaptors function as a complex in vivo.


MATERIALS AND METHODS

Yeast Strains

The double adaptor deletion strain, PSY-17A Deltaada2 Deltaada3 (adeDeltahis4-519 leu2-3, 112 ura3-52), was generated from a cross between PSY316 Deltaada2 (MATalpha ade2-101 Deltahis3-200 leu2-3, 112 lys2 ura3-52; Berger et al.(1992)) and BWG1-7A Deltaada3 (MATa ura3-52 leu2-3, 112 his4-519 ade1-100; Piña et al.(1993)). The resulting diploid was sporulated, and tetrads were dissected using standard techniques (Sherman and Hicks, 1991). Spores that grew poorly on minimal media were tested for deletion of both ADA2 and ADA3 by transformation with ADA2 and ADA3 expression plasmids.

The Deltaada2 derivative of BJ2168 (mata ura3-52 leu2 trp1 prb1-1122 pep4-3 prc1-407) was generated by transformation with pyADA2.KO as described (Berger et al., 1992).

Plasmids and Deletion Mutants

Plasmids were constructed using standard procedures (Ausubel et al., 1994). The deletion mutants of ADA2 or lexA-ADA2 were made as described (Kunkel et al., 1987) using pRS316-ADA2 (Berger et al., 1992) or pRS316-lexA-ADA2 as parental plasmids and the following oligonucleotides for site-directed mutagenesis.

pRS316-lexA-ADA2 was constructed by PCR generation of ADA2 with NotI restriction sites and ligation into pRS316-lexA (Piña et al., 1993) digested with NotI.

GAL4-VP16 expression vectors were described (Berger et al., 1992). The ADA3 expression vector was described (Piña et al., 1993).

For in vitro translation, PCR fragments of ADA3 bearing BamHI restriction sites or ADA2 and GCN5 bearing BglII restriction sites were cloned into SP64 (Pharmacia Biotech Inc.). The three genes were cloned into a yeast expression vector and were functional as assayed for complementation of ada2 or ada3 disruption strains.

SP64-NotI was generated cloning a NotI linker in SP64 digested with SmaI. To generate the ADA2 deletion mutants for in vitro translation, the lexA-ADA2 deletion mutants were digested with NotI and cloned into SP64-NotI.

To generate the deletion mutants of GCN5 for in vitro translation, fragments between residues 1-440, 1-350, 1-250, 1-170, 95-440, 170-440, and 250-440 of GCN5 bearing a NotI restriction site at the 5` end and an EagI restriction site at the 3` end were amplified by PCR. These fragments were digested with EagI and inserted in SP64-NotI opened with NotI.

To generate the two halves of ADA3 for in vitro translation, fragments of ADA3 between residues 1-364 with BamHI restriction sites and 363-702 with a BglII restriction site at the 5` end and a BamHI restriction site at the 3` end were amplified by PCR. These fragments were digested with BamHI or BamHI/BglII and inserted in SP64 digested with BamHI.

pRS426-GAL1-ADA2 and pRS425-GAL1-GCN5 were constructed by PCR generation of ADA2 or GCN5 bearing BglII restriction sites and ligation into the BamHI site of pRS426-GAL1 or pRS425-GAL1 (Mumberg et al., 1994) pRS424-GAL1-HA-ADA3 was constructed in three steps. First, a linker with the sequence encoding the 12CA5 epitope derived from the influenza hemagglutinin protein (Kolodziej and Young, 1991) was inserted into the NotI site of SP64-NotI to generate SP64-HA (only the NotI site downstream of the linker was restored after the ligation); second, ADA3 was PCRed with a NotI restriction site at the 5` end and an EagI restriction site at the 3` end was cloned into the NotI site of SP64-HA to generate SP64-HA-ADA3; finally, SP64-HA-ADA3 was digested with EcoRI and PstI, and HA-ADA3 was cloned into pRS424-GAL1 digested with EcoRI and PstI.

Growth Complementation, Growth Inhibition, and beta-Galactosidase Assays

PSY316 Deltaada2 was transformed as described (Ito et al., 1983) with the ADA2 deletion mutants and plated in fully supplemented SD medium. After 3 days, single colonies were streaked on SD minimal medium and their ability to complement growth was tested.

Growth inhibition by GAL4-VP16 was carried out in BWG1-7A Deltaada2 cotransformed with the ADA2 deletion mutants and GAL4-VP16 high expression plasmid and plated on SD minimal medium.

beta-Galactosidase assays (Rose et al., 1988) were carried out in PSY-17A Deltaada2 Deltaada3 or in PSY316 Deltaada2 transformed with the plasmids described for each experiment. beta-Galactosidase activity was determined as units/mg of protein.

The lacZ reporters used were pLGSD5 (Guarente et al., 1982) for GAL4-VP16 activation, and YEp21 (Brent and Ptashne, 1985) for lexA-ADA2 activation.

Western Blots

Yeast extract prepared as described for the beta-galactosidase assays were analyzed in 8% or 10% SDS-PAGE and immunoprobed with alpha-lexA antisera (Zervos et al., 1993) using standard techniques (Harlow and Lane, 1988). In vivo coimmunoprecipitation experiments were immunoblotted using alpha-ADA2 antisera (Silverman et al., 1994), alpha-GCN5 antisera (Candau et al., 1996), or alpha-HA monoclonal antibody (Babco). Immunodetection was performed using an ECL kit (Amersham Corp.).

In Vitro Translation and Coimmunoprecipitation Assays

In vitro translation experiments were performed as described (TNT kit, Pharmacia). A 20-µl slurry of Gammabind Sepharose beads (Pharmacia) was washed two times with and resuspended in 20 µl of buffer A (10 mM HEPES, pH 7.6, 300 mM potassium acetate, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µM pepstatin A, 0.6 µM leupeptin, 2 µg/ml chymostatin, 2 mM benzamidine). 10 µl of reticulocyte extract containing cotranslated proteins and 2 µl of alpha-ADA2 antisera were added to the beads, mixed, and rotated for 3 h at 4 °C. The reactions were centrifuged for 2 min at 4000 rpm at 4 °C, and the supernatant was removed and washed three times with 1 ml of buffer A. Following the last wash, the beads were resuspended in 50 µl of Laemmli buffer and loaded onto SDS-PAGE as indicated in each case. Input was 50% of the material used in the immunoprecipitation reaction. The gels were dried and exposed overnight on Kodak film.

In Vivo Coimmunoprecipitation Assays

BJ2168 transformed with pRS426-GAL1-ADA2, pRS425-GAL1-GCN5, and pRS424-GAL1-HA-ADA3 or BJ2168 Deltaada2 transformed with pRS425-GAL1-GCN5 and pRS424-GAL1-HA-ADA3 as a negative control were selectively grown in fully supplemented lactate medium. When the cultures reached an optical density = 0.5, galactose was added to final concentration of 2% and incubation was continued 6 h (wild type strain) or overnight (ada2 deletion strain).

50 µl of whole cell extract prepared as described (Berger et al., 1992), except without reducing agents, were incubated with 0.2 µl (to precipitate ADA2 and coprecipitate GCN5) or 2 µl (to coprecipitate HA-ADA3) of alpha-ADA2 antisera and a 40-µl slurry of Gammabind Sepharose beads as described for the in vitro immunoprecipitation. Because ADA2 and GCN5 electrophorese similarly to IgG, the samples used to immunoblot with alpha-ADA2 antisera were electrophoresed in non-reducing conditions, and the samples used to immunoblot with alpha-GCN5 antisera were eluted with 1 M NaCl, 0.2% SDS. Input was 30% of the material used in the immunoprecipitation reaction. The immunoblot probed with alpha-GCN5 showing the elutions from the protein G precipitate was exposed for 20 min, and all other immunoblots were exposed for 3 min.


RESULTS

Generation of Deletion Mutants of ADA2 and Complementation Tests

To study the primary structure and function of the putative adaptors, we created deletion mutations within ADA2. Two series of deletion mutants in ADA2 (Fig. 1) were created by site-directed mutagenesis (Kunkel et al., 1987). In the first series, regions that previously had been noted to be of potential importance (Berger et al., 1992) were removed. Five regions were deleted individually: a cysteine-rich, potential zinc-binding domain that is similar to regions in the putative adaptors CBP and p300 (Delta1; Arany et al.(1994)); a region of sequence similarity with the myb oncogene (Delta2; Lane and Crawford(1979)); a region of similarity with PGM, a Drosophila melanogaster protein (Delta3; Stoffers et al.(1989)); a region of strong negative charge in the middle (Delta4); and a region rich in leucine at the carboxyl terminus (Delta5). The second series was a set of overlapping deletions from the amino to the carboxyl terminus, each removing 200 base pairs (Delta6 through Delta17). Twelve mutants were initially in this series, but two were not used in the functional assays (ADA2 and ADA2) because sequence analysis revealed errors in the junctions of the deletions and the resulting proteins were shorter than predicted (data not shown). However, as shown in Fig. 1, each portion of the gene was deleted in at least one mutant.


Figure 1: Schematic of ADA2 deletion derivatives. Full-length ADA2 comprises amino acids 1-434. Delta1, deletion of the cys-rich region, a putative zinc-binding domain; Delta2, deletion of a region of sequence similarity to the myb oncogene (Lane et al., 1990); Delta3, deletion of a region with similarity with the D. melanogaster PGM gene (Stoffers et al., 1989); Delta4, deletion of a region of strong negative charge; Delta5, deletion of a region rich in leucine residues. Delta6-Delta17 are a set of 200-base pair overlapping deletions.



Several assays were used to determine the effect of the deletions on the function of ADA2. We showed previously that genetic deletion of ADA2 results in defective growth on minimal media (Berger et al., 1992). Thus, as an overall estimate of function, the ability of the ADA2 deletion mutants to complement growth of an ada2 disruption strain was tested. Each deletion mutant was transformed, and each transformant was restreaked onto minimal media along with wild type ADA2 or vector alone, as positive and negative controls for growth ( Fig. 2and Table 1, column 1). Growth of the mutants divided into three groups. Some mutants grew similar to (ADA2and ADA2) or indistinguishable from (ADA2, ADA2, and ADA2) wild type. Five mutants complemented very poorly (ADA2, ADA2, ADA2, ADA2, and ADA2). The remaining mutants gave intermediate growth phenotypes (ADA2, ADA2, ADA2, ADA2, and ADA2). In summary, the carboxyl end of the protein was dispensable for growth. Two regions were crucial for complementation, the amino terminus (Delta6 and Delta7) and a region within the middle of the protein (Delta12, Delta13, and Delta14). Immunoblot analysis demonstrated that the amount of protein of each mutant was similar to wild type, except mutant ADA2, which was not detected (data not shown), even though the mutant protein was stable in vitro (see below). The instability of ADA2 may be significant with regard to the structure and function of the adaptor complex, as will be discussed below.


Figure 2: Growth complementation assay in the ada2 disruption strain. Each ADA2 deletion mutant was transformed into PSY316 Deltaada2, and each transformant was restreaked onto minimal medium. The dividing line shown in the figure separates two different experiments. Full-length ADA2 and vector alone were used as positive or negative controls in each experiment.





In cells containing wild type ADA2, overproduction of GAL4-VP16 results in a strong inhibition of growth and deletion of ADA2 relieves this toxicity (Berger et al., 1992). The growth inhibition by GAL4-VP16 was the basis of the genetic selection used to identify the adaptors ADA2 (Berger et al., 1992), ADA3 (Piña et al., 1993), and GCN5 (Marcus et al., 1994). This assay was used to assess the effect of the deletion mutations on ADA2 function (Table 1, column 2). The results were largely consistent with the growth complementation assay, since the mutations that allowed wild type colonial growth in the first assay (Delta1, Delta5, Delta15, Delta16, and Delta17) did not reduce the ability of ADA2 to complement growth inhibition by GAL4-VP16. Similarly, mutations that resulted in defective growth (Delta6, Delta7, Delta12, Delta13, and Delta14), were defective for growth inhibition by GAL4-VP16. The intermediate group for complementation (mutations Delta2, Delta3, Delta4, Delta9, and Delta10) was somewhat more negative in the growth inhibition assay, particularly Delta2, Delta3, and Delta4. Overall the results were similar in the assays and pointed to two regions crucial for ADA2 function, one at the amino terminus and the second in the middle of the protein.

A third measure of function of the ADA2 mutants is their ability to substitute for wild type ADA2 in transcriptional activation. For example, a total deletion of ADA2 results in a 7-10-fold reduction in activation by GAL4-VP16 (Berger et al., 1992). Each deletion mutant was cotransformed into the ada2 disruption strain with a GAL4-VP16 low copy expression plasmid and a beta-galactosidase reporter plasmid. The level of GAL4-VP16 activation dependent on each of the ADA2 deletion mutants was largely consistent with the results of growth complementation and growth inhibition assays (Table 1, column 3), since the amino terminus (Delta2, Delta3, Delta6, and Delta7) and a middle region (Delta12, Delta13 and Delta14) were required for activity and the carboxyl terminus (Delta15, Delta16, and Delta17) was dispensable. However, this assay seemed to be more stringent than the other two, since certain mutations were intermediate in the previous assays, but negative for GAL4-VP16 activation (Delta2, Delta3, Delta9, and Delta10).

In summary all of these assays suggest that regions of ADA2 required for function are within the amino terminus and the middle of the protein. The carboxyl terminus was dispensable in all assays.

Mapping of ADA2 Interaction with GCN5 in Vitro

ADA2 physically interacts with the putative adaptors GCN5 (Marcus et al., 1994) and ADA3 (Horiuchi et al., 1995) in vitro; however, the significance of these interactions in vivo is not known. We examined the relationship between functional interaction of the adaptors in vivo and physical interaction in vitro, reasoning that if specific mutations in ADA2 affect in vivo and in vitro interactions with other adaptors in a parallel fashion, then this would constitute strong evidence that the adaptors function in a complex in vivo.

To define sequence elements within ADA2 that are required for interaction with GCN5 in vitro, the overlapping ADA2 deletion mutants (Fig. 1) were tested for their ability to coimmunoprecipitate GCN5 (Fig. 3). As shown previously (Marcus et al., 1994), wild type ADA2 co-immunoprecipitated GCN5. Deletion of the amino terminus of ADA2 (Delta6 or Delta7) abolished interaction with GCN5, while deletions downstream of Delta7 did not affect interaction with GCN5. This was consistent with previous results showing that regions contained within the first 166 amino acids (i.e. corresponding to the regions deleted in ADA2 through ADA2) were sufficient to interact with GCN5, and the rest of the protein did not form a complex with GCN5 (Candau et al., 1996).


Figure 3: Effect of deletions in ADA2 on coimmunoprecipitation of GCN5. GCN5 was cotranslated with each ADA2 deletion mutant. Cotranslated proteins were immunoprecipitated with alpha-ADA2 antisera, and S-labeled proteins were visualized by autoradiography after 8% SDS-PAGE. in, input; ppt, precipitate. ADA2 proteins and GCN5 are indicated on the left. The migration of ADA2 varies because of the internal deletions.



To more finely map the region of ADA2 required for interaction with GCN5, the series of smaller deletion mutants, ADA2 through ADA2, was tested in the same assay (Fig. 3). Only Delta1, the Cys-rich region, and Delta3, the region showing similarity to PGM, eliminated interaction. This was consistent with the results of the coarser deletions, since Delta1 and Delta3 lie within the larger deletions Delta6 and Delta7, respectively. The other three small deletions (Delta2, Delta4, and Delta5) did not affect coprecipitation of GCN5. These results support growth complementation and other assays (Table 1) and suggest that mutants containing deletions in the amino terminus of ADA2 (ADA2, ADA2, and ADA2) fail to complement growth because, at least in part, they are unable to interact with GCN5. Mutant ADA2 did not fit this correlation, since coprecipitation was reduced but the mutant was similar to wild type in vivo, which is discussed below.

To map the region of GCN5 responsible for interaction with ADA2, a series of amino- and carboxyl-terminal deletions were made in GCN5 (Fig. 4A). The protein was divided into five regions, based on sequence conservation with the putative human homologue of GCN5 (hGCN5). Since hGCN5 interacts with both the human and yeast ADA2 homologues (Candau et al., 1996), regions of strong sequence similarity between yGCN5 and hGCN5 may be involved in the GCN5/ADA2 interaction. The region between residues 1 and 95 in the yeast protein has low similarity to the equivalent human region, the regions between 95 and 170 and between 250 and 350 have strong similarity, and the region between 170 and 250 has very strong similarity. Finally, the bromodomain, between 350 and 440, not only has strong similarity between yeast and human GCN5 (Candau et al., 1996), it is conserved with other transcription factors (Georgakopoulos and Thireos, 1992). These yeast GCN5 mutants were then tested for their ability to interact with ADA2, using the coimmunoprecipitation assay (Fig. 4B). Neither the amino terminus (amino acids 1-250) or, as previously observed, the carboxyl-terminal bromo domain (amino acids 350-440) of GCN5 were required for the interaction with ADA2. All mutants containing the region between amino acids 250 and 350 interacted with ADA2, while all mutants deleted in this region did not coimmunoprecipitate with ADA2. Thus, the region between 250 and 350 is crucial for this interaction. In summary, the central region of GCN5 (amino acids 250-350) is necessary for the interaction with two separate regions within the amino terminus of ADA2.


Figure 4: GCN5 deletion mutants and interaction with ADA2 in vitro. A, schematic of GCN5 deletion derivatives. Full-length GCN5 comprises amino acids 1-440. Deleted versions of GCN5 are composed of residues 1-350, 1-250, 1-170, 95-440, 170-440, and 250-440, which are each shown relative to full-length GCN5. The percentage of sequence similarity to human GCN5 (Candau et al., 1996) is indicated above the schematic, and ability to bind to ADA2 (from data in Fig. 4B) is indicated on the right. B, ADA2 immunoprecipitation of GCN5 deletion mutants. ADA2 was cotranslated with each GCN5 deletion mutant. Cotranslated proteins were immunoprecipitated with alpha-ADA2 antisera, and S-labeled proteins were visualized by autoradiography after 15% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate GCN5 peptides and ADA2.



Mapping of ADA2 Interaction with ADA3 in Vitro

Next, the region of ADA2 required for interaction with ADA3 was identified, using the in vitro coimmunoprecipitation assay with the ADA2 deletion mutants (Fig. 1). As previously observed (Horiuchi et al., 1995), ADA2 immunoprecipitated ADA3 (Fig. 5A). The amino and carboxyl termini of ADA2 were not required for interaction with ADA3, since mutants ADA2-ADA2 and ADA2-ADA2 immunoprecipitated ADA3. None of the small deletions (Delta1-Delta5) affected interaction. Only deletion of a central region (Delta12, Delta13, or Delta14) of ADA2 abrogated coprecipitation of ADA3. The region of interaction can be delimited to amino acids 201-301, since ADA3 interacted with mutant ADA2, which lacks the second half of the region Delta14 (Fig. 1A). This mapping is consistent with previous results showing that regions contained within ADA2 amino acids 167-434 were sufficient to interact with ADA3, while the ADA2 amino terminus did not form a complex with ADA3 (Candau et al., 1996). Taken together, these results corroborate growth complementation and other assays (Table 1), suggesting that the loss of growth complementation of mutants deleted in this region (ADA2, ADA2, or ADA2) was caused, at least in part, by a failure to bind ADA3.


Figure 5: Interaction between ADA2 and ADA3 in vitro. A, effect of deletions in ADA2 on coimmunoprecipitation of ADA3. ADA3 and each ADA2 deletion mutant were cotranslated and immunoprecipitated with alpha-ADA2 antisera. S-Labeled proteins were visualized by autoradiography after 8% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate ADA2 proteins and ADA3. B, ADA2 immunoprecipitation of ADA3 deletion mutants. ADA2 was cotranslated with each half of ADA3 and immunoprecipitated with alpha-ADA2 antisera. S-Labeled proteins were visualized by autoradiography after 12% SDS-PAGE. in, input; ppt, precipitate. Side arrows indicate ADA3 peptides and ADA2.



To map the region of ADA3 required for interaction with ADA2, the amino- and carboxyl-terminal halves of ADA3 were translated in vitro and tested for interaction with ADA2 (Fig. 5B). The carboxyl-terminal half of ADA3 was immunoprecipitated by ADA2, as previously observed (Horiuchi et al., 1995), while the amino-terminal half of ADA3 did not interact with ADA2. However, the amino terminus of ADA3 was immunoprecipitated by ADA2, in the presence of the carboxyl terminus of ADA3. This result indicates an intramolecular interaction between the two halves of ADA3 and provides a molecular explanation for the previous observation that the two halves of ADA3 complemented growth when cotransformed, but not when singly transformed (Horiuchi et al., 1995).

In summary, the carboxyl terminus of ADA3 interacts with amino acids 201-301 of ADA2.

Dependence of lexA-ADA2 Mutants on ADA3 in Vivo

ADA2 can activate transcription when fused to the bacterial lexA DNA binding domain (Silverman et al., 1994). (^2)The lexA-ADA2 activity was found to be dependent on ADA3 (Silverman et al., 1994), showing functional interaction. We tested whether deletions of regions of ADA2 that were required for growth complementation and interaction with ADA3 in vitro affected lexA-ADA2 dependence on ADA3. A correlation between mutants that failed to immunoprecipitate ADA3 in vitro and mutants that lost dependence on ADA3 in vivo would suggest physiological relevance of the complex.

Activity of lexA-ADA2 decreased in an ada3 disruption strain, and increased when ADA3 was overproduced (Silverman et al., 1994).^2 To understand the basis of the induction of lexA-ADA2 by ADA3, we examined transcriptional activity and the amount of lexA-ADA2 protein in the absence of ADA3, in the presence of endogenous levels of ADA3 and in the presence of overproduced ADA3. The lexA-ADA2 protein and activity were nearly undetectable in the ada3 disruption strain, were induced in ADA3 cells, and were induced further in the presence of high levels of ADA3 (Fig. 6A). In contrast with these effects on lexA-ADA2, lexA alone did not vary in activity or protein levels with alterations in the amount of ADA3. Thus, activity measurements correlated with the amounts of lexA-ADA2 protein, and ADA3 was necessary for stability of lexA-ADA2.


Figure 6: Analysis of the interaction between ADA2 and ADA3 in vivo. A, ADA3 induction of lexA-ADA2 transcriptional activity and protein. Either lexA-ADA2 (lanes 4-6) or lexA (lanes 1-3) was cotransformed with either a plasmid overexpressing ADA3 (lanes 3 and 6) or vector alone (lanes 1, 2, 4, and 5). The yeast strains were either Deltaada2 Deltaada3 (lanes 1, 3, 4, and 6) or Deltaada2 (lanes 2 and 5). In all cases the reporter plasmid contained one lexA binding site upstream of the bacterial lacZ gene. Equal amounts of whole cell extract were tested for beta-galactosidase activity (shown at the bottom) and immunoblotted with alpha-lexA antisera to determine the amount of lexA or lexA-ADA2 protein. B, ADA3 induction of the protein level of lexA-ADA2 deletion mutants. Immunoblot analysis using alpha-lexA antisera of whole cell extract from Deltaada2 Deltaada3 yeast transformed with lexA-ADA2 deletion mutants. Each lexA-ADA2 mutant was cotransformed with vector alone(-) or vector that overproduced ADA3 (+).



Since both activity and amount of lexA-ADA2 were dependent on the level of ADA3 protein, we tested the effect of ADA3 on the lexA-ADA2 deletion mutants. The activity of each lexA-ADA2 deletion mutant was measured in the absence of ADA3, in the presence of endogenous levels of ADA3, or in cells overproducing ADA3 (Table 2). The mutants subdivided into three groups, depending upon the extent of induction by ADA3. This was true whether the activity of lexA-ADA2 in the presence of overproduced ADA3 was compared to activity in the presence of endogenous levels of ADA3 or no ADA3 (Table 2), although the effect was more dramatic when overproduced ADA3 was compared to no ADA3. The induction by ADA3 of mutants lexA-ADA2 was indistinguishable from wild type. Mutants lexA-ADA2 were inducible to a lower extent than wild type. In contrast, mutants lexA-ADA2 were not inducible by ADA3 at all, suggesting that these deletions were in a region that interacted with ADA3.



We then measured the protein level of each lexA-ADA2 mutant fusion (lexA-ADA2). Equal amounts of protein from extracts used for activity determination were used for immunoblot analysis (Fig. 6B). The results paralleled the activity assay. Mutants that were strongly induced for activity showed strong induction of protein levels (lexA-ADA2) and were indistinguishable from wild type. Mutants that were moderately inducible for activity showed moderate induction of protein levels (lexA-ADA2). The three mutants that were not inducible for activity (lexA-ADA2) were also not inducible for the level of protein. Mutants lexA-ADA2 and lexA-ADA2 were stable even in the absence of ADA3, unlike wild type lexA-ADA2 or any of the other mutants, and did not respond to increased levels of ADA3. Mutant lexA-ADA2 was not stable in the presence of any amount of ADA3. The lack of protein for lexA-ADA2 was not caused by an intrinsic instability in the protein, because the mutant was able to be translated and was stable in vitro ( Fig. 3and 5A). Thus the effects of ADA3 on lexA-ADA2 activity and protein suggest an interaction within the Delta12, Delta13, and Delta14 regions of ADA2. Moreover, these three deletion mutants, which were independent of ADA3 in vivo, did not interact with ADA3 in vitro (Fig. 5A) and failed to complement growth ( Fig. 2and Table 1). The congruence of the in vivo assays with the in vitro assay suggests that these deletions define a functional interaction with ADA3. Most important, the consistent results strongly argue that the physical interaction observed in vitro is relevant in vivo.

In Vivo Physical Interaction between the Adaptors

ADA2, ADA3, and GCN5 associated in vitro and functionally interacted in vivo. To determine whether the proteins physically interact in vivo, ADA2 was immunoprecipitated from yeast whole cell extracts, and the presence of ADA3 and GCN5 was determined by immunoblot analysis (Fig. 7). Extracts were prepared from yeast overexpressing all three proteins, or from yeast overexpressing ADA3 and GCN5 but lacking ADA2. As expected, ADA2 immunoprecipitated from extract overexpressing ADA2 (Fig. 7, top panel), but was not detected in the extract lacking ADA2. Both ADA3 (Fig. 7, middle panel) and GCN5 (Fig. 7, lower panel) precipitated from extract containing ADA2, but not from extract lacking ADA2, indicating physical interaction with ADA2. The coimmunoprecipitation is further evidence that the proteins form a complex in vivo.


Figure 7: Immunoprecipitation analysis of the adaptor complex in vivo. ADA2 was immunoprecipitated using alpha-ADA2 antisera from whole cell yeast extracts prepared from either wild type (transformed with plasmids overexpressing ADA2, HA-ADA3, and GCN5) or Deltaada2 (transformed with plasmids overexpressing HA-ADA3 and GCN5) strains. Immunoblots were performed using alpha-ADA2 antisera (upper panel), alpha-HA monoclonal antisera (middle panel), to detect ADA3, or alpha-GCN5 antisera (lower panel). Inputs were 30% of amounts used in immunoprecipitation. Baculovirus-expressed ADA2 served as a size marker.




DISCUSSION

Transcriptional activation mediated by certain acidic activators in yeast appears to require a family of cofactors, which have been termed adaptors (Berger et al., 1992). The function of the putative adaptors may be to promote interaction between activation domains and the basal transcription apparatus (Barlev et al., 1995). Several lines of evidence suggest that the adaptors may function in a complex (Horiuchi et al., 1995; Marcus et al., 1994; Piña et al., 1993; Silverman et al., 1994). In this study we present direct evidence that ADA2 interacts with ADA3 and GCN5 in vivo, using coimmunoprecipitation from extracts prepared from yeast overexpressing the three proteins. We have mapped regions of the adaptors required for the formation of the complex in vitro and have shown that these regions are important for function in vivo.

In previous work we have identified human genes (hADA2 and hGCN5) whose products are similar in sequence and function to yeast ADA2 and yeast GCN5 (Candau et al., 1996). The yeast and human ADA2 proteins contain conserved putative domains, especially at their amino termini. Also, as was observed for yeast ADA2 and GCN5 (Marcus et al., 1994), human ADA2 and GCN5 interacted in a two-hybrid assay in vivo, and yeast ADA2 and human GCN5 interacted as well (Candau et al., 1996). These similarities stimulated a primary structure and functional analysis of yeast ADA2, to dissect the structural requirements for functional interactions between ADA2 and GCN5 or ADA3. An extensive deletion mutagenesis was done through ADA2, and selected regions of possible functional significance were separately deleted. Analysis of these deletions in vivo indicated that two regions were necessary for ADA2 function, including growth complementation of the ada2 disruption strain and GAL4-VP16 activity. These regions are the amino terminus (defined by Delta6 and Delta7) and the middle of the protein (defined by Delta12, Delta13, and Delta14).

We tested whether these regions were important for interaction with two other putative adaptors GCN5 and ADA3. Analysis of the interaction between ADA2/GCN5 and ADA2/ADA3 in vitro showed distinct regions were required for ADA2 interaction with each of these proteins.

The region of ADA2 that forms a complex with GCN5 was previously defined by immunoprecipitation and glutathione S-transferase-ADA2 pull-down assay (Candau et al., 1996). The region of ADA2 between 1 and 166 was sufficient for interaction with GCN5 in the coimmunoprecipitation assay, and between 1 and 180 in a glutathione S-transferase-ADA2 interaction assay with purified recombinant GCN5. The region between 167 and 434 did not interact with GCN5 in either assay.

In this study we have used deletions within the amino terminus of ADA2 to define important subregions for GCN5 interaction in vitro. Deletions that eliminated interaction with GCN5 in vitro (Delta1, Delta3, Delta6, and Delta7) were all in the amino terminus, and deletion of no other portion of ADA2 reduced the interaction with GCN5 and thus was consistent with previous data. The larger deletions (Delta6 and Delta7) overlap the smaller deletions (Delta1 and Delta3), and thus it is not possible to determine from these data whether there are additional small regions in the amino terminus aside from those within Delta1 and Delta3 that contribute to the interaction with GCN5. However, it is clear that regions within both Delta1 and Delta3 are required for in vitro interaction with GCN5, since deletion of either region causes the loss of GCN5 immunoprecipitation. ADA2 complemented growth in vivo, while ADA2 was defective, suggesting interaction of the PGM region (defined by Delta3) with GCN5 in vivo. There may be additional determinants of GCN5 interaction in the region carboxyl to the Cys-rich region, since ADA2, in which the Cys-rich region and additional residues are deleted, is the single most defective mutant in the in vivo assays. The region deleted in ADA2 may contain determinants for GCN5 interaction that are necessary in vitro but not in vivo. Alternatively, the interaction between GCN5 and the mutant ADA2 may be stabilized in vivo by other putative adaptors in the complex, such as ADA5 (Marcus et al., 1994), that are not present in the in vitro assay.

The regions of ADA2 required for interaction with GCN5 are strongly conserved between the human and yeast ADA2 proteins (Candau et al., 1996). This is interesting in light of the previous observation that yeast ADA2 interacts with human GCN5 (Candau et al., 1996), suggesting conservation of the interacting sequences. There is also conservation of sequence within the Cys-rich region of ADA2 and Cys-rich regions in mammalian CBP and p300 (Arany et al., 1994), two putative adaptors for the cAMP response element-binding protein activator (Lundblad et al., 1995). Since the Cys-rich region is implicated in our experiments as being involved in interaction with GCN5, it is possible that the Cys-rich regions in CBP and p300 have a similar role, in interacting with human GCN5 or with other possible homologues of yeast GCN5. Previously it was found that a 133-amino acid region of CBP, including the ADA2-like Cys-rich region, was sufficient for TFIIB interaction using recombinant proteins (Kwok et al., 1994). We have tested interactions between ADA2 and endogenous TFIIB or TBP in yeast nuclear extracts and have observed interaction with TBP, but not TFIIB (Barlev et al., 1995). Thus, it is possible that the Cys-rich regions in ADA2 and CBP have different functions.

Our deletion analysis of GCN5 suggests that the region necessary for its interaction with ADA2 localizes to the middle of the protein, between amino acids 250 and 350, in a region of GCN5 that shares a high degree of sequence similarity with its human counterpart (Candau et al., 1996). Most interesting is the finding of another region of GCN5 (residues 170-250), having exceedingly high conservation (80% similarity) between human and yeast, that is not necessary for the interaction of GCN5 with ADA2 in vitro. It is possible that this region is responsible for interaction of GCN5 with another transcription factor conserved between human and yeast. We are currently investigating this possibility. This factor is not likely to be ADA3, since GCN5 and ADA3 do not interact directly in vitro (Horiuchi et al., 1995).

We have studied the interaction between ADA2 and ADA3 using two assays: induction of lexA-ADA2 by ADA3 in vivo, and the ability of ADA2 to immunoprecipitate ADA3 in vitro. The results indicate that ADA3 interacts with ADA2 in the regions defined by Delta12 and Delta13 (residues 201-301). ADA2, ADA2, and ADA2 lost the ability to coimmunoprecipitate ADA3. The second half of the region defined by Delta14 appears not to contact ADA3, since this region is also deleted in ADA2, which immunoprecipitated ADA3. Our results in vivo are consistent with these results. It has been shown that lexA-ADA2 activity is dependent on ADA3, and we show here that lexA-ADA2 is unstable in the absence of ADA3. Mutant lexA-ADA2 has no activity and is unstable in the presence or absence of ADA3. Deletion of flanking regions (Delta12 or Delta14) results in lexA fusion proteins that are stable independent of ADA3 expression and whose activity is not inducible by ADA3. Mutant lexA-ADA2 and all other mutants are unstable in the absence of ADA3 and have protein that is induced in amount and activity proportionately to increasing ADA3. Thus only deletions Delta12, Delta13, and Delta14 result in lexA fusion proteins that are not inducible by ADA3 in vivo, and this is the only region required for in vitro interaction with ADA3. Thus, overall, the consistency in ADA2 mutant behavior between the in vitro immunoprecipitation and the in vivo induction assays suggests that the interaction observed in vitro between ADA2 and ADA3 also occurs in vivo. Also, we previously observed a correlation between in vitro and in vivo assays in interactions between ADA2 and GCN5. The amino-terminal 166 amino acids of ADA2, but not the remainder of ADA2, interacted with GCN5 in vitro, and lexA-ADA2(1-166) was stabilized by GCN5 in vivo, but GCN5 had no effect on the remainder of ADA2 (Candau et al., 1996).

One manifestation of ADA3 interaction with, and stabilization of, ADA2 is that ADA3 appears to induce the activity of lexA-ADA2. We have observed a similar stabilizing effect of ADA3 on endogenous ADA2. (^3)Thus the level of activity of lexA-ADA2 is proportionate to its concentration, which is directly related to the concentration of ADA3. This also suggests that the amount of lexA-ADA2 in the complex limits the overall activity. This may also be true in the actual activation complex, during interactions with activation domains.

Mutants lexA-ADA2 and lexA-ADA2 were constitutively stable in the presence or absence of ADA3; lexA-ADA2 was unstable in the presence or absence of ADA3; lexA-ADA2 and the remaining mutants were stable in the presence of ADA3 and unstable in its absence. Our interpretation of these data is that the regions flanking the interaction with ADA3 are particularly sensitive to proteolysis, and that these regions may be protected from degradation when ADA3 is present and interacting with the region defined by Delta13. This hypothesis explains the constitutive stability of lexA-ADA2 and lexA-ADA2, since each lacks one of the proteolysis-sensitive regions, and also explains the constitutive instability of lexA-ADA2, since it has both proteolysis-sensitive regions and cannot interact with ADA3.

Consistent with previous results (Horiuchi et al., 1995), we found that ADA2 interacted with the carboxyl terminus of ADA3, but not the amino terminus. However, when we simultaneously translated ADA2 and both halves of ADA3, ADA2 precipitated both the carboxyl terminus, as expected, and the amino terminus of ADA3. This result shows a physical interaction between the two halves of ADA3. Previous studies of the ability of the two halves of ADA3 to complement growth of a ada3 disruption strain showed that, although the two halves of ADA3 were not able to complement growth when transformed independently, they did complement when they were cotransformed (Horiuchi et al., 1995). Here we provide a molecular explanation for this observation, since the two halves of ADA3 apparently interacted with each other to reconstitute the full-length protein.

ADA2 was defective in all assays in vivo, but was able to interact with both ADA3 and GCN5 in vitro. We have previously shown that ADA2 interacts directly with GAL4-VP16 and indirectly with TBP (Barlev et al., 1995). This mutant is likely to interact with GAL4-VP16 because lexA-ADA2 is inactive (we would expect a level of activity similar to wild type in the lexA fusion of mutants unable to interact with activators). Thus, it is possible that ADA2 has lost the ability to interact with TBP.

In summary, we have demonstrated physical interaction between the adaptor proteins in vivo. Interactions among the three adaptors, ADA2, GCN5, and ADA3, were mapped to define sequence elements necessary for their binding. The region of ADA2 necessary for interaction with ADA3 in vitro was required for functional interaction in vivo. These data indicate physiological relevance of the adaptor complex.


FOOTNOTES

*
This work was supported by Grant MCB-9317243 from the National Science Foundation and a Junior Faculty Research Award from the American Cancer Society (to S. B.), Cancer Core Grant CA10815 from the National Institutes of Health, a grant from the Pew Charitable Trust (to Wistar Institute), and a grant from Ministerio de Educacion y Ciencia (Spain) (to R. C.). 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: Wistar Institute, 3601 Spruce St., Rm. 358, Philadelphia, PA 19104. Tel.: 215-898-3922; Fax: 215-898-0663; berger{at}wista.wistar.upenn.edu.

(^1)
The abbreviations used are: RNAPII, RNA polymerase II; TF, transcription factor; TBP, TATA-binding protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

(^2)
S. Berger, unpublished results.

(^3)
N. Barlev, unpublished results.


ACKNOWLEDGEMENTS

We thank Paula Darpino for technical assistance. We gratefully acknowledge the gift of the alpha-lexA antisera from R. Brent. We thank R. Marmorstein and G. Moore for comments on the manuscript.


REFERENCES

  1. Arany, Z., Sellers, W. R., Livingston, D. M., and Eckner, R. (1994) Cell 77, 799-800 [Medline] [Order article via Infotrieve]
  2. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K. (1994) Current Protocols in Molecular Biology , Chap. 3, John Wiley & Sons, Inc., New York
  3. Barlev, N., Candau, R., Wang, L., Darpino, P., Silverman, N., and Berger, S. (1995) J. Biol. Chem. 270, 19337-19344 [Abstract/Free Full Text]
  4. Berger, S. L., Cress, W. D., Cress, A., Triezenberg, S. J., and Guarente, L. (1990) Cell 61, 1199-1208 [Medline] [Order article via Infotrieve]
  5. Berger, S. L., Piña, B., Silverman, N., Marcus, G. A., Agapite, J., Regier, J. L., Triezenberg, S. J., and Guarente, L. (1992) Cell 70, 251-265 [Medline] [Order article via Infotrieve]
  6. Brent, R., and Ptashne, M. (1985) Cell 43, 729-736 [Medline] [Order article via Infotrieve]
  7. Buratowski, S. (1994) Cell 77, 1-3 [Medline] [Order article via Infotrieve]
  8. Candau, R., Moore, P., Wang, L., Barlev, N., Ying, C., Rosen, C., and Berger, S. (1996) Mol. Cell. Biol. 16, 593-602 [Abstract]
  9. Carlson, M., and Laurent, B. C. (1994) Curr. Opin. Cell. Biol. 6, 396-402 [Medline] [Order article via Infotrieve]
  10. Chen, J., Attardi, L., Verrijzer, C., Yokomori, K., and Tjian, R. (1994) Cell 79, 93-105 [Medline] [Order article via Infotrieve]
  11. Cote, J., Quinn, J., Workman, J. L., and Peterson, C. L. (1994) Science 265, 53-60 [Medline] [Order article via Infotrieve]
  12. Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 66, 563-576 [Medline] [Order article via Infotrieve]
  13. Georgakopoulos, T., and Thireos, G. (1992) EMBO J. 11, 4145-4152 [Abstract]
  14. Gill, G., and Tjian, R. (1992) Curr. Opin. Genet. Dev. 2, 236-242 [Medline] [Order article via Infotrieve]
  15. Guarente, L., Yocum, R. R., and Gifford, P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7410-7414 [Abstract]
  16. Harlow, E., and Lane, I. (1988) Antibodies: A Laboratory Manual , pp. 471-510, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. Horiuchi, J., Silverman, N., Marcus, G., and Guarente, L. (1995) Mol. Cell Biol. 15, 1203-1209 [Abstract]
  18. Imbalzano, A. N., Kwon, H., Green, M. R., and Kingston, R. E. (1994) Nature 370, 481-485 [CrossRef][Medline] [Order article via Infotrieve]
  19. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  20. Kim, Y. J., Björklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608 [Medline] [Order article via Infotrieve]
  21. Koleske, A., and Young, R. (1994) Nature 368, 466-469 [CrossRef][Medline] [Order article via Infotrieve]
  22. Koleske, A. J., and Young, R. A. (1995) Trends Biochem. Sci. 20, 113-116 [CrossRef][Medline] [Order article via Infotrieve]
  23. Koleske, A. J., Buratowski, S., Nonet, M., and Young, R. A. (1992) Cell 69, 883-894 [Medline] [Order article via Infotrieve]
  24. Kolodziej, P. A., and Young, R. A. (1991) Methods Enzymol. 194, 508-519 [Medline] [Order article via Infotrieve]
  25. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
  26. Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G., Green, M. R., and Goodman, R. H. (1994) Nature 370, 223-226 [CrossRef][Medline] [Order article via Infotrieve]
  27. Kwon, H., Imbalzano, A. N., Khavari, P. A., Kingston, R. E., and Green, M. R. (1994) Nature 370, 477-481 [CrossRef][Medline] [Order article via Infotrieve]
  28. Lane, D. P., and Crawford, L. V. (1979) Nature 278, 261-263 [Medline] [Order article via Infotrieve]
  29. Lane, T., Ibanez, C., Garcia, A., Graf, T., and Lipsick, J. (1990) Mol. Cell. Biol. 10, 2591-2598 [Medline] [Order article via Infotrieve]
  30. Lewin, B. (1990) Cell 61, 1161-1164 [Medline] [Order article via Infotrieve]
  31. Lundblad, J., Kwok, R., Laurance, M., Harter, M., and Goodman, R. (1995) Nature 374, 85-88 [CrossRef][Medline] [Order article via Infotrieve]
  32. Marcus, G., Silverman, N., Berger, S., Horiuchi, J., and Guarente, L. (1994) EMBO J. 13, 4807-4815 [Abstract]
  33. Mumberg, D., Muller, R., and Funk, M. (1994) Nucleic Acids Res. 22, 5767-5768 [Medline] [Order article via Infotrieve]
  34. Peterson, C. L., Dingwall, A., and Scott, M. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2905-2908 [Abstract]
  35. Piña, B., Berger, S., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989 [Abstract]
  36. Ptashne, M., and Gann, A. A. (1990) Nature 346, 329-331 [CrossRef][Medline] [Order article via Infotrieve]
  37. Reese, J. C., Apone, L., Walker, S. S., Griffin, L. A., and Green, M. R. (1994) Nature 371, 523-527 [CrossRef][Medline] [Order article via Infotrieve]
  38. Roeder, R. G. (1991) Trends Biochem. Sci. 16, 402-408 [CrossRef][Medline] [Order article via Infotrieve]
  39. Rose, M., Winston, F., and Hieter, P. (1988) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  40. Sherman, F., and Hicks, J. (1991) in Guide to Yeast Genetics and Molecular Biology (Guthrie, C., and Fink, G., eds) Vol. 194, pp. 21-37, Academic Press, Inc., San Diego
  41. Silverman, N., Agapite, J., and Guarente, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11665-11668 [Abstract/Free Full Text]
  42. Stoffers, D., Green, C., and Eipper, B. (1989) Proc. Natl. Acad. Sci. U. S. A. 1986, 735-739
  43. Tjian, R., and Maniatis, T. (1994) Cell 77, 5-8 [Medline] [Order article via Infotrieve]
  44. Winston, F., and Carlson, M. (1992) Trends Genet 8, 387-391 [Medline] [Order article via Infotrieve]
  45. Zawel, L., and Reinberg, D. (1993) Prog. Nucleic Acids Res. Mol. Biol. 44, 67-108 [Medline] [Order article via Infotrieve]
  46. Zervos, A., Gyuris, J., and Brent, R. (1993) Cell 72, 223-232 [Medline] [Order article via Infotrieve]

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