The Saccharomyces cerevi"ia SWII, SWI2 (SNF2), SWI3, SNF5, and SNF6 gene products play a crucial role in the regulation of transcription. We provide here direct biochemical evidence that all five SWI/SNF polypeptides are components of a large multisubunit complex. These five polypeptides coelute from a gel-filtration column with an apparent molecular mass of -2 MDa. The five SWI/SNF polypeptides do not copurify when extracts are prepared from swi-or snt mutants. We show that SWI/SNF polypeptides also remain associated during an affinity-chromatography step followed by gel ffltration. Assembly of the SWI/SNF complex is not disrupted by a mutation in the putative ATP-binding site of SWI2, although this mutation eliminates SWI2 function. mM NaCl/0.1% Tween 20/10% (vol/vol) glycerol/pepstatin A at 2 jg/ml/leupeptin at 2 jg/ml/l mM phenylmethylsulfonyl fluoride/0.097 trypsin inhibitor units per ml of aprotinin] with 5 pulses, each 50 seconds in duration on a BioSpec Products (Bartlesville, OK) beadbeater, and clarified by centrifugation at 100,000 x g for 1 hr. An aliquot (0.2 ml; 2-3 mg) was loaded onto a fast protein liquid chromatography Superose 6 gel-filtration column (0.2 ml/min, equilibrated in extraction buffer), and 0.5 ml fractions were collected. Antibodies andImmunoblotting. An epitope-tagged version of SWI2 was created by cloning an oligonucleotide cassette that encodes the hemagglutinin (HA) epitope (6) into the unique Xho I site at the C terminus of SWI2. Likewise, an oligonucleotide cassette that encodes six tandem histidines was cloned adjacent and C-terminal to the sequences encoding the HA epitope. The SWI2-HA and SWI2-HA-6HIS fusion proteins fully complemented a swi2A for defects in growth and transcription of an HO-lacZ fusion gene (C.L.P., data not shown). The SWI2-HA-6HIS fusion gene was integrated at the URA3 locus in strain CY120 (swi2A::HIS3 HO-lacZ) to generate strain CY396 (swi2A: :HIS3 SWI2-HA-6HIS:: URA3 HO-lacZ). Polyclonal antibodies directed against SNF5 and SNF6 proteins were generated by fusing portions of each coding region to glutathione S-transferase and injecting these fusion proteins into rats. A 1750-bp Mse I-Kpn I fragment from SNF5 (7) and a 733-bp Nco I-Pst I fragment from SNF6 (8) were subcloned into the vector pGEX-2TK (International Biotechnologies). For immunoblots, Superose 6 fractions were trichloroacetic acidprecipitated and resuspended in 50 pI of SDS sample buffer; 10-15 pi were then separated on either a 6% or 10%o Laemmli gel. Immunoblots were probed with either rabbit polyclonal a-SWI3 (2), rabbit polyclonal a-SWI1, rat polyclonal a-SNF5, rat polyclonal a-SNF6, or monoclonal antibody 12CA5 (Babco, Emeryville, CA), and developed with a chemiluminescent substrate as described (2).Affinity Purification. Extract (5 ml; 75 mg) from a 1-liter culture of strain CY396 was mixed with 0.5 ml of Sepharose CL-4B (Pharmacia), rocked for 5 min, and centrifuged at 1000 x g for 5 min to remove the resin; the unbound protein was bound batchwise with 0.5 ml of Ni2+-mtrilo...

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.

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