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The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest

The retinoblastoma tumor suppressor protein (RB) binds several cellular proteins involved in cell cycle progression. Using the yeast two-hybrid system, we found that RB bound specifically to the protein BRG1. BRG1 shares extensive sequence similarity to Drosophila brahma, an activator of homeotic gene expression, and the yeast transcriptional activator SNF2/SW12. BRG1 contains an RB-binding motif found in viral oncoproteins and bound to the A/B pocket and the hypophosphorylated form of RB. BRG1 did not bind RB in viral oncoprotein-transformed cells. Coimmunoprecipitation experiments suggested BRG1 associates with the RB family in vivo. In the human carcinoma cell line SW13, BRG1 exhibited tumor suppressor activity by inducing formation of flat, growth-arrested cells. This activity depended on the ability of BRG1 to cooperate and complex with RB, as both an RB-nonbinding mutant of BRG1 and the sequestration of RB by adenovirus E1A protein abolished flat cell formation.

Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells.

This paper describes the successful construction and propagation of a transducing animal virus. A segment of DNA approximately 2 kilobases (kb) in length was removed from the late region of the SV40 genome by sequential cleavages with Hpa II and Bam HI endonucleases (at 0.735 and 0.13, respectively, on the SV40 DNA map). A segment of about 1.5 kb of lambda phage containing ORI (the origin of lambda DNA replication), the two structural genes CII and cro, and four transcriptional promoters, was inserted into the late region of SV40 by the poly(dA:dT) joining procedure. The resulting hybrid DNAs were cloned and propagated as virions in CV-1 monkey kidney cells by mixed infections at 41 degrees C with tsA58, an early mutant of SV40. The location, size, and orientation of the inserted lambda DNA segment was verified by restriction endonuclease digestions and by heteroduplex analysis. Clones with each of the two possible orientations of the lambda DNA segment were isolated. CV-1 cells infected with lambda-SV40 hybrid virus contain little or no lambda-specific RNA or proteins, even though the hybrid virus replicates nearly as well as the helper virus.

Construction, propagation and expression of simian virus 40 recombinant genomes containing the Escherichia coli gene for thymidine kinase and a Saccharomyces cerevisae gene for tyrosine transfer RNA☆

Abstract Recombinant simian virus 40 (SV40) virus genomes have been constructed in vitro by joining SVGT1, the segment of SV40 DNA between map co-ordinates 0.15 and 0.73 (clockwise), to either the Escherichia coli gene for thymidine kinase ( Ecotdk ), or one of the Saccharomyces cerevisae genes for tRNA Tyr ( ScetyrG ); the resulting recombinants were propagated in CV-1 monkey cells at 41 °C using ts A58 as a helper. The sEcotdk gene was obtained first as an approximately 20 kb ‡ DNA segment in a defective transducing phage, φ 80 dtdk 5, then as overlapping 1.85 and 2.35 kb segments in pMB9- Ecotdk , prior to insertion into SVGT1. The ScetyrG segment introduced into SVGT1 was a 1.25 kb DNA fragment contained in λgtl- ScetyrG , a recombinant that had been cloned previously by Olson et al. (1979). After infection of CV-1 monkey cells with the SVGT1- Ecotdk or SVGT1- ScetyrG hybrid viruses, RNA complementary to the exogenous DNA was produced. With SVGT1- Ecotdk the RNA homologous to the Ecotdk segment was heterogeneous, ranging in size from 1 to > 8 kb in length. At least 30 to 40% of this RNA was covalently joined to SV40-specific RNA. No E. coli thymidine kinase enzyme activity could be detected in the infected cells. By contrast, infection with SVTG 1- ScetyrG resulted in the formation of a transfer RNA-sized RNA, complementary to the tRNA Tyr coding sequence of the ScetyrG segment, as well as a population of heterogenous large RNAs. Since the formation of the tRNA-sized RNA occurred after infection with recombinants having the ScetyrG segments in either of the two alternative orientations in SVGT1, it is possible that transcription of the tyrG sequence is initiated within the cloned segment.