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The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb Kpn I fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli , the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS−, and the chloramphenicol resistance (Cm r ) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus , rendering it sensitive to the presence of sucrose.

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.