A widely distributed family of small regulators, called C proteins, controls a subset of restriction-modification systems. The C proteins studied to date activate transcription of their own genes and that of downstream endonuclease genes; this arrangement appears to delay endonuclease expression relative to that of the protective methyltransferase when the genes enter a new cell. C proteins bind to conserved sequences called C boxes. In the PvuII system, the C boxes have been reported to extend from ؊23 to ؉3 relative to the transcription start for the gene for the C protein, an unexpected starting position relative to a bound activator. This study suggests that transcript initiation within the C boxes represents initial, C-independent transcription of pvuIICR. The major C protein-dependent transcript appears to be a leaderless mRNA starting farther downstream, at the initiation codon for the pvuIIC gene. This conclusion is based on nuclease S1 transcript mapping and the effects of a series of nested deletions in the promoter region. Furthermore, replacing the region upstream of the pvuIIC initiation codon with a library of random oligonucleotides, followed by selection for C-dependent transcription, yielded clones having sequences that resemble ؊10 promoter hexamers. The ؊35 hexamer of this promoter would lie within the C boxes. However, the spacing between C boxes/؊35 and the apparent ؊10 hexamer can be varied by ؎4 bp with little effect. This suggests that, like some other activator-dependent promoters, PpvuIICR may not require a ؊35 hexamer. Features of this transcription activation system suggest explanations for its broad host range.The genera hosting restriction-modification (RM) systems appear to comprise the majority of the prokaryotic world. RM systems play a variety of roles, ranging from the simply selfish (44, 54) through defense against bacteriophages (42, 51) to facilitating recombination (34, 36). While much remains to be learned about the functions of RM systems, our understanding of how they are regulated is even more limited. Most, if not all, RM system genes can move from cell to cell, either by residing on a plasmid or because of transduction, transformation, or Hfr-type conjugation. As a result, they all face a critical regulatory problem-how to ensure that a new host's DNA is protectively methylated before endonuclease activity appears. This problem is particularly acute for the type II RM systems, in which the methyltransferase (MTase) and endonuclease (REase) function independently.A subset of type II RM systems includes, in addition to genes for the MTase and REase, a third gene for a conserved regulator called the C (controller) protein. C proteins were originally discovered in the PvuII (58, 59) and BamHI (22, 57) RM systems and were then noted in several other systems as well

The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow. However, relatively little is known about the distribution and movement of restriction-modification systems themselves. We have isolated and characterized the genes for restriction-modification systems from two species of Salmonella, S. enterica serovar Paratyphi A and S. enterica serovar Bareilly. Both systems are closely related to the PvuII restriction-modification system and share its target specificity. In the case of S. enterica serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region. Unlike the chromosomally located Salmonella systems, the PvuII system is plasmid borne. We have completed the sequence characterization of the PvuII plasmid pPvu1, originally from Proteus vulgaris, making this the first completely sequenced plasmid from the genus Proteus. Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in Proteus and Salmonella are completely different. The SptAI and SbaI genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the PvuII genes are adjacent to a mob operon and a XerCD recombination (cer) site.Restriction-modification (RM) systems are nearly ubiquitous among bacteria (both eubacteria and archaea). To date, the only complete bacterial genome sequences lacking candidate RM systems are from the obligate intracellular parasites Chlamydia and Rickettsia. Chlamydia trachomatis is the only known cellular organism that lacks an S-adenosyl-L-methionine (AdoMet) synthetase (63), and a type II RM system with separately active endonuclease and AdoMet-dependent methyltransferase proteins could be dangerous in this context. The other bacterial genomes have between 1 and 22 predicted RM systems (74,75). Even bacteria with the smallest genomes, Mycoplasma and Ureaplasma, have made room for these systems. RM systems provide a defense against DNA bacteriophages, as revealed both by direct experiment and indirectly by the fact that many bacteriophages take specific countermeasures against RM systems (9). In addition to initiating the destruction of some foreign DNAs, RM systems may also promote recombination (6, 51) and improve the spread of some genes by separating them from linked deleterious alleles (4, 42), thus playing both positive and negative roles in modulating the flow of genes among prokaryotes. In the case of type II RM systems, selfish behavior also contributes to their ubiquity (24,45,46). RM system ubiquity is consistent with the selectable phenotypes just summarized, but these phenotypes only help to explain why the systems are maintained once they have entered a given bacterium. It is less clear how these systems move throughout the microbial biosphere. To this end, it would be useful to track the movement of a given RM system by finding very clos...

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Increase in the intracellular inositol triphosphate (IP 3 ) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced a⁄nity to [125 I]Ang II (K d = 0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241^244, 250^251 and 255^256) with that of the AT1 in the mutant M8, not only increased the a⁄nity of the AT2 receptor to [125 I]Ang II (K d = 0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP 3 levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241^256 is su⁄-cient for the AT2-mediated inhibition of AT1-stimulated IP 3 generation. Moreover, these nine mutations are also su⁄cient to render the AT2 with the ability to activate phospholipase C. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

A stop codon at position 322 was introduced to generate a truncated, C-terminal-deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C-terminal-deleted AT2 had reduced a⁄nity to [ 125 I]angiotensin II (K d = 1.7 nM) and enhanced binding of the AT2-speci¢c peptidic ligand [ 125 I]CGP42112A (K d = 0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C-terminal deletion, implying that the C-terminus may have a negative e¡ect on the AT2-mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP-binding domain of the human ErbB3 receptor in yeast two-hybrid assay was abolished by C-terminal deletion. In summary, the C-terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties. ß