The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C ⅐ PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C ⅐ PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5 ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C ⅐ PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C ⅐ PvuII binding, overlapping the ؊10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.The bacterial type II restriction-modification systems include a DNA modification methyltransferase (MTase) and a restriction endonuclease (REase), both of which act independently on the same DNA sequence (65). The REase cleaves duplex DNA sequences in the absence of sequence-specific DNA modification by the MTase. These systems can defend bacterial cells against viral infection, although other functional roles have also been proposed (45). Restriction-modification systems have provided an important focus for studies of molecular recognition. Biochemical and crystallographic analyses are yielding significant insights into the mechanisms of sequence recognition and catalytic activity of these proteins (3,18,50,66).

The effect of enrichment bias on the diversity of 2,4-dichlorophenoxyacetate (2,4-D)-degrading (2,4-D ؉) bacteria recovered from soil was evaluated by comparing the diversity of isolates obtained by direct plating to the diversity of isolates obtained from 85 liquid batch cultures. By the two methods, a total of 159 isolates were purified from 1 g of soil and divided into populations based on repeated extragenic palindromic sequence PCR (rep-PCR) genomic fingerprints. Approximately 42% of the direct-plating isolates hybridized with the tfdA and tfdB genes from Alcaligenes eutrophus JMP134(pJP4), 27% hybridized with the tfdA and tfdB genes from Burkholderia sp. strain RASC, and 30% hybridized with none of the probes. In contrast, the enrichment isolates not only represented fewer populations than the isolates obtained by direct plating but also exhibited, almost exclusively, a single hybridization pattern with 2,4-D catabolic gene probes. Approximately 98% of the enrichment isolates possessed pJP4-type tfdA and tfdB genes, whereas isolates containing RASC-type tfdA and tfdB genes were obtained from only 2 of the 85 enrichment cultures. The skewed occurrence of the pJP4-type genes among the isolates obtained by enrichment suggests that the competitive fitness of 2,4-D ؉ populations during growth with 2,4-D may be influenced either by specific tfd alleles or by genetic factors linked to these alleles. Moreover, the results indicate that evaluation of the diversity and distribution of catabolic pathways in nature can be highly distorted by the use of enrichment culture techniques.

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Hormones and growth factors are generally released from larger precursors by limited proteolysis. The causative agents remain poorly defined with respect to location and properties. One subset of proteases, the glandular kallikreins, have been implicated in a few cases, in part because of their specific association with mature forms of some hormones. However, limited distribution and low copy number in some species cast doubt on this hypothesis, and they may well play other physiological functions that remain to be elucidated.

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SmaI endonuclease recognizes and cleaves the sequence CCC decreases GGG. The enzyme requires magnesium for catalysis; however, equilibrium binding assays revealed that the enzyme binds specifically to DNA in the absence of magnesium. A specific association constant of 0.9 x 10(8) M-1 was determined for SmaI binding to a 22-base duplex oligonucleotide. Furthermore, the KA was a function of the length of the DNA substrate and the enzyme exhibited an affinity of 1.2 x 10(9) M-1 for a 195-base pair fragment and which represented a 10(4)-fold increase in affinity over binding to nonspecific sequences. A Km of 17.5 nM was estimated from kinetic assays based on cleavage of the 22-base oligonucleotide and is not significantly different from the KD estimated from the thermodynamic analyses. Footprinting (dimethyl sulfate and missing nucleoside) analyses revealed that SmaI interacts with each of the base pairs within the recognition sequence. Ethylation interference assays suggested that the protein contacts three adjacent phosphates on each strand of the recognition sequence. Significantly, a predicted protein contact with the phosphate 3' of the scissile bond may have implications in the mechanism of catalysis by SmaI.

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