The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfiteinduced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by derming the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.Cytosine methylation has long been recognized as an important factor in the silencing of genes in mammalian cells. Recent studies have shown that cytosine methylation at single CpG dinucleotides within the recognition sites of a number of transcription factors is sufficient to block binding of the factors to DNA (1-6) and to inhibit transcription (3-5). Therefore, to determine the role of cytosine methylation in specific regulatory mechanisms in vivo, it has become important to know the methylation status of individual CpG dinucleotides in genomic DNA. Genomic sequencing protocols, which have been developed to ascertain the methylation status of selected regions within genes, utilize the Maxam and Gilbert chemical cleavage reactions carried out on genomic DNA (7) with various additional procedures to enhance the signal from the sequence under investigation (8, 9). These protocols are versatile in that they can be adapted for identification of protein-binding sites on genomic DNA in vivo (8, 10) but have two major drawbacks with respect to the identification of 5-methylcytosine residues. First, 5-methylcytosine is identified by the lack of a band in all tracks of a sequencing gel; any background cleavage ladder or close spacing of bands can result in difficulties of interpretation. Second, the sequence obtained represents a population average for many DNA molecules, so that the protocols cannot be adapted for sequencing s...

Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter P ant , and alterations in the sequence of P ant affected the level of resistance expressed by cassette genes. When both P ant and the secondary promoter P2 were present, transcription from both promoters was detected. When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene. In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P ant , and was reduced to different extents by the presence of individual upstream cassettes. In Northern (RNA) blots, multiple discrete transcripts originating at P ant were detected, and only the longer transcripts contained the distal genes. Together, these data suggest that premature transcription termination occurs within the cassettes. The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3 to the coding region) not only function as recombination sites but may also function as transcription terminators.Integrons are genetic elements that contain the determinants of a site-specific recombination system by means of which they are able to capture genes (21). The captured genes, most commonly antibiotic resistance genes, are part of discrete mobile cassettes which contain the gene coding region and a recombination site, known as a 59-base element, located at the 3Ј end of the gene ( Fig. 1) (6-8, 10). The 59-base elements are recombination sites recognized by the integron integrase, and cassettes are inserted at attI (6), a unique integrase recognition site located in the integron adjacent to the integrase gene (19). Once integrated, cassettes are formally part of the integron, and in naturally occurring integrons there appear to be no restrictions on the number or order of inserted cassettes (see reference 10 for a compilation). Moreover, as the cassettes are discrete units that can be independently mobilized by the integron integrase (7), the arrangement of the cassettes in the insert region can be altered by excision of individual cassettes or reassortment (7, 8), or new cassettes can be precisely inserted (6).In general, the initiation codons of the genes found in cassettes are located very close to one boundary of the cassette and a promoter is not included in the cassette. The cassettes are all inserted in the same orientation with respect to their coding regions and are believed to be expressed from a common promoter, P ant , which is the only region with strong similarity to the Escherichia coli promoter consensus present in the conserved segment of the integron (5Ј-conserved segment) located 5Ј to the ...

SummaryAn integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multipie cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the piasmids and transposons that contain them. However, a more general role in evoiution is also likely. Events involving recombination Received 4 July, 1994; revised 17 October, 1994; accepted 19 Oclober, 1994 between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates t>etween different plasmid molecules, and a cassette situated outside the integron context has recently been identified.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991 and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.

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Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5' to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.

Integrons and the site-specific recombination systems encoded by them provide a simple mechanism for the addition of new genes to bacterial chromosomes. Although there is substantial divergence among the four known integron-encoded integrases, they all recognize the recombination sites, known as 59-base elements, that are associated with genes that are packaged in gene cassettes. In contrast, the integron-associated recombination sites, attl sites, are preferentially recognized by the cognate integrase.