WO1999064632A1 - Decouverte de genes d'enzymes de restriction - Google Patents

Decouverte de genes d'enzymes de restriction Download PDF

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Publication number
WO1999064632A1
WO1999064632A1 PCT/US1999/013295 US9913295W WO9964632A1 WO 1999064632 A1 WO1999064632 A1 WO 1999064632A1 US 9913295 W US9913295 W US 9913295W WO 9964632 A1 WO9964632 A1 WO 9964632A1
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dna
genes
seq
expression
gene
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PCT/US1999/013295
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Elisabeth A. Raleigh
Romualdas Vaisvila
Richard D. Morgan
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New England Biolabs, Inc.
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Priority to EP99927501A priority Critical patent/EP1086244A4/fr
Priority to JP2000553622A priority patent/JP2002517260A/ja
Publication of WO1999064632A1 publication Critical patent/WO1999064632A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries

Definitions

  • the invention is generally directed to the field of gene discovery, cloning and expression.
  • a particular aspect of the invention is that it enables direct cloning of intact genes, with a high probability that the orientation of expression is known in advance, and with a low probability of being associated with extraneous possibly toxic genes
  • the invention is limited to genes of a particular kind, since some genes are more likely to be susceptible to cloning and discovery by this method than other genes. Accordingly, the invention is more specifically directed to cloning of genes found within arrays of gene cassettes separated by conserved repeated sequences. Based on present understanding, such arrays are found in prokaryotic organisms and contain genes that have functions that are selectively advantageous to a high level under certain circumstances but are not required under other conditions. Accordingly, some kinds of genes will not be found within these arrays, while other kinds of genes should be enriched in such arrays. Among the genes to be found in such cassette arrays are many genes of commercial interest. The kinds of genes of interest that may be expected in such arrays include:
  • Adhesins which may allow a cell to attach to a particular surface. Enabling specific attachment to a particular surface rather than others has many uses in providing coatings and targeting molecules or organisms to locations of interest. Such adhesins may also mediate pathogenic processes when expressed by pathogenic organisms, and availability of an adhesin may enable competitive exclusion of such pathogenic organisms.
  • Small-molecule modifying enzymes which may convert a toxic or other material abundant in a particular environment to another less toxic to humans or animals, or into a form more useful.
  • toxin molecules that interact with a host organism, which may be useful for synthesis of inhibitors or antagonists of the toxin or for vaccine purposes.
  • Hypervariable regions which show a high level of sequence divergence between closely related strains of the same species, are found at various positions in prokaryotic chromosomes. In some cases, genes present in one strain are absent entirely from a close relative. Examples of this phenomenon include so-called "pathogenicity islands", chromosomal elements that carry genes required for pathogenesis (McDaniel, et al., Proc. Natl Acad. Sci. USA 92(5): 1664-1668 (1995)). Restriction enzyme genes are sometimes found in regions that are hypervariable in this way (Daniel, et al., J. Bacteriol. 170: 1775-1782 (1988); Raleigh, Mol. Microbiol. 6: 1079-1086 (1992); Barcus, et al., Genetics, 140:1187- 1197 (1995)). The mechanism of assembly and variation of these regions may depend on novel genetic mechanisms.
  • Integrons and superintegrons as hypervariable gene regions mobile gene cassettes
  • Integrons are arrays of promoterless gene cassettes, separated by related DNA elements ("59 bp elements") that are sites of action for site-specific integrases related to the lambda integrase (Fig. 1). Each integron has at the 5' end a gene for the relevant integrase. Within the integrase gene is a promoter oriented toward the cassettes, upon which expression of all cassette-borne genes is dependent. Cassettes can be found as extrachromosomal nonreplicating circles, and these can be inserted into the array by the integrase.
  • Characterized integrons are plasmid-borne, and the cassettes specify resistance to drugs or other toxic products (such as mercury). Ordinary integrons are small: up to 8 cassettes have been identified in one ordinary integron, and most have between one and three. It is thought that all the genes are expressed from the single promoter found within the sequence of the flanking integrase (Levesque, et al, Gene 142(l):49-54 (1994); Recchia and Hall, Mol. Microbiol., 15(1): 179-187 (1995)) (Fig. 1); in any event, promoter-like sequences are usually not identified within the gene cassettes.
  • the plasmid location and the multiple-drug resistant character of integrons probably reflect the historical origins of the studies involved: they were found as a result of studies on horizontal transmission of drug resistance in bacteria isolated from clinical settings, where such behavior is selectively advantageous.
  • a superintegron (Mazel, et al., Science, 280(5363):605-608 (1998)) was recently described as a chromosomal array of a large number of gene cassettes mobilizable by a site-specific integrase obtained from an integron.
  • This large array found in Vibrio cholerae, may contain up to a hundred cassettes and may account for as much as 10% of the chromosomE (Barker, et al., J. Bacte ⁇ ol,
  • the Manning laboratory identified this array in the course of studying a pathogenesis-related hemagglutinin (Franzon, et al., Infect. Immun., 61(7):3032-3037 (1993)). Open reading frames within this array are separated by repeated sequences called VCR (for Vibrio cholerae repeats). These repeats are similar to but not the same as the "59 bp elements" of drug-resistance integrons (Mazel, et al., supra (1998)). Manning's laboratory claims to have identified an integrase associated with Vibrio cholerae (Clark, et al., Mol. Microbiol, 26(5):1137-1138 (1997)), and the Davies laboratory has published a description of such a gene from Vibrio cholerae (Mazel, et al., supra (1998)).
  • This superintegron is distinguished from the ordinary integrons in four respects: size, placement of promoters, replicon location, and the nature of the genes found within cassettes. In contrast to the best-studied integron examples, there appear to be 60 to 100 cassettes within the V. cholerae array; and since they are not all oriented in the same direction (Fig. 2), they cannot be expressed from a common promoter. Moreover, the functions encoded by the superintegron are apparently diverse, and some are possibly related to pathogenesis (Mazel, et al., supra (1998)).
  • cassette-borne genes were related to some plasmid- encoded proteins (from database-matching of ORFs 3.1 and 3.2 of the sequence reported in (Barker, et al., supra (1994)), one was a heat-stable toxin (Ogawa and
  • sequences interspersed between gene cassettes are thought to be responsible for acquisition and exchange of gene cassettes among the various replicons on which they are located.
  • sequences designated “59 bp elements” or “VCR elements” are diverse in sequence but display some common features.
  • a consensus sequence was initially deduced for conventional "59 bp elements" (Hall, et al., Mol. Microbiol, 5(8): 1941-1959 (1991)), consisting of:
  • VCR elements were originally said to be unrelated to any other sequence (Barker, et al., supra (1994)) but were subsequently shown to conform with the specifications of the "59 bp elements" except for greater length (Mazel, et al., supra (1998); Clark, et al, supra (1997)): they consist of 124-bp direct repeats of imperfect dyad symmetry, and carry ICS and CS motifs at the ends. VCR elements were found nine times in the original sequence surrounding the putative hemagglutinin gene (Barker, et al, supra (1994)).
  • PCR has been used for characterization of integrons.
  • Some studies employed primers annealing to the conserved integrase genes, or to sull, a conserved gene found at the 3' end of many integrons (e.g.(Levesque, et al., Antimicrob. Agents Chemother., 39(1): 185-191 (1995); Sallen, et al, Microb. Drug Resist., 1(3): 195-202 (1995); Sandvang, et al., FEMS Microbiol Lett.,
  • Restriction enzymes are the workhorses of molecular biology research. They specifically recognize sites in DNA of 4 to 8 basepairs in length, with extremely high selectivity—that is, a site with one mismatch is typically recognized with an affinity one-thousandfold less than the affinity shown for the correct site. This high degree of selectivity is essential for use in practical applications.
  • Enzymes with 8 bp recognition sites (8-cutters, such as Notl, Sfil, Swal, Pad and Pmel) are of particular utility. These enzymes are used for constructing maps of and manipulating DNA from high-complexity sources, such as the genomes of humans and other higher eukaryotes. This utility arises from the rarity of the sites (once per 65,000 bp for palindromic sites), enabling for example the isolation of a whole gene with large introns on a single DNA fragment.
  • the second defect is that is extremely labor-intensive. Each strain must be examined individually, and several of the steps involved are projects in themselves: culture growth, cell lysis, and extract clarification each can be a custom procedure. The quality of crude extract preparations varies greatly among isolates, in the extent of contamination with extraneous nucleases, DNA binding proteins and proteases.
  • Restriction enzymes are found in a wide variety of prokaryotic organisms, many of them with fastidious growth requirements and frequently in low amounts.
  • One method for identifying the presence of a restriction enzyme gene in a clone library is to rely on the presence and expression of a closely-linked gene for a cognate DNA methyltransferase (Wilson, U.S. Patent No. 5,200,333 (1993)).
  • methyltransferase enzymes recognize specific DNA sequences and add a methyl group to an A or C residue within the sequence. This modification prevents cleavage by the endonuclease, thereby protecting the host genome from lethal damage. If such a methyltransferase gene is present in a clone library and effectively expressed, the DNA of that clone will be protected from digestion.
  • plasmid clone DNA is purified from a pool of clones and digested with the desired endonuclease enzyme.
  • the methyltransferase clone will not be digested, while other clones in the library, (which are found in different cells) will be destroyed.
  • Retransformation following such a procedure allows establishment of a selected pool, in which representation of the methyltransferase gene is greatly enriched. If the endonuclease gene is adjacent to the methyltransferase gene, as is often the case, then that gene (or a portion of it) will also be recovered frequently. This method is called the "methyltranf erase selection" method.
  • a second method for identifying the presence of a restriction system gene pair in a clone library is to rely on the presence of conserved polypeptide motif elements found in the DNA methyltransferase proteins (Klimasauskas, et al.,
  • this alternative method is as follows: the polypeptide sequence of the conserved polypeptide motif elements is reverse-translated into a pool of DNA sequences each capable of specifying the polypeptide sequence in question. This pool is called a degenerate pool, because the genetic code is degenerate—several different DNA triplets can specify the same amino acid in many cases.
  • This degenerate pool of oligonucleotides is then used to amplify fragments of DNA from genomic DNA or from a clone library. The sequence of the PCR fragments is then determined, enabling design of further non-degenerate (unique) primers that detect the presence of the proper sequence in the genomic DNA or the clone library by hybridization or PCR.
  • Adjacent DNA sequence can then be obtained by the inverse-PCR method or by Southern blot screening procedures; further sequence can be determined; and finally the complete restriction system can be assembled. This method can be used either alone or in combination with other procedures (below) to isolate the methyltransferase gene and the adjacent endonuclease gene. "Methylase indicator" DNA damage method.
  • a methyltransferase gene cannot be identified, or that a methyltransferase gene can be identified but the open reading frame specifying the endonuclease is uncertain.
  • an additional useful procedure for identifying the gene for the endonuclease specifically can be applied when the endonuclease can be purified in sufficient quantity and purity from the original organism.
  • the endonuclease polypeptide is purified to homogeneity and subjected to N-terminal polypeptide sequencing.
  • the polypeptide sequence is reverse-translated into a pool of DNA primers capable of specifying the appropriate sequence, and these primers are used to amplify a portion of the endonuclease gene from genomic DNA of the original organism or from a clone library.
  • This procedure can be used alone to obtain a portion of an endonuclease gene, or in combination with other methods, such as the degenerate methyltransferase-motif PCR method (Morgan, U.S. Patent No. 5,543,308 (1996)) to obtain portions of genes for both components of the restriction system.
  • the complete genes can be assembled with the assistance of Southern blot or by further direct or inverse PCR methods. If the cognate methyltransferase gene cannot be obtained or cannot be expressed, the stability and utility of solo endonuclease clones will be severely compromised.
  • Such clones can be stabilized with the use of heterospecific methyltransferase genes, which were not associated with the endonuclease in the original host, if they recognize the same or a related sequence and prevent the endonuclease from cleaving its recognition sequence (Wilson and Meda, U.S. Patent No. 5,246,845 (1993)).
  • Another method for identifying the presence of an endonuclease gene in a clone library, independently of the presence of the cognate methyltransferase gene, is to introduce the library into a restrictionless host E. coli strain containing a reporter of DNA damage.
  • This method is related to "methylase indicator method" above, but the strain used contains no restriction activity specific for methylated DNA. In this case, cleavage occurs due to expression of the restriction enzyme, thereby inducing the SOS regulon (and the dinD::lacZ indicator) directly rather than through the action of the methyltransferase and endogenous restriction activities.
  • This indicator can be used to identify restriction endonuclease clones when a modification methyltransferase gene is poorly expressed, so that some DNA damage occurs despite its presence, or without the methyltransferase when conditional activity of the endonuclease can be obtained.
  • the endonuclease in question may be inactive at low growth temperatures but somewhat active at higher growth temperatures. The latter situation obtains, for example, with some restriction endonucleases originally expressed in hyperthermophilic organisms, which normally grow at very high temperatures (Fomenkov, et al., U.S. Patent No. 5,498,535 (1996); Fomenkov, et al., Nucleic Acids Res. 22(12:2399-2403 (1994)).
  • the problem for the experimenter is to obtain sufficient expression from cloned DNA to enable useful amounts of a gene product to be made in the new cellular environment.
  • expression vectors available that provide one or more promoters enabling high- level transcription activity proceeding through the location at which foreign DNA is to be introduced.
  • these vectors are provided with a gene for a regulatory molecule such as a repressor of transcription able to regulate expression from the promoter provided, or are used in host organisms that themselves provide such a regulator.
  • the expression desired can be provided on demand, ie. during induction of specific expression.
  • Many such vectors are described in the art (Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989)).
  • the reverse problem occurs: the product expressed from the cloned DNA is toxic to the cell expressing it for some reason, and ordinary vectors designed for expression at high levels express too much of the toxic product, even in the absence of specific induction. Accordingly, vectors have been described that are designed to express cloned genes at extremely low levels in the absence of induction. The best known of these is the T7 RNA polymerase- dependent expression system designed for use in E. coli (Studier, et al., Meth.
  • cloned genes are expressed from a promoter of transcription that is not recognized at all by any endogenous E. coli RNA polymerase holoenzyme. Instead, the promoter employed is recognized by the RNA polymerase of bacteriophage T7. This polymerase is not encoded in the E. coli genome.
  • This system enables the construction of a clone with toxic properties in the absence of the required RNA polymerase.
  • the clone can then be introduced into a suitable strain into which the T7 RNA polymerase gene has been introduced previously, or the polymerase gene can be introduced by infection with a phage-borne clone. Inhibition of expression from indigenous promoter-like sequences
  • a high level of transcription in the direction opposite that needed for polypeptide expression can interfere with expression in at least two ways. First, it can occlude transcription in the direction needed for expression; and second, it can prevent translation by allowing formation of RNA-RNA hybrids between the RNA used for expression of the toxic protein and the RNA directed in the opposite sense (antisense RNA).
  • Restriction endonucleases which cleave DNA at particular sequences, are normally associated with protective modification methyltransferases. In the present method it is quite likely that the gene for such an endonuclease will be isolated without its partner methyltransferase gene. Very tight regulation of the cassettes thus cloned is therefore critical.
  • a convenient tightly regulated expression plasmid, pLT7K, is available into which pooled PCR fragments can be cloned (Roberts, supra (1999)).
  • pLT7K A convenient tightly regulated expression plasmid
  • two levels of control are available: expression is inducible and inhibition is repressible.
  • a T7 gene 10 promoter reads into one side of the cloning site; Lad provided by the vector represses expression from this promoter, as is expression of the T7 RNA polymerase provided by the host cells used for expression. Further control can be obtained by the use of pLysP, which expresses an inhibitor of T7 RNA polymerase.
  • the ⁇ pL promoter reads into the other side of the cloning site, antagonizing expression from pT7.
  • This antagonistic transcription is regulated by ⁇ cl°5 ' , a thermosensitive repressor. At 40°C and in the absence of IPTG therefore, essentially no expression was observed; at 30°C, some leaky expression is seen; at 30°C in the presence of IPTG, moderate levels of expression can be achieved.
  • This vector has successfully been used to establish the pacIR and nlalllR genes (encoding the restriction enzymes Pad and Nlalll) in the absence of methyltransferase protection, and to express the genes.
  • a general object of the invention is to provide a procedure for obtaining clones of diversity-selected genes.
  • a specific object of the invention is to provide a method for identifying a repeat sequence suitable for identification and cloning of gene cassettes found in arrays and separated by repeat sequences.
  • a specific example of such a repeat sequence family with 74 members is provided together with the sequences of four contiguous DNA stretches comprising one or more cassette arrays.
  • a further specific object of this invention is to provide a procedure for cloning cassettes from such arrays, by PCR directed by oligonucleotides hybridizing with the repeated sequences flanking the cassettes. A specific example of such a PCR procedure is provided.
  • a further specific object of this invention is to provide a procedure for cloning such PCR fragments into an expression vector able to stabilize toxic genes such as restriction enzymes.
  • a specific example of such a gene clonable by this procedure is provided.
  • a further specific object of the invention is to provide a means of identifying particular cloned genes of interest.
  • one method relies on identification by means of protein sequence similarity; a second method relies on an indirect report of gene activity; a third method relies on direct test of biochemical properties.
  • two novel strains that enable provision of indirect report of expressible cloned nuclease genes in the context of the vector pLT7K are provided, together with a method of use.
  • a further specific object of the invention is to provide a method for obtaining expression clones of active restriction enzyme genes without prior knowledge of their biochemical activity or DNA sequence.
  • a specific example of a procedure for obtaining such a clone is provided.
  • these regions provide a mechanism for discovery of diversity- selected genes.
  • the features of these systems enable isolation of DNA enriched for certain kinds of genes including restriction enzyme genes, and also enable the cloning, sequencing and expression of products encoded in this DNA.
  • cassette arrays Three features of cassette arrays are particularly useful for cloning purposes:
  • Each gene (rarely, a pair of genes) is embedded in a predictable sequence context— a particular kind of repeated DNA sequence is found on each side.
  • a second difficulty with these repeat sequences is that individual members of the repeated array display imperfect dyad symmetry elements, making it likely that PCR primers designed will form hairpins or primer dimers and so fail to prime DNA amplification. Accordingly, it is important to design primer that anneal to portions of the repeats that do not display these features. Primers that are able to hybridize with or that enable amplification from many cassettes are provided in accordance with this invention.
  • the present invention provides a method for obtaining expression of cassette-encoded functions even when toxic, by cloning these into an appropriate vector, such as the pLT7K vector described in International Publication No. WO 99/11821 (Roberts, et al., (1999)).
  • This vector has the advantage (in addition to those provided in the original patent) that it can be used in two configurations in this application.
  • the expression condition can be either 30 C + IPTG or 40 C - IPTG; and the repressed condition suitably the reverse. This enables flexibility in screening or selecting for molecules that display activity sensitive to temperature, and in selecting storage conditions for the clone library obtained. Strain enabling indirect report of nuclease activity.
  • a test of function is provided that enables detection of a minority of expression clones of interest in the context of the T7-RNAP dependent regulation required by the vector pLT7K. This test detects nuclease or other DNA damaging activity by SOS induction of dinDr.lacZ alleles. Two strains are provided:
  • the former can be used at either 30°C or 42°C to indicate DNA damage with a dark blue color against a background of lighter blue colonies.
  • the latter can be used at 30°C up to and including 37°C to indicate DNA damage with blue color of any shade against a background of white colonies.
  • cassettes cloned into pLT7K in an orientation such that cassette expression is driven by ⁇ pL and inhibited by pT7 can be screened at 37°C (with or without IPTG) in either strain or 40°C (with or without IPTG) in ER2745 but not ER2746. In each case the presence of activity is indicated when a colony turns bluer than the majority class, and when this property is stable upon reisolation as a single-colony derivative of the original transformant.
  • strains may similarly be used to indicate DNA damage provoked by any agent, including enzymes that are not nucleases, by chemical agents, or by radiation. These strains are most distinctively useful when the damage produced results pursuant to a regulated change in the state of T7 RNA polymerase expression as provided within these strains. Kinds of genes for which this method may be applied.
  • genes for which the invention is useful Some kinds of genes are likely to be present in cassette arrays, while others are unlikely to be present in them.
  • the original cassettes of known function all specified resistance to drugs or other antibacterials. There is no a priori reason to suppose that integrons cannot mediate the spread of functions other than drug resistances.
  • Types of genes likely to be enriched in such arrays include functions useful individually or in pairs, and subject to highly variable selective value. Typically such genes will be subject to strong episodic selection, very important some of the time but not useful at all the rest of the time. In some cases they will be episodically essential— necessary for cell survival: drug resistance factors, restriction-modification systems.
  • Examples would include specific adhesins that allow the cell to attach to a particular surface in a rich environment; specific enzymes that modify an abundant material in the cellular environment to convert it to a form usable as nutrition; or specific toxin molecules that interact with a host organism.
  • Many individual members of a particular species will elaborate gene products that have common general properties (adhesins stick to things).
  • An important feature of relevant gene products, however, is that among the population will be found examples with highly variable specificities (there are many different kinds of specific surfaces to stick to, from rocks to intestinal mucosa to urinary epithelium).
  • Cassette arrays therefore will be enriched for genes that are subject to selection for diversity as described above: that is, genes that are advantageous when rare but of no particular use when frequent in the population; and those episodically required.
  • Types of genes expected to be absent from such arrays include all of the basic components of the cellular maintenance machinery: DNA replicases, basic transcription factors such as vegetative RNA polymerase, the translational machinery, enzymes of small molecule metabolism central to cellular physiology such as those of the tricarboxylic acid cycle. They should be absent for two reasons. First, no selective advantage is expected from maintaining variability as such in the pool of alleles available to a population of cells. Second, many such proteins must maintain (conserve) specific interactions among several different proteins (replicase/RNA polymerase/translation initiation factor interactions for example).
  • Figure 1 is a schematic of the structure of characterized integrons, arrays of gene cassettes (thin lines; fnl, fn2, fn3) separated by repeated sequences (filled boxes; 59 bp elements). These are assembled by the action of a site-specific integrase (large box; intl) by insertion into attl (arrows) of extrachromosomal circles (cassette). Cassettes are transcribed from a promoter within the integrase gene (arrow). Many integrons are associated with a conserved sulfonamide resistance gene (sull) that is not part of the integron itself.
  • sull conserved sulfonamide resistance gene
  • FIG. 2 is a schematic diagram of a fragment of a superintegron identified in Vibrio cholerae. Open reading frames (1-9 and mrhA, mrhB) are separated by repeats (boxes) that are similar to 59 bp elements of integrons
  • Figure 3A-3E is an alignment of some of the PAR elements (SEQ ID NO: 96 through SEQ ID NO: 116), those identified in superintegron contig 1 (SEQ ID NO:l) by the motif search procedure described in Example 1. Consensus lines show bases shared by all (top line), 90% (second line) or the majority (third line) of the elements in the alignment. Individual entries are the same as the majority consensus except for the bp shown.
  • Figure 4. is a dotplot display illustrating an alternative method for identifying repeated sequences.
  • Figure 5 illustrates the self-complementarity of an individual PAR element (SQUIGGLE display of the output of FOLD in the GCG program set).
  • Figure 6 illustrates alignments of subfamilies identifiable in the set of PAR elements herein (SEQ ID NO:5 through SEQ ID NO:78) shown in Table 1.
  • Panels A-D families 1-4.
  • Each family alignment includes PAR2 as an outgroup member, since PAR2 is the most distantly related of the elements identified. Families were identified as bushy groups in a phylogenetic tree generated from the CLUSTAL alignment of the 74 elements.
  • Figure 7 illustrates the location of oligonucleotides used for Southern blots (panel A) and PCR fingerprinting (Panel B) in relation to the majority consensus of all PAR elements and in relation to a typical cassette.
  • Figure 8 illustrates a Southern blot hybridization of a mixture of
  • Oligonucleotides 2-5 (SEQ ID NO:79 through SEQ ID NO:83; Fig 7, see also Table 2) to P. alcaligenes DNA.
  • Figure 9 displays an agarose gel of PCR products generated from chromosomal DNA of isolates of six Pseudomonas species by the use of oligonucleotides 6 and 7 illustrated in Fig. 7.
  • Figure 10 illustrates the scheme for forming a clone library of cassette- encoded open reading frames and expression of their products from pLT7K.
  • the method comprises the following steps, although as the skilled artisan will appreciate, modifications to these steps may be made without adversely affecting the outcome:
  • the desirable genes are those for restriction endonucleases and modification methyltransferases.
  • Types of genes likely to be enriched in cassette arrays include functions useful to the organism individually or in pairs, and subject to highly variable selective value.
  • a function may be identified as likely to be encoded by genes in such arrays when a survey of different isolates of a species determines that the presence of the function, or its specificity, is variable within the collection of isolates. For example, a survey of isolates of Escherichia coli reveals that many isolates but not all isolates express type II restriction enzymes; and that of those that do, the specificity of the enzyme (the sequence recognized) is variable, with many different specificities determined within the species.
  • Candidate functions that will be subject to such variation include, in addition to restriction enzymes, cell surface antigens such as polysaccharide antigens or polypeptide antigens or secreted molecules; adhesins of various sorts such as fimbrial proteins, pilus proteins or outer membrane proteins; transporters of small molecules, especially those with narrow specificity; exported functions such as toxins, hemolysins, hemagglutinins, kinases and signalling molecules; detoxifying enzymes such as drug resistance determinants; catabolic enzymes specific for compounds episodically available (excluding those required for centra] metabolic pathways such as the tricarboxylic acid cycle); enzymes for biosynthesis of rare sugars (excluding those required in all cells, such as ribose, deoxyribose, and sugars of the cell wall), especially of those sugars that form part of the pericellular envelope.
  • cell surface antigens such as polysaccharide antigens or polypeptide antigens or secreted molecules
  • the desirable genes are those for restriction endonucleases and modification methyltransferases.
  • such genes will be subject to strong episodic selection, very important some of the time but not useful at all the rest of the time. Restriction functions can provide a very powerful protection against the invasion of foreign DNA (as when a bacteriophage infects the cell). This protection will only be effective if the host from which the bacteriophage did not carry the same restriction functions— otherwise its DNA would already carry the protective modification pattern of the invaded cell.
  • Genomic DNA is prepared from a strain of interest or from a consortium of strains or from an environmental source by methods known in the art, or DNA of plasmid, cosmid, BAC or PAC clones of genomic DNA from such sources is prepared.
  • PCR conditions are optimized using a non-proofreading DNA polymerase, by varying primer- tempi ate ratio, annealing temperature, magnesium ion concentration and extension time.
  • the DNA preparation is subjected to PCR employing a pair of primers annealing to repeat sequences flanking the cassettes and containing at their 5' ends sites for endonucleases compatible with cloning into a plasmid vector.
  • Preferred primer pairs include those listed in Example 2; other suitable primer pairs may be designed based on sequences listed in Example 1, or based on other particular repeat sequences identified in the literature or by methods described in Example 1.
  • PCR conditions are optimized using a proofreading DNA polymerase, by varying primer-template ratio, annealing temperature, magnesium ion concentration and extension time. PCR fragments are purified away from primers, for example by means of size fractionation using commercially available kits. 5 ) Cassette cloning
  • the PCR fragments are digested with the appropriate restriction endonucleases for cloning, in one preferred procedure with Xhol and Xbal.
  • the digested fragments are ligated into a suitable vector.
  • Preferred vectors for this purpose have two particular properties. First, they contain a cloning site disposed to allow directional cloning of fragments.
  • Directional cloning methods include the process of digesting the vector with two different restriction enzymes such that the single-stranded extension at one end does not hybridize the single-stranded extension at the other end of the vector backbone containing the origin of replication; and then ligating, to that vector backbone, DNA fragments having an extension at one end that hybridizes with one single-stranded extension of the vector backbone, and having an extension at the other end that hybridizes with the other single-stranded extension of the vector backbone.
  • Other directional cloning methods can be envisioned, including for example the use of site-specific recombination enzymes, or hybridization of extensions provided by methods other than restriction enzyme cleavage.
  • preferred vectors contain two independently regulatable expression signals, one on each side of the cloning site described above and directed toward expression of the sequence resident at the cloning site.
  • One preferred vector is pLT7K (Roberts, et al., International
  • vectors include pBR322, pUC19, pACYC184, pSClOl, pBeloBACl l, or their derivatives.
  • the ligated products are transformed into a strain suitable for screening or selecting for cassettes encoding desirable functions.
  • the strain must be compatible with the expression regulation signals provided by the vector chosen and must enable the method to be used for identifying desired cassettes.
  • sequencing large numbers of cloned cassettes and subsequently evaluating the sequence information will identify cassettes of interest by bioinformatic methods.
  • bioinformatic methods include matching the cassette-encoded sequences against public or private databases by means of similarity-determining algorithms such as BLAST or FASTA, or by employing a motif or pattern-based search of the cassette -encoded sequences employing databases such as the PROSITE profiles database or the BLOCKS and PRINTS databases (Patterson, M. and Handel, M. (1998) Trends Guide to Bioinformatics, Elsevier Science, Cambridge, UK). In this case there are few constraints on strain or vector choice.
  • cassettes of interest will be identified by sequence-based methods such as PCR or hybridization with probes. In these cases there are also few constraints on strain or vector choice.
  • cassettes of interest will be identified by activity expressed in vivo.
  • choice of strain and vector is constrained: vector and strain must be compatible, enabling suitable regulation of cassette expression; by the nature of the activity to be expressed will also constrain strain choice.
  • the activities to be expressed are modification methyltransferase activity or restriction endonuclease activity, both of which are amenable to identification by indirect report of activity based on damage inflicted in intracellular DNA and induction of the DNA damage repair response.
  • Two preferred strains ER2745 F ⁇ ' fl ⁇ uA2 [Ion] [dan] ompT lacZ::T7 genel gal sulAll A(mcrC-mrr)114::IS10 R(mcr-73::miniTnlO-TetS)2 R(zgb-210::TnlO -
  • TetS) endAl) dinD2::Mudll734 (KanR, lacZ + ).
  • A(mcrC-mrr)114::lS10 lacZ::T7 genel dinD2: :MudI1734 (KanR, lacZ(ts)) are strains compatible with the vector pLT7K.
  • ER2745 is derived from the particular strain background normally used for
  • T7 RNAP-directed expression and is ultimately a derivative of E. coli B.
  • the protein expression properties of this strain background are well understood.
  • This strain is transformable with DNA, but the level of transformation obtained is less than with other strains.
  • the amount of the indicator lacZ expressed in the absence of DNA damage is relatively high, leading to light-blue colonies on Xgal plates even when no damage has occurred.
  • ER2746 carries a thermosensitive lacZ moiety. This is useful because it lowers the light-blue background color observed on X-gal by the original dinD indicator allele. Discrimination between clones inducing some damage (which are of interest) and those inducing no damage (which are not) is improved in this situation. However, this allele cannot be used to detect DNA damage at high temperature (>37°C), because the lacZ moiety of the indicator fusion is inactive, and will remain white even in the presence of extensive DNA damage. This was demonstrated by testing at various temperatures for induction of blue color by nalidixic acid, a well-characterized DNA damaging agent, on plates containing X-gal.
  • transcriptional fusion of a drug-resistance gene to a damage-inducible promoter should allow selective isolation of clones of interest, rather than the more-laborious screening procedure.
  • Use of a variety of drug concentrations would then allow isolation of clones with different levels of DNA-damaging activity.
  • Introduction of a recD mutation would inactivate the major ATP-dependent double-strand exonuclease of the cell, while an xth mutation would inactivate ExoIII, the major ATP-independent double-strand exonuclease.
  • a triply nuclease-deficient strain should be viable but may not stably maintain the plasmid (Niki, et al., Mol. Gen. Genet. 224(1): 1-9 (1990)).
  • DNA damage-inducing promoters that can be used include those identified by (Lewis, et al., J. Bacteriol, 174:3377-3385 (1992); Lewis, J. Mol. Biol, 241:506-523 (1994)): these are promoters of recA, lexA, uvrA, uvrB, dinG, polB, uvrD, ruvAB, umuDC, sulA, dinH, dinl, sosA, sosB, sosC, sosD.
  • Other SOS-inducible genes identified include recN, dinB and dinF (Walker, Microbiological Review, 48:60-93 (1984)). Some other indicator/reporter genes that can be used were reported in (Fomenkov, et al., supra (1995).
  • transformants are plated onto suitable media.
  • the vector is pLT7K
  • the strain is ER2746
  • plates are
  • Reversing the configuration of expression so that the repressing condition is at 30°C +IPTG and the inducing condition is 40°C - IPTG can be easily accomplished with pLT7K by switching the cloning sites added to the oligonucleotide primers for PCR so that cassettes are in the reverse orientation.
  • strain ER2745 is the preferred strain, since the damage-inducible fusion carries a wild type lacZ allele that enables indication at 40°C. In that case, the colonies desired will be darker blue than the normal light blue color.
  • Further characterization is then carried out on the identified plasmids, either continuing from the replica plate masters or from the archived plasmid DNA following retransformation. Further characterization includes some or all of the following three steps.
  • Crude extracts supernatants are then assayed for nuclease activity in a general screen for 4-6 base cutters, using standard plasmid, phage and viral DNAs such as pUC19, pACYC187, pACYC177, pBR322, M13mpl8 replicative form
  • DNA digestion patterns are resolved by agarose gel electrophoresis using an agarose concentration suitable for visualization of bands between 200 and 0.05 kb (usually 0.7% agarose and 1.3 % agarose), and detected by ethidium bromide staining.
  • DNA digestion patterns are then evaluated and the recognition sequence is determined by methods known in the art. Further purification of the endonuclease thus identified may be required for these methods to be applied.
  • Crude extract supernatants are also assayed in an in vitro screen for enzymes with 8-base sites, using chromsomal DNAs of varying GC-content: Rhodobacter sphaeroides, Escherichia coli and Staphylococcus aureus range from 66% to 34% G+C and are suitable for detecting a variety of enzymes with rare sites.
  • Isolates that yield a positive result on chromosomal digests but not in digests of standard substrates are then further characterized by searching for alternative substrates, guided by the G+C content of the chromosomal DNA yielding a positive result.
  • Pulsed-field gel assay A potentially more-informative assay for 8-base recognition sites relies on separation of total chromosomal fragments on pulsed- field gels. When crude extracts are used for screening procedures, these gels are too cumbersome and too sensitive to other nucleases in the extract to be generally useful.
  • the substrate DNA is obtained by first embedding whole cells in agarose plugs. DNA is released from the cells in situ by means of a series of enzymatic treatments and washes that degrade the cell wall. The restriction endonuclease is then incubated with the plug; this usually takes several hours, since the enzyme must permeate the agarose and the remnants of the previous digestions.
  • the restriction nuclease digestion step consists of inducing expression within the cell, before agarose is added; embedding the cells in agarose and subjecting the cells to electrophoresis on a pulsed-field agarose gel.
  • Controls include: positive control, standard digestion of the host DNA embedded in agarose plugs with purified Pad and Notl; and negative control, samples of the host containing the empty vector, treated in parallel with the experimental samples.
  • Plasmid DNAs prepared from candidate clones obtained by the indirect report assay are fingerprinted by restriction enzyme digestion. Each candidate is digested separately with two to four enzymes with four-base recognition sites: in the preferred example, with Haelll and Msel to yield a patterns characteristic of the cloned cassette.
  • This system enables introduction of primer-binding sites at random locations in plasmids of interest, rapid mapping of the location of the insertion by digestion with rare-cutters that cleave within the transposon, and sequencing of the insertions within the fragment of interest. With these target molecules, about 20% of transposon insertions will be found within the sequence of interest. No more than
  • the target genes (endonucleases recognizing new specificities) can be expected among those not identified by homology search.
  • the desirable function is a methyltransferase gene, which may be selected or screened for by methods known in the art, described above.
  • an endonuclease with suitable specificity may be used if an endonuclease with suitable specificity. This method will be applicable when something is known or suspected about the specificity of potential methyltransferase enzymes and a suitable endonuclease is available. Such an endonuclease may be a heterologous endonuclease recognizing a subset of the relevant sites.
  • the method of may be employed alone, or the degenerate methyltranferase- motif primers may be combined with a repeat-specific primer or primers annealing to the flanking repeats in a single orientation, such as those employed in PCR fingerprinting or cassette cloning as described above.
  • methyltransferase genes include detection of enzymatic activity such as evaluation of 3 H-SAM inco ⁇ oration into specific DNA sequences and may be applied to individual clones or pools of clones.
  • Hybridization detection methods such as colony lifts may be employed to detect the presence of genes with high levels of DNA homology to available methyltransferase genes or to oligonucleotides designed based on the sequences of those genes.
  • This Example outlines the general strategy for identifying a candidate repeated sequence. It also provides a specific repeated sequence family, probes for identification of organisms containing similar repeats and primers for amplification of the gene cassettes.
  • the organisms expressing Pad and Pmel were isolated by at NEB (Polisson, U.S. Patent No. 5,098,839 (1992); Morgan and Zhou, U.S. Patent No. 5,196,330 (1993)). These restriction enzymes are made by particular isolates of Pseudomonas alcaligenes (ATCC No. 55044) (NEB Deposit No. 585, New England Biolabs, Inc.; Beverly, MA) and Pseudomonas mendocina (ATCC No.
  • Chromosomal DNA of P. alcaligenes (ATCC No. 55044) (NEB Deposit No. 585, New England Biolabs, Inc., Beverly, MA) prepared by the procedure described in the manual of Qiagen (Genomic tip 100/G (Cat 10243) was digested with Hindlll to completion.
  • Hindlll fragments were isolated by gel fractionation on agarose gels (0.7%) and fragments between 2 kb and 10 kb were isolated using QIAquick Gel extraction kit (Cat # 28704) according to the instructions of the manufacturer and ligated with Hindlll-digested dephosphorylated pBR322.
  • a library of this size would of course not contain all fragments exactly once and not all fragments in the fraction are 8 kb. Nevertheless, the incidence of PAR-containing fragments in the library is consistent with the estimated size of the putative superintegron (>60 kb; 10% of 800 kb would be 80 kb).
  • Tn7-based transposon an early version of the NEB GPSTM-1 kit, (New England Biolabs, Inc., Beverly, MA, NEB Catalog No.
  • sequence contigs totaling 59.4 kb, containing 74 examples of the repetitive sequence.
  • sequences are SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
  • the specific repeated sequences that are likely to signal the presence of a cassette array can be identified by similarity to those found in known arrays such as the VCR elements of Vibrio cholerae, or by computer- assisted analysis of existing sequence information. These sequences were identified by the following procedure, employing computerized search procedures (both UWGCG SEQED and DNASTAR EDITSEQ programs are suitable): the 5' end of the repeat was found by searching for the sequence TAACWA; the 3' end of the repeats were found by searching for the sequence CGTTRR; and the additional constraint was imposed that the 5' base of the 5' element should be not more than 200 bp from the 3' end of the 3' element. This strategy identified 18 repeated elements in this contiguous stretch of 14.144 kb.
  • Fig 3 shows an alignment of a set of such sequences identified in a part of the P. alcaligenes (ATCC No. 55044) (New England Biolabs, Inc. Beverly, MA, NEB Catalog No. 585) superintegron sequence SEQ ID NO:l.
  • the elements were aligned using the DNASTAR MEGALIGN program, by the CLUSTAL method. The alignment shows a majority consensus (third line), a 90% consensus, at which 16 of the 18 elements are identical (second line) and an identity consensus, with which all elements agree.
  • oligonucleotide probes and primers can be designed for use in Southern blot and PCR experiments, described further below. Examples of these are shown aligned with the consensus of 74 PAR elements (majority rule) in Fig. 7 A (Oligonucleotides 1-5 (SEQ LD NO:79 through SEQ ID NO:83; see Table 2) for Southern blot) and 7B (Oligonucleotides 6 and 7 (SEQ LD NO:84 and SEQ ID NO:85; see Table 2) for PCR).
  • a Southern blot (Fig. 8) was carried out using a mixture of biotin-labeled oligonucleotides (Oligonucleotides 2-5, SEQ ID NO:80 through SEQ LD NO:83; see Table 2) as a probe for repeat sequences (PAR elements), and chromosomal DNA of P. alcaligenes (ATCC 55044) (New England Biolabs, Inc., Beverly, MA, NEB Catalog No. 585) prepared by the procedure of Qiagen (Genomic tip 100/G (Cat 10243). Restriction digests with 8 different restriction enzymes (Sphl,
  • Fig 8 reveals that multiple fragments in each digest hybridized with the probe, confirming that the oligonucleotide recognized a repeated sequence.
  • the minimum sum of sizes of hybridizing bands ranged from -20 (Pstl) to -44 (Ndel) kb, suggesting that a large number of cassettes might be present. Some of these bands may represent doublet or triplet co-migrating species, so the maximum size cannot be reliably estimated.
  • oligonucleotide sequences might be designed based on specific families of PAR elements.
  • a single oligonucleotide such as Oligonucleotide 1 (SEQ ID NO:79; see Table 2) may be used (data not shown), which may be used to prepare a biotin-labeled probe by starting with an unlabeled oligonucleotide, and labeling it by use of a random-priming kit such as NEBlot®
  • the Southern blot procedure separates DNA fragments by size, transfers these to a membrane support, denatures the DNA, hybridizes the probe, then separates the hybridized product from the nonhybridized probe (in this case oligonucleotides) by washing.
  • Alternative derived methods for detecting the presence of hybridized DNA include use of arrays of DNA preparations, not separated by size, adsorbed a membrane (dot blots or slot blots (Moore, supra (1999)) or microtiter plate
  • Alternative possible detection methods include the use of radiolabeled oligonucleotides (labeled with S 35 or P 32 or P 33 ), or of alternative chemical detection methods, such as digoxygenin-based (Roche Molecular Biochemicals Cat #12102201) or fluorescein-based (AP Biotech Cat # RPN 3030) label and detection procedures.
  • Alternative methods of DNA preparation could include purification by detergent/protease treatment followed by precipitation or CsCl centrifugation, or by purification from agarose gels (Moore, supra (1999)).
  • Other commercially available kits that rely on gel filtration may also be employed (e.g. those supplied by 5Prime— >3Prime, or Promega Wizard Genomic DNA Purification Kit, Cat#A1120).
  • a second method for detecting cassette arrays in a population is to employ primers annealing to each end of the repeats separating the cassettes in a PCR experiment (Fig 7B and Fig 9). If the repeats are present and close enough to each other for PCR amplification to be effective, DNA bands representing the cassettes will be observed in ethidium-bromide stained agarose gels following electrophoretic separation.
  • P. maltophila NEB Deposit No. 515 New England Biolabs, Inc., Beverly, MA
  • Pmll P. fluorescens NEB Deposit No. 375
  • PflMI P. putida NEB Deposit No. 372
  • PCR reactions included 100 ng DNA, 0.2 ⁇ mol each oligonucleotide, 1 units of Vent® Exo + polymerase, IX NEB Thermopol buffer in a reaction volume of 50 ⁇ l. Thermal cycling parameters were 15 sec denaturation at 95°C, 1 min annealing at 55°C, 1 min extension time at 72°C. 25 cycles were carried out. Products were subjected to electrophoresis for 1 h at 100 mA in 0.7 % agarose with 10 "4 ⁇ g/ml ethidium bromide.
  • Figure 8 reveals that two of the six species yielded multiple amplification products from this procedure. This confirms the presence of the repeat segments in the correct orientation and at the correct spacing for amplification to occur. It is not possible to assess the number of potential cassettes from this procedure, since some cassettes may be too long to amplify efficiently, especially in the presence of shorter cassettes that would be amplified preferentially. In addition, some amplification products may represent amplification across two cassettes. In this case, the repeat separating them might be more distantly related to the primers than those at the ends of the amplicon.
  • Primers 8-11 are candidate primers for the forward direction, while primers 12 and 13
  • SEQ LD NO:90 and SEQ LD NO:91; see Table 2) are candidate primers for the reverse direction as displayed in Fig. 8
  • Alternative methods of visualization include chemi luminescent detection of affinity-labeled oligonucleotide primers, fluorescent detection of fluorescently labeled nucleotides or oligonucleotide primers inco ⁇ orated during PCR, or autoradiography when using radiolabeled oligonucleotide primers or radiolabeled dNTP.
  • PCR-finge ⁇ rinting strategy it should be possible to apply the PCR-finge ⁇ rinting strategy to mixed populations to identify the presence of cassette arrays in a minority of the population.
  • At least two kinds of applications to mixed populations can be tried: PCR using combinatorial pools of individual strains, and PCR using environmental DNA.
  • Combinatorial pools can be achieved by arraying individual strains in addressable arrays, for example, 96-well plates. Pools can be made combining the individual strains, e.g. all strains in one row in one pool; or all strains in one column in one pool; or all strains in one 2D address from a series of plates. Many such pooling procedures have been worked out and will be familiar to one skilled in the art (e.g. (Chaplin and Brownstein, supra (1999); Green, et al., Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 3, pp. 297-548 (1999)).
  • DNA can be made from these strains individually and the DNA samples then pooled; or the strain cultures can be pooled and DNA made form the pool.
  • Each procedure has disadvantages; in the first instance, a larger number of DNA preps must be made; but in the second procedure, different strains may be differentially subject to cell breakage and DNA extraction, and therefore DNA from some strains will be under-represented relative to others.
  • a positive control a strain known to contain an array (such as P. alcaligenes (ATCC 55044) (NEB Deposit No. 585, New England Biolabs, Inc., Beverly, MA)-can be included in one pool as a single member while the other members are drawn from negative controls— strains known not to contain a responsive array (such as P. lemoignei (NEB Deposit No. 418, New England Biolabs, Inc., Beverly, MA).
  • the positive control can be included in duplicate, in another in triplicate, with corresponding reduction in the representation of the negative control. This will enable assessment of the sensitivity of the overall procedure.
  • a DNA source of great interest is likely to be DNA isolated from environmental samples (e.g. soil, water, filtered air etc) without first obtaining organisms in pure culture.
  • PCR from cassette arrays may be even more desirable as a mechanism for obtaining genes in intact form.
  • the same kinds of positive and negative controls as those described in Cl may be included.
  • other controls should be included.
  • the original environmental sample from which DNA is to be isolated can be divided and a portion doped with a small amount of the positive control strain.
  • DNA extraction from the sample will then include some of the positive control, enabling that portion of the sample to be used as a control for the efficiency of DNA extraction and recovery of known cassettes from a known source.
  • Inclusion of a dilution series of purified positive control DNA in the environmental sample DNA will serve as a control for inhibitory materials in the environmental sample.
  • PCR controls can test for the presence of mitochondria, chloroplasts, and nuclear ribosomal DNA genes by methods known to those skilled in the art (von Wintzingerode, et al., FEMS Microbiol. Rev. 21(3):213-229 (1997); Sekiguchi, et al., Microbiology, 144 (Pt. 9), 2655-2665 (1998)).
  • PCR products can be purified using the QIAquick PCR purification kit (Qiagen Cat No. 28104) or other similar kits.
  • Fragments can be digested to provide ligatable ends compatible with appropriately- digested plasmid or bacteriophage vectors.
  • Xhol and Xbal sites added to the 5' ends of the oligonucleotide primers used for PCR provides directional cloning into pLT7K (Example 2 below) such that a defined orientation is obtained relative to vector-borne expression signals. Accordingly, the use of regulatory signals residing in the vector is feasible. If regulation of expression is not a concern, any vector can be used to clone such cassettes, provided that suitable cloning sites are included at the 5' ends of oligonucleotides used for PCR.
  • Such vectors may be high-copy (e.g.
  • Such vectors may contain expression signals suitable for regulated expression in E. coli (e.g. pLT7K; see Example 2), or may be designed for expression in an organism suitable for further experimental test of a particular cassette (e.g. Bacillus subtilis, Streptomyces coelicolor, Agrobacterium tumefaciens or other prokaryotic organism).
  • a particular cassette e.g. Bacillus subtilis, Streptomyces coelicolor, Agrobacterium tumefaciens or other prokaryotic organism.
  • the ligated fragment pool will normally be recovered as a clone library of fragments consisting of colonies of the recipient organism containing one or more selectable marker of the vector on solid media following transformation by chemical methods or by electroporation (Hanahan, et al., Methods in Enzymol, 204:63-113 (1991)).
  • the cassettes obtained will encode many different sorts of genes.
  • genes encoding functions of one particular kind but with differing specificities have related polypeptide sequences.
  • a particular example of this kind of relationship is the set of genes that encode DNA methyltransferases, which carry out the same reaction (adding a methyl group to a specific base in a specific sequence) but with differing specificities (different particular bases within different particular sequences are modified).
  • These can be tentatively identified by PCR employing primers that anneal to conserved polypeptide motif (Morgan, supra (1996)). Briefly, individual colonies or pools of colonies from step D) can be subjected to degenerate PCR by procedures detailed in Morgan, 1996, with modification.
  • Clones with potential methyltransferase genes can be identified by evaluation using DNA comparison algorithms such as BLAST or FASTA, or by means of programs specifically directed to evaluating such similarities (Posfai, et al., Compt. Appl. Biosci. 10(5):537-544 (1994)).
  • the present procedure will allow isolation in expression-ready form of a large number of cassettes specifying a variety of genes with diversity-selected functions. Accordingly, identification of specific clones expressing functions of the desired type is a critical part of the procedure.
  • This example illustrates one way to identify a particular desired function, a DNA damaging agent, and to refine the functional identification until a site-specific doublestranded DNA endonuclease (a restriction enzyme) has been characterized.
  • a site-specific doublestranded DNA endonuclease a restriction enzyme
  • this example illustrates that the method is useful even when the desired function is toxic to the cell that expresses it.
  • the procedure of this Example is possible specifically because the orientation of the genes is specified in advance, due to the natural orientation of the genes in a cassette array relative to the repeat elements that separate them.
  • the vector employed can be used to regulate the expression of the cloned cassette fragments even when nothing whatever is known about the identity or sequence of the cassettes individually.
  • pLT7K Fig 10
  • two levels of control are available: expression is inducible and inhibition is repressible.
  • a T7 gene 10 promoter reads into one side of the cloning site; expression from this promoter is repressed by Lad provided by the vector, as is expression of the T7 RNA polymerase itself, which is provided by the host cells used for expression. Further control can be obtained by the use of pLysP, which expresses an inhibitor of T7 RNA polymerase.
  • tandem ⁇ pL promoter reads into the other side of the cloning site, antagonizing expression from pT7.
  • This antagonistic transcription is regulated by ⁇ cl ⁇ 57 5 a thermosensitive repressor. At 40°C and in the absence of IPTG therefore, essentially no expression was observed; at 30°C, some leaky expression is seen; at 30°C in the presence of IPTG, moderate levels of expression can be achieved.
  • the T7 RNA polymerase should be expressible after induction; the strain should not contain a lambda lysogen (because it would be induced to express phage-encoded killing functions following DNA damage); it should preferably be highly transformable, in order to obtain a large collection of transformants carrying cloned cassettes; it should express the DNA damage indicator lacZ, preferably only following DNA damage— ie with a clean background of white colonies in the absence of induction; and it should not express the major nonspecific endonuclease of Escherichia coli, Endonuclease I. This last requirement is needed for clear identification of restriction digest banding patterns in agarose gels, resulting from the action of site-specific endonucleases on test DNA substrates.
  • ER2745 and ER2746 were constructed by standard Plvir transduction. These strains provide alternative host backgrounds with differing advantages, both useful for the present goal of identifying cassette clones in pLT7K that cause damage to DNA when expressed.
  • ER2745 was constructed in one step from an existing strain.
  • the existing strain, ER2566, was deficient in all known endogenous restriction systems (enabling efficient cloning), did not express ⁇ -galactosidase, and expressed T7
  • RNA polymerase under lad control from a chromosomal location (not an inducible prophage). It also lacked Endonuclease I, the major nonspecific nuclease of E. coli, and so would be useful for visualizing restriction enzyme activities in crude extracts.
  • the dinD indicator was introduced into this strain by PI transduction from strain ER1992 of Fomenkov, supra (1995)), to form ER2745.
  • ER2746 was constructed in three steps from an existing strain.
  • the existing strain, ER2418 had the desirable property of relatively high induced competence, a property shared by many lined derived from E. coli K12 but not present in lines derived from E. coli like ER2745.
  • T7 RNA polymerase was introduced in two transductional steps: ER2418 x (P1(ER2556) -> TetR (Pro- KanR) to form ER2740; then ER2740 x P1(ER2553) -> Pro+ (KanS TetS Lac- T7RNAP+) to form ER2744. Finally, a dinD indicator allele was introduced into ER2744 from ER2170.
  • Amplified cassettes were then digested with 20 units Xbal and 1 unit Xhol (New England Biolabs Cat. Nos. 145 andl46, Beverly, MA) in IX NEBuffer 2 for 1 h at 37°C. Digested fragments were ligated overnight at 16°C with doubly-digested, dephosphorylated pLT7K. Dephosphorylation was for 1 h at 37°C with shrimp alkaline phosphatase (Amersham #E70092Y); ligation was with NEB Catalog No.
  • the clone library thus recovered under conditions that repress expression of the integron cassettes (40°C -IPTG) to assure viability can then be scored for functional report.
  • Replica plating onto Xgal plates and incubation under semi- inducing (30°C) or inducing (30°C +IPTG) conditions will allow identification of colonies that express DNA damaging functions. Some of these will be restriction enzymes. Individual colonies can then be recovered from master plates that have not been subjected to the damaging condition, to assure recovery of the original sequence.
  • D Assessment of clone identity
  • the DNA damage screen can allow identification of RM genes (Fomenkov, supra (1995); Fomenkov, supra (1994)). However, other sorts of genes will also be obtained; for example, a smgle-strand specific nuclease was among the genes recovered using the Endo-Blue method (Fomenkov, supra (1994))
  • Three procedures can be used to identify RM genes. In the first, cells are induced to express the cassette-encoded genes, crude extracts are made, these extracts are used to digest standard target DNAs, and enzymatic activity is detected by production of discrete bands on agarose gels. In the second, clones are briefly induced to express the cassette-encoded gene, then the whole cells are subjected to pulse-field gel analysis. Discrete bands will result from digestion of the chromosomal DNA of the clone-bea ⁇ ng cells. In the third approach, sequencing of clones to allow classification by homology searches
  • Clones positive m the DNA-damage screen will be grown under non- mducing conditions to late log phase, and shifted to the inducing condition for four hours. This procedure was successful in allowing expression of an amount of Pad similar to that expressed in the native host, P. alcaligenes (D. Byrd, personal communication). Cells are collected by cent ⁇ fugation, resuspended in buffer, lysed by lysozyme-EDTA treatment, clarified by cent ⁇ fugation
  • the substrate DNA is obtained by first embedding whole cells in agarose plugs. DNA is released from the cells in situ by means of a series of enzymatic treatments and washes that degrade the cell wall. The restriction endonuclease is then incubated with the plug; this usually takes several hours, since the enzyme must permeate the agarose and the remnants of the previous digestions.
  • the reestriction nuclease digestion step can be bypassed by inducing expression within the cell, before agarose is added.
  • the candidate clones are known to damage DNA in vivo in regulated manner. Accordingly, a banding pattern should be identifiable using the chromsomal DNA of the cells in which expression of the enzyme is induced. Pad will again be used as a test case. Notl will also be used, since the pattern expected for a total chromosomal digest is already well-known.
  • Critical steps are: quenching endogenous DNA degradation (especially exonuclease activity) at harvest and during the agarose-embedding process; the length of the induction; and the degree of induction.
  • Controls include: positive control, standard digestion of the host DNA embedded in agarose plugs with purified Pad and Notl; and negative control, samples of the host containing the empty vector, treated in parallel with the experimental samples.
  • Improvements in the strain used for this part of the survey include introduction of a recD mutation, which would inactivate the major ATP-dependent double-strand exonuclease of the cell; and introduction of an xth mutation that would inactivate the major ATP-independent double-strand exonuclease.
  • a triply nuclease-deficient strain (endA xth recD) should be viable but may not stably maintain the plasmid (Niki, et al., supra (1990)).
  • Genes obtained can be sequenced. To reduce redundant sequencing efforts, restriction digestion and finge ⁇ rinting of large numbers of candidates can be carried out. The recovered genes into sets with similar finge ⁇ rints, and two of each are sequenced. A minimum of three-fold sequence coverage is usually required in order to have sufficient confidence to carry out preliminary homology searches.
  • Sequencing can be conducted efficiently using the newly available Tn7- based transposition system, GPSTM-1 (New England Biolabs Catalog No. 1700, New England Biolabs, Inc., Beverly, MA).
  • GPSTM-1 New England Biolabs Catalog No. 1700, New England Biolabs, Inc., Beverly, MA.
  • This system enables introduction of primer-binding sites at random locations in plasmids of interest, rapid mapping of the location of the insertion by digestion with rare-cutters that cleave within the transposon, and sequencing of the insertions within the fragment of interest.
  • These target molecules About 20% of transposon insertions will be found within the sequence of interest. No more than 6 suitable insertions are needed in most cases, since cassettes are normally smaller than 2 kb.
  • Methyltransferases are recognizable by bioinformatic methods, since conserved motif elements are always present (see above).
  • Pad and Pmel two enzymes that should be recoverable by the present method, Pad and Pmel, are not adjacent to genes similar to any modification methyltransferase, and indeed so far no protective methyltransferases have been identified in the original hosts. Since these enzymes recognize AT-rich 8-base sites and the host organisms contain GC- rich genomes, host protection may be achieved by means of absence of sites.
  • candidate type II endonuclease genes of special interest will be solo ORFS with no database hits. Candidates adjacent to identifiable methyltranferase genes will be also retained, as will potential isoschizomers, which could have other desirable properties such as those affecting stability.
  • segments are then made into a multiple alignment, for example using the program MEGALIGN (DNASTAR, Madison Wisconsin) and preferably the CLUSTAL method of alignment within it.
  • MEGALIGN DNASTAR, Madison Wisconsin
  • CLUSTAL CLUSTAL method of alignment within it.
  • Segments thus identified can be grouped into families, for example by means of the Phylogeny facility in the MEGALIGN program, and bushy groups, in which there are many interior branches, are chosen as repeat families. These additional families should direct the design of oligonucleotides for use as probes or primers during application of the method.
  • a function of interest is identified in a taxon related to the model organism of interest. This can be for example ability to adhere to a particular tissue, for example red blood cells or the root hairs of plants.
  • a relatively large (>50 members) and diverse collection of isolates within the taxon of interest are collected.
  • the diversity of these isolates is characterized by isolation from locations spanning the extremes of the organism's distribution; these extremes may include spatial (geographic) distribution, thermal tolerance, salt tolerance, pH tolerance, O 2 partial pressure tolerance or requirement or host organism identity.
  • the members of this collection are screened for the presence of the function of interest and its specificity. In this example, it may be done by testing for hemagglutination ability, with red blood cells of sheep, cows, rabbits, pigs, goats, frogs, and humans as examples of different specific targets, or may be tested with one type of red cell in the presence of different mono- or disaccharides, or following various treatments that alter the nature of the red cell surface.
  • the function is identified as variable in the way that is expected of cassette-encoded functions if one or both of two conditions obtains. First, a large fraction (>10%) is different from the rest, in whether the function is present or absent.
  • the specificity of the function varies: for example, some agglutinate sheep red cells, others goat red cells. This criterion is best satisfied if the number of specificities identified is large, for example >4 different specificities in a collection of 50 isolates.
  • Variable functions can also be identified by immunological procedures, for example ELISA assays employing sera from animal or human populations of interest, or monoclonal antibodies recognizing variable epitopes in a compound of interest (e.g. a polypeptide); or by cytotoxicity assays, for example employing tissues of different physical or phylogenetic origins; or assays testing inhibition or stimulation of cellular processes such a DNA synthesis or cAMP hydrolysis directly or indirectly, in a context of tissue- or organism-specific effects; or tests of growth on or transformation of varied potential sources of carbon, nitrogen, or energy; or tests of growth in the presence of or inhibition of varied antimicrobial compounds.
  • immunological procedures for example ELISA assays employing sera from animal or human populations of interest, or monoclonal antibodies recognizing variable epitopes in a compound of interest (e.g. a polypeptide); or by cytotoxicity assays, for example employing tissues of different physical or phylogenetic origins; or assays testing inhibition or stimulation of
  • a preliminary test of the suitability of the method may be carried out by colony PCR, by inoculating a series of small samples of culture medium (for example in microtiter well plates) with portions of isolates of the taxon to be examined (reserving another portion for storage), growing them, boiling them, and carrying out PCR as in Example 1, Part C2.
  • Other primers designed based on these or other repeat families identified from the literature or in step 1 can also be used. Positive isolates identified at this step by the appearance of one or more PCR product are then carried to the next step. 4 ) Cassette isolation
  • DNA preparations from positive isolates is subjected to PCR on a larger scale, employing primer pairs with suitable restriction enzyme cloning sites at the ends as in Example 2: SEQ LD NO:86 with SEQ LD NO:90; SEQ LD NO:86 with SEQ LD NO:91; SEQ LD NO:87 with SEQ LD NO:90; SEQ LD NO:87 with SEQ LD NO:91; SEQ LD NO:88 with SEQ LD NO:90; SEQ LD NO:88 with SEQ LD NO:91; SEQ LD NO:89 with SEQ LD NO:90; SEQ LD NO:89 with SEQ LD NO:91 (see Table 2). Additional primer pairs designed based on additional repeat families may also be designed. Amplification conditions may be adjusted depending on the pairs used.
  • the PCR fragments are digested with Xhol and Xbal if the primers of Example 2 and pLT7K are used; other primers can be used including primers suitable for use with a derivative of pLT7K or similar plasmid carrying other restriction sites at the cloning site.
  • a strain suitable for recovery of cassettes will be one not expressing the function of interest, but in which its presence can be sought.
  • hemagglutinin genes should be expressed in a strain not itself expressinga hemagglutinin that would interfere with the survey.
  • LE392 is an example of an E. coli strain that does not express hemagglutinin activity.
  • the T7 genel construct would need to be introduced into LE392; or alternatively, strains such as ER2645, ER2746, ER2566 or ER2744 could be used if they were shown to lack hemagglutinin activity.
  • the strain may be customized to facilitate expression or report of functionality, for example by expressing a protein export system capable of exporting a class of hemagglutinins sought (eg. fimbriae). 7 ) Cassette identification
  • hemagglutination In the case of hemagglutination, a functional assay is available, so colonies or pools of colonies can be tested for hemagglutination in microtiter wells, following induction of expression as in Example 2.
  • Another method of identification would be to design degenerate primers specific for motifs found in particular classes of expected proteins, for example fimbriae, pili, or outer membrane proteins, and use them to perform PCR on colonies or pools of colonies either alone or in combination with PCR primers specific for the flanking repeats, as described in example 2.
  • expected proteins for example fimbriae, pili, or outer membrane proteins
  • Colonies specifically exhibiting properties expected of desired gene cassettes would then be characterized by methods appropriate to the particular function identified, for example, in a hemagglutination test by competition with small molecules such as various sugars; by its sensitivity to various treatments such as iodination, heating, freezing, treating with acid, alkali, or alkylating agents or with proteases or nucleases ;and by obtaining the sequences of the genes and determining the properties of cells with genes carrying mutations of various sorts including fusions to other reporter molecules such as alkaline phosphatase, beta galactosidase, green flourescent protein or various epitope tags, or obtaining purified preparations of encoded proteins by standard purification methods or by affinity purification by means of polypeptide tags.
  • a hemagglutination test by competition with small molecules such as various sugars; by its sensitivity to various treatments such as iodination, heating, freezing, treating with acid, alkali, or alkylating agents or with proteases or nucle

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Abstract

La présente invention concerne le clonage direct de gènes intacts avec une forte probabilité que l'orientation de l'expression soit connue par avance et avec une faible probabilité pour qu'elle soit associée à des gènes étrangers éventuellement toxiques. En particulier, l'invention concerne l'obtention de gènes codés dans des cassettes d'ADN composées de séquences répétitives flanquant des cadres de lecture ouverts variables. L'invention concerne l'obtention de tels gènes codés en cassette au moyen d'olignucléotides s'hybridant aux éléments répétés, le clonage desdits gènes et leur expression. L'expression peut faire intervenir des vecteurs étroitement régulés et des souches utiles mises au jour. L'invention concerne également des procédés d'identification de gènes à endonucléase de restriction et à méthyltransférase d'ADN en l'absence d'informations préalables sur les séquences ou les spécificités biochimiques de ces enzymes.
PCT/US1999/013295 1998-06-12 1999-06-11 Decouverte de genes d'enzymes de restriction WO1999064632A1 (fr)

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EP1754141A2 (fr) * 2004-06-02 2007-02-21 New England Biolabs, Inc. Fonction d'inference a partir de donnees de sequencage en aveugle
US8227231B2 (en) * 2005-08-04 2012-07-24 New England Biolabs, Inc. Restriction endonucleases, DNA encoding these endonucleases and methods for identifying new endonucleases with the same or varied specificity

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6387681B1 (en) 1999-10-28 2002-05-14 New England Biolabs, Inc. Method for cloning and expression of NHEI restriction endonuclease in E. coli.
EP1754141A2 (fr) * 2004-06-02 2007-02-21 New England Biolabs, Inc. Fonction d'inference a partir de donnees de sequencage en aveugle
EP1754141A4 (fr) * 2004-06-02 2008-01-02 New England Biolabs Inc Fonction d'inference a partir de donnees de sequencage en aveugle
US8227231B2 (en) * 2005-08-04 2012-07-24 New England Biolabs, Inc. Restriction endonucleases, DNA encoding these endonucleases and methods for identifying new endonucleases with the same or varied specificity
EP2540823A1 (fr) * 2005-08-04 2013-01-02 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
EP2574668A1 (fr) * 2005-08-04 2013-04-03 New England Biolabs, Inc. Nouvelles endonucléases de restriction, ADN codant ces endonucléases et procédés pour identifier de nouvelles endonucleases avec la spécificité identique ou variée
US8685689B2 (en) 2005-08-04 2014-04-01 New England Biolabs, Inc. Restriction endonucleases, DNA encoding these endonucleases and methods for indentifying new endonucleases with the same or varied specificity

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