Patent Application
20090068644
The invention relates to the identification and preparation of mismatch specific endonucleases having a high activity and sensitivity and a broad substrate specificity.
In the beginning of the last century, the discovery of the possibility to induce mutations within the DNA by radiations or chemicals has brought a considerable hope to understand the gene function in vivo. Since then, mutagenesis and natural sequence variation have been widely used to identify new functions, genes corresponding to a specific function as well as active sites within a specific protein.
A critical aspect in implementing this approach, in particular in the case of point mutations, is the choice of mutation detection methods that are designed to screen large stretches of DNA without reducing diagnostic sensitivity or specificity, while at the same time providing information about the location of the mutation. Among the most used tools are methods based on imperfectly matched DNA that could be created in vitro by denaturation and renaturation of two DNA molecules. Mismatches are detected in these heteroduplexe molecules using chemicals like groove binders or molecules that can cleave specifically single strand DNA at the mismatch site. Alternatively, single strand specific endonucleases have been used to cleave the DNA at the mismatch site. Most of the endonucleases described this far belong to S1/P1 class of nucleases.
Nucleases such as S1, P1 and mung bean nuclease, belonging to a same family designated as: “S1/P1 nucleases family”, or as: “S1 nucleases family” are known to cut DNA at regions of single-strandedness. These nucleases, however, have acid pH optima in the range of 4.0- 5.0.
This is disadvantageous for mismatch detection, since low pH values favour DNA depurination and destabilizes DNA duplexes, leading to non-specific DNA degradation, and reducing the sensitivity and specificity of the detection.
A few years ago, OLEYKOWSKI et al. (Nucleic Acids Res, 26, 4597-4602, 1998) detected in extracts from various plants a mismatch endonuclease activity having a neutral pH optimum (around pH 8) and performing a single-strand cut on the 3′side of a mismatch site. These authors reported that this mismatch endonuclease activity was associated with mannosyl glycoproteins in extracts of alfalfa sprout, asparagus, celery and tomato. The enzyme from celery, named CEL I, was purified from celery stalks by successive steps of ammonium sulfate precipitation, binding to a concanavalin A-agarose column and elution by [alpha]-d+-mannose, binding to a phosphocellulose column and elution by a linear gradient of KCl, and fractionation by size exclusion chromatography. The preparation of CEL I thus obtained contained several protein bands of 34-39 kDa.
YANG et al., (Biochemistry, 39, 3533-3541, 2000), and PCT application WO 01/62974 describe an improved purification of CEL I by use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of CEL I with endogenous lectins. These documents also disclose the cloning of CEL I cDNA. On the basis of sequence data, CEL I was assigned to a sub-family of the S1/P1 nucleases family, and several potential homologues encoded by the genes BFN1 of Arabidopsis (GenBank nucleotide AY040016), ZEN1 of Zinnia (GenBank (nucleotide) AB003131), and DSA6 of daylily (GenBank (nucleotide) AF082031) were identified.
CEL I endonuclease activity has been shown to be highly specific for base-substitution mismatches and for mismatches resulting from insertion/deletion events, and to be independent of the flanking sequence context. It is thus useful as a mutation detecting reagent in various methods involving mutational screening. The CEL I mismatch detection system is a simple assay that requires PCR amplification of the target sequence, denaturation and annealing to allow formation of heteroduplexes between the wild type and the mutant allele, enzymatic mismatch cleavage, and analysis of the product by gel electrophoresis. It is advantageous over other popular mismatch detection systems, like denaturing HPLC, because of its specificity and sensitivity for detection of mismatches in large stretches of DNA.
By way of example, OLEYKOWSKI et al. and YANG et al. (publications cited above) report its use to detect sequence alterations in the human BRCA1 gene, and SOKURENKO et al. (Nucl. Acids Res., 29, el 11, 2001) disclose its use to detect mutations and polymorphisms in large regions of genomic DNA. CEL I is also used for high-throughput screening in TILLING (Targeting Induced Local Lesions IN Genomes), in which chemical mutagenesis is followed by screening for point mutations, or for detection of polymorphisms in natural populations, also called “Ecotilling” (COMAÏ et al, Plant Journal, 37, 778-786, 2004). It has been used in plants (COLBERT et al., Plant Physiology 126, 480-484, 2001; TILL et al., Genome Research 13, 524-530, 2003; PERRY et al., Plant Physiology 131, 866-871, 2003) as well as in animals such as zebrafish (WIENHOLDS et al., Genome Res., 13, 2700-2707, 2003). PCT application WO 03/066809 proposes to use CEL I in a method for reassorting sequence variations among related polynucleotides, named “Genetic Reassortment by Mismatch Resolution” (GRAMMR).
CEL I has however the disadvantage of having an efficiency of cleavage that varies from one mismatch to another: in the case of a DNA loop with a single nucleotide insertion, OLEYKOWSKI et al. (Nucleic Acids Res. 1998 Oct 15;26(20):4597-602) report that CEL I substrate preference is G>A>C>T; in the case of base-substitution mismatches, CEL I substrate preference is C/C≧C/A˜C/T≧G/G>A/C˜A/A˜T/C>T/G˜G/T˜G/A˜A/G>T/T. Its efficiency is significative on the mismatches C/A, C/C, C/T, G/G. A decrease of the activity is observed for the others, and it is almost inefficient on the mismatch T/T. This variation in the efficiency of cleavage may result in a lower accuracy in the detection of some mutations when the detection of one allele in a pool of DNA is required.
Another inconvenient limiting the use of CEL I is the low yield of the available purification methods. OLEYKOWSKI et al., starting with 7 kg of celery stalk containing about 350 g protein obtained 3 ml CEL I at 0.1 μg/μl; The purification procedure disclosed by YANG et al. and in PCT WO 01/62974 results in 5μg of purified CEL I with a specific activity of 3.1×107 CEL I units/mg protein starting from 105 kg of celery stalk.
It has been proposed, in order to obtain larger amounts of CEL I, to produce it by recombinant DNA technology. PCT application WO 03/066809 proposes a large list of potentially suitable vectors and host cells including almost any known prokaryotic or eukaryotic expression systems; however, the only expression system actually disclosed in this document is a tobamovirus-based vector. The construct resulting from cloning in said vector of the cDNA of CEL I fused to a sequence encoding a 6-Histidine tag has been used to infect tobacco plants. Recombinant CEL I was recovered from intracellular fluid of the infected plants and purified by metal affinity chromatography on nickel-NTA resin. PCT WO 03/066809 is silent about the yield of purified enzyme. It indicates that its activity in a GRAMMR reaction is similar to the one of the native enzyme purified from celery. One of the inconveniences of this system is that viruses tend to recombine, loosing part or completely the expressed gene. This leads to the production of truncated forms in addition of the full-length CEL I, decreasing the specific activity of the enzyme.
PCT Application W02004/035771 relates to a method for producing CEL I in yeast. To this effect, a synthetic gene encoding CEL I was constructed by modifying the native DNA sequence of CEL I according to codon usage in yeast. This document indicates that the recombinant CEL I produced by this synthetic gene is able to recognize all possible mismatch combinations, and exemplifies the recognition and cleavage of the mismatch A/A. On the other hand, it is silent about the mismatch preference of said recombinant CEL I.
It appears from the above that the currently available methods for producing recombinant CEL I do not provide any significant improvement over the production of native CEL I from celery. Further they do not address the problem of the substrate preference of native CEL I.
Also, it would be desirable to have other endonucleases able, like CEL I, to cleave single base pair mismatches in heteroduplex DNA templates under neutral pH, but which have a different mismatch preference, or preferably, that cleave equally well all mismatches. However, such endonucleases have not been identified until now.
The existence of CEL I-like endonuclease activities has been reported in many plants (cf. for instance OLEYKOWSKI et al., 1998 cited above). However, the enzymes responsible for these activities have not been characterized, their biochemical properties, such as substrate preference have not been studied, and their sequences have not been identified. On the other hand, structural homologues of CEL I have been identified in silico (YANG et al., cited above; TILL et al. Nucleic Acids Res., 32, 2632-41, 2004). Three of them (BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily) have been reported to be involved in plant senescence (PEREZ-AMADOR et al., Plant Physiol. 122, 169-180 2000). However, they have not been purified and remain uncharacterised biochemically: in particular, their efficiency to recognise mismatches in heteroduplex DNA in vitro has not been tested. An endonuclease called SP, that has been assigned to the S1/P1 family on the basis of its activity, and that has a neutral pH optimum has been purified from spinach. Like CEL I, this enzyme cleaves insertion/deletions and base-substitution mismatches; however it does not recognize those containing a guanine residue (OLEYKOWSKI et al. Biochemistry, 38, 2200-5, 1999).
Seeking for alternative methods to obtain recombinant CEL I, the inventors have tried to express it in planta via Agrobacterium mediated transient expression.
They have expressed recombinant CEL I in agroinfiltrated tobacco leaves, and have purified it from the leave extract by ammonium sulphate precipitation. They have found that, surprisingly, they obtained a very high yield of recombinant CEL I with a high activity, and furthermore, that said recombinant CEL I preparation recognises the mismatches with a broader specificity and a higher sensitivity than the preparations of CEL I known in the prior art, allowing a clear detection even of mismatches, such as T/T, which were deemed as poorly recognized by CEL I.
In view of these results, the inventors have had the idea to use this method for screening endonucleases identified in silico as belonging to the S1/P1 family, by testing their activity in vitro.
The instant invention thus provides a simple and rapid method to obtain great quantities of endonucleases, in particular S1/P1 nucleases, from a small quantity of starting material, and also provides a method for evaluating the activity in vitro of candidate endonucleases, in particular in order to identify mismatch specific endonucleases.
A mismatch specific endonuclease is defined herein as an endonuclease which is able to cleave specifically all the base-substitution mismatches (i.e., A/A, GIG, C/C, T/T, A/G, A/C, G/T, C/T, G/A, C/A, T/C, T/G), as well as insertion/deletions of one or more nucleotides.
An object of the present invention is thus a method for producing a recombinant endonuclease, wherein said method comprises:
Said plant cells may be part of a cell suspension, or of a tissue or organ culture. In this case, the enzyme can be collected from the supernatant and/or from the cultured cells or tissue or organ. Preferably, they will be part of a whole plant or of an organ detached therefrom; in this case, the transient transformation with the Agrobacterium strain will be performed by agroinfiltration.
Agroinfiltration is a transient expression method based on the delivery of Agrobacteria containing a gene of interest into intact plant tissue.
A DNA construct comprising a gene of interest is cloned into a binary vector and transferred into a chosen Agrobacterium strain, and the transformed Agrobacteria are grown to log phase or to saturation and collected in the same way as for conventional Agrobacterium mediated transformation. Classically, agroinfiltration is performed by applying a suspension of the transformed Agrobacterium cells either by injection into an organ (generally the leaves) of the chosen plant using a syringe without a needle, or by vacuum infiltration for a few minutes. After release of the vacuum, the organ, or the entire plant is placed in a growth chamber. The expressed protein of interest is extracted from the infiltrated organ, usually one to four days post-infiltration. Agroinfiltration protocols are disclosed in various publications, for instance, KAPILA et al., (Plant Sci.122, 101-108, 1997); BENDAHMANE et al., (Plant Cell, 11, 781-792, 1999); SCOFIELD et al., (Science. , 274, 2063-5, 1996); TANG et al., (Science., 274, 2060-3, 1996); MARILLONNET et al., (Proc Natl Acad Sci USA, 101, 6852-7, 2004); WROBLEWSKI et al., (Plant Biotech. J., 3, pp. 259-273, 2005).
The classical protocols used for Agrobacterium mediated transient expression, and in particular for agroinfiltration can be used in the practice of the present invention. A large choice of Agrobacterium strains, of binary vectors, and of regulatory elements controlling the expression of the gene of interest, is available, and one of skill in the art can choose among them the more appropriate, for instance according to the host plant that one intends to use.
In the experimentations disclosed in the Examples below, the inventors have used a pBIN19-derived binary vector, pBIN61, and agrobacterium strain C58C1 harboring the hypervirulence pCH32 plasmid, and the cDNA or the genomic coding sequences have been expressed under CaMV 35S promoter. However, other binary vectors, other strains of Agrobacterium and other constitutive or inducible promoters can be used with the same result.
Advantageously, agroinfiltration will be performed in the leaves, which can optionally be detached from said plant immediately before, or immediately after, the infiltration.
Host plants that can be used in the method of the invention include any plant that is compatible with Agrobacterium transformation. Preferred plants include in particular those of the genus Nicotiana, in particular Nicotiana benthamiana and Nicotiana tabacum.
According to a preferred embodiment of the invention, said endonuclease is isolated from an agroinfiltrated plant organ, in particular an agroinfiltrated leaf by a process comprising the following steps:
Said protein precipitate is resuspended in an appropriate buffer, for instance a buffer comprising Tris HCl (pH 8), PMSF and 10% glycerol. It can be used directly, or stored at −80° C. until use.
Alternatively, the total extract obtained after the first step indicated above can be used as such, without performing precipitation steps with ammonium sulfate.
Optionally, the endonucleases produced by the method of the invention can be further purified, by any appropriate method known in itself, such as column affinity purification where CEL I is tagged with a tag that has an affinity to a specific component in the column. According to a preferred embodiment, CEL I of the invention is provided with a 6-Histidine tag, and purified by metal affinity chromatography on nickel-NTA.
The invention also provides a method for testing whether a candidate endonuclease is a mismatch-specific endonuclease, wherein said method comprises:
a) producing said candidate endonuclease under recombinant form by the method of the invention, as defined above;
b) testing said recombinant endonuclease for its ability to degrade single stranded DNA;
c) testing said recombinant endonuclease for its ability to cleave a test heteroduplex DNA fragment at a pre-defined mismatch site;
d) testing said recombinant endonuclease for its ability to cleave heteroduplex DNA fragments carrying all the types of mismatches (i.e., the base substitution mismatches A/A, GIG, C/C, T/T, A/G, A/C, G/T, C/T, G/A, C/A, T/C, T/G, as well as insertion or deletion mismatches) If an endonuclease passes the tests of steps b) c) and d) (i.e., if it is able to degrade single stranded DNA, to cleave a test heteroduplex DNA fragment at a pre-defined mismatch site, and to cleave heteroduplex DNA fragments carrying all the types of mismatches), it is considered as a mismatch-specific endonuclease.
The invention also provides a method for screening mismatch-specific endonucleases, wherein said method comprises:
a) producing candidate endonucleases under recombinant form by the method of the invention, as defined above;
b) testing said recombinant endonucleases for their ability to degrade single stranded DNA;
c) testing the recombinant endonucleases able to degrade single stranded DNA, and testing them for their ability to cleave a test heteroduplex DNA fragment at a known and well characterized mismatch site;
d) selecting the recombinant endonucleases able to cleave a test heteroduplex DNA fragment at a known and well characterized mismatch site, and testing them for their ability to cleave heteroduplex DNA fragments carrying all the types of mismatches.
e) selecting the recombinant endonucleases that pass the tests of steps b) c) and d).
According to a preferred embodiment of the above defined methods, they further comprise a step consisting of testing said endonuclease(s) for its (their) sensitivity by testing their ability to detect a mutant allele in a DNA pool, in presence of an excess of the wild-type allele, and selecting the endonucleases that are able to detect said mutant allele in the presence of at least 9-fold excess of the wild-type allele (i.e., one mutant allele in a pool of 10), preferably in the presence of at least 14-fold excess, still more preferably in the presence of at least 19-fold excess, and by order of increasing preference, 29-fold excess, 39-fold excess, 49-fold excess, 59-fold excess of the wild-type allele.
Preferably, the above defined tests are performed in a reaction mixture having a pH from 7 to 8, advantageously from 7.4 to 7.8, and containing from 5 to 20 mM, advantageously 10 mM MgCl2. Advantageously, said reaction mixture also comprises from 0.5 mM to 2 mM, and preferably 1 mM DTT. The inventors have also observed that addition of PEG-8000, at from 2% to 10% (w/v), and in particular 5% of the final reaction mixture, increased the global activity of the endonucleases.
The candidate endonucleases that can be tested by the methods of the invention, can be found among those of the S1/P1 family. In the PFAM database (BATEMAN et al., Nucleic Acids Res. 32, D138-41, 2004), this family is designated as PFAM 02265. One can for instance use the profile HMM (Hidden Markov Models) built from PFAM 02265 as a probe to screen the available DNA sequence data banks, using the HMMER software, to identify candidate endonucleases in different plants.
Alternatively, the InterPro IPR003154 code (corresponding to S1/P1 nucleases) can be used for screening the content of databases, for example using the following address:
http://www.ebi.ac.uk/interpro/ISpy?ipr=IPR003154
Screening the Trembl/Swissprot database with this code identified 43 proteins (Table 1).
This analysis is preferably completed by performing a Blast on the databases (blastp or tblastn), using a reference protein sequence (for example the CEL I sequence), and selecting the best hits.
The examples below will describe with more details the testing of five candidate endonucleases from Arabidopsis thaliana.
The inventors found that one of these endonucleases, which is represented in the annexed sequence listing under SEQ ID NO: 2 (the corresponding DNA sequence is represented under SEQ ID NO: 1), and which corresponds to BFN1 of Arabidopsis thaliana, has a different specificity, and a far greater sensitivity than CEL I.
This discovery of the properties of BFN1 as a mismatch-specific endonuclease, which were unknown until now, allows to propose its use as a mutation detecting reagent, for detecting mismatches resulting from base substitutions, as well as from insertion/deletions of one or more nucleotides.
BFN1, as well as any mismatch-specific endonucleases that can be identified according to the method of the invention can be obtained in great quantities by the method of production of endonucleases.
The present invention also encompasses recombinant endonuclease preparations obtainable by the method of production according to the invention. These are in particular recombinant CEL I preparations and recombinant BFN1 preparations.
The recombinant CEL I preparations of the invention have a different mismatch specificity and a higher sensitivity than the CEL I preparations of the prior art. The recombinant CEL I preparations of the invention, have the following mismatch preference: T/G˜A/G˜G/G˜G/T˜T/T˜G/A≧A/A˜C/C≧T/C˜C/T>A/C˜C/A, while the mismatch preference of the CEL I preparations of the prior art is C/C≧C/A˜C/T≧G/G>A/C˜A/A˜T/C>T/G˜G/T˜G/A˜A/G>T/T. The recombinant CEL I preparations of the invention can recognize a mutant allele in the presence of a 23-fold excess of the wild type allele, while the CEL I preparations of the prior art do not efficiently recognize a mutant allele when diluted over a 8-fold dilution.
The recombinant BFN1 preparations of the invention have also a different mismatch specificity than both the CEL I preparations of the prior art and the recombinant CEL I preparations of the invention. The recombinant BFN1 preparations of the invention, have the following mismatch preference: G/G˜G/A˜A/G˜G/T˜T/G>T/T˜A/A˜C/C˜T/C≧C/T˜A/C—C/A. The mismatch preferences for each of these enzymes are summarized in Table 2 below.
The recombinant BFN1 preparations of the invention further have a higher sensitivity than both the CEL I preparations of the prior art and the recombinant CEL I preparations of the invention. The recombinant BFN1 preparations of the invention can recognize a mutant allele in the presence of a 59-fold excess of the wild type allele.
The recombinant BFN1 or CEL I endonuclease preparations of the invention can be used, as a mutation detecting reagent, in any method involving mismatch screening, as mentioned above. They are particularly advantageous in genotyping, in TILLING, High-throughput TILLING, Ecotilling, GRAMMR, etc.
Methods for using the endonuclease preparations of the invention involve basically the same steps as those of the prior art, i.e., PCR amplification of the target sequence, denaturation of the amplification product and annealing to allow formation of heteroduplexes between the wild type and the mutant allele, cleavage of the heteroduplexes by the endonuclease, and analysis of the cleavage products. A mix of different endonucleases can advantageously be used for cleaving the heteroduplexes when performing these methods.
Due to their high sensitivity, the endonuclease preparations of the invention also enable to perform high-throughput methods for identifying mutations in a sample. For example, endonuclease preparations according to the invention can be used to perform methods such as described in WO 01/75167, with a far larger number of samples, since it is possible to pool many samples together for analysis.
According to the invention, the endonuclease preparations described above can be used to screen one or more mutations in a target gene, in a large number of samples from any organism or cell-line derived therefrom, by performing the following steps:
a) amplifying said target gene or a part thereof from each individual of said population,
b) ordering said amplification products in a 2- or 3-dimensional matrix, comprising lines, rows (2-D matrix) and columns (3-D matrix),
c) pooling said amplification products such as to obtain different pools, each pool representing a row, a line or a column of said matrix,
d) adding to each pool a reference amplification product obtained from a non-mutated gene, and incubating such pools in conditions enabling the formation of heteroduplexes,
e) incubating each pool with an endonuclease preparation according to the invention, and
f) detecting the presence of heteroduplexes in said incubated pools.
Alternatively, the above method can be performed by first ordering the samples in the matrix, then pooling them (with addition of the reference), and performing the amplification, incubation and detection steps.
Depending on the numbers of samples to screen, the matrix can be a 2 or 3-D matrix. For example, if 576 samples are to be screened, a 24×24 matrix can be used. If 13824 samples are to be screened, a 3-D matrix may thus be more appropriate (24×24×24). Only 72 reactions would be needed to screen this population, and the mutated genes would be individualized by the column, row and line pools to which it belongs.
The reference amplification product corresponds to an amplification product obtained from the reference gene (compared to which mutations are searched), amplified with the same primers as the target gene in the population. It is important to add a reference amplification product. Indeed, although it is very unlikely that all samples harbour the exact same mutation as compared to the reference gene, this will ensure that heteroduplexes may be formed if the pool contains a target gene that harbours a mutation.
The present invention will be further illustrated by the additional description which follows, which refers to examples illustrating the preparation and properties of recombinant CEL-I endonuclease, the testing of five candidate endonucleases from Arabidopsis thaliana, and the identification of BFN1, (hereinafter also designated as ENDO1) as a mismatch specific endonuclease . It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in any way a limitation thereof.
On the top of the gel is indicated the homoduplexes size (661 bp). The arrow at 405 bp shows the fragment labeled with the FAM fluorochrome. The arrow at 256 bp shows the fragment labeled with the ROX fluorochrome.
1: D100; 2: D500; 3: D1000; 4: no enzyme.
A: Terese; B: rms1.13; C: T+rms1.13.
Cloning of the cDNA of CEL I
Extraction of RNA from Celery
Young leaf tissues (1 g) were ground in liquid nitrogen using a pestle and mortar. The powder was suspended in 10 ml Trizol (Gibco) extraction buffer. The suspension was mixed with 2 ml of chloroform and centrifuged at 12 000 rpm at 4° C. for 15 min. The supernatant was mixed with an equal volume of isopropanol and total RNA were precipitated by centrifugation at 12 000 rpm for 10 minutes at 4° C. The pellet was washed with 80% ethanol, air-dried and resuspended in 200 μl of DEPC water.
DNase treatment was carried out to hydrolyse DNA contaminating the RNA preparation. 10 μg of total RNA were incubated with 10 units of DNase following the manufacturer conditions (Promega).
The reaction was incubated at room temperature for 15 minutes then stopped by addition of EDTA to 25 mM final concentration and by heat inactivation of the DNase at 65° C. for 10 minutes.
CEL I cDNA Synthesis
Ten microliters of DNAse treated-RNA was used for first strand cDNA synthesis. First strand cDNA synthesis was primed with 2 picomoles of 20 mers oligo dT primer. The reaction mix of 50 μl (10 μl of 5× Superscript buffer [GIBCO-BRL], 5 μl of 100 mM DTT, 5 μl of 5 mM dNTP) was heated at 70° C. for 10 min and then cooled on ice. 1 μl of Superscript reverse transcriptase (200 units/μl; GIBCO-BRL) and 1 μl RNase inhibitor (37 units/μl; Pharmacia) were added and the reaction was incubated at 42° C. for 1 hour. PCR amplification was used to convert the first strand cDNA into double strand DNA by PCR amplification using two primers specific to 5′ and 3′ UTR of CEL I (see Table 3 below). Cloning of CEL I and expression in tobacco leaves Full length CEL I open reading frame was PCR amplified and inserted between the 35S promoter and the transcriptional terminator of CaMV in the binary vector pBin19 to create pBIN35S-CELI. Another construct pBIN35S-CELI8His was also constructed. pBIN35S-CELI8His is identical to pBIN35S-CELI except that a Histidine tag of 8 amino acids was inserted at C-terminal of CEL I protein. The oligonucleotides used to create pBIN35S-CELI and pBIN35S-CELI8His are indicated in table 3 below.
These constructs were transformed into Agrobacterium strain C58C1 carrying the virulence helper plasmid pCH32 (Hamilton et al. PNAS, 93(18): 9975-9979, 1996). pCH32 expresses VirG and VirE and was used to enhance T-DNA transfer. Agrobacterium cells were inoculated into 5 mL of L broth medium (Sambrook et al. 1989) supplemented with 50 μg/mL kanamycin and 5 μg/mL tetracycline and grown at 28° C. overnight. L broth medium (50 mL) supplemented with 50 μg/mL kanamycine, 5 μg/mL tetracycline was then inoculated with the 5-mL overnight cultures and grown at 29° C. for 2 days. Cells were precipitated and resuspended to a final concentration of 0.5 OD6 00 in a solution containing 10 mM MgCl2, 10 mM MES, pH 5.6, and 150 μM acetosyringone. The cultures were incubated at room temperature for 2 hr before agroinfiltration into Nicotiana benthamiana leaves.
The Agrobacterium suspension was injected in the leaves using a syringe without needle.
Agroinfiltrated Nicotiana benthamiana plants were incubated for at least 48 hours at 24° C., 16 hours of light, 60% of humidity. To test for the efficiency of agroinfiltration, plants were also agroinfiltrated with a construct expressing the green fluorescent protein (GFP). Before harvesting the leaves, the intensity of the expression of the GFP for each leaf was checked using an UV lamp. The plant leaves were harvested only if the GFP was expressed. Preparation of the protein from tobacco leaves by ammonium sulfate precipitation Agroinfiltrated tobacco leaves were harvested and weighted. Two grams of agroinfiltrated leaves were homogenized in 7 ml of buffer containing 0.1 M Tris-HCl pH8, 200 μM PMSF, 0.125 mM β-mercaptoethanol and 10% of glycerol and then centrifuged at 3000 g for 25 minutes to pellet the cellular debris. To the supernatant 100% ammonium sulfate was added to the final concentration of 30%, and the samples were then incubated on ice for 1 hour, and centrifuged at 30 000 g for 30 minutes at 4° C. to pellet proteins that precipitate at 30% ammonium sulfate. To the supernatant 100% ammonium sulfate was added to obtain 80% final concentration and the samples were again incubated on ice for 1 hour and centrifuged as above to pellet the proteins that precipitate at 80% ammonium sulfate. The pellet containing the proteins precipitated at 80% of ammonium sulfate was resuspended in 600 μl of homogenization buffer. The protein concentration was determined by the kit Coomassie Plus Reagent Assay (Pierce). The homogenized pellet contains 141 g protein/μl. Thus, 8400 kg proteins were recovered from 2 grams of agroinfiltrated tobacco leaves. The homogenized pellet was diluted at 1 μg/μl in a buffer containing 50 mM Tris-HCl pH8, 10% glycerol and 100 μM PMSF, aliquoted and stored at −80° C.
Five grams of agroinfiltrated tobacco leaves were collected and homogenized in 15 ml iced buffer containing Na-Phosphate (100 mM), Tris HCl pH 8 (10 mM), NaCl (200 mM), Sodium methabisulfite (0.2%), PMSF (1 mM), βMercaptoethanol (10 mM). After homogenization, the sample was centrifugated at 6000 g (Beckman, rotor JA 20) for 10 minutes at 4° C. Imidazole (10 mM) was added to the supernatant and the pH was adjusted to 9 with NaOH. The solution was centrifugated at 42 000 g (Beckman, rotor JA 20) for 60 minutes at 4° C. The supernatant was mixed with 1 ml of Ni-NTA agarose (Quiagen) pre-equilibrated with homogenization buffer+10 mM imidazole pH9 (buffer B). The mix was homogenized for 2 hours at 4° C. to allow the protein to bind to the Ni-NTA agarose beads. The beads were packed in a 1 ml polypropylene column (Quiagen) and the resin was washed with 20 ml of the buffer B. The protein was eluted with 5 ml (5×1 ml) of buffer B+250 mM imidazole pH9. Aliquots of the fractions were kept to follow the activity of the enzyme during the purification. To avoid any inhibition of the enzyme activity by high concentration of imidazole, the eluted fractions were dialysed against 4 liters of buffer containing Tris-HCl pH8, 0.1 M, PMSF 100 μM and ZnCl2 2 μM overnight at 4° C. Thus, 1000 μg proteins were recovered. The homogenised pellet was diluted at 3 μg/μl in a buffer containing 50 mM Tris-HCl pH8, 10% glycerol and 100 μM PMSF, aliquoted and stored at −80° C. (Dilution D10 000).
The activity of the recombinant Cel I on degradation of single strand DNA was carried out as described previously (SUNG SC, LAKKOWSKI M Sr.(1962) J Biol Chem. 1962 Feb;237:506-11). Thirty micrograms of Dnase, Rnase and protease free Pea genomic DNA was incubated with 2 μg of protein extract in a buffer containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM DTT and 5% PEG-8000. To stop the reaction equal volume of 20 mM LaCL3 in 0.2N HCl was added. The samples were centrifuged at 21000 g for 40 min and the absorbance at 260 nm of the supernatant was measured using spectrophotometer to determine the amount of DNA that had become acid-soluble.
To test if the CEL I endonuclease produced in tobacco can recognize single point mutations, the activity of the recombinant CEL I preparation obtained by ammonium sulphate precipitation was tested on heteroduplex DNA from two clones that differ in a single point mutation: C-G to A-T transition (Bendhamane et al., Plant Cell, 11, 781-792, 1999). PCR was carried out on the two clones, using two oligonucleotides R21 and R22 (R21 5′ GAC ATA TGG ACT ACA GAA GCT TGG G 3′ SEQ ID N°8; R22 5′ GTT CAC GGG TCA CAT CAT GCA TTC C 3′ SEQ ID N°9). The PCR amplification and the reconstitution of heteroduplex DNA was carried out using the following program: denaturation for 2 min at 95° C. followed by 7 cycles with 20 sec at 94° C., Tm (55° C.) +3° C. to Tm −4° C. for 15s, −1° C. per cycle, gradient to 72° C. at 0.5° C. /sec and a an extension at 72° C. for 1 min, then 44 cycles with 20 sec at 94° C., Tm −5° C. for 30° C., gradient to 72° C. at 0.5° C./sec, and a extension at 72° C. for 1 min, a final extension at 72° C. for 5 min and a denaturation step at 94° C. for 10 min followed by a ramp to 40° C. for 20 sec and −0.3° C. per cycle.
The PCR products (a mix of wild type and mutant DNA, or just wild type or mutant DNA) were incubated with the CEL I preparation (stock solution at 1 μg/μl diluted at 1/100, 1/500, or 1/1000) as follows: For example, 10 μl of the PCR product (500 ng) was incubated with 2.5 μl of the reaction buffer (Hepes 10 mM, MgSO4 10 mM, Triton ×100 0.002%, KCl 10 mM) and 2.5 μl of the diluted CEL I preparation in a total volume of 25μl, for 30 minutes at 37° C. The reaction was stopped by 5 μl of EDTA 500 mM and the digestion products were analysed on a 3% agarose gel.
As shown on
PCR products obtained using fluorescently labelled primers and digested in the same way as above were analysed on acrylamide gel on ABI377 sequencer (
As shown in
These results show that the CEL I protein produced in planta is able to recognize mismatches in DNA.
To test whether the recombinant CEL I purified from tobacco could be used to detect single point mutations in pea, different pea rms and le mutants, characterised previously by Catherine Rameau (MORUS et al Plant Physiol 2001, 126 :1205-1213. ; RAMEAU et al, Plant Physiol 2002, 115 :458-467), were used as a test; rms 1.11 contains G---->A at different positions in the sequence of the gene; rms 1-12 contains G---->A; rms 1-13 contains G--->A. All were mutated for G---->A on the same gene rms I but at different positions.
To amplify the wild type and the mutant alleles of the rms loci (rms1-13, rms1-10, rms1-12) and le locus, fifty nanograms of pea genomic DNA was used as template. The primers used in this PCR amplification are summarised in Table 4.
The program of the PCR amplification was 94° C. 1 min, (94° C. 15s, 55° C. 15s, 74° C. 1 min, X35) 74° C. 7 min, 8° C. The PCR products were analysed on agarose gel and digested, as disclosed in Example 2 above, with different dilutions of recombinant CEL I preparation obtained by ammonium sulphate precipitation. As shown on
To test whether the recombinant CEL I prepared according to the invention cuts preferentially a type of mismatches, like the CEL I purified from celery, a series of mutants based on the Rx gene were created. For that purpose, we have designed different primers (called Rx-A, T, G or C) containing each, a different point mutation. Each of these primers allows to introduce at a given position of the Rx gene, one of the 4 bases (A, T , G or C). Heteroduplexes are created by mixing the amplification products obtained with the different primers.
The PCR mix (in total volume of 50 μl) contained template DNA (50 ng), dNTP (0.2 mM), 5 ,l PCR tampon Pfu (10 ×, Stratagene), primer Rx 21 (0.4 μM), primer Rx-A or Rx-T or Rx-G or Rx-C (0.4 μM), Pfu (5 U, Stratagene, 2.5Unit/μl). The program used for PCR amplification was 94° C. 1 min, (94° C. 15s, 55° C. 15s, 74° C. 2min, ×35) 74° C. 7 min, 8° C. overnight. Labeled oligonucleotides with IRD700 and IRD800 fluorophore (MWS(®) were used for the PCR to allow mismatch detection on LICOR4300 (LICOR (®).
The PCR products were analysed on agarose gel, and cloned in pGEM 3Zf. All the clones have been sequenced to make sure that the correct mutation has been inserted. The combinations of these constructs as template in PCR amplification were used to reconstitute all the types of mismatches.
Mismatched PCR product were incubated as disclosed in Example 2 above, with a recombinant CEL I preparation obtained from tobacco leaves by ammonium sulphate precipitation as disclosed in Example 1 above.
The products of the digestions were analyzed on acrylamide gel. A denaturating 6.5% acrylamide gel has been used, and electrophoretic conditions were: 1500V, 40 W, 40 mA, 45° C. with a scan speed of 1.
The results are shown on
These results show that the CEL I preparations of the invention recognize all types of mismatches and particularly mismatches reported in the prior art as weakly recognized by CEL I purified directly from celery. Indeed, as shown in Table 2 above, CEL I preparation of the invention recognizes mismatches weakly recognized by CEL I preparation of the prior art, like T/T, G/A, A/G, G/T, T/G with very high specificity, and the mismatch preference of this enzyme is as follows: T/G˜A/G˜G/G˜G/T>T/T˜G/A˜A/A˜C/C>T/C˜C/T˜A/C˜C/A.
This specific activity was also enhanced when the CEL I reaction buffer was supplemented with 5% PEG (data not shown).
To verify that the recombinant CEL I of the invention can be used for high throughput genotyping, it was tested whether it can recognize mutations in a pool of individuals.
Genomic DNA with a known SNP (C-->T), corresponding to the dwarf pea mutant Le1, and genomic DNA from wild type pea cultivar Torstag, have been used to amplify the Le1 locus. These genomic DNA have been used as controls for homoduplex and heteroduplex formation.
To create heteroduplex DNA Genomic DNA derived from pea plant homozygote for the le1 locus was diluted with genomic DNA derived from pea plant homozygote for the Le1 locus in different proportions and 30 ng was used for PCR amplification using the primers Le2462 labelled with TET fluorochrome (5′-TGATATTGTCGTGCAATATGATGAAAC-3′ SEQ ID N°14) and Le3082 labelled with ROX (MWG®) fluorochrome (5′-ATACCTATTTAGCCCACTTGGACAC-3′ SEQ ID N°15). The PCR reactions were carried out as follows: 94° C. 1 min, ° (94° C. 15s, 55° C. 15s, 74° C. 1 min ) X35, 74° C. 7 min. Heteroduplex DNAs reconstituted from the PCR products were used as template in the mismatch detection assay as described above.
The results of this pooling experiment using the protein produced in tobacco are presented in
To conclude, the protein produced in planta has a high sensitivity, allowing the identification of a known SNP within amplified DNA sequences coming from genomic DNA of at least 24 individuals.
An analysis of a family of genes from Arabidopsis coding for endonucleases has been done, using the protein profile PF02265 from the database PFAM. This profile HMM was used as a probe to target all the 27117 predicted proteins in Arabidopsis genome and the totality of the genome translated in the 6 different frameshifts to avoid the effect of the automatically structural annotation. In this analysis we identified 5 candidate genes At1g11190, At1g68290, At4g21590, At4g21585 and At4g21600.
The cDNA of each candidate gene was PCR amplified and inserted between the 35 S promoter and the transcriptional terminator of CaMV in the binary vector pBin61 to create pBIN35S-ENDO1, -ENDO2,-ENDO3,-ENDO4 and -ENDO5 that correspond to At1g11190, At1g68290, At4g21585, At4g21590 and At4g21600, respectively (Table 5). These constructs were transformed into Agrobacterium strain C58C1 carrying the virulence helper plasmid pCH32 (HAMILTON CM, et al. (1996) Proc Natl Acad Sci USA. ,93(18):9975-9). pCH32 expresses VirG and VirE and was used to enhance T-DNA transfer, Agrobacterium cells were inoculated into 2 mL of L broth medium (SAMBROOK et al. 1989) supplemented with 50 μg/mL kanamycin and 5 μg/mL tetracyclin and grown at 28° C. overnight. Cells were precipitated and resuspended to a final concentration of 0.50 D600 in a solution containing 10 MM MgCl2, 10 mM MES, pH 5.6, and 150 μM acetosyringone. The cultures were incubated at room temperature for 2 hr before agroinfiltration into Nicotiana benthamiana leaves, Agroinfiltrated Nicotiana benthamiana plants were incubated for at least 24 hours at 24° C., 16 hours of light, 60% of humidity. To test for the efficiency of agroinfiltration, plants were also agroinfiltrated with a construct expressing the green fluorescent protein (GFP). Before harvesting the leaves, the intensity of the expression of the GFP for each leaf was checked using an UV lamp. The plant leaves were harvested only if the GFP was expressed. The candidate proteins were extracted by ammonium sulfate precipitation as disclosed in Example 1.
As a simple test to see if the candidate protein is active, we incubate some supercoiled plasmid with different dilutions of protein extract (80% ammonium sulfate precipitate). As a result of the presence of an endonuclease in the protein extract, the supercoiled structure should be relaxed and some new DNA bands will appear when you run the incubation medium on an agarose gel. Using the different dilutions we can compare the different endonucleases and see which one is the most active. The control was always recombinant Cel I endonuclease prepared by ammonium sulfate precipitation as disclosed in Example 1 above.
To screen for candidate proteins able to cleave heteroduplex DNA at the mismatch site, we can also use a quick characterisation system based on three consecutive steps.
In the first step the candidate proteins were tested for their ability to degrade single strand DNA. This condition was tested because the DNA at the mismatch site in heteroduplex DNA is single stranded. Thus, an endonuclease that is not able to digest single strand DNA is predicted not able to cleave DNA at the mismatch site. Second, the candidate protein should cleave a test heteroduplex DNA at known and well characterised mismatch site. Third, proteins that pass test 1 and 2 were evaluated for their efficiency to cleave heteroduplex DNA fragments carrying all the types of mismatches. This was carried out using DNA toolbox which consists of a set of well characterised plasmid constructs that contain at specific position of the insert, each of the four possible nucleotides.
The activity of the candidate proteins on degradation of single strand DNA was carried out as described in Example 2. In this analysis all the five candidate proteins showed nuclease activity and were classified from the most active to the less active as follows: ENDO1, ENDO5, ENDO2, ENDO3 and ENDO4.
To test if the candidate proteins produced in tobacco can recognize single point mutations, we tested the activity of the protein extracts on heteroduplex DNA created as disclosed in Example 3. The PCR products containing each or both of the alleles were incubated with 1/1000 dilution of the protein extracts derived from leaves agroinfiltrated either with the candidate endonuclease gene or with the GFP as control, as follows: 500 ng of the PCR products were incubated with tobacco protein extract in 25 μl final volume containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 1 mM DTT and 5% PEG-8000 for 30 minutes at 37° C. The reactions were stopped with EDTA at the final concentration of 80 mM and analysed on a 3% agarose gel If the two oligonucleotides were fluorescently labelled the digestion products were analysed on acrylamide gel, on a 377Abi DNA sequencer. In this experiment, we predicted that if the protein extract contained a mismatch specific endonuclease, the heteroduplex DNA would be cleaved at the mismatch site, thereby releasing two bands of 256 bp and 405 bp.
The results are shown on
From this biochemical analysis, we concluded that the five endonucleases exhibit mismatch specific cleavage activities. Moreover, the three enzymes, ENDO1, ENDO5, ENDO2, which have higher single strand DNA specific-nuclease activity, cleave also with higher efficiency heteroduplex DNA at the C-A/T-G mismatch site. Furthermore, among these three endonucleases, ENDO1 and ENDO5 were the most active ones. Thus, these two nucleases were selected for further accurate characterisation.
The main goal of this task is to identify an endonuclease that cleaves mismatches that CEL-I does not recognise efficiently. This was carried out using DNA toolbox which consists of a set of well characterised plasmids constructs that contain at specific position of the insert each of the four possible nucleotides. The combination of these constructs as template in PCR amplification was used to reconstitute all the types of mismatches.
Mismatched PCR product were incubated as above (see example 5) with the candidate endonuclease and analysed on the LICOR sequencing machine.
The results are shown on
In this analysis, ENDO5 like CEL-I recognises weakly the T/T type of mismatches. In contrast, ENDO1 recognises nearly all the type of mismatches with high efficiency. From this analysis, we can conclude that ENDO1 is able to recognise mismatches undetected by CEL-I.
The sensitivity of ENDO1 has been evaluated as described in Example 3, with dilutions of 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 50 or 60 fold with DNA carrying the wild-type allele.
The results of this pooling experiment are presented in
In identical experiments, CEL-I purified from celery can detect one allele in a maximum of 16, with low sensitivity, and a correct sensitivity is obtained only when the dilution is inferior or equal to 8-fold.
In conclusion, endonucleases according to the invention and particularly ENDO1 have a much less background noise than Cel I, can be used at very high dilution compared to Cel I and have better specificity and activity than Cel I endonuclease.
A series of mutants based on Rx gene were created. We have designed different plasmids containing each one type of mismatch in order to show the specificity of ENDO1. The PCR mix contains oligonucleotides specific to the plasmids. Labeled oligonucleotides with ROX and FAM fluorophore (MWG®) were used for the PCR to allow mismatch detection on ABI377 MWG®.
As shown in
Anytime we have a mismatch in our samples (resulting from the formation of heteroduplex between two strands of DNA coming from 2 different plasmids, as for example on lines 2, 3, 4 or 6, 7 and 9) the ENDO 1 recognizes and cuts at the site of the mismatch, resulting in the appearance of two bands corresponding to the two products, each labelled with one fluorophore. For the ENDO 1 digestion, a decrease in the background can be seen and some mismatches can be detected only with ENDO 1 (line 9 for example)
Two plasmids containing a wild form and a mutated form of Rx gene were used. They differ only by one known point mutation. The PCR mix contains oligonucleotides specific to the plasmids. Labelled oligonucleotides with ROX and FAM fluorophores (MWG®) were used for the PCR to allow mismatch detection on ABI377 MWG®.
As shown on
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2004/009159 | Jul 2004 | EP | regional |
| PCT/EP2004/009166 | Jul 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/009220 | 7/29/2005 | WO | 00 | 8/21/2007 |
