WO2002070684A2 - Gene library for screening methods - Google Patents

Abstract

The present invention relates to a method for generating a gene library characterised in that it contains substantially all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of different fragments. The present invention also relates to a gene library obtainable by said method. The gene library of the invention is particularly useful for the 'yeast two hybrid' (Y2H) screening.

Description

Gene library for screening methods

The present invention relates to a method for generating a gene library characterised in that it contains substantially- all genes of a desired source, e.g. organism, in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of different fragments. The present invention also relates to a gene library obtainable by said method. The gene library of the invention is particularly useful for the „yeast two hybrid" (Y2H) screening.

The yeast two hybrid screening technology is based on the screening of gene libraries in order to pick up gene fragments encoding a protein (or protein fragment) which preferably binds to a desired protein. This technology makes use of genetic methods in the yeast Saccharomyces cerevisiae allowing to isolate those fragments contained within a gene library which encode specifically binding proteins. However, the 2YH screening requires high quality gene libraries. In the case of organisms having a genome which contains only low amounts of non-coding regions and (almost) no introns (prokaryotes and low eukaryotes) a suitable gene library can be generated by fragmenting genomic DNA and ligating the fragments in suitable vectors. However, in the case of higher eukaryotes genomic DNA is not suitable for the generation of Y2H libraries due to the presence of large amounts of non-coding regions and introns. Accordingly, one has to switch to cDNA as a basis for the libraries. However cDNA based libraries exhibit a variety of disadvantages, e.g.:

- the preparation of cDNA by reverse transcription results in an irregular representation of genes due to the fact that the efficiency of reverse transcription differs depending on various technical features of the mRNA (length, secondary structure etc.) . This applies to random priming as well as to priming using oligo dT primer;

- the cDNA library is restricted to genes which are sufficiently expressed in the tissue used as a source for the mRNA. In other words, in the cDNA library weakly expressed genes are not present or at least under-represented whereas highly expressed genes are over-represented. In order to overcome this problem, i.e. in order to ensure that every gene is present in the library, the cDNA libraries have to comprise very high amounts of individual clones;

- in order to ensure that all genes of an organism are present in the cDNA library different tissues have to be used as sources for the mRNA resulting in an increase of the Y2H screening region by some orders of magnitude .

Due to these limitations Y2H screenings for higher eukaryotes are much more complex compared to Y2H screenings for prokaryotes or lower eukaryotes. Due to this fact the use of this approach in a large scale, e.g. in order to assay all possible protein interactions of a genome (proteoms) is limited. In order to overcome this problem recently several groups have started to clone the open reading frames of all genes of an organism specifically and individually. This can be done by the use of PCR using specific primers to obtain the open reading frame of each gene as a PCR-product. This has been done for the yeast Saccharomyces cerevisiae (Hudson et al., Genome Res. 1997, 7(12): 1169-1173). In this case, genomic DNA was used as template. Alternatively, full length cDNAs found in cDNA libraries can be used as template. Efforts to obtain the complete collection of expressed genes have been initiated in the genome of man (Strausberg et al . , Science 1999 , 286 ( 5439 ) : 455 - 457 ) .

Such collections of full length genes allow Y2H screenings and recently attempts were made to determine all protein interactions within yeast. However, unfortunately, there are lots of interactions which cannot be determined within the full length protein by use of the Y2H system due to the fact that in yeast many foreign proteins, e.g. human proteins, do not exhibit in the correct conformation which is achieved in the natural (physiological) setting, e.g., by posttranslational modifications or the binding of specific ligands. Accordingly, since there is a high probability that the protein expressed in yeast does not exhibit its correct conformation, many of the possible interactions cannot be determined. This is one of the major drawbacks when using full length gene libraries. In addition, this approach does not allow any conclusions as regards the region within the full length protein which is responsible for the binding.

Therefore, it is the object of the present invention to provide a gene library which overcomes the above discussed limitations and failings of the gene libraries of the prior art, i.e. which contains all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of different fragments.

According to the invention this is achieved by the subject matters as defined in the claims.

Thus, in one aspect, the present invention relates to a method for generating a gene library characterised in that it contains substantially all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of different fragments, said method comprising

(a) generating by in vitro amplification a pool of DNA molecules encoding the full length open reading frames of substantially all genes of the desired source and mixing the different DNA molecules of the pool of DNA molecules in such a way that they are present in substantially equimolar amounts;

(b) fragmentation of the DNA sequences obtained in (a) ; and

(c) cloning of the DNA fragments of (b) into a vector.

The gene library obtainable by this method (which has a much lower complexity compared to a cDNA library) does not contain DNA sequences corresponding to non-coding region or introns, allows to screen all the proteins of a desired organism and does not exhibit the restrictions of gene libraries based on full length genes as regards the possible protein bindings. In addition it allows to determine the region of the protein which is responsible for binding. Starting material for this approach may be the aforementioned collections of expressed genes as templates to generate PCR-products encompassing the open reading frames using specific templates. However, it is equally possible to use reverse-transcribed mRNA from a collection of (human) tissues as a template.

As used herein, the term „each gene is represented by a sufficient amount of different fragments" means that the gene is not present in the gene library as a full length gene but in form of fragments altogether representing the entire gene. This means a set of fragments which in their entirety cover the entire sequence of the gene. Preferably, these fragments may be overlapping, such that for each part of the open reading frame at least two or more fragments can be found.

The generation of a pool of DNA molecules encoding the full length open reading frames of substantially all genes of the desired source by in vitro amplification, e.g. PCR, wherein the different DNA molecules of the DNA molecules are present in substantially equimolar amounts can be carried by the person skilled in the art by routine methods, e.g. by the methods described in the Example below. First, the sequences of all known open reading frames of a genome are obtained from a publicly available source, such as the Swissprot database. In the next step, DNA oligonucleotide primers are synthesized which direct the amplification of the open reading frame.

These primers are then used in separate PCR reactions for each gene using preferably cDNA as template.

The fragmentation of the DNA molecules can be carried by a variety of well known methods, e.g sonication, mechanical shearing (e.g. in a syringe) or the use of frequently cutting restriction enzymes. Preferably, the fragments should have a minimum length of 100 bp. Particularly preferred are fragments having a length in the range of 100 to 1000 bp. In a preferred embodiment, the fragmentation of the DNA molecules is random fragmentation, e.g. ultrasonication. Particularly preferred is fragmentation by ultrasonication. Insertion of fragments into a suitable vector requires the polishing of the termini of the fragments prior to cloning. Suitable methods for polishing, i.e. for preparing blunt ends, include treatment with Klenow DNA polymerase or Pwo polymerase.

Subsequently, the polished fragments are inserted into a vector, preferably a recombinant vector suitable for yeast two hybrid screening. Suitable vectors are for example: pGAD424, pAD-GA 4-2-l, pBD-GAL4, pBD-GAL4 Cam, pCMV-AD, pCMV-BD, pMyr, pSos, pACT2, pAS2-l, pHISi, pLexA, pM, pHISi-1, pB42AD, pVP16, pGADIO, pGBKT7, pLacZi, p8op-lacZ, pAD GH, pGilda, pAD GL, pGADT7, pGBDU, pDBLeu, pPCLeu, pPC86 and pDBTrp. The ligation mixture is transformed in a suitable host, e.g. E. coli, according to methods which are well known to those skilled in the art; see, for example, the techniques described in Maniatis et al . , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. After in vivo amplification, the vector DNA is isolated from the transformants, purified and introduced into the final host for the expression library, e.g. yeast. In yeast, a number of vectors containing constitutive or inducible promoters may be used (c.f. above). For a review see, e.g., Current Protocols in Molecular

Biology, Vol. 2, 1988, Eds. Asubel et al . , Greene Publish Assoc. & Wiley Interscience, Ch. 13; Grant et al . , 1987, Expression and Secretion Vectors for Yeast, in: Methods in Enzymology, Eds. Wu & Grossman, Acad. Press, N.Y., Vol. 153, pp. 516-544. Particulary preferred are Y2H vectors, e.g. pGAD424. Introduction of the above described vectors into the host cell can be effected by well known methods, e.g. calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, or other methods. Three transfection methods are prefered for yeast: protoplast transformation, lithium acetate mediated transformation and electroporation. Such methods are described in many standard laboratory manuals.

Finally, the present invention also relates to a gene library characterised in that it contains substantially all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of differing fragments and wherein said gene library is obtainable by the method of the invention described above. Uses of such a gene library include Y2H screening.

The following example illustrates the invention. Example

Generation of a yeast two hybrid (Y2H) library

The DNA sequences encoding open reading frames (ORF) corresponding to nuclear receptors and cofactors which were inserted in an already available collection of pENTRY-plasmids (RZPD, Heidelberg) were amplified by PCR using the following primers with attBl-sequences (5'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC G-3') (SEQ ID NO. 1) and attB2-sequences (5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC-3') (SEQ ID NO. 2) , respectively. The attBl sites are of use for recombinational cloning of the open reading frame into other vectors and are used to "PCR out" the inserts. PCR products were separated on an 1%-agarose gel and the sizes of the fragments were determined by comparison with comigrating size markers. Subsequently, the gene fragments were purified using the QIAquick PCR purification kit from Qiagen, Hilden (Cat. No. 28106) and the amount of the amplified fragments was determined according to the optical density at 260 run. Then, the molar concentrations of the various purified fragments was determined (taken into account the sizes and the amounts of the fragments) as well as the dilution factor required for ensuring that all PCR products are present in substantially equimolar amounts. Then, the various PCR products were diluted according to the various dilution factors and equal volumina of the diluted solutions were mixed. The PCR products of the mixture were subjected to ultrasonication in such a way that fragments in the range of about 100 to 1000 bp were produced. Sonication was done in a Sonoplus UM20170 (Bandelin, Berlin) instrument for 5 minutes using the following settings: 100%, pulse 0.9, time: 5 min.. The fragments obtained were separated on a preparative agarose gel and fragments having the desired sizes were isolated from the gel using the Jetsorb Gel Extraction Kit (Cat. 110300, Genomed, Bad Oeynhausen) . The isolated fragments were pooled, i.e. three pools comprising fragments having lengths of 100 to 300 bp, 300 to 600 bp and 600 to 1000 bp, respectively, were generated. Then, the termini of the sonicated fragments were polished by use of Pwo polymerase (Roche Diagnostics) in a mix containing 300 ng of DNA, 2.5 units of Pwo polymerase, 4.5 μl of incubation buffer supplied by the manufacturer of the Pwo polymerase (Roche Diagnostics) , 8 μl of 200 μM solutions of both desoxyGTP, desoxyATP, desoxyCTP and desoxyTTP, and water to a final volume of 45 μl for 30 min. at 72°C. Y2H-vector (pGAD424) (Clontech, Palo Alto, USA) was linearized using the restriction endonuclease Smal and, subsequently, dephosphorylated using shrimp phosphatase. Each pool of PCR fragments described above was inserted separately into the vector and E. coli (E. coli DH5alpha) were transformed with the ligation mixtures. Subsequently, transformants were plated on LB plates containing ampicillin and the DNA was prepared from the colonies of transformants using the Plasmid Mini Kit from Qiagen, Hilden (Cat. No. 12143) to yield the gene library. In a subsequent experiment, the gene library was used for Y2H screening.

Claims

1. A method for generating a gene library characterised in that it contains substantially all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of different fragments, said method comprising

(a) generating by in vitro amplification a pool of DNA molecules encoding the full length open reading frames of substantially all genes of the desired source and mixing the different DNA molecules of the pool of DNA molecules in such a way that they are present in substantially equimolar amounts;

(b) fragmentation of the DNA sequences obtained in (a) ; and (c) inserting the DNA fragments of (b) into a vector.

2. The method of claim 1, wherein the fragmentation is random fragmentation .

3. The method of claim 2, wherein the random fragmentation is achieved by ultrasonication and wherein after ultrasonication the fragments are polished.

4. The method of any one of claims 1 to 3, wherein the fragments have a length in the range of 100 to 1000 bp.

5. The method of any one of claims 1 to 4, wherein the vector of step (c) is a Y2H vector.

6. A gene library characterised in that it contains substantially all genes of a desired source in substantially equimolar amounts, wherein each gene is represented by a sufficient amount of differing fragments and wherein said gene library is obtainable by the method of any one of claims 1 to

7. Use of the gene library of claim 6 for Y2H screening.