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WO2000042167A1 - Systeme permettant d'identifier des modulateurs de gene articule autour de cellules eucaryotes - Google Patents

Systeme permettant d'identifier des modulateurs de gene articule autour de cellules eucaryotes Download PDF

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WO2000042167A1
WO2000042167A1 PCT/AU1999/001131 AU9901131W WO0042167A1 WO 2000042167 A1 WO2000042167 A1 WO 2000042167A1 AU 9901131 W AU9901131 W AU 9901131W WO 0042167 A1 WO0042167 A1 WO 0042167A1
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sequence
expression
gene
target
reporter
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PCT/AU1999/001131
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Gregory Martin Arndt
David George Atkins
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Johnson & Johnson Research Pty. Limited
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Priority to AU22684/00A priority Critical patent/AU2268400A/en
Publication of WO2000042167A1 publication Critical patent/WO2000042167A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates generally to eukaryotic cell-based systems for screening molecules which are capable of modulating gene expression. More particularly, the present invention relates to high throughput phenotypic screening systems for detection of candidate suppression molecules, which systems rely on the use of target-reporter gene sequence fusion constructs.
  • a valuable complementary approach to gene function identification involves the study of cells in which the expression of a putative gene has been eliminated or suppressed.
  • the elimination of gene expression is most commonly achieved through the use of mutagenesis or gene disruption.
  • These techniques can be laborious and are usually restricted to organisms with well-defined genetics.
  • tram-acting strategies such as antisense RNA, ribozymes, and dominant negative proteins have been shown to be widely applicable in suppressing specific gene expression.
  • the full potential of these technologies has yet to be realised due to the inability to select the most effective agents of this type for in vivo studies.
  • the ideal screening system should be based on an organism that is easy to handle, readily transformable, inexpensive and has the potential for application to high throughput screening.
  • the candidate that satisfies these requirements are the yeasts and several groups have made progress in defining conditions in which yeasts can be used as hosts for studying gene-delivered modes of gene suppression (Arndt G.M et al, Mol. Gen. Genet. 1995, 248:293-300; Ferbeyre G et al, Gene, 1995, 155:45-50).
  • a requirement of any yeast- based screening system is the use of a target gene that when down-regulated confers a readily scorable cellular phenotype. Ideally, a quantitative relationship would exist between the degree of target gene suppression and the expression of the external phenotype.
  • lacZ gene used both as a target gene sequence and a reporter sequence, could be regulated using lacZ gene antisense RNA in the fission yeast Schizosaccharomyces pombe (Arndt G.M et al, Mol. Gen. Genet. 1995, 248:293-300).
  • lacZ gene antisense RNA in the fission yeast Schizosaccharomyces pombe (Arndt G.M et al, Mol. Gen. Genet. 1995, 248:293-300).
  • due to the low steady-state level of lacZ target mRNA it was not possible to adequately test the relationship between antisense RNA effectiveness and the external blue colony colour phenotype encoded by the lacZ gene.
  • yeast cell-based system for identifying gene suppression constructs Another yeast cell-based system for identifying gene suppression constructs
  • GSCs trans-acting molecules capable of suppressing the expression of l ⁇ cZ gene, again used both as a target and a reporter sequence
  • PCT/AU95/00235 published as WO 95/29254.
  • antisense sequences derived from the l ⁇ cZ gene were used to transform a yeast strain expressing target lacZ gene at a high level and effective GSCs identified by screening transformants for changes in the lacZ gene-encoded blue colour colony phenotype.
  • This system however is not capable of usefully identifying differences in the intensity of blue colony colour nor is it capable of being applied equally to transformants expressing low and high levels of ⁇ -galactosidase, to the extent that it can be effectively used for high through-put screening of expression libraries.
  • FIGURES Figure 1 shows a strategy for construction of random fragment expression libraries and screening in fission yeast.
  • This schematic uses the SV40 promoter-driven ura4-lacZ fusion gene as an example (top of the diagram).
  • the fusion gene is used as a template for PCR to amplify the SV40 promoter-wra ⁇ target sequence (step 1).
  • This DNA is randomly fragmented using DNasel and the resulting fragments size-selected, end-filled and ligated to Bglll linkers (step 2).
  • step 3 The fragments are then cloned into the kanR plasmid pBGS to construct an intermediate library (step 3), and then sub-cloned from this library into the yeast expression plasmid pGTl 18 to produce the yeast expression library (step 4).
  • This library DNA is then used to transform the SUL strain, containing the SV40 promoter-wra -/ ⁇ cZ fusion gene integrated at the ura4 locus, and transformants selected for leucine prototrophy and screened for their blue colony colour phenotype following overlay with agarose-Xgal medium (step 5).
  • the light blue transformants are assayed for b-galactosidase activity and whole cells used as templates to amplify inserts by PCR for sequencing (step 6).
  • FIG. 1 The resulting plasmid with insert is referred to as a gene suppression construct (GSC).
  • Figure 2 shows inserts contained on GSCs derived from the ura4 gene-specific expression library. The top of the schematic shows the region of the ura4-lacZ fusion gene containing the SV40 early promoter and the ura4 sequence. The inserts contained on GSCs derived from the DL6 library are aligned below the target gene. To the left of each insert is the transformant designation, while to the right is indicated the degree of suppression of ⁇ -galactosidase activity in comparison to the vector control. The direction of the arrows represents the orientation of the insert in the GSC. The T indicates the transcription initiation site and 'A' represents the ATG codon.
  • Figure 3 shows alignment of the c-myc-specific inserts contained on GSCs identified from the DL7A library screen in yeast. At the top of the schematic is indicated the region of the c-myc-lacZ fusion gene used as a template for construction of the DL7A library. The exon 2 and exon 3 boundaries are marked. The inserts contained on each GSC are aligned below the target gene. All abbreviations are as referred to in Figure 2.
  • Figure 4 shows single and chimeric inserts on GSCs derived from the DL7B c- wyc-specific expression library.
  • the target gene region at the top of the diagram is as described in Figure 3.
  • the inserts from the DL7B library are indicated relative to the target gene and categorised into 4 sub-groups as indicated.
  • the arrows indicate the orientation of this DNA in the GSC.
  • the regions X and Y represent potential sub-regions of accessibility in the c-myc mRNA.
  • the number and letter designation within square brackets above or below fragments in the chimeric inserts define the position and orientation, respectively, within the GSC insert (with A indicating antisense and S representing sense orientations). Hatched boxes differentiate this fragment from others derived from the same target site in the chimeric inserts.
  • Figure 5 shows the alignment of inserts derived from the ura4 gene-specific GSCs identified in yeast transformants using 5-FOA selection.
  • the schematic at the top of the diagram represents the region of the ura4-lacZ fusion gene containing the SV40 early promoter and the ura4 sequences.
  • the inserts contained on the GSCs from the DL6 random expression library identified in yeast using 5-FOA selection are aligned below the target gene sequences. These inserts are categorised as antisense or sense according to their orientation in the yeast expression plasmid pGTl 18. All abbreviations are as referred to in Figure 2.
  • Figure 6 shows the inserts contained on the GSCs specific for Hradl and their alignment with the target gene sequence and the level of suppression of the fusion gene target in yeast.
  • the labels "ATG” and “TGA” rerfer to the translation initiation and stop codons, respectively.
  • Figures 7 and 8 summarise the arrangement of inserts derived from the Hrad27
  • GSCs and the level of suppression of the fusion gene target in yeast.
  • the target gene sequence fused to the amino terminal end of the lacZ reporter is indicated at the top of the diagram.
  • the labels “ATG” and “TGA” refer to the translation initiation and stop codons, respectively.
  • “EVS” indicates the penultimate codon relative to the stop codon
  • the screening method of the present invention is particularly but not exclusively useful for screening gene-encoded suppression molecules such as for example antisense, sense or ribozyme constructs, or transdominant polypeptides and small peptides.
  • gene-encoded suppression molecules such as for example antisense, sense or ribozyme constructs, or transdominant polypeptides and small peptides.
  • other classes of suppression molecules may also be advantageously screened by the methods of the present invention.
  • the methods of the present invention may be used to screen for molecules which modulate target gene expression in ways other than suppression.
  • the rationale behind the most preferred format of the method of the present invention is the production in the yeast cell of a single mRNA transcript encoding both the target nucleic acid product or a portion thereof and a reporter molecule or a sequence encoding a reporter molecule.
  • the reporter molecule will be encoded by a nucleotide sequence placed downstream of the nucleotide sequence encoding the target gene product.
  • a molecule such as a genetic sequence capable of modulating the expression of the target gene sequence is then tested by reference to a change in the detectable reporter molecule.
  • the level of expression of the reporter nucleic acid sequence is titrated for each fusion gene-expressing yeast strain following quantitation of the reporter nucleic acid product activity by for example conventional assays such as solution enzyme assay.
  • a method for determining whether, or to what degree, a molecule specifically modulates the expression of a target nucleic acid sequence in a eukaryotic cell which comprises the steps of (a) determining for the target sequence the conditions required for the expression of the reporter sequence or for the activity of its expression product in the Schizosaccharomyces pombe cell of claim 2, (b) contacting the Schizosaccharomyces pombe cell with the molecule under said conditions, and (c) detecting or quantitating a phenotypic change in the Schizosaccharomyces pombe cell resulting from the modulation of the expression of the reporter sequence, thereby determining whether, or to what degree, the molecule specifically modulates the expression of the target sequence in a eukaryotic cell.
  • the phenotypic changes are for example changes in colour, size, shape or texture of the yeast cells of colonies of yeast cells.
  • the phenotypic change is blue colour or size of transformant yeast colonies resulting from the expression of ⁇ -galactosidase or orotidine monophosphate (OMP) decarboxylases reporter molecules, respectively.
  • the molecules capable of modulating target nucleic acid expression are preferably genetic constructs such as antisense, sense nucleic acid sequences or ribozymes relative to a target gene, or other nucleic acid sequences such as oligonucleotides, random nucleic acid sequences or nucleic acid sequences selected for cleavage in vitro.
  • the principles of the present invention extend to non- nucleic acid molecules such as transdominant polypeptides, small peptides and chemical compounds as well as to synthetic nucleic acid molecules or nucleic acid analogue molecules.
  • modulating expression include in their scope up- regulating and down-regulating expression of a target sequence as well as activity of a product of a target sequence.
  • molecules which modulate expression or activity may be agonists or antagonists.
  • the present invention extends to identifying agents, such as for example, ribozymes, antisense and sense nucleotide molecules whose activity is regulated by cellular factors including switching mechanisms and intracellular address signals. Additionally, in some circumstances, the level of effect on expression may be enhanced by increasing the level of modulating molecules such as antisense, sense or ribozyme molecules.
  • gene is used in its broadest sense to include a genomic gene sequence as well as a genetic sequence comprising only the coding portions of a gene (i.e. exons), a cDNA sequence corresponding to a mRNA transcript and a synthetic gene sequence.
  • a "gene” as contemplated herein especially in relation to a target gene includes a naturally occurring gene, a partial gene, a synthetic gene and a fusion between a target gene and another gene or genetic sequence.
  • a “gene”, therefore, is considered herein to include any target nucleotide sequence and may be of eukaryotic, prokaryotic or viral origin.
  • protein as used in the context of the present invention is intended to encompass polypeptides and peptides.
  • the target nucleic acid sequence is "exogenous" to S. pombe meaning it is a heterologous gene which has been introduced by transformation, conjugation, electroporation or other means to the yeast cell.
  • the target gene may alternatively be "endogenous".
  • Particularly preferred endogenous or homologous S. pombe genes are those which encode cell cycle proteins, modulate cell cycles and/or are involved in programmed cell death. Such genes and in particular antagonists thereof identified in accordance with the present invention may be useful in the treatment of cancers in mammals such as humans.
  • Other important endogenous target genes are S. pombe homologues of mammalian (e.g. human) genes.
  • a particularly useful yeast in accordance with this aspect of the present invention has S. pombe genes replaced by mammalian (e.g. human) homologues or comprise homologous animal, mammalian or plant target genes or have homologous functions to animal, mammalian or plant target genes.
  • the target nucleic acid sequences include eukaryotic, prokaryotic and viral genes.
  • eukaryotic genes include mammalian growth factors and cytokines and their receptors and cancer specified genes and plant genes.
  • prokaryotic genes include antimicrobial resistance genes and pathogen-specific genes.
  • viral genes include retroviral genes such as those of HIV and Hepatitis.
  • a nucleic acid fusion construct capable of being expressed when introduced into Schizosaccharomyces pombe, consisting essentially of a target nucleic acid sequence and a reporter nucleic acid sequence, wherein (a) the target and reporter sequences are situated with respect to each other so that the expression of the reporter sequence is modulated by any molecule which also modulates the expression of the target sequence, and (b) the expression of the reporter sequence confers on Schizosaccharomyces pombe a detectable phenotype.
  • the reporter nucleic acid sequence product may be any molecule capable of giving an identifiable signal and may be an enzyme, preferably ⁇ -galactosidase or orotidine monophosphate (OMP) decarboxylase.
  • OMP orotidine monophosphate
  • Other reporter sequences which tolerate 5' fusions can also be used, for example those which encode luciferase or green fluorescent protein.
  • Examples of other reporter molecules which may be used are chloramphenicol acetyl transferase (CAT), other enzymes confering antibiotic resistance, ⁇ -glucuronidase, fluorescent proteins, essential growth factors and cell cycle proteins.
  • CAT chloramphenicol acetyl transferase
  • the target-reporter nucleic acid fusion construct may be integrated into the chromosome of S pombe under the control of an endogenous promoter or exogenous promoter or may exist as an extrachromosomal, replicating element. Any number of exogenous promoters may be used such as an SV40 promoter.
  • a suitable endogenous constitutive promoter is S. pombe adh 1 promoter.
  • the genetic sequences to be tested may be introduced to the yeast cell by any number of means including transformation, conjugation, electroporation, amongst others.
  • the genetic sequence may be expressed under an endogenous or exogenous promoter or may be introduced without a promoter.
  • the present invention permits the rapid screening of genetic sequences having an effect on expression of a wide range of target genes.
  • the present invention is particularly applicable for the development of a disease model system suitable for screening for useful genetic sequences to target viral, cancer and aberrant "self genes.
  • the method of the present invention may also be useful as a drug screening system.
  • receptor genes are engineered and expressed in a fusion construct with a reporter or a reporter gene sequence.
  • the modified yeast is then used in high throughput assays for agonists or antagonists of the receptors.
  • the present invention is concerned with the improvement of the Schizosaccharomyces pombe gene suppressor screening system, particularly the phenotypic detection system, to permit the identification of GSCs for any target gene sequence including genes of mammalian origin.
  • Particularly preferred embodiments of the present invention exploit the capacity of a reporter gene sequence to be expressed despite additional sequences at its 5 ' end and are based on the observation that the nature of the target sequence in the fusion construct governs the level of expression of the reporter gene product, eg ⁇ -galactosidase or orotidine monophosphate (OMP) decarboxylase, and thus affect the expression of the desired transformant yeast colony phenotype, eg, blue colour or colony size.
  • OMP orthophosphate
  • the improvement comprising the present invention relies on pre-determining the conditions necessary for expression of reporter gene sequences, eg. ⁇ -Galactosidase or orotidine monophosphate (OMP) decarboxylase , for each target/reporter gene fusion construct.
  • reporter gene sequences eg. ⁇ -Galactosidase or orotidine monophosphate (OMP) decarboxylase
  • OMP orotidine monophosphate
  • Those conditions include but are not limited to X-gal concentration, detergent content and time of incubation, so that the screening system can be used to detect blue colour changes in transformants expressing low as well as high levels of ⁇ -galactosidase.
  • a random fragment expression library derived from the target gene sequence is then expressed in the fusion gene-containing yeast strain and screened using the lacZ gene-encoded phenotype.
  • Transformants containing effective GSCs may be identified on the basis of a light blue or white phenotype, however, less advantageously measurement of ⁇ -galactosidase enzyme activity may also be used for detection.
  • S. pombe ura4 gene sequence which encodes orotidine monophosphate (OMP) decarboxylase.
  • This enzyme is involved in the uracil biosynthetic pathway.
  • Yeast strains containing null mutations in this gene are auxotrophic for uracil and able to grow in the presence of the pyrimidine analog 5- fluoro-orotic acid (5-FOA).
  • wild type strains are prototrophic for uracil and sensitive to 5-FOA.
  • the association of a negative selection phenotype (resistance to 5- FOA) with the loss of ura4 gene function provided the basis for testing this genetic marker as an alternative reporter gene in gene fusions for use in screening random fragment expression libraries to isolate effective GSCs. This approach is discussed in more detail below.
  • the improved phenotypic cell-based screening methods of the present invention provide for a significant increase in the number of yeast transformants and therefore the number of expression constructs, that can be screened using the desired phenotype, such as change in the colony colour or size, and are high throughput with the capability to screen complex libraries, such as those having more than 10 ⁇ constructs. It is also amenable to automation and has the potential to develop therapeutic GSCs as well as providing tools for rapid insight into functional genomic studies.
  • Example 1 Yeast strains, constructs and expression libraries
  • Yeast media and methods All yeast strains and transformants were maintained on standard YES or EMM media (Moreno, S. et al, Meth. Enzymol, 1991. 194:795-823). Yeast cells were transformed with plasmid DNA by electroporation (Prentice, H.L. Nucleic Acids Res., 1992, 20:621). Plasmid DNA was isolated using a glass bead method (Hoffman, C.S. and Winston, F., Gene, 1987, 57:267-272) and recovered into DH5 ⁇ cells using standard electroporation procedures (Sambrook et al, Molecular cloning: a laboratory manual.
  • b-galactosidase enzyme activity was determined using a cell permeabilization protocol as previously described (Arndt, G.M et al., Mol. Gen. Genet., 1995, 248:293-300).
  • PCR amplification of plasmid DNA within whole yeast cells involved treating cells with 10 mg/ml zymolyase-20T (ICN Biomedicals, Aurora,
  • Yeast strains and plasmids All routine DNA manipulations were completed using standard protocols (Sambrook et al, Molecular cloning: a laboratory manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • Plasmid pGT5 was derived from pNEB195 by deletion of the BamHl site located in the polylinker 5' to the ura4 5' DNA flank.
  • the adhl promoter and ura4 3' processing signal sequences were derived as a PCR fragment by amplification of plasmid pURAS (Patrikakis, M., et al., Curr. Genet.
  • strain ⁇ S (h ⁇ , ura4::adhl-lacZ, leul-32) was identified as an ascospore isolate of this cross and characterised for expression of the lacZ gene.
  • the SV40 early promoter was PCR amplified from pSVB (Clontech) using the following 5' and 3' primers, respectively: (5')
  • TGGGGATCCAAGCTTAATTCGAGCTCGGTACAGCTTGG 3'
  • AAGGGATCCCTACTTCTGGAATAGCTCAGAGG 3'
  • the 350 bp fragment was digested with BamHl, gel-purified and subcloned into a pGEM3Zf(-)-based plasmid, containing the adhl promoter and ura4 3' processing signal (Patrikakis, M., et al., Curr. Genet. 1996, 30:151-158), in place of the adhl promoter.
  • a Hindll fragment containing the SV40 promoter and ura4 3' sequence was subcloned into plasmid pGT5 between the ura4 5' and 3' flanking sequences to produce plasmid p40F.
  • the 3.1 kb lacZ fragment used in constructing the fusion gene was prepared by PCR amplification, from pGEM- BGal containing the 3.5 kb N tl fragment from pSVB, using the primers (5') GACGGATCCCGGGTCGTTTTACAACGTCGTGAC (3') and (5')
  • the ura4 test gene sequence was amplified by PCR, from the template pR ⁇ P4 (Maundrell, K., Gene 1993, 123:127-130), using the primers (5') GAGAAGCTTGGATCCGCAAAGTTATGGATGCTAGAG (3') and (5') TTGGGATCCAGAATGCTGAGAAAGTCTTTGCTG (3'). This fragment was digested with BamHl and cloned into pI ⁇ TVEC in the sense orientation between the SV40 promoter and the lacZ sequences to produce pivURA4s. D ⁇ A sequencing confirmed that the ura4 gene was in-frame with the lacZ gene.
  • Plasmid pivURA4s was digested with Pacl-Pmel to release the SV40 promoter- ura4-lacZ-ura4 3' fusion cassette flanked by ura4 chromosomal sequences.
  • Strain HS was transformed with this D ⁇ A and integrants selected on EMM medium. Southern analysis confirmed integration of the fusion cassette at the ura4 locus.
  • pINTVEC was partially digested with BamHl and H dIII to remove the SV40 early promoter, and then ligated to the 745 bp adhl promoter fragment (Patrikakis, M., et al., Curr. Genet.
  • the region of the c-myc cDNA used in constructing the c-myc-lacZ fusion gene was amplified from the Quick-Screen human cDNA library (Clontech, Palo Alto, CA) using the following 5' and 3' primers, respectively: (5') CGAGGATCCTTGCAGCTGCTTAGA (3') and (5') TAGGGATCCCGCACAAGAGTTCCGTAG (3').
  • the 1379 bp fragment was digested with BamHl, gel-purified and cloned into the pGEM3Zf(-) plasmid. The complete sequence of this amplified product was determined.
  • the c-myc sequence was subcloned as a BamHl fragment into pSIV in the sense orientation between the adhl promoter and the lacZ gene to produce pAMLls.
  • DNA sequencing confirmed that the c-myc gene was in frame with the lacZ gene.
  • Plasmid pAMLls was digested with Pacl-Pmel to release the c-myc-lacZ fusion gene flanked by ura4 chromosomal sequences.
  • Strain NCYC 1913 was transformed with this DNA and integrants selected on EMM medium containing 1 mg/ml 5-fluoroorotic acid. The integration was confirmed by Southern analysis.
  • the yeast shuttle plasmid pGTl 18 was used in constructing all random fragment expression libraries. This vector was created by modifying pREPl (Maundrell, K., J. Biol. Chem. 1990, 265:10857-10864) to include a single ATG translation start codon in the nmtl promoter region, a unique Bglll cloning site between the nmtl promoter and 3' processing signal, and TAA stop codons in three reading frames downstream of the Bglll site.
  • pREPl Moundrell, K., J. Biol. Chem. 1990, 265:10857-10864
  • the SV40 promoter-wra4 portion of the fusion expression cassette was PCR amplified from pivURA4s using the primers (5') TGGAAGCTTAATTCGAGCTCGGTACAGCTTGG (3') and (5') TTGGGATCCAGAATGCTGAGAAAGTCTTTGCTG (3').
  • This DNA was partially digested with DNasel and fragments greater than 300 bp size-selected using CL-4B Sepharose. Fragments were end-filled with T4 DNA polymerase and Klenow fragment and ligated to Bglll linkers (New England Biolabs).
  • pBGS 5g/II-digested pBGS
  • pBS8(+) plasmid pBS8(+)
  • a library of 11,000 clones was generated in DH5a by amplifying single colonies on LB plates containing 50 ⁇ g/ml kanamycin.
  • the Bglll inserts from this intermediate library were subcloned into pGTl 18. Transformation of DH10B cells (BRL) produced the random fragment expression library designated DL6 which contains 20,000 independent clones with 90% of these having inserts.
  • a region including 20 bp upstream of the adhl transcription start site and the entire c-myc region was PCR-amplified from pAMLs using the primers (5') TGGCCTTCGCTTTTCTTTAAGCAAGAG (3') and (5 * ) TAGGGATCCCGCACAAGAGTTCCGTAG (3').
  • DNasel 5'
  • fragments were size-selected to isolate those greater than and less than 300 bp. In separate reactions, these two pools of fragments were end-filled and ligated to Bglll linkers. After Bglll digestion, DNA fragments were ligated independently to BgHl- digested pBGS, to produce two intermediate libraries in DHIOB cells.
  • the Bglll inserts contained in each of these libraries were subcloned into pGTl 18. Transformation of DH10B cells produced two yeast expression libraries designated DL7A and DL7B containing, respectively, large and small inserts derived from the c-myc target sequence.
  • the DL7A library contained 230,000 clones of which 40% contained inserts.
  • the DL7B library was composed of 1.8x10 ⁇ clones with 96% of these containing inserts. (iv) Screening random fragment expression library in yeast.
  • the transformed cell mixture is inoculated into selective liquid EMM medium and these cultures are grown at 30°C for approximately 72 hours. This allows recovery of the cells from electroporation, accumulation of the plasmid encoded RNA and turnover of the fusion protein.
  • Transformations are plated to selective solid EMM medium and grown for approximately 4 to 5 days at 30°C. The plating density is maintained at up to 1000 colonies per plate (diameter of 14cm). These colonies are overlay ed with an agarose-Xgal medium and then scored for differences in blue colony colour using the vector alone transformant as a control.
  • the agarose-Xgal medium is composed of 0.5M sodium phosphate, 0.5% agarose and 2% dimethylformamide. In addition, this medium also contains sodium dodecyl sulfate (SDS) and X-gal. The concentrations of these latter components are pre-determined for each fusion gene-expressing yeast strain following quantitation of the ⁇ -galactosidase activity displayed in the standard liquid solution enzyme assay.
  • This strategy provides the flexibility to detect blue colony colour for yeast transformants expressing low or high levels of ⁇ -galactosidase activity. For example, detection of the blue colony colour for the ura4-lacZ fusion gene- expressing strain requires 0.01% SDS and 200 ⁇ g/ml X-gal and incubation at 37°C for 3 hours. In contrast, strain AML1 expressing the c-myc-lacZ fusion gene produces detectable blue colour using 0.01% SDS and lOOO ⁇ g/ml when incubated at 37°C overnight.
  • transformants containing inserts were then single colony purified and assayed in triplicate for ⁇ -galactosidase activity. Select transformants displaying greater than 20% suppression of ⁇ -galactosidase activity were re-assayed in triplicate.
  • the PCR fragments were purified using Wizard PCR columns (Promega) and sequenced in both directions using the following sequencing primers: (5') AAGCTTTTATAGTCGC (3') and (5') AAGCTTTACCCGGGGA (3'). All fragments were sequenced using an Applied Biosystems 373 DNA sequencer.
  • the level of the fusion protein expressed in vivo can be controlled using two different promoter systems: the fission yeast adhl promoter (as used with AML1) and the mammalian SV40 early promoter (as used with the ura4-lacZ fusion strain described below).
  • the S. pombe ura4 sequence as a test gene.
  • the ura4-lacZ fusion gene-expressing strain SUL was transformed with the DL6 expression library derived from the SV40 early promo ⁇ cr-ura4 sequences contained in the fusion target gene.
  • a total of 47,000 yeast transformants were screened for changes in their blue colony colour and 291 light blue and white transformants were identified.
  • 170 were assayed for ⁇ -galactosidase activity in triplicate and all exhibited suppression of the ura4-lacZ target gene as measured by a reduction in ⁇ -galactosidase activity of between 29% and 77%.
  • the specific level of suppression was shown to be reproducible by re-assaying 44 of these transformants for ⁇ -galactosidase activity.
  • 20 light blue transformants were assayed for ⁇ -galactosidase activity following growth in the presence of thiamine and, in each case, suppression of ⁇ -galactosidase activity was dependent on transcription of the plasmid insert. Plasmids were then isolated from 17 different primary transformants, re-transformed into SUL and three independent transformants for each plasmid assayed for ⁇ -galactosidase activity. All plasmid re-transformants showed the same level of suppression of activity as the primary yeast transformant, indicating that the observed suppression was contingent on the presence of the specific GSC.
  • PCR fragments amplified from inserts contained on plasmids of 44 light blue transformants were sequenced and a subset of these are shown aligned with the target SV40 promoter-wra4 sequence in Figure 2.
  • a total of 29 independent antisense inserts and 2 sense inserts were identified which suppressed ura4-lacZ gene expression.
  • transformant 1-E6 contained two GSCs each with a ura4-spcc ⁇ c insert in the antisense orientation.
  • the top 26 antisense insert-containing GSCs produced a level of suppression equal to or greater than that obtained using the full length antisense RNA complementary to the entire ura4-lacZ fusion transcript (data not shown).
  • the S. pombe ura4 gene encodes the enzyme orotidine monophosphate (OMP) decarboxylase which is involved in the uracil biosynthetic pathway.
  • OMP orotidine monophosphate
  • Yeast strains containing null mutations in this gene are auxotrophic for uracil and able to grow in the presence of the pyrimidine analog 5-fluoro-orotic acid (5-FOA).
  • wild type strains are prototrophic for uracil and sensitive to 5-FOA.
  • the association of a negative selection phenotype (resistance to 5-FOA) with the loss of ura4 gene function provided the basis for testing this genetic marker as an alternative reporter gene in gene fusions for use in screening random fragment expression libraries to isolate effective GSCs.
  • the ura4 gene-specific library (DL6) was used to transform SUL and 4.5x10 cells plated to EMM media containing 0.5 mg/ml 5-FOA and 2.5 mg/L uracil. A total of 46 large 5-FOA-resistant colonies were assayed for ⁇ -galactosidase activity to quantitate the level of suppression of the fusion gene. This analysis indicated that 75%> of these transformants displayed reproducible levels of suppression ranging from 21% to 72%. Plasmids were isolated from 15 transformants and re-transformed into the ura4-lacZ gene-expressing strain.
  • the ura4 gene was fused to the amino terminus of the lacZ reporter gene.
  • the newly created ura4-lacZ gene has the potential to be used in the present system as a unique dual reporter for constructing fusion gene targets.
  • GSCs could be identified to the amino terminally fused target sequence by selection for 5-FOA resistant transformants. The level of suppression could then be quantitated using ⁇ -galactosidase enzyme assays.
  • a screen for GSCs specific for the human c-myc gene was performed.
  • a portion of the human c-myc cDNA was fused 5' to the lacZ gene and the resulting c-myc-lacZ fusion gene expressed from the ura4 locus in strain AML1.
  • the DL7A expression library containing c-myc-derived inserts greater than 300 bp in size, was used to transform AML1 and from 14,000 transformants screened, 3% exhibited a light blue phenotype. Of these transformants, 28 were assayed for ⁇ -galactosidase activity and all displayed suppression ranging from 20% to 72%>.
  • the inserts contained on the GSCs were PCR amplified, sequenced and aligned with the target gene in Figure 3. Two types of inserts were identified including 19 different antisense and 3 sense inserts.
  • the antisense inserts ranged in size from 593 bp to 1081 bp and transformants containing these GSCs showed reductions in ⁇ -galactosidase activity ranging from 43% to 72%.
  • a total of 85% of these inserts contained the ATG codon, suggesting that this region may be an important component of the long antisense RNAs.
  • a second c-wyc-specific expression library containing inserts ranging in size from 100 bp to 300 bp was constructed. Following transformation of the DL7B library into AML1 and screening of 16,000 transformants, a total of 315 light blue transformants were identified, ⁇ -galactosidase assays on 56 of these transformants showed that the GSCs suppressed c-myc-lacZ gene expression by 20% to 81%. Sequencing of the inserts contained on the resident GSCs revealed both single fragment-containing inserts and chimeric inserts ( Figure 4).
  • RNAs are commonly used tools for suppressing the expression of specific genes or controlling viral replication.
  • the efficacy of this approach is dependent on a number of parameters, one of which is the accessibility of the antisense RNA to the target mRNA in vivo. This accessibility is determined by both the intracellular localisation of the two interacting RNAs as well as the ability of these RNAs to hybridise.
  • a number of in vitro strategies have been developed to identify regions of a target mRNA accessible to hybridisation with antisense sequences (Milner, N., et al., Nature Biotech., 1997, 15:537-541; Patzel, V. and Sczakiel, G., Nature Biotech. 1998, 6:64-68).
  • RNAs or oligonucleotides do not always function in vivo. This is not surprising given the fact that the complexity of RNA structures in vivo is determined by long-range secondary and tertiary interactions and RNA binding proteins.
  • the fission yeast genetic screen offers the distinct advantage of identifying effective antisense RNAs within the cellular milieu. By restricting the size of the inserts, it is possible to identify sub-regions of a target mRNA which are accessible in vivo. In the present study, we demonstrated this feature of the genetic screen by identifying two potential accessible regions in the c- myc target RNA.
  • the advantages of such antisense RNAs is the ability to hybridise with more than one region of the target mRNA and the potential for one of the antisense sequences to act as a facilitator of other antisense sequences contained on the same chimeric RNA (Nesbitt, S. and Goodchild, J. Antisense Res. Develop., 1994, 4:243-249).
  • a biological approach such as the one we have described is the only way to select such effective and novel chimeric gene constructs.
  • the S pombe genetic screen described herein provides a method for the identification of GSCs and other molecules capable of modulating target gene expression, for any target sequence in a eukaryotic cell environment.
  • Candidate genes need only to be characterised at the primary nucleic acid sequence level and be amenable to fusion to the N-terminus of the lacZ gene such that ⁇ -galactosidase activity is maintained.
  • sequence information is not limiting and the lacZ gene, for example, can be incorporated into thousands of functional fusions (Burns, N., et al, Genes Develop, 1994, 8:1087-1105; Dang, V-D., et al, Yeast, 1994, 10:1273-1283).
  • the screening method of the present invention is both rapid and simple and, as such, is particularly advantageous in identification of hundreds of different GSCs from a pool of hundreds of thousands of gene constructs. Every stage of the genetic screen is amenable to robotic automation and therefore large numbers of target gene sequences can be screened for effective gene-specific GSCs in a high throughput fashion.
  • the populations of GSCs obtained confer a wide range of levels of, for example, suppression and provide the potential to quantitatively modulate the expression of a specific target gene.
  • the more effective GSCs and other molecules capable of modulating target gene expression have the potential to completely suppress gene expression at the mRNA level and produce phenocopies of homozygous null mutants.
  • the universal nature of the fusion approach provides a valuable tool for identifying GSCs and other molecules capable of modulating target gene expression, useful in validating potential novel drug targets and better understanding the biological function of unknown genes.
  • Hradl and Hrad27 These genes play potential roles in cell cycle checkpoint functions.
  • Figure 6 shows the inserts contained on the GSCs specific for Hradl and their alignment with the target gene sequence. The level of suppression of ⁇ -galactosidase activity displayed by these GSCs ranged from 21% to 45%.
  • Figures 7 and 8 summarise the arrangement of inserts derived from the Hrad27 GSCs and the level of suppression of the fusion gene target in yeast.
  • the target gene sequence fused to the amino terminal end of the lacZ reporter is indicated at the top of the diagram.
  • the labels “ATG” and “TGA” refer to the translation initiation and stop codons, respectively.
  • “EVS” indicates the penultimate codon relative to the stop codon.
  • the expression library construction process was modified from that indicated in Figure 1. More specifically, the random fragments derived from each of the target genes were cloned directly into the yeast expression plasmid pGTl 18 without first constructing an intermediate library (step 3 in Figure 1).

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Abstract

Cette invention a trait à des produits de recombinaison de fusion d'acide nucléique pouvant être exprimés une fois introduits dans Schizosaccharomyces pombe. Ces produits de recombinaison de fusion sont principalement constitués d'une séquence nucléotidique cible et d'une séquence nucléotidique reporter, ces séquences, cible et reporter, étant situées l'une par rapport à l'autre de manière que l'expression de la séquence reporter soit modulée par toute molécule modulant également l'expression de la séquence cible. L'expression de la séquence reporter confère un phénotype décelable à Schizosaccharomyces pombe. Cette invention porte également sur des techniques permettant de déterminer si, ou à quel degré, une molécule module de manière spécifique une séquence nucléotidique cible dans une cellule eucaryote.
PCT/AU1999/001131 1999-01-08 1999-12-21 Systeme permettant d'identifier des modulateurs de gene articule autour de cellules eucaryotes WO2000042167A1 (fr)

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WO2002046369A3 (fr) * 2000-12-08 2003-03-13 Septegen Ltd Essai a base de levure

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0590721A1 (fr) * 1992-09-29 1994-04-06 ENICHEM S.p.A. Procédé d'expression de récepteurs du système nerveux humain ches la levure schizosacchoromyces pombe
WO1995029254A1 (fr) * 1994-04-20 1995-11-02 Gene Shears Pty. Ltd. SYSTEME D'EXPRESSION DE GENES $i(IN VIVO)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0590721A1 (fr) * 1992-09-29 1994-04-06 ENICHEM S.p.A. Procédé d'expression de récepteurs du système nerveux humain ches la levure schizosacchoromyces pombe
WO1995029254A1 (fr) * 1994-04-20 1995-11-02 Gene Shears Pty. Ltd. SYSTEME D'EXPRESSION DE GENES $i(IN VIVO)

Non-Patent Citations (1)

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Title
KUDLA B. ET. AL.: "Construction of an expression vector for the fission yeast Schizzosaccharomyces pombe.", NUCLEIC ACID RESEARCH, vol. 16, no. 17, 1988, pages 8603 - 8617 *

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WO2002046369A3 (fr) * 2000-12-08 2003-03-13 Septegen Ltd Essai a base de levure

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