GB2326413A - Screening apoptosis inhibitors using the BAX gene - Google Patents
Screening apoptosis inhibitors using the BAX gene Download PDFInfo
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Abstract
A method of screening cDNA encoding putative apoptosis inhibitors in yeast cells expressing the pro-apoptotic BAX gene is described. Saccharomyces cerevisiae are transformed with vectors containing the BAX gene and a promoter, and candidate cDNA's. BAX gene expression is induced by exposing the cells to a substance which activates the promoter. cDNA is recovered from those cells which do not display apoptosis and introduced into a mammalian cell, for example COS cells, which are then challenged with a proapoptotic protein. Those cells which do not exhibit apoptosis confirm the inhibitory capacity of the cDNA. The restoration of cell growth in yeast expressing BAX genes by Bcl-2 and Bcl-xL is described. The BAX-delta gene lacks the region coding for a C-terminal membrane anchor. Bax-delta expressing cells can be recovered by transfection with a human cerebellum cDNA library but not by Bcl-2 or Bcl-xL.
Description
Organic Compounds NEW SCREEN
The present invention relates to a method of screening and isolating apoptosis inhibitors to genes obtainable by this method and to proteins encoded by said genes.
Programmed cell death is an essential feature of all multicellular organisms, crucial for development and useful in disease control. In mammalian cells, the most common form of programmed cell death is apoptosis. Although this describes a very specific series of events only seen in animals, the term apoptosis is often used interchangeably with programmed cell death even to describe similar events occurring in plant cells. This type of cell death is distinct from necrosis and plays a crucial role in different physiological situations. A malfunctioning apoptotic pathway contributes to a variety of diseases which include cancer and neurodegeneration. Whereas too much cell death can cause degenerative disorders, such as neurodegenerative diseases and neuropathies, too little cell death may lead to proliferative disorders, such as cancer, or autoimmune diseases. Cells undergoing apoptosis generally display shrinkage, loss of cell-cell contact, chromatin condensation, and internucleosomal degradation of DNA to oligonucleosomal fragments. The degradation of DNA may be a slow process occurring days after cessation of cellular activities accompanying apoptosis. In certain circumstances it is evident that apoptosis is triggered by or is proceeded by changes in protein synthesis. Cells can also undergo apoptosis in response to environmental conditions for example the disappearance of a stimulus such as interleukin-3 withdrawal from mature lymphocytes, appearance of a stimulus, such as glucocorticoid hormones for immature thymocytes or the removal of a colony stimulating factors from hemopoietic precursors. However, although it is known how to induce apoptosis in different types of cells, the mechanism of this process is still unclear. Accordingly, a need exists to identify genes and their expression products which play key roles in the apoptotic process. Such progress is expected to bring about new treatments for those diseases in which apoptosis, or the lack thereof, plays a major role.
An ever growing number of genes has been identified which are involved in regulation of apoptosis. One group of such genes belongs to the Bcl-2 family. The members of this family probably control a distal step in the pathway which culminates in programmed cell death. In general, expression of family members Bcl-2 and Bcl-x(L) has been shown to aid survival of cells (for example, following withdrawal of growth factors). In contrast, elevated levels of another member of the family, Bax, inhibits cell growth and promotes death. Apoptosis is also induced by staurosporine, a compound which inhibits protein kinase C and a wide range of other kinases (Tamaoki, T. (1991)
Methods-Enzymol. 201 340-7). It has been postulated that the ratio between antiapoptotic proteins, such as Bcl-2 or Bcl-x(L), relative to pro-apoptotic proteins, such as
Bax, determines whether a cell lives or dies.
The precise way in which the Bcl-2 family of proteins regulates apoptosis remains an enigma. However, it is believed that some form of regulation occurs through physical interactions between death-effecting and death-inhibiting molecules.
The known inhibitors of apoptosis in mammalian cells Bcl-2 and Bcl-x(L), overcome growth arrest in yeast. The effects of Bax and its rescuing partners Bcl-2 and
Bcl-x(L), on the yeast Saccharomyces cerevisiae, is discussed in "Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in
Saccharomyces cerevisiae Greenhalf W. et al. FEBS Letters 380 (1996) 169-175, the entire contents of which are incorporated herein by reference thereto.
It is clear from the above that the ability to control apoptosis in cells by preventing it from occurring will provide therapeutic methods for neurodegenerative diseases and neuropathies which are characterised by cell loss or degeneratiops.
Diseases associated with apoptosis include Alzheimer's disease, Huntington's disease, spinal muscular dystrophy and amyotrophic lateral sclerosis.
The present invention is concerned with a method of screening and isolating apoptosis inhibitors, with genes obtainable by this method and to proteins encoded by said genes. These inhibitors are useful for treatment of neurodegenerative diseases and neuropathies as above discussed.
A further object of the present invention relates to the introduction into a cell of a heterologous nucleic acid, hereinafter referred to as cDNA, encoding an apoptosis inhibitor of the invention and thereafter examining the resultant physiological impact, so as to develop agents that inhibit apoptosis.
Generally, host cells suitable for production of a protein isolated through use of the invention include eukaryotic cells, for example animal cells particularly mammalian cells.
Yeast cells are also particularly suitable for protein production. The vector containing the cDNA encoding a protein of the invention can be propagated in prokaryotic cells, such as gram-positive and gram-negative bacteria, such as e.g. E. coli K-12 strains, DH5a and HB 101, or Bacilli. A protein of the invention can be produced directly in recombinant cell culture or as a fusion with a signal sequence, preferably a hosthomologous signal. Higher eukaryotic host cells and yeast cells such as e.g.
Saccharomyces cerevisiae are preferred. An especially preferred host for production of the protein of the invention is Saccharomyces cerevisiae, particularly the following strains SE312 (MATa, leu2, lys2, his4, ura3, pral and prbl), TFY2 (MATa, his, ura3-52, trpl-285, acel, LEU2::YlpCL:CUP1 'Gal+) and W3116 each of which is referred to in Greenhalf et al.
The invention relates to a method of screening and isolation of nucleic acid sequences which potentially encode apoptosis inhibitors comprising the steps of (a) preparing a test system comprising a host cell comprising a Bax expression cassette (b) introducing a test nucleic acid into the test system (c) identifying a test nucleic acid which inhibits apoptosis.
In particular the present invention relates to a method for the screening and isolation of nucleic acids encoding an apoptosis inhibitor, comprising the steps of:
(a) growing a host yeast cell comprising a BaxA expression cassette operatively linked to a first promoter, the expression of said cassette being capable of inducing apoptosis in the host yeast cell in the absence of an apoptosis inhibitor, and a cDNA;
(b) exposing the host yeast cell to a condition or substance(s) which activates the first and second promoter;
(c) isolating the cDNA sequence from host yeast cells which do not display apoptosis; and
(d) confirming anti-apoptotic capacity of the cDNA sequence by introducing the cDNA sequence into a mammalian cell lacking an apoptosis inhibitor, the cDNA sequence being introduced in a manner such that it is capable of being expressed, and thereafter exposing the cell to a pro-apoptotic protein.
It will be understood that cells which do not exhibit apoptosis, confirm the antiapoptotic capacity of the cDNA sequence. Suitable cells in which the anti-apoptotic capacity of the cDNA sequence can be confirmed are for example COS1/ COS7 cells pre B cells, T cells and neuronal cells.
The BaxA expression cassette operatively linked to a first promoter and the DNA operatively linked to a second promoter are suitably introduced into the host cell using one or more expression vectors. According to a first method a single plasmid may carry the BaxA expression cassette operatively linked to a first promoter and the cDNA subsequently transformed operatively linked to a second promoter. According to a second method, the host may be co-transformed with a first plasmid carrying the BaxA expression cassette operatively linked to a first promoter and a second plasmid carrying the cDNA operatively linked to a second promoter. Transformation is carried out using standard methods known to one skilled in the art.
It will be understood that in order to identify and isolate nucleic acid encoding an apoptosis inhibitor according to the present invention, it is necessary to ensure that the nucleic acid encoding the apoptosis inhibitor is expressed to an extent sufficient to inhibit growth attenuation caused by expression of the BaxA expression cassette in yeast and it is similarly necessary to ensure that BaxA expression cassette is expressed to an extent sufficient to cause growth attenuation in the host in the absence of expression of the apoptosis inhibitor. Hereinafter we will define the extent to which the nucleic acid encoding the apoptosis inhibitor is required to be expressed as "an apoptosis inhibitory extent" and the extent to which the BaxA expression cassette is required to be expressed as "an apoptosis causing extent" will be defined. These extents can easily be determined by routine methods by those skilled in the art.
Expression and cloning vectors (referred to hereinbelow) usually contain a promoter that is recognised by the host organism and is operably linked to the nucleic acid which is to be expressed or cloned. An expression vector additionally comprises expression control sequences essential for the transcription and translation of the DNA.
Thus an expression vector refers to a recombinant DNA construct, such as a plasmid, a phage, recombinant virus or other vector which, upon introduction into a suitable host cell, results in expression of the cDNA. Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and/or prokaryotic cells.
Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be amplified by insertion into the host genome.
Advantageously, expression and cloning vectors contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
Typical selection markers for yeast are ADE2, LEU2, URA3, HIS3, HIS4, LYS2, TRP1 and TRP2.
Since the amplification of the vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid.
Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding an apoptosis inhibitor of the invention, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G41 8 or hygromycin. The mammalian cell transfectants are placed under selection pressure which only those transfectants are uniquely adapted to survive which have taken up and are expressing the marker.
Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to the nucleic acid which is to be expressed or cloned. Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA to be expressed or cloned by removing the promoter from the source
DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.
Promoters suitable for use with prokaryotic hosts include, for example, the - lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA to be expressed or cloned, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a
Shine-Delgarno sequence operably linked to the DNA to be expressed or cloned.
Transcription from vectors in mammalian host cells may be controlled by a promoter compatible with the host cell systems, e.g. a promoter derived from the genome of a virus. Suitable plasmids for expression in mammalian cells, are vectors containing e.g. the promoter of cytomegalovirus (CMV) for example pcDNA3 a mammalian expression vector derived from pRc/CMV which has a SV40 origin of replication allowing transient episomal relication in mammalian cells, RSV, SV40 virus or MMTV LTR. Promoters particularly suitable for use with a yeast host are any of the known yeast promoters for example Gal (i.e. GAL1/GAL10), ura3, cycle, arg3, trpl, alcohol dehydrogenase (adh) 1 or phosphoglycerate kinase (pgk) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or functional fragments of these promoters. Preferably the first and second promoters are the same.
Normally the vector into which the nucleic acid of the invention is introduced is a plasmid or a phage or a virus. Where the vector encodes for an apoptosis inhibitor and is to be used in a method of treatment as hereinafter described, it will be appreciated that the vector should be innocuous. Preferably the vector is a plasmid. The vector may be a yeast integrative plasmid (Ylp), which means it is maintained in yeast only after chromosomal integration for example pRS305 (ATCC77140) a yeast integrating vector with the Saccharomyces cerevisiae LEU2 gene and the ampR gene as yeast and E. coli selectable markers respectively, and pPFY7 which is a similar plasmid also carrying the yeast LEU2 gene. The LEU2 gene is the preferred site for integration, a unique BstElI site lies within LEU2 and is used for direct integration. Alternatively the vector may be any known yeast episomal plasmid which is present in a high copy number, 25-100 copies per cell, and transform yeast very well for example pDP34 (a YEp vector containing URA3 and dLEU2 as yeast selection marker. In a still further embodiment of the present invention any known yeast expression vector including for example pYEUra3 which is a yeast centromere plasmid present in single or low copy number, having an inducible GAL1/GAL10 promoter carrying the URA3 gene for selection in yeast and confers ampicillin resistance for selection in E.coli. may be used. Any promoter fragment is suitable.
Transcription of a DNA encoding a protein according to the invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.
The various DNA segments of the vector DNA are operatively linked, i.e. they are contiguous and placed into a functional relationship to each other employing conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the required vectors. If desired, analysis to confirm correct sequences in the constructed vectors is performed in a manner known in the art. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing expression and function of the expressed or cloned protein are known to those skilled in the art. Nucleic acid presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), in situ hybridisation, using an appropriately labelled probe based on the nucleic acid sequence of an apoptosis inhibitor of the present invention, binding assays, immunodetection and functional assays.
Nucleic acids of the invention can be incorporated into vectors for further manipulation. Such vectors are also provided herein. Specifically, the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of the above mentioned DNAs of the invention, particularly such DNA designated as being preferred.
The hybrid vectors of the invention comprise an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restriction sites.
Preferably, a hybrid vector of the invention comprises an above described nucleic acid insert operabiy linked to an expression control sequence, in particular those described hereinafter.
Vectors typically perform two functions in collaboration with compatible host cells.
One function is to provide for replication and expression of gene constructs in a suitable host, either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors) as above discussed. The other function is to facilitate the cloning of a nucleic acid that encodes a protein of the invention, i.e. to produce useable quantities of the nucleic acid (cloning vectors). A cloning vector comprises the DNAs as described above, an origin of replication or an autonomously replicating sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites.
The invention further provides host cells capable of producing a protein of the invention and containing heterologous (foreign) DNA encoding said protein.
The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g. those mentioned above, that are transfected with an appropriate expression vector.
A protein of the invention may also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein(s) or fusion protein encoded by the DNA of the invention is (are) expressed.
The first and second promoters used in the method of identifying and isolating are preferably the same.
Stably transfected cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, cells are transfected with a reporter gene to monitor transfection efficiency. To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of apoptotic inhibitor-encoding nucleic acid to form protein of the invention. The precise amounts of DNA encoding a protein of the invention may be empirically determined and optimised for a particular cell and assay.
Host cells are transfected or transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique, by electroporation or lipofectin-mediated. The DNA to be expressed can be amplified by
PCR and be directly transfected into the host cells without any replication component.
Successful transfection or transformation is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press).
The above-mentioned properties may be detected, and optionally quantified, in vitro or in vivo according to methods well-known to those skilled in the art, e.g. using the assays decribed in more detail in the Examples. Briefly, the ability of a protein of the invention to suppress the growth of tumor cells may be determined in culture by a colony formation assay.
Such assay enables growth suppression or cell death in its most general sense to be measured as it scores for either growth arrest and/or apoptosis. Apoptotic phenomena can be investigated using the TUN(N)EL staining procedure with single cells. The transactivation potential may be determined according to the CAT assay protocols described in G.P. Zambetti et al, Genes & Develop. 6, 1143-1152 (1992) incorporated herein by reference.
The invention further provides a method for the treatment of neurodegenerative diseases or neuropathies characterised by apoptosis said method comprising the steps of: a) preparing a non-pathogenic vector comprising a nucleic acid coding for an apoptosis inhibitor; and
b) administering the vector to a site undergoing apoptosis.
The vector can be administered to the central nervous system at a site which is undergoing apoptosis. Furthermore, the invention provides a method for treatment of a neurodegenerative disease or neuropathy comprising introducing into a mammalian cell, particularly a human neuronal cell, in which cell death cell cycle arrest is aberrant, a nucleic acid encoding an apoptosis inhibitor. It will be appreciated by the man skilled in the art that the present invention also embodies a method for the treatment of neurodegenerative diseases or neuropathies characterised by apoptosis wherein a protein of the invention, or a compound that mimics such a protein, is administered to a mammalian cell in a mammal in which cell death or cell cycle arrest is aberrant.
The following Examples serve to illustrate the present invention, but should not be construed as a limitation thereof. The invention particularly relates to the specific embodiments (e.g. the proteins, peptides nucleic acids, vectors, host cells, methods for their preparation, assays and uses thereof) as described in these Examples.
Example 1: Chemical synthesis of the human bax-aipha (bax) gene, using veast-biased codons
The 595 bp gene coding (SEQ ID No. 1) for the human Bax protein (Oltvai, Z. N.
Milliman, C. L. and Korsmeyer, S. J. Cell 74, 609-619, 1993; SEQ ID No. 2) is chemically synthesized using yeast codon bias. In essence, the gene is assembled from complementary deoxyoligonucleotides, each 70 to 80 base long and having 20 bp overlaps), via (i) 5' to 3' chain extension followed by (ii) polymerase chain reaction (PCR) amplification (Current Protocols, Asubel et al.). All PCRs are performed using the Vent polymerase (New England Biolabs).
The complete bax gene (a Bglll-Xbal fragment) is constructed in three parts. The first segment (a Bglll-Sall fragment) is assembled from oligonucleotides with SEQ ID Nos. 36). The second segment (a Sall-BamHI fragment) is assembled from oligonucleotides with SEQ ID Nos. 7-10). The third segment (a BamHI-Xbal fragment) is assembled from oligonucleotides with SEQ ID Nos. 11-14). Each segment is first subcloned in pUC19 or pUC19Bgl (Bglll site introduced at the Smal site of pUCl9 using linkers) and the authenticity of the clones are first confirmed by DNA sequencing. Finally, the 3 segments are ligated into pUC19Bgl (pUC19 modified at the BamHI site by Bglll linkers) which is digested with Bglll and Xbal. One correct clone is referred to as pUC1 9Bgl/bax.
Example 2: Subcloning of the human bax gene in a yeast integrating vector
A -675 bp Sall-BamHI GAL 10 promoter fragment (West R. Jr et al M. Mol. Cell. Biol. 4, 2467-2478, 1984), the -600 bp Bglll-Xbal bax gene sequence (SEQ ID No. 1; PCR primers SEQ ID No. 15-16 using pUC19Bgl/bax as template) and a -300 bp Xbal-Sacl fragment containing the SUC2 transcription terminator (Taussig, R. et al. Nucleic-Acids
Res, 11:1943-1954, 1983; SEQ ID Nos. 17-18) are subcloned in pRS305 (ATCC77140; GenBanklEMBL accession number: U03437) between its Sall and Sacl restriction sites.
The vector pRS305 is a yeast integrating vector with the Saccharomyces cerevisiae
LEU2 gene and the ampR gene as yeast and E. coil selectable markers. The plasmid pRS305/GALlOp-bax-SUC2tis thus obtained. The yeast expression cassette of human
Bax-alpha is depicted in SEQ ID No. 19.
Example 3: Subcloning the truncated human bax gene, Bax-delta, which lacks the region coding for the C-terminal membrane anchor, in pBlueScript KS+
The 522 bp gene (SEQ ID No. 20) coding for the Bax-delta protein (i.e. full-length human Bax SEQ ID No. 2, without its C-terminal membrane anchor, SEQ ID No. 21 is amplified by the polymerase chain reaction (PCR) using pRS305/bax as a template and two primers (SEQ ID Nos. 22-23). The gene is isolated as a Bglll-Xbal fragment.
The 3'-end of the bax-delta gene codes for the peptide QGGWDGLLSYFGT. The fragment is subcloned in pBlueScript KS+ (Stratagene) which is digested with Bglll and
Xbal, to yield the plasmid pBluKS+/bax-delta.
Example 4: Subcloning of bax-delta in a yeast integrating vector
The plasmid pBluKS+/bax-delta is digested with Bglll-Xbal. The 522 bp bax-delta gene fragment is isolated. A -675 bp Sall-BamHI GAL 10 promoter (as above), the -522 bp
Bglll-Xbal bax-delta gene sequence and a -300 bp Xbal-Sacl fragment containing the
SUC2 transcription terminator are subcloned in pRS305 (see Example 2) between the Sall and Sacl restriction sites (see Example 2). The plasmid pRS3051GAL10p-bax-delta- SUC2t is thus obtained. The yeast expression cassette of human Bax-alpha is depicted in SEQ ID No. 24.
Example 5: Construction of yeast expression plasmids for human Bcl-2 and Bcl- xL
The complete coding region of the human Bcl-xL protein (Boise, L. H. et al C. B. Cell 74, 597-608, 1993) is cloned from a thymus cDNA library (Clontech) as a Bglll-Xbal fragment by PCR using the primers with SEQ ID Nos. 25-26.
The complete coding region of the human Bcl-2 protein (C.M. Croce et al. Proc. Natl.
Acad. Sci. USA 83, 5214-5218, 1986; Proc. Natl. Acad. Sci. USA 87, 3660-3664, 1990) is cloned as a Bglll-Xbal fragment by PCR using a plasmid clone of human B cell
CLL/lymphoma 2 BCL2 (ATCC 79804) as a template and two primers (SEQ ID Nos. 2728).
DNA sequencing confirmed the authenticity of the isolated sequences. The construction of the Bcl-xL and the Bcl-2 expression cassettes, under the control of the GAL 10 promoter, is performed as described in Example 2. The two cassettes, however, are subcloned in the CEN plasmid pDP83 (containing the yeast URA3 gene as a selectable marker) and the yeast 2-micron vector pDP34 (containing the yeast URA3 gene as a selectable marker). The resultant plasmids are referred to as pDP83/GALlOp-BcI-xL- SUC2t, pDP83/GAL I Op-Bcl-2-SUC2t, pD P34/GAL 1 Op-8cl-xL-SUC2t and pDP341GALlOp-Bc1-2-SUC2t, respectively.
Example 6: Integration of bax and bax-delta genes into the yeast genome
A gene pulser is employed for transformation of yeast cells by electroporation [Becker,
D. M.et al. Methods in Enzymol., 194: 182-187, 1991]. The strain is used for integrations of the human bax-aipha and the bax-delta genes into the yeast chromosome. Correct gene integration and gene replacement events are verified by PCR with primers flanking the insertion sites. Subsequently, the amplified fragments are analysed by agarose gel electrophoresis. Strains containing the correct integrations are referred to as HT444 bax and HT444 baxdel.
Example 7: Both bax and bax-delta genes, when expressed in yeast, stop cell growth
The protocols for monitoring induction of cell death have been described earlier (Greenhalf W et al as above).
The yeast cell culture media is minimal medium SD (as above) and rich medium YEP (2% bacto-peptone, 1% yeast extract Difco). The supplements are added to the SD medium as required for growth of a specific strain. The carbon sources which are used for growth are 2% (w/v) glucose or 2% (w/v) galactose.
Cells are suspended in water and 5p1 aliquots are spotted on appropriate minimal medium or rich medium plates.
Example 8: Both bax and bax-delta genes, when expressed in yeast1 kill yeast cells
The protocols for monitoring induction of cell death have been described earlier (see
Greenhalf W et al above).
Two stains (methylene blue and erythrosine B) are used for identifying cell death (i.e. cells which have lost membrane integrity). The cells are initially grown in SD-Glucose (in which case the cells were washed 3 times in sterile water before induction with galactose).
In order to stain with methylene blue, cells are first resuspended in an appropriate volume of 0.9 M KCI and were then treated with the dye solution at a final concentration of 0.5 g/l (Pierce, J., J. Inst. Brew. 76, 442-443, 1970).
The medium used for staining with erythrosine B (Bonneau, M et al, M. Anal. Biochem.
193, 225-230, 1991; Serva,) contains agarose (2% w/v) in SD, an appropriate carbon source and 7.5 pM erythrosine B. Cells are first spread on microscope slides and then incubated at 300C in a humidifying chamber.
Methylene blue stained cells are counted using a haemocytometer. The average and standard error of mean (SEM) is calculated separately for blue (dead) and white ("live") cells in each quadrant. The percentage of blue cells are then defined as lying between the extremes (EG. the highest value would be (Blue + SEMblue)l (Blue + SEMblue +
White - SEMWhjte). The experiments are repeated and the final range is defined as the overlap of results from different experiments.
TABLE 1. Inhibition of cell growth and induction of cell death (as monitored by methylene blue staining), after induction of Bax in HT444~bax*. The strain HT444~bax was grown in liquid medium using a combination of different pre- and main-cultures. The media SD-Glucose, SD-Galactose, YEP-Glucose and YEP-Galactose have been described above and, where necessary, are supplemented with the appropriate amino acids and uracil. Although the effects described here pertain to single-copy expression of Bax, no significant differences are observed on expressing the protein from the multicopy episomal plasmid pDP34.
Preculture Induction Methylene Blue Growth in Bax Medium Medium Staining inducing Medium After 24 hours (%) YEP-Glu YEP-Gal 22-35 Growth for 2 to 5 hours YEP-Glu SD-Gal 50-70 No Growth SD-Glu ~ SD-Gal - 80-90 No Growth *The number of cells which were examined varied but in all cases exceeded 500.
TABLE 2. Comparison of the strains HT444~bax and HT444~baxdel: inhibition of cell growth and induction of cell death, as monitored by erythrosine B staining. The strains
HT444~bax and HT444~baxdel were pre-cultured in SD-Glucose liquid medium for 16 h and were then induced for Bax and Baxdel expression in SD-Galactose for different time periods. The effects described here pertain to single-copy expression of Bax and
Baxdel, under the control of the strong GAL 10 promoter, from the yeast genome.
Strains Erythrosine B Staining Growth in SD-Gal Medium (% viability)* 8 h 24 h 8 h 24 h HT444~bax 14.5 *2.64 8.98*1.96 No Growth No Growth HT444 baxdel 59.4*6.1 51.5 i4.25 No Growth No Growth *The number of cells which were examined varied but in all cases exceeded 500.
Example 9: The bcl-2 and bcl-xL genes, encoding Bax inhibitory proteins, restore yeast cell growth in Bax-expressing cells
The plasmids pDP34/GAL lOp-Bcl-xL-SUC2t (Example 18) and pDP34/GAL10p-Bcl-2-
SUC2t (Example 5) are transformed in the strain HT444~bax. Relative growth rates, in the presence of galactose, are compared in Table 3. Similar results are obtained when plasmids pDP83/GAL1Op-Bcl-xL-SUC2t and pDP83/GAL1Op-Bcl-2-SUC2t (Example 5) are transformed in the stain HT444-bax.
Example 10: The bcl-2and bcl-xL genes do not restore yeast cell growth in Baxdelta expressing cells
The plasmids pDP34/GAL lOp-Bcl-xL-SUC2t and pDP341GALlOp-Bc1-2-SUC2t (Example 5) are transformed in the strain HT444~baxdel. Relative growth rates, in the presence of galactose, are compared in Table 3. Similar results are obtained when plasmids pDP83/GAL1 Op-Bcl-xL-SUC2t and pDP83/GAL1 Op-Bcl-2-SUC2t (Example 5) are transformed in the strain HT444~baxdel.
TABLE 3. Relative growth rates of strains HT444~bax and HT444~baxdel in the presence/absence of plasmids encoding bcl-2 and bcl-xL
Strain Plasm it Relative growth rates in SD medium glucose alactose HT444~bax pDP34 +++ HT444~bax pDP34/GAL10p-bcl-xl-SUC2t +++ HT444~bax pDP34/GAL 10p-bcl2-SUC2t +++ ++ HT444 baxdel PDP34 HT444~baxdel pDP34/GAL10p-bcl-xl-SUC2t HT444~baxdel pDP34/GAL10p-bcl2-SUC2t Example 11: Transfection of a cDNA library from the human cerebellum restores growth in Bax-delta expressing yeast cells
A cDNA library from the human brain cerebellum (Clontech), constructed in the CENplasmid (CEN-4) pYEUra3 (i.e. a yeast single-copy vector), was transformed by the lithium acetate protocol (Gietz, D. et al. Nucleic Acids Res. 20, 1425, 1992) in selective
SD medium containing glucose. Plates containing confluent colonies are replica-plated onto selective SD medium containing galactose. Colonies which grow in galactosecontaining medium contain plasmids encoding genes that negate the effect of
Bax~delta.
SEQ ID No. 1
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 596 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: a Bglll-Xbal DNA fragment, coding for the human Bax protein, chemically synthesized using yeast-biased codons Bglll 5' AT AGATCT A
ATGGACGGTTCCG GTGAACAACCAAGAGGTGGGGGTCCAACCTCCTCTGAACAAA TCATGAAGACTGGTGCCTTGTTGCTTCAAGGTTTCATCCAAGACAGAGCTGGTAGA ATGGGTGGTGAAGCTCCAGAATTGGCCTTGGACCCAGTTCCACAAGACGCTTCCA CCAAGAAGTTGTCTGAATGrrTGAAGAGAATCGGTGACGAATTGGACTCTAACATG
GAATTGCAAAGAATGATTGCCGCTGTCGACACCGACTCCCCAAGAGAAGTCTTCTT CAGAGTCGCCGCTGACATGTTCTCTGACGGTAACTTCAACTGGGGTAGAGTTGTC
GCCTTGTTCTACTTCGCCTCTAAGTTGGTCTTGAAGGCCTTGTGTACCAAGGTCCC AGMTTGATCAGMCCATCATGGGTTGGACI GTTGGAC7GGACTTCTTGAGAGAAAGATTGT TGGGTTGGATCCAAGACCAAGGTGGTTGGGACGGTTTGTTGTCCTACTTCGGTAC TCCAACTTGGCAAACCGTTACCATCTTCGTTGCCG GTGTCTTGACCGCCTCTTTGA CCATCTGGAAGAAGATGGGTTGA TCTAGA AT 3'
Xbal
SEQ ID No. 2
Sequence type: the human Bax-alpha protein
Sequence length: 194 amino acid residues
Immediate experimental source: translated from the DNA sequencee
Features: human Bax-alpha MDGSGEQPRGGGPTSSEQIMKTGALLLQGFIQDRAG RMGGEAPELALDPVPQDASTK
KLSECLKRIGDELDSNMELQRMIAAVDTDSPREVFFRVAADMFSDGNFNWGRVVALFY FASKLVLKALCTKVPELI RTI MGWTLDFLRERLLGWIQDQGGWDGLLSYFGTPTWQTV
TI FVAGVLTASLTIWKKMGSR
SEQ ID No. 3
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 85 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (first part: Bglll-Sall fragment), yeastbiased codons 5' ATAGATCTAATGGACGGTTCCGGTGAACAACCAAGAGGTGGGGGTCCMCCTCCT
CTGMCMATCATGMGACTGGTGCCTTGT 3' SEQ ID No. 4
Sequence type: DNA coding for the human Bax-aipha protein
Sequence length: 85 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (first part: Bglll-Sall fragment), yeastbiased codons 5' CAAGGCCAATTCTGGAGCTTCACCACCCATTCTACCAGCTCTGTCTTGGATGAAAC CTTGAAGCAACAAGGCACCAGTCTTCATG 3'
SEQ ID No. 5
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 85 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (first part: Bgill-Sall fragment), yeastbiased codons 5' AAGCTCCAGAATTGGCCTTGGACCCAGTTCCACAAGACGCTTCCACCAAGAAGTTG
TCTGAATGTTTGAAGAGAATCGGTGACGA 3' SEQ ID No. 6
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 68 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (first part: Bglll-Sall fragment), yeastbiased codons 5' GTGTCGACAGCGGCAATCATTCTTTGCAATTCCATGTTAGAGTCCAATTCGTCACC
GATTCTCTTCAA 3' SEQ ID No.7 Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 67 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (middle part: Sall-BamHI fragment), yeast-biased codons 5' CTGTCGACACCGACTCCCCAAGAGAAGTCTTCTTCAGAGTCGCCGCTGACATGTTC
TCTGACGGTAA3' SEQ ID No. 8
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 70 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (middle part: Sall-BamHI fragment), yeast-biased codons 5' CCAACTTAGAGGOGAAGTAGAACAAGGCGACAACTCTACCCCAGTTGAAGTTACCG
TCAGAGAACATGTC 3'
SEQ ID No.9 Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 70 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (middle part: Sall-BamHI fragment), yeast-biased codons 5'
CTACTTCGCCTCTAAGTTGGTCTTGAAGGCCTTGTGTACCAAGGTCCCAGAATTGA TCAGAACCATCATG 3' SEQ ID No. 10
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 68 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (middle part: Sall-BamHI fragment), yeast-biased codons 5'
TTGGATCCAACCCAACAATCTTTCTCTCAAGAAGTCCAAAGTCCAACCCATGATGGT TCTGATCAATT 3' SEQ ID No. 11
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 52 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (third part: BamHI-Xbal fragment), yeast-biased codons 5' TTGGATCCAAGACCAAGGTGGTTGGGACG GTTTGTTGTCCTACTTCGGTACT 3' SEQ ID No. 12
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 50 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (third part: BamHI-Xbal fragment), yeast-biased codons 5' AACGAAGATG GTAACG GTTTGCCAAGTTGGAGTACCGAAGTAG GACAACA 3' SEQ ID No. 13
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 50 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (third part: BamHI-Xbal fragment), yeast-biased codons 5' AAACCGTTACCATCTTCGTTGCCGGTGTCTTGACCGCCTCTTTGACCATC 3'
SEQ ID No. 14
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 53 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: DNA coding for the human Bax protein (third part: BamHI-Xbal fragment), yeast-biased codons 5' ATTCTAGAACCCATCTTCTTCCAGATGGTCAAAGAGGCGG SEQ ID No. 15
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 29 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' end sense strand of the coding sequence of the human bax-alpha gene; an extra nucleotide after the Bglll site 5' AT AGATCTAATGGACGGTTCCGGTGAACA3'
Bglll SEQ ID No. 16
Sequence type: DNA coding for the human Bax-alpha protein
Sequence length: 31 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' end anti-sense strand of the coding sequence of the human bax-alpha gene; contains the Xbal site 5' TC TCTAGA TCAACCCATCTTCTTCCAGATGG 3'
Xbal
SEQ ID No. 17
Sequence type: DNA with restriction site
Sequence length: 29 bases
Strandedness: single
Topology: linear
Source: yeast genomic DNA
Immediate experimental source: synthetic
Features: sense strand of the yeast SUC2 gene transcriptional terminator 5' AT TCTAGA AGGTTATAAAACTTATTGTC 3'
Xbal
SEQ ID No. 18
Sequence type: DNA with restriction site,
Sequence length: 28 bases
Strandedness: single
Topology: linear
Source: yeast genomic DNA
Immediate experimental source: synthetic
Features: anti-sense strand of the yeast SUC2 gene transcriptional terminator 5' TA GAGCTC GGTCCATCCTAGTAGTGT 3'
Sacl
SEQ ID No. 19
Sequence type: DNA containing the human Bax expression cassette for expression in yeast
Sequence length: 1607 bases
Strandedness: single
Topology: linear
Source: human and yeast
Immediate experimental source: synthetic
Features: a Sall-Sacl DNA fragment containing the Sall-BamHI GAL 10 promoter fragment (33-707 bases), the human bax gene (716-1291 bases) and the terminator from the yeast Sus2 gene (1301-1601 bases).
Sall 5' GTCGACG GTATC G ATAAG CTTGATATC GAATTC CTTGAATTTTCAAAAATTCTTACTT
TTT-Tf-TGGATGGACGCAAAGAAG7AATAATCATATTACATGGCATTACCACCAT ATACATATCCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAA
GAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCT TAACTGCTCATTG CTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGC CCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTG AAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACT AGCTTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTC
AAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCT
TATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATAT
AAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATT
ACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACT
TTAACGTCAAGGAGAAAAAACCCCGGATCTAATGGACGGTTCCGGTGAACAACCAA GAGGTGGGGGTCCAACCTCCTCTGAACAAATCATGAAGACTGGTGCCTTGTTGCTT
CAAGGTTTCATCCAAGACAGAGCTGGTAGAATGGGTGGTGAAGCTCCAGAATTGG
CCTTGGACCCAGTTCCACAAGACGCTTCCACCAAGAAGTTGTCTGAATGmTGAAG
AGAATCGGTGACGAATTGGACTCTAACATGGAATTGCAAAGAATGATTGCCGCTGT CGACACCGACTCCCCAAGAGAAGTCTTCTTCAGAGTCGCCGCTGACATGTTCTCTG
ACGGTAACTTCAACTGGGGTAGAGTTGTCGCCTTGTTCTACTTCGCCTCTAAGTTG
GTCTTGAAGGCCTTGTGTACCAAGGTCCCAGAATTGATCAGAACCATCATGGGTTG
GACTTTG GACTTCTTGAGAGAAAGATTGTTG G GTTG GATCCAAGACCAAG GTGGTT GGGACGGTTTGTTGTCCTACTTCGGTACTCCAACTTG GCAAACCGTTACCATCTTC GTTGCCGGTGTCTTGACCGCCTCTTTGACCATCTGGAAGAAGATGGGTTGATCTAG
AACGGTACTAGAGGTTATAAAACTTATTGTCTTTTTTATTTTTTTCAAAAGCCATTCT AAAG G G CTTTAGCAAC GAGIGAC GAATGTAAAAC7MGA7CAAAGAATAACCT
CCAAACCATTGAAAATGTATTTTTA rr IrrATTTTCTCCCGACCCCAGTTACCTGGAA TTTGTTCTTTATGTACTTTATATAAGTATAATTCTCTTAAAAATTTTTACTACTTTGCA ATAGACATCATtTTTTCACGTAATAAACCCACAATCGTAATGTAGTTG CCTTACACTA CTAGGATGGACCGAGCTC 3'
Sacl
SEQ ID No. 20
Sequence type: DNA coding for the Bax-delta protein
Sequence length: 522 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: a Bglll-Xbal DNA fragment coding for the Bax-delta protein (i.e. human Baxalpha from which the C-terminal membrane anchor is deleted) Bglll 5' AT AGATCT A ATGGACGGTTCCGGTGAACAACCAAGAGGTGGGGGTCCAACCTCCTCTGAACAAA
TCATGAAGACTGGTGCCTTGTTGCflCAAGG7CATCCAAGACAGAGCTGGTAGA ATGGGTGGTGAAGCTCCAGAATTGGCCTTGGACCCAGTTCCACAAGACGCTTCCA CCAAGAAGTTGTCTGAATG7GAAGAGAATCGGTGACGAATTGGACTCTAACATG GAATTGCAAAGAATGATTGCCGCTGTCGACACCGACTCCCCAAGAGAAGTCTTCTT
CAGAGTCGCCGCTGACATGTTCTCTGACGGTAACTTCAACTGGGGTAGAGTTGTC
GCCTTGTTCTACTTCGCCTCTAAGTTGGTCTTGAAGGCCTTGTGTACCAAGGTCCC
AGAATTGATCAGAACCATCATGGGTTGGACTTTGGACTTCTTGAGAGAAAGATTGT
TGGGTTGGATCCAAGACCAAGGTGGTTGGGACGGTTTGTTGTCCTACTTCGGTAC
TTGA TCTAGA AT 3'
Xbal
SEQ ID No. 21
Sequence type: the Bax-delta protein
Sequence length: 167 amino acid residues
Immediate experimental source: translated from the DNA sequencee
Features: human Bax-alpha without the membrane anchor MDGSGEQPRGGGPTSSEQIMKTGALLLQGFIQDRAGRMGGEAPELALDPVPQDASTK
KLSECLKRlGDELDSNM ELQRMlAAVDTDSPREVFFRVAADM FSDGNFNWGRVVALFY FASKLVLKALCTKVPELI RTIMGWTLDFLRERLLGWIQDQGGWDGLLSYFGT
SEQ ID No. 22
Sequence type: DNA with restriction site
Sequence length: 29 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' end sense strand of the coding sequence of the bax-delta gene; an extra nucleotide after the Bglll site 5' AT AGATCT A ATGGACGGTTCCGGTGAACA 3' Bglll SEQ ID No. 23
Sequence type: DNA with restriction site
Sequence length: 31 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' anti-sense strand of the coding sequence of the bax-delta gene containing an Xbal site 5' AT TCTAGA AGTACCGAAGTAGGACAACAAAC-3' (reverse primer)]
Xbal
SEQ ID No. 24
Sequence type: DNA containing the Bax-delta expression cassette for expression in yeast
Sequence length: 1529 bases
Strandedness: single
Topology: linear
Source: human and yeast
Immediate experimental source: synthetic
Features: a Sall-Sacl DNA fragment containing the Sall-BamHI GAL 10 promoter fragment (33-707 bases), the human bax-delta gene (716-1216 bases) and the terminator from the yeast SUC2 gene (1223-1527 bases).
Sall 5'
GTCGACGGTATCGATAAGCTTGATATCGAATTCCTTGAATTTTCAAAAATTCTTACTT
TTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCAT
ATACATATCCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAA GAGCCCCATTATCTTAGCCTAAAAAAACCTTCTC7G GAACTTTCAGTAATACG CT
TAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGC CCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTG
AAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACT
AGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTC AAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTrFTTAGCCT TATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATAT AAATG CAAAAACTG CATAAC CAC7AACTAATAC7CAACATtTTC G G7GTAfl ACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACT TTAACGTCAAGGAGAAAAACCCCGGATCTAATG GACGGTTCCGGTGAACAACCAA GAGGTGGGGGTCCAACCTCCTCTGAACAAATCATGAAGACTGGTGCCTTGTTGCTT
CAAGGTTTCATCCAAGACAGAGCTGGTAGAATGGGTGGTGAAGCTCCAGAATTGG CCTTGGACCCAGTTCCACAAGACGCTTCCACCAAGAAGTTGTCTGAATG7GAAG
AGAATCGGTGACGAATTGGACTCTAACATGGAATTGCAAAGAATGATTGCCGCTGT
CGACACCGACTCCCCAAGAGAAGTCTTCTTCAGAGTCGCCGCTGACATGTTCTCTG ACGGTAACTTCAACTGGGGTAGAGTTGTCGCCTTGTTCTACTTCGCCTCTAAGTTG
GTCTTGAAGGCCTTGTGTACCAAGGTCCCAGAATTGATCAGAACCATCATG GGTTG
GACTTTGGACTTCTTGAGAGAAAGATTGTTGGGTTGGATCCAAGACCAAGGTGGTT
GGGACGGTTTGTTGTCCTACTTCGGTACTTCTAGAACGGTACTAGAGGTTATAAAA
CTTATTGTCTTTTTTATTTTTTTCAAAAGCCATTCTAAAGGGCTTTAGCAACGAGTGA CGAATGTAAAACTTTATGATTTCAAAGAATAACCTCCAAACCATTGAAAATGTATf
TATTTTTATTTTCTCCCGACCCCAGTTACCTGGAATTTGT AAGTATAATTCTCTTAAAAATTTTTACTACTTTGCAATAGACATCATTTTTTCACGTAA
TAAACCCACAATCGTAATGTAGTTGCCTTACACTACTAGGATGGACCGAGCTC 3' Sacl SEQ ID No. 25
Sequence type: DNA with restriction site
Sequence length: 40 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' end sense strand of the coding sequence of the human bcl-xL gene; an extra nucleotide after the Bglll site 5' AT AGATCT ACGCGTGATGTCTCAGAGCAACGGGAGCTGG 3' Bglll SEQ ID No. 26
Sequence type: DNA with restriction site
Sequence length: 35 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' anti-sense strand of the coding sequence of the human bcl-xL gene containing an Xbal site 5' TA TCTAGA 7CCGACTGAAGAGTGAGCCCAGCAG 3' (reverse primer)]
Xbal SEQ ID No.27 Sequence type: DNA with restriction site
Sequence length: 28 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' end sense strand of the coding sequence of the human bcl-2 gene; an extra nucleotide after the Brill site 5'
AT AGATCT AATGGCGCACGCTGGGAGAA 3'
Bglll
SEQ ID No. 28
Sequence type: DNA with restriction site
Sequence length: 27 bases
Strandedness: single
Topology: linear
Source: human
Immediate experimental source: synthetic
Features: 5' anti-sense strand of the coding sequence of the human bcl-2 gene containing an Xbal site 5' AT TCTAGA CTTGTGGCCCAGATAGGCA 3' (reverse primer)]
Xbal
Claims (3)
- Claims 1. A method for the screening and isolation of nucleic acids encoding an apoptosis inhibitor, comprising the steps of: (a) growing a host yeast cell comprising a BaxA expression cassette operatively linked to a first promoter, the expression of said cassette being capable of inducing apoptosis in the host yeast cell in the absence of an apoptosis inhibitor, and a cDNA; (b) exposing the host yeast cell to a condition or substance(s) which activates the first and second promoter; (c) isolating the cDNA sequence from host yeast cells which do not display apoptosis; and (d) confirming anti-apoptotic capacity of the cDNA sequence by introducing the cDNA sequence into a mammalian cell lacking an apoptosis inhibitor, the cDNA sequence being introduced in a manner such that it is capable of being expressed, and thereafter exposing the cell to a pro-apoptotic protein.
- 2. A cDNA sequence obtainable according to the method claimed in claim 1.
- 3. A protein encoded by a cDNA sequence as claimed in claim 2.
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GB9713021A GB2326413A (en) | 1997-06-20 | 1997-06-20 | Screening apoptosis inhibitors using the BAX gene |
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GB9713021A GB2326413A (en) | 1997-06-20 | 1997-06-20 | Screening apoptosis inhibitors using the BAX gene |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001002550A2 (en) * | 1999-07-01 | 2001-01-11 | Janssen Pharmaceutica N.V. | Cell death related drug targets in yeast and fungi |
WO2002064766A2 (en) * | 2000-12-22 | 2002-08-22 | Janssen Pharmaceutica N.V. | Bax-responsive genes for drug target identification in yeast and fungi |
WO2007093807A2 (en) | 2006-02-15 | 2007-08-23 | Morvus Technology Limited | Apoptosis methods, genes and proteins |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995005760A1 (en) * | 1993-08-26 | 1995-03-02 | Ionel Marin Olteanu | Footwear with electronic device |
WO1995028497A1 (en) * | 1994-04-13 | 1995-10-26 | La Jolla Cancer Research Foundation | Interaction of proteins involved in a cell death pathway |
-
1997
- 1997-06-20 GB GB9713021A patent/GB2326413A/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995005760A1 (en) * | 1993-08-26 | 1995-03-02 | Ionel Marin Olteanu | Footwear with electronic device |
WO1995028497A1 (en) * | 1994-04-13 | 1995-10-26 | La Jolla Cancer Research Foundation | Interaction of proteins involved in a cell death pathway |
Non-Patent Citations (1)
Title |
---|
Biosis Acc. No.199800268468 & Molecular Cell Vol.1 (3) 1998 Qunli X & Reed J C pages 337-346 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001002550A2 (en) * | 1999-07-01 | 2001-01-11 | Janssen Pharmaceutica N.V. | Cell death related drug targets in yeast and fungi |
WO2001002550A3 (en) * | 1999-07-01 | 2001-11-15 | Janssen Pharmaceutica Nv | Cell death related drug targets in yeast and fungi |
WO2002064766A2 (en) * | 2000-12-22 | 2002-08-22 | Janssen Pharmaceutica N.V. | Bax-responsive genes for drug target identification in yeast and fungi |
WO2002064766A3 (en) * | 2000-12-22 | 2003-06-26 | Janssen Pharmaceutica Nv | Bax-responsive genes for drug target identification in yeast and fungi |
US7101990B2 (en) | 2000-12-22 | 2006-09-05 | Janssen Pharmaceutica N.V. | Bax-responsive genes for drug target identification in yeast and fungi |
WO2007093807A2 (en) | 2006-02-15 | 2007-08-23 | Morvus Technology Limited | Apoptosis methods, genes and proteins |
WO2007093807A3 (en) * | 2006-02-15 | 2007-11-29 | Morvus Technology Ltd | Apoptosis methods, genes and proteins |
Also Published As
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Date | Code | Title | Description |
---|---|---|---|
WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |