Patent Application
20050191639
The present invention relates generally biotechnology, and, more particularly, to a method of isolating genes involved in aging and/or aging-associated diseases and/or oxidative stress by mutation or transformation of a yeast cell, subsequent screening of the mutant or transformed cells that are affected in aging and isolation of the affected gene or genes, and the use of these genes to modulate aging and aging-associated diseases in a eukaryotic cell and/or organism.
Aging is a process in which all individuals of a species undergo a progressive decline in vitality leading to aging-associated diseases (AADs) and ultimately to death. The process of aging is influenced by many factors, including metabolic capacity, stress resistance, genetic stability, and gene regulation (Jazwinski, 1996). The final life span of an organism is also affected by the sum of deleterious changes and counteracting repair and maintenance mechanisms (Johnson et al., 1999).
Several approaches have been used to study aging. These include the identification of key genes and pathways important in aging, the study of genetic heritable diseases associated with aging, physiological experiment, and advanced molecular biology studies of model organisms. Among these organisms, Caenorhabditis elegans, Drosophila melanogaster and the budding yeast Saccharomyces cerevisiae have a life span that can be influenced by single gene mutations or overexpression of a particular protein (Johnson et al., 1999). Especially S. cerevisiae has been used as one of the model organisms to study the aging process (Gershon and Gershon, 2000). Yeast life span is defined as the number of daughter cells produced by mother cells before they stop dividing. This yeast cell divides asymmetrically, giving rise to a larger mother cell and a smaller daughter cell, leaving a circular bud scar on the mother cell's surface at the site of division. Thus, the age (counted in generations) of a mother cell can simply be determined by counting the number of bud scars on its surface. However, counting of the bud scars is labor intensive and time consuming and cannot be used as such as a screening method to isolate cells with an increased life span. Methods to isolate mutant yeasts with an increased life span have, amongst others, been described in PCT International Publication No. WO 95/05459 and U.S. Pat. No. 5,874,210. The latter patent describes a method to isolate a mutation which increases the number of divisions of yeast cells, comprising the labeling of the cell surface of the yeast cell with a fluorescent marker, thereby generating fluorescent yeast cells, culturing the yeast cells under conditions for growth of yeast cells for a period of time greater than the chronological life span of the strain, selecting the fluorescent cells by fluorescence-activated cell sorting and replating the fluorescent yeast cells. However, although this method may indeed give an enrichment of strains that survive longer, there is no direct selection for strains with an increased number of divisions. Non-dividing or slower dividing cells that also survive may be selected too.
Disclosed herein is a method for specific isolation of old yeast mother cells, with an increased number of divisions by staining the bud-scar chitin with fluorescein isothiocyanate (FITC)-wheat germ agglutinin (WGA) lectin and sorting by a FACS apparatus, after initial enrichment of the mother cells through magnetic-based sorting. The process is presented in
Much attention has been focused on the hypothesis that oxidative damage plays an important role in aging (Shan et al., 2001; Hamilton et al., 2001) and a generally accepted relation exists between oxidative stress and aging (Tanaka et al., 2001). Moreover, mutations in genes related to protection against oxidative stress have a clear influence on life span, both in S. cerevisiae and Caenorhabitis elegans (Laun et al., 2001; Ishii, 2001). The method disclosed herein is also suitable as an indirect selection for genes involved in oxidative stress. This is especially useful in cases where screening of libraries in an endogenous system is difficult or impossible, such as the screening of mammalian or plant libraries. Screening of such libraries may lead to new genes involved in protection against oxidative stress in general, but also, in the case of mammalian cells, to genes involved in AADs and/or diseases caused by oxidative stress, especially neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease (Calabrese et al., 2001).
A frequently practiced strategy in searching genes responsible for aging is by selecting survivals after the exposure of cells to stresses. Then, because of the complexity of the process, the question constantly remaining is whether the genes picked up are in response to the stress treatment rather than involved in aging. The invention described herein, however, provides an alternative that allows direct hunting of genes with potential anti-aging functions from various libraries or library combinations of eukaryotic organisms. Yeast lines are selected in a more natural condition, in addition to advantages of high throughput, high efficiency, and short time investment. The invention has great potential for rational drug design and development of therapies and prevention in the field of age-related diseases.
It is a first aspect of the invention to provide a method to screen genes involved in aging and/or AADs and/or oxidative stress, comprising a) mutation or transformation of a yeast cell, b) cultivation of the cell, c) enrichment of the population for mother cells, d) labeling the mother cells with a WGA-based label, and e) isolation of the highly labeled cells.
To obtain a sufficient distinction between old cells and young cells, it is essential to use a marking of the bud scars that is sufficiently linear with the number of scars and is not or only weakly interacting with other cell wand compounds. Surprisingly, we found that WGA can bind with the chitin in the bud scar, without major interference with other cell compounds, so that the amount of WGA bound is a reliable measurement of the number of bud scars. The WGA bound is then measured using a WGA-based label. A WGA-based label, as used herein, may be any kind of label that allows quantifying the amount of WGA bound to the cell and may be, as a non-limiting example, WGA coupled to a stain or a detectable antibody that binds to WGA. Detectable antibodies are known to the person skilled in the art and may be, as a non-limiting example, rabbit antibodies that can be detected by a labeled anti-rabbit antibody. The labeling of mother cells with a WGA-based label may be a one-step process, wherein labeled WGA is bound to the cell, or a two-step process, wherein in a first step, WGA is bound to the bud scars and in a second step, the bound WGA is labeled. A preferred embodiment is a method according to the invention, wherein the WGA-based label is FITC-labeled WGA.
Preferably, the isolation of highly stained cells is based on FACS sorting. Methods for the enrichment of the population of mother cells are known to the person skilled in the art and may be based on, as a non-limiting example, staining of the cell wall of the cells at a certain point in the growth phase followed by continuation of the culturing and sorting of the stained cells. Alternatively, the cells may be antibody labeled.
Preferably, the enrichment of the population of mother cells is a magnetic-based sorting. Instead of being based on a global cell wall labeling as described above, the enrichment of the population of mother cells may be based on the labeling of a fraction of the mother cells, such as a bud-scar-based labeling. In fact, the enrichment of the mother cells may be carried out by a first WGA-based labeling and sorting, wherein the enriched mother cells are subjected to a second WGA-based labeling and sorting. The labeling method in the first and second round may be different.
Methods to mutate yeasts are known to the person skilled in the art and include, but are not limited to, chemical and physical mutagenesis, such as ethyl methane sulphonate (EMS) treatment or UV treatment. Methods to transform yeast are also known to the person skilled in the art and include, but are not limited to, protoplast transformation, lithium acetate-based transformation and electroporation. The yeast transformation may be carried with one or more nucleic acids, up to a complete library. The nucleic acid used is not necessarily yeast nucleic acid, but may be from any origin, as long as it is functionally expressed in yeast. Preferred examples of nucleic acids are mammalian nucleic acids, such as human nucleic acid, and plant nucleic acid, wherein the nucleic acids are cloned in a yeast expression vector. Preferably, the yeast is transformed with an expression library. The nucleic acid that is transcribed into mRNA does not necessarily translate into protein, but may exert its effect as antisense RNA. Indeed, it is an additional advantage of the method that it can detect in one screening experiment both the effect of overexpression of a protein, as well as the effect of down-regulation of a protein by blocking the translation of an endogenous messenger by a homologous antisense RNA resulting from the expression library.
Another aspect of the invention is a gene or functional gene fragment isolated with the method of the present invention. The functional fragment may encode for a polypeptide that directly affects aging and/or an AAD and/or oxidative stress, or it may be transcribed into antisense RNA, which affect aging and/or an AAD and/or oxidative stress by silencing an endogenous gene. Preferably, the gene or functional gene fragment is selected from the nucleic acid listed in Table 2. More preferably, the gene or functional gene fragment comprises a sequence as represented in SEQ ID NOS:1, 3, 5, 7, 8, 9, 11, 13, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53. Even more preferably, the gene or gene fragment consists essentially of one of the foregoing sequences. Even more preferably, the gene or functional gene fragment consists of one of the foregoing sequences.
A preferred embodiment is a gene fragment, isolated with the method of the present invention, consisting essentially of SEQ ID NO:11, preferably consisting of SEQ ID NO:11. Another preferred embodiment is a gene fragment, isolated with the method of the present invention, consisting essentially of SEQ ID NO:16, preferably consisting of SEQ ID NO:16.
Still another aspect of the invention is the use of a gene or functional gene fragment isolated with the method according to the invention to modulate aging and/or to modulate the development of AADs and/or to protect against oxidative stress. Preferably, the modulation is an inhibition of aging. Preferably, the gene or gene fragment is selected from the nucleic acids listed in Table 2. More preferably, the gene or gene fragment comprises a sequence as represented in SEQ ID NOS:1, 3, 5, 7, 8, 9, 11, 13, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53. Even more preferably, the gene or gene fragment consists essentially of one of the foregoing sequences. Even more preferably, the gene or gene fragment consists of one of the foregoing sequences.
A preferred embodiment uses a functional gene fragment consisting essentially of SEQ ID NO:11, preferably consisting of SEQ ID NO:11. Another preferred embodiment is the use of a gene fragment, isolated with the method, consisting essentially of SEQ ID NO:16, preferably consisting of SEQ ID NO:16.
Another aspect of the invention is a polypeptide encoded by a gene or functional gene fragment isolated with a method according to the invention. Preferably, the modulation is an inhibition of aging and/or inhibition of the development of an AAD. Preferably, the polypeptide is enclosed by a nucleic acid listed in Table 2. More preferably, the polypeptide is encoded by a nucleic acid comprising SEQ ID NO:1, 3, 5, 7, 8, 9, 11, 13, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53. Even more preferably, the polypeptide is encoded by a nucleic acid consisting essentially of one of the foregoing sequences. Even more preferably, the polypeptide is encoded by a nucleic acid consisting of one of the foregoing sequences. Even more preferably, the polypeptide comprises SEQ ID NOs:2, 4, 6, 10, 12, 14, 18, or 20. Even more preferably, the polypeptide consists essentially of SEQ ID NOS:2, 4, 6, 10, 12, 14, 18 or 20. Even more preferably, the polypeptide consists of SEQ ID NOS:2, 4, 6, 10, 12, 14, 18 or 20.
A preferred embodiment is a polypeptide essentially consisting of SEQ ID NO:12, preferably consisting of SEQ ID NO:12. Still another preferred embodiment is a polypeptide encoded by a nucleic acid essentially consisting of SEQ ID NO:16, preferably consisting of SEQ ID NO:16.
Still another aspect of the invention is the use of a polypeptide encoded by a gene or functional gene fragment isolated with a method according to the invention to modulate aging and/or to modulate the development of an AAD and/or to protect against oxidative stress. Preferably, the modulation is an inhibition of aging and/or inhibitor of the development of an AAD. Preferably, the polypeptide is encoded by a nucleic acid selected from the nucleic acids listed in Table 2. More preferably, the polypeptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, 8, 9, 11, 13, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53. More preferably, the polypeptide comprises SEQ ID NO:2, 4, 6, 10, 12, 14, 18 or 20. Even more preferably, the polypeptide consists essentially of SEQ ID NO:2, 4, 6, 10, 12, 14, 18 or 20. Most preferably, the polypeptide consists of SEQ ID NO:2, 4, 6, 10, 12, 14, or 20. A preferred embodiment is the use of a polypeptide essentially consisting of SEQ ID NO:12, preferably consisting of SEQ ID NO:12, to modulate aging and/or to modulate the development the development of an AAD. Preferably, the modulation is an inhibition of aging and/or an inhibition of the development of an AAD. Still another preferred embodiment is the use of a polypeptide encoded by a nucleic acid comprising SEQ ID NO:16, preferably consisting essentially of SEQ ID NO:16, more preferably consisting of SEQ ID NO:16, to modulate aging and/or to modulate the development the development of an AAD.
Still another aspect of the invention is the use of an antisense RNA encoded by a gene or a functional gene fragment, isolated with a method according to the invention, to modulate aging and/or to modulate the development of an AAD. In such an application, the gene or functional gene fragment is operationally linked to a promoter in such a way that an antisense RNA, complementary to the mRNA encoding the polypeptide normally encoded by the gene or gene fragment, is transcribed. Preferably, the gene or functional gene fragment encoding the antisense RNA comprises SEQ ID NO:7, 8, or 15. Even more preferably, the modulation of aging is an inhibition of aging and/or an inhibition of the development the development of an AAD.
Definitions
“Gene” as used herein refers to a region of DNA that is transcribed into RNA and subsequently preferentially, but not necessarily, translated into a polypeptide. The term is not limited to the coding sequence. The term refers to any nucleic acid comprising the region, with or without the exon sequences, and includes, but is not limited to, genomic DNA, cDNA and messenger RNA. As, on the basis of these sequences, it is evident for the person skilled in the art to isolate the promoter region, the term “gene” may include the promoter region when it refers to genomic DNA.
“Nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, methylation, “caps” substitution of one or more of the naturally occurring nucleotides with an analog.
“Functional fragment of a gene” involved in aging is every fragment that, when tested with the method according to the invention, still gives a positive response. Typically, functional fragments are fragments that have deletions in the 5′ and/or 3′ untranslated regions. Alternatively, the functional fragment may be an antisense fragment encoding an RNA that is silencing an endogenous gene or functions as RNAi. As the coding sequence on its own is also considered as a functional fragment, it is evident for the person skilled in the art that it may be functional when it is placed between suitable heterologous 5′ and 3′ untranslated sequences.
“Polypeptide” refers to a polymer of amino acids and does not refer to a specific length of the molecule. This term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation.
“Aging” as used herein includes all forms of aging, particularly also AADs. AADs are known to the person skilled in the art and include, but are not limited to, arteriosclerosis, Parkinson's disease and Alzheimer's disease.
The invention is further described with the aid of the following illustrative Examples.
Materials and Methods to the Examples
Strains and Media
The following S. cerevisiae strains were used: INVSc-1 (Invitrogen, San Diego, Calif.); BY4741 and BY4742 (Euroscarf, Frankfurt, Germany), as well as the BY4742-derived Δfob1 strain (Euroscarf; Accession No. Y14044). Strains were grown at 30° C. in rich YPD medium (2% dextrose, 2% bactopeptone and 1% yeast extract) or minimal SD medium (0.67% yeast nitrogen base without amino acids, 2% dextrose and 0.077% complete supplement mixture—uracil). The INVSc-1 and BY4741 strains used for library screening were grown in S-glycerol, S-galactose or S-raffinose media, where dextrose is replaced with 3% glycerol, 2% galactose or 2% raffinose, respectively. S-glycerol was used to induce expression of genes cloned in pEX2, whereas S-galactose was used to induce expression of genes cloned in pSCGAL10-SN. Media were solidified with 2% agar.
Cloning and Overexpression of a Human cDNA Library
To recover mRNA from various responses, a pool of equal proportions of human HEPG2 cells, subjected to different treatments, was used for library construction. These treatments included heat shock for 1.5 hours at 42.5° C., 1 mM dithiothreitol, 100 U/ml interleukin-6 and 10−7 M dexamethasone. Construction of cDNA libraries was carried out essentially as described previously (Declercq et al. 2000). cDNA was cloned at the site of SfiI/NotI in the vectors pEX2 (BCCM/LMBP Plasmid Collection, Ghent University, Belgium; Accession No. 2890) and pSCGAL10-SN (BCCM/LMBP Plasmid Collection, Accession No. 2471). cDNA expression is driven by the cytochrome c promoter in pEX2 and by the GAL10 promoter in pSCGAL10-SN. Yeast strain INVSc-1 was used as the host for pEX2 library transformation. The pSCGAL10-SN library was transformed to the BY4741 strain. Transformations were performed as described previously (Gietz and Woods, 2001). Approximately 3.5×105 colonies from each transformation were produced.
Magnetic Sorter-Based Preparation of Yeast Mother Cells (M-cell)
Cells were cultured at 30° C. in liquid medium, such as minimal SD medium or in the specific induction medium, to OD600 of 0.7-1 and were collected by centrifugation. All cells harvested were used as M-cells. The biotin labeling of M-cells was carried out essentially as described previously (Smeal et al., 1996). Before labeling, M-cells were washed twice with cold phosphate-buffered saline (PBS; pH 8.0), resuspended in PBS to a concentration of 2.5×107 cells/ml and then incubated with 0.1 mg/ml Sulfo-NHS-LC-Biotin (Pierce Chemical Company, Rockford, Ill.) for 30 minutes at room temperature under gentle shaking. The free biotin reagent was removed by two washings with PBS. Biotinylated M-cells were grown in liquid medium for a desired number of generations (up to G7 in our conditions; culture was not allowed to exceed OD600=1).
The separation of mother cells from the daughter cells they produced was carried out via magnetic cell sorting. This was realized by coupling the biotinylated mother cells to magnet beads by incubating 107 mother cells with 80 μl of Anti-Biotin MicroBead (Miltenyi Biotec, Germany) in 1 ml PBS pH 7.2 for 1 hour at 4° C. Unbound beads were removed by washing twice with PBS. M-cells were isolated with a magnetic sorter according to the supplier's protocol (Miltenyi Biotec). When needed, these sorted M-cells can be further grown in liquid medium for additional generations and isolated again by the magnetic sorting system.
The purity of sorted mother cells was determined on the basis of streptavidin binding. About 107 biotinylated cells were stained with 3 μg streptavidin-conjugated R-phychoerthrin (PE) (Molecular Probes) in 1 ml of PBS pH 7.2 for 1 hour at room temperature in total darkness. Then cells were washed twice with PBS and suspended in 2 ml of PBS pH 7.2. The yeast cells with more bud scars were recognized as a high intensity of FITC signals.
WGA-Based Bud-Scar Staining
The bud scars of yeast cells were stained with fluorescein isothiocyanate (FITC)-labeled WGA lectin (Sigma). The staining was carried out by adding 107 yeast cells together with 12 μg WGA-FITC in 1 ml of PBS pH 7.2 for 1.5 hours in the dark at room temperature. After two washing steps with PBS to remove the free WGA-FITC reagent, yeast cells were resuspended with PBS to a concentration of 0.5×107 cell/ml for FACS analysis.
Propidium Iodide (PI) Staining
PI (Sigma) was freshly dissolved in PBS buffer to a final concentration of 1 mg/ml as stock solution. For staining, yeast cells were suspended in PBS pH 7.2 to approximately 107 cell/ml and then, 3 μl of PI stock solution was added into 1 ml yeast cell suspension. The sample was run within five to ten minutes on a flow cytometer (Becton Dickinson), which is capable of measuring red fluorescence (with a band pass filter>650). No washing steps were included.
Set-Up of Becton Dickinson FACScan
Analysis of FITC, PE and PI labeling of the cell population was accomplished at an excitation wavelength of 488 nm, using a 15 mWatt argon ion laser. FITC emission was measured as a green signal (530 nm peak fluorescence) by the FL1 detector, PE was measured as an orange signal (575 nm peak fluorescence) by the FL2 detector, and PI was measured as a red signal (670 nm peak fluorescence) by the FL3 detector. The FACScan flow cytometer (Becton Dickinson) was operated according to the standard protocol of the supplier. For multi-color staining, electronic compensation was used among the fluorescence channels to remove residual spectral overlap. A minimum of 10,000 events was collected on each sample. Analysis of the multivariate data was performed with CELLQuest software (Becton Dickinson Immunocytometry System).
Transformation and Aging Assay in Nematode
The expression vector of human ferritin fragment (FTL) for C. elegans was derived from L4759 by replacing the GFP with FTL fragment.
Wild-type C. elegans strain (N2) was used as host for FTL expression. The animals were cultured and handled as described (Brenner, 1974). The transient overexpression of human FTL was carried out according to Jin (1999) using an Eppendorf FemtoJet-TransferMan NK injection system (Eppendorf, Leuven, Belgium). Twenty-five to thirty worms were injected with plasmid carrying the human FTL gene or control plasmid. Plasmid pRF4, which carries the dominant rol-6 (su1006) allele was coinjected to mark transformed progeny. After a one-hour recovery period in M9 buffer, injected animals were allowed to lay eggs for approximately 40 hours on plates containing nematode growth medium (NGM) and a lawn of E. coli bacteria (OP50) as food. Transformed eggs were predominantly laid during the last 20 hours resulting in a fairly synchronous experimental cohort. Subsequently, the injected animals were removed and progeny (F1) was allowed to grow at 24° C. Fourth stage larvae or young adults showing the Roller phenotype were transferred onto separate plates (NGM+OP50) containing 300 μM 5-fluoro-2′-deoxyuridine (FUDR, Sigma) to prevent progeny (F2) production. Live/dead scoring was carried out daily. Lifespan is defined as the day when the first transformed larvae hatched until their death.
Construction of a Full Ferritin Clone
A ferritin PCR fragment (end to stop cordon) was generated from the hepatoma cDNA library by using specific primers (5′-ctacgagcgtctcctgaagatgc-3′ (SEQ ID NO:54) and 5′-cgcggatccaagtcgctgggctcagaaggctc-3′ (SEQ ID NO:55)). This fragment was cloned directly into the TOPO vector (Invitrogen, The Netherlands) and then digested with NotI, generating a NotI fragment. Subsequently, the NotI fragment was inserted in the NotI site of ferritin light fragment clone (PGAL 10-FL), resulting a 750 bp full ferritin clone in pSCGal-SN-10.
To use yeast as an aging model, the first step needed is the development of a system, which allows the isolation of a relatively pure population of old yeast cells. The method for distinguishing and separation of S. cerevisiae cells between generations is based on the fact that daughter cells have a wall that is newly formed and do not have any detectable wall remnants of the mother cells. Cells from an overnight culture of S. cerevisiae strain INVSc-1 in minimal SD medium were covalently coated with biotin and designated as mother cells (M-cells). The M-cells were inoculated into fresh medium and allowed to grow for five to six generations as determined by the cell density that is measured by a UV-visible spectrophotometer (Shimadzu). After loading with anti-biotin beads, M-cells were sorted out using a magnetic sorter or MACS (Materials and Methods).
The purity of the collected M-cells was determined by staining with streptavidin-PE, which specifically binds to biotin coated on the cell wall of M-cells, followed by flow cytometric analysis. Due to the reaction of biotin with streptavidin-PE, high-density staining of biotinylated M-cells was shown. As shown in
Wheat germ agglutinin (WGA, Triticum vulgare) is the first lectin of which the amino acid sequence was completely determined (Wright, 1984). WGA is a mixture of several isolectins (Rice and Etzler, 1975). Sharing similar carbohydrate binding properties with other lectins, WGA reacts strongly with the chitobiose core of asparagines-linked oligosaccharides, especially with the Manβ(1,4)GlcNAcβ(1,4)GlcNAc trisaccharide (Yamamoto et al., 1981).
One of the most striking features of the cell surface during aging, S. cerevisiae is the accumulation of chitin-containing bud scars. To verify whether WGA can be used for specific labeling of chitin in yeast bud scars, the yeast strain INVSc-1 (pEX2) was incubated with the FITC-conjugated WGA. The enriched, magnetically sorted M-cells were subjected to WGA reaction.
Under a fluorescence microscope, it was found that the major part of the fluorescent signal for WGA-FITC staining was co-localizing with the bud scar rings (
It has been reported that overexpression of certain human genes in yeast might have an influence in the frequency distribution of the yeast population (Gershon and Gershon, 2000). This overexpression of a single gene, which modulates the longevity in a single-cell system, has opened up the field of aging study to the power of yeast genetics. To screen human genes that might be involved in aging processes, a cDNA library from hepatoma cells was constructed and transferred into the yeast strain INVSc-1 (pEX2) (see Materials and Methods). The transformed yeast population was first labeled with biotin and then cultured in a Bioreactor (AppliTek), for about 14 generations, as deduced from the cell density. According to the method described above, the initial biotinylated M-cells were isolated by magnetic beads described herein and then labeled with WGA-FITC. By flow cytometric analysis (
One of the colonies contained a gene fragment encoding ferritin light (FL) chain (M1147.1; Afl 19897.1). To verify whether the overexpression of this gene could influence the life-span of the yeast cell or not, an analysis of cell death using PI staining was performed in this ferritin L chain clone (CI2-FL) using its parent line of INVSc-1 (pEX2) as a control. Ten million M-cells for each cell line were isolated. As shown in
To confirm the usefulness of the method, additional screening experiments were set up using the same outline as described above, both using the pEX2 library and the pSCGAL10-SN library. The results of the additional screening experiments are listed in Table 2 and identified by their GenBank® accession number. Several results of the first screening have been confirmed, illustrating the usefulness and the reliability of the method.
One of the colonies contained a gene fragment encoding ferritin light (FTL) chain (M1147.1; Afl19897.1) cloned in pSCGAL10-SN. The plasmid was indicated as pGAL10-FL. Ferritin is ubiquitously distributed in the animal kingdom. It is composed of two subunits, the heavy chain (H) and the light chain (L). Ferritin plays a major role in the regulation of intracellular iron storage and homeostasis. One of the functions is to limit iron availability for participation in reactions that produce free oxygen radicals, which have the potential to damage lipids, proteins and DNA. Indeed, several reports have implicated that ferritin is involved in the protection against oxidative stress, such as stress induced by hydrogen peroxide. However, there is not such ferritin-like protein present in yeast and anti-oxidative activity of ferritin fragments was never demonstrated. To test whether the human ferritin fragment plays a role as an antioxidant in yeast, we examined the partial-ferritin L clone (CI2-ferritin), which was isolated by the method according to the invention, against H2O2 stress.
The condition for treatment of the cells was essentially the same as described by Jamieson et al. (1994). Exponential phase cultures of strain BY4741 that contained the empty vector pSCGAL10-SN (Control) and the ferritin expression vector (FTL indicated as CI2-ferritin), respectively, were grown aerobically in S-galactose medium at 30° C. The cell cultures were then challenged to a lethal concentration of H2O2 (1 mM). Cell survival was monitored by taking samples at 0, 30 and 60 minutes, diluting the samples in the same medium and plating aliquots on YPD plates.
The experiment showed that, compared with the control line, ferritin cells are significantly more resistant to treatment with 1 mM H2O2 (
Although on the cellular level there might be some conserved mechanism of aging processes throughout evolution (Martin et al., 1996), it is easy to imagine that in different species some underlying distinctive ways of intercellular regulation also contribute to reach their fate (Guarente 2001). In this sense, results from other organisms may provide a closer vision on the postulated function of the human FTL gene involved in aging. Therefore, it was tested whether FTL might affect lifespan in C. elegans, a multicellular organism, too. Indeed, as shown in
A frequently practiced strategy in searching genes responsible for aging is by selecting survivals after exposure of cells to stresses. The question constantly exists that the genes picked up might be in response to the stress treatment rather than involved in aging because of the complicity of the process. The screening method described here, however, provides an alternative that allows direct hunting of genes with potential anti-aging functions from various libraries or library combinations of eukaryotes. Yeast lines are selected in a more native condition and also with advantages of high throughput, high efficiency, and short time consumption. The invention has a great potential in application in rational drug design and therapies development in the field of age-related diseases preventing/treatments.
Based on the fact that a parental yeast strain and its direct derivative have a similar cell cycle rate, a mixed culture method has been developed to verify the long-living character of a transformed yeast strain when these strains are grown together in the same culture.
Two (or possibly more than two) yeast strains with a similar growth rate are initially mixed in the same culture in an equal ration (50% each in the case of two strains). The strains can be distinguished from each other by the use of a selective marker. The initial inoculated cells, called mother cells (M-cells), are labeled with biotin and are grown together in the same culture during their entire life span. Mother cells at different generation points are sampled and collected by a magnetic system (MACS) similar to the method described in Example 1. The ratio of living M-cells from the two strains is determined by the use of the selective marker. If the two strains have a similar life span, the ratio of two viable strains will stay the same at different generation time points; otherwise, the ratio will change. This method is essentially based on the screening method, wherein the identification of the long-living cells is not carried out by WGA staining but by direct count of the number of living mother cells of the transformed stain(s), compared to the number of living mother cells of the parental strain.
FOB1 is required for the replication fork block. An FOB1 mutation results in a decreased rDNA recombination rate and an increase in yeast life span of 70%. The growth rate of the Δfob1 mutant strain, as measured, is similar to its parental strain. Therefore, the long-living Δfob1 strain with its parental strain BY4742 was used to develop the mixed-growth system.
The initial mother cells were prepared as follows: a first pre-culture was made by inoculating BY4742 and Δfob1 cells (from freshly grown on a SD plate) in 5 ml of SD medium, respectively. The culture was incubated at 30° C. on a shaker at 250-300 rpm overnight. A second pre-culture was made by inoculating the first pre-culture into 5 ml of SD medium at a cell density of OD600=0.001˜0.005. These cells were incubated until the culture reached a cell density of OD600=0.5˜0.7. Cells were collected by centrifugation of the culture at 4° C. for five minutes at 3000 rpm. The cell pellet was washed twice with pre-cooled PBS (pH 8) and resuspended in PBS at a cell density of OD600=5 (approximately 5×107 cells/m.). The biotinylation of cells was performed in an Eppendorf tube, in 1 ml reaction volume consisting of 0.5 ml of the above-mentioned cells (2.5×107 cells) and 0.5 ml of 1 mg/ml biotin (Sulfo-NHS-LC-Biotin). The mixture was incubated for 30 minutes at room temperature with a gentle shaking. The biotinylated cells were centrifuged for 5 minutes at 13000 rpm and washed twice with 1 ml of cold PBS to get rid of free biotin. These cells were used as initial mother cells (M-cells).
A 100 ml mixed-growth culture of BY4742 and Δfob1 was set up by inoculating 1×107 biotinylated M-cells from each strain (mother cells) at the ratio of 1:1 in a SD medium. The mixed-growth culture was incubated at 30° C. on a shaker at 250-300 rpm. The culture density was not allowed to exceed OD600>1.
After growing several generations (up to seven generations in this condition), the M-cells were labeled with anti-biotin microbeads and isolated using the magnetic system (MACS). The purity of M-cells was determined by FACS (fluorescence-activated cell sorter) after staining M-cells with streptavidin conjugated with FE. Using these conditions, more than 90% M-cells could be obtained. After the final magnetic sorting, the ratio of viable M-cells was measured.
Mixed M-cell samples were plated at about 500 cells per plate on YPD and YPD/geneticin plates to determine the ratio of mother cells of the two strains at different generation points. Plates were incubated for three days at 30° C. The ratio of BY4742 and Δfob1 mother cells was monitored by counting the colonies on the two kinds of plates. The total viable number of M-cells could be determined on the YPD plate, while the number of viable Δfob1 M-cells could be derived from the YPD/geneticin plate.
As shown in
A kinetic analysis for growth rate of the ferritin yeast (FTL) and its parental strain INVSc-1 (with a geneticin-selectable marker) revealed a similar rate. About an equal amount of two strains was mixed, as described above, but using S-glycerol medium to obtain induction of the ferritin expression. This mixed culture was subjected to a mixed-growth experiment for determining their life span differences. After examination of the longevity of a mixed-growth of these two cell types by the mixed-growth system and subsequent plating, we found that the ferritin line was predominant in the viable M-cell group after a growth of ten generations (
Iron is an essential nutrient for virtually every organism because it is required as an essential cofactor for many proteins. However, excess iron can generate, via the Fenton reaction, highly toxic-free radicals generating oxidative damage to the cell. Thus, cellular iron concentration must be tightly controlled. To exam whether expression of human ferritin in yeast could protect cell death upon excess iron, the life span analysis of ferritin strains was carried out by micromanipulator as described previously (Kennedy et al., 1994) with the following slight modifications. Cells were pre-grown on non-inducing SD medium (2% glucose), shifted to inducing S-raffinose (2% raffinose) medium with 500 μM ferric ammonium citrate (FAC) and 80 μM ferrichrome (Sigma), and grown for at least two generations. Cells were taken from this logarithmically growing liquid culture and transferred at low density on S-raffinose with 500 μM FAC and 80 μM ferrichrome plate (2% agar). The cells were then incubated at 30° C. overnight. Virgin daughter cells were isolated as buds from populations by micromanipulator and used as the starting mother cells for life span analysis. For each successive bud removed from these mother cells, they were counted one generation older. Cells were grown at 30° C. during the day and at cold room overnight. Each experiment includes at least 60 cells. The statistical analysis of life span was carried out by a Wilcoxin's test. The life span of full ferritin and partial ferritin yeast strains were significantly extended by 10 to 15% compared to their parent strain BY4741 (
(1)antisense
Table 2: Results of further screening experiments. The results are grouped in mitochondrial functions, ribosomal proteins, and other genes with known function, unknown functions and chromosomal fragments. The results of the first screening are not repeated in this table; however, several genes, like the ferritin fragment, have been identified in more than one screening experiment. The sequences are identified by their GenBank® accession number. The length of the isolated fragment may differ from the GenBank® sequence and is normally shorter. Where relevant, the fragment is indicated, using the nucleotide's numbers of the GenBank® sequence.
| Number | Date | Country | Kind |
|---|---|---|---|
| 01204600.9 | Nov 2001 | EP | regional |
This application is a continuation of International Application Number PCT/EP02/13549, filed Nov. 28, 2002, International Publication Number WO 03/045988 A2, published in English on Jun. 5, 2003, the contents of the entirety of which is incorporated by this reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP02/13549 | Nov 2002 | US |
| Child | 10852705 | May 2004 | US |
