ES2301375A1 - Method for the regulated expression of genes in eukaryotic cells and elements for performing said method - Google Patents

Abstract

The invention relates to a method and group of elements for the regulated expression of genes in eukaryotic cells in response to an exogenous effector molecule. The regulation method involves the dual inducing action of the exogenous effector molecule on the activity of a transcriptional activator and on the intracellular levels thereof. The activator regulates the expression of the target gene to be expressed by activating the promoter responsible for controlling said target gene. The invention is suitable for use in fields involving the use of eukaryotic organisms as models for studying the function of genes thereof or as biotechnological tools for producing biomolecules of scientific, therapeutic or industrial interest. The aforementioned technology is particularly suitable for use in the regulated expression of genes in eukaryotic microorganisms such as Saccharomyces cerevisiaeyeast.

Description

Regulated gene expression procedure in eukaryotic cells and elements for its realization.

Fields of application

The present invention can be applied to the regulated expression of endogenous or exogenous genes in eukaryotic cells, in order to study the biological effects of such expression in an organism, or in order to obtain the products encoded by these genes at a scale that allow its extraction and later use. The fields of application of the described technology will therefore be preferably those of the use of eukaryotic organisms as models for the study of the function of genes of the same or different organism, and as biotechnological tools for the production of biomolecules of scientific, therapeutic interest. or industrial The technology described in the regulated expression of genes in eukaryotic microorganisms such as Saccharomyces cerevisiae yeast has a special relevance.

State of the art

The study of gene functions constitutes a preponderant part of the investigations Biologicals that are carried out today. Sequencing of the complete genome of a large number of organisms, including the human being is providing a growing volume of information that you need of the appropriate means to be transformed into useful knowledge. Among the tools that serve to analyze the function of the new genes identified are Find the heterologous expression, which allows you to study the biological effects of the expression of these genes, and on the other obtain your products, with which to carry out all kinds of tests biochemicals These tests often require obtaining a considerable amount of protein, especially when needed crystallize it to solve its three-dimensional structure, or when must be administered to laboratory animals in tests Pharmacological Once a specific protein becomes product with commercial value will be necessary to produce large scale to meet market demand.

In any gene expression system, the amount of protein produced will be determined mainly by the promoter activity of the transcription of the system, which will be responsible for the synthesis of protein-specific RNA. The DNA sequence that promotes the transcription of the gene to be expressed is called a promoter, and its activity can be constitutive or regulable, the adjustable systems being the most attractive by allowing greater control over the expression of the gene under study, or the production of The protein of interest. In this sense, expression systems have been developed in eukaryotic organisms based on promoters adjustable by chimeric proteins, such as the fusion between the TetR protein (which responds to the tetracycline antibiotic) and the transcriptional activation domain derived from the Herpes simplex virus VP16 protein (Gossen and Bujard 1992). Similarly, a eukaryotic expression system based on a chimeric transactivator resulting from the fusion between the nuclear receptor of the insect hormone ecdysone and the VP16 protein has been developed, which responds to the hormone by activating the transcription of promoters recognizable by the receptor. hormonal (No, Yao et al . 1996). There are several published patents that propose eukaryotic expression systems based on chimeric transcriptional activators, formed by DNA binding modules, and transcription activation modules that respond to exogenous effector molecules, such as that described in WO0111037, which incorporates as elements of chimeric transactivator to the regulatory protein of S. cerevisiae Gal4 and to that already described VP16, or that described in US5599904, which incorporates a modular transactivator similar to the previous one but which responds to retinoic acid. Other patent eukaryotic expression systems use similar response elements to exogenous molecules, such as that based on a transactivator that responds to cumic acid (US2004205834).

In all recombinant protein production processes, microorganisms continue to be the most widely used expression systems, mainly due to their easy handling and culture, and the high production yield achieved with them. Among these organisms is the yeast S. cerevisiae , an organism very easy to grow, extremely manipulable from the genetic point of view, and whose genome has been sequenced in its entirety, circumstances that allow it to be used in biological research as a model eukaryotic organism for the study of cellular, genetic and molecular functions, both endogenous and other organisms. Yeast is a host organism traditionally used in the expression of proteins, for analytical purposes and also for biotechnological production, with numerous heterologous protein production processes based on S. cerevisiae (Hinnen, Meyhack et al . 1989; Goeddel 1990; Gellissen , Melber et al . 1992; Hinnen, Buxton et al . 1995; Gellissen and Hollenberg 1997; Cereghino and Cregg 1999; Porro, Sauer et al . 2005), although in recent times the biotechnological use of yeasts as expression systems It has spread to other organisms, such as Pichia pastoris, Kluyveromyces lactis, Candida boidinii, Arxula adeninivorans and Yarrrowia lipolytica , among others (Bergkamp, Kool et al . 1992) (Buckholz and Gleeson 1991) (Gellissen, Weydemann et al . 1992; Faber , Harder et al . 1995) (Gellissen, Kunze et al . 2005) (Reiser, Glumoff et al . 1990).

Heterologous yeast expression systems are generally based on replicative plasmids, in which it is possible to insert the DNA sequence of the protein gene that is intended to be expressed, and which can be introduced by transformation into the appropriate yeast strain ( Parent, Fenimore et al . 1985; Rine 1991; Schneider and Guarente 1991; Romanos, Scorer et al . 1992). These plasmids have their own origin of replication, and have genes whose presence in the host strain is necessary for it to survive under certain culture conditions, and which can therefore be used as selection markers of the transformed strains. In addition, expression plasmids incorporate promoter and transcription terminator DNA sequences, operational signals recognizable by the cellular RNA synthesis machinery that are necessary for the expression of genes from organisms other than yeast. Yeast plasmids also carry replication and selection sequences in Escherichia coli that allow their spread in this bacterium, facilitating the necessary donation procedures.

Although constitutive promoters can be used, it is convenient to use adjustable or inducible promoters so that the protein is only overexpressed when biomass is being acquired in the culture medium. In this way, mutants that do not express the recombinant protein are prevented from growing faster because they do not have to endure a metabolic ballast of overproducing the protein all the time, since this makes them quickly dominate the culture, drastically lowering yields. Promoters with a very low baseline level also allow regulated expression of proteins that are toxic to the host cell. With this objective, plasmid-based yeast expression systems have been developed with adjustable promoters, such as that of the MET3 gene, negatively regulated by the amino acid methionine (Mountain, Bystrom et al . 1991), that of the PHO5 gene, negatively regulated by phosphate inorganic (Kramer, DeChiara et al . 1984), that of the CUP1 gene, positively regulated by Cu 2+ ions (Macreadie, Horaitis et al . 1991), or that of the GAL1 and GAL10 genes, which are transcribed in a manner divergent from the same promoter region between them (West, Yocum et al . 1984; Yocum, Hanley et al . 1984).

Among the systems with adjustable promoters, those based on GAL1-10 have proven to be the most potent in terms of levels of expression obtainable (Schneider and Guarente 1991). The activity of the GAL1-10 promoter is activated by the presence of galactose in the culture medium, and is repressed by glucose. Under natural conditions, the activation of the GAL1-10 promoter depends on the Gal4 activator protein, product of the gene ( Johnston 1992 ). Since galactose is an expensive compound, and glucose difficult to avoid in most industrial culture media, the use of expression systems based on GAL1-10 may be inconvenient outside the laboratory. In this sense, an expression system in S. cerevisiae has been described that uses elements of the classical GAL system, combining them with other heterologous elements described above (a hormonal receptor, the VP16 protein), to configure an expression system that responds to the β-estradiol hormone (Louvion, Havaux-Copf et al . 1993).

In the present invention a system is described of expression derived from several of the elements described above, assembled in new regulatory circuits that enable the highly regulated expression of genes of interest in research and biotechnology. The regulatory procedures of this invention are based on the use of an exogenous effector molecule that operates on a double level: on the activity of the activating elements of transcription and its intracellular levels. Is achieved thus improve previously existing procedures by reducing basal expression and increase induction levels.

Brief Description of the Invention

The object of the present invention is a procedure for the regulated expression of genes in cells eukaryotic, through the use of an exogenous effector molecule that allows to induce both intracellular concentration and intrinsic activity of a positive transcription regulator which operates on a specific promoter sequence and directs its through the expression of the gene that you want to regulate. The activity of promoter depends on the positive regulator being in a activated state and the intracellular concentration of said positive regulator; both parameters are dependent on the presence of the exogenous effector molecule. The double effect of exogenous effector molecule promotes low levels of basal expression when it is not present in the middle, together with high rates of activation when it is present. The regulator of the transcription must be able to recognize the effector molecule and join a specific DNA sequence to activate the transcription of promoters close to said sequence. This can be achieved by the modular combination of three domains protein: one capable of recognizing a sequence in DNA, another able to activate transcription, and a third party able to recognize the effector molecule and that works by regulating the activity of one of the other two previous domains.

When the sequence transcript that code the regulator is placed under its own control by genetic engineering techniques, you get a system that positive feedback in response to the effector molecule. How the activity of the regulator in turn depends on the molecule effector, the result is that the amount of active regulator present in the cell grows in response to the presence of said effector molecule.

A more sophisticated system variant than described here uses two positive regulators that respond to the same effector molecule, but they recognize different DNA sequences. One of them expresses itself constitutively and transcriptionally regulates the expression of the second, which in its instead transcriptionally regulates the protein expression of interest; both regulators are activated by the same molecule effector It is achieved that both the quantity and the activity of the second regulator are controlled by the same molecule effector and can act synergistically. This system allows higher levels of expression of the gene of interest, from very low baseline levels, without the need for a coupled transcription repressor.

A possible development of this invention uses the yeast Saccharomyces cerevisiae and positive regulators that respond to the hormone β-estradiol, either the human estrogen receptor (hER) or the chimeric protein Gal4-ER-VP16, formed by the binding domain to Gal4 DNA, the hER β-estradiol response domain and the transcription activation domain derived from a viral protein (VP16) (Louvion, Havaux-Copf et al . 1993). In the presence of β-estradiol, the resulting protein strongly promotes the transcription of DNA sequences located downstream of promoters recognizable by Gal4, which normally activates the expression of yeast genes responsible for galactose assimilation in culture media. with this sugar as a source of carbon (Johnston 1992). The invention is, however, applicable to other positive transcription regulators that recognize other DNA sequences and respond to other effector molecules.

The described system has several important advantages, since it achieves high levels of induction of gene expression, from very low basal expressions, in response to very small concentrations of the effector molecule, which is also not metabolized in the process. Induction of gene expression occurs with a minimum of interference with the general yeast metabolism, requires the addition of very small amounts of the inducer, and is virtually independent of the culture medium used. Expression systems that use β-estradiol as an effector molecule can easily be combined with others, such as the repressible-inducible by tetracycline (Tet-Off and Tet-On) (Fishman, Kaplan et al . 1994; Belli, Gari et al . 1998), in studies of substitution of cellular functions of the product of one gene by those of another. In addition, yeast systems based on Gal4-ER-VP16 are compatible with the use of gene constructs under previously recognized promoters by Gal4.

Brief description of the figures

Figure 1: General scheme of the invention. A exogenous effector molecule simultaneously induces, well directly, either through intermediary elements, the transcriptional activity and intracellular levels of a transcription activator, whose coding sequence is is under the control of promoter 1. The activator sensitive to the effector molecule regulates, through promoter 2, the levels of expression of the target gene of regulation, which is under the control of this second promoter.

Figure 2: Feedback loop scheme positive controlled by an exogenous effector molecule. Molecule exogenous effector triggers positive feedback to simultaneously activate the activity of the activator and its own synthesis, what is achieved by placing the coding sequence of the activator under its own transcriptional control.

Figure 3: Application of the scheme of Figure 2 to S. cerevisiae yeast by using the Gal4-ER-VP16 chimeric activator controlled by β-estradiol. The activator is capable of activating the GAL1 promoter thanks to the DNA binding domain of Gal4 and the transcription activation domain of VP 16, but only in the presence of β-estradiol, which is detected by the hormone binding domain of the estrogen receptor. In the presence of β-estradiol, the activator activates its own synthesis by being under the control of the GAL1 promoter, as well as transcription of the target gene, which is also under the control of the GAL1 promoter.

Figure 4: Regulatory Cascade Scheme controlled by an exogenous effector molecule. The effector molecule exogenous induces the activity of a primary regulator that activates the transcription of the target gene, through an adjustable promoter that is recognized by said primary regulator. Simultaneously the same exogenous effector molecule induces an increase in intracellular levels of the primary activator, when activating a secondary activator that positively regulates the gene promoter which encodes the primary trigger. The secondary trigger is is permanently expressed in the cell when its expression placed under the control of a constitutive promoter.

Figure 5: Application of the scheme of Figure 4 to the S. cerevisiae yeast by using the human estrogen receptor (hER) as the primary activator and the chimeric activator Gal4-ER-VP16 as a secondary activator. The activity of both regulators is induced by β-estradiol. The regulation target gene is under the control of a promoter that contains one or more estrogen response elements (ERE), which allows its recognition by the estrogen receptor when it is active. Estrogen receptor expression is under the control of the GAL1 promoter, which in turn is regulated by the chimeric activator Gal4-ER-VP16, whose expression is constitutive as it is under the control of the ADH1 gene promoter .

Figure 6: Results of Example 1. Quantification of the β-galactosidase activities of the cultures of Saccharomyces cerevisiae strain W303-1A transformed only with the reporter plasmid p416GAL1lacZ (Without activator), with the reporter plasmid and the plasmid pADH1-GAL4ERVP16 constitutively expressing the chimeric activator (with constitutive Gal4ERVP16), or with the reporter plasmid and the plasmid expressing the chimeric activator under control of the GAL1 promoter (with self-regulated Gal4ERVP16). The values corresponding to the average obtained from four assays of the β-galactosidase activity achieved by each culture after 6 generations in the presence or absence of β-estradiol (11 µM) are shown. Error bars indicate the standard deviation.

Figure 7: Results of Example 2. Quantification of the β-galactosidase activities of cultures of S. cerevisiae strain W303-1A transformed only with the reporter plasmid pERECYC1lacZ (Without activator); of cultures in galactose medium of said strain transformed with the reporter plasmid and with the plasmid pGAL1-ER, which under these conditions constitutively expresses the estrogen receptor (with constitutive hER); or of cultures of strain Y01044 (lacking the Gal4 activator and the Gal4-Gal80 repressor) transformed with the reporter plasmid, plasmid pADH1-GAL4ERVP 16, which constitutively expresses the chimeric activator, and plasmid pGAL1-ER, which expresses the receptor estrogen when the GAL1 promoter is activated (with Gal4ERVP16 and hER in cascade). The values corresponding to the average obtained from four assays of the β-galactosidase activity achieved by each culture after 6 generations in the presence or absence of β-estradiol (1 µM) are shown. Error bars indicate the standard deviation.

Detailed description of the invention

The present invention consists of a Regulated gene expression procedure comprising:

one.

A transcription promoter sequence that directs the expression of the gene that encodes the protein of interest. This sequence can be a complete promoter of the organism used, which contains both the binding sequences of the transcriptional regulator and the sequences that direct the initiation of transcription. In the case of S. cerevisiae, highly regulated promoters such as the GAL1-GAL10 genes can be used , whose regulatory sequences (GAL UAS) are simultaneously recognized by the repression system that keeps baseline transcription levels low and by the positive regulator. Alternatively, the promoter sequence may consist of a chimeric promoter that combines regulatory sequences and assembly regions of the transcriptional machinery ( core promoter elements ) from different sources.

2.

A positive transcription regulator or activator, which specifically recognizes the regulatory sequences of the regulated promoter described above. This positive regulator may be a transcriptional factor of the organism used or it may come from a different organism and be expressed heterologously in the working organism. It can also consist of a chimeric protein that combines protein domains with different functionality: DNA binding domain that recognizes the regulatory sequence, transcription activation domain and allosteric regulatory domain that responds to an effector molecule. In the case of promoters whose regulatory sequences are the UAS GALs of S. cerevisiae , chimeric regulators containing the DNA binding domain of the Gal4 protein, fused to transcription activation domains and regulatory domains of different origin, may be used, as is the case with the chimeric protein Gal4-ER-VP16 (Louvion, Havaux-Copf et al . 1993).

3.

An effector molecule that modulates the activity of the positive transcription regulator as well as its intracellular concentration (Figure 1). The effector molecule can regulate acting positively on the activity of the regulator and its abundance, or it can do so negatively, being necessary in this case to remove the effector molecule to achieve positively regulate the expression of the protein of interest. The effector molecule can modulate the activity of the regulator by direct interaction with it, through a regulatory site, or it can do so through one or more intermediate elements that transmit the signal originating in the effector molecule to the regulator. In the case of using a nuclear receptor as a regulator, the effector molecule is the corresponding ligand, usually a hormone, which is recognized by the hormone binding domain. This is also the case of the GAL4-ER-VP16 chimeric protein, which contains the estrogen binding domain of the human estrogen receptor, and whose activity strictly requires the presence of estrogens such as β-estradiol. The effector molecule also controls the intracellular levels of the positive regulator. For this, the expression of the positive regulator must be under the control of the effector molecule. In the simplest model covered by the present invention, the expression of the positive regulator is under its own control, as the regulatory sequences recognized by the positive regulator are found in the promoter that regulates its expression. The global system takes the form of a loop that is positively fed back after being triggered by the presence of the effector molecule (Figure 2). If GAL4-ER-VP16 protein is used as activator and β-estradiol as effector molecule, positive feedback is achieved by placing GAL4-ER-VP16 expression under the control of GAL-UAS sequences such as those contained in the promoter. of the S. cerevisiae GAL1 gene, which are recognized by the regulator itself in the presence of the effector molecule (Figure 3).

The positive feedback loop system works optimally when the basal levels of expression of the activator, in the absence of the effector molecule, are low, as a consequence of the action of a repressor. The need for the basal levels of the primary activator to be reduced decreases if it is under the transcriptional control of a second activator (secondary activator), which is constitutively expressed, but whose activity responds to the presence of the same effector molecule. In this case, instead of a loop with positive feedback, we have a regulatory cascade that depends on the exogenous effector molecule (Figure 4). If β-estradiol is used as an exogenous effector molecule, the primary regulator may be the human estrogen receptor itself, with the ability to recognize in the DNA the sequence of estrogen response elements (ERE). The promoter that directs the expression of the protein of interest must therefore contain ERE sequences. The expression of the primary regulator, in this case the human estrogen receptor, must be under the control of the secondary activator, which in turn must also respond to β-estradiol. As a secondary regulator, the Gal4-ER-VP 16 protein can therefore be used, provided that the primary regulator is under the control of the UAS GAL that recognizes the Gal4 DNA binding domain. The secondary activator can be expressed by a constitutive promoter, such as that of the S. cerevisiae ADH1 gene. The overall result is a regulatory cascade that responds to β-estradiol by amplifying the response to it (Figure 5). Given the great evolutionary conservation of eukaryotic transcriptional machinery and the fact that this β-estradiol-induced cascade regulation system is virtually independent of any specific host regulator, it is highly likely that this system can be easily adapted to others. eukaryotes that lack estrogen regulation.

The different genetic elements that constitute the regulatory systems described, both the promoters and coding sequences that direct the expression of the activators, and the promoters under whose expression the target genes are located, can be expressed from one or several plasmids. These plasmids can be non-replicative and introduced into cells by transient transfection, or they can be replicative in nature and introduced into the cell stably by transfection, transformation or conjugation. The number of copies that each plasmid reaches in the host cell can be stable, for example single copy, or it can be multiple and vary depending on the physiology of the culture. In the case where S. cerevisiae is used as an expression organism, stable plasmids must carry autonomous replication sequences, which may be accompanied by centriomerican sequences, or may carry replicative sequences derived from the 2 micron circle; in the first case the resulting plasmids will have a stable and close to one copy number, while in the second case the copy number will be multiple and variable within the culture cell population (Futcher 1988; West 1988). Plasmids containing the genetic elements of the regulatory systems described in this invention may contain more than one of these elements. In the case of using similar promoters to direct the transcription of more than one of the elements of the system, a bidirectional promoter located in a plasmid can be used and divergently directs the transcription of two of the genes. In the case of the positively feedback loop regulatory system, the bidirectional promoter can direct the transcription of the activator coding sequence and, divergently, direct the transcription of the target gene. The application of this scheme to S. cerevisiae can be achieved by placing the divergent promoter GAL1-GAL10 on the plasmid, under whose control the coding sequence of an activator that recognizes the GAL-UAS sequences present in said promoter and in the direction would remain in one direction. opposite the target gene subject to regulation. What is described for Saccharomyces cerevisiae circular centromeric plasmids is also applicable to artificial yeast chromosomes (YAC) (Burke 1990).

Alternatively, the genetic elements that make up the gene regulation systems described in this invention may be integrated into the chromosomal genome of the organism chosen to perform the expression. In this case it would be necessary to use techniques that allow the integration of the transcriptional units that direct the expression of the activators and the target gene, by molecular recombination procedures. This recombination may be random (illegitimate recombination), may be mediated by recombinases (site-specific recombination) or may respond to sequence homology (homologous recombination). In the case of S. cerevisiae being used as an expression organism, the homologous recombination integration procedure is highly successful. The transcriptional units that express the genetic elements of the regulatory system can therefore be integrated into this yeast if they are flanked by sequences of more than 40 base pairs in length that have homology with the locus chosen for integration (Rose 1990).

The present invention has been developed in the yeast Saccharomyces cerevisiae but is applicable to other eukaryotic organisms using analogous elements and transcription factors, already known in the state of the art.

Examples of embodiment of the invention Example 1 Β-estradiol-induced expression of E. coli β-galactosidase in S. cerevisiae by a positively feedback loop system

The present Example shows the ability of the described procedure to induce the expression of the β-galactosidase of the bacterium Escherichia coli in the yeast Saccharomyces cerevisiae by adding to the medium micromolar concentrations of β-estradiol, starting from very basal levels of expression reduced

For Example plasmid pGAL1-GAL4ERVP16 was constructed by inserting into the centromeric vector yeast expression p414GAL1 (Mumberg, Muller et al. 1994) the DNA fragments corresponding to the DNA-binding domain of the Gal4 protein S cerevisiae (93 first amino acid residues of the N-terminal end), the hormone binding domain of the human estrogen receptor (residues 282 to 756 of the protein) and the transactivation domain of the VP16 protein of Herpes simplex virus (residues 424 to 490 of the protein). These three domains were inserted in phase as a translational fusion, between the GAL1 promoter and the CYC1 terminator present in the vector. Plasmid pADH1-GAL4ERVP16 was also constructed, by inserting the GAL4ERVP16 translational fusion into the vector p413ADH1, between the ADH1 promoter and the CYC1 terminator present in the vector (Mumberg, Muller et al . 1995).

The centromeric plasmid p416GAL1lacZ was used as the β-galactosidase expression vector, in which the E. coli lac Z gene is placed under the control of the S. cerevisiae GAL1 promoter (Mumberg, Muller et al . 1994).

The W303-1A line ( MATa, ade2, his3, leu2, trpl, ura3 ) of S. cerevisiae was transformed with plasmids pGAL1-GAL4ERVP16 and p416GAL1lacZ, with pADH1GAL4ERVP16 and p416GAL1lacZ, or only with p416 manipulation standard yeasts (Rose 1990). The transformants were grown at 30 ° C, in liquid medium SC free of uracil, tryptophan or histidine (depending on the combination of plasmids) to maintain the selective pressure in favor of the maintenance of the introduced plasmids, and in continuous orbital agitation. As a control, transformants that exclusively carried plasmid p416GAL1lacZ were used. The 5 ml cultures were carried out in the presence of 1 µM? -Estradiol (SIGMA E-1024-1G), by adding 2 µL of a 2.5 mM solution of?-Estradiol dissolved in ethanol . Equal volume of ethanol was added to controls without β-estradiol. After 6 generations of culture (approximately 14 hours) and reaching an optical density between 0.8 and 1.0 (at 600 nm), the cells were collected by centrifugation. The β-galactosidase activity was tested following standard procedures for yeasts (Guarente 1983). The experiments were performed in quadruplicate.

The results obtained are shown in the Figure 6. The presence of the positive regulator under the control of the positive feedback loop did not involve any increase in the basal expression of β-galactosidase, while the addition of the effector molecule (β-estradiol) produced an induction of 274 times at the level of expression. By cons, when it was expressed constitutively the positive regulator (pADH1GAL4ERVP16) the baseline expression in the absence of β-estradiol was 27 times higher and the induction obtained by adding β-estradiol was only 32 times.

Example 2 Β-estradiol-induced expression of E. coli β-galactosidase in S. cerevisiae by a regulatory system with two cascading transcriptional activators

The present Example shows the ability of the described procedure to induce high levels of E. coli β-galactosidase expression in S. cerevisiae yeast by adding micromolar concentrations of β-estradiol to the medium, starting from reduced basal levels of expression, in the absence of mechanisms to repress the expression of positive regulators.

To perform the Example, plasmid pGAL1-ER was constructed by inserting the human estrogen receptor cDNA into the centromeric yeast expression vector p414GAL1, between the GAL1 promoter and the CYC1 terminator present in the vector (Mumberg, Muller et al . nineteen ninety five).

The centromeric plasmid pERECYC1lacZ was constructed as the β-galactosidase expression vector, in which the Escherichia coli lac Z gene is placed under the control of the chimeric promoter composed of an estrogen response sequence (ERE) and the basal elements of the CYC1 promoter of S. cerevisiae . For this, the ERE sequence was inserted into the vector p416CYC1lacZ, upstream of the CYC1 promoter (Mumberg, Muller et al . 1995).

Line Y01044 ( MATa, his3, leu2, met15, ura3, gal4 ) of S. cerevisiae , which lacks the Gal4 activator and therefore the Gal4-Gal80 repressor, was transformed with plasmids pADH1-GAL4ERVP16, pGAL1-ER and pERECYC1lacZ, by standard procedures of genetic manipulation of yeasts (Rose 1990). The transformants were grown at 30 ° C, in liquid medium SC free of uracil, tryptophan or histidine (depending on the combination of plasmids) to maintain the selective pressure in favor of the maintenance of the introduced plasmids, and in continuous orbital agitation. As a control, transformants of strain W303-1A that exclusively carried the plasmid pERECYC1lacZ were used. As constitutive control, cells of the W303-1A line transformed with pERECYC1lacZ and pGAL1-ER, grown in medium with galactose as the sole carbon source were used to achieve continuous expression of the GAL1 promoter. The 5 ml cultures were performed in the presence of 11 µM? -Estradiol, by adding 2 µL of a 2.5 mM solution of β-estradiol dissolved in ethanol. Equal volume of ethanol was added to controls without β-estradiol. After 6 generations of culture (approximately 14 hours) and reaching an optical density between 0.8 and 1.0 (at 600 nm), the cells were collected by centrifugation. The β-galactosidase activity was tested following standard procedures for yeasts (Guarente 1983). The experiments were performed in quadruplicate.

The results obtained are shown in the Figure 7. The presence of the genetic elements of the waterfall regulator in the absence of the effector molecule was not an increase some of the basal expression of the β-galactosidase, while the addition of the effector molecule (β-estradiol) produced a 135 times induction in the expression level of this enzyme, reaching levels of β-galactosidase 16 times higher than the previous Example. However, when cultures constitutively expressed the estrogen receptor, the accumulation of β-galactosidase in the absence of β-estradiol was significantly superior and the level of induction by the effector molecule did not exceed 4 times.

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Claims (24)

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1. Procedure for the regulated expression of genes introduced by recombinant DNA technology in eukaryotic cells characterized by the use of

to.

to the less a heterologous transcriptional activator, whose concentration intracellular and intrinsic activity are regulated simultaneously by the same exogenous effector molecule.

b.

a promoter that is the target of said activator and that directs the expression of said introduced recombinant genes.

2. Method for the regulated expression of genes according to claim 1 characterized in that the expression of the heterologous transcriptional activator is controlled by at least one other heterologous transcriptional activator whose intrinsic activity is regulated by the same exogenous effector molecule. 3. Method for the regulated expression of genes according to claim 1 characterized in that the expression of the heterologous transcriptional activator is controlled by a promoter that is inducible by the same heterologous transcriptional activator in the presence of the exogenous effector molecule. Method for the regulated expression of genes according to claims 1 to 3 characterized in that at least one of the heterologous transcriptional activators is a chimeric polypeptide consisting of

to.

A DNA binding domain

b.

A transcription activator domain

C.

A sensor domain of the exogenous effector molecule that regulates the chimeric polypeptide activity.

5. Procedure for the regulated expression of genes according to claim 4 wherein the DNA sequence coding for the chimeric polypeptide comprises a sequence of DNA coding for a sensor domain that comes from the sequence of the hormone binding domain of an estrogen receptor. 6. Method for the regulated expression of genes according to claim 4 wherein the DNA sequence encoding the chimeric polypeptide comprises a coding sequence for a DNA binding domain that comes from the GAL4 gene sequence of Saccharomyces cerevisiae . 7. Procedure for the regulated expression of genes according to claim 4 wherein the DNA sequence coding for the chimeric polypeptide comprises a sequence of DNA coding for a transcription activating domain that  It comes from the VP16 gene of the Herpes simplex virus. Method for the regulated expression of genes according to any one of claims 1 to 3 characterized in that at least one of the heterologous transcriptional activators is an estrogen receptor. 9. Method for the regulated expression of genes according to any one of claims 1, 2, 3 and 8 characterized in that at least one of the heterologous transcriptional activators is the human estrogen receptor. 10. Method for the regulated expression of genes according to any one of claims 1 to 5, 8 and 9 characterized in that the exogenous effector molecule has estrogenic activity. Method for the regulated expression of genes according to any one of claims 1 to 5, and 8 to 10 characterized in that the exogenous effector molecule is β-estradiol or any of its derivatives. 12. The method of claim 1 to 3 characterized in that the sequence of the target promoter of at least one heterologous transcriptional activator is derived from the GAL1-GAL10 promoter of Saccharomyces cerevisiae . 13. The method of claims 1 to 11 characterized in that the target promoter of at least one heterologous transcriptional activator contains at least one estrogen response element that allows it to be recognized by the estrogen receptor DNA binding domain. 14. Method for the regulated expression of genes according to any one of claims 1 to 13 wherein the promoter or terminal promoters that direct the expression of the recombinant genes contain sequences from the GAL1-GAL10 promoter of Saccharomyces cerevisiae . 15. Procedure for the regulated expression of genes according to any one of claims 1 to 13 wherein the promoter or terminal promoters that direct the expression of the recombinant genes contain sequences of at least one element of estrogen response that allows it to be recognized by the domain binding to estrogen receptor DNA.

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16. Procedure for the regulated expression of genes according to any one of claims 1 to 15 wherein the coding sequences of the transcriptional activators heterologists are integrated into at least one plasmid. 17. Procedure for the regulated expression of genes according to any one of claims 1 to 16 wherein the coding sequences of the transcriptional activators heterologists are integrated into any of the chromosomes of the host cell of the expression system. 18. Procedure for the regulated expression of genes according to any one of claims 1 to 17 wherein at least one terminal promoter and the gene or genes controlled by it they are integrated into a plasmid vector or a chromosome artificial. 19. Procedure for the regulated expression of genes according to any one of claims 1 to 18 wherein at least one terminal promoter and the gene or genes controlled by it they are integrated in any of the cell's chromosomes host 20. Eukaryotic cells containing the coding sequences of transcriptional activators heterologists to use them in the procedure of any one of claims 1 to 19. 21. Cells of claim 20 wherein the Organimo is from the ascomycete group. 22. Cells of claim 21 wherein the organ is Saccharomyces cerevisiae . 23. DNA vectors containing the bidirectional promoter GAL1-GAL10 of Saccharomyces cerevisiae that on the one hand control the expression of the chimeric heterologous transcriptional activator of claim 6 and divergently direct the expression of the recombinant gene. 24. Use of the procedure of any one of claims 1 to 19 to control the expression of polypeptides for use in research, diagnosis, enzymes industrial, therapeutic targets, or in metabolic engineering for overproduce or generate vaccines or new metabolites of use Industrial or pharmaceutical.