MXPA96006257A - Bts1 geranylgeranyl disphosphate synthase in saccharomyces cerevisiae - Google Patents

Abstract

The present invention refers to yeast geranylgeranyl diphosphate synthase proteins, and to nucleic acid molecules codifying said proteins. Mehtods for producing geranylgeranyl diphosphate and farnesyl diphosphate are also included.

Description

SYNASE PROTEINS OF GERANILGERANIL DIFOSFATE. MOLECULES OF NUCLEIC ACID, AND USES OF THEM This application claims priority of the Request Provisional of the United States of America with serial number 60 / 008,301, filed on December 7, 1995, entitled "BTS1 Geranylgeranyl Diphosphate Synthase in Saccharomyces Cerevisiae". This invention was made in part with government support under the number CA46128, issued by NCI, National Institutes of Health. The government has certain rights to this invention.

FIELD OF THE INVENTION The present invention relates to yeast geranylgeranyl diphosphate synthase nucleic acid molecules, and to proteins encoded by those nucleic acid molecules. The present invention also includes methods for producing those nucleic acid molecules and those proteins.

BACKGROUND OF THE INVENTION Protein prenylation is a post-translational lipid modification that involves the covalent attachment of isoprenoid groups to cysteine residues at or near the carboxyl termini of the proteins. The binding of a lipophilic isoprenoid group to proteins is thought to increase their hydrophobi, allowing otherwise hydrophilic proteins to associate with the membranes. It is estimated that up to 0.5 percent of the total cellular proteins are prenylated. Known prenylated proteins include small GTP binding proteins of the Ras superfamily, nuclear sheets, yeast coupling pheromone factor A, and trimeric G proteins. These proteins are coupled in a variety of cellular processes, which include cell growth control, signal transduction, cytokinesis, and intracellular membrane trafficking. Two different isoprenoid groups, farnesyl (15 carbon atoms) and geranylgeranil (20 carbon atoms), bind post-translationally to the proteins. Farnesyl is added to proteins that end in a CAAX motif (where C is cysteine, A is an aliphatic amino acid, and X can be methionine, cysteine, alanine, glutamine, phenylalanine, or serine), while geranylgeranil is transferred to proteins that end in CAAL (where L is leucine), CC, or CXC motifs (X is any amino acid). The best known prenylated proteins are geranylgeranylated. The farnesyl and geranylgeranyl groups are bound to the proteins from all-trans farnesyl diphosphate (FPP), and all-trans geranylgeranyl diphosphate (GGPP), respectively. These lipid precursors are intermediaries in the biosynthetic path of isoprenoid. This trajectory consists of a series of reactions by means of which the mevalonate becomes a different family of lipophilic molecules that contain a structure of five repetitive carbon atoms. Isoprenoids are subsequently incorporated into a large number of end products, which include: sterols, ubiquinones, dolicholes, tRNAs, and prenylated proteins. FPP is the product of the farnesyl diphosphate synthase reaction. This enzyme, which is the most abundant and widely occurring prenyltransferase, catalyzes the formation of FPP by sequential addition of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), and geranyl diphosphate (GPP). In some organisms, GGPP is synthesized by a GGPP synthase (GGPPS) that catalyzes the stepwise additions of IPP to DMAPP, GPP and FPP. This type of GGPP synthase activity has been detected in mammalian tissue. However, eukaryotic geranylgeranyl diphosphate synthases are known that synthesize GGPP by the addition of a single molecule of IPP to FPP. But, due to its low activity and the problems of separating this enzyme from the FPP synthase, its purification has been difficult.

GGPP is the substrate for two different protein phenyltransferases, geranylgeranyl type I transferases (GGTase-I), and Type II (GGTase-II). GGTase-I catalyzes the transfer of a geranylgeranyl group from the GGPP to the proteins that end in a CAAL motif, while the GGTase-II binds the geranylgeranil to the CC or XCC terminal residues. Their protein substrates include members of the Ras family of small GTP binding proteins. GGPP and FPP are important intermediates in the formation of a variety of derivatives that have important uses in the production of anti-cancer compounds, anti-tumor compounds, anti-cholesterol compounds, and anti-ulcer compounds. For example, GGPP and FPP can be used in the prenylation of ras oncogene protein to inhibit neoplastic transformation. Taxol, a potent agent against cancer, is a derivative of GGPP for which there is currently a lack of effective biosynthetic production methods for cost. Accordingly, the isolation of GGPP synthases to be used in the modulation of the biosynthetic pathways of GGPP and FPP is both desirable and commercially valuable. Although previous investigators have identified GGPP synthases in organisms such as bacteria, archaebacteria, rodents, bovine and filamentous fungi, the GGPP synthases have been difficult to isolate. Prior to the present invention, a GGPP synthase has never been identified in yeast. Accordingly, there is a need for the isolation of genes encoding eukaryotic GGPP synthases for use in the production of GGPP in large quantities, in an effective cost-effective manner.

COMPENDIUM OF THE INVENTION The present invention relates to geranylgeranyl diphosphate synthase nucleic acid molecules d yeast, and to proteins encoded by those nucleic acid molecules d. One embodiment of the present invention is an isolated nucleic acid molecule encoding a yeast geranylgeranyl diphosphate synthase protein. A preferred embodiment of the present invention is an isolated nucleic acid molecule encoding a protein of syntans of geranylgeranyl diphosphate of Saccharomyces. A more preferred embodiment of the present invention is an isolated nucleic acid molecule encoding a geranylgeranyl diphosphate synthase protein of Saccharomyces cerevisiae. Other embodiments of the present invention include a recombinant molecule encoding a yeast geranylgeranyl diphosphate synthase protein, and a recombinant cell that is capable of expressing a nucleic acid molecule encoding a yeast geranylgeranyl diphosphate synthase protein. Another embodiment of the present invention is an isolated protein comprising a yeast geranylgeranyl diphosphate synthase protein. Preferably, this protein is a geranylgeranyl diphosphate synthase protein of Saccharomyces, and more preferably a geranylgeranyl diphosphate synthase protein of Saccharomyces cerevisiae. Yet another embodiment of the present invention is a method for producing geranylgeranyl diphosphate. This method includes culturing a recombinant cell that is capable of expressing a yeast geranylgeranyl diphosphate synthase protein. Another embodiment of the present invention relates to a method for producing farnesyl diphosphate. This method includes culturing a recombinant cell with a reduced ability to express a yeast geranylgeranyl diphosphate synthase protein.

DESCRIPTION OF THE FIGURES Figure 1 shows that the deletion of a mutant of GGTase-II by a gene encoding a geranylgeranyl diphosphate synthase of Saccharomyces cerevisiae, depends on the dosage of the gene.

Figure 2 shows that overexpression of a geranylgeranyl diphosphate synthase gene from Saccharomyces cerevisiae increases the membrane-bound group of two GGPP-dependent membrane proteins. Figure 3 schematically illustrates the strategy for sequencing a geranylgeranyl diphosphate synthase nucleic acid molecule of Saccharomyces cerevisiae isolated from the present invention. Figure 4 shows that a diploid yeast cell containing a copy of an altered GGPP synthase gene is sensitive to cold for growth. . Figure 5 is a Western blot showing that the membrane binding of a small GTP binding protein, Sec4p and Yptlp, is defective in a yeast cell containing a copy of an altered GGPP synthase gene. Figure 6 is a reverse phase high performance liquid chromatography elution profile of a radiolabelled frenyltransferase reaction mixture, illustrating that expression of the gene encoding a geranylgeranyl diphosphate synthase results in the production of GGPP . Figure 7 shows the saturation curves of [3 H] GGPP, which demonstrate that GGTase-II in a mutant of GGTase-II, has a reduced affinity for GGPP.

DETAILED DESCRIPTION OF THE INVENTION The present invention includes isolated geranylgeranyl diphosphate synthase nucleic acid molecules (GGPP synthase), and isolated geranylgeranyl diphosphate synthase proteins. Also included is the use of these proteins and nucleic acid molecules to produce geranylgeranyl diphosphate (GGPP). One embodiment of the present invention is an isolated nucleic acid molecule encoding a geranylgeranyl diphosphate synthase. This nucleic acid molecule can be an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a yeast geranylgeranyl diphosphate synthase protein. Although the nucleic acid molecules and the geranylgeranyl diphosphate synthase proteins have previously been identified in other organisms, before the present invention, none have been identified in the yeast. Moreover, as described in more detail below, a yeast geranylgeranyl diphosphate synthase nucleic acid molecule and protein identified herein are significantly different from the nucleic acid molecules and the diphosphate synthase proteins of the present invention. geranylgeranil previously reported from other organisms. In another aspect of the present invention, this nucleic acid molecule has a sequence that is more than about 35 percent similar to the nucleic acid sequence of SEQ ID NO: 1, which encodes a Saccharomyces GGPP synthase gene. cerevisiae Preferably, a nucleic acid molecule of the present invention has a sequence that is more than about 50 percent similar to SEQ ID NO: 1, more preferably more than about 75 percent similar to SEQ ID NO: 1 , and still more preferably more than about 90 percent similar to SEQ ID NO: 1. In a further embodiment, a nucleic acid molecule of the present invention comprises the nucleic acid sequence of SEQ ID NO: 1. The degree to which a nucleic acid molecule is similar to another nucleic acid molecule can be determined by conventional methods known in the art. For example, several computerized databases, such as NBLAST and EMBL / GenBank, allow for comparisons of nucleic acid sequences and assessments of the similarity between those sequences. These databases can directly compare these sequences, and determine the percentage of similarity between the sequences. The similarities (ie, the nucleic acid residues that match) between two sequences can be intercalated across all the nucleic acid molecules, or they can be grouped (i.e., localized) in different regions of the nucleic acid molecules .

The comparison of a GGPP synthase nucleic acid molecule of the present invention, with known GGPP synthase nucleic acid sequences, reported in GenBank, indicates that the coding region represented in SEQ ID N0: 1 is very similar to that of the GGPP synthase of the fungus, Neurospora crassa, which is only about 31 percent similar to the GGPP synthase gene of Neuroepora crassa. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural place (i.e., that has been subjected to human manipulation). As such, "isolated" does not reflect the degree to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA, or DNA or RNA derivatives. It should be noted that the term "an" or "an" entity refers to one or more of that entity; for example, a gene refers to one or more genes, or at least one gene. As such, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" can be used interchangeably. Although the phrase "nucleic acid molecule" refers primarily to the physical nucleic acid molecule, and the phrase "nucleic acid sequence" refers primarily to the nucleotide sequence on the nucleic acid molecule, the two phrases can be used in an interchangeable manner, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, which is capable of encoding a GGPP synthase protein. In another embodiment, an isolated nucleic acid molecule of the present invention hybridizes under stringent hybridization conditions, with a geranylgeranyl diphosphate synthase gene from Saccharomyces cerevisiae. In a preferred embodiment, this geranylgeranyl diphosphate synthase gene from Saccharomyces cerevisiae comprises the nucleic acid sequence of SEQ ID NO: 1. Preferably, this isolated nucleic acid molecule of the present invention encodes a geranylgeranyl diphosphate synthase protein. As used herein, stringent hybridization conditions refer to conventional hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify molecules having similar nucleic acid sequences. Stringent hybridization conditions typically allow the isolation of nucleic acid molecules having at least about 70 percent identity of the nucleic acid sequence, with the nucleic acid molecule that is being used as a probe in the reaction of the nucleic acid. hybridization. These conventional conditions are described, for example, in Sambrook et al., 1989, Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press Reference Sambrook et al., Ibid. , is incorporated as a reference to the present in its entirety. Examples of these conditions include, but are not limited to, the following: oligonucleotide probes of about 18 to 25 nucleotides in length, with Tm of about 50 ° C to about 65 ° C, for example, can be hybridized to molecules of nucleic acid typically immobilized on a filter (eg, nitrocellulose filter) in a solution containing 5X SSPE, 1 percent Sarcosyl, 5 Denhardts, and 0.1 milligram / milliliter denatured salmon sperm DNA at 37 ° C for about 2 to 1 hours. The filters are then washed three times in a wash solution containing 5X SSPE, 1 percent Sarcosil at 37 ° C for 15 minutes each. The filters can be further washed in a wash solution containing 2 SSPE, 1 percent Sarcosyl at 37 ° C for 15 minutes per wash. Randomly primed DNA probes can be hybridized, for example, in nucleic acid molecules typically immobilized on a filter (eg, nitrocellulose filter) in a solution containing 5X SSPE, percent Sarcosyl, 0.5 percent Blotto (dry milk in water), and 0.1 milligram / milliliter of denatured salmon sperm DNA at 42 ° C for about 2 to 12 hours. The filters are then washed twice in a wash solution containing 5X SSPE, 1 percent Sarcosyl at 42 ° C for 15 minutes each, followed by two washings in a wash solution containing 2X SSPE, 1 per cent of Sarcosilo at 42 ° C for 15 minutes each. As used herein, a GGPP synthase gene includes all nucleic acid sequences related to a natural GGPP synthase gene, such as the regulatory regions that control the production of the GGPP synthase protein encoded by that gene (such as, but not limited to, the transcriptional, translational or post-translational control regions), as well as the coding region itself. In a similar manner, a nucleic acid molecule of the present invention may include one or more regulatory regions, full length or partial coding regions, or combinations thereof. The reference herein to a nucleic acid sequence refers to the identified sequence, as well as its complement. For example, the nucleic acid sequence SEQ ID NO: 1 represents the genomic DNA sequence of the coding strand of the nucleic acid molecule encoding the GGPP synthase, the production of which is described in the examples. The complement of SEQ ID NO: 1 refers to the nucleic acid sequence of the complementary strand for the strand having SEQ ID NO: 1, which can be easily determined by those skilled in the art. The complement of SEQ ID NO: 1 is represented herein as the nucleic acid sequence, SEQ ID NO: 4. In the same manner, a nucleic acid sequence complement of any nucleic acid sequence of the present invention, refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (ie, can form a double helix with) the strand for which the sequence is cited. Accordingly, a double-stranded nucleic acid molecule of the present invention for which a strand of that nucleic acid molecule is represented by SEQ ID NO: 1, also comprises a complementary strand, SEQ ID NO: 4, which has a sequence that is a complement to that SEQ ID N0: 1. As such, the nucleic acid molecules of the present invention, which may be double-stranded or single-stranded, include nucleic acid molecules that form stable hybrids under stringent hybridization conditions, either with a given sequence denoted in the present, and / or with the complement of that sequence. It should be noted that, since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO: 1 (as well as other nucleic acid and protein sequences presented herein), at best, represents a sequence of apparent nucleic acid of the nucleic acid molecule encoding a GGPP synthase protein of the present invention. The reference herein to a nucleic acid molecule refers to the identified molecule, as well as allelic variants thereof. As used herein, an allelic variant of a nucleic acid molecule is a nucleic acid molecule that occurs essentially in the same place (or places) in a genome as the identified molecule, but which, due to natural variations caused, for example, by mutation or recombination, has a similar sequence, but not identical. Allelic variants typically encode proteins that have an activity similar to that of the protein encoded by the gene with which they are being compared. Allelic variants may also comprise alterations in the non-translated 5 'and 3' regions of the gene (e.g., in the regions of regulatory control). The allelic variants also include variants based on the degeneracy of the genetic code. Accordingly, any degenerate nucleic acid sequences encoding a GGPP synthase protein of the present invention are incorporated herein. Allelic variants are well known to those skilled in the art, and would be expected to be found within a given organism where the genome is diploid and / or between a group of two or more organisms. A GGPP synthase nucleic acid molecule of the present invention can be obtained from its natural source, either as a whole (ie, complete) nucleic acid molecule, or a portion thereof. A GGPP synthase nucleic acid molecule can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning), or chemical synthesis. The reference herein to a nucleic acid molecule refers to the identified molecule, as well as its homologs. A homologue of a nucleic acid molecule can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., Ibid.). For example, nucleic acid molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques, and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule. for inducing mutations, restriction enzyme dissociation of a nucleic acid fragment, ligation of nucleic acid fragments, amplification with polymerase chain reaction and / or mutagenesis of selected regions of a nucleic acid sequence, synthesis of mixtures of oligonucleotides and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Homologs of the nucleic acid molecule can be selected from a mixture of modified nucleic acids by classifying the function of the protein encoded by the nucleic acid (e.g., the GGPP synthase activity or the ability to cause a immune response against at least one epitope of a GGPP synthase protein), and / or by hybridization to a GGPP synthase gene. The present invention also includes nucleic acid molecules which are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other nucleic acid molecules, preferably longer, of the present invention, such as those comprising synthase genes. of GGPP from Saccharomyces cerevisiae, or other yeast GGPP synthase nucleic acid molecules. The oligonucleotides of the present invention can be RNA, DNA or derivatives of any of them. The minimum size of these oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present invention. The characteristics of the minimum size are described here. The size of the oligonucleotide must also be sufficient to use the oligonucleotide according to the present invention. The oligonucleotides of the present invention can be used in a variety of applications, including, but not limited to, probes to identify additional nucleic acid molecules, as primers to amplify or extend the nucleic acid molecules, or in therapeutic applications to inhibit the production or activity of the GGPP synthase protein. These therapeutic applications include the use of these oligonucleotides, for example, in antisense-based technologies, triplex formation, ribozyme and / or RNA drug. The present invention, therefore, includes those oligonucleotides and their use in a method of the present invention. Knowledge of the nucleic acid sequences of certain GGPP synthase nucleic acid molecules of Saccharomyces cerevisiae of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, ( b) obtain nucleic acid molecules that include at least a portion of those nucleic acid molecules (e.g., nucleic acid molecules that include full length genes, full length coding regions, regulatory control sequences, coding regions) truncated), and (c) obtaining GGPP synthase nucleic acid molecules for other yeast species, particularly, because, as described in detail in the example section, the isolation of GGPP synthase nucleic acid molecules is described. of Saccharomyces cerevisiae of the present invention. These nucleic acid molecules can be obtained in a variety of ways, including the classification of appropriate expression libraries with antibodies of the present invention.; traditional cloning techniques using oligonucleotide probes of the present invention to classify appropriate libraries or DNA, and amplification with polymerase chain reaction of the appropriate libraries or DNA, using the oligonucleotide primers of the present invention. Preferred libraries for classifying, or from which the nucleic acid molecules are amplified, include libraries of yeast cDNAs, as well as libraries of genomic DNA. In a similar manner, the preferred DNA sources for classifying, or from which the nucleic acid molecules are amplified, include yeast cDNA, and genomic DNA. Techniques for cloning and amplifying genes are described, for example, in Sambrook et al., Ibid. The present invention also includes nucleic acid molecules that encode a protein having at least a portion of SEQ ID NO: 2, including nucleic acid molecules that have been modified to accommodate the codon usage properties of the cells in where they are going to express those nucleic acid molecules. As used herein, "a portion" of a given sequence may refer to a part or all of that sequence, within the size limitations for the proteins and nucleic acid molecules encoded by those sequences, as stipulated with detail later. As described hereinabove, the isolated nucleic acid molecules of the present invention have the additional feature of encoding an isolated protein comprising a GGPP synthase protein, and preferably a yeast GGPP synthase protein. In accordance with the present invention, an isolated, or biologically pure, protein is a protein that has been removed from its natural environment. As such, "isolated" and "biologically pure" does not necessarily reflect the degree to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, it can be produced using recombinant DNA technology, or it can be produced by chemical synthesis. A further embodiment of the present invention is an isolated geranylgeranyl diphosphate synthase protein. This protein can be an isolated protein comprising a yeast geranylgeranyl diphosphate synthase protein. As noted above, although the nucleic acid molecules and the geranylgeranyl diphosphate synthase proteins have previously been identified in other organisms, prior to the present invention, none have been identified in yeast. A preferred yeast from which the GGPP synthase proteins of the present invention are isolated (including isolation of the native protein or production of the protein by recombinant or synthetic techniques), includes Saccharomyces. More preferably, a GGPP synthase protein of the present invention is isolated from Saccharomyces cerevisiae. As discussed hereinabove, a geranylgeranyl diphosphate synthase protein has the property of catalyzing stepwise additions of IPP in DMAPP, GPP and FPP, to form GGPP. The GGPP synthase can also catalyze the addition of a single molecule of IPP to FPP to form GGPP. The methods for identifying a GGPP synthase protein of the present invention are further described in the examples section below. In another aspect, a GGPP synthase protein of the present invention can include a protein comprising an amino acid sequence that is at least about 45 percent, preferably at least about 55 percent, and more preferably at least about 75 percent, and still more preferably at least approximately 90 percent similar to the amino acid sequence of SED ID NO: 2, which is an amino acid sequence of GGPP synthase of Saccharomyces cerevisiae. The degree to which an amino acid sequence is similar to another amino acid sequence can be determined in the same manner as for the nucleic acid sequences. For example, several computerized databases, such as Swiss-Prot, allow direct comparisons of amino acid sequences, and evaluations of the percentage of similarity between those sequences. A particularly preferred GGPP synthase protein of the present invention is a protein comprising SEQ ID NO: 2 (including, but not limited to, the encoded protein, full-length proteins, processed proteins, fusion proteins, and multivalent proteins), as well as a protein that is a truncated homolog of a protein comprising SEQ ID NO: 2. A still more preferred protein includes PGGPPS335. The isolated proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. Examples of the methods for producing this protein are described herein, including in the Examples section. In a further embodiment, the isolated geranylgeranyl diphosphate synthase protein nail of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions, with a geranylgeranyl diphosphate synthase gene from Saccharomyces cerevisiae. Preferably, an isolated GGPP synthase protein of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a Saccharomyces cerevisiae GGPP synthase gene comprising the nucleic acid sequence of SEQ ID NO: l. More preferably, a GGPP synthase protein of the present invention includes a protein encoded by at least a portion of SEQ ID NO: 1, and as such, has an amino acid sequence that includes at least a portion of the SEQ ID NO: 2. As used herein, a GGPP synthase protein can be a protein of the entire length, or any homologue of that protein. Examples of the GGPP synthase homologs include GGPP synthase proteins wherein the amino acids have been deleted (eg, a truncated version of the protein, such as a peptide), have been inserted, inverted, substituted and / or derived (eg, by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitoylation, amidation and / or glycerophosphatidyl inositol addition), such that the homologue functions as a GGPP synthase, and / or includes at least one epitope capable of elicit an immune response against a GGPP synthase protein. That is, when the homologue is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce a humoral and / or cellular immune response against at least one epitope of a GGPP synthase protein. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. The GGPP synthase protein homologs may be the result of a natural allelic variation or a natural mutation. The GGPP synthase protein homologs of the present invention can also be produced using techniques known in the art, including, but not limited to, direct modifications to the protein, or modifications to the gene encoding the protein, using, for example. , recombinant or classical DNA techniques to perform random or directed mutagenesis. The minimum size of a GGPP synthase homolog of the present invention is a sufficient size to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., to hybridize under stringent hybridization conditions), with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. As such, the size of the nucleic acid molecule encoding that protein homologue depends on the composition of the nucleic acid, and the percentage of similarity between the nucleic acid molecule and the complementary sequence. It should also be noted that the degree of similarity required to form a stable hybrid may vary depending on whether similar sequences are intercalated through all the nucleic acid molecules, or are grouped (i.e., located) in different regions on the nucleic acid molecules. The minimum size of these nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length, if the nucleic acid molecules are rich in GC, and at least about 15 to about 17 bases in length if they are rich in AT. As such, the minimum size of a nucleic acid molecule used to encode a GGPP synthase protein homolog of the present invention is from about 12 to about 18 nucleotides in length. There is no limit, other than a practical limit, on the maximum size of this nucleic acid molecule, in which the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions of the same. In a similar manner, the minimum size of a GGPP synthase protein homolog of the present invention is from about 4 to about 6 amino acids in length, the preferred sizes depending on whether full-length, fusion portions are desired, multivalent, or functional proteins. It should be appreciated that the present invention also includes syngetic mimetics of GGPP synthase proteins of the present invention, which can be used in accordance with the methods described for the GGPP synthase proteins of the present invention. As used herein, a "mimetic" of a GGPP synthase protein of the present invention refers to any compound that is capable of mimicking the activity of that GGPP synthase protein, often because the mimetic has a structure which mimics the GGPP synthase protein. Mimetopes can be, but are not limited to, peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and / or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (eg, carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. These mimetics can be designed using computer-generated structures of the proteins of the present invention. Mimetopes can also be obtained by the generation of random samples of molecules, such as oligonucleotides, peptides, or other organic molecules, and the classification of these samples by affinity chromatography techniques, using the corresponding binding partner. One embodiment of the isolated protein of the present invention is a fusion protein that includes a domain containing GGPP synthase protein linked to a fusion segment. The inclusion of a fusion segment as part of a GGPP synthase protein of the present invention can improve the stability of the protein during production, storage and / or use. In addition, a fusion segment can function as a tool to simplify the purification of a GGPP synthase protein, such as to make possible the purification of the resulting fusion protein, using affinity chromatography. A suitable fusion segment can be a domain of any size that has the desired function (for example, that imparts greater stability and / or simplifies the purification of a protein). It is within the scope of the present invention to use one or more fusion segments. The fusion segments can be linked to the amino and / or carboxyl terminus of the GGPP synthase-containing domain of the protein. The linkages between the fusion segments and the GGPP synthase-containing domains of the fusion proteins may be susceptible to dissociation in order to make possible forward forward recovery of the GGPP synthase-containing domains of those proteins. The fusion proteins are preferably produced by culturing a recombinant cell transformed with a nucleic acid molecule that encodes a protein that includes the bound fusion segment, either the carboxyl and / or amino terminal end of a domain containing synthase. of GGPP. Preferred fusion segments for use in the present invention include a metal binding domain (eg, a poly-histidine segment capable of binding to a divalent metal ion); an immunoglobulin binding domain (e.g., Protein A, Protein G, B cell, Fc receptor, or complement protein antibody binding domains); a sugar binding domain (eg, a maltose binding domain, from a maltose binding protein); a glutathione binding domain; and / or a "tag" domain (eg, at least a portion of β-galactosidase, a strip label peptide, other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include glutathione-S-transferase, and a poly-histidine segment. A particularly preferred fusion segment of the present invention is a polyhistidine segment. The translation of SEQ ID NO: 1 suggests that the nucleic acid molecule nGGPPS1005 encodes a GGPP synthase protein of the entire length of about 335 amino acids, referred to herein as PGGPPS335. NGGPPS1005 represents the open reading frame, excluding the stop codon (stop), which corresponds to a genomic nucleic acid sequence comprising the nucleic acid molecule nGGPPS1600 of the present invention, whose nucleic acid sequence is represented in this by SEQ ID NO: 3. As such, the open reading frame within SEQ ID NO: 3 has an initiation codon (initial) extending from about nucleotide 301 to about nucleotide 303 of SEQ ID NO. : 3, and a stop codon extending from about nucleotide 1306 to about nucleotide 1308 of SEQ ID NO: 3. The complement of SEQ ID NO: 3 refers to the nucleic acid sequence of the complementary strand for chain having SEQ ID NO: 3, and is represented herein as SEQ ID NO: 5. The deduced amino acid sequence of PGGPPS335 is depicted herein as SEQ ID NO: 2. Based on that amino acid sequence , PGGPPS335 has an estimated molecular weight of approximately 38,627 daltons. It is predicted that the amino acid sequence of PGGPPS335 is completely hydrophilic, without significant hydrophobic spaces. One embodiment of the present invention includes a recombinant molecule comprising a nucleic acid molecule encoding a GGPP synthase protein of the present invention. A recombinant molecule, also referred to as a recombinant vector, of the present invention, includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule to a host cell. This vector contains heterologous nucleic acid sequences, i.e., nucleic acid sequences that are not naturally found adjacent to the nucleic acid molecules of the present invention, and that are preferably derived from a different species of the species, from which nucleic acid molecules are derived. The vector can be RNA or DNA, either priocariótico or eucariótico, and typically is a virus or a plasmid. The recombinant molecules can be used in the cloning, sequencing and / or otherwise manipulation of the GGPP synthase nucleic acid molecules of the present invention. A recombinant molecule of the present invention can be used in expression of nucleic acid molecules of the present invention. Preferred recombinant vectors are capable of replicating in a transformed cell. Suitable and preferred nucleic acid molecules for inclusion in the recombinant molecules of the present invention are as described herein for the appropriate and self-preferred GGPP synthase nucleic acid molecules. A particularly preferred nucleic acid molecule for inclusion in the recombinant molecules, and particularly in the recombinant molecules of the present invention, includes nGGPPS1005. A recombinant cell capable of expressing a nucleic acid molecule of the present invention is included in the present invention. A recombinant cell of the present invention includes suitable host cells, to be transformed with a nucleic acid molecule of the present invention. The host cells can be untransformed cells, or cells that are already transformed with at least one nucleic acid molecule. The host cells of the present invention can be endogenously (ie, naturally) capable of producing GGPP synthase proteins of the present invention, or they can be capable of producing those proteins after being transformed with at least one nucleic acid molecule of the present invention. The host cells of the present invention can be any cell capable of producing at least one protein of the present invention, including bacterial, fungal (including yeast), insect, and other animal and plant cells. Preferred host cells include yeast cells and bacterial cells. The most preferred host cells include Saccharomyces cells and Escherichia cells. A particularly preferred host cell is Saccharomyces cerevisiae. Another particularly preferred host cell is Escherichia coli. A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules. A molecule The recombinant of the present invention is a molecule that can include at least one of any nucleic acid molecule described hitherto operatively linked to at least one of any transcription control sequence capable of effectively regulating the expression of the nucleic acid molecules in the cell that is going to transform. Details regarding the production of a recombinant molecule containing a GGPP synthase nucleic acid molecule from Saccharomyces cerevisiae, are described in the Examples section herein. The phrase "operably linked" refers to the insertion of a nucleic acid molecule into an expression vector in such a manner that the molecule can be expressed when it is transformed into a host cell.As used herein, an "expression vector" is a DNA or RNA vector that is capable of transforming a host cell, and of effecting the expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be prokaryotic or eukaryotic, and are typically viruses or plasmids. The expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal (including yeast), insect, other animal and plant cells. The preferred expression vectors of the present invention can direct gene expression in yeast cells, and more preferably in the specific cell types described heretofore. The transformation of a recombinant molecule into a cell can be carried out by any method, by which a recombinant molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroincorporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell can remain unicellular, or it can grow to a tissue, organ, or a multicellular organism. The transformed nucleic acid molecules of the present invention may remain extrachromosomal, or may be integrated into one or more sites within a chromosome of the transformed (i.e., recombinant) cell, such that their ability to express themselves is retained. Preferred and preferred nucleic acid molecules with which a cell is transformed are as described herein for the suitable and preferred GGPP synthase nucleic acid molecules themselves. Particularly preferred nucleic acid molecules for inclusion in the recombinant cells of the present invention include nGGPPS1005"The recombinant molecules of the present invention can also (a) contain secretory signals (ie, signal segment nucleic acid sequences) to make It is possible for an expressed GGPP synthase protein of the present invention to be secreted from the cell that produces the protein and / or (b) to contain fusion sequences that lead to the expression of nucleic acid molecules of the present invention, such as fusion. Examples of suitable signal segments and fusion segments encoded by the fusion segment nucleic acids are described herein. Eukaryotic recombinant molecules can include intervening and / or untranslated sequences that surround and / or remain within the nucleic acid sequences of the nucleic acid molecules of the present invention. Suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility glycoprotein and viral envelope signal segments. The nucleic acid molecules of the present invention can be operably linked to expression vectors containing regulatory sequences, such as transcription control sequences, translation control sequences, replication origins, and other regulatory sequences that are compatible with the cell recombinant, and which control the expression of nucleic acid molecules of the present invention. In particular, the recombinant molecules of the present invention include transcription control sequences. The transcription control sequences are sequences that control the initiation, elongation and termination of transcription. Particularly important transcription control sequences are those that control the initiation of transcription, such as the promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. Those skilled in the art are aware of a variety of these transcription control sequences. Preferred transcriptional control sequences include those that function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp / lpp, rrnB, bacteriophage lambda (?) (such as? pL and? pR, and fusions that include these promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, coupling factor a, Pichia alcohol oxidase, subgenomic promoters of alpha viruses (such as Dindbis virus subgenomic promoters), and antibiotic resistance gene, baculovirus, Heliotis zea insect virus, vaccinia virus, herpes virus, raccoon pox virus, other smallpox virus, adenovirus , cytomegalovirus (such as early promoters of intermediaries), simian virus 40, retroviruses, actin, long terminal retroviral repeat, Rous sarcoma virus, heat attack transcription control sequences, fos fato and nitrate, as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. More preferred transcription control sequences include those that function in yeast, and include, but are not limited to, different galactose promoters or combinations, such as Gall, Gal 7, GallO and GAP / GAL (GAP: glyceraldehyde dehydrogenase). 3-phosphate). Additional suitable transcription control sequences include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., interferon-inducible or interleukin-inducible promoters). The transcription control sequences of the present invention may also include naturally occurring transcription control sequences, which are naturally associated with a yeast, such as a Saccharomyces cerevisiae molecule, prior to isolation. One skilled in the art can appreciate that the use of recombinant DNA technologies can improve the expression of transformed nucleic acid molecules by manipulation, for example, of the number of copies of the nucleic acid molecules within a host cell, of the the efficiency with which these nucleic acid molecules are transcribed, the efficiency with which the resulting transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of the nucleic acid molecules of the present invention include, but are not limited to, nucleic acid molecules operably bonding with high copy number plasmids, integration of the nucleic acid molecules into one or more chromosomes of the host cell, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (eg, promoters, operators, enhancers), substitutions or modifications of translation control signals (e.g., Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the use of the host cell codon, deletion of sequences that destabilize the transcripts, and use of control signals that temporarily separate recombinant cell growth of the production of the recombinant enzyme during the fermentation. The activity of an expressed recombinant protein of the present invention can be improved by fragmentation, modification or derivation of nucleic acid molecules encoding that protein. In accordance with the present invention, the recombinant cells of the present invention can be used to produce one or more proteins of the present invention, by culturing those cells under conditions effective to produce that protein, and recovering the protein. Effective conditions for producing a protein include, but are not limited to, appropriate conditions of medium, bioreactor, temperature, pH and oxygen, which allow the production of the protein. An appropriate or effective means refers to any medium wherein a cell of the present invention, when cultured, is capable of producing a GGPP synthase protein of the present invention. This medium is typically an intermediate medium comprising assimilable sources of carbon, nitrogen and phosphate, as well as salts, minerals, metals and other suitable nutrients, such as vitamins. The medium can comprise complex nutrients, or it can be a defined minimal medium. The cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch fermentors, batch feed, cell recycle, and continuous fermentors. The culture can also be conducted in shake flasks, test tubes, microtiter plates, and petri dishes. The culture is carried out at a temperature, pH, and oxygen content, appropriate for the recombinant cell. These culture conditions are within the experience of an ordinary expert in this field. Examples of suitable conditions are included in the Examples section. Depending on the vector and the host system used for production, the resulting proteins of the present invention can remain inside the recombinant cell; they can be secreted into the fermentation medium; they can be secreted into a space between two cell membranes, such as the periplasmic space in E. coli, or can be retained on the outer surface of a cell membrane.

The phrase "protein recovery" can simply refer to the collection of all the fermentation medium containing the protein, and does not need to involve additional steps of separation or purification. The proteins of the present invention can be further purified using a variety of conventional protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, filtration chromatography. of gel, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Another embodiment of the present invention relates to a method for producing geranylgeranyl diphosphate (GGPP). As described above, GGPP is the source of geranylgeranyl groups that are used to pre-screen the proteins in the isoprenoid biosynthetic pathway. GGPP is an important intermediary in the formation of a variety of derivatives that have important uses in the production of anti-cancer compounds, anti-tumor compounds, anti-cholesterol compounds and anti-ulcer compounds. This method for producing GGPP includes culturing a recombinant cell, such that GGPP is produced, wherein the cell comprises an isolated nucleic acid molecule encoding a geranylgeranyl diphosphate synthase of the present invention. This recombinant cell can include bacterial, fungal (including yeast), insect or other animal and plant cells. Preferred recombinant cells include yeast cells and bacterial cells. The most preferred recombinant cells include yeast of the genus Saccharomyces and bacteria of the genus Escherichia. A particularly preferred recombinant cell is Saccharomyces cerevisiae. Another particularly preferred host cell is Escherichia coli. Another embodiment of the present invention relates to a method for producing farnesyl diphosphate (FPP), which comprises culturing a recombinant cell having a reduced ability to produce geranylgeranyl diphosphate synthase, such that farnesyl diphosphate is produced. As discussed hereinabove, FPP, like GGPP, is a precursor in the isoprenoid biosynthetic pathway. The formation of GGPP can be catalyzed by additions by passage of IPP to DMAPP, GGPP and FPP, or in eukaryotes, by the addition of a single molecule of IPP to FPP. Accordingly, since the production of FPP precedes the production of GGPP in the biosynthetic path, the reduction of the production of a GGPP synthase of the present invention can be useful to improve the production of FPP. More preferably, a recombinant cell having a reduced ability to produce a geranylgeranyl diphosphate synthase of the present invention, contains an endogenous nucleic acid molecule encoding a GGPP synthase of the present invention, which has been modified in such a way that the production of GGPP synthase is reduced. This modification can include a mutation in the GGPP synthase nucleic acid sequence of the present invention, resulting in this mutation the expression of a GGPP synthase protein having a reduced enzymatic activity. The reduction of the enzymatic activity of GGPP synthase can result in reduced production of GGPP, and FPP accumulation within the recombinant cell. This mutation in a GGPP synthase nucleic acid sequence can be in any portion of the GGPP synthase gene, such as in the regulatory regions that control the production of the GGPP synthase protein encoded by that gene (such as, but not limited to, the regions of control of transcription, translation or post-translation), as well as the coding region itself. In an alternative way, a recombinant cell having a reduced ability to produce a geranylgeranyl diphosphate synthase of the present invention, does not contain an endogenous nucleic acid molecule encoding a GGPP synthase of the present invention, but does contain recombinant nucleic acid molecules that encode portions of the synthetic pathway of GGPP, including a GGPP synthase of the present invention that has been modified, such that the production of GGPP synthase is reduced. These modifications have been discussed previously in this. This reduction in the GGPP synthase, therefore, decreases the production of GGPP, and increases or improves the production or accumulation of FPP. A GGPP synthase nucleic acid molecule with a reduced ability to produce GGPP synthase protein, can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as mutagenesis. directed to the site, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme dissociation of a nucleic acid fragment, ligation of nucleic acid fragments, amplification with polymerase chain reaction and / or mutagenesis of selected regions of a nucleic acid sequence, synthesis of mixtures of oligonucleotides and ligation of mixed groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecules with a reduced ability to produce GGPP can be selected from a mixture of modified nucleic acids, by sorting by the function of the protein encoded by the nucleic acid (eg, the ability to convert FPP to GGPP) and / or by hybridization with an isolated GGPP synthase gene of the present invention. This cell containing a modified recombinant GGPP synthase gene is exemplified in Example 4. The following examples are provided for the purposes of illustration, and are not intended to limit the scope of the present invention. The following strains were used in the Examples 1-8 later: ANY119 (MATa, bet2-l, ura3-52, his4-619), NY648 (MAT a / a, leu2-3, 112 / leu2-3, 112, ura3-52 / ura3-52) , NY180 (MATa, ura3-52, leu2-3, 112), SFNY26-6A (MATa, his4-619), and SFNY368 (MATa, ura3-52, leu2-3, 112, ÜRA3:: BTS1). The yeast strains were cultured at 25 ° C or at 37 ° C, either in YP or in minimal selective medium that was supplemented with 2 percent glucose.

Example 1 The following example demonstrates the isolation of a plasmid (pSJ28) which is a suppressor of the bet-2-1 mutant. GGTase-II is composed of three subunits BET2, BET4 and MRS6). Bet2p, the β subunit of this enzyme complex, forms a complex with Bet4p, the a subunit. Mrs6p is an escort protein that presents the protein substrate to the Bet2p-Bet4p complex. During geranylgeranylation, the Bet2p-Bet4p complex binds to, and transfers the GGPP to, Yptlp, Sec4p and other small GTP binding proteins. bet2-l is a temperature sensitive mutant for the β subunit of Saccharomyces cerevisiae, of GGTase-II. The mutant grows at 25 ° C (permissive temperature), but dies at 37 ° C. To isolate genes whose products can interact with the Bet2 protein (Bet2p), a yeast genomic library was prepared by ligation of the genomic DNA that was prepared from the mutant bet2-l (ANY119). This DNA was partially digested with Sau3A, and inserted into the BamHI site of pRS316 (CEN, URA3). The library was used to transform ANY 119, and the transformants (1 x 106) were selected on minimal medium lacking uracil. After a three-day incubation at 25 ° C, the cells were stamped on YPD plates, and incubated overnight at 37 ° C. 11 positive transformants were obtained. The plasmids (pSl-pSll) recovered from these transformants were amplified in Escherichia coli, and retested in ANY119. The growth of mutant cells containing six of these plasmids (group A) was indistinguishable from those of wild type at 37 ° C (data not shown). The other five (group B), however, are not suppressed so well. The restriction analysis indicated that the plasmids in group A contained the gene structuring BET2. Since the genomic library was prepared from Jbet2-2 mutant cells, the restoration of growth observed at 37 ° C is not a true complement. The plasmids of group B contained a 2.0 kb overlapping region of DNA. Accordingly, the gene that suppresses the bet2-l mutant is located within this 2.0 kb fragment. The smallest group B plasmid (pS8) that was isolated contained a 2.8 kb insert (Figure Ib). To analyze the ability of this graft to suppress bet2-l, this fragment was cloned into a high capacity copy vector URA3 (pRS426) to generate the plasmid, pSJ28. When pSJ28 was transformed into Jet2-1 mutant cells, the suppression was significantly improved (Figure 1, compare b and e with the mutant alone in a). In fact, the growth of the mutant was restored to that of the wild type (Figure 1, compare c and d), suggesting that the deletion depended on the dosage of the gene. As demonstrated in this example, in an attempt to identify new genes whose products can interact with Bet2p, the present inventors isolated a suppressor of the bet2-l mutant. The examples below show that this suppressor gene, called BTS1, encodes a geranylgeranyl diphosphate synthase, a previously unidentified prenyltransferase from the isoprenoid biosynthetic pathway of yeast. The BTS1 gene product works on this trajectory to convert FPP to GGPP.

Example 2 The following example demonstrates that plasmid pSJ28 increases the membrane-linked group of two GGPP-dependent proteins in bet2-l mutant cells. The membrane association of Yptlp and Sec4p, two small GTP binding proteins, that regulate intracellular membrane trafficking, is defective in bet2 mutant cells. This defect is a consequence of the failure to geranylgeranilar these proteins. Accordingly, the lethal phenotype of the bet2-l mutant is likely to be a consequence of the inability of these proteins to bind to the membranes. Since the plasmid pSJ28 suppresses the growth defect of the bet2-l mutant at 37 ° C, the present inventors believed that it would also cure the defect of membrane binding observed in these cells. To solve this possibility, pSJ28 was transformed into bet2-l. When the distribution of Yptlp and Sec4p was examined in these transformants, and compared with the mutant and the wild type, it was discovered that pSJ28 improves the membrane association of these small GTP binding proteins (Figure 2, compare the amount in lysate (T) with supernatant (S) and granule (P) fractions The presence of pSJ28 did not lead to an increase in residual GGTase-II activity, which can be measured in bet2 mutant cells. , the restoration of the membrane association of Yptlp and Sec4p is not a consequence of the increased activity of GGTase-II. example 3 The following example illustrates the cloning and sequencing of the bet2-l suppressor gene. To locate the nucleic acid molecule encoding the suppressor within the 2.8 kb genomic fragment described above, subclones of pSJ28 were constructed, and inserted into pRS316 (URA3, CEN). The suppression studies revealed that the Sacl site contained within this fragment is critical for its activity. The smallest region of the DNA capable of suppressing bet2-l, was found to be a 1.6 kb SspI-NruII fragment, referred to herein as nGGPPS1600 (SEQ ID NO: 3). This region of DNA was sequenced in both directions, using the strategy shown in Figure 3. The DNA sequence of the BTS1 gene was determined by the dideoxynucleotide chain termination method. The reactions were performed using the Sequenase protocol (U.S. Biochemical Corp.), and the data was analyzed with GCG software. Homology searches were performed with the EMBO / GenBank and Swiss-Prot databases. We identified an open reading frame of 1005 base pairs that extends the Sacl site. The nucleic acid molecule encoding this open reading frame is referred to as BTS1 (Bet2 Suppressor), or nGGPPS1005 (SEQ ID NO: 1). The BTSI product, also referred to herein as Btslp, or PGGPPS335 (SEQ ID NO: 2), was predicted to encode a protein of 335 amino acids with a calculated molecular mass of 38,627 daltons. Above all, the amino acid composition of Btslp is hydrophilic, and no significant hydrophobic stretches were observed. The comparison of the amino acid sequence of Btslp predicted with the Swiss-Prot protein sequence database revealed that Btslp and the gene product N. crassa albino-3 identified above (Carattoli et al., J. Biol. Chem., 1991, Volume 266: 5854- 5859) are 40 percent identical at the amino acid level, with the most conserved region located at half of these proteins. The albino-3 gene encodes a geranylgeranyl diphosphate synthase in the carotenoid biosynthetic pathway of N. crassa. Btslp also contains five conserved regions found in other FPP and GGPP synthases, including the aspartate-rich sequences proposed as involved in linkage and catalysis. These comparisons indicated that BTS1 encodes a previously unidentified GGPP synthase, a prenyltransferase of S. cerevisiae Example 4 The following example demonstrates that the BTS1 nucleic acid molecule, or nGGPPS1005, is not essential for the vegetative growth of yeast cells, but in its absence, growth is impaired. To investigate if BTS1 is required for the vegetative growth of yeast cells, a copy of this gene was altered in diploid cells, and a tetradic analysis was performed. To alter the BTS1, a 1.7 kb Dral-NruI fragment containing the BTS1 gene was separated from the pS8 and cloned into the PvuJJ site of pUCllβ to generate pSJ30. The alteration arising from the BTSl plasmid was constructed by replacing a 0.65 kb SacI-EcoRI fragment in pSJ30 with a 1.2 kb SacI-EcoRI fragment containing the URA3 gene. The resulting plasmid (pSJ31) was digested with SspI and BglII, and transformed into NY648. The transformants were sporulated, and the tetradic analysis was performed. After three days at 25 ° C, in the 48 tetrads examined, four viable spores were obtained. However, two of the colonies in each of the tetrads exhibited a growth defect at 25 ° C. The large colonies were Ura-, and the small colonies were Ura +, indicating that they contained the altered BTSl gene. To confirm that the small colonies contained the altered BTSl nucleic acid molecule, yeast genomic DNA prepared from NYTY180 (above), or SFNY368 (below), was examined by DNA-DNA hybridization. Genomic DNA digested with BglII was fractionated on a 0.8 percent agarose gel, and transferred to a BioTrans membrane (ICN). The spot was probed with a radiolabelled 0.65 kb Sacl-EcoRI fragment, containing BST1, prepared by random primer labeling, and visualized by autoradiography. The above results demonstrate that BTS1 is not essential for the vegetative growth of yeast cells, but that in their absence, growth is impaired. The growth of the altered strain (SFNY368 or? BTS1) was further examined at different temperatures. As shown in Figure 4, the BTS1 cells (Figure 4, a and d), grew as well as the wild type at 30 ° C (Figure 4, b and c). However, at lower temperatures (25 ° C and 14 ° C) a growth defect arose. Only the small colonies appeared after three days at 25 ° C (Figure 4, a and d), while at 14 ° C, the cells did not survive (Figure 4, a and d). This result clearly showed that the SFNY368 is sensitive to cold for growth. Because each of the subunits of GGTase-II is essential, it would be expected that BTS1 would also be required for the vegetative growth of yeast cells. Surprisingly, the strain? BTS1, was only sensitive to cold for growth. In addition, the growth of this strain was not impaired at 30 ° C or at higher temperatures. When the membrane association of Yptlp and Sec4p was examined in? BTS1 cells cultured at 30 ° C, a small fraction of each of these proteins was membrane bound. Accordingly, BTS1 depleted cells are able to pre-screen proteins at a level that is sufficient to sustain cell growth at higher temperatures. When these cells were changed to 14 ° C, less membrane-bound Yptlp and Sec4p were detected, implying that the growth ceases as a consequence of the failure to pre-harvest those essential proteins.

Example 5 The following example shows that the BTS1 gene product is required for membrane binding of Yptlp and Sec4p. Yptlp and Sec4p are two small GTP binding proteins that regulate intracellular membrane trafficking. Like many small GTP binding proteins, they are synthesized in the cytosol, but become bound to a membrane to perform their function. The ability of Yptip and Sec4p to bind to membranes is conferred by the addition of the geranylgeranyl fraction of 20 carbon atoms. The geranylgeranilation of these proteins is catalyzed by a protein prenyltransferase that uses GGPP as a lipid donor. If BTS1 encodes a GGPP synthase, altering this gene should result in depletion of GGPP. Consequently, the geranylgeranylation of Yptlp and Sec4p will be abolished. To test this hypothesis, we examined the membrane association of these proteins in the SFNY368 strain? BTS1. The SFNY368 was cultured at 30 ° C for 12 hours, until the A600 was at 1.0 before changing the cells at 14 ° C for another 12 hours. Aliquots of cells were removed at each point of time, converted to spheroplasts, used, and centrifuged at 450 x g to remove unbroken cells and nuclei. Subsequently, these lysates were centrifuged at 100,000 x g for one hour to obtain a supernatant and the granule fractions, and the distribution of Yptlp and Sec4p in each of these fractions was examined by Western blot analysis. The wild type NY180) and the strain of? BTS1 (SFNY368) were grown overnight at 30 ° C in a YPD medium, until a first exponential phase. An aliquot of cells was pelleted (150 A599 units), and washed once with ice cold 10 mM sodium azide. The remaining cells were changed at 14 ° C, and the incubation was continued for 12 hours before the cells were harvested. To generate spheroplasts, the cells were resuspended in 0.7 milliliters of ice cold 10 mM sodium azide, and mixed with an equal volume of 2 x spheroplast medium (2.8 M sorbitol, 100 mM Tris-HCl (pH 7.5), 20 mM sodium azide), containing 100 units of cimolyase. After one hour of incubation at 25 ° C, the spheroplasts were harvested by centrifugation in a clinical centrifuge during a centrifugation at 1,400 rpm for 5 minutes, washed, and lysed in 1.4 milliliters of ice cold lysis buffer (sorbitol 0.8 M, 10 mM trietanolic amine (pH 7.2), 1 mM EDTA). The cell debris was removed during a 3 minute centrifugation at 450 x g, and the supernatant from this centrifugation was centrifuged at 100,000 x g for 1 hour to generate a soluble fraction. The granule was resuspended in a volume of lysis buffer equal to the supernatant. The samples were electrophoresed and subjected to Western blot analysis using antibodies against Yptlp or against Sec4p (dilution of 1: 2000). In the wild-type cells (Figure 5, compare the amount in the lysate (T) with the supernatant (S) and the granule (P)), the larger phase of Yptlp and Sec4p was bound with membrane at both points of time, and the change in temperature did not affect its membrane association (Figure 5, compare 14 ° C and 30 ° C). However, in SFNY368, most of Yptlp and Sec4p was soluble at both temperatures (Figure 5, compare the amount in the lysate (T) with the supernatant (S) and the granule (P)), although this defect was more pronounced at 14 ° C. Accordingly, the membrane association of these small GTP binding proteins is defective in the BTS1 cells, demonstrating that the BTS1 encodes a GGPP synthase.

Example 6 The following example further demonstrates that BTS1 encodes a geranylgeranyl diphosphate synthase. To demonstrate that BTSl encodes a geranylgeranyl diphosphate synthase, the gene was cloned into a püCllβ vector to be expressed in E. coli. The sequence of the BTSl open reading frame was generated by polymerase chain reaction, using two primers that overlapped with the initiation codon or the region of 100 base pairs downstream from the stop codon. The EcoRI and ClaJ sites were also incorporated at the 5 'and 3 * ends, respectively. The product of the polymerase chain reaction was digested with EcoRI and ClaJ, and cloned into the pUCllβ expression vector. The resulting genetic fusion encodes a Btel protein, with six amino acids with additional NH2 terminals, from β-galactosidase. This construction was then transformed into bacterial cells JM101, and expressed. Crude extracts of E. coli containing pUCllβ (control), or pUC118 / BTSl, were tested for the activity of prenyltransferase in the presence of [1-14C] IPP, using DMAPP or FPP as the allyl substrate, and the mixture of reaction was analyzed by high performance liquid chromatography. The standard assay mixture contained 20 mM BHDA buffer (pH 7.0), 10 mM β-mercaptoethanol, 1 mM MgCl 2, 0.1 percent (w / v) bovine serum albumin, 200 μM DMAPP or FPP, [1- 1 C] IPP 20 μM (10 μCi / micromol, purchased from Amersham), and 70-80 micrograms of protein in a total volume of 200 microliters. DMAPP, FPP and GGPP were synthesized. After 10 minutes at 37 ° C, 200 microliters of CH30H-HC1 (4: 1) was added, and the incubation was continued for 30 minutes. The reaction mixture was extracted with one milliliter of ligroin, and 0.5 milliliters of the ligroin layer was mixed with 10 milliliters of Cytoscint-ES (ICN) for measurement of radioactivity in a Packard-TriCarb 4530 liquid scintillation spectrometer. The products were analyzed using high performance liquid chromatography. For product analysis, bovine serum albumin was omitted from the conventional assay mixture, but 10 mM sodium fluoride was present, to suppress phosphatase activity. After one hour of incubation at 37 ° C, the reaction was terminated by the addition of EDTA (12.5 mM, final concentration). Unlabeled GGPP (25 micrograms) was added, and 150 microliters of the mixture was injected onto a Shodex Asahipak ODP-50 column (4.6 millimeters (internal diameter) per 250 millimeters). After two minutes fractions were collected, and the radioactivity in each fraction was determined by a liquid scintillation counter after the addition of 15 milliliters of Cytoscint-ES. The observed prenyltransferase activity was dependent on the presence of FPP, since no accounts were obtained when the pUC118 / BTSl extract was tested in the absence of FPP (not shown). The radioactive product from this incubation was coeluted with unlabeled synthetic GGPP, indicating that it is GGPP (Figure 6). No conversion of FPP into GGPP was seen with pUCllβ control. Both extracts also showed low levels of activity in the conversion of DMAPP to an acid-labile product. However, because the degree of conversion was the same for both samples, this activity might not be due to Btslp (not shown). In summary, it was found that bacterial lysates expressing Btslp contain an activity that synthesizes GGPP from IPP and FPP. Accordingly, BTS1 encodes a geranylgeranyl diphosphate synthase.

Example 7 The following example shows the mechanism by which the overexpression of BTS1 (GGPP synthase) suppresses the lethality of the bet2-l mutant. One possibility for the suppression mechanism BTS1 is that BTS1 suppresses by increasing the intracellular group of GGPP, thus compensating for a mutant GGTase-II that has a lower affinity for GGPP. To test this hypothesis, the activity of wild type GGTase-II and the bet2-l mutant were measured in the presence of different concentrations of GGPP. As a control, the activity of the Jet4-2 mutant extracts was also evaluated. BET4 encodes the a subunit of GGTase-II, and extracts prepared from that mutant are empty of GGTase-II activity. Yeast cells were cultured in YPD medium at ° C until the last logarithmic phase. Cells were harvested, lysed with glass beads, and centrifuged at 100,000 x g for 45 minutes. The soluble fraction was collected and tested for the activity of GGTase-II. Prenylation assays were performed in a 50 microliter reaction containing 50 mM Tris-HCl (pH of 7.5), 10 mM MgCl 2, 5 mM dithiothreitol, 25 micrograms of extract, 0.4 μM of recombinant Yptlp, and different concentrations of [3 H] GGPP (American Research Lab, 17,500 dpm / pmol). The reaction mixture was incubated at 30 ° C for 30 minutes before being terminated with 1 M HCl in ethanol (1 milliliter), and filtered on a Whatman GF / A filter. Unlike bet2-l, overexpression of BTS1 does not suppress the temperature-sensitive growth defect of the bet4-2 mutant (not shown in the data). As shown in Figure 7, the GGTase-II activity of the wild-type extract was saturated to approximately 0.8 μM of GGPP. At this concentration, the activity of the bet2-l mutant extract was about 5 to 10 percent of the wild-type. This activity was significantly improved when the GGPP concentration was increased beyond 2 μM, and the saturation was reached at 6 μM. In contrast, the GGTase-II activity of the bet4-2 mutant extract could not be compensated by increasing the GGPP concentration. The Kj values, calculated from GGTase-II in the bet2-l mutant and the wild-type, were approximately 3.6 and 0.4 μM, respectively. Accordingly, it appears that GGTase-II in the bet2-l mutant has a reduced affinity for GGPP, which results in a decrease in prenylation activity. By increasing the amount of GGPP that is added to the assay, the prenylation activity is efficiently restored. This result provides a clear explanation for the deletion of the GGTase-II mutant, bet2-l, by GGPP synthase (BTS1). The deletion of the bet2-l mutant by BTS1 could be explained in several ways. The BTS1 gene product can itself have GGTase-II activity, or it could directly interact with GGTase-II to stimulate its activity. In any situation, it would be expected that overexpression of BTSl would increase the activity of GGTase-II. However, the present inventors did not observe this. Alternatively, deletion may simply be a consequence of increasing the intracellular group of GGPP. Since in vitro prenylation studies have shown that the GGTase-II mutant has a low affinity (greater K ,,,) for GGPP, which is compensated by higher concentrations of GGPP, without being bound by theory, the present inventors They believe that this alternative explanation is correct. According to this model, additional copies of BTSl should result in higher intracellular concentrations of GGPP, and a better suppression of bet2-l, explaining in this way the reason why the suppression of bet2-l by BTSl depends on the dosage of the gene. Since BTS1 is not essential for the growth of yeast cells, one would expect that the synthase gene could be duplicated. Nevertheless, DNA hybridization experiments argue against this possibility. Another explanation for eliminating the use of BTSl is that GGTase-II could use FPP as an alternative substrate. However, since GGTase-II can not transfer FPP to Yptlp, this possibility is not very likely either. In addition, extracts prepared from? BTSl cells do not support the transfer of [3H] FP onto Yptlp. Accordingly, it is more possible that another prenyltransferase, such as hexaprenyl diphosphate synthase, can produce small amounts of GGPP as an intermediate product during the elongation of GGPP to longer polyisoprenoid chains. In strain BTS1, GGPP can be formed in this manner, making it possible for yeast cells to survive at certain temperatures in the absence of geranylgeranyl synthase. Although different modalities of the present invention have been described in detail, it can be seen that efforts in this field will be able to think of modifications and adaptations of those modalities. It should be expressly understood, however, that those modifications and adaptations are within the scope of the present invention, as stipulated in the following claims.

SEC. INFORMATION ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: '.A) LENGTH: 1005 base pairs (B) TYPE: nucleic acid (C) CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1..1005 (xi) SEQUENCE DESCRIPTION: SEC. ID NO: l: ATG GAG GCC AAG ATA GAT GAG CTG ATC AAT AAT GAT CCT GTT TGG TCC 48 Met Glu Ala Lys He Asp Glu Leu He Asn Aan Aep Pro Val Trp Ser 1 5 10 15 AGC CAA AAT GAA AGC TTG ATT TCA AAA CCT TAT AAT CAC ATC CTT TTC 96 be Gln Asn Glu Ser Leu He Ser Lye Pro Tyr Aßn HIB He Leu Leu 20 25 30 AAA CCT CGC AAG AAC TTT AGA CTA ATA TTA ATA GTT CAA ATT AAC AGA 144 Lys Pro Gly Lys Aßn Phe Arg Leu Asn Leu He Val Glp He Asn Arg 35 40 45 GTT ATG AAT TTG CCC * AAA GAC CAG CTG GCC ATA GTT TCG CAA ATT GTT 192 Val Met Aan Leu Pro Lys Aßp Gln Leu Ala He Val Val Gln He Val 50 65 60 GAG CTC TTG CAT AAT TCC AGC CTT TTA ATC GAC GAT ATA GAA GAT AAT 240 Glu Leu Leu His Aßn Ser Ser Leu He Asp Asp Lie Glu Aßp Asn 65 70 75 80 GCT CCC TTG AGA AGG GGA CAG ACC ACT TCT CAC TTA ATC TTC GGT GTA 2T8 Wing Pro Leu Arg Arg Gly Gln Thr Thr Ser Hiß Leu He Phe Gly val 05 90 95 CCC TCC ACT ATA AAC ACC GCA AAT TAT ATG TAT TTC AGA GCC ATG CAA 336 Pro Be Thr He Asn Thr Wing Asp Tyr Met Tyr Phe Arg Wing Met Gln 100 IOS 110 CTT GTA TCG CAG CTA ACC AAA GAG CCT TTG TAT CAT AAT TTG ATT 384 Leu Val Ser G n Leu Thr Thr Lyß Glu Pro Leu Tyr Hiß Asp Leu He US 120 125 ACG ATT TTC AAC GAA GAA TTG ATC AAT CTA CAT AGG GGA CAA GGC TTG 432 Thr He Phe Asn Glu Glu Leu He Asm Leu His Arg Gly Gln Gly Leu 130 135 ICO GAT ATA TAC TGG AGA GAC TTT CTG CCT GAA ATC ATA CCT ACT CAG GAG 480 Aep He Tyr Trp Arg Asp Phe Leu Pro Glu He He Pro Thr Gln Glu 145 150 155 160 ATG TAT TTG AAT ATG GTT ATG AAT AAA ACA GGC GGC CTT TTC AGA TTA 528 Met Tyr Leu Asn Met Val Met Aßn Lye Thr Gly Gly Leu Phe Arg Leu 165 170 175 ACG TTG AGA CTC ATG GAA GCG CTG TCT CCT TCC TCA CAC CAC GGC CAT 576 Thr Leu Arg Leu Met Glu Ala Leu Ser Pro Ser Ser Hiß Hie Gly His 180 185 190 TCG TTG GTT CCT TTC ATA AAT CTT CTC GGT ATT ATT TAT CAG ATT AGA 62 Ser Leu Val Pro Phe He Aen Leu Leu Gly He lia Tyr Gln He Arg 195 200 205 GAT GAT TAC TTG AAT TTG AAA GAT TTC CAA ATG TCC AGC GAA AAA GOC bll Aßp Aßp Tyr Leu Asn Leu Lys Aßp Phe Gln Met Ser Ser Glu Lys Gly 210 215 220 TTT GCT GAG GAC ATT ACÁ GAG GGG AAG TTA TCT TTT CCC ATC GTC CAC 720 Phe Wing Glu Asp He Thr Glu Gly Lyß Leu Ser Phe Pro He Val Hiß 225 230 235 240 GCC CTT AAC TTC ACT AAA ACG AAA GGT CAA ACT GAG CAA CAC AAT GAA 768 Ala Leu Aßn Phe Thr Lye Thr Lys Gly Gln Thr Glu Gln Hiß Aßn Glu 245 250 255 ATT CTA AGA ATT CTC CTG TTG AGG ACA AGT GAT AAA GAT ATA AAA CTA 816 He Leu Arg He Leu Leu Arg Thr Ser Aßp Lys Asp He Lyg Leu 260 265 270 AAG CTG ATT CAA ATA CTG GAA TTC GAC ACC AAT TCA TTG GCC TAC ACC Tó4 Lys Leu He G n He eu Glu Phe Aep Thr Asp Ser Leu Wing Tyr Thr 275 2T0 285 AAA AAT TTT ATT AAT CAA TTA CTG AAT ATC ATA AAA AAT GAT AAT GAA 912 Lys Asn Phe He Asn Gln Leu Val Aßn Met He Lye Aßn Aep Asn Glu 290 295 300 AAT AAG TAT TTA CCT GAT TTG GCT TCG CAT TCC GAC ACC GCC ACC AAT 960 Asn Lyß Tyr Leu Pro Asp Leu Wing be His Ser Asp Thr Wing Thr Aen 305 310 315 320 TTA CAT GAC GAA TTG TTA TAT ATA ATA GAC CAC TTA TCC GAA TTG 1005 L Hie Aßp Glu Leu Leu Tyr He He Asp His Leu Ser Glu Leu 325 330 335 SEC. INFORMATION ID NO: 2: (i) CHARACTERISTIC OF THE SEQUENCE ÍA} LONGI TUD: 335 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 2: Het Glu Wing Lys He Asp Clu Leu He Asn Asn Aep Pro Val Trp Ser 1 5 10 15 Being Gln Asn Glu Being Leu He Being Lyß Pro Tyr Aßn Hiß He Leu Leu 20 25 30 Lyß Pro Gly Lyß Asn Phe Arg Leu Aßn Leu He Val Gln He Aßn Arg 35 40 45 to Met Asn Leu Pro Lys Asp Gln Leu Ala He Val Ser Gln He Val SO 55 60 Glu Leu Leu His Asn Ser Ser Leu Leu He Asp Asp He Glu Asp Aen 65 70 75 80 Ala Pro Leu Arg Arg Gly Gln Thr Thr Ser His Leu He Phe Gly Val 85 90 95 Pro Ser Thr He Asp Thr Wing Aen Tyr Met Tyr Phe Arg Wing Met Gln 100 105 110 Leu Val Ser Gln Leu Thr Thr Lys Glu Pro Leu Tyr His Asn Leu He 115 120 125 Thr He Phe Aßn Glu Glu Leu He Aßp Leu His Arg Gly Gln Gly Leu 130 135 140 Aep lie Tyr xrp Arg Asp Phe Lt * u Px.o Glu He He Tro Thr Cln Glu 145 150 155 160 Met Tyr Leu Asn Met val «et Asn Lyß Thr Gly Gly Leu Phß Arg Leu 165 170 175 Thr Leu Arg Leu Mßt Glu Ala Leu Ser Pro Ser Ser Hiß 'Hiß Gly Hie 180 185 190 be Leu val Pro Phe He Aen Leu Leu Gly He He Tyr Gln He Arg 195 200 205 Asp Aßp Tyr Leu Aßn Leu Lys Aßp Phe Gln Met Ser Ser Glu Lyß Gly 210 21S 220 Phe Ala Glu Aßp He Thr Glu Gly Lye Leu Ser Phe Pro He Val Hie 225 230 235 240 Ala Leu Aßn Phe Thr Lys Thr Lys Gly Gln Thr Glu Gln Hiß Asp Glu 245 250 255 He Leu Arg He Leu Leu L «= * u Arg Thr Ser Asp Lyo Aßp He Lys T, PU 260 265 270 Lys Leu He Gln He Leu Glu Phe Asp Thr Asn Ser Leu Wing Tyr Thr 275 280 285 Lys Aßn Phe He Asn Gln Leu Val Aßn Met lie Lyß Asn Asp Asn Glu 290 295 300 Aßn Lys TJ * Leu Pro? Sp Leu Wing Sor Hiff Ser Asp Thr Wing Thr Aen 305 310 315 320 Leu His Aep Glu Leu Leu Tyr He He Asp His Leu Ser Glu Leu 325 330 335 SEC. ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1569 base pairs (B) TYPE: nucleic acid (C) CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ) (ix) DESCRITION OF THE SEQUENCE: SEC. ID NO: 3: AAT TTACAT ATAGATATAG GACAAGCCCG CATTTTCATA CTGAAAGGTA AACTTCTATT 60 ATTATAGTGG TATCCAACGT TCACCGCTTC CAGCATAGCA GAAATTACGT GTTTTTGCAT 120 ATGTTATGCT GATCATTGTA TGCTTACTAC CATTTTTCTT TGCTTCGCCT TGCCTTCTTT 180 GACGTTTTTT TGAAGCAAAA AAAAAGTCAA GACAGATGTG CTTACAAAAC CATGTAAGGC 240 TCATTTTCAA AGAAGCTACT AATAGAAAGA GAACAAAGAG TTTACGAGTC TGGAAAATCA 300 ATGGAGGCCA AGATAGATGA GCTGATCAAT AATGATCCTG TTTGGTCCAG CCAAAATGAA 360 AGCTTGATTT CAAAACCTTA TAATCACATC CTTTTGAAAC CTGGCAAGAA CTTTAGACTA 420 AATTTAATAG TTCAAATTA? CAGAGTTATG AATTTGCCCA AAGACCAGCT GGCCATAGTT 480 TCGCAAATTG TTGAGCTCTT GCATAATTCC ACCCTTTTAA TCGACGATAT AGAAGATAAT 540 GCTCCCTTGA GAAGGGGACA GACCACTTCT CACTTAATCT TCGGTGTACC CTCCACTATA 600 AACACCGCAA ATTATATGTA TTTCAGAGCC ATGCAACTTG TATCGCAGCT AACCACAAAA 660 GAGCCTTTGT ATCATAATTT GATTACGATT TTCAACGAAG AATTGATCAA TCTACATAGG 20 GGACAAGGCT TGGATATATA CTGGAGAGAC TTTCTGCCTG AAATCATACC TACTCAGGAG 780 ATCTATTTGA ATATGGTTAT GAATAAAACA GGCGGCCTTT TCAGATTAAC GTTGAGACTC 840 ATGGAAGCGC TGTCTCCTTC CTCACACCAC GGCCATTCGT TGGTTCCTTT CATAAATCTT 900 CTGGGTATTA TTTATCAGAT TAGAGATGAT TACTTGAATT TGAAAGATTT CCAAATGTCC 960 AGCGAAAAAG GCTTTGCTGA GG? CATTACA GAGGGGAAGT TATCTTTTCC CATCGTCCAC 1020 GCCCTTAACT TCACTAAAAC GAAAGGTCAA ACTGAGCAAC ACAATGAAAT TCTAAGAATT 1080 CTCCTGTTGA GCACAAGTGA TAAAGATATA AAACTAAAGC TüATTCAAAT ACTGGAATTC 1140 GACACCAATT CATTGGCCTA CACCAAAAAT TTT T ATC AATTAGTGAA TATGATAAAA 1200 AATGATAATG AAAATAAGTA T7TACCTGAT TTGGCTTCGC ATTCCGACAC CGCCACCAAT 1260 TTACATGACG AATTGTTATA TATAATAGAC CACTTATCCG AATTGTGAAA TAAATTGATC 1320 AATCAAATTA GTGGAGGAAG ATAGTCAGAA ATAAAGCCTT CTCTCCTCCT CTTTCGCATC 1380 TATACATACG ATTTCATATA TACGTTTCAT TGCATCATCT TTTGATATAT CTCAAAAAGA 1440 AATT ATAGC CTTTATATTT TTTCCACGAT ISOO TTCTGAAACT CCTTTTTATC AGCACCGTTA ATGCTAGCGG TTACTGTCAA ATCGCCGGTA 1560 AATTCGCGA 1569 SEC. INFORMATION ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1005 base pairs (B) TYPE: nucleic acid (C) CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ) (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 4: TACCTCCCCT TCTATCTACT CCACTAGTTA TTACTAGGAC AAACCAGGTC GGTTTTACTT 60 TCG ACTAAA GTTTTGGAAT ATTAGTGTAG GAAAACTTTG GACCGTTCTT GAAATCTGAT 120 TTAAATTATC AAGTTTAATT GTCTCAATAC TTAAACGGGT TTCTGGTCGA CCGGTATCAA 180 AGCGTTTAAC AACTCGAGAA CGTATTAAGG TCGGAAAATT AGCTGCTATA TCTTCTATTA 240 CGAGGGAACT CTTCCCCTGT CTGGTGAAGA GTGAATTAGA AGCCACATGG GAGGTGATAT 300 TTGTGGCGTT TAATATACAT AAAGTCTCGG TACGTTGAAC ATAGCGTCGA TTGGTGTTTT 360 CTCGGAAACA TAGTATTAAA CTAATGCTAA AAGTTGCTTC TTAACTAGTT AGATGTATCC 420ACCTATATAT GACCTCTCTG AAAGACGGAC TTTAGTATGG ATGAGTCCTC 480 TACATAAACT TATACCAATA CTTATTTTGT CCGCCGGAAA AGTCTAATTG CAACTCTGAG 540 TACCTTCGCG ACAGAGGAAG GAGTGTGGTG CCGGTAAGCA ACCAAGGAAA GTATTTAGAA 600 GACCCATAAT AAATAGTCTA ATCTCTACTA ATGAACTTAA ACTTTCTAAA GGTTTACAGG 660 TCGCTTTTTC CGAAACGACT CCTGTAATGT CTCCCCTTCA ATAGAAAAGG CTAGCAGGTG 720 CGGCAATTGA AGTGATTTTG CTTTCCAGTT TGACTCGTTG TGTTACTTTA AGATTCTTAA 780 GAGGACAACT CCTGTTCACT ATTTCTATAT TTTGATTTCG ACTAAGTTTA TGACCTTAAG 840 CTGTGCTTAA GTAACCGGAT GTGGTTTTTA AAATAATTAG TTAATCACTT ATACTATTTT 900 TTACTATTAC TTTTATTCAT AAATGGACTA AACCGAAGCG TAAGCCTGTG c GGTGGTTA 960 AATGTACTGC TTAACAATAT ATATTATCTG GTCAATAGGG TTAAC 100 & SEC. INFORMATION ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1569 base pairs (B) TYPE: nucleic acid (C) CHAIN: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: TTATAATGTA TATCTATATC CTGTTCGGGC GTAAAAGTAT GACTTTCCAT TTGAAGATAA 60 TAATATCACC ATAGGTTGCA AGTGGCGAAG GTCGTATCGT CTTTAATGCA CAAAAACGTA 120 TACAATACGA CTAGTAACAT ACGAATGATG GTAAAAAGAA ACGAAGCGGA ACGGAAGAAA 160 CTGCAAAAAA ACTTCGTTTT TTTTTCAGTT CTGTCTACAC GAATGTTTTC GTACATTCCG 240 AGTAAAAGTT TCTTCGATGA TTATCTTTCT CTTGTTTCTC AAATGCTCAG ACCTTTTAGT 300 TACCTCCGGT TCTATCTACT CGACTAGTTA TTACTAGGAC AAACCAGGTC GGTTTTACTT 360 TCGAACTAAA GTTTTGGAAT ATTAGTGTAG GAAAACTTTG GACCGTTCTT GAAATCTGAT 420 TTAAATTATC AACTTTAATT GTCTCAATAC TTAAACGGGT TTCTGGTCGA CCGGTATCAA 480 AGCCTTTAAC AACTCGAGAA CGTATTAAGG TCGGAAAATT AGCTGCTATA TCTTCTAT A 540 CGAGGGAACT CTTCCCCTGT CTGGTGAAGA CTGAATTAGA AGCCACATGG GAGGTGATAT 600 TTGTGGCGTT TAATATACAT AAAGTCTCGG TACGTTGAAC ATAGCGTCGA TTGGTGTTTT 660 CTCGGAAACA TAGTATTAAA CTAATCCT ?? ? AGTTGCTTC TTAACTAGTT AGATGTATCC 720 CCTGTTCCGA ACCTATATAT GACCTCTCTG AAAGACGGAC TTTAGTATGG ATGAGTCCTC 780 TACATAAACT TATACCAATA CTTATTTTGT CCGCCGGAAA AGTCTAATTG CAACTCTGAG 840 TACCTTCGCG ACACAGGAAG GAGTGTGGTC CCGGTAAOCA ACCAAGGAAA GTATTTAGAA 900 GACCCATAAT AAATAGTCTA ATCTCTACTA ATGAACTTAA ACTTTCTAAA GGTTTACAGG 960 TCGCTTTTTC CGAAACGACT CCTGTAATGT CTCCCCTTCA ATAGAAAAGG GTAGCAGGTG 1020 CGGGAATTGA AGTGATTTTG CTTTCCAGTT TCACTCGTTG TGTTACTTTA AGATTCTTAA 1080 GAGGACAACT CCTGTTCACT ATTTCTATAT TTTGATTTCG ACTAAGTTTA TGACCTTAAG 1140 CTGTGGTTAA GTAACCGGAT GTGGTTTTTA AAATAATTAG TTAATCACTT ATACTATTTT 1200 TTACTATTAC TTTTATTCAT AAATGGACTA AACCGAAGCC TAAGGCTCTG GCGGTGGTTA 1260 AATGTACTGC TTAACAATAT ATATTATCTG GTGAATAGGC TTAACACTTT ATTTAACTAG_1320_TT? CTTt ?? T C? CCTCCTTC TATCACTCTT Titt r ^? A GAGAG AGGA GAAAGCGTAG J.380 ATATGTATGC TAAAGTATAT ATGCAAAGTA ACGTAGTAGA AAACTATATA GAGTTTTTCT 1440 AGAGAATCAA GCGTTTATCA GTTTAGAAGT TTAAATATCG GAAATATAAA AAACGTGCTA 1500 AAGACTTTGA GGAAAAATAG TCGTGGCAAT TACGATCGCC AATGACAGTT TAGCGGCCAT 1560 TTAAGCGCT 1569

Claims (42)

1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a yeast geranylgeranyl diphosphate synthase protein.

2. An isolated nucleic acid molecule as described in claim 1, wherein the nucleic acid sequence encodes a geranylgeranyl diphosphate synthase protein of Saccharomyces.

3. An isolated nucleic acid molecule as described in claim 1, wherein the nucleic acid sequence encodes a geranylgeranyl diphosphate synthase protein of Saccharomyces cerevisiae.

4. An isolated nucleic acid molecule as described in claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 1.

5. A recombinant molecule comprising a nucleic acid molecule as described in claim 1, operably linked to a transcription control sequence.

6. A recombinant cell comprising a nucleic acid molecule as described in claim 1, wherein said cell is capable of expressing this nucleic acid molecule.

7. An isolated nucleic acid molecule encoding a geranylgeranyl diphosphate synthase, wherein the nucleic acid molecule has a sequence that is more than about 35 percent similar to the nucleic acid sequence of SEQ ID NO: l , wherein SEQ ID NO: l encodes a geranylgeranyl diphosphate synthase of Saccharomyces cerevisia.

8. An isolated nucleic acid molecule as described in claim 1, wherein said nucleic acid molecule has a sequence that is more than about 50 percent similar to the nucleic acid sequence of SEQ ID NO: 1.

9. An isolated nucleic acid molecule as described in claim 7, wherein said nucleic acid molecule has a sequence that is more than about 75 percent similar to the nucleic acid sequence of SEQ ID NO: 1.

10. An isolated nucleic acid molecule as described in claim 7, wherein said nucleic acid molecule has a sequence that is more than about 90 percent similar to the nucleic acid sequence of SEQ ID NO: 1.

11. An isolated nucleic acid molecule as described in claim 7, wherein said nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 1.

12. A recombinant molecule comprising a nucleic acid molecule as described in claim 7, operably linked to a transcription control sequence.

13. A recombinant cell comprising a nucleic acid molecule as described in claim 7, said cell being capable of expressing that nucleic acid molecule.

14. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, insect cells, animal cells and plant cells. .

15. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein said cell is a yeast cell.

16. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein said cell is Saccharomyces.

17. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein the cell is Saccharomyces cerevisia.

18. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein said cell is a bacterial cell.

19. A recombinant cell comprising a nucleic acid molecule as described in claim 13, wherein this cell is Escherichia coli.

20. Nucleic acid molecule nGGPPS1005 *

21. Nucleic acid molecule nGGPPS1600.

22. An isolated protein comprising a yeast geranylgeranyl diphosphate synthase protein.

23. An isolated protein as described in claim 22, where the yeast is Saccharomyces.

24. An isolated protein as described in claim 22, wherein the yeast is Saccharomyces cerevisia.

25. An isolated protein as described in claim 22, wherein the protein is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1.

26. An isolated protein as described in claim 22, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2.

27. An isolated geranylgeranyl diphosphate synthase protein, wherein said protein comprises a sequence of amino acid which is at least about 45 percent similar to an amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is an amino acid sequence of geranylgeranyl diphosphate synthase of Saccharomyces cerevisiae.

28. An isolated geranylgeranyl diphosphate synthase protein as described in the claim 27, wherein this protein has a sequence that is more than about 55 percent similar to the amino acid sequence of SEQ ID NO: 2.

29. An isolated geranylgeranyl diphosphate synthase protein as described in the claim 27, wherein this protein has a sequence that is more than about 75 percent similar to the amino acid sequence of SEQ ID NO: 2.

30. An isolated geranylgeranyl diphosphate synthase protein as described in the claim 27, wherein this protein has a sequence that is more than about 90 percent similar to the amino acid sequence of SEQ ID NO: 2.

31. An isolated geranylgeranyl diphosphate synthase protein as described in claim 27, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2.

32. A geranylgeranyl diphosphate synthase protein isolated as described in claim 27, wherein this protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions in a geranylgeranyl diphosphate synthase gene of Saccharomyces cerevisiae.

33. An isolated geranylgeranyl diphosphate synthase protein as described in the claim 32, wherein this gene of geranylgeranyl diphosphate synthase of Saccharomyces cerevisiae comprises the nucleic acid sequence of SEQ ID NO: 1.

34. The protein GGPPS3

35. 35. A method for producing geranylgeranyl diphosphate, which comprises culturing a recombinant cell, wherein said recombinant cell comprises an isolated nucleic acid molecule encoding a yeast geranylgeranyl diphosphate synthase protein.

36. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein said cell is selected from the group consisting of bacterial cells, fungal cells, insect cells, animal cells and plant cells.

37. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein said cell is a yeast cell.

38. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein this cell is Saccharomyces.

39. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein this cell is Saccharomyces cerevisiae.

40. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein said cell is a bacterial cell.

41. A method for producing geranylgeranyl diphosphate as described in claim 35, wherein this cell is Escherichia coli.

42. A method for producing farnesyl diphosphate, which comprises culturing a recombinant cell, wherein this recombinant cell has a reduced ability to produce geranylgeranyl diphosphate synthase, whereby, this recombinant cell produces farnesyl diphosphate.