# Patent

## Title
Compositions and methods using the yeast YLR110C promoter

## Abstract

The invention provides novel yeast promoters useful for controlling the expression of homologous and heterologous nucleic acid molecules in yeast cells. The yeast promoters are induced by a fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both. Therefore, expression of nucleic acid molecules encoding a polypeptide under the control of the novel yeast promoters may be regulated by varying the level of a fermentable carbon source, or a non-fermentable carbon source, or both.

## Background
This application is a continuation of application Ser. No. 10/776,213, filed Feb. 12, 2004, which is a continuation of application Ser. No. 09/743,194, filed Jan. 8, 2001, now U.S. Pat. No. 6,716,601 which is the National Stage of International Application No. PCT/SE00/02277, filed Nov. 17, 2000 the entire content of which is hereby incorporated by reference in this application.


BACKGROUND OF THE INVENTION
The controlled production in yeast of an enormous variety of useful proteins or polypeptides can be achieved using recombinant DNA technology. Yeast cells can be transformed with yeast expression vectors, which contain homologous or heterologous nucleic acid molecules encoding polypeptides (coding sequences). The yeast cells can then produce large quantities of the useful proteins or polypeptides in yeast cell culture.
Expression of the nucleic acid molecule encoding a polypeptide by the yeast expression vector is initiated at a region known as the promoter, which is recognized by and bound by RNA polymerase. The RNA polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5′ to 3′ end into messenger RNA, which is in turn translated into a polypeptide having the amino acid sequence for which the DNA codes. The present invention provides novel yeast promoters useful for, inter alia, controlling the expression of homologous and heterologous nucleic acid sequences encoding proteins and polypeptides in yeast cells.
SUMMARY OF THE INVENTION
It is an object of the invention to provide novel yeast promoters, yeast expression vectors, and transformed yeast cells. It is a further object of the invention to provide a method for producing proteins and polypeptides in yeast cell culture.
In one embodiment of the invention a yeast promoter which comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide is provided. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule encoding a polypeptide.
As used herein, the term “promoter” refers to a nucleic acid sequence which is cable of initiating transcription of a nucleic acid molecule encoding a polypeptide (coding sequence); a “yeast promoter” is capable of initiating transcript of a coding sequence in yeast cells; and “promoter activity” refers to the level or amount of transcription initiation of a coding sequence, and encompasses any level above background (i.e., the level or amount that occurs in the absence of a promoter; a background level, which is normally zero).
Another embodiment of the invention provides a yeast promoter which comprises an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule encoding a polypeptide.
Yet another embodiment of the invention provides a yeast promoter fragment which comprises at least 17 contiguous nucleotides of a polynucleotide. The polynucleotides are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The fragment has promoter activity as determined by cloning the fragment into a yeast expression vector, wherein the fragment is operably linked to a reporter gene, transforming yeast cells with the yeast expression vector, growing the yeast cells in yeast cell culture under conditions favorable for expression of the reporter gene, and assaying the yeast culture for a reporter protein expressed by the reporter gene. The expression of the reporter gene indicates the fragment has promoter activity.
Still another embodiment of the invention provides a yeast expression vector comprising a yeast promoter. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule encoding a polypeptide.
A further embodiment of the invention provides a yeast expression vector where activity of the promoter is controlled by varying the level of a non-fermentable carbon source, such as ethanol, in a medium of yeast cells in culture. The yeast cells are transformed with said yeast expression vector.
In yet another embodiment of the invention, a yeast expression vector comprising a yeast promoter which comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide is provided. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4. Promoter activity is controlled by varying the level of a fermentable carbon source in a medium of yeast cells in culture, where the yeast cells are transformed with the yeast expression vector. The fermentable carbon source can be glucose.
Another embodiment of the invention provides a yeast expression vector comprising a yeast promoter. The yeast promoter comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4. Promoter activity is controlled by varying the level of a fermentable carbon source and a non-fermentable carbon source, such as ethanol, in a medium of yeast cells in culture, where the yeast cells are transformed with the yeast expression vector. The fermentable carbon source can be glucose. The non-fermentable carbon source can be ethanol.
Still another embodiment of the invention provides a yeast cell transformed with a yeast expression vector. The yeast expression vector comprises a yeast promoter. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule encoding a polypeptide.
Yet another embodiment of the invention provides a method for producing a polypeptide. A yeast expression vector is constructed where a polynucleotide encoding the polypeptide is controlled by a yeast promoter. The yeast promoter comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule encoding a polypeptide. A culture of yeast cells is transformed with the yeast expression vector. The yeast cells are maintained in culture so that the polypeptide is expressed. The polypeptide is then recovered.
Still another embodiment of the invention provides a method for producing a polypeptide. A nucleic acid molecule encoding the polypeptide is cloned into an expression vector selected from the group consisting of pYLR110P+luc, pYMR251AP+luc, pYMR107P+luc, pZEO1P+luc, pYLR110P, pYMR251AP, pYMR107P, and pZEO1P. The nucleotide acid molecule is operably linked to a promoter of the expression vector. A culture of yeast cells is transformed with the yeast expression vector. The yeast cells are maintained in culture so that the polypeptide is expressed and the polypeptide is then recovered.
Another embodiment of the invention provides a method for producing a polypeptide. A yeast expression vector is constructed where a nucleic acid molecule encoding the polypeptide is controlled by a yeast promoter. The yeast promoter comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4. Yeast cells are transformed with the yeast expression vector and are maintained in culture medium. The expression of the nucleic acid molecule encoding the polypeptide is controlled by varying the level of a fermentable carbon source, such as glucose, in the culture medium. The polypeptide is then recovered.
Still another embodiment of the invention provides a method for producing a polypeptide. A yeast expression vector is constructed where a nucleic acid molecule encoding the polypeptide is controlled by a yeast promoter. The yeast promoter comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The promoter is operative when operably linked to a nucleic acid molecule. A culture of yeast cells is transformed with the yeast expression vector. The yeast cells are maintained in culture medium and the expression of the nucleic acid molecule encoding the polypeptide is controlled by varying the level of a non-fermentable carbons source, such as ethanol, in the culture medium. The polypeptide is then recovered.
Another embodiment of the invention provides a method for producing a polypeptide. A yeast expression vector is constructed where a nucleic acid molecule encoding the polypeptide is controlled by a yeast promoter. The yeast promoter comprises at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The promoter sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4. A culture of yeast cells is transformed with the yeast expression vector. The yeast cells are maintained in culture medium and the expression of the nucleic acid encoding the polypeptide is controlled by varying the level of a fermentable carbon source, such as glucose, and a non-fermentable carbon source, such as ethanol, in the culture medium. The polypeptide is then recovered.
Yet another embodiment of the invention provides a method of identifying a promoter fragment with promoter activity by generating a fragment comprising at least 17 contiguous nucleotides of an isolated and purified polynucleotide. The polynucleotides are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The fragment is cloned into a yeast expression vector, so that the fragment is operably linked to a reporter gene. Yeast cells are transformed with the yeast expression vector and grown in yeast cell culture under conditions favorable for expression of the reporter gene. The yeast culture is assayed for a reporter protein expressed by the reporter gene. Expression of the reporter gene indicates the fragment has promoter activity.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a map of YEp13 expression vector.
FIG. 2 schematically illustrates construction of YLR110C and YMR251WA promoter constructs.
FIG. 3 is a map of pPRB1P.
FIG. 4 is a map of pPRB1P+luc.
FIG. 5 is a map of pYLR110P+luc.
FIG. 6 is a is a map of pYMR251AP+luc.
FIG. 7 is a map of pYMR107P+luc.
FIG. 8 is a map of pZEO1P+luc.
FIG. 9 is a map pYLR110P.
FIG. 10 is a map of pYMR251AP.
FIG. 11 is a map of pYMR107P.
FIG. 12 is a map of pZEO1P.
FIG. 13 schematically illustrates the YLR110C promoter region.
FIG. 14 schematically illustrates the YMR251WA promoter region.
FIG. 15 schematically illustrates the YMR107W promoter region.
FIG. 16 schematically illustrates the ZEO1 promoter region.



DETAILED DESCRIPTION OF THE INVENTION
Novel yeast promoters whose activity can be controlled by a fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both have been identified. The yeast promoters are useful for, inter alia, the high level production of proteins or polypeptides in yeast cell culture.
Yeast Promoters
The isolated and purified promoter polynucleotides of the invention are shown in SEQ ID NO:1 (the YLR110C promoter), SEQ ID NO:2 (the YMR251WA promoter), SEQ ID NO:3 (the YMR107W promoter), and SEQ ID NO:4 (the ZEO1 promoter). Yeast promoters comprising as little as 17 nucleic acids have been determined to function as promoters. The yeast promoters of the invention comprise at least 17, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 or 700 contiguous nucleic acids of an isolated and purified polynucleotide up to the maximum length provided in any one of the sequences presented herein, that is, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
Preferably, the promoter polynucleotides are isolated free of other components, such as proteins and lipids. The polynucleotides can be made by a cell and isolated or can be synthesized in the laboratory, for example, using an automatic synthesizer or an amplification method such as PCR.
Naturally occurring variants and artificial sequence variants (that is, those which do not occur in nature) of the promoters are included in the invention. Variants of the promoters and/or fragments thereof have, along their entire length, sequence identity of at least 90%, and preferably greater than 95% as determined by the Smith-Waterman homology search algorithm as implemented in MPsrchTM program (University of Edinburgh) using an affine gap search with the following search parameters: gap open penalty: 12, gap extension penalty: 1.
Fragments of the full-length promoters are also functional as promoters. A promoter fragment of at least 17 contiguous nucleotides may occur at any position along the full-length promoter as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. Accordingly, promoter activity of 17 or more contiguous nucleotides occurring anywhere along the full-length promoter can be analyzed. Fragments of 17, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700, nucleotides of the promoters may be constructed by, for example, subjecting an isolated promoter to restriction endonucleases, to 5′- or 3′-deletion mutagenesis, to PCR, or to site specific deletion. A combination of these methods can also be used to generate fragments of a promoter.
The invention further embodies a hybrid promoter, i.e., a promoter that comprises more than one promoter or more than one fragment of a promoter from which it was derived. The promoter fragments can be derived from more than one of the promoter sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The promoters and fragments can be constructed as described above, ligated together, and cloned into a yeast expression vector. Where a promoter comprises nucleotides from at least two polynucleotides selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, at least 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous nucleotides are derived from each of the polynucleotides to form a promoter of at least 17 nucleotides. Alternatively, each of the full-length promoters can be combined with another full-length promoter or with fragments of another promoter.
The yeast promoters, fragments of the promoters, and hybrid promoters are useful for controlling expression of a protein or polypeptide when the yeast promoter is operably linked to a nucleic acid molecule encoding the protein or polypeptide.
Determination of Promoter Activity
Promoters and fragments of promoters can be assayed for promoter activity by cloning a fragment of a promoter, or a full-length promoter, or a hybrid promoter into a yeast expression vector so that is operably linked to a reporter gene, i.e., a coding sequence for a reporter protein. The yeast expression vector is transformed in yeast cells, which are grown in yeast cell culture under conditions favorable for expression of the reporter gene, for example, under conditions providing a fermentable and/or non-fermentable carbon source. Expression of the reporter gene, as determined by an assay for the amount of a reporter protein expressed by the reporter gene, indicates that the promoter has activity.
For example, to determine if a promoter has activity, i.e. is operative, expression of a reporter gene by a promoter of the invention may be compared to expression of the reporter gene by a reference promoter such as PBR1 (Cottingham et al. (1991) Eur J Biochem 196(2):431–8; Sleep et al. (1991) Biotechnology 9(2):183–7; Finnis et al. (1992) Yeast 8(1):57–60; Meldgaard et al. (1995) Glycoconj J 12(3):380–90; Bach et al. (1996) Receptors and Channels 4(2):129–39. A promoter, a fragment of a promoter, or a hybrid promoter of the invention is operative if it expresses at least 25% of the amount of a reporter protein as the full-length PBR1 promoter in a medium containing a non-fermentable carbon source, or a fermentable carbon source, or both. Preferably, an operative promoter expresses at least 50%, 75%, 100%, 200%, 300%, 400%, or more of the amount of a reporter protein as the full-length PBR1 reference promoter.
Assays for promoter activity are useful for identifying yeast promoters with high activity and the specific nucleotide sequences of the promoters that are necessary for promoter activity.
Yeast Expression Vectors
The yeast promoters of the invention, which comprise isolated and purified polynucleotides selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 or fragments thereof, can be used to construct yeast expression vectors.
Yeast expression vectors are any vectors capable of autonomous replication within a yeast host organism or capable of integrating into the yeast genome. Yeast expression vectors are useful for introducing foreign DNA into yeast cells. Typical yeast expression vectors include yeast integrative plasmids (YIp), yeast replicating plasmids (YRp), yeast expression plasmids (YXp), yeast centromere-containing plasmids (YCp), and yeast episomal plasmids (YEp). Preferably, a yeast expression vector can be selected and maintained in both yeast and E. coli. 
Yeast expression vectors, typically plasmids, incorporate the yeast promoters of the invention to control expression of nucleic acid molecules encoding heterologous or homologous proteins or polypeptides. The nucleic acid molecules are operably linked to a promoter in the yeast expression vector. A wide range of heterologous eukaryotic and prokaryotic proteins or peptides may be expressed by the vectors of the invention.
Expression vectors incorporating the promoters can be constructed by inserting into a vector a nucleic acid molecule encoding a protein or polypeptide (coding sequence) which is to be expressed. The coding sequence can be inserted at a restriction site which is provided downstream of a translation start codon controlled by the promoter. The coding sequence must be inserted in the correct translational reading frame.
Alternatively, the polynucleotide can itself be provided with a translational start codon followed directly by a coding sequence. Where the promoter does not contain a translational start codon, a restriction site is provided so that the coding sequence can be inserted in the correct reading fame and so that its translational start codon is correctly positioned in relation to the promoter. The coding sequence can encode heterologous or homologous or eukaryotic or prokaryotic polypeptides or proteins. In a preferred embodiment the coding sequence encodes a fusion protein. The coding sequence may further comprise a signal sequence.
In addition to the promoters of the invention, other components can be added to the expression vectors of the invention. For example, yeast selective markers, such as LEU2 or TRP1, which allow for selection of yeast cells that have been effectively transformed by the vector can be added. A yeast replication origin, such as the replication origin of the 2-micron plasmid or the autonomous ARS replication segment can be added. Upstream activating sequences and transcription terminator sequences may be added. Further, at least a portion of a bacterial plasmid, such as found in YEp13, can be added to enable the yeast expression vector to be manipulated in an intermediate bacterial host system, such as Escherichia coli. 
The expression vector may also comprise a reporter gene which encodes, for example, β-galactosidase or luciferase. The reporter gene can be under the control of a promoter of the invention. Where the reporter gene, i.e., coding sequence, is linked to a gene encoding a desired protein, assaying the level of expression of the reporter protein can quickly and easily determine the level of expression of the desired protein.
The expression vectors of the invention can be used to direct the fermentable carbon source- and/or non-fermentable carbon source-induced high level expression of proteins or polypeptides in yeast. The promoters of the invention can be induced by the presence of a fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both. That is, the promoters have greater promoter activity in the presence of a fermentable carbon source, or a non-fermentable carbon source, or both than in the absence of a fermentable carbon source, or a non-fermentable carbon source, or both. Promoters YLR110C, as shown in SEQ ID NO:1; YMR251WA, as shown in SEQ ID NO:2; and ZEO1, as shown in SEQ ID NO:4, can be induced by a fermentable carbon source, such as glucose, or by a non-fermentable carbon source, such as ethanol, or by both. Promoter YMR107W, as shown in SEQ ID NO:3, can be induced by a non-fermentable carbon source, such as ethanol. Thus, the amount of expression of a homologous or heterologous nucleic acid molecule encoding a protein operably linked to the promoters of the invention can be controlled by varying the amount of an available fermentable carbon source, such as glucose, or a non-fermentable carbon source, such as ethanol, or both.
Transformed Yeast Cells
Yeast cells can be transformed with the yeast expression vectors of the invention. Transformation can be accomplished by well known methods, including, but not limited to electroporation, calcium phosphate precipitation, and microinjection. The yeast expression vectors of the invention can be used to transform yeast cells, including, but not limited to Saccharomyces cerevisiae, S. uvarum, S. carlsbergensis, Saccharomycopsis lipolytica, Schizosaccharomyces pombe, and Kluyveromyces lactis. 
Transformed yeast cells containing a yeast expression vector can be grown in an appropriate medium for the yeast. A fermentable or non-fermentable carbon source can be added to the yeast culture medium in order to control the activity of the promoter.
Methods of Production of Proteins
Yeast cells transformed with expression vectors comprising a promoter of the invention can be used to produce proteins and polypeptides. Under proper cell culture conditions, preferably in the presence of a fermentable or non-fermentable carbon source, or both, the promoters of the invention will control expression of a nucleic acid molecule encoding a polypeptide operably linked to the promoter.
The protein or polypeptide can be retained within the yeast cell. The yeast cells can be then harvested, lysed, and the protein obtained and substantially purified in accordance with conventional techniques. Such techniques include, but are not limited to chromatography, electrophoresis, extraction, and density gradient centrifugation.
In a preferred embodiment of the invention, the protein or polypeptide to be recovered will further comprise a signal peptide capable of transporting the protein or polypeptide through the membrane of a transformed yeast cell. The protein or polypeptide can be recovered from the culture medium by, for example, adsorption or precipitation.
Further, the proteins and polypeptides may be produced as a fusion protein, which includes not only the amino acid sequence of the desired protein, but also one or more additional proteins. Affinity purification protocols can be used to facilitate the isolation of fusion proteins. Typically, a ligand capable of binding with high specificity to an affinity matrix is chosen as the fusion partner for the desired protein. For example, fusion proteins made with glutathione-S-transferase can be selectively recovered on glutathione-agarose and IgG-Sepharose can be used to affinity purify fusion proteins containing staphylococcal protein A.
Preferably, the protein or polypeptide of interest can be separated from the remainder of the fusion protein. The fusion protein can be constructed so that a site for proteolytic or chemical cleavage is inserted between the protein of interest and the fusion partner. For example, sites for cleavage by collagenase, Factor Xa protease, thrombin, and enterokinase, have been inserted between the fusion partner and the protein of interest. The protein of interest can be also cleaved from the remainder of the fusion protein by chemical cleavage by, for example, hydroxylamine, cyanogen bromide (CNBr), or N-chlorosuccinamide.
The following are provided for exemplification purposes only and are not intended to limit the scope of the invention described in broad terms above. All references cited in this disclosure are incorporated by reference.
EXAMPLE 1
Preparation of Yeast Samples
S. cerevisiae Strain 11C
This example describes the growth of haploid Saccharomyces cerevisiae strain 11C. It has the genotype: ade2-161, trp1-Δ63, ura3-52, lys2-801, leu2Δ1 &/or leu2-3 &/or leu2-112, his3Δ200 &/or his4-519. 11C was generated by crossing the strains YPH500 (Mat a ura3-52 lys2-801 ade2-161 trp1-Δ63 his3Δ200 leu2Δ1) (Sikorski and Hieter. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27) and AH22 (MATa leu2-3 leu2-112 his4-519) (Hinnen et al. (1978) Transformation of yeast. Proc. Natl. Acad. Sci. USA 75: 1929–1933).
Three sterile 500 ml conical flasks, each containing 100 ml sterile YPD broth (Sigma, Cat No. Y-1375) were inoculated with sterile 10 μl loops of differing quantities of the S. cerevisiae strain 11C from a freshly streaked YPD plate (Sigma, Cat No. Y-1500), and grown in an orbital shaker at 30° C., 200 rpm, overnight. The growth of 11C in the three flasks was measured by absorbance at 600 nm. One flask was deemed to be at the late exponential growth phase (1.98 ODU ml at 600 nm), and this culture was used to inoculate (50 ml o/n culture per flask) 2 identical 5 L sterile conical flasks (labeled E and L), each containing IL sterile YPD broth to a final concentration of  ̃0.1 ODU ml. Flasks E and L were grown in an orbital shaker at 30° C., 200 rpm. 10 ml samples were collected at times indicated below (Table 1). The samples were treated as follows: their growth was determined (A600 nm), the possibility of contamination was checked (using a light microscope), cells were harvested in a benchtop centrifuge ( ̃2000×g for 5 minutes), and the supernatant removed and frozen at −20° C. (samples labeled E0–E3, and L0–L5).







TABLE 1







Growth of cultures E and L as measure by absorbance at 600 nm.










Time
Time after inoculation
Growth of flask E
Growth of flask L


Point
(min)
(ODU)
(ODU)













T0
0
0.099
0.099


T1
310
0.37
0.36


T2
410
0.71
0.72


T3
455
0.97
0.92


T4
775
—
3.64


T5
1420
—
6.05









After 455 minutes, a time deemed to be late exponential growth phase in glucose, flask E (i.e. early) was harvested ( ̃2000×g for 5 minutes), split into 50 ml aliquots, and frozen at −80° C. After 1420 minutes, a time deemed to be growth on ethanol, flask L (i.e. late) was harvested ( ̃2000×g for 5 minutes), split into 50 ml aliquots, and frozen at −80° C.
Determination of Glucose and Ethanol Concentration
Supernatant samples (E0–E3, and L0–L5) were defrosted, and their ethanol and glucose contents were measured using ethanol (Boehringer, Cat. No. 176290) and glucose (Boehringer, Cat. No. 176251) detection kits according to manufacturers instructions. The concentrations determined are shown below in Table 2.







TABLE 2







Glucose and Ethanol concentrations in supernatants of cultures E and L at


different time points.











Time after
Glucose level in
Ethanol level in


Sample
inoculation (min)
media (g L−1)
media (g L−1)













E0
0
20.0
0.0


E1
310
21.8
0.3


E2
410
21.8
0.8


E3
455
21.2
0.87


L0
0
20.0
0.0


L1
310
22.2
0.36


L2
410
22.0
0.62


L3
455
20.0
0.87


L4
775
11.8
5.2


L5
1420
0.0
11.8









It can seen in Table 2 that at the point of culture harvest for E (E3, 455 minutes), the cells were still utilizing glucose as a carbon source, while at the point of culture harvest for L (L5, 1420 minutes), glucose was exhausted, and the cells were utilizing ethanol as a carbon source. Calibration values used to calculate glucose concentrations are shown in Table 3. Calibration values used to calculate ethanol concentrations are shown in Table 4.







TABLE 3







Glucose standards










GLUCOSE STANDARDS g/l
OD A340














0
0



0.2
0.246



0.4
0.461



0.6
0.726



0.8
0.967



1
1.227

















TABLE 4







Ethanol standards










ETHANOL STANDARDS g/L
OD A340














4.72
0.041



9.44
0.083



18.88
0.166



37.76
0.322



56.6
0.534



75.5
0.664



94.4
0.846










EXAMPLE 2
Analysis of RNA Levels from Yeast Dimorphic Growth Samples
Total RNA Isolation
Total RNA was isolated from 300 ml of culture using the hot phenol protocol. The frozen yeast pellets were resuspended in lysis buffer (4 ml) (0.5 ml Tris-CL (1M, pH 7.5), 1.0 ml EDTA (0.5 M), 2.5 ml 10% SDS, and 46.0 ml ddH2O) and an equal volume of acid phenol was added and vortexed. Following incubation at 65° C. for one hour (with occasional vigorous vortexing) the mixture was placed on ice for 10 minutes then centrifuged (10 minutes). The aqueous layer was transferred to a fresh centrifuge tube and mixed with an equal volume of phenol at room temperature. The mixture was centrifuged and an equal volume of chloroform was mixed with the aqueous layer in a fresh centrifuge tube. Following centrifugation the aqueous layer was transferred to a fresh centrifuge tube and sodium acetate (to a final concentration of 0.3M) and two volumes of 100% ethanol was added to precipitate the RNA. The mixture was placed at −20° C. for 30 minutes then centrifuged for 10 minutes to pellet the RNA. The RNA pellet was washed 2–3 times with 70% ethanol then allowed to dry at room temperature. The pellet was resuspended in ddH2O (200–500 μL). The RNA was quantitated by measuring OD 260–280. Yield of total RNA was  ̃4.5 mg from each culture.
Poly A+ RNA Purification
Poly A+ RNA was purified from total RNA using Qiagen Oligotex mRNA Midi Kit (Qiagen, Cat. No. 70042). 2 mg of total RNA was used as starting material and made up to a volume of 500%1 with DEPC treated H2O. To this 500 μl buffer OBB (2× binding buffer) and 55 μl oligotex suspension was added. The “Ologotex mRNA Spin Column Protocol” from the kit protocol booklet was followed. The pelleted mRNA was washed in 200 μl 75% ethanol, dried and resuspended in 10 μl DEPC treated H2O. Yield of Poly A+ RNA was  ̃8 μg for each sample.
cDNA Synthesis
cDNA was synthesized using the protocol for GeneChip Expression Analysis Manual using reagents from Gibco BRL Life Technologies Superscript Choice System cat. No. 18090-019. For each sample 5 μg Poly A+ RNA was added to 100 pmol of T7-(dT)24 primer (sequence: GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24, HPLC purified) (SEQ ID NO:15) in a total of 8 μl (made up to volume with DEPC treated H2O). The reaction mixture was incubated for 10 minutes at 70° C. in a Perkin Elmer PE9600 thermalcycler then put on ice. The following reagents were added to the reaction mixture: 4 μl 5× first strand cDNA buffer; 2 μl 0.1M DTT; and 1 μl 10 mM dNTP mix. The reaction mixture was mixed and incubated at 37° C. for 2 minutes in a Perkin Elmer PE9600 thermocycler. 5 μl SuperScript II reverse transcriptase was then added. The mixture was incubated at 37° C. for 1 hour in a Perkin Elmer PE9600 thermocycler.
The first strand cDNA reaction was placed on ice and the following reagents added: 91 μl DEPC treated H2O; 30 μl 5× second strand reaction buffer; 3 μl 10 mM dNTP mix; 1 μl 10 units/μl E. coli DNA ligase; 4 μl 10 units/μl E. coli DNA Polymerase I; and 1 μl 2 units/μl RNase H. The mixture was incubated at 16° C. for 2 hours in a Perkin Elmer PE9600 thermalcycler. 2 μl 5 units/μl T4 DNA Polymerase was then added. The mixture was incubated for a further 5 minutes at 16° C. in a Perkin Elmer PE9600 thermalcycler. 10 μl 0.5M EDTA was then added.
The double stranded DNA was cleaned up by phenol extraction. The reaction product transferred to a 1.5 ml eppendorf tube and 162 μl Tris pH 8.0 saturated phenol was added. The tube was mixed by vortexing, the tube was then centrifuged in a microfuge at 13,000 rpm for 5 minutes. The top fraction was recovered and cDNA precipitated by addition of 60 μl 7.5M ammonium acetate plus 400 μl absolute ethanol. This was immediately centrifuged in a microfuge at 13,000 rpm for 20 minutes. The supernatant fraction was discarded, the pellet was washed in 75% ethanol and then air-dried. The pellet was resuspended in 20 μl DEPC treated H2O
Synthesis of Biotin-Labeled cRNA by In Vitro Transcription (IVT)
Reagents from Ambion MEGAscript T7 kit, cat. No. 1334, were used for the synthesis of biotin-labeled cRNA by in vitro transcription (IVT). The NTP Labeling mix comprised 7.5 mM ATP; 7.5 mM GTP; 5.625 mM UTP; 1.875 mM Biotin-16-UTP (Enzo cat No. 42814); 5.625 mM CTP; and 1.875 mM Biotin-11-CTP (Enzo cat No. 42818). The IVT Labeling reaction comprised: 14.5 μl NTP Labeling mix; 2 μl 10× Ambion Transcription Buffer; 1.5 μl Double strand cDNA (from above); and 2 μl Ambion T7 Enzyme Mix.
The reaction mixture was incubated for 6 hours at 37° C. in a Perkin Elmer PE9600 thermalcycler. The biotinylated cRNA was cleaned up using Qiagen RNeasy kit, cat No. 74103. The RNeasy kit protocol was followed exactly. RNA was eluted in 2 aliquots of 30 μl DEPC treated H2O. The RNA was precipitated by addition of 6 μl 3M sodium acetate pH 5.5 plus 75 μl absolute ethanol. The RNA was allowed to precipitate overnight at −20° C. Samples were centrifuged in a microfuge at 13,000 rpm for 20 minutes to pellet the RNA. The supernatant fraction was discarded and the pellet was washed in 1 ml of 75% ethanol and then allowed to air dry. The pellet was then resuspended in 20 μl DEPC treated H2O. The yield of cRNA was  ̃40 μg for each sample.
cRNA Fragmentation
11 μg of cRNA was fragmented. 8 μl of 5× Fragmentation buffer (200 mM Tris-Acetate pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) plus 11 μg cRNA made up to 20 μl with DEPC treated H2O was used. The reaction mixture was incubated 94° C. for 35 minutes in a Perkin Elmer PE9600 thermal cycler.
Hybridization to GeneChip Microarray
The hybridization mix comprised: 20 μl (11 μg) of fragmented cRNA; 2.2 μl of control oligo B2 (50 pmol/μl) (sequence: 5′ Biotin-GTCAAGATGCTACCGTTCAG 3′ HPLC purified) (SEQ ID NO:16); 2.2 μl Herring Sperm DNA (10 mg/ml); 110 μl 2× Buffer (2M NaCl, 20 mM Tris pH 7.6, 0.01% Triton X-100); and 85.6 μl DEPC treated H2O. The hybridization mix heated to 95° C. in a Techne hot block for 5 minutes, followed by incubation at 40° C. for 5 minutes. The hybridization mix was clarified by centrifugation in microfuge at 13,000 rpm for 5 minutes.
200 μl of supernatant to added to the GeneChip cartridge (GeneChip cartridge was previously pre-wetted with 200 μl 1× Buffer and incubated for 10 minutes at 40° C. in the rotisserie box of a GeneChip hybridization oven 320 (cat No. 800127) at maximum rpm. The sample was hybridized to the microarray overnight at 40° C. in a GeneChip hybridization oven in the rotisserie at maximum rpm.
Washing and Staining of Probe Arrays
The hybridization mix was recovered from the GeneChip cartridge and put back in the tube containing the remainder of the sample. 200 μl 6× SSPE-T (6× SSPE plus 0.005% Triton X-100) was applied to the chip and pipetted in and out twice. This process was repeated twice more. Another 200 μl 6× SSPE-T was applied to the cartridge and the cartridge was then incubated for 1 hour at 50° C. at maximum rpm in the GeneChip hybridization oven. The 6× SSPE-T was removed and 200 μl 0.5× SSPE-T was added to cartridge. The cartridge was incubated for 15 minutes at 50° C. at maximum rpm in the GeneChip hybridization oven. The 0.5× SSPE-T was removed and the cartridge was re-filled with 200 μl 6× SSPE-T.
The stain solution comprised: 190 μl 6× SSPE-T; 10 μl of 20 mg/ml acetylated BSA; and 2 μl 1 mg/ml conjugated streptavidin:phycoerythrin (Molecular Probes cat. No. S-866). 200 μl 6× SSPE-T was removed from the GeneChip cartridge and 200 μl of stain solution added. The cartridge was incubated at ambient temperature in a GeneChip hybridization oven at maximum rpm in the rotisserie for 10 minutes. The stain solution was removed and the cartridge was washed by adding 200 μl 6× SSPE-T and pipetting this in and out of the cartridge twice. This process was repeated six times. The cartridges were then completely filled with 6× SSPE-T and any bubbles removed. Hybridization, washing and staining was repeated using the same hybridization mixes until both samples had been hybridized to each of the four yeast chip sub-set arrays.
Data Collection
Data was collected by scanning the hybridized chips on a Hewlett-Packard GeneArray scanner. A “halo” effect (appearance of stain non-specifically across the array image) was seen on one of the scanned images: yeast growing in glucose rich media, sub-set C array. Scanning of this array was aborted after one scan and the chip was washed twice with 200 μl 6× SSPE-T and then re-filled as before. This array was then re-scanned three times and the data collected was the average of these three scans. All other arrays were scanned four times without problems and the data collected was the average of the four scans.
EXAMPLE 3
Isolation of Promoters and Construction of Expression Vectors.
PCR Amplification of Promoter Regions from Genomic DNA
Based on the Saccharomyces cerevisiae genomic sequence in the GenEMBL nucleotide database oligonucleotide primers were designed to amplify the genomic sequence 5′ to the following ORFs: YLR110C (Johnston et al. (1997) Nature 1997 May 29;387(6632 Suppl):87–90), YMR251WA (common name HOR7) (Bowman et al. (1997) Nature May 29;387(6632 Suppl):90–3), YMR107W (Bowman et al. (1997) Nature May 29;387(6632 Suppl):90–3), and YOL109W (common name ZEO1) (Dujon et al. (1997) Nature May 29;387(6632 Suppl):98–102). The region amplified was the non-coding region separating the selected ORF and the next predicted Saccharomyces cerevisiae ORF in the 5′ direction, with a minimum length of 500 bp.
Sequence of Oligonucleotide Primers Used to Amplify Promoter DNA
HindIII, NheI and NdeI cloning sites underlined.
YLR110C-F ATGCAAGCTTCGCGGCCGCCGTCTGATTTCCGTTT SEQ ID NO:5
YLR110C-R CCAGGCCGCATATGTCATATAGTGTTTAAG SEQ ID NO:6
YMR251WA-F AGCTAAGCTTCGCGGCCGCCTTTCGATTAGCACGCAC SEQ ID NO:7
YMR251WA-R AGATACCTTCATATGTTATTATTAGTC SEQ ID NO: 8
YMR107W-F AGCTAAGCTTCGCGGCCGCGCAGAAATGATGAAGG SEQ ID NO:9
YMR107W-R ATCCATCCCATATGTGATATCTCGATTAG SEQ ID NO:10
ZEO1-F AGCTAAGCTTCGCGGCCGCGGAGGTCTGCTTCACG SEQ ID NO:11
ZEO1-R TACGATCGCATATGTAATTGATATAAACG SEQ ID NO:12
PCR reactions were set up for each primer pair as follows: For YMR251WA and ZEO190 μl of Reddy-Load PCR (1.1×) mix, 3.5 mM MgCl2, (Advanced Biotechnologies, cat.no. AB-0628); 2 μl of forward primer (100 μM); 2 μl of reverse primer (100 μM); 1 μl of S. cerevisiae genomic DNA (Promega G310A, lot 8347702, 276 μg/ml); and 5 μl of H2O were combined.
For YLR110C and YMR107W 90 μl of Reddy-Load PCR (1.1×) mix, 1.5 mM MgCl2, (Advanced Biotechnologies, cat.no. AB-0575); 2 μl of forward primer (100 μM); 2 μl of reverse primer (100 μM); 1 μl of S. cerevisiae genomic DNA (Promega G310A, lot 8347702, 276 μg/ml); and 5 μl of H2O were combined.
The thermocycling was carried out as follows: For the YMR251WA promoter: 94° C. for 5 minutes followed by 30 cycles of: 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute; followed by 72° C. for 5 minutes. The reaction mixtures were then held at 4° C. For the YMR107W and ZEO1 promoters: 94° C. for 5 minutes followed by 30 cycles of: 94° C. for 30 seconds, 45° C. for 30 seconds, 72° C. for 1 minute; followed by 72° C. for 5 minutes. The reaction mixtures were then held at 4° C. For the YLR110C promoter: 94° C. for 5 minutes followed by 30 cycles of: 94° C. for 30 seconds, 50° C. for 30 seconds, 72° C. for 1 minute; followed by 72° C. for 5 minutes. The reaction mixtures were then held at 4° C.
The PCR solutions were loaded onto an LMP gel and the bands were purified using Wizard PCR Preps (Promega, cat. no. A7170) according to protocol, eluted in 50 μl, ethanol precipitated, and resuspended in 20 μl. A map of the YLR110C promoter region is shown in FIG. 13 and SEQ ID NO:29. A map of the YMR251WA promoter region is shown in FIG. 14 and SEQ ID NO:30. A map of the YMR107W promoter region is shown in FIG. 15 and SEQ ID NO:31. A map of the ZEO1 promoter region is shown in FIG. 16 and SEQ ID NO:32.
Cloning Promoter Regions into a Yeast Vector Containing the Luciferase Gene
The PCR products representing the regions upstream of the YLR110C and YMR251WA ORFs were cloned into the suitably digested YEp13-based multicopy yeast expression vector pPRB1P+luc. A map of YEp13 is shown in FIG. 1. The Accession number for YEp13 is U03498. A map of pPRB1P is shown in FIG. 2. The sequence of pPRB1P is shown in SEQ ID NO:27. A map of pPRB1P+luc is shown in FIG. 3 and the sequence is shown in SEQ ID NO:28. The PRB1 promoter was removed from the vector by digesting with the restriction enzymes HindIII and NdeI. The digested backbone was then ligated with a HindIII/NdeI digested PCR product. See FIG. 4.
The PCR products described below, and maxi-prepped pPRB1P+luc were digested as follows. 60 μl of pPRBP1+luc (328 μg/ml), 10 μl of Hind III (Life Technologies, cat.no. 15207-012, 10 units/μl), 10 μl NdeI (Amersham, cat.no. E0216Y, 20 units/μl), 10 μl NEBuffer 2 (NEB, cat.no. 007-2), and 10 μl of H2O. 14 μl YLR110C, 2 μl of Hind III (Life Technologies, cat.no. 15207-012, 10 units/μl), 2 μl Nde I (Amersham, cat.no. E0216Y, 20 units/μl), and 2 μl NEBuffer 2 (NEB, cat.no. 007-2). 14 μl YMR251WA, 2 μl of Hind III (Life Technologies, cat.no. 15207-012, 10 units/μl), 2 μl Nde I (Amersham, cat.no. E0216Y, 20 units/μl), and 2 μl NEBuffer 2 (NEB, cat.no. 007-2). The solutions were allowed to react at 37° C., for 4 hours.
The double digested pPRB1P+luc backbone was purified on an LMP gel using Wizard PCR preps (Promega, cat. no. A7170), and then ethanol precipitated. The remaining digestion products were also ethanol precipitated. The pPBR1P+luc digests were resuspended in 60 μl of H2O and the PCR product digests were resuspended in 20 μl.
Ligation reactions were then carried out between each promoter region and the digested pPRBP1+luc at 16° C. overnight. The PCR products representing the regions upstream of the following ORFs; YMR107W and ZEO1, were prepared, restricted, and ligated essentially as described above, however BCL restriction buffer B and different amounts of PCR product/volumes were used.
Transformation of Ligation Products into E. coli 
The products of the ligations described above were transformed into E. coli (Invitrogen's One-Shot TOP10 Competent cells, cat.no. C4040-10) according to manufacturers protocol. In each case 5 μl of the ligation product was added to the cell suspension. The total final cell suspension was plated out onto L-amp plates and incubated overnight at 37° C.
Colonies were picked from the plates and PCR screened using the PCR primers used to amplify the promoters originally. Two positive colonies from each ligation were grown in 5 ml overnight cultures and their plasmids were purified (Promega Wizard Plus SV Mini-preps, cat. no. A1330). The eluted DNA was ethanol precipitated and resuspended in 20 μl of water. Analytical restriction digests were carried out to confirm the presence of the correct promoter. Clones containing all four promoter constructs were obtained.
The new constructs were named as follows:
pPRB1+luc backbone+YLR110C promoter=pYLR110P+luc SEQ ID NO:19
pPRB1+luc backbone+YMR251WA promoter=pYMR251AP+luc SEQ ID NO:20
pPRB1+luc backbone+YMR107W promoter=pYMR107P+luc SEQ ID NO:21
pPRB1+luc backbone+ZEO1 promoter=pZEO1P+luc SEQ ID NO:22
Maps of pYLR110P+luc, pYMR251AP+luc, pYMR107P+luc, and pZEO1P+luc are shown in FIGS. 5, 6, 7, and 8, respectively. Plasmid DNA (pYLR110P+luc and pYMR251AP+luc) was prepared for transformation into yeast and sequencing using the QIAGEN Plasmid Maxi kit (Cat.no. 12162). The DNA concentrations of the maxi-preps (measured by absorbance at 260 nm) were: pYLR110P+luc 463 μg/ml; pYMR251AP+luc 346 μg/ml; pYMR107P+luc  ̃300 μg/ml; and pZEO1P+luc  ̃720 μg/ml. The remaining plasmids were transformed into yeast as Wizard Plus SV Mini-prep DNA, and maxi-prep DNA was obtained for sequencing using the Gibco BRL Concert Plasmid Maxi kit (Cat no.11452).
Sequencing of Promoter Constructs
DNA of each of the four promoter constructs were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, part no. 4303153) was used to carry out the sequencing reactions. Each reaction contained 8 μl of Reaction Mix and 1 μg of 3.2 μM primer. The volumes of template DNA and H2O added are as follows: 1.1 μl of pYLR110P+luc template and 9.9 μl of water; 1.4 μl of pYMR251AP+luc template and 9.6 μl of water; 2.0–6.0 μl of pYMR107P+luc template and 9.0–5 μg of water; and 0.5–1.5 μl of pZEO1P+luc template and 10.5–9.5 μl of water.
The thermocycling protocol is described in the ABI protocol, the PCR products were ethanol precipitated by adding 3M NaOAc and absolute Ethanol, standing at room temperature for 15 minutes, centrifuging for 20 minutes and washing with 250 μl of 70% ethanol. The precipitated DNA was resuspended in 3 μl of loading dye and 2 μl of each suspension was analyzed on an PE-ABI 377 automated sequencer.
The following promoter constructs pYLR110P+luc and pYMR251AP+luc were each sequenced using four primers:
YEp13 F2: CCTCAATTGGATTAGTCTCA—SEQ ID NO:13—aligns to the YEp13 backbone, 290 bp 5′ of the Hind III site.
Luc R1: CACCTCGATATGTGCATCTG—SEQ ID NO:14—aligns to the Luc ORF, 150 bp 3′ of the NdeI site.
Forward PCR primer: forward primer used to PCR clone promoter, i.e., SEQ ID NO:5 and SEQ ID NO: 7.
Reverse PCR primer: reverse primer used to PCR clone promoter, i.e., SEQ ID NO:6 and SEQ ID NO:8.
The remaining promoter constructs (pYMR107P+luc and pZEO1P+luc) were each sequenced using primers Yep13 F2 and Luc R1. Combining the data from all primers completely sequenced the promoter regions and spanned the cloning sites of the original vector.
Deviations from Published Genomic Sequences
All sequences differ by a few base pairs around the ATG, this results from the creation of an NdeI site at the 3′ end of the promoter. In addition, the following further alterations from published sequences were identified.
pYLR110P+luc: A substitution of a C for a T had taken place at a base pair 361 of the sequence.
pYMR107P+luc: In the initial construct (for which luciferase reporter data is described), a cloning artifact led to the junction between the promoter region and the LUC ORF in pYMR107W+luc to have the sequence: CATATATG (where ATG is the luciferase translational start site). This sequence was modified by site directed mutagenesis to create the sequence CATATG, which generates a novel NdeI site at the promoter/luciferase junction. Subsequent luciferase expression analysis confirmed that expression from the NdeI site modified pYMR107P+luc construct did not differ significantly from the original construct, therefore the sequence of the corrected CATATG construct is included herein.
Other Modifications
pYMR107P+luc: Cloning artifacts created an additional HindIII site and linker to the 5′ (i.e. outside) of the pYMR107P+luc and promoters:
Instead of:










hindIII NotI       promoter 5′




 AAGCTT-CGCGGCCGCG-NNNNNNN
SEQ ID NO:17






The sequence is:








hindIII     hindIIINotI        promoter 5′








AAGCTT-AGCT-AAGCTT-CGCGGCCGCG-NNNNNNN
SEQ ID NO:18.






EXAMPLE 4
Luciferase Assays of Promoter Activity
Transformation of S. cerevisiae with Promoter Constructs.
S. cerevisiae strain 11C was transformed with five promoter constructs. This strain carries six metabolic markers, Ade, Trp, Ura, Lys, Leu and His. It has the genotype: ade2-161, trp1-D63, ura3-52, lys2-801, leu2D1 &/or leu2-3 &/or leu2-112, hisD200 &/or hisD200. 11C was generated by crossing the strains YPH500 (Mat a ura3-52 lys2-801 ade2-161 trp1-D63 hisD200 leu2D1) and AH22 (MATa leu2-3 leu2-112 his4-519 can1.
11C cells were streaked from a glycerol stock onto a YPD plate and grown at 30° C. for two days. The cells were transformed with the five plasmids, pYLR110P+luc, pYMR251AP+luc, pYMR107P+luc, & pZEO1P+luc and pPRB1P+luc to act as a control. The transformations were carried out using the Quick and Easy method (Gietz, R. D. and R. A. Woods, 1994, Molecular Genetics of Yeast: Practical Approaches pp. 121–134. 10 ml of plasmid was added to the transformation mix in each case. The whole transformation mixes were plated out onto -Leu plates and incubated at 30° C. for three days. Three individual colonies from each transformation plate were picked and used to inoculate 10 ml YPD cultures. The 10 ml cultures were incubated in an orbital shaker set to 200 rpm and 30° C. Cells were harvested from the cultures at two points. First, at a point at which the OD of the culture was close to 1.0, at which time a 4 ml sample was taken. Second, a 3 ml sample was taken after an incubation time of 45 hours. The ODs and incubation time of each sample is shown in Table 5. For all harvested samples, the cells were immediately spun down at 3000 rpm and 4° C., washed in 5 ml of dH2O, repelleted and frozen at −20° C.











TABLE 5








Incubation time at




Clone
OD at time of harvesting
harvesting of first sample
OD at time of harvesting


Plasmid
number
first 4 ml sample
(hours)
second 3 ml sample



















pPRB1P +
7
0.98
24.5
4.80


luc
8
0.68
28
5.56



9
1.15
28
5.66


pYLR110P +
8
1.12
28
5.50


luc
9
0.46
28
4.38



10
1.16
24.5
5.51


pYMR251AP +
8
1.20
24.5
4.99


luc
9
1.05
27
4.71



10
1.15
27
5.18


pYMR107P +
1
1.06
27
5.47


luc
2
0.49
28.5
4.54



3
0.97
25.5
5.58


pZEO1P +
1
1.02
28.5
4.84


luc
2
0.62
28.5
4.97



3
0.42
28.5
4.31









Analysis of Luciferase Activity

All of the samples were analyzed for luciferase activity, using the LucLite Luciferase Reporter Gene Assay Kit (Packard, cat.no 6016911). The cells were prepared by resuspending in PBS and diluting to a final concentration of 6×106 cells/ml. 100 ml of each cell suspension was pipetted into wells in duplicate on two 96 well plates, so that each well contained 6×105 cells. The plates were incubated at 30° C. for 10 minutes. 100 ml of a 1 in 2 dilution of reconstituted substrate was added to each well, and the plate was further incubated at room temperature for 10 minutes. The luminescence was then measured using the Packard TopCount. The luminescence readings obtained after 0.03 min are shown below in counts per second (CPS) in Table 6.











TABLE 6









Clone
First sample
Second sample
















Plasmid
number
Readings
(CPS)
Average
Average
Readings
(CPS)
Average
Average



















pPRB1P + luc
7
3589
35690
35790
34898
20322
20975
20648
19867



8
2549
25276
25387
24495
52997
51778
52388
51607



9
2413
27797
25967
25075
49192
46971
48081
47300


pYLR110P + luc
8
5235
53618
52986
52094
41789
38904
40346
39565



9
10529
99776
102537
101645
85562
84468
85015
84234



10
10753
109226
108379
107486
22507
22436
22471
21690


pYMR251AP + luc
8
7199
69797
70895
70003
40869
40202
40536
39755



9
9885
98389
98621
97729
51159
49828
50493
49712



10
8321
87546
85378
84485
70091
74576
72334
71553


pYMR107P + luc
1
9046
8650
8848
6790
29413
28505
28959
28124



2
3996
4009
4002
1945
24391
23915
24153
23318



3
3018
3236
3127
1069
23866
23408
23637
22802


pZEO1P + luc
1
6413
63162
63649
61592
47469
45769
46619
45784



2
1957
18329
18954
16897
44910
42982
43946
43111



3
8757
90317
88944
86887
142414
142262
142338
141503









The results are summarized in Table 7.











TABLE 7







Luciferase
Luciferase




Expression
Expression


Promoter
mRNA levels
Glucose
Ethanol


















PRB1
Ethanol Induced
1.00
1.00


YLR110C
Highly Ethanol and
3.03
1.22



Glucose Induced


YMR251WA
Highly Ethanol and
2.92
1.35



Glucose Induced


YMR107W
Ethanol Induced
0.21
0.95


ZEO1
Very Highly Ethanol
3.62
2.89



and Glucose Induced









Three promoters give higher levels of expression than PRB1 at both ODs, these are: YLR110C, YMR251WA, and ZEO1. The promoter showing the greatest fold induction is YMR107W.
Creating Vectors with Promoters but without the Luciferase Gene
Based on the analysis of luciferase expression four further promoter constructs have been made. The lack the luciferase gene and can be used to clone nucleic acid molecules encoding polypeptides of interest downstream of the promoters such that they drive expression of the nucleic molecules of interest. The sequences of these four plasmids are named: G1: pYLR110P (SEQ ID NO:23) (map at FIG. 9); G2: pYMR251AP (SEQ ID NO:24) (map at FIG. 10); G3 pYMR107P (SEQ ID NO:25) (map at FIG. 11); and G4: pZEO1P (SEQ ID NO:26) (map at FIG. 12). These were constructed by digesting pPRB1P (SEQ ID NO:27) with HindIII and NdeI to obtain the vector. The promoter+luc construct was digested with HindIII and NdeI to obtain the promoter fragment. The vector and promoter DNA was purified from LMP agarose using PCRpreps. The vector and promoter was ligated and used to transform E. coli. Correct recombinants were screened for.
EXAMPLE 5
Isolation of Active Promoter Fragments
Operative fragments of the YLR110C, YMR251WA, YMR107W and ZEO1 promoters can be generated using restriction endonucleases, 5′ or 3′ deletion mutagenesis, PCR, site specific deletion, or a combination thereof. For example, purified pYLR110P+luc, pYMR251AP+luc, pYMR107P+luc or pZEO1P+luc plasmids, as generated in Example 3, can be subjected to restriction endonucleases to generate fragments of the YLR110C, YMR251WA, YMR107W or ZEO1 promoters. Restriction endonuclease sites, preferably unique restriction endonuclease sites, within the promoter sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 can be identified that generate fragments of the promoter upon restriction endonuclease digestion. Such fragments are preferably, 17, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 nucleotides in length.
The fragments generated by restriction endonuclease digestion of the promoters shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 can be separated by agarose gel electrophoresis. The agarose gel band corresponding to the desired promoter fragment can be cut out of the agarose gel. The fragment can be isolated and purified from the agarose gel by, for example, electroelution or kits such as QIAquickTM gel extraction kit or QIAEX® II Gel Extraction System (Qiagen Cat. No. 28704 and 20021).
The purified promoter fragment can be ligated into the isolated and purified HindIII, NdeI, double-digested pPRBP1+luc backbone such that the promoter fragment is operably linked to a luciferase gene and transformed into E. coli, as described in Example 3. The new expression vector comprising a fragment of YLR110C, YMR251WA, YMR107W, or ZEO1 promoter region can be isolated and purified from E. coli, sequenced, and transformed into yeast as described in Example 3.
To analyze promoter activity, luciferase assays as described in Example 4, can be conducted using S. cerevisiae cultures that have been transformed with the expression vector comprising a fragment of the YLR110C, YMR251WA, YMR107W, or ZEO1 promoter operably linked to a luciferase gene and S. cerevisiae cultures that have been transformed with pPRB1P+luc. The S. cerevisiae cultures are grown in medium containing a non-fermentable carbon source, such as ethanol, or a fermentable carbon source, such as glucose, or both. Cells are obtained from the cultures and analyzed for luciferase activity as described in Example 4.
A promoter fragment is operative if it expresses at least 75% of the luciferase activity as the PRB1 promoter. Preferably, an operative promoter fragment expresses at least 100%, 200%, 300%, 400%, or more of the luciferase activity as the PRB1 promoter.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 Polynucleotide sequence of promoter YLR110C
SEQ ID NO:2 Polynucleotide sequence of promoter YMR251WA
SEQ ID NO:3 Polynucleotide sequence of promoter YMR107W
SEQ ID NO:4 Polynucleotide sequence of promoter ZEO1
SEQ ID NO:5 Forward PCR primer for YLR110C
SEQ ID NO:6 Reverse PCR primer for YLR110C
SEQ ID NO:7 Forward PCR primer for YMR251WA
SEQ ID NO:8 Reverse PCR primer for YMR251WA
SEQ ID NO: 9 Forward PCR primer for YMR107W
SEQ ID NO:10 Reverse PCR primer for YMR107W
SEQ ID NO:11 Forward PCR primer for ZEO1
SEQ ID NO:12 Reverse PCR primer for ZEO1
SEQ ID NO:13: Yep13 Forward PCR primer
SEQ ID NO:14: Luc R1 Forward PCR primer
SEQ ID NO:15 Primer used in cDNA sequencing
SEQ ID NO:16 Control oligonucleotide used in GeneChip Microarray assay
SEQ ID NO:17 Original pYMR107P+luc sequence
SEQ ID NO:18 Modified pYMR107P+luc sequence
SEQ ID NO:19 Nucleotide sequence of pYLR110P+luc
SEQ ID NO:20 Nucleotide sequence of pYMR251AP+luc
SEQ ID NO:21 Nucleotide sequence of pYMR107P+luc
SEQ ID NO:22 Nucleotide sequence of pZEO1P+luc
SEQ ID NO:23 Nucleotide sequence of pYLR110P
SEQ ID NO:24 Nucleotide sequence of pYMR251AP
SEQ ID NO:25 Nucleotide sequence of pYMR107P
SEQ ID NO:26 Nucleotide sequence of pZEO1P
SEQ ID NO:27 Nucleotide sequence of pPRB1P
SEQ ID NO:28 Nucleotide sequence of pPRB1P+luc
SEQ ID NO:29 YLR110C promoter region
SEQ ID NO: 30 YMR251WA promoter region
SEQ ID NO:31 YMR107W promoter region
SEQ ID NO:32 ZEO1 promoter region

## Claims

1. An isolated and purified polynucleotide consisting of SEQ ID NO:1 wherein the polynucleotide is operative as a promoter to express a nucleic acid molecule encoding a polypeptide when operably linked to said nucleic acid molecule.

2. A yeast expression vector comprising the polynucleotide of claim 1.

3. The yeast expression vector of claim 2 wherein the yeast expression vector is selected from the group consisting of pYLR110P+luc and pYLR110P.

4. A yeast cell transformed with the yeast expression vector of claim 2.

5. A yeast cell transformed with the yeast expression vector of claim 3.

6. A method for producing a polypeptide comprising the steps of:
(a) constructing a yeast expression vector wherein the expression of a nucleic acid molecule encoding the polypeptide is controlled by the polynucleotide of claim 1;
(b) transforming a culture of yeast cells with the yeast expression vector;
(c) maintaining the yeast cells in culture so that the polypeptide is expressed; and
(d) recovering the polypeptide.

7. A method for producing a polypeptide comprising the steps of:
(a) cloning a nucleic acid molecule encoding the polypeptide into an expression vector selected from the group consisting of pYLR110P+luc and pYLR110P, wherein the nucleic acid molecule is operably linked to a promoter of the expression vector;
(b) transforming a culture of yeast cells with the yeast expression vector;
(c) maintaining the yeast cells in culture so that the polypeptide is expressed; and
(d) recovering the polypeptide.

8. A method for producing a polypeptide comprising the steps of:
(a) constructing a yeast expression vector wherein the expression of a nucleic acid molecule encoding the polypeptide is controlled by the polynucleotide of claim 1;
(b) transforming a culture of yeast cells with the yeast expression vector;
(c) maintaining the yeast cells in culture medium and controlling the expression of the nucleic acid molecule encoding the polypeptide by varying the level of a fermentable carbon source in the culture medium; and
(d) recovering the polypeptide.

9. The method of claim 8 wherein the fermentable carbon source is glucose.

10. A method for producing a polypeptide comprising the steps of:
(a) constructing a yeast expression vector wherein the expression of a nucleic acid molecule encoding the polypeptide is controlled by the polynucleotide of claim 1;
(b) transforming a culture of yeast cells with the yeast expression vector;
(c) maintaining the yeast cells in culture medium and controlling the expression of the nucleic acid molecule encoding the polypeptide by varying the level of a non-fermentable carbon source in the culture medium; and
(d) recovering the polypeptide.

11. The method of claim 10 wherein the non-fermentable carbon source is ethanol.

12. A method for producing a polypeptide comprising the steps of:
(a) constructing a yeast expression vector wherein the expression of a nucleic acid molecule encoding the polypeptide is controlled by the polynucleotide of claim 1;
(b) transforming a culture of yeast cells with the yeast expression vector;
(c) maintaining the yeast cells in culture medium and controlling the expression of the nucleic acid molecule encoding the polypeptide by varying the level of fermentable carbon source and a non-fermentable carbon source in the culture medium; and
(d) recovering the polypeptide.

13. The method of claim 12 wherein the fermentable carbon source is glucose.

14. The method of claim 12 wherein the non-fermentable carbon source is ethanol.

15. A method of identifying a promoter fragment, wherein the fragment has promoter activity comprising the steps of:
(a) generating a fragment comprising at least 17 contiguous nucleotides of an isolated and purified polynucleotide which is SEQ ID NO:1;
(b) cloning the fragment into a yeast expression vector, wherein the fragment is operably linked to a reporter gene;
(c) transforming yeast cells with the yeast expression vector;
(d) growing the yeast cells in yeast cell culture under conditions favorable for expression of the reporter gene; and
(e) assaying the yeast culture for a reporter protein expressed by the reporter gene;
wherein expression of the reporter gene indicates the fragment has promoter activity.