WO1999063074A2

WO1999063074A2 - Altering the ligand-binding characteristics of a nucleic acid ligand binding sequence by altering the nucleotide composition of its flanking sequences

Info

Publication number: WO1999063074A2
Application number: PCT/US1999/012515
Authority: WO
Inventors: Michael J. Lane; Albert S. Benight; Brian D. Faldasz
Original assignee: TM Technologies Inc
Current assignee: Luminex Molecular Diagnostics Inc
Priority date: 1998-06-04
Filing date: 1999-06-04
Publication date: 1999-12-09
Anticipated expiration: 2000-12-04
Also published as: WO1999063074A3

Abstract

This invention relates to a method of improving the relative expression level of any eukaryotic, prokaryotic, viral or any gene of interest in eukaryotic or prokaryotic cell lines and related methods and products. The invention described herein alters the ligand-binding characteristics of a nucleic acid sequence of any given length without the use of small molecule pharmaceuticals and teaches that the binding affinity of a ligand, such as RNA polymerase and/or transcription factors, for its ligand binding site may be modulated solely by the nucleotide composition of the polynucleotide sequence(s) flanking an adjacent ligand binding site.

Description

METHOD FOR ALTERING THE RELATIVE EXPRESSION LEVEL OF ANY GENE AND RELATED METHODS AND PRODUCTS

Background of Invention The ability of a gene to be expressed in the environment of a given cell has important applications in the manufacture of protein products from that gene. For example, it has been found that some proteins which serve as therapeutic molecules in treatment of human disorders cannot be produced unless they are made in particular types of cells. This is due to the processing of the particular protein in that cellular environment. This fact has engendered the need to construct a variety of different expression vectors (e.g., baculovirus, retrovirus, etc.) which can be employed in specific cell lines (or bacteria) when manufacturing such gene products on a commercial scale.

The cost of large scale production of protein products for human therapeutic use is a significant factor in the cost of the protein pharmaceutical. Consequently, issues in manufacturing which reduce bioprocessing costs are of great interest (Genetic Engineering

News Feb. 1 1995, p. 10). Accordingly, recovery levels has been targeted as an important issue in bioprocess manufacturing.

To address this issue, a means to improve cellular expression of a given biopharmaceutical or to reduce and perhaps inhibit entirely the expression of a repressor polypeptide that interferes with the expression of the desired protein pharmaceutical without altering process validated manufacturing and product purification steps is desirable.

The present inventors have previously found that the relative binding of a DNA binding ligand to a site on a duplex DNA is affected by DNA base pair stability of sequences immediately flanking the ligand's binding site (see, e.g., Benight et al. (1995) Adv. Biophys. Chem.. 5: 1-55). Recent work, however, has demonstrated that this effect is completely general for any ligand and leads to the conclusion that the relative binding of a promoter, enhancer or any regulatory polynucleotide sequence by a ligand, wherein the ligand is at least one transcription factor and/or RNA polymerase can be altered by modifying the composition of the nucleotides in the polynucleotide sequence immediately flanking the binding agent's binding site.

Methods have been developed in the past to alter the ligand binding properties of nucleic acid constructs (see, e.g., U.S. Patent No. 5,306,619; 5,578.444; 5,693.436. 5.716,780: 5,726,014; and 5,738,990). The methods described therein screen for small molecule pharmacological agents which bind to a test sequence flanking an adjacent DNA-binding protein site, the cognate site, and in turn alter the binding affinity of the DNA binding protein for its cognate site. In theory this method can be used to alter the expression level of any gene by screening for small molecule pharmacological agents that would bind to polynucleotide sequences adjacent to promoter sites which in turn would either increase or decrease the binding affinity of RNA polymerase and/or other transcription factors for the promoter thereby either increasing or decreasing expression of the gene linked to the promoter (U.S. Patent No. 5,578,444). The experiments described in U. S. Patent No. 5,578,444; 5,693,436; 5,716,780; 5,726,014; and 5,738,990 further teach that a ligand binds to its cognate site with "indifference to the nucleotide sequences flanking the screening site." In other words, the binding affinity of a ligand for its binding site is independent of the nucleotide composition of the flanking sequences placed adjacent to the ligand binding site.

Summary of the Invention

The present inventors have determined a method of improving the expression of a gene from any given gene locus. The gene may be prokaryotic, eukaryotic, viral or any gene of interest. In contrast to the prior art which describes the utilization of small molecules which bind to polynucleotide sequences adjacent to promoter sequences and in turn alter the binding characteristics of RNA polymerases and/or transcription factors that transcribe genetic information from DNA to RNA (US. Patent No. 5,578,444), the invention described herein alters the ligand-binding characteristics of a nucleic acid sequence of any given length without the use of small molecule pharmaceuticals and teaches that the binding affinity of a ligand, such as RNA polymerase and/or transcription factors, for its ligand binding site may be modulated solely by the nucleotide composition of the polynucleotide sequence(s) flanking an adjacent ligand binding site. In one aspect, this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence (promoter) is flanked by an adjacent heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition 5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and/or a heterologous duplex flanking polynucleotide sequence 3' of the promoter having the nucleotide composition

S ' -ATATAAATTAAATATAA-S ' ( SEQ ID NO : 2 ) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively higher binding affinity when compared to the adjacent native duplex polynucleotide flanking sequences adjacent to the promoter, and thus improves the transcription level of the gene of interest that is linked downstream (3') of the promoter. Such heterologous adjacent duplex flanking sequences will convert weak promoters to strong promoters and convert already strong promoters to even stronger promoters. The promoter sequence utilized may be a pre-existing promoter sequence available in several commercially available expression vectors. For example, the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:l) and/or 3' (SEQ ID NO:2) side of the promoter by methods well known to those skilled in the art. The heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively higher binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a higher frequency compared to the adjacent native duplex polynucleotide flanking sequences.

In another aspect, this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence (promoter) is flanked by an adjacent heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition

5 ' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and/or a heterologous duplex flanking polynucleotide sequence 3' of the promoter having the nucleotide composition 5 ' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ I D NO : 4 ) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively lower binding affinity when compared to the adjacent native duplex polyoucleotide flanking sequences adjacent to the promoter, and thus decreases or significantly reduces the transcription level of the gene of interest that is linked downstream (3') of the promoter. Such heterologous adjacent duplex flanking sequences will convert strong promoters to weak promoters and convert already weak promoters to even weaker promoters. The promoter sequence utilized may be a preexisting promoter sequence available in several commercially available expression vectors. For example, the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:3) and 3' (SEQ ID NO:4) side of the promoter by methods well known to those skilled in the art. The heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively lower binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a decreased frequency compared to the adjacent native duplex polynucleotide flanking sequences.

In a preferred aspect of this invention, the improved DNA expression vehicle has a novel hetrologous duplex polynucleotide promoter sequence specific for a biologically functional DNA of interest. The sequence of the duplex polynucleotide promoter sequence bound by transcription factor(s) and/or RNA polymerase for the gene of interest that is to be expressed can be determined by methods well known to those skilled in the art (e.g., DNase I footprinting or band shifting). The duplex polynucleotide promoter sequence itself can then be amplified by the polymerase chain reaction and native duplex polynucleotide flanking sequences 5' and/or 3' adjacent to the promoter can be replaced by a heterologous duplex polynucleotide sequence 5' of the promoter having the nucleotide composition

5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and/or a heterologous duplex flanking polynucleotide sequence 3 ' of the promoter having the nucleotide composition 5 ' -ATATAAATTAAATATAA-3 ' ( SEQ ID NO : 2 ) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively higher binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter and thus improves the transcription level of the gene of interest that is linked downstream (3') of the promoter. In still another preferred aspect of this invention, the improved DNA expression vehicle has a novel hetrologous duplex polynucleotide promoter sequence specific for a biologically functional DNA of interest. The sequence of the duplex polynucleotide promoter sequence bound by transcription factor(s) and/or RNA polymerase for the gene of interest that is to be expressed can be determined by methods well known to those skilled in the art (s g., DNase I footprinting or band shifting). The duplex polynucleotide promoter sequence itself can then be amplified by the polymerase chain reaction and native duplex polynucleotide flanking sequences 5' and/or 3' adjacent to the promoter can be replaced by a heterologous duplex polynucleotide sequence 5 ' of the promoter having the nucleotide composition

5' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and/or a heterologous duplex flanking polynucleotide sequence 3' of the promoter having the nucleotide composition 5 ' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO: 4 ) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively lower binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter and thus lowers the transcription level of the gene of interest that is linked downstream (3') of the promoter. In another aspect, this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence is flanked by an adjacent heterologous duplex flanking polynucleotide sequence 5' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the polynucleotide sequence having the nucleotide composition 5 ' -AATTAAATAATAAATTA-3 ' (SEQ ID NO : l ) and/or an adjacent heterologous duplex flanking polynucleotide sequence 3' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to nucleotide sequence having the nucleotide composition

5'-ATAT AATTAAATATAA-3' (SEQ ID NO: 2) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively higher binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter, and thus improves the transcription level of the gene of interest that is linked downstream (3') of the promoter. Such heterologous adjacent duplex flanking sequences will convert weak promoters to strong promoters and convert already strong promoters to even stronger promoters. The promoter sequence utilized may be a pre-existing promoter sequence available in several commercially available expression vectors. For example, the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:l) and 3' (SEQ ID NO:2) side of the promoter by methods well known to those skilled in the art. The heterologous duplex flanking sequences adjacent to the promoter se_quence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively higher binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a higher frequency compared to the adjacent native duplex polynucleotide flanking sequences.

In still another aspect, this invention features an improved recombinant DNA expression vehicle whose polynucleotide promoter sequence is flanked by an adjacent heterologous duplex flanking polynucleotide sequence 5' of the promoter sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the polynucleotide sequence having the nucleotide composition

5 ' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and/or a heterologous duplex flanking polynucleotide sequence 3' of the promoter sequence, the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to nucleotide sequence having the nucleotide composition

5 ' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : 4 ) which allow transcription factors and/or RNA polymerases to bind the promoter with relatively lower binding affinity when compared to the native duplex polynucleotide flanking sequences adjacent to the promoter, and thus decreases or significantly reduces the transcription level of the gene of interest that is linked downstream (3') of the promoter. Such heterologous adjacent duplex flanking sequences will convert strong promoters to weak promoters and convert already weak promoters to even weaker promoters. The promoter sequence utilized may be a preexisting promoter sequence available in several commercially available expression vectors. For example, the duplex polynucleotide flanking sequences adjacent to the promoters in these expression vectors will be replaced by the heterologous duplex polynucleotide flanking sequences on the 5' (SEQ ID NO:3) and 3' (SEQ ID NO:4) side of the promoter by methods well known to those skilled in the art. The heterologous duplex flanking sequences adjacent to the promoter sequence will allow transcription factors and/or RNA polymerase to contact the promoter with relatively lower binding affinity as compared to the adjacent native duplex polynucleotide flanking sequences and initiate transcription at a decreased frequency compared to the adjacent native duplex polynucleotide flanking sequences. In still another aspect of this invention, the improved DNA expression vehicle can also have, in addition to the heterologous duplex polynucleotide sequence of nucleotide composition

5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) flanking the 5' side of a pre-existing promoter or novel heterologous promoter, and/or a heterologous duplex flanking polynucleotide sequence of nucleotide composition

5 ' -ATATAAATTAAATATAA-3 ' (SEQ ID NO : 2 ) flanking the 3' side of a pre-existing promoter or novel heterologous promoter, DNA sequence elements which act in conjunction with the promoter and contact polypeptide molecules which either induce or repress expression of the gene of interest from the promoter. These unique DNA sequence elements allow the promoter to be regulated by one of several factors which may include pH of the growth medium, growth temperature or composition of the growth medium of the unicellular organism transformed by the improved DNA expression vector. Thus, in the presence of repressors or absence of inducers, expression of the biologically functional gene of interest does not occur. Subsequent to induction or derepression of the promoter, expression of the gene of interest is high.

In another aspect of this invention, the improved recombinant DNA expression vehicle comprises (i) a duplex polynucleotide enhancer sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex flanking sequence or a pair of heterologous duplex flanking sequences; or (ii) a duplex polynucleotide proximal - promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iii) a duplex polynucleotide operator sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iv) a duplex polynucleotide long terminal repeat sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; and (v) a duplex polynucleotide promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of adjacent naturally occurring duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences. The heterologous duplex polynucleotide sequence 5' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition

5 - -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) or a polynucleotide sequence at least 80% homologous to it. The heterologous duplex flanking polynucleotide sequence 3' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition

5 ' -ATATAAATTAAATATAA-3 ' (SEQ ID NO : 2 ) or a polynucleotide sequence at least 80% homologous to it. The duplex polynucleotide enhancer or duplex polynucleotide proximal-promoter sequence may be located at a great distance either 5' or 3' from the transcription start site of the gene whose expression is to be improved. In this particular aspect of the invention the duplex polynucleotide promoter sequence is located 5' relative to the gene whose expression is to be improved. The resulting improved DNA expression vehicle will increase the expression of the desired gene.

In another aspect of this invention, the improved recombinant DNA expression vehicle comprises (i) a duplex polynucleotide enhancer sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex flanking sequence or a pair of heterologous duplex flanking sequences; or (ii) a duplex polynucleotide proximal promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iii) a duplex polynucleotide operator sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; or (iv) a duplex polynucleotide long terminal repeat sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of naturally occurring adjacent duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences; and (v) a duplex polynucleotide promoter sequence whose naturally occurring adjacent duplex polynucleotide flanking sequence or a pair of adjacent naturally occurring duplex polynucleotide flanking sequences may be replaced by a heterologous duplex polynucleotide flanking sequence or a pair of heterologous duplex polynucleotide flanking sequences. The heterologous duplex polynucleotide sequence 5' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition

5'-GGGCCGCGGGCGGGCGA-3' (SEQ ID NO: 3) or a polynucleotide sequence at least 80% homologous to it. The heterologous duplex flanking polynucleotide sequence 3 ' of the enhancer, proximal-promoter, operator, long terminal repeat or promoter is comprised of the nucleotide composition

5 ' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : ) or a polynucleotide sequence at least 80% homologous to it. The duplex polynucleotide enhancer or duplex polynucleotide proximal-promoter sequence may be located at a great distance either 5' or 3' from the transcription start site of the gene whose expression is to be improved. In this particular aspect of the invention the promoter is located 3' relative to the gene whose expression is to be altered and the resulting improved DNA expression vehicle will synthesize anti-sense mRNA of the gene to which the promoter is associated. The anti-sense mRNA will contact and duplex with the endogenous sense mRNA of the corresponding endogenous gene and prevent or significantly reduce the expression of the gene product.

In a preferred embodiment the improved DNA expression vehicle is used in a compatible host cell to enhance the expression of a biologically active polypeptide molecule. The host cell is initially transfected with the improved DNA expression vehicle and cells which stably integrate the expression vehicle into their genomes can be further selected by methods well known to those skilled in the art. The stable transformants can then be grown under appropriate nutrient conditions in which the intact biologically functional mRNA is expressed constitutively and produces the biologically functional polypeptide molecule which is further purified to homogeneity. In another preferred embodiment the improved DNA expression vehicle is used in a compatible host cell to significantly reduce or eliminate the expression of a biologically active polypeptide molecule. The host cell is initially transfected with the improved DNA expression vehicle and cells which stably integrate the expression vehicle into their genomes can be further selected by methods well known to those skilled in the art. The stable transformants can then be grown under appropriate nutrient conditions in which the biologically functional gene product is not expressed. In yet another preferred embodiment the improved DNA expression vehicle is used in a compatible host cell to enhance expression of a biologically active polypeptide in a regulatable manner. The pre-existing duplex polynucleotide promoter sequence and its adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences will be replaced with a duplex polynucleotide sequence comprising a duplex polynucleotide regulatable promoter flanked on the 5' side by a heterologous duplex polynucleotide flanking sequence having the nucleotide composition

5 ' - AATTAAATAATAAATT A- 3 ' ( SEQ ID NO : l ) and/or on the 3' side by a heterologous duplex flanking polynucleotide sequence having the nucleotide composition 5 ' -ATATAAATTAAATATAA-3 ' ( SEQ ID NO : 2 ) which confer a relatively higher binding affinity for the transcription factors and/or RNA polymerases compared to the native duplex polynucleotide flanking sequences, and thus increase the frequency of transcription initiation. When utilizing a regulatable promoter, the expression of the biologically functional polypeptide, however, can be either turned-on or turned-off due to the presence of inducible DNA sequence elements.

In still another preferred embodiment the improved DNA expression vehicle is used in a compatible host cell to reduce or eliminate the expression of a biologically active polypeptide in a regulatable manner. The pre-existing duplex polynucleotide promoter sequence and its adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences will be replaced with a duplex polynucleotide sequence comprising a duplex polynucleotide regulatable promoter flanked on the 5' side by a heterologous duplex polynucleotide flanking sequence having the nucleotide composition

5 ' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and/or on the 3' side by a heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5 ' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : 4 ) which confer a relatively lower binding affinity for the transcription factors and/or RNA polymerases compared to the native duplex polynucleotide flanking sequences and thus decrease expression of the undesired gene. Alternatively, the relevant pre-exisung duplex polynucleotide promoter sequence and adjacent duplex polynucleotide flanking_s.equences can be replaced by a heterologous duplex promoter sequence and adjacent duplex polynucleotide flanking sequence or a pair of duplex polynucleotide flanking sequences specific for a gene of interest whose biologically functional protein product is to be reduced or eliminated. When utilizing a regulatable promoter, the expression of the biologically functional polypeptide, however, can be either turned-on or turned-off due to the presence of inducible DNA sequence elements.

Detailed Description of the Invention

A "polynucleotide" includes multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g. adenine (A) or guanine (G)). The term "polynucleotide" as used herein refers to both polyribonucleotides and polydeoxyribonucleotides. Polynucleotide sequences can be obtained from existing nucleic acid sources (e.g., genomic DNA or cDNA), but can also be synthetic (e.g., produced by oligonucleotide synthesis) DNA or RNA.

A "ligand" includes transcription factor(s), RNA polymerase(s) or any compound which when bound to a regulatory sequence, promoter, enhancer, or proximal-promoter sequence, operator or long terminal repeat is able to initiate or inhibit transcription. The transcription factors) may be of prokaryotic (e.g., sigma factor of E. Coli) or eukaryotic (e.g., general transcription factors (GTFs), AP-1 or SP-1) origin. RNA polymerases may be of prokaryotic (e.g., RNA polymerase of E. Coli), eukaryotic (e.g., RNA polymerase II) or viral (e.g., bacteriophage T7) origin. Other ligands may include the lambda cl repressor protein. A "flanking sequence" is a polynucleotide sequence located adjacent to a promoter sequence. A flanking sequence or flanking sequences may be 5', 3' or 5' and 3' of the promoter sequence.

A "regulatory sequence" or "polynucleotide regulatory sequence" is a polynucleotide sequence which when contacted by a ligand regulates the transcription of a biologically functional gene associated with the regulatory sequence.

A "promoter", "polynucleotide promoter sequence" or "promoter sequence" is any DNA sequence which transcription factor(s) and/or RNA polymerase contacts. The promoter

- 11 - determines the polarity of the transcript by specifying which strand will be transcribed. Promoters can be classified according to their "strength," that is the relative frequency of transcription initiation (times per minute) at each promoter. Thus, RNA polymerase initiates transcription at a high frequency at strong promoters and at low frequency at weak promoters.

A "regulatable promoter" is one whose transcription activity can be regulated by changes in pH of the growth medium, growth temperature or composition of the growth medium.

An "enhancer" is any regulatory DNA sequence to which a protein or proteins contact, thereby influencing the rate of transcription of a biologically functional gene associated with the enhancer. Contact of the enhancer by the protein or proteins may either stimulate or decrease the rate of transcription of the associated gene. Enhancers may be located at a great distance either 5' or 3' from the transcription start site of the biologically functional gene it controls. Enhancers may also be regulate transcription of its associated gene when placed in either orientation i.e., 5'→ 3' or 3'→ 5' relative to the gene whose transcription it controls.

A "proximal-promoter sequence" or "proximal-promoter element" is any regulatory sequence that is located close to (within 200 base pairs of) a promoter and binds a protein or proteins thereby modulating the transcription of the biologically functional gene associated with the promoter. The proximal-promoter sequence can occur 5' or 3 ' of the transcription start site of the biologically functional gene.

An "operator" is a short polynucleotide DNA sequence in a bacterial or viral genome which contacts a protein or proteins and regulates transcription of an associated biologically functional gene.

A "Long Terminal Repeat" or "LTR" is a regulatory polynucleotide sequence of viral origin (DNA tumor viruses or retroviruses) comprising integration signals for integrating into the host genome, an enhancer, a promoter and a polyadenylation site.

"Sequence identity or homology", as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is compared by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g., those arising from deletions or insertions in one of the sequences, are counted as mismatches. The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment parameters include GAP Penalty = 10, Gap Length Penalty = 10. For DNA alignments, the pairwise alignment parameters can be Htuple = 2, Gap penalty = 5, Window = 4, and Diagonal saved = 4. For protein alignments, the pairwise alignment parameters can be Ktuple = 1, Gap penalty = 3, Window = 5, and Diagonals Saved = 5.

An additional non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natn. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natn. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al, (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score =100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.nchi.nlm.nih.gov. Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The following two examples illustrate the basic principle of the present invention by which the expression level of any gene can be either increased or decreased. Although pBluescript SK (+/-), a commercially available vehicle from Stratagene which contains the T7 promoter, is used to illustrate the utility of the present invention, the scope of the present invention is not limited in any way to this particular vector.

Initiation of transcription with T7 RNA polymerase is highly specific for the T7 bacteriophage promoter. Cloning vectors have been developed e.g., pBluescript SK (+/-), which direct transcription from the T7 promoter through polylinker cloning sites. Expression vectors containing the T7 promoter allow in vitro synthesis of defined RNA transcripts and in conjunction with the T7 RNA polymerase gene in vivo synthesis of defined RNA transcripts from a cloned DNA sequence. Under optimal conditions, greater than 700 moles of T7 RNA transcript can be synthesized per mole of DNA template (Noren, C. J. et al, 1990). RNA produced using the T7 RNA polymerase is biologically active as mRNA (Krieg, P. A. and Melton, D. A., 1984) and can be accurately spliced (Green, M. R. et al., 1983). The intent of the present invention is to further increase the synthesis of defined RNA transcripts.

EXAMPLE 1: The T7 promoter consensus sequence flanked by a heterologous adjacent polynucleotide sequence or a pair of heterologous adjacent polynucleotide sequences to increase transcription from the promoter

The present inventors have previously found that the relative binding of a DNA binding ligand to a site on a duplex DNA is affected by DNA base pair stability of sequences immediately flanking the binding site of the ligand (Benight et al., (1995) Adv.Biophys.Chem. 5:1-55). Thus, in order to improve the output of mRNA transcript driven by the T7 promoter in pBluescript SK(+/-), the polynucleotide sequences immediately flanking the T7 promoter have been replaced by heterologous polynucleotide flanking sequences that increase the binding affinity of T7 RNA polymerase to the T7 promoter polynucleotide sequence by standard molecular cloning techniques well known to those skilled in the art.

In the commercially available pBluescript SK (+/-) vehicle the T7 promoter consensus sequence:

5 ' -TAATACGACTCACTATAGGGAGA-3 ' ( SEQ ID NO : 5 ) is flanked on the left (5' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -TTGTAAAACGACGGCCAGTGAATTG-3 ' ( SEQ ID NO : 6) and on the right (3' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -CGAATTGGGTACCGGGCCCCCCCTCG-3 ' ( SEQ ID NO : 7 )

In a preferred embodiment of this invention the flanking sequences described above are excised by standard molecular cloning techniques and replaced by heterologous flanking polynucleotide sequences that confer a higher binding affinity for the T7 RNA polymerase as compared to the pre-existing sequences flanking the T7 promoter. The resulting stronger T7 promoter has a higher relative frequency of transcription initiation as compared to the T7 promoter flanked by the preexisting polynucleotide sequences in the commercially available vector. A method of obtaining the optimum sequence composition of the heterologous polynucleotide flanking sequence has been described by the present inventors in US Provisional Application No. 60/068,616. The present inventors find that the following heterologous polyoucleotide sequence located 5' of the T7 promoter:

5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and the following heterologous polynucleotide sequence located 3' of the T7 promoter:

5 ' -ATATAAATTAAATATAA-3 * ( SEQ ID NO : 2 ) confers a higher relative binding affinity for T7 RNA polymerase to the T7 promoter and thus increases the frequency of transcription initiation from the T7 promoter as compared to the original pBluescript SK (+/-) vector.

EXAMPLE 2: The T7 promoter consensus sequence flanked by a heterologous adjacent polynucleotide sequence or a pair of heterologous adjacent polynucleotide sequences to decrease transcription from the promoter

Given that the relative binding of a DNA binding ligand to a site on a duplex DNA is affected by DNA base pair stability of sequences immediately flanking the binding site of the ligand (Benight et al., (1995) Adv.Biophys.Chem. 5:1-55), the output of mRNA transcript driven by the T7 promoter in pBluescript SK(+/-) can also be decreased. To this extent the sequences immediately flanking the T7 promoter have been replaced by heterologous polynucleotide flanking sequences that decrease the binding affinity of T7 RNA polymerase to the T7 promoter polynucleotide sequence by standard molecular cloning techniques well known to those skilled in the art.

5 ' -TAATACGACTCACTATAGGGAGA-3 ' ( SEQ ID NO : 5 ) is flanked on the left (5' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -TTGTAAAACGACGGCCAGTGAATTG-3 ' ( SEQ ID NO : 6 ) and on the right (3' of the T7 promoter sequence) by the polynucleotide sequence: 5 ' -CGAATTGGGTACCGGGCCCCCCCTCG-3 ' ( SEQ I D NO : 7 ) In a preferred embodiment of this invention the flanking sequences described above are excised by standard molecular cloning techniques and replaced by heterologous flanking polyoucleotide sequences that confer a lower binding affinity for the T7 RNA polymerase as compared to the pre-existing sequences flanking the T7 promoter. The resulting weak T7 promoter has a lower relative frequency of transcription initiation as compared to the T7 promoter flanked by the pre-existing polynucleotide sequences in the commercially available vector. A method of obtaining the optimum sequence composition of the heterologous polynucleotide flanking sequence has been described by the present inventors in US Provisional Application No. 60/068,616. The present inventors find that the following heterologous polynucleotide sequence located 5' of the T7 promoter:

5 ' -GGGCCGCGGGCGGGCGA-3 * (SEQ ID NO : 3 ) and the following heterologous polynucleotide sequence located 3' of the T7 promoter:

5 * -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : 4 ) confers a lower relative binding affinity for T7 RNA polymerase to the T7 promoter and thus decreases the frequency of transcription initiation from the T7 promoter as compared to the original pBluescript SK (+/-) vector.

While the above examples have been restricted to the use of the T7 promoter, the scope of this invention is not limited to promoter sequences, but can be used to modify the flanking polynucleotide sequence or a pair of flanking polynucleotide sequences of any regulatory DNA sequence that alters the relative frequency of transcription initiation as compared to the preexisting flanking polynucleotide sequence or a pair of flanking polynucleotide sequences. Enhancers and proximal-promoter sequences are examples of such sequences. Replacing the native flanking polynucleotide sequence or a pair of flanking polynucleotide sequences adjacent to an enhancer can alter the frequency of transcription initiation of the promoter that the enhancer regulates. Several commercially available expression vectors are available which contain promoter-enhancer sequences that regulate the expression of a biologically functional DNA cloned into the vector. pcDNA 3.1(+/-) from Invitrogen® is an example of such an expression vector. The expression of the desired cloned gene can be further improved by replacing the existing polynucleotide flanking sequences with heterologous flanking sequences which confer a higher binding affinity of the promoter-enhancer for transcription factor(s) and/or RNA polymerase, thereby increasing the frequency of transcription initiation and in turn increasing cellular output of the desired product. Thus, the ability to increase the cellular output of a biopharmaceutical by simply altering the polynucleotide sequences flanking the promoter of the desired biologically functional gene is of great economic value. However, the ability to alter the expression of a gene simply by altering the polynucleotide sequences flanking its promoter and/or enhancer also offers great promise in the treatment of human pathological conditions. For example, the present invention can be utilized to increase the expression of a gene whose protein product can be increased to treat a disease in vivo. The STATs (Signal Transducers and Activators of Transcription) are a family of latent cytoplasmic proteins that are activated to participate in gene control when cells encounter various extracellular signals. In a recent report by Kumar et al. (1998) the STATs have been shown to be involved in the induction of cell death (apoptosis). Apoptosis is initiated by activation of a cascade of enzymes that cleave cellular proteins, resulting in the efficient termination of the cell. STATs activate genes containing GAS (gamma-activated sequence) elements. Thus, the present invention can be utilized to replace the polynucleotide flanking sequence or a pair of polynucleotide flanking sequences adjacent to the GAS element with a heterologous polynucleotide flanking sequence or a pair of heterologous polynucleotide flanking sequences which confer a relatively higher binding affinity for the STATs as compared to the naturally occurring flanking sequences and thereby increase the expression of genes that which will induce premature death of unwanted cells or cells that have lost control of their own growth. Alternatively, this invention can also be utilized to reduce and perhaps even entirely inhibit the expression of undesirable genes whose protein products can lead to the manifestation of pathological conditions. Once the promoter of the disease causing gene is isolated, the naturally occurring adjacent flanking sequence or a pair of naturally occurring adjacent flanking sequences can be excised and replaced by a heterologous flanking polynucleotide sequence or a pair of heterologous polynucleotide flanking 'sequences which confers a relatively lower binding affinity of the transcription factor(s) and/or RNA polymerase for the promoter as compared to the naturally occurring adjacent polynucleotide flanking sequence or a pair of adjacent polynucleotide flanking sequences. The resulting promoter will have a lower relative frequency of transcription initiation as compared to the promoter flanked by the original polynucleotide sequences in the genome and thus reduce or perhaps entirely inhibit the expression of the unwanted gene product. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. The contents of all publications cited herein are hereby incorporated by reference.

Claims

What is claimed is:

1. A DNA sequence comprising a duplex polynucleotide regulatory sequence adjacent to a heterologous duplex flanking polynucleotide sequence 5' of the duplex polynucleotide regulatory sequence having the nucleotide composition

5 - -AATTAAATAATAAATTA-3 ' (SEQ ID NO : l ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence having the nucleotide composition

5 ' -ATA AAATTAAATATAA-3 ' ( SEQ ID NO: 2 ) wherein the duplex heterologous flanking polynucleotide sequences confer a relatively higher binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

2. A DNA sequence comprising a duplex polynucleotide regulatory sequence adjacent to a heterologous duplex flanking polynucleotide sequence 5' of the duplex polynucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5 ' -A A AAATTAAAT TAA-3 ' ( SEQ ID NO : 2 ) wherein the duplex heterologous flanking polynucleotide sequence confers a relatively higher binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

3. A DNA sequence comprising a duplex polynucleotide regulatory sequence adjacent to a heterologous duplex flanking polynucleotide sequence 5' of the duplex polynucleotide regulatory sequence having the nucleotide composition

5' -GGGCCGCGGGCGGGCGA-3' (SEQ ID NO : 3 ) and a heterologous duplex flanking polynucleotide sequence 3 'of the duplex polynucleotide regulatory sequence having the nucleotide composition

5' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : 4 ) wherein the duplex heterologous flanking polynucleotide sequences confer a relatively lower binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

4. A DNA sequence comprising a duplex polynucleotide regulatory sequence adjacent to a heterologous duplex flanking polynucleotide sequence 5' of the duplex polynucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5' -GGGCCGCGGGCGGGCGA-3' (SEQ ID NO: 3 ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5' -AGCGCGCGGCGGGCGGG-3 ' ( SEQ ID NO : ) wherein the duplex heterologous flanking polynucleotide sequences confer a relatively lower binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

5. The DNA sequence of claim 1, wherein the duplex polynucleotide regulatory sequence is a polynucleotide promoter sequence.

6. The DNA sequence of claim 5, wherein the duplex regulatory polynucleotide promoter sequence is of eukaryotic, prokaryotic or viral origin.

7. The DNA sequence of claim 5, wherein the promoter is a regulatable promoter.

8. The DNA sequence of claim 7, wherein the regulatable promoter is regulated by a factor selected from pH, growth temperature or composition of growth medium.

9. The DNA sequence of claim 7 associated with its naturally occurring duplex polynucleotide enhancer sequence, its naturally occurring duplex polynucleotide proximal- promoter sequence or its naturally occurring duplex polynucleotide operator sequence.

10. A recombinant DNA expression vehicle comprising the DNA sequence of claim 1.

11. A recombinant DNA expression vehicle comprising the DNA sequence of claim 2.

12. A recombinant DNA expression vehicle comprising the DNA sequence of claim 3.

13. A recombinant DNA expression vehicle comprising the DNA sequence of claim 4.

14. The recombinant DNA expression vehicle of claim 10, wherein an intact biologically functional DNA is located 3' of the duplex polynucleotide promoter sequence.

15. The recombinant DNA expression vehicle of claim 10, wherein an intact biologically functional DNA is located 5' of the duplex polynucleotide promoter sequence.

16. The recombinant DNA expression vehicle of claim 10, wherein the ligand which contacts the duplex polynucleotide regulatory sequence, duplex polynucleotide promoter sequence, duplex polyoucleotide enhancer sequence, duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof is a transcription factor and optionally an RNA polymerase, a compound which contacts a duplex regulatory polynucleotide sequence and initiates expression of a biologically functional gene associated with the duplex regulatory polynucleotide sequence, and any combination thereof.

17. A method of constructing a recombinant DNA expression vehicle, the method comprising the steps of: (a) incorporating into the vehicle a duplex polynucleotide DNA sequence having: (i) a duplex polynucleotide sequence encoding a protein to be expressed thereby; (ii) a duplex polynucleotide regulatory sequence of eukaryotic, prokaryotic or viral origin for contacting of a ligand thereto, operably linked to the coding sequence; and (iii) a 5' duplex polynucleotide flanking sequence, heterologous to the duplex polynucleotide regulatory sequence, having the nucleotide composition 5 ' -AATTAAATAATAAATTA-3 ' ( SEQ ID NO : l ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence having the nucleotide composition

5 ' -ATATAAATTAAATATAA-3 ' ( SEQ ID NO : 2 ) wherein the duplex heterologous flanking polynucleotide sequences confer a relatively higher binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

18. A method of constructing a recombinant DNA expression vehicle, the method comprising the steps of

(a) incorporating into the vehicle a duplex polynucleotide DNA sequence having: (i) a duplex polynucleotide sequence encoding a protein to be expressed thereby;

(ii) a duplex polynucleotide regulatory sequence of eukaryotic, prokaryotic or viral origin for contacting of a ligand thereto, operably linked to the coding sequence; and (iii) a 5' duplex polynucleotide flanking sequence, heterologous to the duplex polynucleotide regulatory sequence, having the nucleotide composition

5' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence having the nucleotide composition 5' -AGCGCGCGGCGGGCGGG-3 ' (SEQ ID NO : ) wherein the duplex heterologous flanking polynucleotide sequences confer a relatively lower binding affinity for a ligand(s) to the duplex polynucleotide regulatory sequence as compared to the naturally occurring duplex flanking polynucleotide sequences.

19. The method of claim 17, wherein the heterologous 5' duplex flanking polynucleotide sequence of step (iii) is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5 ' -AATTAAATAATAAATTA-3 * ( SEQ ID NO : l ) and a heterologous duplex flanking polynucleotide sequence 3' of the duplex polynucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5 ' -ATATAAATTAAATATAA-3 ' ( SEQ ID NO : 2 )

20. The method of claim 18, wherein the heterologous 5' duplex flanking polynucleotide sequence of step (iii) is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition 5' -GGGCCGCGGGCGGGCGA-3 ' ( SEQ ID NO : 3 ) and a heterologous duplex flanking polynucleotide sequence 3 ' of the duplex polyriucleotide regulatory sequence, wherein the heterologous duplex flanking polynucleotide sequence is at least 80% homologous to the heterologous duplex flanking polynucleotide sequence having the nucleotide composition

5'-AGCGCGCGGCGGGCGGG-3' (SEQ ID NO: 4)

21. The method of claim 17, wherein the duplex polynucleotide regulatory sequence is selected from the group consisting of a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

22. The method of claim 18, wherein the duplex polynucleotide regulatory sequence is selected from the group consisting of a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

23. The method of claim 19, wherein the duplex polynucleotide regulatory sequence is selected from the group consisting of a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

24. The method of claim 20, wherein the duplex polynucleotide regulatory sequence is selected from the group consisting of a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

25. A method of improving the expression of an intact biologically functional DNA which comprises the steps of:

(a) transforming under transforming conditions a compatible unicellular organism with the recombinant DNA expression vehicle of claim 17; (b) growing the unicellular organism under appropriate nutrient conditions in which the unicellular organism expresses the intact biologically functional DNA and produces the biologically functional polypeptide molecule; (c) and recovering the biologically functional polypeptide molecule.

26. A method of reducing the expression of an intact biologically functional DNA which comprises the steps of:

(a) transforming under transforming conditions compatible unicellular organism with the recombinant DNA expression vehicle of claim 18;

(b) growing the unicellular organism under appropriate nutrient conditions in which the unicellular organism expresses the intact biologically functional DNA and produces the biologically functional polypeptide molecule; and recovering the biologically functional polypeptide molecule.

27. A method of improving the expression of an intact biologically functional DNA which comprises the steps of:

(a) transforming under transforming conditions a compatible unicellular organism with the recombinant DNA expression vehicle of claim 19;

(b) growing the unicellular organism under appropriate nutrient conditions in which the unicellular organism expresses the intact biologically functional DNA and produces the biologically functional polypeptide molecule;

(c) and recovering the biologically functional polypeptide molecule.

28. A method of reducing the expression of an intact biologically functional DNA which comprises the steps of:

(a) transforming under transforming conditions compatible unicellular organism with the recombinant DNA expression vehicle of claim 20; (b) growing the unicellular organism under appropriate nutrient conditions in which the unicellular organism expresses the intact biologically functional DNA and produces the biologically functional polypeptide molecule; and recovering the biologically functional polypeptide molecule.

29. A method of expressing a protein in a system having a DNA sequence and a duplex polynucleotide regulatory sequence, the DNA sequence having a polynucleotide sequence which encodes a biologically functional protein, a duplex polynucleotide regulatory sequence associated with the DNA sequence thereto, and a duplex polynucleotide sequence flanking the duplex polynucleotide regulatory sequence, the method comprising, expressing the biologically functional protein in the presence of the DNA sequence wherein the duplex polynucleotide sequence flanking the duplex polynucleotide regulatory sequence is heterologous to the duplex polynucleotide regulatory sequence and selected to enhance contact of a ligand to the duplex polynucleotide regulatory sequence.

30. The method of claim 29, wherein the duplex polynucleotide regulatory sequence is selected from the group consisting of: a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

31. A method of isolating duplex polynucleotide molecules with relatively high or relatively low binding affinity to a given ligand from a plurality of duplex polynucleotide jnolecules, the method comprising the steps of:

(a) providing a plurality of different duplex polynucleotide molecules wherein each of the duplex polynucleotide molecules has the same duplex regulatory polynucleotide sequence and a randomly synthesized duplex flanking polynucleotide sequence or a pair of randomly synthesized duplex polynucleotide sequences adjacent to the regulatory duplex polynucleotide sequence; (b) exposing the duplex polynucleotide molecules to a ligand selective for the duplex regulatory polynucleotide sequence; (c) isolating duplex polynucleotide molecules which bind to the ligand.

32. The method of claim 31 , wherein the duplex polynucleotide regulatory sequence is s selected from the group consisting of: a duplex polynucleotide promoter sequence, a duplex polynucleotide enhancer sequence, a duplex polynucleotide operator sequence and a long terminal repeat, or any combination thereof.

33. The method of claim 32, wherein the step of isolating the duplex polynucleotide o molecules which contact the ligand includes affinity purification or band shifting in order to separate duplex polynucleotide molecules bound to a ligand from non-contacted duplex polynucleotide molecules.