WO1993003138A1

WO1993003138A1 - A method for over-expressing nucleic acids using an enhancer sequence from potato virus x

Info

Publication number: WO1993003138A1
Application number: PCT/US1992/005403
Authority: WO
Inventors: Joseph G. Atabekov; Sergei Yu Morozov
Original assignee: Scientific Dimensions Usa, Inc.
Priority date: 1991-06-27
Filing date: 1992-06-26
Publication date: 1993-02-18
Also published as: AU2305592A; RU2005788C1

Abstract

A method is described for efficiently expressing the products of selected nucleic acid sequences in cell-free systems, in cells, or in transgenic plants. The method is comprised of inserting the selected nucleic acids downstream from the αβ sequence derived from potato virus X (PVX) or from subfragments of the αβ sequence that retain enhancer function, and then introducing the hybrid construct into the genome of a cell or a plant. A phagemid (pKX3αβ) was constructed in which the αβ sequence was inserted 5' to several cloning sites into which the selected nucleic acid can be inserted. This phagemid can be maintained in Escherichia coli as a double-stranded plasmid; or it can be isolated as single-stranded phage, or it can be used to selectively synthesize RNA transcripts for hybridization probes, in vitro translation, protoplast electroporation and cell micro-injection.

Description

AMETHODFOROVER-EXPRESSINGNUCLEICACIDSUSINGANENHANCERSEQUENCEFROMPOTATOVIRUS

FIELD OP THE INVENTION

The present invention relates to the use of cis- acting nucleic acid seguences to enhance the translation of isolated nucleic acid fragments. It also relates to the development of transgenic plants and cells that express the products of foreign DNA seguences in detectable, biologically effective concentrations.

BACKGROUND OF THE INVENTION

The explosive progress in recombinant technigues have made it possible to obtain biologically important proteins in sufficient guantities for performing pioneering clinical studies, for developing new therapies, and for commercial exploitation. They have also led to the production of transgenic prokaryotic and eukaryotic animals and plants that express foreign genes and exhibit traits encoded by those genes. These transgenic organisms have been the subject of a host of studies and discoveries relating to normal physiological mechanisms and pathological states. They have also been instrumental in the development of effective treatments for different diseased states.

For many reasons, plants have been an especially fertile system in which to conduct these studies. The tissues of many plant species have been genetically and phenotypically transformed by the introduction of exogenous genes. In some plant species, genetically altered tissues or cells may be induced to develop into transgenic plants which can transmit the introduced traits to their progeny through normal Mendelian inheritance. The potential commercial utility of transgenic plants is enormous. Instead of slowly and painstakingly breeding plants over several generations to develop strains that possess certain desirable features (e.g. size, timing of crop, size of crop, resistance to plant diseases, etc.), with the advent of methods for producing transgenic plants it is possible to produce such strains in just one generation simply by inserting the genes that encode these traits.

There are several technigu.es for producing transgenic cells and plants. One method involves exploiting the unique properties of the plant pathogen Acrrobacterium tumefaciens. Natural (or wild-type) A. tumefaciens harbors a tumor-inducing plasmid. Acrrobacterium introduces this plasmid into the genome of the infected plant host. The genetically altered host cells become tumorous and produce plant metabolites called opines on which the Aσrobacteriu has the unique ability to feed. By removing the genes responsible for tumor induction and opine production and replacing them with selected exogenous chimeric genes, the natural pathogenic properties of A_j_ tumefaciens can be exploited to introduce foreign genes into plant tissues.

Other techniques for producing transgenic cells and plants involve the direct introduction of nucleic acids into the cytoplasm of plant cells. Microinjection is one of the more popular methods for introducing foreign DNA sequences into a subject cell. Another technique is electroporation, in which cells (usually protoplasts) are exposed to an electric shock that disrupts the cellular membranes and causes the transient formation of pores through which diffusible substances of appropriate size can penetrate into the cell. Plant protoplasts that are subjected to electroporation can incorporate nucleic acid constructs from the surrounding medium. Finally, a variety of vectors (nucleic acid constructs derived principally from plasmids and viruses that possess a variety of properties, such as the ability to direct the production of proteins and nucleic acids that result in replication and expression, that enable them to replicate in cells and express encoded proteins) have been developed that have greatly facilitated the expression of• recombinant nucleic acids and proteins.

Once introduced, the exogenous nucleic acids (The term nucleic acid is meant to encompass both RNA and DNA sequences, since it is currently a routine matter to generate a DNA sequence using the RNA as a template, and vice versa.) may be incorporated into the genome of the plant and expressed in the same way as the plant's intrinsic genes, generation after generation. Some times the exogenous nucleic acids are not incorporated into the host cell genome. While for some applications non-incorporation might be desirable, for the purpose of producing a cell line that permanently expresses the transgene from generation to generation it is far preferable that the transgene be inserted into the host genome.

Since the foreign genes exert their effects primarily through their gene products, it is important that they be expressed at biologically effective levels (i.e., the gene products of the introduced sequence must be produced in detectable quantities and/or they must have a measurable phenotypic effect) . To this end, many scientists have focused on optimizing the transcription and translation of foreign cDNA seguences that are introduced into plants.

Many nucleic acid seguences that regulate the rates of transcription and translation have been identified. For example, the 5' untranslated leaders of various plant and animal viral mRNAs have been shown to enhance the translation of foreign eukaryotic and prokaryotic genes, both in vivo and in vitro . Gallie D.R. , et al . (1987) Nucleic Acids Res. 15, 8693-8711; Jobling S.A. and Gehrke L. (1987) Nature 325, 622-625; Dolph P.D., et al . (1988) J. Virol. 62, 2059-2066; Ruiz-Linares A., et al . (1989) Nucleic Acids Res. 17, 2463-2476; Carrington J. C. and Freed D. D. (1990) J. Virol. 64, 1590-1597. Commonly used plant cell transcription promoters include the nopaline synthase promoter from the T-DNA of Aσrobacterium tumefaciens and the 35S promoter from the cauliflower mosaic virus.

Although these promoters are effective in any plant cells, the level of transcription varies considerably. The degree of enhancement depends on many factors, such as the number of the insertion events, the location of the insertion site or sites the genome of the transformed cell, the absence or presence of cis-or trans-acting regulatory factors. No one enhancer is equally effective in all circumstances. Therefore, a considerable amount of effort has been expended to identify new enhancers, to chart their properties and optimize their effects.

The traditional protocols for identifying, amplifying, sequencing and packaging DNA fragments used to produce transgenic organisms were time consuming. Each step often required that the DNA fragments be excised and inserted into a particular environment (e.g., plasmids were used for cloning and amplifying, and viral particles were used for sequencing) . The use of phagemids (recombinant nucleic acid constructs that are capable of being passaged and amplified in bacteria and also of being packaged into viral particles) , of which PTZ18R (Pharmacia-LKB) is an example, has greatly reduced the effort for certain applications. We have recently demonstrated that a region called the aβ seguence derived from potato virus X (PVX) is an efficient enhancer of gene expression in cell free systems. Smirnyagina et al . (1991), Biochimie 73: 587- 598) . Our invention involves the use of this sequence and its homologues to enhance the expression of exogenous nucleic acids in cell free systems and in transgenic cells and plants.

OBJECTS AND SUMMARY OF THE INVENTION

For the reasons mentioned above, it is an object of this invention to utilize subfragments of the PVX αβ seguence to provide improved new methods and nucleic acid constructs for the efficient expression of selected nucleic acids in cell free systems, in cells, and in transgenic plants.

It is a further object of this invention to provide a vector and a method for reducing the number of steps required for producing transgenic cells and plants, which include forming ah appropriate construct, sequencing the construct; performing site specific mutations; efficiently transforming cells; efficiently expressing the selected gene or DNA, and generating clones to maintain, and from which to harvest, the chimeric construct.

It is another object of this invention to provide a phagemid having the following properties:

(i) selected nucleic acids can be inserted in the vicinity of the efficient α?-leader translational enhancer, or of a fragment having enhancer functions;

(ii) the phagemid can be used for the in vitro synthesis of mRNA transcripts of isolated genes attached to the 5^•'-proximal translational enhancer sequence;

(iii) selected genes or cDNAs that have been spliced downstream from the translational enhancer sequence can be excised together with the enhancer and this chimeric construct can easily be inserted into vectors that contain eukaryotic transcription signals in order to obtain high-level gene expression in transgenic plants or animals; and

(iv) the α3-seguence can be used as a specific tag for monitoring the transcription level of newly introduced genes in the cases when the transgenic cells are constructed to amplify authentic host genes.

The present invention provides an improved method for the enhanced expression of selected gene products, wherein selected nucleic acids are ligated to the PVX mRNA aβ enhancer fragments or to fragments thereof having enhancer function, forming constructs which may be either translated in vitro in cell-free systems, or used to produce transgenic cells, animals and plants that express the gene product.

The present invention also provides a phagemid (pXK3α,9) , constructed by inserting the aβ sequence derived from PVX mRNA into the phagemid pTZ18R (Pharmacia) , that can be maintained in Escherichia coli as a double-stranded plasmid, or isolated as single- stranded phage, or used to selectively synthesize RNA transcripts for hybridization probes, in vitro translation, protoplast electroporation and cell micro- injection. This pXK3α_/3 is a versatile cloning and expression vector that allows nucleic acid sequences such as genes or cDNAs that are inserted downstream from the α,9-leader to be sequenced and/or mutated, or excised as a cassette that contains the αβ sequence together with the gene or cDNA in preparation for microinjection, and/or inserted into plant- and animal-specific vectors. This invention further provides additional phagemids derived from the PXK3α,*3. One of them (pKX3 ) contains the a region of the aβ seguence, the other one (pKX3,9) the β region. It also provides a variety of subfragments derived from the aβ sequence that possess enhancer functions.

This invention also provides a method and a product for producing transgenic cells and transgenic plants.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. (Published) Nucleotide Sequences of the aβ sequence.

Fig. 2. (Published) Cell-free translation of the neomycin phosphotransferase I (NPTI) gene ligated to the PVX aβ seguence (α,9-NPTI) or to the tobacco mosaic virus Ω enhancer (Ω-NPTI) in different protein synthesizing systems: rabbit reticulocyte lysate (RRL) , wheat germ lysate (WG) or Krebs-2 ascite lysate (KA) .

A. Translation in RRL: α_/3-NPTI: 10 μg/ l, 20 μ/ml, 40 μ/ml (lanes

1, 2, 3);

Ω-NPTI: 10 μg/ml, 20 μg/ml, 40 μg/ml

(lanes 4, 5, 6) ;

No enhancer-NPT-1: 40 μg/ml (lane 7) ; no

RNA added (lane 8) .

B. Translation in KA:

Ω-NPTI: 40 μg/ml, 20 μg/ml (lanes 1, 2) ; No enhancer-NPTI: 40 Ωg/ml (lane 3); α3-NPTI: 40 μg/ml, 20 μg/ml (lanes 4, 5).

C. Translation in WG:

No enhancer-NPT-1: 80 μg/ml, 120 μg/ml (lanes 1, 2): α3-NPT-l: 80 μg/ml, 120 μg/ml (lanes 3,4); no RNA added (lane 5) 8

Fig. 3. (Published) Nucleotide sequences of the 5'- untranslated leaders of NPT II gene transcripts. Fig. 4. (Published) SDS-PAGE of [³⁵S]-labelled translation products (rabbit reticulocyte lysate) of synthetic RNA transcripts of NPT II gene. Lane 1, control NPT II transcript; lane 3, aβ-containing transcript; lane 3, no RNA added. Concentration of transcripts was 20 μg/ml. Fig. 5. (Published) Nucleotide seguences of the 5'- untranslated leaders of Bacillus thuringiensis toxic protein gene transcripts. Fig. 6. (Published) SDS-PAGE of [³⁵S]-labelled translation products (rabbit reticulocyte lysate) of synthetic RNA transcripts of B. thuringiensis insect control protein gene. Lanes 1 and 2, control transcript (20 and 40 μg/ml, respectively); lanes 3 and 4, αβ- containing transcripts (20 and 40 μg/ml, respectively) . Fig. 7. (Published) Nucleotide sequences of the 5'- untranslated leaders of GUS gene transcripts. Fig. 8. Published) SDS-PAGE of [³⁵S]-labelled translation products (rabbit reticulocyte lysate) of synthetic RNA transcripts of GUS gene. Lanes 1 and 2, control transcript (46 nt leader) (20 and 40 μg/ml, respectively) . Lanes 3 and 4, control transcript (12 nt leader) (20 and 40 μg/ml, respectively) . Lanes 5 and 6, αβ-containing transcript (20 and 40 μg/ml, respectively. Fig. 9. The DNA sequences of the polylinker regions of pXK3α and pXK3J phagemids (A) and nucleotide sequences of the leader regions of the corresponding in vitro RNA transcripts. Fig. 10. Autoradiogram of an 8-20% gradient polyacrylamide-SDS gel containing [³⁵S]-labelled translation products (rabbit reticulocyte lysate) directed by an uncapped RNA transcript of pTMV-CP (lane 1), pXK3α/ϊTMVCP (lane 2), PXK39TMVCP (lane 3), pXK3αTMVCP (lane 4). The concentration of transcripts was 40 ug/ml. The positions of endogenous product (E) and TMV coat protein (CP) are indicated.

Fig. 11. Schematic map of phagemid pXK3Q._/3. The numbers correspond to the parental plasmid pTZ18R.

Fig. 12. Nucleotide sequence of the region of pXK3α3 that contains the T7 promoter, the α3-leader and the polylinker.

Fig. 13. a.) Schematic maps of RNA transcripts wherein different subfragments of the aβ region were spliced to the NPT1 gene. b.) Efficiency of translation of the RNA transcripts in 13.b.

DETAILED DESCRIPTION OF THE INVENTION

We have reported the complete nucleotide seguence of PVX genomic RNA (Skryabin et al . (1988) Nucleic Acids Res. 16, 10929-10930). The 5' leader of potato virus X (PVX) genomic RNA (the 83 nucleotide sequence adjacent to the cap structure) has been shown to consist of two distinct sub-sequences, the α-sequence (41 nucleotides 3' to the cap that lack G) and the β-sequence (42 nucleotides upstream from the first AUG) . (Fig. 1, from Smirnyagina et al . (1991) Biochimie 73: 587-598) Computer-based folding predictions suggest that the 5'- proximal region of the αβ sequence is unstructured. In addition, the αβ sequence contains segments that are apparently complementary to the 3'-terminal region of 18S rRNA. Recombinant DNA technigues well known to those skilled in the art were used throughout the studies. These techniques include cloning and production of single strand phagemid DNA (Vieira J. and Messing J. (1987) Methods Enzymol. 154, 3-11) , transformation (Mandel M. and Higa A. (1970) J. Mol. Biol. 53, 154-160), seguencing (Sanger F. et al . (1977) Proc Nat. Acad. Sci. USA 74, 5463-5467) , screening, agarose gel electrophoresis (Dretzen G. et al . (1981) Anal. Biochem. 12, 295-302), polyacrylamide gel electrophoresis (Maxam A.M. and Gilbert W. (1980) Methods Enzymol. 65, 499-503), restriction enzyme mapping (Smith H. 0. and Bernstiel M. L., (1976) Nucl. Acids Res. 3, 2387-2394), modification of DNA fragments (Norrander J. et al . (1983) Gene 26, 101-107), preparation of plasmid DNA (Birnboim H.C. (1984) Methods Enzymol. 100, 243-250) , Southern blotting (Southern E.M. (1975) J. Mol. Biol. 98, 503-520) and filter hybridization (Grunstein M. and Wallace J. (1980) Methods Enzymol. 154, 3-11).

The αβ sequence enhanced the expression of several reporter genes that were inserted 3' to the αβ seguence and translated in cell free systems.

The αβ sequence enhanced the translation of a neomycin phosphotransferase I (NPT1) gene in cell-free systems from rabbit reticulocytes (RRL) , wheat germ (WG) and Krebs-2 ascites cell extracts (KA) by 6 to 40-fold (Fig. 2, taken from Smirnyagina et al . , (1991) Biochimie 73: 587-598). The PVX αβ sequence was about as effective as the TMV Ω sequence in enhancing the expression of a reporter gene. (Fig. 2) However, although the TMV sequence is slightly more efficient, it cannot be used for constructing phagemids similar to the PVX-based pXK3α,9 because the lac Z sequence of the resulting construct contains termination codons in all three reading frames. In a competitive translation experiment (data not shown) , PVX mRNA strongly inhibited TMV mRNA and some other plant virus mRNAs in RRL and WG systems. This competitive ability did not correlate with the presence of the aβ seguence in the competing mRNA. The evidence suggests that the aβ seguence together with about 150 nucleotides of the coding seguence is responsible for the translation competitive ability of PVX RNA.

Enhancement was also observed when the aβ was attached to three other reporter genes: neomycin phosphotransferase II (NPTII, Figs. 3 and 4) Bacillus thuringiensis insect central protein (Figs. 5 and 6) and 3-glucuronidase (GUS, Figs. 7 and 8) . These experiments tested the properties of different chimeric constructs and the influence of gene coding sequences and the non- viral spacer sequences located between aβ sequence and AUG-initiator on the translational enhancement.

For the experiments with the neomycin phosphotransferase type II (NPT II) gene, the Bglll- and BamHl-cut restriction fragment was excised from the pNEO plasmid (Pharmacia-LNB) and spliced into the BamHl cloning site of a phagemid which is also a subject of this invention, pXK3α,5, whose construction is described below. The 5' leader of the transcript of the resulting plasmid includes a relatively long spacer (38 nucleotides in length) located between the AUG initiation codon and the 5'-terminal aβ sequence (Fig. 3) . In the control construct the reporter gene was inserted into the same sites of the pTZlδR polylinker. The plasmids were transcribed without m⁷GpppG, and the resulting transcripts were translated in the rabbit reticulocyte lysate.

The expression of the NPT II gene was found to be enhanced several fold (Fig. 4) . These results demonstrate that a high level enhancement in the translation of a foreign gene can occur irrespective of the length of a spacer upstream from the initiation codon.

DNA fragments containing Bacillus thuringiensis coleopteran-specific toxic protein gene (Fig. 5) (Dobrzhanskaya et al . , unpublished data) and β- glucuronidase (GUS) gene (Fig. 7) were fused into BamHl- and Hindlll-cut pXK3α,9. The translation in rabbit reticulocyte lysates of both reporter genes was considerably enhanced when the αβ seguence was present (Figs. 6 and 8) .

The α sub-region by itself can enhance the translation of reporter genes to a level comparable with that achieved using the αβ seguence (Figs. 9 and 10) . A DNA fragment that contained the entire α sequence and 8 base pairs of the β sequence (residues 1-50 of the PVX genome) was excised from pXK3α3 by co-digestion with EcoRI and Muni. The fragment was purified by gel electrophoresis and then fused to an EcoRI-cut pTZ18R. The blue colonies that were obtained after transformation of E. coli strain XL-IB were screened by hybridization and sequence analysis. The DNA sequence corresponding to α-sequence and the polylinker in resulting PXK3α phagemid are presented in Fig. 9A. The large DNA fragment isolated during electrophoretic separation of the fragments of an EcoRI-Muni-cut pXK3α_/3 was purified, religated and used to transform XL-IB culture. The DNA sequence of the resulting plasmid pXK33 was verified by sequencing (Fig. 9A) .

To measure the enhancer properties of isolated α- and β- sequences, a tobacco mosaic coat protein (TMV-CP)- reporter gene was inserted into the Pst I site of pTX18R (control), pXK3α,5, pXK3α and pXK3_/3. The sequences of the 5' leaders of the corresponding RNA transcripts are shown in Fig. 9B.

The expression of the TMV-CP gene in rabbit reticulocyte lysates was found to be enhanced significantly by the aβ seguence as well as by the α sequence alone. No enhancement was observed when the TMV-CP was coupled to the β seguence (Fig. 10) .

Example

We have produced a versatile phagemid expression vector (pXK3α3, Figs. 11 and 12) that allows selected nucleic acid fragments to be inserted downstream from the aβ sequence, sequenced, transcribed in vitro or in vivo, mutated at will and/or excised as a cassette (together with the aβ seguence) for insertion into plant- and animal-specific vectors.

The phagemid was constructed as follows: A double- stranded DNA corresponding to the aβ sequence was chemically synthesized and fused to an EcoRI- and S al- cut pTZ18R phagemid (Pharmacia-LKB) . The resulting pXK3α/3 contained the aβ sequence flanked by an EcoRI site at its 5' border and multiple cloning sites at its 3' border (Figs. 11 and 12) . It also retained all the properties of the parental vector: pKX3α3 can be maintained in E. coli as double-stranded plasmid, or it can be isolated as single-stranded phage following infection of XL-IB or NM522 cultures with the M13K07 helper phage (Pharmacia-LKB) . The phagemid also contains the viral T7 promoter (Fig. 12) which can be used to selectively synthesize RNA transcripts for hybridization probes. Finally, pK 3aβ can be used for in vitro translation, protoplast electroporation and cell micro- injection. Foreign genes or cDNAs can easily be inserted into any of the cloning sites, and the desired chimeric constructs can easily be cloned, isolated and maintained in Escherichia coli strains XL-IB (Stratagene) or NM522 (Pharmacia-LKB) . The Escherichia coli clones that contain an insert can easily be identified by exploiting the fact that the multiple cloning sites are located within the lac Z gene of the pXK3α/3 phagemid. The successful insertion of a selected gene or cDNA will disrupt the lac Z gene. Escherichia coli clones that contain the unmodified pXK3α? can be distinguished from those that contained a modified phagemid by the blue color which the former give on appropriate X-gal indicator plates. This property facilitates cloning by using blue/white screening of recombinants.

Finally, the αβ sequence is also efficient as a translational enhancer in vivo (in protoplasts) and can be used for super-production of proteins in transgenic plants and probably in yeast cells and transgenic animals.

Example

In addition to the αβ sequence itself, several subfragments derived from therefrom are capable of enhancing translation in cell free systems. The deletion of a segment located in the middle of the α region actually enhanced expression (Figure 13) . Furthermore, the insertion of four bases into the β seguence resulted in a sharp six-fold decrease in the enhancement properties of the αβ sequence (Figure 13) . Thus, it seem that long term structural interactions between the different elements of the αβ sequence may play a role in its ability to enhance translation in cell free systems. Example

Modifications and variations of the methods and constructs used throughout this disclosure will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Atabekov, Joseph G. Morozov, S. Yu.

(ii) TITLE OF INVENTION: A Method for Overexpressing Nucleic Acids in Cell Free Systems, Cells and Plants Using a Multifunctional Phagemid that Contains an Enhancer Sequence Derived from Potato Virus X

(iii) NUMBER OF SEQUENCES: 15 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Patrick J. Birde

(B) STREET: One Broadway (C) CITY: New York

(D) STATE: NY

(E) COUNTRY: USA

(F) ZIP: 10004

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version

#1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: May 8, 1992

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Birde, Patrick J

(C) REFERENCE/DOCKET NUMBER: 1036-24 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 425-7200

(B) TELEFAX: (212) 425-5288

(C) TELEX: 422141 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 83 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Potato Virus X

(viii) POSITION IN GENOME:

(B) MAP POSITION: 1-83

(C) UNITS: bp

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GAAAACUAAA CCAUACACCA CAACACAACC AAACCCACCA CGCCCAAUUG UUACACACCC 60 GCUUGGAAAA GCAAGUCUAA CAA

83 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGAAUUCGA GCUCGGUACC CGGGGAUCUG AUCAAGAGAC AGGAUGAGGA UCGUUUCGC 59 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 123 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Potato Virus X

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGAAUUCGA AAACUAAACC AUACACCAAC AACACAACCA AACCCACCAC GCCCAAUUGU 60

UACACACCCG CUUGGAAAAG UAAGUCUAAC GGGGAUCAAG AGACAGGAUG AGGAUCGUUU 120 CGC

123 (2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: GGGAAUUCGA GCUCGGUACC CGGGGAUCCG GGAGGAAGAA AA 42 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 124 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus thuringiensis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGAAUUCGA AAACUAAACC AUACACCAAC AACACAACCA AACCCACCAC GCCCAAUUGU 60

UACACACCCG CUUGGAAAAG UAAGUCUAAC GAGCUCGGUA CCCGGGGAUC

CGGGAGGAAG 120

AAAA

124 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGGCGAAUUC GAGCUCGGUA CCGGAUCCCC GGGUGGUCAG UCCCUU

46 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGGAAUUCUA CC 12

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GGGAAUUCGA AAACUAAACC AUACACCAAC AACACAACCA AACCCACCAC GCCCAAUUGU 60

UACACACCCG CUUGGAAAAG UAAGUCUAAC GGGAUCC 97 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AATTCGAAAA CTAAACCATA CACCAACAAC ACAACCAAAC CCACCACGCC CAATTCGAGC 60

TCGGTACCCG GGGATCCTCT AGAGTCGACC TGCAGGCATG CAAGCTT 107

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: AATTGTTACA CACCCGCTTG GAAAAGTAAG TCTAACGGGG ATCCTCTAGA GTCGACCTGC 60 AGGCATGCAA GCTT

74 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 67 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGAAUUCGA GCUCGGUACC CGGGGAUCCU CUAGAGUCGA CCUGCAGGUC GAGGAUUCGU 60 AUUAAAU

67 2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 136 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: tabacco mosaic virus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAAUUCGA AAACUAAACC AUACACCAAC AACACAACCA AACCCACCAC GCCCAAUUGU 60

UACACACCCG CUUGGAAAAG UAAGUCUAAC GGGGAUCCUC UAGAGUCGAC CUGCAGGUCG 120 AGGAUUCGUA UUAAAU

136 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 118 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGGAAUUCGA AAACUAAACC AUACACCAAC AACACAACCA AACCCACCAC GCCCAAUUCG 60 AGCUCGGUAC CCGGGGAUCC UCUAGAGUCG ACCUGCAGGU CGAGGAUUCG UAUUAAAU 118 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AAUUGUUACA CACCCGCUUG GAAAAGUAAG UCUAAGGGGA UCCUCUAGAG UCGACCUGCA 60

GGUCGAGGAU UCGUAUUAAA U

81 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 144 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA ^* (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Potato Virus X (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TAATACGACT CACTATAGGG AATTCGAAAA CTAAACCATA CACCACAACA CAACCAAACC 60

CACCACGCCC AATTGTTACA CACCCGCTTG GAAAAGCAAG TCTAACGGGG ATCCTCTAGA 120 GTCGACCTGC AGGCATGCAA GCTT 144

Claims

2-1 CLAIMS

We claim:

1) A vector for cloning purified nucleic acid fragments and expressing them in cells, plants and cell- free systems, comprising a subfragment of a Potato Virus X aβ enhancer seguence having enhancer function, covalently linked to an isolated purified nucleic acid not of Potato Virus origin.

2) A vector according to claim 2 that is a member of the group that includes plasmids, phagemids, and phages.

3) A vector according to claim 2 in which the isolated purified nucleic acid not of Potato Virus X origin is covalently linked 3' to the subfragment of the aβ enhancer sequence.

4) A phagemid according to claim 2 wherein said phagemid further comprises a nucleic acid sequence derived from a pTZlδR phagemid.

5) A phagemid according to claim 2 wherein said phagemid is selected from the group that includes the phagemids pXK3α3, pXK3α, and pXK3_/3.

6) A cell that comprises the isolated, purified nucleic acid sequence covalently linked to the aβ subfragment according to claim 1.

7) The cell of claim 6 in which the isolated, purified nucleic acid fragment is covalently linked 3' to the subfragment of the aβ sequence.

8) The cell of claim 7 in which the isolated, purified nucleic acid sequence covalently linked to a subfragment of the aβ sequence is incorporated into the cell's genome.

9) A plant comprising a cell according to claim 7.

10) A plant comprising a cell according to claim 8.

11) A seed comprising a cell according to claim 7.

12) A seed comprising a cell according to claim 8.

13) A vector according to claim 2 having inserted therein a nucleic acid sequence having substantially the same sequence and substantially the same enhancing activity as the PVX a sequence.

14) A vector according to claim 13 having inserted therein a subfragment of the PVX a sequence having substantially the same enhancing activity as the PVX a sequence.

15) A vector according to claim 2 having inserted therein a nucleic acid sequence having substantially the same sequence and substantially the same enhancing activity as the PVX β sequence.

16) A vector according to claim 15 having inserted therein a subfragment of the PVX β sequence having substantially the same enhancing activity as the PVX β sequence.

17) In a method causing expression of a recombinant nucleic acid, said system being capable of a predetermined rate of polypeptide expression, an improvement comprising covalently linking said recombinant nucleic acid 3' to a subfragment of a Potato Virus X aβ enhancer seguence having enhancer function.

18) A method according to claim 17 in which the constructs are introduced into cells via the group of techniques that includes infection, transfection, transformation, microinjection, electroporation, and calcium precipitation.

19) A method for cloning and expressing purified isolated nucleic acid sequences in cell free systems, cells or plants, comprising: a) a member of a group of plasmids, phagemids and phages that includes the phagemids pXK3a3, pXK3α and pXK3/3; b) having inserted therein a nucleic acid fragment covalently linked to a nucleic acid fragment that contains a region having substantially the same sequence and substantially the same expression- enhancing effect as the group of sequences that includes the Potato Virus X aβ sequence and the a sequence; c) further comprising cloning any of the group of constructs described in 19.b. into a suitable bacterium; d) further comprising sequencing the constructs to confirm that they are indeed the constructs described in 19.b. ; e) further comprising amplifying and purifying the constructs; f) further comprising introducing the construct into cells; g) further comprising using probes that are complementary to any part of the aβ sequence as a specific tag for monitoring the transcription level of newly introduced genes.

20) A vector for cloning purified nucleic acid fragments and expressing them in cells, plants and cell- free systems, comprising a Potato Virus X aβ enhancer sequence having enhancer function, covalently linked to an isolated purified nucleic acid not of Potato Virus origin.