US20030045701A1

US20030045701A1 - Polynucleotide constructs and methods of expression in a host cell

Info

Publication number: US20030045701A1
Application number: US10/214,655
Authority: US
Inventors: Johnway Gao; Daniel Anderson; Brian Hooker; Rodney Skeen
Original assignee: Individual
Current assignee: Battelle Memorial Institute Inc
Priority date: 2000-03-27
Filing date: 2002-08-07
Publication date: 2003-03-06
Also published as: WO2001073083B1; WO2001073083A2; EP1268824B1; US6465635B2; US20020102636A1; WO2001073083A3; ATE288495T1; AU2001247842A1; DE60108729D1; DE60108729T2; EP1268824A2

Abstract

The invention includes polynucleotide constructs that contain a polynucleotide promoter sequence from the phosphoglycerate kinase 1 (pgk1) gene from Rhizopus oryzae. The invention also includes methods of expressing a polypeptide in a host cell using a polynucleotide construct containing a 3-phosphoglycerate kinase (pgk) gene promoter sequence operably linked to a polynucleotide encoding a polypeptide of interest. The invention also includes a system for producing a polypeptide. The system has a first polynucleotide sequence containing a 3-phosphoglycerate kinase gene promoter sequence isolated from a fungal species, and a second polynucleotide sequence which has been isolated from a second species and which encodes a polypeptide. A host cell contains the first polynucleotide sequence operably linked to the second polynucleotide sequence. The host cell is from a different species than the fungal species from which the first polynucleotide sequence is isolated.

Description

RELATED PATENT DATA

This application is a continuation-in-part of U.S. application Ser. No. 09/536,034 which was filed on Mar. 27, 2000 and which is hereby incorporated by reference.[0001]

CONTRACTUAL ORIGIN OF THE INVENTION

[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of contract number DE-AC0676RLO1830, awarded by the U.S. Department of Energy.

TECHNICAL FIELD

The invention pertains to recombinant polynucleotide constructs and methods of expression in a host cell.

BACKGROUND OF THE INVENTION

Introduction of a polynucleotide sequence that encodes a protein into a host cell can allow the host cell to express the encoded protein from the polynucleotide sequence. Recombinant polynucleotide introduction can allow host cells to produce engineered proteins or proteins native to other organisms. Recombinant polynucleotides can also be introduced to increase copy numbers of sequences native to the host. Expression of proteins from a host cell using recombinant polynucleotides is useful for a variety of applications including protein function studies, production and isolation of a desired protein, conferring a desired phenotype to the host, regulating cell functions, and enhancing production of a protein native to the host cell.

The ability of a host cell to transcribe an introduced coding sequence can depend upon the presence of a functional promoter linked operably to the coding sequence. Additionally, the level of expression from the coding sequence can depend upon the specific promoter sequence utilized. Some promoters are constitutive while others are regulated. Additionally, some promoters function in only specific cell types, specific tissue types or specific organisms. It would be desirable to provide promoters that can function in multiple species and thereby allow recombinant polynucleotide molecules to be constructed for introduction and expression in a variety of hosts.

SUMMARY OF THE INVENTION

In one aspect, the invention encompasses a polynucleotide construct that contains a polynucleotide promoter sequence from the phosphoglycerate kinase 1 (pgk1) gene from Rhizopus oryzae, and also contains a polynucleotide sequence that encodes a polypeptide of interest.

In one aspect the invention encompasses a method of expressing a polypeptide in a host cell. A polynucleotide construct is formed containing a fungal 3-phosphoglycerate kinase (pgk) gene promoter sequence operably linked to a polynucleotide encoding a polypeptide of interest. The polynucleotide construct is introduced into a host cell.

In one aspect the invention encompasses a system for producing a polypeptide. This system includes a first polynucleotide sequence containing a 3-phosphoglycerate kinase gene promoter sequence isolated from a fungal species. The system also includes a second polynucleotide sequence which has been isolated from a second species and which encodes a polypeptide. A host cell contains the first polynucleotide sequence operably linked to the second polynucleotide sequence. The host cell is from a different species than the fungal species from which the first polynucleotide sequence is isolated.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described below with reference to the following accompanying drawings. [0009]
FIG. 1 is a flowchart diagram illustrating a particular aspect of the present invention. [0010]
FIG. 2 schematically depicts a method of isolating a promoter encompassed by the present invention. [0011]
FIG. 3 schematically depicts a method of vector construction according to one aspect of the present invention. [0012]
FIG. 4 schematically depicts formation of a plasmid vector according to one aspect of the present invention. [0013]
FIG. 5 shows the results of selective media screening of non-transformed (Panel A) and transformed (Panel B) tobacco callus cultures. [0014]
FIG. 6 shows the results of PCR analysis of DNA samples from cultured transgenic tobacco cells, comparing transgenic cell DNA samples (Lanes 1-10) to a positive control (Lane P) and a non-transformed cell culture sample (Lane C). Lane S corresponds to a DNA size-marker sample. [0015]
FIG. 7 shows the result of PCR analysis for detection of the presence of a polynucleotide construct according to one aspect of the present invention. The resulting PCR analysis of genomic DNA from transformed maize callus tissue (Lane 3) was compared to a positive control (Lane 2) and a non-transformed maize sample (Lane 4). [0016] Lane 1 corresponds to a polynucleotide size marker sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention encompasses recombinant gene expression involving an isolated polynucleotide promoter sequence. For purposes of the present description the term “expression” can refer to the combined process of transcription and translation, or can refer to a portion of the combined transcription and translation process. The term “isolated” can refer to a naturally occurring molecule such as, for example, a polynucleotide or a polypeptide that has been recovered from the organism which produced it, or alternatively can refer to a synthetic molecule. The invention also encompasses formation of polynucleotide constructs containing isolated polynucleotide promoter sequences, encompasses polynucleotide constructs, and encompasses host cells containing the polynucleotide constructs. [0017]
A process encompassed by the present invention is described generally with reference to the block diagram of FIG. 1. At an initial step (A) an isolated polynucleotide comprising a promoter sequence is provided. The isolated promoter sequence can be a promoter sequence isolated from, for example, a species within the phylogenetic kingdom Fungi. [0018]
In particular embodiments of the present invention, the isolated promoter utilized in step (A) of FIG. 1 is isolated from the fungal species [0019] Rhizopus oryzae. The isolated promoter can comprise an isolated polynucleotide promoter sequence from a 3-phosphoglycerate kinase gene (pgk), for instance pgk1. In a preferred embodiment, the isolated promoter is a functional promoter comprising the sequence set forth in SEQ ID NO:7. Alternatively, the isolated promoter can comprise a portion of the sequence set forth in SEQ ID NO:7. For example, an isolated promoter can be provided in step (A) which comprises a fragment of SEQ ID NO:7 which is unique relative to other known promoter sequences. The unique fragment can comprise, for example, at least nucleotides 1187-1254 of SEQ ID NO:7.
As will be understood by those of ordinary skill in the art, the promoter sequence set forth in SEQ ID NO:7 can be manipulated by deleting, inserting or modifying one or more nucleic acids within the sequence utilizing conventional methods to result in a sequence that retains promoter functionality. The retained functionality can, in certain instances, even be enhanced by such modification. Accordingly, the invention encompasses utilizing such modified sequences for the isolated promoter in step (A). [0020]

Referring to FIG. 2, an exemplary method for isolation of a promoter sequence for use in step (A) of FIG. 1 is shown. Genomic DNA can be isolated from, for example, cultured R. oryzae cells. The isolated genomic DNA can be purified and used for subsequent isolation of the R. oryzae pgk1 promoter. For instance, the genomic DNA (FIG. 2, top) can be digested with restriction enzymes to produce fragments of the genomic DNA. Such fragments can be self ligated to form circular DNA shown in FIG. 2 (center). Inverse PCR can then be performed to introduce desired restriction enzyme sites into the fragments. Exemplary reverse primers appropriate for use during such inverse PCR process are set forth in SEQ ID NOS:1 and 2. Exemplary forward primers for utilization during such inverse PCR process are set forth in SEQ ID NOS:3, 4, 5 and 6.

TABLE 1


PCR Reaction Primer Pairing for
Restriction Enzyme Digested Genomic DNA

Inverse PCR

Restriction

Primer Pairing

Reaction	Enzyme	Reverse primer	Forward primer

1	Bgl II	SEQ ID NO.:1	SEQ ID NO.:5
2	Bgl II	SEQ ID NO.:2	SEQ ID NO.:5
3	EcoR I	SEQ ID NO.:1	SEQ ID NO.:3
4	EcoR I	SEQ ID NO.:2	SEQ ID NO.:3
5	Hinc II	SEQ ID NO.:1	SEQ ID NO.:4
6	EcoR I	SEQ ID NO.:2	SEQ ID NO.:4
7	Kpn I	SEQ ID NO.:1	SEQ ID NO.:6
8	Kpn I	SEQ ID NO.:2	SEQ ID NO.:6
9	Nco I	SEQ ID NO.:1	SEQ ID NO.:4
10	Nco I	SEQ ID NO.:2	SEQ ID NO.:4
11	Sph I	SEQ ID NO.:1	SEQ ID NO.:3
12	Sph I	SEQ ID NO.:2	SEQ ID NO.:3
13	Xmn I	SEQ ID NO.:1	SEQ ID NO.:3
14	Xmn I	SEQ ID NO.:2	SEQ ID NO.:3

Table 1 shows exemplary restriction enzymes (column 2) that can be utilized during digestion of genomic DNA, and shows pairing of the exemplary primers for utilization during the inverse PCR process. It is to be understood that the invention encompasses use of alternative or additional restriction enzymes during the digestion and also encompasses use of alternative primers and primer pairing. [0022]
As shown in FIG. 2, the restriction enzyme digestion, self ligation, and subsequent inverse PCR can be used to produce the isolated pkg1 promoter for utilization in step (A) of FIG. 1. [0023]
Referring again to FIG. 1, a polynucleotide sequence that encodes a protein can be provided in step (B) and can be utilized in conjunction with the isolated promoter provided in step (A) for a polynucleotide construct formation step (C). The encoding polynucleotide provided in step (B) of is not limited to any specific polynucleotide sequence. The coding sequence can comprise sequence encoding any polypeptide of interest (see below), or can encode two or more polypeptides of interest. The phylogenetic origin of the coding sequence can be any desired species, either prokaryotic or eukaryotic. In particular embodiments, the native origin of the coding sequence can be a different species than the originating species of the isolated promoter, can be a different species than the originating species of the eventual host (discussed below), or both. [0024]
Polynucleotide construct formation step (C) can comprise fusing of the isolated promoter provided in step (A) with the coding sequence provided in step (B). Preferably, the isolated promoter is operably linked to the provided coding sequence. Operable linkage of a polynucleotide to a promoter to form a recombinant polynucleotide construct in step (C) can allow expression of the polynucleotide and production of the encoded polypeptide to be controlled by the promoter. [0025]
Recombinant DNA construct formation step (C) can additionally comprise formation of a vector. For purposes of the present invention, a vector can comprise a plasmid, cosmid, phage or yeast artificial chromosome (YAC). A particular vector to be formed in step (C) can be determined based on the intended use of the vector. For example, vector formation in step (C) can be determined by appropriateness of the vector for introduction to a host in step (D) (discussed below). [0026]
Once the desired polynucleotide construct is formed in step (C), the construct can be utilized for introduction into a host cell in step (D). Introduction into a host cell is not limited to a specific method of introduction. In particular embodiments, introducing a construct into a host cell can comprise transformation of a host cell utilizing one or more of electroporation, sonication, T-DNA mediated transformation, particle-bombardment mediated transformation, microinjection, virus-mediated transformation, whiskers mediated transformation, liposome mediated transformation, chemical mediated transformation and plasmid transformation. [0027]
The host cell into which the polynucleotide construct is introduced in step (D) is not limited to a particular type of cell. Such host cell can be prokaryotic or eukaryotic. Additionally, the host cell can be from a different species than the species from which the isolated promoter has been isolated, can be different from the species from which the coding polynucleotide is isolated, or can be different from both the originating species of the promoter and the originating species of the coding polynucleotide. [0028]
In particular embodiments, the host cell can be a species belonging to a phylogenetic kingdom that is different from the phylogenetic kingdom from which at least one of the isolated promoter and the isolated coding polynucleotide originate. For example, when the isolated promoter comprises a polynucleotide promoter sequence isolated from the kingdom Fungi, the host cell can comprise, for example, a cell belonging to a species within the kingdom Plantae. [0029]
Alternatively, when an isolated promoter is isolated from a species within a particular phylogenetic kingdom, the host cell can belong to a species within the same phylogenetic kingdom. When belonging to the same kingdom, the host cell species can be the same or can be different from the species from which the promoter is isolated. For example, when the isolated promoter provided in step (A) is a fungal promoter originating in the species [0030] Rhizopus oryzae, the host cell in step (D) can also be Rhizopus oryzae, can be a second species within the Rhizopus genus, or can belong to an independent genus or even an independent phylum within the Fungi kingdom.
The invention also encompasses introducing the constructs formed in step (C) into a prokaryotic host such as bacteria. The introduction of a construct of the present invention into bacteria can be useful for manipulation or amplification of the construct. [0031]
As shown in FIG. 1, once the recombinant polynucleotide construct has been introduced into the host cell, the polypeptide encoded by the polynucleotide can be expressed by the host cell in an expression step (E). Alternatively, the host cell can be utilized to assist transformation of a subsequent host cell, or to amplify or manipulate the polynucleotide construct (not shown). [0032]
In embodiments of the present invention wherein the encoded polypeptide is expressed in the host cell, the expressed polypeptide is not limited to any particular type of peptide and can be, for example, an intracellular peptide, an excreted or extracellular peptide, or a transmembrane peptide. Alternatively, the expressed peptide can confer resistance to the host cell. Such conferred resistance can be resistance to one or more of an antibiotic, a herbicide, a toxin, a parasite and a pathogen. The invention also encompasses introducing constructs where the coding sequence codes for a protein that can regulate or control expression of native genes within the host cell, or can regulate or control expression of introduced (heterologous) genes. Additionally encompassed are constructs, and hosts containing the constructs, where the protein encoded by the construct confers a new (non-native) phenotype to the host cell. Alternatively, the expressed polypeptide can have a sequence, a function, or an effect that is similar to or identical to one or more native polypeptides produced by the host. [0033]

EXAMPLES

Example 1

Expression from an Isolated Promoter in a Yeast Host Cell [0034]
The 3-phosphoglycerate kinase gene 1 (pgk1) was isolated from [0035] Rhizopus oryzae utilizing the exemplary method discussed above with reference to FIG. 2. The isolated pgk1 promoter was initially inserted into transient vector pGEM-T (Promega, Madison, Wis.) to form pGA2086 which was utilized for initial amplification, cloning, and sequencing of the promoter. The determined sequence of the isolated R. oryzae pgk1 promoter is set forth in SEQ ID NO:7.
The pgk1 promoter was subsequently cloned from pGA2086 utilizing a forward primer having the sequence set forth in SEQ ID NO:8, and a reverse primer having the sequence set forth in SEQ ID NO:9. Utilization of the specified forward and reverse primer introduced a Spe I restriction enzyme site at the 5′ end of the promoter sequence and introduced a BamH I restriction enzyme site at the 3′ end of the promoter sequence. The forward and reverse primers were used in conjunction with PCR to produce a polynucleotide comprising the entire SEQ ID NO:7 and having appropriate ends to allow insertion into vector pGA2028D to form plasmid vector pGA2174 as shown in FIG. 3. [0036]
As shown in FIG. 3, vector pGA2028D comprises an [0037] E. coli polynucleotide coding sequence which encodes the β-glucuronidase (gus) protein. Formation of plasmid vector pGA2174 included operably linking the isolated pgk1 promoter to the coding portion of the E. coli β-glucuronidase gene.
Plasmid vector pGA2174 was introduced into a Saccharomyces hybrid yeast strain (obtained from James R. Mattoon of University of Colorado) utilizing plasmid transformation of the Saccharomyces host. After transformation, the Saccharomyces cells were plated onto appropriate selective media containing glucose, and incubated at 30° C. for 4 days. [0038]

Transformed colonies were selected for determining activity of the introduced pgk1 promoter utilizing glucuronidase (GUS) activity analysis. Protein samples were collected from isolated colonies by suspending a single transformed colony and subsequently disrupting the cells. After disruption, the sample was centrifuged and the supernatant containing protein was analyzed for protein content and GUS activity.

TABLE 2


GUS expression under the control of Rhizopus oryzae 3-phosphoglycerate
kinase
1 promoter in transformed Saccharomyces colonies.

	GUS Specific Activity	Average Activity
Clone No.	(units/mg)	(units/mg)

C*	5	501 ± 74
1	503
2	459
3	420
4	556
5	529
6	464
7	509
8	471
9	497
10	517
11	444
12	722
13	507
14	524
15	482
16	456
17	629
18	373
19	504
20	473

The result of the GUS activity analysis is summarized in Table 2. The GUS specific activity (units of glucuronidase activity per milligram of total protein, wherein one unit of glucuronidase activity is the amount of glucuronidase that converts one pmole of 4-methylumbelliferul-beta-D-glucuronide (MUG) to 4-methylumbelliferone) is reported for twenty isolated transformed colonies in [0040] column 2. The measured GUS activity was compared to a control assay which utilized non-transformed cells (C*). The results shown in Table 2 indicate that the pgk1 promoter isolated from Rhizopus oryzae is able to function as a promoter in other fungal species.

Example 2

Expression in a Plant Cell Protoplast Host Utilizing an Isolated Fungal Promoter [0041]
Protoplasts were prepared from [0042] Nicotiana tabacum cell suspension cultures. Super-coiled plasmid pGA2174 (discussed above) was combined with salmon sperm DNA as a carrier, and was utilized for electroporation transformation of the protoplasts. The protoplasts were subsequently cultured for 48 hours at 28° C. in modified Murashige and Skoog (MS) medium in the presence of sucrose, +/−starch. The protoplasts were then tested for GUS activity.
The GUS activity of the protoplasts was analyzed by extracting protein samples from the protoplasts using sonication of suspended protoplasts, centrifugation, and collection of the protein in the resulting supernatant. The supernatant was assayed for GUS activity as described above in Example 1. [0043]

The results of the GUS activity analysis are shown in Table 3. Eight transformant protoplast samples (1-4 grown in the absence of starch and samples 5-8 grown in the presence of starch) were compared to a control (C; non-transformed protoplast sample). As indicated by the results shown in Table 3, the isolated R. oryzae pkg1 promoter is able to function as a promoter in plant protoplast cultures.

TABLE 3


GUS expression under the control of Rhizopus oryzae 3-phosphoglycerate
kinase
1 promoter in electroporated Nicotiana tabacum cells.

		GUS Specific	Average Activity
Test No.	Culture Medium	Activity (units/mg)	(units/mg)

C*	MS/Sucrose	10	—
1		53	70 ± 13
2		77
3		84
4		66
5	MS/Sucrose/Starch	35	52 ± 18
6		42
7		59
8		74

Example 3

Expression Utilizing an Isolated Fungal Promoter in a Dicot Plant Cell Culture [0045]
A vector was constructed for introducing a recombinant construct comprising the isolated [0046] R. oryzae pkg1 promoter operably linked to a resistance gene into a dicot type angiosperm plant cell host. The pkg1 promoter was initially recovered from plasmid pGA2086 (discussed above) utilizing a forward primer having the sequence set forth in SEQ ID NO:10, and a reverse primer having the sequence set forth in SEQ ID NO:11. Utilization of the specified primers allowed BamH I restriction enzyme sites to be incorporated at both the 5′ and the 3′ ends of the isolated promoter.
After PCR utilizing the specified primers, the recovered promoter was cloned into transient cloning vector pGEM-T (Promega, Madison, Wis.) for amplification, and was subsequently cloned into a Ti (tumor inducing) plasmid vector to form plasmid pGA2182 as shown in FIG. 4. Insertion of the isolated pgk1 promoter into the Ti plasmid vector operably linked the [0047] R. Oryzae pgk1 promoter to the coding region of the neomycin phosphotransferase II gene (npt II), thereby forming a recombinant pgk1-npt II construct. As shown in FIG. 4, pGA2182 comprises a left border (BL) T-DNA sequence and a right border (BR) T-DNA sequence (T-DNA being the portion of the Ti plasmid that is transferred to a plant cell host). The isolated pgk1 promoter is inserted such that the promoter, operably linked to the npt II coding region, can be transferred between the BL and BR to a plant cell host.
After construction of Ti plasmid vector pGA2182, the vector was utilized to directly transform [0048] Agrobacterium tumefaciens LBA4404 using the freeze-thaw method.
Bacteria mediated transformation was utilized to introduce the recombinant pgk1-npt II construct into tobacco cell cultures by co-cultivation of the tobacco cells with transformed Agrobacterium. After transformation, the plant cells were plated onto selective media and incubated at 29° C. [0049]
As shown in FIG. 5, non-transformed control tobacco cells (panel A) were unable to grow on the selective media. The transformed tobacco cells (panel B) were able to form plant callus cell colonies which appeared within about 3-5 weeks of incubation. The results of the selective media screening process indicate that the isolated fungal pgk1 promoter is able to function as a promoter in transformed tobacco calli. [0050]
Transformants were analyzed for insertion of the recombinant pgk1-npt II construct into the genomic DNA of the transformed tobacco cells. After selective media screening, tobacco calli transformants were utilized for producing subcultures for genomic DNA preparation. Genomic DNA was prepared utilizing the hexadecyltrimethyl ammonium bromide (CTAB) mini-preparation method. Once obtained, the resulting DNA samples were used for PCR amplification of the pgk1-npt II construct utilizing the forward primer having the sequence set forth in SEQ ID NO:10 and a reverse primer having the sequence set forth in SEQ ID NO:12. The specified primers allowed cloning and amplification of a polynucleotide fragment (approximately 1.8 kb) containing the pgk1 promoter and a portion of the npt II gene. [0051]
FIG. 6 shows the obtained PCR results obtained from 10 transgenic cell culture DNA samples (lanes 1-10) as compared to a control non-transformed tobacco calli sample (lane C) and a positive control which was prepared by PCR treatment of the Ti plasmid pGA2182 (lane P) utilizing the forward primer SEQ ID NO:10 and the reverse primer SEQ ID NO:12. A DNA size-marker sample is shown in Lane S. The results shown in FIG. 6 indicate that the pgk1 promoter-npt II coding region construct was integrated into the genomic DNA of the transgenic tobacco cells. [0052]

Example 4

Expression Utilizing an Isolated Fungal Promoter in a Monocot Plant Host [0053]
The constructed plasmid vector pGA2182 (discussed above) containing the pgk1 promoter operably linked to the npt II gene coding region was utilized for transformation of maize suspension cultures by particle-acceleration (bombardment) transformation. The maize suspension cultures to be transformed were prepared from embryos which were produced by cross-pollinating by hand maize inbred lines B73 and A188. Type II callus lines (from immature embryos of the hybrid cross) were utilized to start maize suspension cultures which were subsequently maintained in the dark at 25° C. These suspensions were subjected to bombardment procedures and, directly after bombardment, were transferred to osmotic medium and acquiesced. At 16 hours post-bombardment the embryos were transferred to selective medium containing kanamycin. After 14 days of growth on selective medium each callus piece was split into smaller tissue portions and transferred onto a second selective medium containing kanamycin for an additional 4 weeks. [0054]
The presence of positive callus lines after two selections indicates that the fungal pgk1 promoter is able to function as a promoter within the transformed maize callus line tissue. [0055]
After the second selection, seven positive callus lines were analyzed for genomic integration of the recombinant pgk1-npt II construct. Genomic DNA was extracted from the positive callus lines using the DNeasy Plant Mini Kit (QIAGEN, Valencia, Calif.). PCR was performed utilizing the forward primer having the sequence set forth in SEQ ID NO:10, and the reverse primer having the sequence set forth in SEQ ID NO:12. [0056]
The results of the genomic integration analysis are set forth in FIG. 7. The PCR analysis of a positive transformant maize line M32 is shown in [0057] Lane 3. PCR analysis of a non-transformed maize line M50B is shown in Lane 4. The positive control (Lane 2) was prepared by PCR analysis of plasmid pGA2182. As discussed above, the PCR utilizing the specified primers allows production of a polynucleotide fragment (approximately 1.8 kb restriction fragment) containing the isolated pgk1 promoter and a portion of the npt II gene. A sample containing DNA size-markers is shown in Lane 1. The results shown in FIG. 7 indicate that the positive transformant maize line M32 contains the recombinant pgk1 promoter-npt II construct integrated within the genomic DNA of the transformed cells.
The combined results of the examples set forth above indicate that the pgk1 promoter isolated from [0058] R. oryzae can be utilized as a functional promoter in other fungal species and also in species belonging to other phylogenetic kingdoms, including both dicot species and monocot species of angiosperms.
In compliance with the statute, the invention has been described in language more or less specific as to structural and methodical features. It is to be understood, however, that the invention is not limited to the specific features shown and described, since the means herein disclosed comprise preferred forms of putting the invention into effect. The invention is, therefore, claimed in any of its forms or modifications within the proper scope of the appended claims appropriately interpreted in accordance with the doctrine of equivalents. [0059]
1 12 1 35 DNA artificial sequence synthetic oligonucleotide primer 1 gctctagaag gttgaggtcg cgaatagaga gcttg 35 2 35 DNA artificial sequence synthetic oligonucleotide primer 2 gctctagaac ggtaggaaga gcttgaacga tacga 35 3 36 DNA artificial sequence synthetic oligonucleotide primer 3 gatgcatgct ctcaaaccag tggctgctga ggttga 36 4 36 DNA artificial sequence synthetic oligonucleotide primer 4 gatgcatgcg cctttggtac tgctcaccgt gctcac 36 5 33 DNA artificial sequence synthetic oligonucleotide primer 5 gatgcatgct gttaaagtaa ggttctctta taa 33 6 33 DNA artificial sequence synthetic oligonucleotide primer 6 gatgcatgct atttgaattc gatgccttct cta 33 7 1257 DNA Rhizopus oryzae 7 tctcaaacca gtggctgctg aggttgaaaa gcttttggga aaacctgtcg aattcaagct 60 tctgaggatg ctattttagc tgcaaagcat ggttgtcaag gtattgtcat ctctaaccat 120 ggtggtagac aattggattt cgcaccttct gctattgaaa ttttgcctga agtcacagct 180 gctcttaagc gaggaagaat taatgaagaa tttgaggttt atattgatgg aggtattcga 240 cgaggtagcg atatttttaa agcaattgct ttaggcgcca aaggtgtagg cattggacgt 300 ccttctctgg taagcttgcg taccttttaa agcaattgat atttaatgtg cgactagtat 360 gctatgtctt cttatggtga tgctggtgta gaaaggctct tggaattatt gcaggtaaat 420 ttttttcaca attttggaaa gaagctaact gaaaaataga atgagtttga aatgatcatg 480 cgcttaatgg gcgtaacatc gattgagcag attaagccag agatggtaga tactcgtaat 540 ctcaaggatc actttgcttc tattcccaag gattatttgg cagaattagc ctatgatcca 600 atgcaaccta gatctcattt ttctaagcta taaaacagga aaagatatcc tttaaataaa 660 gaacagcaat gttttctttc atcattgatg tacatacatt atatttgtac tatatgagca 720 caacaatcta tttatactgc cttattacag tttagacaac ttactgatac tcattgatta 780 ttctttgtac gcagacacta accaaacaca ttagttttat accaacctgt gaattttata 840 attcagcttg cttaagcttt ccaagtggca tggttttttc tctgatgtcg cgatatcaaa 900 attcaaaacc gtatcacatc tatcaaacaa aaaaaaaggt gattccaaga tgttatcaga 960 aatatcaagc taggcttact tttatgttat tcgtttagga agcttcattt tttagaagaa 1020 gaaaaaaaag agttgaaaat acaaggaaac aagttaaatg ttatacaata agcatgtgct 1080 ttcataacag aaaggctcct ttagtttaat gaaatcccga tttccatcga acaattaccc 1140 aataatatta tttgggaaag tgaagtcatg tatgattatt tttttctaat atttatgtca 1200 agcaaatact tttcttctct tctctttaat ttcaaatacc aatttttcaa cagaatg 1257 8 36 DNA artificial sequence synthetic oligonucleotide primer 8 tctagaacta gtatgctatg tcttcttatg gtgatg 36 9 39 DNA artificial sequence synthetic oligonucleotide primer 9 ggtaccggat cctctgttga aaaattggta tttgaaatt 39 10 36 DNA artificial sequence synthetic oligonucleotide primer 10 gcatgcggat cctctcaaac cagtggctgc tgaggt 36 11 44 DNA artificial sequence synthetic oligonucleotide primer 11 gactagtgga tccgggccat tctgttgaaa aattggtatt tgaa 44 12 25 DNA artificial sequence synthetic oligonucleotide primer 12 caccatgata ttcggcaagc aggca 25

Claims

1. A polynucleotide construct comprising:

a promoter comprising at least nucleotides 1187-1254 of SEQ ID No.:7; and

a polynucleotide sequence that encodes a polypeptide of interest.

2. A vector comprising the construct of claim 1.

3. The vector of claim 2 wherein the vector is a Ti plasmid.

4. The vector of claim 3 further comprising:

a T-DNA left border sequence; and

a T-DNA right border sequence.

5. A cell comprising the construct of claim 1.

6. A prokaryotic cell comprising the construct of claim 1.

7. A bacterial cell comprising the construct of claim 1.

8. A eukaryotic cell comprising the construct of claim 1.

9. A plant cell comprising the construct of claim 1.

10. A polynucleotide construct within a eukaryotic host cell, comprising:

a polynucleotide sequence encoding a protein other than a phosphoglycerate kinase; and

a phosphoglycerate kinase gene promoter sequence isolated from a first species, the first species belonging to a first phylogenetic kingdom, the promoter sequence being operably linked to the polynucleotide sequence, the eukaryotic host cell belonging to a second species within a second phylogenetic kingdom that is different from the first phylogenetic kingdom.

11. The polynucleotide construct of claim 10 wherein the first phylogenetic kingdom is fungi.

12. The polynucleotide construct of claim 10 wherein the second phylogenetic kingdom is plantae.

13. The polynucleotide construct of claim 10 wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID NO.:7.

14. The polynucleotide construct of claim 10 wherein the protein confers a resistance to the host cell.

15. The polynucleotide construct of claim 14 wherein the resistance is a resistance to at least one of an antibiotic, a herbicide, a pathogen, a toxin and a parasite.

16. The polynucleotide construct of claim 10 wherein the polynucleotide construct is integrated into the genomic DNA of the eukaryotic host cell.

17. A method of expressing a polypeptide in a host cell comprising:

providing a polynucleotide construct having a polynucleotide encoding a polypeptide of interest operably linked to a fungal phosphoglycerate kinase (pgk) gene promoter sequence; and

introducing the polynucleotide construct into a host cell.

18. The method of claim 17 wherein the pgk promoter sequence comprises at least 100 nucleotides as set forth in SEQ ID NO.7.

19. The method of claim 17 wherein the host cell is a yeast.

20. The method of claim 19 wherein the yeast is a species of the genus Saccharomyces.

21. The method of claim 17 wherein the host cell is a plant cell.

22. The method of claim 21 further comprising growing the plant cell in a cell culture.

23. The method of claim 21 further comprising growing the plant cell in plant tissue.

24. The method of claim 21 wherein the plant cell is an angiosperm plant cell.

25. The method of claim 24 wherein the plant cell is a monocot plant cell.

26. The method of claim 25 wherein the plant cell is a maize cell.

27. The method of claim 24 wherein the plant cell is a dicot plant cell.

28. The method of claim 27 wherein the plant cell is a tobacco plant cell.

29. The method of claim 17 wherein the introducing comprises transformation of the host cell utilizing at least one of electroporation, T-DNA mediated transformation, particle-bombardment mediated transformation, microinjection, freeze-thaw transformation, bacterial mediated transformation, virus-mediated transformation, chemical mediated transformation, whiskers mediated transformation, and plasmid transformation.

30. A system for producing a polypeptide comprising:

a first polynucleotide sequence isolated from a fungal species; the first polynucleotide sequence comprising a promoter sequence of a phosphoglycerate kinase gene;

a second polynucleotide sequence isolated from a second species, the second polynucleotide sequence comprising a coding region that encodes the polypeptide;

a host cell containing the first polynucleotide sequence operably linked to the second polynucleotide sequence, the host cell being of a species that is different from the fungal species from which the first polynucleotide sequence is isolated.

31. The system of claim 30 wherein the fungal species is from the genus Rhizopus.

32. The system of claim 30 wherein the host cell is a plant cell.

33. The system of claim 30 wherein the host cell is a yeast cell.

34. The system of claim 30 wherein the polypeptide confers a resistance to the host cell.

35. A method of producing a protein in a plant cell comprising:

creating a polynucleotide construct by operably linking an isolated fungal 3-phosphoglycerate kinase gene (pgk) promoter sequence to a polynucleotide sequence encoding at least one peptide of interest;

transforming the plant cell with the construct; and

utilizing the fungal pgk promoter to express the at least one peptide of interest within the plant cell.

36. The method of claim 35 wherein the polynucleotide sequence encodes a peptide foreign to the plant cell.

37. The method of claim 35 wherein the polynucleotide sequence encodes a peptide native to the plant cell.

38. The method of claim 37 wherein a non-transformed plant cell produces the peptide at a first level and the transforming the plant cell increases production of the peptide to a second level.

39. A plant cell comprising a promoter sequence isolated from Rhizopus Oryzae.

40. The plant cell of claim 39 wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID. No 7.

41. The plant cell of claim 39 wherein the promoter sequence comprises the first 1254 nucleotides of SEQ ID No:7.

42. The plant cell of claim 39 wherein the promoter sequence is integrated into the plant cell genomic DNA.

43. A yeast cell comprising at least one promoter sequence isolated from Rhizopus Oryzae.

44. The yeast cell of claim 43 wherein the promoter sequence comprises at least 100 nucleotides of SEQ ID. No 7.

45. The yeast cell of claim 43 wherein the promoter sequence comprises the first 1254 nucleotides of SEQ ID No:7