Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
2001-07-09
2002-05-28
McKelvey, Terry (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S320100, C435S410000, C536S024100
Reexamination Certificate
active
06395965
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to novel promoters isolated from Chlorella virus. The novel promoters are useful for expression of heterologous genes in host cells.
BACKGROUND OF THE INVENTION
Genetic engineering allows for isolation of a structural gene from one organism and expression of that gene in a different organism. Expression of a gene includes both transcription of the nucleic acid into mRNA and translation of the mRNA into protein. In order for the structural gene to be expressed in a new organism, the gene must be linked to a regulatory sequence in the proper location to signal transcription of the gene. The regulatory sequence generally includes a promoter sequence upstream from the structural gene. A promoter sequence is a DNA sequence which directs transcription of a structural gene. The nucleic acid sequence of the structural gene is transcribed into messenger RNA (mRNA) and then translated into a sequence of amino acids characteristic of a specific polypeptide or protein. Typically, a promoter sequence is located in the 5′ region of a gene, upstream from the transcriptional start site of the structural gene.
A promoter may be inducible or constitutive. In response to an inducing agent, the activity of an inducible promoter is increased, thereby increasing the rate of transcription of an operably linked coding sequence. In contrast, the rate of transcription of a gene under control of a constitutive promoter is not regulated. It is noted, however, that a constitutive promoter can be made an inducible promoter by the addition of an operator sequence. For example, the lac operator is added to the T7 bacteriophage promoter, changing it from a constitutive promoter to one induced by IPTG (Rosenberg, et al., U.S. Pat. No. 4,952,496).
Although not under the control of an inducing agent, some constitutive promoters provide higher levels of transcription than others. High activity promoters providing of high levels of gene transcription can have significant advantage in commercial production of a gene product.
In general, the ability of a promoter to direct transcription outside of its natural host varies. Moreover, the transcription rate of a particular promoter can also vary with the particular host in which the promoter is functioning. Therefore, new promoters capable of promoting high levels of transcription in a wide variety of host cells are needed.
The Chlorella viruses are a group of viruses which infect certain strains of unicellular, eukaryotic, Chlorella-like green algae. (Van Etten, 1995
, Mol. Cells
. 5:99-106; Van Etten, et al., 1991
, Microbiol. Rev
. 55:586-620). These viruses are among the largest and most complex viruses known, generally 150-190 nm diameter polyhedrons containing greater than 300 kb of double stranded DNA. The Chlorella virus genome has the potential to encode several hundred gene products.
Chlorella virus methyltransferase promoters have been isolated and shown to function in prokaryotic and eukaryotic host cell systems. These methyltransferase promoters function well in some bacterial and higher plant cells. See, for example, U.S. Pat. No. 5,563,328; and Mitra, et al., 1994
, Plant Molec. Biol
, 26: 85-893 (“Mitra”).
The present invention provides novel promoter sequences isolated from Chlorella virus that can induce a high level of gene expression in prokaryotic or eukaryotic cells, and over a wide range of temperatures, e.g., about 21° C. to 37° C.
SUMMARY OF THE INVENTION
The present invention provides novel promoter sequences isolated from Chlorella virus (SEQ ID NOS: 1-7). The invention includes gene constructs comprising a promoter sequence of the invention operably linked to a DNA sequence of a structural gene. The invention further provides vectors and host cells for expressing a product encoded by the structural gene of a gene construct of the invention, and cells transformed with a heterologous gene operably linked to the promoter.
The promoters of the invention include inducible Chlorella promoters, rendered inducible, for example, by fusion to an operator sequence. In a preferred embodiment, the Chlorella promoters of the invention are fused to a lac operator. The Chlorella promoters of the invention show very strong promoter activity over a wide range of temperatures, for example, from about 21° C. to about 37° C.
In one embodiment, the structural gene is a non-Chlorella virus DNA sequence encoding a protein for production in a host cell. According to this embodiment, the non-Chlorella virus DNA sequence can be any suitable structural gene which encodes a peptide, protein, hormone, enzyme, etc. Examples of suitable structural genes include glucagon like peptide 1 (GLP-1), growth hormone releasing factor (GRF), parathyroid hormone (PTH), interlinking peptides, amidation code sequences, carbonic anhydrase, beta-galactosidase, chloramphenicol acetyltransferase (CAT), glutathione acetyltransferase, and the like.
A gene construct of the invention is introduced into an appropriate host cell for expression of the gene product. Host cells are transformed directly or through a vector. In one embodiment, a suitable vector for a prokaryotic cell such as
E. coli
strains HB101 or JM109 is the plasmid pKK232-8.
The promoters of the invention are useful in a wide variety of prokaryotic and eucaryotic host cells. In one embodiment, the promoters of the invention are used to promote high levels of gene expression in plants, including tobacco, wheat, and other plants.
The invention further provides a process for producing a protein composition. According to this embodiment, a protein product is produced in a host cell transformed with a gene construct of the invention. The gene construct includes a promoter sequence of the invention operably linked to a structural gene encoding the protein to be produced in the host cell. The invention also provides methods for screening and isolating a promoter sequence having strong transcriptional properties, including truncated versions of the Chlorella virus promoters shown below.
REFERENCES:
patent: 4912046 (1990-03-01), Henner et al.
patent: 4952496 (1990-08-01), Rosenberg et al.
patent: 5436128 (1995-07-01), Harpold et al.
patent: 5563328 (1996-10-01), Mitra et al.
patent: 5595887 (1997-01-01), Coolidge et al.
Deboer et al. “The tac promoter: A functional hybrid derived from the trp and lac promoters,”Proc. Nat'l. Acad. Sci. USA, 80:21-25, (1983).
M.V. Graves et al. “Characterization of the gene encoding the most abundant in vitro translation product from virus-infected Chlorella-like algae,” Elsevier Science Publishers B.V., New York, NY,Gene, 113:149-55 (1992).
Gruber et al. “Vectors for Plant Transformation”,Methods in Plant Molecular Biology and Biotechnolgy, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, Florida, 89-119 (1993).
Horsch et al. “A Simple and General Method for Transferring Genes into Plants,”Science, 227:1229-1231 (1985).
Itakura et al. “Expression inEscherichia coliof a Chemically Synthesized Gene for the Hormone Somatostatin,”Science, 198:1056-1063 (1977).
Lu et al. “Analysis of 45 kb of DNA Located at the Left End of the Chlorella Virus PBCV-1 Genome,”Virology, 206:339-352 (1995).
W.R. McClure “Mechanism and Control of Transcription Initiation in Prokaryotes,”Ann. Rev. Biochem.54:171-204 (1985).
Miki et al. “Procedure for Introducing Foreign DNA Into Plants”,Methods In Plant Molecular Biology and Biotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton, Florida, 67-88 (1993).
A. Mitra et al. “The Chlorella virus adenine methyltransferase gene promoter is a strong promoter in plants,”Plant Molecular Biology, 26:85-83 (1994).
A. Mitra et al. “A Chlorella virus gene promoter functions as a strong promoter both in plants and bacteria,” Academic Press, New York, NYBiochem. Biophys. Res. Commun., 204:187-194 (1994).
Narva et al. “Molecular cloning and characterization of the gene encoding the DNA methyltransferase, M. CviBIII, from Chlorella virus NC-1A,”Nucleic Acids Research15:9807-9823 (1987).
M.C. O'Neill“Escherichia coliP
Foley & Lardner
McKelvey Terry
Restoragen, Inc.
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