Nuclear-encoded transcription system in plastids of higher...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...

Reexamination Certificate

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C800S278000, C800S298000, C435S320100, C435S419000, C435S430000, C435S468000, C536S024100

Reexamination Certificate

active

06472586

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to plant genetic engineering and particularly to plastid transformation in higher plants. The invention provides a novel promoter sequences useful for the expression of foreign genes of interest in various plant species.
BACKGROUND OF THE INVENTION
Chloroplast genes are transcribed by an RNA polymerase containing plastid-encoded subunits homologous to the &agr;, &bgr; and &bgr;′ subunits of
E. coli
RNA polymerase. The promoters utilized by this enzyme are similar to
E. coil &sgr;
70
-promoters, consisting of −35 and −10 consensus elements (G. L. Igloi and H. Kossel, Crit. Rev. Plant Sci. 10, 525, 1992; W. Gruissem and J. C. Tonkyn, Crit. Rev. Plant. Sci. 12:-19, 1993) Promoter selection by the plastid-encoded RNA polymerase is dependent on nuclear-encoded sigma-like factors ((Link et al. 1994, Plant promoters and transcription factors, Springer Verlag, Heidelberg, pp 63-83). In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter (L. A. Allison and P. Maliga,
EMBO J
., 14:3721-3730; R. Iratni, L. Baeza, A. Andreeva, R. Mache, S. Lerbs-Mache,
Genes Dev
. 8, 2928, 1994). These factors mediate nuclear control of plastid gene expression in response to developmental and environmental cues.
There has been speculation of the existence of a second transcription system in plastids. However, direct evidence to support such a speculation has heretofore been unavailable. Identification of a novel second transcription system in plastids represents a significant advance in the art of plant genetic engineering. Such a system enables greater flexibility and range in plant species available for plastid transformation, and facilitates tissue specific expression of foreign proteins and RNAs via constructs that can be manipulated by recombinant DNA techniques.
SUMMARY OF THE INVENTION
This invention provides DNA constructs and methods for stably transforming plastids of multicellular plants. The DNA constructs of the invention extend the range of plant species that may be transformed and facilitate tissue specific expression of foreign genes of interest.
According to one aspect of the invention, DNA constructs are provided that contain novel promoter sequences recognized by a
n
uclear
e
ncoded
p
lastid (NEP) RNA polymerase. The DNA construct contains a transforming DNA, which comprises a targeting segment, at least one cloning site adapted for insertion of at least one foreign gene of interest, the expression of the foreign gene of interest being regulated by a promoter recognized by a NEP polymerase, and a plastid selectable marker gene.
The use of promoter elements recognized by
p
lastid
e
ncoded
p
lastid (PEP) RNA polymerase for enhancing expression of foreign genes of interest is another aspect of the instant invention. Like the constructs described above, these constructs also contain a targeting segment, and a cloning site for expression of a foreign gene of interest.
The promoters recognized by plastid encoded plastid RNA polymerase have been well characterized in photosynthetic tissues such as leaf. In contrast, the nuclear-encoded polymerase transcription system of the present invention directs expression of plastid genes also in roots, seeds and meristematic tissue. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). In a preferred embodiment of the invention, efficient plastid transformation in these crops will be greatly facilitated, through the use of the NEP plastid transcription system, promoters and polymerases of the present invention.
The NEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof, such as those described in U.S. Pat. No. 5,451,513 and pending U.S. application Ser. No. 08/189,256, and also described by Svab & Maliga.,
Proc. Natl. Acad. Sci. USA
, 90, 913 (1993), the disclosures of which are all incorporated herein by reference. To obtain transgenic plants, plastids of non-photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, to express proteins of interest, expression cassettes are constructed for high level expression in non-photosynthetic tissue, using the NEP promoter transcribed from the nuclear-encoded polymerase. In another aspect of the invention, PEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof.
In yet another aspect of the invention, the NEP transcription system also may be combined with the &sgr;
70
-type system through the use of dual NEP/PEP promoters.


REFERENCES:
patent: 5451513 (1995-09-01), Maliga et al.
patent: 5576198 (1996-11-01), McBride et al.
patent: 5877402 (1999-03-01), Maliga et al.
Shinozaki et al, 1986. Intron in the gene for the ribosomal protein S16 of tobacco chloroplast and its conserved boundary sequences. Mol. Gene. Genet. 202:1-5.*
Zoubenko, O. V. et al., “Efficient targeting of foreign genes into the tobacco plastid genome. ” 1994, Nucleic Acids Research, vol. 22, pp. 3819-3824.*
Staub et al., “Long Regions of Homologous DNA Are Incorporated into the Tobacco Plastic Genome by Transformation”; The Plant Cell, Jan. 1992, vol. 4, pp. 39-45.
Mullet, John E., “Dynamic Regulation of Chloroplast Transcription”; Plant Physiol. 1993, vol. 103, pp. 309-313.
Tonkyn et al., “Differential expression of the partially duplicated chloroplast S10 ribosomal protein operon”; Mol. Gen. Genet. 1993, vol. 241, pp. 141-142 and 148-152.
Svab et al., “High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene”; Proc. Natl. Acad. Sci. USA, Feb. 1993, vol. 90, pp. 913-917.
Antonio Vera et al., “Chloroplast of rRNA transcription from structurally different tandem promoters: an additional novel-type promoter”, Curr Genet (1995) 27:280-284.
Uwe Klein et al., “Two types of chloroplast gene promoters inChlamydomonas reinhardtii”; Proc. Natl. Acad. Sci. USA, vol. 89, pp. 3453-3457, Apr. 1992.
Michael C. Little et al., “Chloroplast rpoA, rpoB, and rpoC Genes Specify at Least Three Components of a Chloroplast DNA-dependent RNA Polymerase Active in tRNA and mRNA Transcription”; The Journal of Biological Chemistry, vol. 263 No. 28, Issue of Oct. 5, pp. 14302-14307, 1988.
Silva Lerbs-Mache, “The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: Single-subunit enzyme or catalytic core of multimeric enzyme complexes?”; Proc. Natl. Acad. Sci. USA, vol. 90, pp. 5509-5513, Jun. 1993.
Thomas Pfannschmidt et al., “Separation of two classes of plastid DNA-dependent RNA polymerases that are differentially expressed in mustard (Sinaais alba L.) seedlings”; Plant Molecular Biology 25: 69-81, 1994.
W.R. Hess et al., “Chloroplast rps 15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase”; The EMBO Journal, vol. 12 No. 2, pp. 563-571, 1993.
Robert F. Troxler et al., “Evidence That &sgr; factors Are Components of Chloroplast RNA Polymerase”; Plant Physiol. (1994) 104:753-759.
Rabah Iratni et al., “Regulation of rDNA transcription in chloroplasts: promoter exclusion by constitutive repression”; Genes & Development 8:2928-2938 (1994).
Lori A. Allison et al., “Light-responsive and transcription-enhancing elements regulate the plastid psbD core promoter”; The EMBO Journal, vol. 14 No. 14, pp. 101-110, (1995).
Eric Sun et al., “In Vitro Analysis of the Pea Chloroplast 16S rRNA Gene Promoter”; Molecular and Cellular Biology, Dec. 1989, vol. 9, pp. 5650-5659.
Wataru Sakamoto et al., “In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of &bgr;-glucuronidase translational fusions”; Proc. Natl. Acad. Sci. USA, vol. 90, pp. 497-501, Jan. 1993.

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