MXPA98001108A - Transcription systems, encoded in nuclear form, in plant plants superior - Google Patents

Abstract

The present invention provides novel DNA constructs and methods for stably transforming the plastids of higher plants. The herein described constructs contain unique promoters, which are transcribed by the plastid polymerases coded in nuclear form as the plastid polymerases encoded by the plastid. The use of the novel builders of the invention facilitates the transformation of a larger number of plant species and enables the specific expression of tissues of a transformed DNA in the plastids of multicellular plants.

Description

TRANSCRIPTION SYSTEMS. CODIFIED IN NUCLEAR FORM. IN PLASTIDS OF SUPERIOR PLANTS FIELD OF THE INVENTION The present invention relates to the genetic engineering of plants and particularly to the transformation of plastids or plastids in higher plants. The invention provides 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 subunits encoded with plastids, homologous to the α, β and β1 subunits of the E RNA polymerase. coli The promoters used by this enzyme are similar to the s70 promoters of E. coli, consisting of -35 to -10 consensus elements (GL Igloi and H. Kossel, Crit. Rev. Plant Sci., 10, 525, 1992. Gruissem and JC Tonkyn, Crit. Rev. Plant. Sci. : -19, 1993). The selection of promoters by the RNA polymerase encoded by plastids is dependent on the sigma-type factors encoded in nuclear form (Link et al., 1994, Plant promoters and transcription factors, Springer Verlag, Heidelberg, pages 63-83). In addition, the transcription activity of some promoters is modulated by transcription factors encoded in nuclear form, which interact with elements upstream of the core promoter (LA Allison and P. Maliga, EMBO J., 14: 3721-3730 R. Iratni, L. Baeza, A. Adreeva, R. Mache, S. Lerbs-Mache, Genes Dev., 8, 2928, 1994). These factors mediate the nuclear control of plastid gene expression in response to the ranks of development and the environment. It has been a speculation the existence of a second transcription system in the plastids. However, direct evidence to support such speculation so far is not available. The identification of a novel second system of transcription in plastids represents a significant advance in the technique of genetic engineering of plants. Such a system makes possible a greater flexibility and greater number of plant species available for the transformation of plastids, and facilitates the specific expression of foreign protein tissues and RNA by means of builders 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 increase the number of the species of silver that can be transformed and facilitate the specific expression of foreign gene tissues of interest. According to one aspect of the invention, DNA constructs are provided which contain novel promoter sequences recognized by the RNA polymerase of nuclear-encoded plastids (NEP). The DNA construct contains a transforming DNA, which comprises a target segment, at least one cloning site adapted for the insertion of at least one foreign gene of interest, the expression of the foreign gene of interest is regulated by a promoter recognized by the NEP polymerase and a selectable marker gene for plastids. The use of promoter elements recognized by plastid-encoded plastid RNA polymerase (PEP) to improve the expression of foreign genes of interest is another aspect of the present invention. Like the constructs described above, these builders also contain a target segment and a cloning site for the expression of an alien gene of interest. The promoters recognized by the polymerase of Plastid RNA encoded by these plastids have been well characterized in photosynthetic tissues, such as leaves. In contrast, the transcription system of the nuclear-encoded polymerase of the present invention is directed to the expression of plastid genes also in the roots, seeds and meristematic tissues. In most plants, which include corn, cotton and wheat, regeneration of plants is achieved through somatic embryogenesis (ie, involving the meristematic tissue). In a preferred embodiment of the invention, the efficient transformation of plastids in these crops will be greatly facilitated through the use of the plastid transcription system of the NEP, the promoters and the polymerases of the present invention. The NEP promoters of the invention are incorporated into the plastid transformation vectors and protocols, currently available for use, such as those described in U.S. Patent No. 5,451,513 and the pending US patent application. . 08 / 189,256, and are also described by Svab &; Maliga , Proc. Nati Acad. Sci. , E.U.A., 90, 913 (1983), the descriptions of all these documents are incorporated herein by reference. To obtain transgenic plants, the plastids of the non-photosynthetic tissues are transformed with selectable marker genes, expressed from the NEP promoters and transcribed by the nuclear-encoded polymerase. Similarly, to express proteins of interest, expression cassettes are constructed for high level expression in a non-photosynthetic tissue, using the NEP promoter transcribed from the nuclear-encoded polymerase.

In another aspect of the invention, the PEP promoters of the invention are incorporated into the plastid transformation vectors and protocols, currently available, for their so. In yet another aspect of the invention, the transcription system of the NEP can also be combined with the s70-type system through the use of double NEP / PEP promoters.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The deletion of rpoB from the tobacco plastid genome by the replacement of the target gene. (A) Homologous recombination (diagonal lines) by means of plastid DNA sequences flanking aadA in plasmid pLAA57, results in the replacement of rpoB (Sac I to Sma I fragments) in the wild-type plastid genome ( ptADN) with the aadA sequences, which supply the plastid genome? rpoB (? rpoB ptDNA). The abbreviations rpoB, rpoCl, rpoC2 are plastid genes that encode the ß, ß 'and ß "subunits of the E-type RNA polymerase, coli, aadA is a chimeric gene resistant to spectinomycin. restriction: P, Pst I; Sm, Sma I; Se, Sa I. (B) Pigment deficiency is associated with the deletion of rpoB Total cell DNA was isolated from green leaf tissue ((strips 1, 3, 5) and white (strips 2, 4, 6) from three independently transformed lines (Line Nt-pLAA57-llB, strips 1 and 2, line Nt-pLAA57-16B, strips 3 and 4, line Nt-pLAA57- 18C, strips 5 and 6) and the wild-type green leaf (Nt, band 7) .The DNA was digested with PstI and the gel spot was hybridized with a DNA fragment (nucleotide posns 22883-24486 of the ptDNA, numbering according to K. Shinozaki, et al., (EMBO JS 2043, 1986)) which contains part of rpoCl (thick black line in Figure A.) The probe hybridizes to a fragment of 0.0 kb from the wild type genome and a 4.2 kb fragment from the? RpoB ptDNA. (c) DNA gel stain analysis confirms the lack of ptDNA copies in white shoots of the Nt-pLAA57-10A line (belt 2) and white seed progeny of the grafted chimeric plant from the same line ( strip 3) The DNA of the wild-type green leaf tissue was loaded into strip 1. Note the absence of the 9.0 kb fragment of wild type ptDNA in the plants rpoB. The stain was prepared as for Figure IB. Figure 2. The deletion of rpoB results in a poor pigment phenotype. (A) Green plants are shown, wild type (left),? RpoB deficient in pigment (right), and chimeric (center). (B) a chimeric plant that blooms in the greenhouse. Note that the margin of the white sheet indicates the plastids of? RpoB in the second sheet layer that forms the cells of the germination line.

Figure 3. (A) The plastids (P) in the mesophilic cells of the leaf of rpoB plants lack organized photosynthetic membranes. Abbreviations; N, nucleus; V, vesicle, M, mitochondrion. (B) For comparison, an electron micrograph of a chloroplast of wild type leaves (Cp) with thylakoid membranes (T) is shown. The amplification of both (A) and (B) is 7,8000 times. Figure 4. (Top) The accumulation of mRNA from the plastid for (A) photosynthetic genes and (B) genes from genetic systems in plants? RpoB. The gel spots were prepared with total cellular RNA (A, 3 μg per strip; B, 5 μg per strip) from wild-type leaf tissues (strips 1, 3, 5) and leaf tissues? RpoB, and hybridized to the indicated plastid gene sequences. (Bottom) The spots shown above were retested with the 25S rDNA sequences. Hybridization signals were quantified with the Molecular Dynamics Phosforolmager apparatus and normalized to the 25S rRNA signal. The excess of folds of the wild-type signal intenss over those of? RpoB for each probe is shown below the strips. Figure 5. The transcription of plants? RpoB starts from a promoter that does not conform to the rules. (A) The preparer extension analysis was used for the map of the 51 ends of the transcripts of rbcL and 16SrDNA in wild type plants (strips 1, 3) and? RpoB (strips 2, 4). The primary transcripts were marked by circles (open for the wild type, closed for? RpoB), the transcripts processed, by a triangle. Transcripts of unknown origin were marked with a star. The escalas of accompanying sequences (order, loading GATC) were generated using the same primers that served in the extension reactions of the preparer. The numbers behind each extension product mark the distance from the first nucleotide of the coding sequence for rbcL and from the first nucleotide of the mature 16 rDNA. (B) Mapping of the primary transcripts for 16S rRNA in the wild-type (strip 2) and the rpoB (strip 3) plants. RNA from the total leaf (20 μg) was excised in vitro, and the 16S rRNA species were identified by the protection of the RNA after hybridization with a complementary RNA probe. The finished protected products were marked as in (A). Strip 1 contains the RNA standards of the indicated sizes. (C) The RNA sequence of the upstream region 16SrDNA with the initiating transcripts of the promoters for the polymerases encoded with plastids (type s70, Pl) and encoded in nuclear form (P2) (the designation of Pl and P2 is based in A. Vera and M. Sugiura, Curr Genet, 27, 280, 1995). The consensus s70 promoter elements (-35 and -10) are in tables. The initiation sites were marked by circles, as in (A) and (B). The numbering starts from the first nucleotide upstream of the coding region 16SrDNA (-1 = nucleotide 102757) in the tobacco plastid genome). Figure 6. The accumulation of plastid mRNA in wild-type and? RpoB tobacco leaves. The spots for the plastid genes (see Example I) were grouped as follows. (A) mRNA is significantly more abundant in wild type leaves than in rpoB plants. (B) The levels of the mRNA are comparable in the leaves of wild type and? RpoB or (C) are greater in the leaves? RpoB. The gel spots were prepared with the total cellular RNA (3 μg per strip) from the tissue of wild-type leaves (fascia 2) and of? RpoB (fascia 2), and were hybridized to the plastid gene sequences indicated . (Bottom panel) To control the load, the spots shown above were retested with the 25S rDNA sequences. Figure 7. Mapping of the initiation sites of the transcription atpB in leaves of wild type tobacco and type? RpoB. (A) Extension analysis of the preparer. The trainer extension products labeled at the end, from the wild type (wt) and the? RpoB (T57) type samples were operated together with the homologous sequence obtained using the same preparer. The numbers along the sequence refer to the distance from the translation initiation codon of ATG. The primary transcripts of the NEP and PEP promoters were marked by full and open circles, respectively. (B) The in vitro auction and the protection test of the RNases to identify the 5 * ends of the primary transcript. The strips were loaded with? RpoB RNA samples (T57; 1, 2) and wild type (4, 5) with (2, 4) and without (1,5) protective antisensible RNA. The molecular weight markers (MW) (nucleotides 100, 200, 300, 400 and 500) were loaded into strip 3. The 5 'end of transcript in (A) corresponds to the size of the protected fragment in parentheses: -254 ( 277nt), -289 (311). Note the artifact slightly below the 200 nt marker, which is present in the unprotected RNA samples. (C) Physical map of the rbcL intergenic region. Map position of the 5 'ends of the primary transcript for the promoters of atpB NEP and PEP were labeled as in (A). Figure 8. Mapping of the atpl transcription initiation sites in wild-type and? RpoB tobacco leaves. (A) Trainer extension analysis. The preparation extension products labeled at the end of the wild type (wt) and? RpoB samples were operated together with the homologous sequence obtained using the same preparer. The numbers along the sequence refer to the distance from the initiation codon of the ATG translation. The primary transcripts of the NEP and PEP promoters were marked by full and open circles, respectively. (B) (B) Screening in vitro and the RNase protection assay to identify the ends 51 of the primary transcript. The strips were loaded with? RpoB (T57; 1, 2) and wild type (4, 5) RNA samples with (2, 4) and without (1.5) antisense complementary protective RNA. The markers of molecular weights (MW) (nucleotides 100, 200, 300, 400 and 500) were loaded into strip 3. The 5 'end of transcript in (A) corresponds to the size of the protected fragment in parentheses: -130 ( 235nt), -207, 209, 212 (303, 305, 309, not resolved). (311) Note the artifact slightly below the 200 nt marker, which is present in the unprotected RNA samples. (C) Physical map of the intergenic region rps2 -atpl. Map position of the 5 'ends of the primary transcript for atpl promoters NEP and PEP were labeled as in (A). Figure 9. Mapping of clpD transcription initiation sites in wild type and? RpoB tobacco leaves. (A) Trainer extension analysis. The preparation extension products labeled at the end of the wild type (wt) and? RpoB samples were operated together with the homologous sequence obtained using the same preparer. The numbers along the sequence refer to the distance from the initiation codon of the ATG translation. The primary transcripts of the NEP and PEP promoters were marked by full and open circles, respectively. (B) Screening in vitro and the RNase protection assay to identify the ends 51 of the primary transcript. The strips were loaded with? RpoB (T57; 1, 2) and wild type (4, 5) RNA samples with (2, 4) and without (1.5) antisense complementary protective RNA. The markers of molecular weights (MW) (nucleotides 100, 200, 300, 400 and 500) were loaded in strip 3. The 5 'end of transcription in (A) corresponds to the size of the fragment protected in parentheses: -53 ( 96nt), -95 (138 nt), -173 (216nt) and -511 (69 nt). Note the artifact slightly below the 200 nt marker, which is present in the unprotected RNA samples. (C) Physical map of the clpP intergenic region. Map position of the 5 'ends of the primary transcript for the clpP promoters NEP and PEP were labeled as in (A). Figure 10. Mapping of the initiation sites of accD transcription in wild type and? RpoB tobacco leaves. (A) Trainer extension analysis. The preparation extension products labeled at the end of the wild type (wt) and? RpoB samples were operated together with the homologous sequence obtained using the same preparer. The numbers along the sequence refer to the distance from the initiation codon of the ATG translation. The primary transcripts of the NEP and PEP promoters were marked by full and open circles, respectively. (B) (B) Screening in vitro and the RNase protection assay to identify the 5 'ends of the primary transcript. The strips were loaded with? RpoB RNA samples (T57; 1, 2) and wild-type (4,5) with (2,4) and without (1,5) protective antisensing RNA. The molecular weight markers (MW) (nucleotides 100, 200, 300, 400 and 500) were loaded into strip 3. The transcription end 51 in (A) corresponds to the size of the protected fragment 103 nt. Note the artifact slightly below the 200 nt marker, which is present in the unprotected RNA samples. (C) Physical map of the intergenic region accD - rbcL. Map position of the 5 * ends of the primary transcript for the NEP promoter PaccD-129 was labeled. Figure 11. Alignment of the DNA sequences flanking the transcription initiation sites of the NEP promoter. Nucleotides with more than 6 correspondences are framed. The consensus sequence adjacent to the transcription initiation site is shown below. The position of the 5 'ends are marked by the full circles. Note that the 51 ends for Prpsl2-152 and Prpsl6-107 are not capped and may not be primary transcripts.

Figure 12. The poly erases NEP and PEP, through the recognition of different promoters, provide a mechanism for the selective transcription of the plastid genes. Note that some genes have only PEP promoters (photosystem 1 and photosystem 11), others have both PEP promoters and NEP (most of the governing genes) or only NEP promoters (accD). Figure 13. A schematic diagram of a chimeric plastid gene, expressed from a NEP promoter.

DESCRIPTION OF THE INVENTION Several reports have suggested the existence of an RNA polymerase encoded in nuclear form, with an additional localized plasmid (reviewed in Gruissem and Tonkyn, 1993, Igloi and Kossel, 1992, Mullet, 1993, Link, 1994). Deleting the ropB gene encoding the essential β subunit of the E. coli type tobacco RNA polymerase. The existence of a second system of transcription of plastids, which is encoded by the nucleus, has been established. (Allison et al., 1996, EMBO J. 15: 2802-2809). The deletion of rpoB supplied photosynthetically defective, pigment-deficient plants. An examination of the plastid ultrastructure in mesophilic cells of plant leaves? rpoB, revealed proplastid type organelles, which lack the arrangements of the stacked thylaoid membranes that are characteristic of photosynthetically active chloroplasts. Transcripts for the photosynthetic rbcL, psbA and psbD genes are low, while the mRNAs for the accumulated rpll6, atpl and 16SrADN genes are approximately at wild type or higher levels of these levels. The lack of transcription accumulation for the photosynthetic genes was due to the lack of promoter activity of the s70 type. While in wild-type tobacco leaves the ribosomal RNA operon is normally transcribed from a s7 ^ type promoter, in the rpoD plants the rRNA operon was transcribed from a non-s70 promoter. The rRNA operon is the first unit of transcription for which both the polymerase of the RNA of a plastid-encoded plastid and that encoded in nuclear form (PEP and NEP, respectively) were identified. An analysis of the promoter regions of other genes has revealed that the rRNA operon is not unique. It is a member of a large class of plastid genes that has at least one promoter for each of the PEP and NEP, with a potential for expression by either of the two plastid RNA polymerases. In addition, the plastid genes have been identified, which were transcribed exclusively by the NEP. Also, the data suggest that additional gene-specific mechanisms regulate NEP transcript levels in different types of plastids.

A transcription start site of the NEP has been identified approximately 62 bases upstream by the mature 5 'term of the 16S rRNA. The sequence surrounding the initiation site is highly conserved among the numerous species of plants examined, and bears no resemblance to the consensus sequence of the PEP promoter. NEP promoter consensus sequences important for the recognition of the nuclear-encoded polymerase and the ligand (analogous to the -10 and -35 sequences of the E. coli-type transcription start site) are preferably located within about 50 nucleotides in any direction of the start site of NEP transcription. As described in more detail in Example 1, there are several different NEP promoters, and these NEP promoters are sometimes found in conjunction with the PEP promoters. The polymerases of the invention can be purified by chromatography, using standard models. NEP polymerase activity in column fractions can be assayed using the DNA segments comprising the promoter region of NEP as standards in the in vitro transcription reactions. Alternatively, the segments of the NEP promoter can be linked to a matrix, which can be separated by some resources (for example, small magnetic spheres). The DNA bound to the matrix is incubated with an extract of plants under conditions in which the polymerase encoded in nuclear form is expected to bind to DNA. The matrix / DNA / polymerase complex is then separated from the plant extract and the bound protein can then be isolated and characterized. The protein purified by any of the aforementioned protocols can be used to produce antibodies for the probe expression libraries, in order to isolate the nuclear genes or the cDNAs encoding the nuclearly encoded polymerase. As an alternative approach to NEP isolation or polymerase, proteins with specific affinity for the promoter fragment can be isolated and the amino acid sequence of the N-terminus can be determined by micro-sequence. The amino acid sequence can then be used to design the appropriate PCR primers for the isolation of the gene. The activity of the s70-type transcription system, which encodes with plastids, previously known, has been well characterized in photosynthetically active tissues, such as the leaf. In contrast, the transcription system of the polymerase encoding the nuclear form of the present invention directs the expression of plastid genes also in roots, seeds and atheromatous tissue. In most plants, which include corn, cotton, and wheat, regeneration of the plant is achieved through somatic erythrogenesis (ie, involving the meristematic tissue). Efficient transformation of the plastid in these crops will be enabled, or greatly facilitated, through the use of the plastid transcription system of the NEP of the present invention. The NEP promoters of the invention are incorporated into the plastid transformation vectors and protocols, currently available for use, such as those described in U.S. Patent No. 5,451,513 and the pending US patent application. . 08 / 189,256, and are also described by Svab &; Maliga , Proc. Nati Acad. Sci. , E.U.A., 90, 913 (1983), the descriptions of all these documents are incorporated herein by reference. To obtain transgenic plants, the plastids of the non-photosynthetic tissues are transformed with selectable marker genes, expressed from the NEP promoters and transcribed by the nuclear-encoded polymerase. Similarly, to express proteins of interest, expression cassettes are constructed for high level expression in a non-photosynthetic tissue, using the NEP promoter transcribed from the nuclear-encoded polymerase. The NEP transcription system can also be combined with the s70 type system, through the use of double NEP / PEP promoters. In some cases, the expression of transgenes from NEP promoters in photosynthetic tissue may also be convenient. The detailed description set forth in the following Examples I-III, presents preferred methods for obtaining and using the DNA constructs of the present invention and for practicing the methods of the invention. Any techniques of molecular cloning and recombinant DNA, not specifically described, are carried out by standard methods, as generally indicated, for example, in Ausubel (Ed.), Current Protocole in Molecular Biology, John Wiley & Sons, Inc. (1994). The following non-limiting Examples describe the invention in more detail.

EXAMPLE I Demonstration of a Second System of Transcription of Distinctive Plastids by the Suppression of rpoB To establish the existence of an RNA polymerase not similar to E. coli, in plastids, the gene for one of the essential subunits of the E. coli type enzyme was deleted from the tobacco plastid genome. The levels of the mRNA were then assessed in the mutant plastids. The data indicate that, in the absence of the E. coli enzyme encoded with plastid, expression of some photogenes, was drastically reduced. In contrast, the transcript levels for the plastid genes encoding the gene expression apparatus are similar to the levels in the wild-type plants. Therefore, the non-E.coli type RNA polymerase selectively transcribes n subset of plastid genes. This second transcription apparatus does not start from the typical E70 s promoters. coli, but recognizes a novel promoter sequence.

Materials and Methods for Example I Construction of Plasmid. Plasmid pLAA57 is a derivative of pBSKS + (Stratagene), which carries a Sacl to Bam Hl fragment (nucleotides 22658 to 29820) of the ptDNA. An internal DNA fragment Sac I to Sma I within the ptDNA insert, between nucleotides 24456 and 28192, was replaced by the spectinomycin-resistant chimeric gene (aadA). This gene is identical to that described (Z. Svab and P. Maliga, Proc. Nati, Acad. Sci. USA, 90, 913, 1993), except that the region psbA 3 • is shorter and is contained in the fragment Sba 1 to Dral as described (JM Staub and P. Maliga, Plant J., 6, 547, 1994).

Plant transformation. For the transformation of the plastid, tungsten particles were coated (Z. Svab and P. Maliga, Proc. Nati, Acad. Sci. USA, 90, 913, 1993) with pLAA57 DNA, and introduced into the leaves of the plants of Nicotiana tabacum using the Biolistics PDSlOOOHe gun from DuPont at 77 kg / cm2. The transgenic shots were selected aseptically in the RMOP medium (2. Svab, P. Hajdukiewics, P. Maliga, Proc. Nati, Acad. Sci., USA 87.8526, 1990) containing 500 mg / ml dihydrochloride Spectinomycin. The transgenic slices were guided and maintained in an RM medium consisting of MS salts solidified with agar (T. Murashigs and F. Skoog, Physiol, Plant., 15, 493, 1962) containing 3% sucrose. Electron Microscopy The electron microscopy was done in fully expanded wild type and? RpoB, the cuttings grew in sterile cultures in the RM medium with 3% sucrose. The tissue was fixed for 2 hours in 2% glutaraldehyde, 0.2 M sucrose, 0.1M phosphate buffer (pH 6.8) at room temperature, and washed three times in 0.2M sucrose, 0.1M regulator. phosphate. The fixed tissues were subsequently fixed in 1% osmium tetroxide, with 0.2 M sucrose, dehydrated in a series of graduated ethanol, embedded in Spurr epoxy resin (hard), sectioned and stained with uranyl acetate and loin citrate for transmission electron microscopy.

Gel spots The DNA of the total leaf was prepared as described (IJ Mettler, Plant Mol, Biol. Rep., 5, 346, 1987), digested with the Pst I restriction endonuclease, separated on 0.7% agarose gels. and transferred to Hybond N (Amersham) using the Posiblot Transfer apparatus (Stratagene). Hybridization to a randomly labeled fragment in select form was carried out in the Rapid Hybridization Buffer (Amersham) overnight at 652C. The total leaf RNA was prepared using TRIzol (GIBCo BRL), following the manufacturer's protocol. The RNA was electrophoresed in 1% agarose / formaldehyde gels, then transferred to a nylon membrane and tested as for DNA spots.

Synthesis of Probes. The double-stranded DNA probes for psbA, atpl and rplld were prepared by randomly labeling 32P in select form of the DNA fragments generated by the PCR. The preparation sequence used for the PCR, together with its positions within the tobacco PDNA (K. Sinozaki, et al., EMBO J. S5, 2043, 1906) is as follows: psbA, preparatory 5 '= 5' -CGCTTCTGTAACTGG -3 '(complementary to nucleotides 1550 to 1536 of the ptDNA), 3 * preparatory = 5' -TGACTGTCAACTACAG '3 • (nucleotides 667 to 682); atpl 51 preparatory = 5 '-GTTCCATCAATACTC-3' (complementary to nucleotides 15985 to 15971), 3 'preparatory = 5'-GCCCCGGCTAAAGTT-3' (nucleotides 15292 to 15306); rplld 5 'preparatory = 5-TCCCACGTTCAAGGT-3' - (complementary to nucleotides 84244 to 84230), 3 • -preparatory = 5'-TGAGTTCGTATAGGC-3 * (nucleotides 83685 to 83699). To generate probes for rbcl, psbD / C and 16S rRNA, the following restricted DNA fragments are labeled 32P; rbcL, a fragment of Bam Hl (nucleotides 58047 to 59285 in the PDADN); psbD / C a SacII to the Hind III fragment of the tobacco psbD / C operon (nucleotides 34691-36393); 16S rRNA, an Eco Rl fragment to Eco RV (nucleotides 138447 to 140855 n the ptDNA). The probe for the 25 S rRNA of tobacco is from plasmid pKDRl (D. Dempsey, KW Wobbe, DF Klessig, Mol. Plant Path, 83, 1021, 1993) containing an Eco Rl fragment of 3.75 kb from a 25S site. 18S of tobacco cloned in plasmid pBR325. When gel spots are hybridized to the 25S rRNA, the double-stranded DNA probe labeled 32P was mixed with the unlabeled plasmid pKDRl which corresponds to a 2-fold excess of the amount of RNA present in the filter. Normalization of DNA levels by plastid genome copy number. To test whether the plastid genome copy number changed in the estimated differences in gene expression, the total cell RNA and DNA were prepared from equal amounts of leaf tissue from wild type plants and? RpoB. To compare the number of plastid genome copies per sheet mass equivalent, DNA gel spots were carried out with an equal volume of each DNA preparation and were rounded with an Eco Rl to Eco RV radio- labeled (from nucleotides 138447 to 140845 from ptDNA (K.

Shinozaki, et al. , EMBO J. S. 2043, 1986) of the 16SrDNA sequence. The quantification of 1 phosphoric image analysis showed an equal number of plastid genome copies in each sample. The amount of 16S rRNA from equal tissue samples, as measured by the gel spots of the RNA in an equal volume of each RNA preparation, was reduced by 2.5 times in the? RpoB plants. This value is similar to the estimated 3-fold reduction when normalized with the cytoplasmic signal of 25S rRNA (Figure 3B).

Preparation extension reactions. Preparatory extension reactions were carried out at 3 μg (wild type) or 10 μg (? RpoB) of the total leaf RNA, as described (LA Allison and P. Maliga, EMBO J., in press) using the following primers : 165 rRNA: 5 * -TTCATAGTTGCATTACTTATAGCTTC-3 '(complementary to nucleotides 102757-102732); rbcL: 5 • -ACTTGCTTTAGTCTCTGTTTGTGGTGACAT (complementary to nucleotides 57616-57587). The sequence scales were generated with the same trainers using the sequence II (USB) equipment.

Identification of primary transcripts by the in vitro auction. RNA from the total leaf (20 μg) of wild-type plants and? RpoB was screened in the presence of [a-32P] GTP (J.C. Kennell and D.R. Pring, Mol.Gen. Genet, 216, 16, 1989). The 16S labeled rRNAs were detected by the ribonuclease protection (A. Vera and M. Sugiura, Plant Mol. Biol. 19, 309, 1992) using the RPAII (Ambion) kit. To prepare the complementary protective RNA, the upstream region SrDNA (nucleotides 102526-102761 of the ptDNA were amplified by PCR using the following primers: the 5 'terminal preparer was 5'-CCTCTAAGACCCTAAGCCCAATGTG-3', which corresponds to the nucleotides 102526 and 102541 of the PDNA (K. Shinozaki et al., EMBO J. 5, 2043, 1980) underlined) plus a Kbal site; the 3 'preparer was 5'-CCGGTACCGAGATTCATAGTTGCATTAC-3' complementary to nucleotides 102761 to 102742 of the PDNA (underlined) plus a Kpn I site. The amplified product was cloned as an Xba I to Kpn I fragment in Xba I and Kpn I- restricted pBSKS + vector (Stratagene). To generate an unlabeled RNA complementary to the 5 'end of 16S rRNA, the resulting plasmid was linearized with Xbal, and transcribed in a Megascript reaction (Ambion) with the T3 RNA polymerase. The markers (nucleotides 100, 200, 300, 400 and 500) were prepared with the set of RNA Century marker markers (RNA Century Markers Set Table) (Ambion) following the manufacturer's protocol. The 72 nucleotide marker was the mature processed transcription of the plasmid trpV gene and was generated by the protection of the RNAse.

Results? Discussion The interruption of the polymerase activity of E. coli type RNA in tobacco plastids results in a pigment deficient phenotype. To avoid the disruption of plastid genes for other functions, the rpoB gene was chosen for deletion, since it is the first reading frame of an operon that exclusively encodes subunits of the E-type plastid polymerase. coli (K. Shinozaki, et al.), EMBO J. 5, 2043, 1986). Deletion was accompanied by replacing the majority of the rpoB coding region (3015 of 3212 base pairs) and 691 bp of the upstream non-coding sequence, with a chimeric spectinomycin resistance gene (aadA) (Z. Svab and P. Maliga , Proc. Nati, Acad. Sci. USA, 90, 913, 1993) in a cloned plastid DNA fragment (ptDNA). The resulting plasmid was introduced by bombardment of particles in tobacco chloroplasts, where the aadA gene integrated into the plastic genome via flank plastid DNA sequences, as illustrated in Figure IA. Since the genetic system of plastids is highly polyploid, with each cell of the leaf containing up to 10,000 identical copies of the pDNA, the selective amplification of the transformed genome copies was carried out by cutting the tissue bombarded in the medium containing the Spectinomycin (P. Maliga, Trenas Biotechnol 11, 101, 1993). From the initial round of the selection, several spectinomycin-resistant plants were obtained, which exhibit sectors of white-leaf tissue (Figure 2A). DNA gel spot analysis of white and green sectors indicated that the pigment deficiency correlates with the deletion of rpoB in three independently transformed lines (Figure IB). The majority of DNA samples from the pigment deficient tissue, for example band 4 in Figure IB, contained a mixture of wild-type and transformed genome copies. The complete absence of copies of wild type ptDNA was critical for the interpretation of the data. Therefore, to obtain plants containing only transformed plastid genomes, shoots of the white tissue sectors were regenerated. This procedure provided uniformly white plants (Figure 2A) that do not contain the wild-type ptDNA as judged by the gel spot analysis of the DNA (Figure 1C9.) The regeneration of these white leaves in the spectinomycin-free medium provided exclusively shoots. pigment deficient, confirming the complete absence of wild-type plastid genomes in all leaf layers and cell types.

It is difficult to obtain seeds of tobacco plants that grow in a sterile culture. Fortuitously, during the regeneration of the plant of the primary transformants, we obtained a periolinal chimeric ion homoplast (S. Poethig, Trends Genetics 5, 273, 1989) for the mutation of plastids in leaf layer L2 (Figure 2A). This line was grafted with wild-type tobacco and brought to maturity in the greenhouse (Figure 2B). The seeds of the self-pollinated flowers gave rise to uniformly white seedlings, in which no wild-type plastid genome could be detected by the DNA spot analysis of gal (Figure IC).

Plastids on leaves of rpoB plants lacking thylakoid membranes. Pigment-deficient plants were unable to grow photoautotrophically. However, if a medium with sucrose is maintained to compensate for its lack of photosynthesis, they grow normally but at a reduced rate, compared to wild type plants and do not exhibit notable changes in organ morphology. Also, the rpoB seedlings germinated with high efficiency and were developed in plants. These observations indicate that the E. coli plastid RNA polymerase is not required to maintain the functions of non-photosynthetic plastids, necessary for the growth and differentiation of plants.

An examination of the plastid ultrastructure in the mesophilic cells of the leaves of the rpoB plants revealed that the mutant plastids were smaller and rounder than the wild-type chloroplasts, averaging 2 to 5 μm in length, compared to 5 at 9 μm for the wild-type chloroplasts. The rpoB plastids are thus larger than the undifferentiated proplastides, whose average size is 1 μm (M. R. Thomas and R. J. Rose, Plants 158, 329, 1983). In addition, rpoB plastids typically contain multiple vesicles of irregular size and configuration, and lack the arrangements of the stacked thylakoid membranes, which are characteristic of photosynthetic active chloroplasts (Figure 3). The transcription of plastid genes is maintained in plastidiums rpoB. In the absence of the β subunit, no transcription is expected from the s70 type plastid promoters. To determine if any transcription activity is maintained in the plastids? rpoB, the accumulation of the RNAs was examined by the analysis of the gel spot of the RNA. Transcripts were investigated for two different classes of plastid genes (E. Shinozaki et al., EMBO J. 5, 2043, 1986). The first group includes genes that encode subunits of the photo-synthetic apparatus: the psbD / C operon, which encodes D2 and CP43 subunits of photosystem II; rbcL, which encodes the large subunit of ribulose-1, 5-bisphosphate-carboxylase; and psbA, which encodes the DI subunit of the reaction center of the photo-system II. The second group contains genes for the components of the gene expression apparatus: rpll6, which encodes the ribosomal protein subunit, and the 16SrDNA gene. All quantification of plastid RNA is normalized to cytosplasmic 25S ribosomal RNA levels. Surprisingly, the accumulation of mRNA was detected for all genes examined. However, the effect of the deletion of rpoB on the transcription accumulation was drastically different for the two classes of genes. Stable-state mRNA levels of the photosynthetic genes psbD / C, rbcL and psbA were reduced by 40 to 100 times compared to the wild-type levels (Figure 4A, the signals were visible in all strips? long exposure). In contrast, the transcription levels for the polymerase genes encoded in nuclear form were much less affected. A 3-fold reduction for the 16S rRNA was measured, and a real increase for the multiple transcripts arising from the poly-cistronic operon containing the rpll6 gene was also observed (Figure 4B). These data indicate that, while the expression of the genes encoding the photosynthetic apparatus is defective in the? RpoB plants, the RNAs for genes involved in the governance functions accumulate approximately at wild type levels, or higher.

The 16SrADN gene was transcribed from a novel promoter in? RpoB plants. The accumulation of the plastid RAs confirmed that there is activity of the RNA polymerase in the plastids lacking the β subunit of the E. coli type enzyme. However, the migration of plastid genes to the nucleus has been documented (SL Baldauf and JD Palmer, Nature 334, 262 1990; JS Gantt, SL Baldauf, PJ Calie, NF Weeden, JD Palmer, EMBO J. 10, 3073, 1991, MW Gray, Curr Op. Genet, Dev 3, 884 1993). Therefore, the transcription in plastids? RpoB can still conceivably start from the promoters of type s70 if there is a nuclear copy of rpoB, whose product can be imported into plastids and assembled in the functional enzyme of E. coli type. To establish if the plasmid transcripts detected in the? RpoB plants are transcription products of the promoter of type s70 > the ends of the 5 'transcript for four genes were mapped, rbcL (K. Shinozaki and M. Sugiura, Gene 20, 91, 1992), 16SrDNA (A. Vara and M. Sugiura, Curr. Genet 27, 280, 1995) , psbA (M. Sugita and M. Sugiura, Mol.Gen. Genet, 195, 308, 1984) and psbD (WB Yao, BY Meng, M. Tanaka, M. Sugiura, Nucí, Acids Res. 17 9583, 1989) , for which the transcription initiation sites have been previously established. None of the 5 'ends mapped to the initiation sites of the s70 type promoter (data is shown for rbcL and 16SrDNA in Figure 5A). Therefore, it was concluded that the residual RNA polymerase activity in the? RpoB plastids is not due to an E-type enzyme. coli, but represents a second unique system of transcription of plastids. This distinctive RNA polymerase enzyme is referred to as the polymerase of RNA encoded Plastids in Nuclear form (NEP), to distinguish it from the enzyme of E. coli type which is designated polymerase of Plastidium RNA encoded in the form of Plastids (PEP) . Since the tobacco plastid genome has been completely sequenced and since the few unidentified reading frames do not carry sequence similarity to the known RNA polymerase subunits (K. Shinozaki, et al., EMBO J. 5, 2043, 1986), the transcription by the polymerase of the RNA encoded in nuclear form depends on the nuclear gene products.

In the absence of transcription of s70-type promoters in the? RpoB plants, the question remains: which promoters were the source of the plastid RNAs. The 16S rRNA end 5 'detected in the? RpoB plants mapped 62 nucleotides upstream of the mature 5' terminus of 16S rRNA (Figure 5A). This endpoint 51 was determined to be a primary transcript by the in vitro splice (Figure 5B). A prominent primary transcript with a similar 5 'end was recently reported in tobacco cell proplastides cultured heterotrophically and designated P2 (A. Vera and M. Sugiura, Curr, Genet 27, 280, 1995: This transcript is also present in levels very low in wild-type leaf cells (A. Vera and M. Sugiura, Curr. Genet, 27, 280, 1995; Figure 5A, longer exposure, not shown) .The sequence surrounding the start site is highly conserved among all the species of plants examined, and has no resemblance to the sequence of consensus s70 (A. Vera and M. Sugiura, Curr, Genet 27, 280 1995.) Based on this prominent use in plants? rpoB, it was concluded that this unique promoter is used by the transcription apparatus of the NEP.

In contrast to the 16S rRNA, the main transcripts for the photosynthetic genes rbcL and psbD / C mapped to the processed ends, previously characterized (data shown for rboL, Figure 5, L. Hanley-Bowdoin, EM Orozco, NH Chua, Mol. Cell Biol. 5, 2733, 1985, JE Mulklet, EM Orozco, N. -H. Chua, Plant.Mol. Biol., 39, 1985. S. Reinbothe, C. Reinboths, C. Heintren, C. Seidenbecher, B. Parthier, EMBO J. 12, 1505, 1993). The additional minor transcription ends upstream of the processed terminals. Therefore, the low levels of transcription accumulation for these photosynthetic genes are the result of the upstream promoter activity and the subsequent process of reading through the RNAs to deliver the correct size transcripts. Proposed functions for the traceability of plastids. In plants? RpoB there is accumulation of RNAs transcribed by the NEP system. This indicates a function for the RNA polymerase encoded in nuclear form in maintaining the expression of the governing genes of plastids. Apparently, these levels of expression are sufficient to support the growth and differentiation of non-photo-autotrophic plants. In contrast, the E. coli PEP RNA polymerase is required to deliver the high levels of the transcripts of the plastid gene necessary for the development of the photosynthetically active chloroplasts. The proposed function for the AQRN polymerase encoded in nuclear form implies a high demand for its function during the early phases of chloroplast development, before the PEP RNA polymerase is active (JE Mullet, Plant Physiol., 103, 309 , 1993). The regulation of the development of RNA polymerase encoded in nuclear form is supported by the observation that the P2 promoter of the nuclear-encoded polymerase of the 16SrDNA gene is more active in proplastidia of cultured tobacco cells than in leaf chloroplasts. (A. Vera and M. Sugiura, Curr Genet, 27, 280, 1995).

EXAMPLE II Transcription by two different RNA polymerases is a general regulatory mechanism of gene expression in higher plants As described in Example I, the accumulation of transcripts in plants lacking the polymerase of PEP leads to the identification of a NEP promoter for the plastid ribosomal RNA operon (Allison et al., 1996, EMBO J. 14: 3721-3730). To facilitate the mapping of additional NEP promoters, the accumulation of mRNA was examined in? RpoB plants for most classes of plastid genes. The novel promoter sequences described herein can be used to extend the number of species within such plastid transformation is feasible and to drive the expression of foreign genes of interest in a tissue-specific manner.

Materials and Methods for Example II RNA Gel spots. The total leaf RNA was prepared using the TRIzol (GIBCo BRL), following the manufacturer's protocol. The RNA was subjected to electrophoresis in 1% agarose / formhyde gels, then transferred to Hybond N (Amersham) with the use of the Posiblot transfer device (Stratagenus). Hybridization to the preparative labeled fragment in a random manner was carried out in a rapid hybridization regulator (Amersham) overnight at 65 ° C. Double-stranded DNA probes were prepared by 32P labeling of random preparation of the DNA fragments generated by the PCT. The sequence of preparers used for the PCT, along with their positions within the tobacco PDNA (Shinozaki, et al., 1986, eupra), are as follows: Position Gene Sequence 5 'nucleotide in the plastid DNA accD 60221 GGATTTAGGGGCGAA 60875 GTGATTTTCTCTCCG atpB 56370 (C) AGATCTGCGCCCGCC 55623 CCTCACCAACGATCC ATPL 15085 (C) GTTCCATCAATACTC 15292 GCCGCGGCTAAAGTT clpP 73621 (C) GACTTTATCGAGAAAG 73340 GAGGGAATGCTAGACG ndhA 122,115 (C) GATATAGTGGAAGCG 121602 GTGAAAGAAGTTGGG ndhB 97792 (C) CAGTCGTTGCTTTTC 97057 CTATCCTGAGCAATT ndhF 113366 (C) CTCCCCTTCTTCCTC 112749 CTCCGTTTTTACCCC ORF1901 129496 (C) GTGACTATCAAGAGG 128,895 GACTAACATACGCCCG ORF2280 92881 GCTCGGGAGTTCCTC 93552 TGCTCCCGGTTGTTC petB 78221 GGTTCGAAGAACGTC 78842 CCCCCAGAAATACCT psaA 43457 (C) TTCGTTCGCCGGAACC 42743 GATCTCGATTCAAGAT psbB 75241 GGAGCACATATTGTG 75905 CGATTATTGCCGATG psbE 66772 (c ) CAATATCAGCAATGCAGTTCATCC 66452 CCAATCCTTCCAGTAGTATCGGCC rpsl4 36621 CACGAAGTATCTGTCCGGATAGTCC rpl33 / rpll8 70133 GGAAAGATGTCCGAG 70636 GTTCACTAATAAATCGAC rpS16 mRNA was tested with the EcoRI fragment isolated from plasmid PJS40, which contains sequences between the nuclei Otomies 4938/5363 and 6149/6656 of lptDNA of tobacco (Shinozaki et al., 1986, supra). The probe for the 25S rRNA of tobacco was from the plasmid pKDR1 (Dempssy et al., Mol.

Plant Path. 83: 1021, 1993) containing an EcoRI fragment of 3.75 kb of 25S / 18S site of the cloned tobacco in plasmid pBR325. When gel spots were hybridized to 25S rRNA, the double-stranded DNA probe, labeled with 32P, was mixed with unlabelled pKDRI plasmid, which corresponds to a 2-fold excess of the amount of RNA present in the filter.

React the extension of the Preparer. The extension reactions of the preparer were carried out in 10 μg (wild type) or 10 μg (? RpoB = of the total leaf RNA, as described (Allison and Maliga, 1995, EMBO J. 15: 2802-2809). The primers are listed below The underlined oligonucleotides were also used to generate the backtack builders.

Gene Position of nucleotide 5 'Sequence in plastid DNA accD 50758 CCGAGCTCTTATTTCCTATCAGACTAACC atpl 56736 CCCCAGAACCAGAAGTAGTAGGATTGA clpP # l 74479 GGGACTTTTGGAACACCAATAGGCAT elpP # 2 74947 GGGAGCTCCATGGGTTTGCCTTGG ORF1901 31451 CTTCATGCATAAGGATACTAGATTACC ORF2280 87419 GGGAGCTCTACATGAAGAACATAAGCC rps2 16921 CCAATATCTTCTTGTCATTTCTCTC rpsl6 5185 CATCGTTTCAAACGAAGTTTTACCAT The sequence scales were generated with the same trainers, using the Sequences II (USB) equipment. Identification of the primary transcription by the auction n vitro. The total leaf RNA (20 mg) from the wild-type and? RpoB plants were topped in the presence of [a-32P] GTP (Kennell and Pring, 1989, Mol. Gen. Genet., 216: 16-24) . The labeled RNAs were detected by the protection of the ribonuclease (Vera and Sugira, 1992, supra) using the RPAII (Ambion) equipment. To prepare the complementary protection RNA, the upstream region 16SrDNA (nucleotides 102526-102761 of the pDNA) was amplified by PCR using the primers listed below. The 5 'primers were designed to add a Sbal restriction site (underlined) upstream of the amplified fragment. The 3 'primers were designed to add the Kpnl site (underlined) downstream of the amplified sequence. The amplified product was cloned as a Sbal fragment in the vector (Stratagene) pBSKS + restricted in Xbal- and Kpnl. To generate the complementary RNA without labeling to the 5 'end of the RNA, the resulting plasmid was linearized with Xbal and transcribed in a Megascript reaction (Ambion) with the T3 RNA polymerase. Markers (nucleotides 100, 200, 300, 400 and 500) were prepared with the Century Marker Pattern Set of the RNA (Ambion), following the manufacturer's protocol.

Gene position 5 'nucleotide sequence in the plastid DNA accD 59756 CCGAGCTCTTATTTCCTATCAGACTAACC 59576 CCGGTACCATAGGAGAAGCCGCCC ATPS 56750 CCGAGCTCGTAGTAGGATTGATTCTCA 57131 (C) CCGGTACCGGAGCCAATTAGATACAAA ATPL 15895 CCCACCTCTGACTTGGAAACCCCC 16277 (C) CCCAATTCTAGTATTCGCAATTTGT CLPF 74462 GGGAGCTCCAGGACTTCGCAAACC 74752 (C) GGGGTACCAATACGCAATGGGG 74947 GGGAGCTCCATGGGTTTGCCTTGG 75080 (C) GGGGTACCGCTAATTCATACAGAG ORF1901 31424 GGGAGCTCCGACCACAACGACCG 31724 (C) GGGGTACCCTTACATGCCTCATTTC ORF2280 87419 GGGAGCTCTACATGAAGAACATAAGCC 57154 GCCCTACCCTGCCTAAGGGCATATCGG Analysis of the DNA sequence. The sequence analysis of the DNA was carried out using the Wisconsin Sequence Analysis Package (Genetics Computer Group, Inc.).

Results and Discussion Based on the accumulation of mRNAs in wild-type and rpoB-type leaves, the plastid genes can be divided into three classes. The first class includes genes for which mRNAs accumulate at high levels in wild-type leaves, and at very low levels in the leaves of plants rpoB (Figure 6A). The genes that belong to this class are: psaA (photosystem I gene), psbB and psbE (photosystem II genes), petB (cytochrome b6 / f complex gene), ndhA (chain NADH dehydrogenase homologue) Respiratory, Matsubayashi et al., 1987 Mol. Gen. Genet. 210: 385: 393) and rpsl4 (ribosomal protein gene). The second class includes the plastid genes for which mRNAs accumulate to almost equal levels in the wild type and rpoB type leaves (Figure 6B). This class includes atpB (ATP synthase gene), ndhF (NADH respiratory chain dehydrogenase homolog gene, Matsubayashi et al., 1987, supra), rpsl6 (ribosomal protein gene) and ORF1901 (a gene with a unknown function; Wolfe et al., 1992, J. Mol. Biol. 223: 95-104). The third class includes genes for which there is a more significant mRNA in the rpoB leaves than in the wild type plant leaves (Figure 6C). Typical of this class are rpl33 and rplld (ribosomal protein genes), accD (acetyl-CoA carboxylase subunit coding, Sasaki et al., 1993 Plant Physiol. 108: 445-449) and ORF2280 (putative ATPase with unknown function, Wolfe 1994, Curr, Genet 25: 379-383). Two additional genes of this class, ndhB (respiratory chain NADH dehydrogenase homolog, Matsubayashi et al., 1987, supra) and clpP (which encodes the proteolytic subunit of the ATP-dependent protease, Clp; Maurizi et al., 1990 J. Biol Chem. 265: 12546-12552; Gray et al., 1990 Plant Mol. Biol. 15: 747-950) form a subgroup of this class that demonstrates significant levels of mRNA in wild-type leaves. The atpD and atpl synthase genes have both NEP and PEP promoters.

The RNA gel spot analysis identified a number of genes and operons for which high levels of transcription are maintained in the leaves? RpoB. To identify additional NEP promoters, the 5 'end of several transcripts has been mapped by the extension extension analysis. The 5 'ends may be those of the primary transcripts that identify a promoter, or generated by the RNA process. Since the triphosphate groups retain the transcripts of primary plastids at their 5 'ends, the specific transfer [32 P] GMP to these RNA molecules by the enzyme guanylyltransferase allowed accurate discrimination between the primary transcripts and the processed ends. For the tobacco atpS operon, the 5 'ends of transcription have been identified by Orozco et al. (1980, Curr, Conet, 17: 65-71.) At nucleotide positions -611, -502, -488, -289 and -255, upstream of the translation initiation codon (Figure 7c). The 5 'ends are numbered relative to the translation initiation codon (ATG) when the nucleotide directly upstream of A is in the -1 position. The preparation extension analysis, identified at each of these 5 'ends in our wild-type plants (Figure 7A). In the? RpoB sample only the RNA species -289 are present, whose 5 'end is a substrate for the guanylyltransferase (Figure 7B). Therefore the -289 RNA is transcribed from a NEP promoter, PatpB-289. Interestingly, the -289 transcript is present in wild type leaves, although it is less abundant than in the plants rpoB. The transcripts -255, -488 and -611 are absent in the plants rpoB (Figure 7A). DNA fragments containing these promoters (but not PatpB-289) are recognized by the E. coli RNA polymerase (Orozco et al., 1990, supra) and are transcribed by the PEP in the plastids. The atpA operon includes the genes atpl, -atpH-atpF-atpA (Figure 8c). In wild-type tobacco leaves, the 5 'ends of the mRNA are mapped to three regions upstream of atpl: the -209 region, with the 5' ends mapping the nucleotides -212, -209 and -207 the ends 51 at nucleotides -130 and -85. On the sheets ? rpoB only transcript -207 can be detected (Figure 8A). This transcription can be capped in the sample of the RNA? RpoB (Figure 8B), therefore, it is transcribed from a NEP promoter. A signal in this position was also obtained in the in vitro spiking reaction of the wild-type RNA samples. The transcripts -209 and -212 may be due to the activity of an overlap of the PEP promoter, or the formation of multiple transcripts from the NEP promoter in wild-type plants. The transcript -130 which is present only in 1 wild type leaf RNA, can also be topped (Figure 8A, 8D). Since they are sequences similar to the elements -10 / -35 at the correct spacing upstream of this 5 'end, it is transcribed by the polymerase of the PEP.

A promoter of clpP NEP is highly expressed in chloroplasts The clpP protease subunit gene also belongs to the class which has both promoters, NEP and PEP. Expanded preparation analysis in the wild type plants identified the 5 'ends of the RNA at the nucleotide positions -53, -95 and -173, while in the? RpoB plants the 5' ends mapped to the nucleotide positions - 53, -173 and -511 (Figure 9A). in the in vitro auction reaction it was verified that each of them are primary transcripts (Figure 9B). Three of the transcripts are derived from the NEP promoters. The PolpP-53 promoter is highly expressed in both wild type and? RpoB type plants, and thus represents a distinct class of NEP promoters with a potential for high level expression in different tissue types. The PolpP-53 promoter is well preserved in spinach (Westhoff, 1985, Mol.Gen Gene.S. 201: 115-123). Additional clpP promoters for the NEP are PclpP-173 and PclpP-511. Since the PclpP-511 transcript accumulates only in those plants, rpoB (Figure 9A) is a candidate of the regulated NEP promoter. Note also that PclpP-511 is located within the region encoding psbd and its expression can be affected by the convergent psbB PEP promoter (Figure 9C).

The only PEP promoter directly upstream of clpP is PclpP-95. The RNAs of this promoter accumulate only in the wild-type leaves and the PclpP-95 has upstream sequences evocative of the conserved elements -10 - / - 35 (not shown).

The accD gene is transcribed exclusively from a NEP promoter.

For the lipid biosynthetic gene accD, mRNA accumulates at high levels only in plants? RpoB. A major transcript is initiated at nucleotide position -129 (Figure 10A), which can be capped in vitro (Figure 10B). Therefore, this RNA is transcribed from a NEP promoter. Since PaccD-129 does not have a significant activity in mesophilic leaf cells, photosynthetically active, it serves as a candidate for the regulated NEP promoter, with a distinct tissue-specific expression pattern. The NEP promoters share a loose consensus adjacent to the transcription initiation site.

The sequences flanking the transcription initiation sites are aligned to identify conserved NEP promoter elements (Figure 11). Nine promoters identified in this study and Prrn-62, the NEP promoter described in Allison et al., Are included in the sequence alignment. (1996, supra). The sequences for PORF2280-1577, and PORF1901-41 for which the 5 'ends are shown to be primary transcripts by in vitro spiking are also included (data not shown). Both of these promoters are active in the? RpoB leaves, but not in the leaves of wild type plants. Also included in the sequence alignment are the NEP tentative promoters for rps2 and rpsl6, for which there is more mRNA in the leaves? RpoB. The ends 51 of these transcripts are mapped by the extension extension analysis. The in vitro spiking assay failed due to the low abundance of the mRNAs (data not shown). The multiple sequence alignment of the regions immediately flanking the 5 'ends of the NEP is identified as a consensus of loose nucleotides around the transcription initiation site (Figure 11). The conservation of additional nucleotides upstream and downstream is also evident. The impact is the lack of sequence conservation between PclpP-53 and other NEP promoters, which is the only NEP promoter highly active in chloroplasts. Given the lack of sequence similarity, this sequence is not included in the alignment. The sequences around the start site of the PcllP-53 transcript are shown separately at the bottom of Figure 11.

The poly erases of the NEP and PEP, through the recognition of the different promoters, provide a mechanism for the selective transcription of plastid genes (Figure 12). The data provided here demonstrates that some genes have only PEP promoters or NEP promoters while others have regulatory sequences both PEP and NEP.

EXAMPLE III NEP Promoters for Gene Expression Selectable Markers For versatility and universal applications, the expression of selectable marker genes for plastid transformation is convenient in all tissue types at a high level. The selectable marker genes in the plastid transformation vectors, currently used, are expressed from the PEP promoters recognized by the RNA polymerase encoded with plastids. The PEP polymerase transcribes photosynthetic genes and some governing genes, therefore it seems to be the RNA polymerase dominant in leaf tissues, photosynthetically active. Efficient transformation of plastids has been achieved in tobacco based on the formation of chloroplasts in leaf cells. However, regeneration of the plant is not feasible, or it is not practical for the leaves of most agronomically important cereal crops, which include corn, rice, wheat and in cotton. In these crops, transgenic plants are typically obtained by the transformation of embryogenic cells from tissue cultures or small plant tissues. Since these tissues are a non-photosynthetic expression of marker genes by NEP promoters, which are active in non-green tissues, they appear to be particularly advantageous and will facilitate the transformation of plastids in all types of non-photosynthetic tissues.

A particularly suitable promoter for promoting the expression of marker genes is the PclpP-53 promoter. This promoter is highly expressed in the propplasties of plants? RpoB, therefore it can also be highly expressed in the proplastidia of the embryogenic cell cultures that supply transgenic cereal plants. The expressed marker genes of these promoters will also be suitable for selecting the transformants of the plastids in bombardment leaf cultures, since this promoter was found to be active in the chloroplasts. The marker genes expressed from promoters, such as the PclpP-53 promoter, will have a wide application in obtaining the transformed plastids.

Selectable marker genes will be constructed using the principles outlined in U.S. Patent No. 5,451,513 and pending U.S. Patent Application Serial No. 08 / 189,256, the subject of which is incorporated herein by reference. A transformation of the DNA construct is illustrated in Figure 13. More specifically, the promoter will be cloned upstream of a DNA segment encoding a plastid selectable marker. The signals for translation will be provided by the incorporation of suitable DNA sequences between the promoter fragment and the coding region of the selectable marker. Untranslated segments 31 of a plastid gene to provide signals for the termination of transcription and stabilize the chimeric mRNA, will be cloned downstream of the selectable marker. the use of the 3 'untranslated region of genes expressed from NEP promoters is preferred since the requirements for the termination of transcription for the NEP and PEP polymerases may be different. PclpP-53 is a particularly strong NEP promoter. However, plants with transformed plastids can be obtained with weak promoters as well.

There are several examples for such weak NEP promoters in the preceding examples.

Expression of Transgenes of Tissue Specific Plastids. Driven by Promoters of the NEP The tissue-specific expression of plastid transgenes is convenient in many applications. The tissue-specific expression of a protein that makes plant tissues repellent or toxic to root nematodes may be convenient at the roots. However, the expression of the same protein in the leaves will drain plant sources and may affect the use of parts of the plants in series. Since more often expressed in non-green tissues, the NEP promoters described in this application, and the expressed promoters of the NEP polymerase, in general, are a rich source of tissue-specific promoters for the expression of transgenes.

Several of the NEP promoters, for example PclpP-511, are highly expressed in plant proplastids? RpoB. The proplastidia are present in the edible part of the cauliflower. Therefore, the high-level expression of foreign genes in cauliflower is anticipated from this promoter in the edible parts of the plant.

The plaD gene encodes a subunit of prokaryotic acetyl-CoA carboxylase, an enzyme involved in lipid biosynthesis. Interestingly, in wild type leaves, the level of the mADN of accD is low while it is high in the proplastidios of plants? RpoB. This observation suggests that PaccD-129 is active in non-green plastids of tissues actively involved in lipid biosynthesis, such as developed seed plastids, which are rich in oil.

Claims (13)

1. A DNA construct, to stably transform plastids of multicellular plants, which comprises transforming the DNA that has a target segment, which performs the insertion of the transformation DNA into the plastid genome by homologous recombination, a selectable marker gene, which confers a selectable phenotype to the cells of the plant containing the transformed plastids and a cloning site for the insertion of an additional expressible DNA, which encodes a foreign gene of interest, in which the enhancement comprises a 5 'promoter element, which it is recognized and transcribed by a polymerase of the RNA of a plastid encoded in nuclear form.

2. The DNA constructor, according to the claim 1, in which this constructor is incorporated into a suitable vector for the transformation of the plastids.

3. The DNA construct according to claim 1, wherein the 5 'promoter element is recognized and transcribed by an RNA polymerase of a plastid encoded by plastid.

4. The DNA construct according to claim 1, wherein the promoter element is selected from the promoter elements of the plastid genes, selected from the group consisting of. Prrn-62, PORF2280-1577, PatpB-289, PORF1901-41, Prbs2-152, Prpsl6-107, PatpI-207, Pclp-511, Pclp-173, Pclp-53 and PaccD-129.

5. The DNA construct according to claim 2, wherein the promoter element is Pclp-95.

6. A constructor of DNA, to stably transform the plastids of a plant cell and for the expression of at least one additional gene product there, this constructor comprises: a) a target segment, comprising a DNA sequence substantially homologous to a predetermined plastid genome sequence, with a plastid which is to be transformed, this target segment enables homologous recombination with the predetermined genomic sequence of the plastid; b) a selectable marker gene, disposed within the target segment, this selectable marker gene confers a selectable, non-lethal phenotype to cells containing plastids with the DNA construct; c) an additional DNA segment, comprising a transcription unit of a gene encoding a protein or a precursor thereof; and d) a promoter sequence, which is operably linked to the transcription unit, this promoter sequence is recognized by a polymerase encoded in nuclear form, the gene encoding the protein is regulated by the promoter.

The DNA construct according to claim 6, wherein the transcription unit encodes a selectable marker gene.

The DNA construct according to claim 7, wherein the selectable marker gene is regulated by a promoter transcribed by a plastid polymerase encoded in nuclear form.

9. The DNA construct according to claim 6, wherein the transcription unit encodes an information gene.

10. The DNA construct according to claim 6, in which this constructor is incorporated into a suitable vector for the transformation of the plastids.

11. A multicellular plant, stably transformed with the DNA construct of claim 1.

12. A multicellular plant, stably transformed with the DNA construct of claim 2.

13. A method for obtaining a plant cell or a multicellular plant, the plastids of this cell have been stably transformed by at least one foreign gene of interest, this method comprises administering to a plant cell a DNA construct, which includes: a) a target segment, comprising a DNA sequence substantially homologous to a predetermined plastid genome sequence, with a plastid to be transformed, this target segment enables homologous recombination with the predetermined genomic sequence of the plastid; b) a selectable marker gene, disposed within the target segment, this selectable marker gene confers a plastid selectable phenotype to cells containing plastids, with this DNA construct; c) and an alien gene of interest, this gene is regulated by a promoter recognized by a plastid RNA polymerase, encoded in nuclear form; d) selecting the cells which express said phenotype; and e) regenerating a silver from the cell, which contains the stably transformed plastids.