| PATENT ASSIGNEE'S COUNTRY | Japan. USA |
| UPDATE | 12.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 12.12.00 |
| PATENT TITLE |
Porphyrin-accumulating type herbicide resistance gene |
| PATENT ABSTRACT |
Provided are DNA fragments and biologically functional equivalents thereof that confer resistance to porphyrin-accumulating type herbicides upon plant and algal cells, plasmids containing these DNA fragments or biologically functional equivalents thereof, microorganisms containing these DNA fragments or biologically functional equivalents thereof, methods for conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells using these DNA fragments or biologically functional equivalents thereof, and herbicide-resistant plants or algae into which these DNA fragments or biologically functional equivalents thereof have been introduced and in which they are expressed. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 20.01.98 |
| PATENT REFERENCES CITED |
Sato et al., Am. Chem. Soc. Symposium Series 559:91-104(1994). Oshio et al., Zeitschrift Fur Naturforschung 48:339-344 (1993). Kataoka et al., J. Pesticide Science 15:449-451 (1990). Shibata et al., Research in photosynthesis 3;567-570(1992). Sato et al., Abstracts of ACS Nat'l meeting; Abstr. #112 (1993). Pornprom et al., Pesticide Biochemistry and Physiology 50: 107-114 (1994). Takahashi et al. The EMBO J. 1991. vol. 10:2033-2040. Napoli et al. The Plant Cell. 1989. vol. 2: 278-289. Tapperman et al. Plant Molecular Biology. 1990. vol. 14: 501-511. Sato et al. Am. Chem. Soc. Symposium Series. 1994. vol. 559: 91-104. Shibata et al. Research in Photosynthesis. vol. III. pp. 567-570. Edited by Murata, 1992. Gordon-Kam et al. The Plant cell. 1990. vol. 2: 603-618. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated, purified DNA, having the following characteristics: a) said DNA comprises a nucleotide sequence of a DNA fragment having a molecular size of approximately 3.4 kb, said DNA fragment being obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides; b) said DNA contains restriction sites for XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, and XhoI, and has a restriction site map as shown in FIG. 1(a); and c) said DNA confers resistance to porphyrin-accumulating type herbicides in algal cells when expressed therein. 2. The isolated, purified DNA according to claim 1, having the following characteristics: a) said DNA comprises a nucleotide sequence of a DNA fragment having a molecular size of approximately 9.9 kB; and b) said DNA contains restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, and HindIII, and has a restriction site map as shown in FIG. 1(b). 3. The isolated, purified DNA according to claim 1, having the following characteristics: a) said DNA comprises a nucleotide sequence of a DNA fragment having a molecular size of approximately 10.0 kB; and b) said DNA contains restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, and KpnI, and has a restriction site map as shown in FIG. 1(c). 4. The isolated, purified DNA according to claim 1, having the following characteristics: a) said DNA comprises a nucieotide sequence of a DNA fragment having a molecular size of approximately 13.8 kB; and b) said DNA contains restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, BamHI, SalI, HindIlI, and KpnI, and has a restriction site map as shown in FIG. 1(d). 5. An isolated, purified DNA comprising the nucleotide sequence shown in SEQ. ID. NO.: 1. 6. A plasmid containing the DNA of any one of claims 1, 2, 3, 4 or 5. 7. A microorganism containing: (a) the plasmid of claim 6, or (b) the DNA of any one of claims 1, 2, 3, 4 or 5. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to DNA fragments that confer resistance to porphyrin-accumulating type herbicides on plant and algal cells, plasmids and microorganisms that contain these DNA fragments, methods for conferring resistance to porphyrin-accumulating type herbicides onto plant and algal cells by using these DNA fragments, and plants and algae into which these DNA fragments have been introduced for the purpose of conferring resistance to such herbicides thereon. 2. Description of Related Art A group of widely-known compounds used as active ingredients of some varieties of commercially- and otherwise-available herbicides exhibit herbicidal activity in the presence of light, but exhibit no herbicidal activity in darkness. This has led to their common designation as light-dependent or porphyric herbicides. It has recently been shown that these herbicides induce high levels of porphyrin accumulation in plants and algae, and thus they are now designated as "porphyrin-accumulating type herbicides" [Zoku,Iyakuhin-no-Kaihatsu, (translation: "The Development of Medical Drug Products; continuation") vol. 18; Development of Agricultural Chemicals II, chapter 16, section 16-1, Hajime Iwamura, Tamio Ueno & Katsuzo Kamoshita, eds., Hirokawa Shoten, Tokyo, pubs.) or simply "porphyric herbicides". It was reported by Matringe, M., Camadro, J. M., Labbe, P. & Scalla, R. (Biochem J. 260:231 (1989)) and by Matringe, M., Camadro, J. M., Labbe, P. & Scalla, R. (FEBS Lett. 245:35 (1989)) that porphyrin-accumulating type herbicides (referred to below also as porphyric herbicides) inhibit isolated protopor-phyrinogen oxidase (referred to below as "protox"). Since most crop plants do not exhibit resistance to these porphyric herbicides, it is not possible to use these herbicides on farmland when such crops are under cultivation. If it were possible to develop crops resistant to porphyric herbicides, such herbicides could be used on these crops. This would make crop management easier, and increase the value of these herbicides in agricultural applications. For this reason, it is desirable to develop a method for conferring resistance to porphyrin-accumulating type herbicides upon crop plants. SUMMARY OF THE INVENTION With this goal in mind, the present inventors have investigated a mutant strain, designated RS-3, of the unicellular green alga Chlamydomonas reinhardtii which displays specific resistance to porphyric herbicides. Wild-type strains of this alga are normally highly sensitive to porphyric herbicides. The present inventors have discovered that inhibition by porphyric herbicides of protox activity in chloroplast fragments isolated from the RS-3 strain of Chlamydomonas reinhardtii was significantly lower than in chloroplast fragments from the wild-type strain. The inventors therefore constructed a genomic DNA library from total nuclear DNA isolated from the RS-3 mutant strain, and succeeded in isolating clones that contain a gene responsible for resistance to porphyric herbicides. Thus, the inventors were able to obtain DNA fragments that can confer resistance to porphyrin-accumulating type herbicides onto plant and algal cells. Accordingly, it is an object of the present invention to provide an isolated, purified DNA fragment that confers resistance to porphyrin-accumulating type herbicides when expressed in plant or algal cells, plasmids and microorganisms containing said DNA fragment. A DNA fragment according to the present invention preferably has a nucleotide sequence of one or more portions of DNA comprising the genome of an alga, or has a nucleotide sequence highly homologous to the nucleotide sequence of DNA comprising one or more portions of the genome of an alga. Additional objects of the present invention are a method for conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragment into said plant or algal cells, wherein said DNA fragment is expressed; and plants or algae into which said DNA fragment has been introduced, wherein said DNA fragment is expressed, thereby conferring herbicide resistance upon said plants or algae. Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics: a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides; b) containing restriction sites for XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, and XhoI, and having a restriction site map as shown in FIG. 1(a); c) having a molecular size of approximately 3.4 kb; and d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof. A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics: a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides; b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI and HindIII, and having a restriction site map as shown in FIG. 1(b); c) having a molecular size of approximately 9.9 kB; and d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof. Another object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics: a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides; b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, and KpnI, and having a restriction site map as shown in FIG. 1(c); c) having a molecular size of approximately 10.0 kb; and d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof. A further object of the present invention is to provide an isolated, purified DNA fragment having the following characteristics: a) comprising a nucleotide sequence derived from a DNA fragment obtained from a strain of the unicellular green alga Chlamydomonas reinhardtii that exhibits resistance to porphyrin-accumulating type herbicides; b) containing restriction sites for EcoRI, XhoI, PstI, PstI, PstI, PstI, PstI, BamHI, SalI, SalI, XhoI, HindIII, BamHI, SalI, HindIII, and KpnI, and having a restriction site map as shown in FIG. 1(d); c) having a molecular size of approximately 13.8 kb; and d) which confers resistance to porphyrin-accumulating type herbicides in plant or algal cells when expressed therein, or a biologically functional equivalent thereof. Further objects of the present invention are to provide plasmids and microorganisms containing any of the foregoing DNA fragments or biologically functional equivalents thereof, a method of conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing said DNA fragments or biologically functional equivalents thereof into plant or algal cells in a functionally operable manner so that said DNA fragments or biologically functional equivalents thereof are expressed in said plant or algal cells, and the expression of the DNA fragment confers resistance to porphyrin-accumulating type herbicides upon the transformed plant or algal cells. It is preferred that cells cultured in vitro that have been transformed by the DNA fragments of the invention in a functionally operable manner are resistant to a porphyrin-accumulating type herbicide at a concentration of at least 0.01 .mu.M, preferably at a concentration of at least 0.03 .mu.M, most preferably at a concentration of at least 0.1 .mu.M herbicide. When compound A or compound B are used as the test compounds, the range of concentration is preferably 0.01 to 0.3 .mu.M, more preferably 0.03 to 0.6 .mu.M, most preferably 0.1 to 0.3 .mu.M. Otherwise the range is between 0.01 to 30 .mu.M, more preferably 0.03 to 10 .mu.M, most preferably 0.1 to 3 .mu.M. The concentration of herbicide used to test resistance of transformed plants or tissues therefrom is to the high end of these ranges or even higher, and can be determined by the ordinarily skilled artisan by experimentation typical in the art. The herbicide used for testing herbicide resistance of cells in vitro or of whole transformed plants or algae is preferably a N-phenyl-tetrahydrophthalimide compound. N-(4-chloro-2-fluoro-5-propargyloxy)phenyl-3,4,5,6-tetrahydrophthalimide (compound A) or 7-fluoro-6-[(3,4,5,6,)-tetra-hydrophthalimido]-4-(2-propynyl)-1,4-benzoxaz in-3(2H)-one (referred to below as "compound B") are especially preferred for this purpose. Another object of the present invention is to provide plants or algae into which have been introduced in a functionally operable manner said DNA fragments or biologically functional equivalents thereof. A still further object of the present invention is to provide an isolated, purified genomic DNA fragment comprising the nucleotide sequence shown in SEQ ID NO:1; plasmids and microorganisms containing said DNA fragment; a method of conferring resistance to porphyrin-accumulating type herbicides upon plant or algal cells, comprising introducing the cDNA corresponding to the mRNA encoded by said DNA fragment that confers porphyric herbicide resistance on plant or algal cells, wherein said cDNA is expressed; and plants or algae into which cDNA corresponding to the mRNA encoded by said DNA fragment having the nucleotide sequence shown in SEQ ID NO:1 has been introduced, wherein said cDNA is expressed. Yet further objects of the present invention include the use of any of the DNA fragments or biologically functional equivalents thereof disclosed herein as a genetic marker (for herbicide resistance), to produce a recombinant plasmid or transformed microorganism, to produce a probe useful in identifying related DNA sequences that confer resistance to porphyrin-accumulating type herbicides in plant and algal cells, and to produce plants or algae resistant to porphyrin-accumulating type herbicides. The DNA fragments and biologically functional equivalents thereof of the present invention are hereinafter referred to as the "subject nucleic acid fragments" or "subject DNA fragments". Specific individual fragments will be designated by their restriction sites and molecular sizes (kb). The present invention includes plasmids containing the above-mentioned DNA fragments or their biologically functional equivalents (hereinafter referred to as the "subject plasmids"), microorganisms containing these DNA fragments or their equivalents (hereinafter referred to as the "subject microorganisms"), plants or algae containing these DNA fragments or their equivalents (hereinafter referred to as the "subject plants"), and methods for conferring resistance to porphyrin-accumulating type herbicides upon plant and algal cells by using these DNA fragments or their equivalents. With regard to the terminology used herein, the term "DNA fragments" refers not only to the subject DNA fragments, but also to degenerate isomers and genetically equivalent modified forms of these fragments. "Degenerate isomers" is taken here to mean isomers whose nucleotide base sequence is degenerately related to the original fragments; that is, all nucleic acid fragments including the corresponding mRNA or corresponding cDNA, that contain essentially the same genetic information as the original fragments. "Genetically equivalent modified forms" is taken here to mean DNA fragments that may have undergone base changes, additions, or deletions, but which essentially contain the same inherent genetic information as the original fragments. Specific examples of the latter include DNA fragments whose nucleotide sequence shows high homology to the subject nucleic acid fragments, which are readily isolated using conventional DNA-DNA or DNA-RNA hybridization techniques, or that are amplified using known PCR (Polymerase Chain Reaction) methods, and which possess the ability to confer resistance to porphyrin-accumulating type herbicides when introduced by conventional transformation techniques into plant or algal cells normally sensitive to these herbicides. The phrase "porphyrin-accumulating type herbicide" or the phrase "porphyric herbicides" refers to light-dependent herbicides, i.e., compounds that kill sensitive plants in the presence of light, but which exhibit no herbicidal activity in darkness, and which induce the accumulation of high levels of porphyrins in plants to which they have been applied. These herbicides include, for example, oxadiazon, flupropacil, [N-(4-chloro-2-fluoro-5-propargyloxy)phenyl-3,4,5,6-tetrahydrophtalimide (referred to below as compound A), the diphenyl ether herbicides such as acifluorfen, lactofen, oxyfluorfen, as well as the following: pentyl [2-chloro-5-(cyclohex-1-ene-1,2-dicarboximido)-4-fluor-phenoxy]acetate, 7-fluoro-6-[(3,4,5,6,)-tetra-hydrophthalimido]-4-(2-propynyl)-1,4-benzoxaz in-3(2H)-one (referred to below as "compound B"), 6-[(3,4,5,6-tetrahydro)pthalimido]-4-(2-propynyl)-1,4-benzoxazin-3(2H)-one , 2-[7-fluoro-3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]perhy droimidazo [1,5-a]pyridine-1,3-dione, 2-[(4-chloro-2-fluoro-5-propargyloxy)phenyl]perhydro-1-H-1,2,4-triazolo-[1 ,2-a]pyridazine-1,3-dione, 2-[7-fluoro-3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]5,6,7, 8-1,2,4-triazolo[4,3-a]pyridine-3H-one, 2-[3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-1-methyl-6-tri fluoromethyl-2,4(1H,3H)-pyrimidinedione, 2-[6-fluoro-2-oxo-3-(2-propynyl)-2,3-dihydrobenzthiazol-5-yl]-3,4,5,6-tetr ahydrophthalimide, 1-amino-2-[3-oxo-4-(2-propynyl)-3,4-dihydro-2H-1,4-benzoxazin-6-yl]-6-tri- fluoromethyl-2,4(1H,3H)-pyrimidinedione, as well as analogs of these compounds. The subject nucleic acid fragments may be constructed by the artificial synthesis of their nucleotide sequences; however, they are more typically isolated from a mutant strain of the unicellular green alga Chlamydomonas reinhardtii, designated RS-3, that is resistant to porphyrin-accumulating type herbicides. Said mutant strain RS-3 is stored at the Chlamydomonas Genetics Center (address: DCMB Group, Department of Botany, Box 91000, Duke University, Durham, N.C., 27708-1000, USA) under the entry number CC-2674. Thus, the mutant strain RS-3 is publicly available for distribution. As will be described below, the microorganisms that host the plasmids containing the subject nucleic acid fragments are also on deposit under the terms of the Budapest Treaty, and are thus freely available as well. The plasmids hosted by these microorganisms can be readily extracted using conventional techniques and the subject fragments recovered by reference to the restriction maps shown in FIGS. 1(a)-1(d). It would be possible, for example, to introduce specific alterations into these fragments using PCR or other site-directed mutagenesis techniques, or to use the subject nucleic acid fragments or their corresponding cDNAs, PCR products, or oligonucleotides as probes to isolate other DNA fragments exhibiting high homology to the subject nucleic acid fragments, and thus to generate homologs as discussed above. Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. It should be understood, however, that the following detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawings, all of which are given by way of illustration only and are not limitative of the present invention, in which: FIGS. 1(a)-1(d) shows restriction site maps of cloned DNA fragments of various sizes that confer resistance to porphyrin-accumulating type herbicides. The sizes of the fragments are indicated by the numbers (kb) in FIG. 1(e). Abbreviations: B, BamHI; S, SalI; P, PstI; X, XhoI; E, EcoRI; H, HindIII; K, KpnI; C, ClaI. FIG. 1(a): 3.4 kb DNA fragment designated as Xho3.4; FIG. 1(b): 9.9 kb DNA fragment designated as Hind9.9; FIG. 1(c): 10.0 kb DNA fragment designated as Hind10.0; FIG. 1(d): 13.8 kb DNA fragment designated as Eco13.8; FIG. 1(e): approximately 40 kb DNA fragment harbored by cosmid clone 2955 (Cos2955). FIG. 2 shows the structure of a pBS plasmid containing an Xho3.4 fragment insert. Distances between restriction sites (kb) are shown by the numbers in the insert. FIG. 3 shows the structure of a pBS plasmid containing a Hind10.0 fragment insert. Distances between restriction sites (kb) are shown by the numbers in the insert. FIGS. 1(c) and 2 show the PstI sites. FIG. 4 shows the structure of a pBS plasmid containing an Eco13.8 fragment insert. Distances between restriction sites (kb) are shown by the numbers in the insert. FIGS. 1(d) and 2 show the PstI sites. DETAILED DESCRIPTION OF THE INVENTION The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery. The contents of each of the references cited herein are herein incorporated by reference in their entirety. OVERVIEW The present nucleic acid fragments are obtained by conventional genetic engineering protocols as described in publications such as Molecular Cloning, 2nd Edition, by J. Sambrook, E. F. Frisch, and T. Maniatis, Cold Spring Harbor Publications (1989). Specifically, genomic DNA is extracted from the mutant strain RS-3 according to a protocol such as that described by E. H. Harris, The Chlamydomonas Source Book, pp. 610-613 (Chapter 12), Academic Press, San Diego (1989). Namely, C. reinhardtii cells are lysed and the DNA is extracted by treatment with a protease and surface active agents such as SDS or Sarkosyl. Genomic DNA is subsequently extracted by conventional techniques involving phenol-chloroform extraction, centrifugation, etc., to remove proteins, after which the DNA is recovered by ethanol precipitation. The DNA thus obtained can be further purified by sodium iodide-ethidium bromide density gradient centrifugation, and the lowermost, major band corresponding to nuclear genomic DNA recovered. Nuclear genomic DNA thus obtained is partially digested using an appropriate restriction enzyme such as Sau3AI. Linkers or adaptors are attached to both ends of the DNA fragments thus obtained using T4 DNA ligase. If necessary, excess free linkers or adaptors can be removed by gel filtration, and the fragments can then be inserted into an appropriate commercially available cosmid vector or a phage vector such as those derived from .lambda. phage. Phage particles generated by in vitro packaging are transfected into E. coli and allowed to form colonies or plaques on solid media. A genomic DNA library can be obtained by isolating and maintaining individual E. coli clones harboring hybrid cosmids or by conventional methods for isolating and maintaining E. coli clones or phage particles in a mixture. Since no porphyric herbicide resistance gene had been previously isolated and characterized from any plant or algal species prior to the present invention, it was not feasible to screen the genomic DNA library described above by synthesis of an oligonucleotide probe corresponding to the deduced nucleotide sequence of such a gene, labeling this probe with a radioisotope or fluorescent tag, and using this to select genomic DNA clones containing the subject DNA fragments. Therefore, the genomic clones containing subject DNA fragments were screened by transforming a strain of Chlamydomonas reinhardtii sensitive to porphyric herbicides with the genomic DNA from the cosmid library using normal transformation techniques for this organism (Kindle, K., Proc. Natl. Acad. Sci. USA Vol. 87, p. 1228 (1990); Boynton J. E. and Gillham, N. W., Methods In Enzymology: Recombinant DNA, Part H, R. Wu, Ed., Academic Press, San Diego, Calif., Vol. 217, p. 510, (1993)) to isolate hybrid cosmids containing nuclear genomic DNA fragments capable of conferring resistance to a porphyric herbicide. A restriction map of the hybrid cosmid clone thus obtained was determined, various restriction fragments were subcloned into the pBluescript vector, and subclones that conferred resistance to porphyric herbicides onto normally sensitive Chlamydomonas strains were selected. Using the subject DNA fragments and the subject plasmids as starting material, the nucleotide sequence of the 3.4 kb fragment was determined by the method of Sanger (Sanger, F. and Coulson, A. R. J. Mol. Biol., Vol. 94, p. 441 (1975); Sanger, F., Nicklen, and Coulson, A. R. Proc. Natl. Acad. Sci. USA, Vol. 74, p. 5463 (1977)) or improved versions of this method. Sequences of the larger 9.9, 10.0, and 13.8 kb fragments or the cDNAs corresponding to these fragments can be determined by the same methodology. The transcriptional initiation site of the porphyric herbicide resistance gene can be localized in one or more of these overlapping fragments using the primer extension technique described by Bina-Stem, M. et al. (Proc. Natl. Acad. Sci. USA, Vol. 76, p. 731, (1977)) and Sollner-Webb and Reeder, R. H. (Cell, Vol. 18, p. 485 (1978)), or by the S1 mapping technique described by Berk, A. J. and Sharp, P. A. (Proc. Natl. Acad. Sci. USA, Vol. 75, p. 1274 (1978)). Typically, promoter sequences that are responsible for the regulation of gene expression are found in a region approximately 1 kb to 10 kb upstream of the transcription initiation site. The promoter region of the gene which confers resistance to porphyric herbicides can be determined by using standard Chlamydomonas transformation techniques (Kindle, K., Proc. Natl. Acad. Sci. USA, Vol. 87, p. 1228 (1990)) and chimeric reporter constructs. For example, various lengths of the region upstream of the transcription start site can be joined to an appropriate heterologous reporter gene such as GUS or one encoding enzymatically-determined antibiotic resistance. By introducing these constructs into Chlamydomonas reinhardtii using transformation and monitoring reporter gene expression, the promoter region of the gene conferring resistance to porphyric herbicides can ultimately be determined. In addition, a transcription terminator sequence is expected to be present within one or more of the overlapping cloned genomic DNA fragments downstream of the poly-A addition signal found in the 3' non-coding region downstream of the stop codon. Herbicide-resistant transformants were obtained from the 13.8 and 10.0 kb fragments from RS-3 at about 80-fold higher frequency than from the 3.4 kb fragment. This is consistent with the 13.8 and 10.0 kb fragments containing the entire coding sequence plus upstream and downstream regulatory elements and integrating non-homologously and randomly in the nuclear genome of the herbicide-sensitive recipient strain. In contrast, the low transformation frequency observed with the 3.4 kb fragment is consistent with this fragment containing only a portion of the RS-3 gene which must integrate into the herbicide-sensitive RS-3 gene of the recipient by homologous recombination to be expressed. Nuclear transformants of Chlamydomonas reinhardtii arise much more frequently by random non-homologous recombination than by homologous recombination as has been demonstrated by experiments with the nuclear nit-l gene by Sodeinde, O. A. and Kindle, K. L., (Proc. Natl. Acad. Sci. USA Vol. 90, p.9199 (1993)). The foregoing will be described in detail in the Examples presented below, although the present invention is not limited to these Examples. |
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