Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/822—Reducing position variability, e.g. by the use of scaffold attachment region/matrix attachment region (SAR/MAR); Use of SAR/MAR to regulate gene expression

Definitions

• the invention relates to the field of plant molecular biology, in particular to the technolog transferring an exogenous gene into a plant cell and conferring a stable phenotype associated said gene on a transgenic plant.
• Transgene or “exogenous gene” are terms used in the art to denote a gene which has b transferred to a host cell or host plant from a source other than the host cell or host pl
• a transgene may herein refer to a gene normally locatable in an host plant or plant c to which is added one or more flanking regions comprising stabilizing DNA segments. Thu gene endogenous to a host plant cell or host plant may be modified with flanking regi comprising stabilizing DNA segments whereby the endogenous gene becomes an exogenous g or transgene with the meaning of the instant application.
• the terms “transge and "exogenous gene” have the same meaning. Transfer of an exogenous gene to a host pl cell can be accomplished by a variety of means known in the art. Most classes of plants h been transformed and regenerated to yield adult plants expressing a phenotype associated with transgene.
• the process of producing a transgenic plant typically includes exposing plant cells, which be in the form of individual cells, protoplasts or excised tissue, to DNA comprising the exogen gene, in order to introduce the exogenous gene into the host cells. Only a fraction of the c exposed to the DNA are actually transformed. The recipient cells are then cultured in vitro order to proliferate the transformants and to identify and select for those which express transgene. Frequently a selectable marker is introduced, together with the exogenous DNA, that transformants can be selected by their ability to grow under conditions that inhibit gro of nontransformed cells, or which favor growth of transformed cells. However, it is also possi in some cases to identify directly the transformed cells or callus containing them. Further st include techniques of regeneration to produce differentiated shoots, roots or embryos from whi ultimately, whole plants can be obtained. Such steps are known to the skilled man.
• Primary transformants are those cells or proliferated tissue (e.g., callus colonies) which initially observable, directly or indirectly, as possessing the exogenous gene after transformation step. Most commonly, possession of the exogenous gene is observed indire as expression of a co-transformed selectable marker. In some instances the phenotype associat with expression of the exogenous gene will be observable in the primary transformant.
• selectable marker such as antibiotic resistance
• culture in the presence of the selecti agent the antibiotic, ensures that only cells expressing the resistance phenotype will grow. In t absence of selection, however, it has frequently been observed that descendants of prima transformants lose the phenotype associated with the transgene.
• explants of primary transformant callus often fail to display the phenotype of the exogenous gene, in t absence of continued selection pressure.
• whole plants are regenerated fro transformed tissue or callus, some of the regenerated plants fail to have the phenotype of t exogenous gene, and the same phenomenon is sometimes observed in the progeny of selfe transformed plants.
• the loss of phenotype is du to loss of the exogenous gene itself, or to loss of ability to express the exogenous gene.
• the lo of phenotype whatever the mechanism, results in a gradual decline in overall transformatio efficiency, i.e., the total number of transformants declines over time with respect to the numb of initial transformants.
• the present invention provides a means for stabilizing transforman against loss of phenotype associated with the exogenous gene, so that higher overa transformation efficiency is obtainable.
• LIS Li-3,5-diiodosalicylate
• endonuclease digestion Such procedures leave residual DNA segments bound to the nuclea scaffold.
• DNA segments have been termed scaffold attachment regions (SAR) and matri associated regions (MAR).
• SAR scaffold attachment regions
• MAR matri associated regions
• MAR DNA segments are considered to be functionally similar i nature, independent of how they are obtained.
• MAR is used herein to refer to DN segments isolated from nuclear scaffold or nuclear matrix preparations after endonucleas treatment. MARs typically bind reversibly to nuclear matrix or scaffold preparations. Binding is saturab indicating binding to a limited number of specific sites.
• MARs can be of any size, however, th are generally of about 1 kb or less in size and are generally AT rich. They do not necessari share extensive sequence homology, although certain sequence motifs have been observed in so MARs. Many MARs possess a topoisomerase II cleavage site consensus sequence.
• MARs are believed to function in vivo as structural attachment points linking chromosomal D to structural elements of the nucleus.
• Models of chromosome structure have been proposed, which chromosomal regions between two adjacent MARs form a loop of DNA between t anchor points of the MARs.
• MAR attachment facilitates transcripti of nearby genes, by locating those genes close to nuclear pores or channels where polymeras transcription factors, substrates, etc., may concentrate.
• anchorage to the nucle matrix serves to separate or isolate groups of genes on separate chromatin loops, acting boundary elements to limit the influence of nearby transcription units on one another, common called position effects.
• MARs appear to function across species boundaries, although matrix binding specificity m be diminished, for example when using animal MARs in plants. Functional association betwe MARs and DNA replication has also been implicated in studies showing that some matrix-bindi sequences of maize DNA may function as ARS (autonomous replicating sequence) elements yeast.
• ARS autonomous replicating sequence
• MARs function both as insulators wh bracketing a gene together with its enhancers, to maintain activity of the enhancers by isolati them from chromosomal position effects, and as barriers when interposed between a gene and enhancer.
• Tobacco MARs (termed SARS therein) were isolated fro flanking regions of three root-specific genes.
• An "endogenous" assay for MARs was disclose based on their ability to bind to nuclear scaffold preparations.
• scaffold-associated DNA region located downstream of the pea plastocyanin gene was isolat and characterized by Slatter, R.E. et al. (1991) Plant Cell 1:1239-1250.
• the SAR was linked a downstream repeated sequence and had a sequence rich in A and T sequences, sever topoisomerase II binding sites and several ARS sequences.
• Plant Cell 4:463-471 used a tobacco-derived SAR to analyze the effe of flanking a reporter gene in transgenic plants, similar to the experiment described by Phi- V et al. (1990).
• Qualitatively similar results were obtained showing an effect of a tobacco SA flanking a transgene on variance of expression among independent tobacco transformant
• no increase of average expression level was observed.
• the effect of reducing varian was not observed for constructs containing a mammalian ⁇ -globin SAR instead of the tobac SAR. All effects heretofore observed have related to phenomena occurring within a sing generation.
• the basis of the present invention is that there exist certain stabilizing DNA segments whic when introduced together with an exogenous gene into plant host cells, serve to stabilize t exogenous gene from one generation of cells to the other, or from one generation of transgen plants to another. Stabilization has occurred when the phenotype associated with the exogeno gene (the "transgene phenotype") is retained from one generation to the next at a higher frequen than is observed in the absence of the stabilizing DNA. Since the rate of loss of the transge phenotype is most apparent by comparison with primary transformants, stabilization is al defined as a reduction of the frequency of loss of the transgene phenotype over one or mo generations when compared with the number of primary transformants.
• Stabilizing DNA segments include, but are not limited to, MAR and SAR segments as identifi by art-known measures of binding with nuclear matrix or scaffold. Stabilizing DNA segmen also include certain repeated sequences, locus control regions (LCR), also known as loc activating regions (LAR) and certain other sequences such as DNAse hypersensitive regions (HR In the absence of binding assays, suitable candidate stabilizing DNA segments can be identifi by various structural attributes such as possession of one or more topoisomerase II binding sit hypersensitivity to DNAse, having high A & T content and ARS consensus sequences. stabilizing DNA segment can have one or more of these structural features, however, the absen of one or more such features would not rule out the segment as having the stabilizing functi
• LCR locus control regions
• LAR loc activating regions
• HR DNAse hypersensitive regions
• a functional test is described herein which exploits the property of a stabilizing DNA segm to separate adjacent genes, on a vector into independently expressed units.
• a stabilizing D segment interposed between two genes or transcription units oriented in tandem on a vect effectively prevents the depressed expression of the downstream gene which occurs in the absen of a stabilizing DNA segment between the genes, as disclosed herein.
• the invention provides vectors for achieving stable transgene expression over ma cell generations by providing stabilizing DNA segments in one or both the 3'- and 5'-flanki regions of the transgene.
• the invention also includes vectors for achieving independe expression of at least two exogenous genes on the same vector by providing a stabilizing D segment interposed between the exogenous genes.
• Also included as part of the invention is a method for transforming plant cells with an exogeno gene to enhance the stability of the phenotype associated with the exogenous gene, by introduci into the plant cell a vector having at least one stabilizing DNA segment present in a flanki region of an exogenous gene.
• the vector will possess more than o stabilizing DNA segment such that the exogenous gene(s) lies between the stabilizing DN elements.
• Figure 1 is a diagram of the plasmids pZO1071 and pZO1051 described in Example 2
• Figure 2 is an autoradiograph of DNA after electrophoresis in a nuclear matrix bindi assay, as described in Example 9.
• Figure 3 is a graph showing saturation of MAR binding to nuclear matrix as the amou of MAR DNA is increased. See Example 9.
• Figure 4 is a graph showing competition of tomato MAR binding to tomato nucle matrices as amounts of competitor MAR DNA is added. Each test represents 100 ng labell tomato MAR per assay. pBR322 vector fragments
• Figure 5 is an autoradiograph of an electrophoretic gel showing MAR binding to nucle matrix and nuclear shell preparations in presence and absence of R. coli DNA. See Example
• FIG. 6 is a diagram of a T-DNA vector, pZU043A carrying the TSWV nucleoprotei gene controlled by a CaMN 35S promoter. See Example 15.
• Figure 7 is an autoradiograph of a Southern blot showing the presence of TSWV DN in various transformant lines, as described in Example 15.
• Figure 8 is a diagram of T-DNA vectors carrying various MAR segments, as describe in Example 17.
• SEQ ID No. 1 shows the nucleotide sequence of MRS5 given in Table IA
• SEQ ID No. 2 shows the nucleotide sequence of MRS4 given in Table IB
• SEQ ID No. 3 shows the partial nucleotide sequence of MRS 3 given in Table 1C
• SEQ ID No. 4 shows the nucleotide sequence of MAR1 given in Table 5
• a SEQ ID No. 5 shows the nucleotide sequence of MAR2 given in Table 5B
• SEQ ID No. 6 shows the nucleotide sequence of the SARLa region from a soybean sma heatshock gene (MAR3) given in Table 5C
• SEQ ID No. 7 shows the nucleotide sequence of the primer 1929ECOR
• SEQ ID No. 8 shows the nucleotide sequence of the primer 1929ECOU
• Stabilization is the term used herein to denote increased retention of a phenotype associate with an exogenous gene, over one or more plant cell and/or plant generations, when comparin transformants having a "stabilized exogenous gene” of the invention with those having a cont exogenous gene.
• the comparison is made between the number of transformants having phenotype associated with the exogenous gene ("transgene phenotype") at a given time, and number of transformants having the transgene phenotype at a later time, after correcting for to cell proliferation. Therefore, it is not the quantitative expression level which is to be measu (although that may incidentally be affected), but the rate of retention of the transgene phenoty itself in individual cell lines and/or plants descendant from primary transformants.
• a stabilized exogenous gene is an exogenous gene which includes one or preferably m stabilizing DNA elements in its 3'- and 5' flanking regions.
• stabilizi DNA elements can be provided such that all genes are flanked, and stabilized by a single pair stabilizing DNA segments, or they may be individually flanked, or one gene can be flanked wh another is left unflanked.
• the unflanked gene may be less stable than t flanked gene, which may be desired if, for example, the unflanked gene were only useful a marker for initial transformant selection, but of no value to the whole plant.
• Stabilizing DNA segments include MAR, SAR, LCR or LAR, HR and the like as describ supra, repetitive elements as described infra, and other DNA segments sharing structural a functional features therewith. Some stabilizing DNA segments also exert a shielding effect wh interposed between two tandemly oriented genes on a single vector, such that the downstre gene is less affected by expression of the upstream gene than would be observed if no stabilizi DNA segment were present.
• a demonstration of the shielding effect of MRS elements provided herein, providing a means for recognizing a stabilizing DNA segment.
• Other means recognizing a stabilizing DNA segment include the various matrix binding and scaffold bindi assays known in the art. Stabilizing DNA elements can be obtained from any eukaryotic prokaryotic cell type.
• Preferred sources are eukaryotic cell types from animal or plant sourc and most preferred are stabilizing DNA segments compatible with the host cell, such D segments may be endogenous to the host cells. Stabilizing DNA segments display a range effectiveness and specificity. Any detectable level of stabilization is operative, thereby reduci the need to screen and evaluate large numbers of transformants to find satisfactory performe
• a gene of the host pla provided with a different promoter so that the timing, tissue specificity, expression leve inducibility or other aspect of gene control was altered such that the plant's transgene phenotyp was distinct from the wild type would be an exogenous gene under the definition.
• the gene a parasite or pathogen of the host plant or from other sources such as a nonpathogen to the ho plant is also an exogenous gene.
• the phenotype associated with the exogenous gene is any trait or characteristic conferred on the transgenic plant or host plant cells, by the exogenou gene.
• a phenotype can range from a measurable amount of the protein or RNA encoded by th exogenous gene, to a physical or agronomic trait. In most cases, more than one phenotype ca be detected for a given exogenous gene.
• the exogenous gene encodes a insecticidal protein such as the Bacillus thuringiensis toxin
• the phenotypes include presence o the protein in plant tissues and resistance to certain insects.
• the phenotypes include presence of the RNA in plant tissue and resistance to certain viruses.
• one or more exogenous genes may b introduced in a single stabilized gene cassette, in order to provide an easily measured phenotyp linked to a difficultly measurable phenotype.
• An example is kanamycin resistance linked to gene for fungal resistance. Therefore, a phenotype associated with an exogenous gene include a phenotype associated with a linked exogenous gene.
• exogenous genes and their associated phenotypes include, but are not limited to: a) antisense RNA to confer virus resistance or to modify expression of an endogenou gene of the host plant; b) viral coat protein and/or RNA, or other viral or plant genes to confer virus resistance c) fungus resistance, possibly conferred by a wound induced gene; d) insect resistance conferred by an insecticidal toxin or other protein; e) flower color or flower pattern conferred by genes affecting pigment production; f) yield improvement; g) drought resistance; h) self-incompatibility; i) male sterility j) delayed or accelerated maturation; k) protein production, for example, conferred by mammalian genes encodin therapeutically useful protein;
• a stabilizing DNA segment can inserted in the 5'-flanking region upstream of promoter sequences, or in the 3'-flanking reg which may or may not lie downstream of polyadenylation signal sequences, if present.
• the e distance of the stabilizing DNA segment from either end of the exogenous gene is not criti
• the construct includes T-DNA borders of Agrobacterium tumefaciens, the stabiliz DNA segments should be inserted so as to be between the T-DNA borders, to ensure integrat of the stabilizing DNA segments.
• the effect of one or more stabilizing DNA elements is stabilize those genes lying in close association or proximity therewith.
• the prefer construction is to place the stabilizing DNA elements so that they flank only the genes wh expression is to be stabilized.
• stabilizing DNA segments do not necessarily h restriction sites at or near their ends, it is a matter of ordinary skill to modify the ends using, e ligation of oligonucleotide linkers, or primers for polymerase chain reaction incorporatin restriction site sequence, to facilitate inserting the stabilizing DNA segments at desired sites a vector.
• Orientation of a stabilizing DNA segment with respect to the orientation of the g to be stabilized is not a critical factor as regards the stabilization function.
• the stabilizing D elements flanking a given gene need not be identical, nor must they be obtained from the s source organism.
• the stabilizing DNA elements need not be exogenous, but can instead obtained from the host organism. Although there is superficial similarity between animal M and SAR sequences and their plant counterparts, plants are the preferred source for stabilizi DNA element, the most preferred being plants of the host plant species or closely related speci
• Stabilizing DNA segments can be isolated by a variety of methods. First, it is possible to analy the 3' or 5' flanking regions of any stable gene, preferably, but not limited to, those flanki regions lying within about 500 kbp either side of the coding region. Candidates are identifia by such criteria as DNAse .hypersensitivity, topoisomerase II binding sites, ability to bind mat or scaffold preparations, and the like. An alternative method is simply to clone fragments o stable gene, then select the fragments that display such properties as DNAse hypersensitivi locus control regions, matrix binding, scaffold binding and the like.
• Yet another alternative is isolate plant nuclei, extract the nuclei with LIS, then treat with an endonuclease including, f example, a restriction endonuclease, extract DNA remaining bound to matrix or scaffold phenol extraction, and clone the resulting MARs.
• Another procedure is to isolate plant nucle matrices or scaffolds, then screen for DNA fragments preferably limited to a size approximately 0.1 - 1.0 kb capable of binding to the matrices or scaffolds. Those able to bi tightly are then cloned.
• an isolated locus control regi (LCR) from, e.g., chicken, is used to isolate LCR binding protein.
• the gene encoding an L protein is then cloned and expressed in a suitable system to produce sufficient amounts to usable to identify plant DNA segments capable of binding to the protein.
• whe candidate stabilizing DNA segments are cloned, their ability to stabilize a transgene through ma cell generations is testable using a marker transgene in a suitable transgenic host plant.
• the te host plant preferably displays a low frequency of stable expression in the absence of a stabilizi DNA segment. For example, lettuce displays a frequency of stable expression about 15% primary transformants, when not transformed with a stabilizing DNA segment.
• Transformation can be carried out by any means known in the art. These include, but are n limited to, direct transfer of DNA into whole cells, tissues or protoplasts, optionally assisted chemical or physical agents to increase cell permeability to DNA, e.g., treatment wi polyethylene glycol, dextran sulfate, electroporation and ballistic implantation of DNA-coat particles. Transformation is also mediated by Agrobacterium strains, notably A. tumefaciens a A. rhizogenes, and also by various genetically engineered transformation plasmids which inclu portions of the T-DNA of the tumor-inducing plasmids of Agrobacteria. The T-DNA borders c be incorporated into other transformation constructs, to facilitate integration of a stabili exogenous gene lying between the T-DNA border elements. Other means for effecting entry DNA into cells include viral vectors and agroinfection.
• DNA constructs suitable for transformation include at a minimum the exogenous gene (prom and coding sequence) to be transferred, flanked by at least one stabilizing DNA segment, but involve other elements as well.
• the stabilized exogenous gene can be inserted into a vec flanked by T-DNA borders, combined with a marker gene, all according to techniques know the art.
• the choice of construct will be influenced by the method of transformation adopted. example, if a ballistic transformation is desired, use of a vector may be superfluous, w flanking T-DNA borders (in addition to the stabilizing DNA segments) may be desired as a me of promoting genomic integration of the transgene.
• vectors suitable for plant transformation include, but are not limited to:pCGN15 pART27, ⁇ OCA18, pCVOOl, pCV002, MON200, pGV3850, pGV260, pGPTV vectors, and Mi Ti plasmids. References describing the foregoing and other vectors suitable for use in invention are also included: (Mini-Ti) Framond de, A . (May 1983) Bio/Technology, pp. 2 269; (pCV001/pCV002) Koncz, C. and Schell, J. (1986) Mol. Gen. genet. 204:383-396; Klee, et al.
• Virtually all plants of agronomic or horticultural value are known to be both transformable regenerable.
• the techniques vary in individual detail from species to species, as is underst by those skilled in the art.
• the nature of applicable transformation methods to be used fo given plant species may be affected by the type of regeneration protocol that can be used in given instance. For example, where regeneration cannot be obtained from protoplasts, the met of transformation must be suitable for whole cells or tissues.
• the plant species is diffic to transform using Agrobacterium, the alternative of ballistic transformation may be preferre All such considerations are matters well-known to those of skill in the art.
• plants suitable for use in the invention include, but are not limited to, those pla that are members of Solanaceae, Apocynaceae, Chenodiaceae, Polygonaceae, Boraginace Compositae, Rubiaceae, Scrophulariaceae, Caprifoliaceae, Leguminosae, Araccae, Morace Euphorbiaceae, Brassicaceae, Primulaceae, Violaceae, Protulaceae and Rosaceae.
• plants of the following families are suitable for use in the invention: polypodiaceae, umbellife liliaccae, crucifereae, gramineae, geranaceae, ranunculceae, begoniaceae, labiatae, caryophyllace balsaminaceae, papilionaceae, gesneriaceae, violaceae, araliaceae, as well as plants common known as: fern, carrots, leek, asplenium, radishes, celery, onions, fennel, wheat, rye, barley, co (maize), soybean, oats, rices, geraniums, violets, windflowers, ornamental asparagus, begoni flame nettle, lark spur, carnations, gilliflowers, Busy Lizzy, lupin, crowflower, sage, bell flowe soap herbs and Panax ginseng.
• tomat melon watermelon, pepper, lettuce, beans, brassica including rapeseed plants, cabbages, broccol cauliflowers, sunflowers, sugar beet, violas, begonia, pelargonium peltatum, pelargoniu hortorum, com (maize), sweet corn, Cyclamen and Impatiens.
• a library of Zea mays L. (A3780) genomic DNA is constructed in pTZ19R (Pharmacia) restricting genomic DNA with EcoRI plus Hindlll, separating the resulting fragments on 0.6 low-melting point agarose, excising the region containing fragments from 1 to 3 kb in siz diluting, melting and ligating to EcoRI plus Hindlll cut pTZ19R, and transforming in I c C600 cells. Unless otherwise specified, methods for cloning and preparing plasmid DNA a essentially as described (Maniatis et al. (1982) Molecular Cloning, Cold Spring Harb Laboratory).
• the maize inserts are cut at their Hindlll sites, made bl by treatment with T4 DNA polymerase, then EcoRI linkers are ligated using T4 DNA lig Excess linkers are removed by treatment with EcoRI. which also frees each fragment from plasmid vector. Each fragment is excised from low-melt agarose and ligated to pZO1 (Example 2), which has been cut with EcoRI and treated with calf intestinal alkaline phosphat (CAP) and excised from low-melt agarose (see Fig. 1). In general, only one orientation of e insert in pZO1071 is recovered.
• CAP calf intestinal alkaline phosphat
• MRS5 is given in Table IA (SEQ ID NO Table IB gives the sequence of MRS4 (SEQ ID NO:2) and Table IC gives a partial sequenc MRS3 (SEQ ID NO:3).
• the EcoO 1091 site of pUC19 (Yanisch-Perron, C. et al. (1985) Gene 33:103-119) is conven to a Bglll site by filling in and ligation of a Bglll linker to give pZO919.
• the NPT II ge cassette is assembled by treating pZO919 with Bglll and CAP and ligating to it a 1.7 kb Bgi to Smal fragment, consisting of the 35S promoter (AM to Ddel, Franck A. et al. (1980) C 21:285-294), maize AdhlS intron 2 (Freeling, M. and Bennett, D.C. (1985) Ann. Rev.
• Plasmid pZO921 consists of the particular orientation of t cassette in pZO919 in which the NOS terminator is closest to the EcoRI site of the multi cloning site.
• the ⁇ -glucuronidase (GUS) cassette consists of the 35S promoter (Ddel to D Franck et al.
• the orientation of the multiple cloning site of pZO919 is reversed by replacing its PvuII fragm with the corresponding PvuII fragment (ca. 300 bp) from pUC18 to form pZO930.
• the G cassette is then inserted again as an EcoRI to Hindlll fragment to form pZO1068.
• NPT II cassette from pZO921 is cloned as a 1.9 kb BamHI to BglHI piece into the Bglll site pZO1068.
• the orientation of the NPT II cassette for which the 35 S promoter is closest to Hindlll site is named pZO1071 (see Fig. 1).
• Table 2 shows the results of a transient assay in electroporated BMS protoplasts.
• the G activity of pZO1071 is set to 1.00. Results are given for each MRS cloned into the EcoRI s of either pZO1051 or pZO1071.
• pZO1051 When electroporated into BMS protoplasts, pZO1051 has o about 20% as much GUS activity as pZO1071. (Conversely, when NPT II activity is measu pZO1071 has somewhat less activity than pZO1051, data not shown.)
• the gene which downstream in the direction of transcription shows reduced expression. The upstream gene activity unchanged from that found when another cassette is not present on the same plasm Each MRS is tested for its ability to relieve the inhibition.
• results from electroporating plasmids into tobacco protoplasts a obtained.
• the effect of the orientation of the MRS relative to the GUS cassette is also examine Table 3 shows that the GUS activity from pZO1051 is also much reduced compared to pZO107 in tobacco protoplasts, that MRS3, MRS4, and MRS5 can relieve this inhibition, and that the may be a modest preference for orientation of MRS4 and MRS3.
• MRS3 the (a) orientatio occurs when the original EcoRI site is proximal to the promoter.
• MRS4 the (a) orientatio occurs when the original Hindlll site is proximal to the promoter.
• MRS5 the (a) orientatio occurs when the original Hindlll site is proximal to the promoter.
• Plasmids pZO1071, pZO1051, pZO1442, pZO1443, and pZO1464 are electroporated into B protoplasts, which are plated on filters, then cultured on suitable agar containing medium wi layer of feeder cells and kanamycin, 75mg/L. Extracts are prepared from about twenty kanam resistant calli for each construct. To determine that each callus is a true transformant, NP activity is confirmed by ELISA (5 Prime, 3 Prime, Inc.). GUS activity is meas spectrophotometrically and normalized to total protein.
• the frequency of GUS-expressing calli is thought to be highest for those constructs contai MRS3, 4 or 5, and the levels of GUS activity found are more uniform as well.
• GUS-expressing calli are maintained for several months on kanamycin containing medium periodically assayed for GUS activity.
• Individual calli transformed with pZO1051 or pZO10 are found to have lost GUS expression, and the fraction of such calli is found to increase ov several months.
• transformants of [one or more of] pZO1442, pZO1443, and pZO14 are found to maintain GUS expression at a significantly higher frequency.
• Example 6 A similar experiment to that of Example 6 is performed, except that a regenerable maize cell li is transformed, either via electroporation or the ballistic method, depending on the cell lin Stable calli recovered after growth on selective media are transferred to suitable regenerati medium for shoot initiation, the shoots are moved to rooting medium, finally resulting in plan which are grown to maturity, characterized for GUS and NPT II expression, and out-crossed selfed. The resulting first progeny generation is also characterized for transgene expression. T expression and heritability of the transgenes continue to be followed for succeeding generation
• MAR1 (maize 0.8kb AT rich region) and MAR2 (maize 1.25 kb region wi ARS3), found within a 5kb maize EcoRI fragment originally cloned by R. Berlani et al. (19 Plant Mol. Biol. ⁇ :161-172) have nuclear matrix binding activity.
• the sequence of the MA fragment is given in Table 5A (SEQ ID NO:4) and that portion of MAR2 not previoul sequenced is given in Table 5B (SEQ ID NO:5) along with the published portion of MA named ARS3 (Berlani et al. 1988 Plant Molecular Biology U_: 173-182).
• MA is subcloned from pZMA321 as an EcoRI -Hindlll fragment into pT7T3-18U (Pharmacia) to fo pZO1927.
• MAR2 is subcloned from pZMA321 as a Hindlll fragment into pT7T3-18U to fo PZO1929.
• MAR3 is directly cloned into EcoRI sites followi restriction with EcoRI.
• pZO1927 and pSVB20-SARL are cut w EcoRI, treated with T4 DNA polymerase I, Hindlll linkers ligated, followed by restriction w Hindlll.
• MAR2 is cloned directly into Hindlll sites following restriction with Hindlll.
• Plasmids pZO 107 land pZO1051 are constructed according to Example 2 and each MAR fragm is cloned into the EcoRI sites. These plasmids are contructed according to Example 2. For o orientation of each MAR at EcoRI, a second copy is also cloned into the Hindlll site to gi plasmids in which the GUS cassette is bound by a pair of MARs.
• Example 3 Preparation and electroporation of protoplasts of maize black mexican sweet (BMS) suspensi cells are decribed in Example 3.
• Table 6 shows the mean results of a number of transient assa in electroporated BMS protoplasts.
• the GUS activity of pZO1071 is set to 1.00. Results given for each MAR cloned into the EcoRI site of either pZO1071 or pZO1051.
• MAR1 t A orientation occurs when the Clal site is proximal to the promoter.
• MAR2 the orientation occurs when the pair of Sac II sites are proximal to the promoter.
• For MAR3 the orientation occurs when the EcoRV site is distal to the promoter.
• Protoplasts are isolated from 20 grams leaves, resuspended in W5 medium, Menczel et al. (198 Theor. Appl. Genet. 59:191-195, spun down (7' 80 g) and resuspended in 15 ml IB (20 mM hep pH 7.4, 0.05 mM spermine, 0.125 mM spermidine, 20 mM KCl, 1% thiodiethanol, 1 M hexyle glycol, 0.5 mM EDTA, 0.5% Triton-X-100,TM 0.2 mM PMSF, 5 mg/ml aprotinin, 10 mM E [trans-epoxy succinyl-L-leucyclamide[4 guanidino] butane]).
• the protoplasts are homogeniz by vortexing 20" and the resulting homogenate is centrifuged for 7' (80 g). The supernatant centrifuged (10' 300 g) and the resulting pellet (crude nuclei) is purified on a 15% perc gradient made in IB (15' 600 g). An interphase and/or a "smear" on the wall of the tube mig appear, both fractions do contain many contaminations and few nuclei. Purified nuclei in t pellet fraction are resuspended and washing in IB buffer without Triton,TM followed centrifugation (10' 400 g).
• Portions of 12 X IO 6 nuclei are washed in 10 ml WB (3.75 mM Tris pH 7.4, 20 mM KCl, mM EDTA, 1% thiodiethanol, 0.05 mM Spermine, 0.125 mM Spermidine, 0.1% digitoni tracylol 1 mg/ml) and spun down for 10' (400 g).
• Washed pellets are resuspended in 100 ⁇ l and the nuclear matrix is stabilized by incubating for 20' at 42°C in a shaking water bat Histone proteins are extracted by incubating the stabilized nuclei with 10 ml LIS-HLE buffer ( mM Hepes pH 7.4, 0.1 M LiAc, 1 mM EDTA, 4 mg/ml LIS [3',5'-diiodosalicylate], 0.1 digitonin, 25 ⁇ g/ml tracylol, 1.5 mM PMSF) for 5' at room temperature.
• 10 ml LIS-HLE buffer mM Hepes pH 7.4, 0.1 M LiAc, 1 mM EDTA, 4 mg/ml LIS [3',5'-diiodosalicylate], 0.1 digitonin, 25 ⁇ g/ml tracylol, 1.5 mM PMSF
• the chromosomal DN which is not bound to the skeleton of the nuclei ( ⁇ 90%), looped out; after centrifugation ( 13.000 g) the extracted nuclei appear as a fluffy pellet, consisting of a stabilized nuclear skelet (the nuclear matrix or nuclear halos) associated with looped out chromosomal DNA.
• the matrices are washed 3 times with 12 ml DB (20 mM Tris pH 7.4, 20 mM KCl, 70 mM NaCI, mM MgCl 2 , 0.05 mM Spermine, 0.125 mM Spermidine, 0.2 mM PMSF); the last matrix pell is resuspended in 9.6 ml DB containing 1200 units restriction enzyme. Looped out DNA removed by digestion for 60' at 37°C. At that stage the matrices are suitable for DNA bindi assays to select MAR sequences.
• a nuclear matrix MAR binding system prepared from IO 6 nuclei, is incubated overnight in D buffer at 37°C with 5 ng ⁇ - 32 P-end labelled digested plasmid carrying potential MAR sequence Binding of MAR DNA fragments will take place either at "empty" matrix binding sites or "occupied” matrix binding sites ("displacement" binding of endogenous MARs). After bindin the mixture is spun down; the pellet (containing the nuclear matrix MAR binding syste associated with bound labelled DNA MAR fragments) is washed once with DB and spun agai The two supernatant fractions (containing the unbound labelled DNA fragments) are pooled a the final pellet is resuspended in 200 ⁇ l DB.
• DNA is isolated from both the supernatant and t pellet fractions by a SDS/proteinase K treatment, followed by a phenol/chloroform extraction, t ether extractions and an ethanol precipitation. Both the DNAs isolated from the supernatant- a the pellet-fractions are electrophoresed on horizontal agarose gels, the gels are dried a autoradiographic exposure is performed for 1-3 days at -70°C with intensifying screens. DN fragment end-labelling, DNA extractions, electrophoresis and X-ray exposures are all perform according to Maniatis (Maniatis et al. 1989).
• Binding in such a system is specific as only the MAR-containing fragment is bound to the pel fraction when the proper nonspecific E. coli competitor DNA quantity is applied (see Fig. Binding in such a system is also saturable as liquid scintillation counting of a bound tomato M fragment in experiments with increasing concentrations of added end-labelled restriction fragme containing that tomato MAR [1-250 ng], show that between 50 and 100 ng fragment maximum binding level is obtained (see Fig. 3).
• MAR binding sites in nuclear matrices are nonselective for a specific MAR fragment as ot MAR fragments can compete for binding effectively, consequently MAR binding is a reversible.
• this is demonstrated for the binding of a tomato MAR; it can be compet to approximately 20% by a 6-fold excess of rat MAR. This suggests that MAR matrix interacti is conserved during evolution.
• nuclear matrix skelet internal matrix consisting of residual nucleoli and granular clusters of electron dense clust embedded in a highly branched network of thin filaments
• nuclear shells s Fig. 5
• the only difference is that binding at the nuclear shell binding sites is more rapi competed by nonspecific R. coli DNA, demonstrating that the number of MAR binding sites nuclear shells is lower relative to the nuclear matrix.
• Nuclear envelopes are isolated from tomato protoplasts according to Kaufmann and Shaper (19 Exp. Cell Res. 155:477-497; Lam Bl-like molecules are isolated from those envelopes described by Aebi et al. (1986) Nature 323:560-564. Lamin Bl is coupled to inert columns, e. sepharose CL 4B, using cyanogen bromide, which are used as selection tools for potential M sequences.
• Tomato nuclear chromosomal DNA is isolated according to Bernatzki, R. and Tanksley, (1986) Plant Mol. Biol. Reporter 4:37-41. Mbol digested tomato DNA is passed over a Lam Bl affinity column and specifically bound DNA fragments are eluted, cloned and furth characterized for nuclear matrix binding in the nuclear matrix MAR binding system as describ in Example 9.
• Example 13 Isolation of plant MAR DNA sequences from a potential chromatin lo surrounding a stable transgene
• TSWV tomato spotted wilt virus
• Transformants selected for kanamycin resistance, are analyzed for both the cop number of the TSWV transgene by Southern blotting hybridization and for expression of t TSWV nucleocapsid gene by specific ELISA assays. Transformants, containing one single cop of the TSWV nucleocapsid gene, are selfed and tested for gene expression stability in t successive generations by ELISA analysis.
• a genomic cosmid DNA library is constructed fro purified tomato chromosomal DNA, isolated from a stable transgenic S3 line using the restrictio endonuclease Mbol.
• Colony hybridization screening with the TSWV nucleocapsid gene as probe results in th identification of clones carrying the transgene; using chromosome walking techniques (Mania 1989), 100 kbp regions upstream and 100 kpb regions downstream of the transgene are identifie and further characterized.
• This 200 kbp region includes part of an euchromatin ("open") loo most probably this region includes a complete euchromatin loop, as the sizes of average chromati loops are 80-90 kbp (Jackson et al. 1990).
• Subclones of this 200 kbp region are tested f specific nuclear matrix binding in the nuclear matrix MAR binding system, as described i Example 9.
• tomato chromosomal DNA fragments are identified as specific MAR DN sequences.
• Example 14 Isolation of plant MAR DNA sequences flanking 5' and 3' of nuclear genes
• a genomic cosmid DNA library is constructed (using the restriction endonuclease Mbol) fro purified tomato chromosomal DNA.
• Colony hybridization screening results in a clone containin the tomato plastocyanin gene with about 20 kbp flanking region from both sides.
• the 5' regio is subcloned and tested for specific binding in the nuclear matrix MAR binding system described in Example 9. An approximately 1 kbp 5' upstream tomato chromosomal D subfragment is identified as a specific MAR DNA sequence.
• Chromatin is organized in topologically constrained DNA loops by the anchoring of specific M sequences to the external (shell) and internal nuclear matrix. Loops carrying potential acti genes (called dispersed euchromatin) are thought to contain regions which are accessible transcription factors, RNA polymerases and other components required for transcription, where inactive loop (regions) (called condensed heterochromatin) are inaccessible.
• a transgene in stable transgenic (plant) line is supposed to be integrated in an at least partly dispersed, op euchromatin loop region, which is transcriptionally active.
• Hypersensitive chromosomal DN regions are defined as DNA regions showing an increased sensitivity toward nuclease digestio Such hypersensitive DNA regions contain cis-acting regulator sequences like LCR sequenc Therefore screening for DNAse I hypersensitive DNA regions is a tool for preselecting chroma conformation-regulating cis-acting elements.
• Protoplasts are isolated as described in Example 8, with the modification that the cell w degradation enzymes are applied in (lower) range concentrations, resulting in permeable ce which are intermediate forms between in vivo cells and protoplasts.
• Cells/protoplasts are wash in W5 medium twice, followed by resuspension in nuclease buffer (0.05 M Tric-HCl, pH 7.8, mM MgCl 2 , 0.01 M 2-mercaptoethanol, 10 ⁇ g/ml BSA). Cells are treated with varyi concentrations of DNAsel (IO '3 - IO '5 U/ml) for 10' at room temperature.
• DNA is isolated usi a standard phenol/chloroform method, digested with appropriate restriction enzymes and blott to Hybond N + .
• Hybridizations are carried out with probes derived from the 200 kbp genom region surrounding the stable TSWV nucleocapsid gene isolated from a stable tomato transgen line (described in Example 12).
• Hypersensitive regions are identified as those regions which show a decrease in amou of probe able to hybridize with the expected DNA restriction fragment when a series of increasi DNAse I concentrations is applied to the permeable tomato cells.
• identification HR can also be performed by analyzing additional hypersensitive related DNA subfragmen essentially as described by Forrester, W.C. et al. (1990) Genes and Development 4:1637-1649
• transgene instability is observed upon introduction of the TS nucleoprotein gene cassette.
• the first signs of "silencing" of transg expression are encountered during ELISA analyses of the progeny populations revealing abnor non-Mendelian segregation ratios.
• Combined ELISA and southern blot analysis of nine individ S2 progeny plants (transformant line 27-5) descending from one primary transformant result the identification of certain plant individuals that carry an inactivated transgene.
• Digestion w Xbal releases the complete TSWV nucleoprotein gene cassette of 1.6 kb, while digestion Hindlll generates border fragments, the number of which correlates with the number of T-D copies integrated into the genome.
• transgene itself segregates normally at DNA level; only one-third of die individuals analyzed have "lost" die transgene thro segregation, but others may harbor transgenes that have been inactivated, resulting in abnor segregation ratios at the protein level.
• Example 18 Stabilization of gene expression over generations using MAR sequences
• T-DNA constructs are transformed to tomato and lettuce, in which t NPTII selection marker and the TSWV nucleoprotein gene cassette are flanked by MA sequences isolated from rat, soybean and tomato (pMARr, pMARs and pMARt, respectivel
• MA sequences isolated from rat, soybean and tomato pMARr, pMARs and pMARt, respectivel
• pMARr, pMARs and pMARt respectivel
• the pMARc construct carri boundary elements consisting of random DNA sequences (e.g., vector DNA) of about equal leng to the MAR elements, but that do not exhibit any affinity to plant nuclear matrices.
• TSWV nucleocapsid protein Upon Agrobacterium-mediated transformation of tomato and lettuce, primary transforman accumulating TSWV nucleocapsid protein are analyzed for their T-DNA copy number throu Southern blot analysis. Only transformants that carry a single copy of the T-DNA are maintain and self-pollinated. From the resulting SI progeny population, ten individual plants expressi die nucleoprotein transgene are once more self-pollinated to produce S2 lines. Nonsegregati S2 lines in which the transgene has been "fixed" in die homozygous state, are identified by mea of ELISA analysis for accumulation of TSWV nucleoprotein. These "homozygous" S2 lines a maintained by repeated selfing and monitored for stability of transgene expression over successi generations.
• the toma as well as the soybean MAR reduce the instability of transgene expression in lettuce exemplified by the pMARc transformant lines.
• Apparendy, plant-derived MAR sequences c be used in heterologous transformation systems to stabilize transgene expression over generation SEQUENCE LISTING
• MOLECULE TYPE DNA (genomic)
• GATAGACCTC AACAGAAAAC TGTTGAGTAA CGGCAGCAAG TGATTGAGTT CAGTAGTTCC 900
• MOLECULE TYPE DNA (genomic)
• CGAGGTACTG CAGAAAAAAG AACCGCAAAA TCCGATCCAA TTTTTCGTAA TCGATTAGTT 240
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• AACGCTAACC CCGGCTTGAA GTGTGCTTAA AGTTTGTAAA TTTCAGTTTC CGCCTATCCA 480
• AAAAGCTT 1268 (2) INFORMATION FOR SEQ ID NO: 6:
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• HYPOTHETICAL NO
• ANTI-SENSE NO
• ORIGINAL SOURCE
• MOLECULE TYPE DNA (genomic)

Landscapes

• Genetics & Genomics (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Cell Biology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=21960497

Family Applications (1)

Country Status (6)

Families Citing this family (7)

Family Cites Families (3)

• 1994
  • 1994-04-18 JP JP6522764A patent/JPH08508649A/ja active Pending
  • 1994-04-18 IL IL10933394A patent/IL109333A0/xx unknown
  • 1994-04-18 WO PCT/EP1994/001193 patent/WO1994024293A1/en not_active Application Discontinuation
  • 1994-04-18 AU AU66790/94A patent/AU6679094A/en not_active Abandoned
  • 1994-04-18 EP EP94914395A patent/EP0699239A1/en not_active Withdrawn
  • 1994-04-19 ZA ZA942685A patent/ZA942685B/xx unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events