WO2002066661A1 - Method for producing and transforming clusters of mitochondria - Google Patents

Abstract

The invention relates to a method for producing clusters of mitochondria in order to simplify the subsequent transformation of said mitochondria. The production of the clusters of mitochondria includes the expression, in the cells containing the mitochondria, of a first protein component which is capable of anchoring in the outer membrane of the mitochondria, and of a second protein component which can sterically protect the determinants on the surface of the mitochondria.

Description

 Process for the creation and transformation of mitochondrial conglomerates

The invention relates to a method for producing mitochondrial conglomerates to facilitate the subsequent transformation of the mitochondria, wherein the production of the mitochondrial conglomerates involves the expression of a first protein component which is capable of being anchored in the outer mitochondrial membrane and a second protein component capable of sterically shielding determinants on the surface of mitochondria in which the cells containing mitochondria are comprised.

The expression of the protein components mentioned, which are components of a common chimeric (fusion) protein, leads to the formation of mitochondrial conglomerates which, thanks to their size, can be transformed with foreign nucleic acids in comparison to individually present mitochondria.

Mitochondria, like plastids, are organelles from eukaryotic cells. Both the mitochondria and the plastids, especially chloroplasts, contain their own DNA. Generally are

Mitochondria stretched cylindrical, rarely spherical, with a diameter of 0.5 to 1 μm. Like their prokaryotic ancestors, mitochondria and chloroplasts divide by constriction. A ring of tubulin-related proteins is formed, which constricts with GTP consumption. For chloroplasts, this ring consists of FtsZ. Less is known about the division of mitochondria. In the fully known genomes of Caenorhabditis elegans and Saccharomyces cerevisiae, no ftsz homologues were found that could be responsible for mitochondrial division, but in the unicellular algae Mallomonas splendens and the unicellular red algae Cyanidioschyzon merolae. In yeast and animals, the mitochondria are constricted with the participation by Dynamin. Dynamins were previously known to be associated with endocytosis, where they lead to constriction of the endocytosis vesicles.

The mitochondrion is surrounded by two highly specialized membranes, the outer membrane and the inner membrane, which are crucial for its activity. Each of the two lipid bilayers contains a unique protein pattern, and together they enclose and define two separate mitochondrial compartments: the inner matrix space and the narrow space between the two membrane systems (intermembrane space).

There are no publications on successfully transformed plant mitochondria. The transformation of mitochondria is of great interest, especially for plant biotechnology and molecular plant breeding, since the transformation of cell organelles, which is already possible with larger chloroplasts, offers clear advantages over the manipulation of the nucleus. In an organelle transformation, for example, a higher expression of the transgene is observed, which is usually only inherited from the mother, which is a further advantage of the organelle transformation since, for example, the transgene is hardly disseminated by pollen (Bogorad L. (2000) Trends Biotechnol. 18: 257-263; Daniell H. (1999) Nat. Biotechnol. 17: 855-856).

Compared to transgenic plastids, mitochondria would also offer the advantage that they are also productive in large quantities in seeds.

It is therefore an object of the present invention to produce enlarged mitochondria or mitochondrial conglomerates, that is to say aggregations, mergers or associations of mitochondria, which are accessible to transformation processes. In transformation processes that are used for the transfer of Foreign genes on mitochondria are suitable, for example micromanipulation or bombardment with DNA.

These and other tasks that result from the following description are solved by the method defined in the main claim. Preferred embodiments are specified in the subclaims.

It has now surprisingly been found that the expression of a (fusion) protein which comprises two components, namely a first, which causes the protein to be anchored in the outer mitochondrial membrane, and a second, which is able to determine determinants on the Sterically shielding the surface of mitochondria, which causes the formation of mitochondrial conglomerates. The phenomenon now observed for the first time is not a random event, but a reproducible way of providing larger mitochondrial structures that can be genetically manipulated using conventional transformation methods due to their size.

The possibility of larger mitochondrial associations by targeted expression of a protein, comprising an anchoring component (targeting component) for the outer mitochondrial membrane and a steric

Manufacturing shielding component is described here for the first time.

Mitochondrial lumps were developed by Rubino et al. (2001, Journal of General Virology 82: 29-34) after transient expression of the 36K viral protein, fused to GFP, described in Nicotiana benthamicana, but the size changes referred to there as "dumping" are not genetically manipulable conglomerates in the The meaning of the present invention by Rubino et al. (2001, vide supra) EM recordings only show that individual mitochondria get bigger, change their structure and can clump together, with a maximum of two clumped mitochondria being shown.

In contrast to this, the method according to the invention effects a conglomeration of several (usually at least 3 or more) mitochondria, as is necessary for the use of the mitochondria for genetic manipulation.

The EM recordings by Rubino et al. (2001, vide supra) rather suggest that the mitochondria are destroyed in their function and structure and that this destruction is due to the expression of the 36K viral protein. In contrast to the fusion protein according to the present invention, the 36K viral protein appears to cause the mitochondrial structure to dissolve. So it happens in the experiments by Rubino et al. an expansion of the intermembrane space, an entry of cytosol into the intermembrane space and the first signs of mutivesicular bodies (MVB) can be observed. This is not surprising given the actual function of the 36K viral protein, which consists in the dissolution of the outer mitochondrial membrane to form multivesicular bodies. The fact that Rubino et al. only a transient, but no stable expression of the 36K fusion protein in the plant cell is possible, furthermore indicates that the mitochondria observed there are also no longer functionally active. Such mitochondria, even if they should aggregate occasionally, could not be used for the production of transgenic plants. The clumps of Rubino et al. (2001, vide supra) are therefore not conglomerates in the sense of the present invention. Looking at the publication by Rubino et al. from 2001 further in the context of an earlier publication by the same authors, namely Rubino et al. 2000, Journal of General Virology 81; 279-286, see above), it is clear from this that the Rubino et al. 2001, vide supra, the described effect is due solely to the 36K virus protein. For example, the previous publication already indicated that CIRV (Carnation Italian Ringspot Virus) -induced multivesicular bodies (MVBs) are similar to structures that are produced by other tombus viruses. In addition, it can be seen from the discussion in the earlier publication that identical structures were induced with unfused 36K protein as with the 36K-GFP fusion protein. In this context it is also mentioned that other tombus virus proteins, such as 33K, also generate MVBs, inter alia from peroxisomes, that is to say completely different membranes or organelles than mitochondria.

It is therefore clear that the mitochondrial clumping described in the prior art is due solely to the 36K viral protein or other proteins from tombus viruses and the effect based on these viral proteins is a destruction of membranes, be it the outer mitochondrial membrane or that of peroxisomes . Such viral proteins, in particular proteins of the tombus virus family, which bring about the dissolution and destruction of membranes are excluded from use in the context of the present invention.

The fusion proteins used in the context of the invention do not destroy the mitochondrial membrane.

The transgenic plants in which the mitochondrial conglomeration takes place preferably do not show any visible phenotype. Without wishing to be bound by any hypothesis, it is currently assumed that the expression of the protein components according to the invention inhibits mitochondrial division, which results in mitochondrial conglomerates which could be termed “giant mitochondria” and which can be genetically manipulated due to their size .

The ("giants") mitochondria produced according to the invention are significantly larger than the naturally occurring mitochondria and consist of several mitochondria. The mitochondria according to the invention preferably have a size of at least 2 μm, 3 μm, more preferably at least 4 μm, 6 μm and particularly preferably of at least 7 μm, 8 μm Most preferably, the mitochondria have a size of at least 9 μm, 10 μm or more in diameter, and the particularly preferred mitochondria are therefore comparable in size to the cell nucleus.

In a preferred embodiment, the anchoring component is the enzyme hexokinase (HK) or parts of this enzyme which are capable of anchoring in the outer mitochondrial membrane. Other preferred anchoring components are porins or parts thereof which are capable of anchoring in the outer mitochondrial membrane. In principle, any protein or part of a protein is suitable which has a cytoplasmic localization with anchoring in the outer mitochondrial membrane.

The hexokinase Hxk 1 from Nicotiana tabacum, for example, is an anchoring component. The sequence of Hxk 1 was published under accession no.

AF 118133 published, whereby the description of the sequence in the database shows that Hxk 1 is a chloroplast protein. The sequence of the hexokinase from tobacco was obtained by using a previously characterized hexokinase from spinach (Accession No. AF 118132) a cDNA library from tobacco was screened (Wiese et al. (1999) FEBS Lett. 461: 13-18). The tobacco hexokinase obtained was obviously not subsequently characterized, but only the description of the spinach hexokinase was taken over, which indicates a localization of the hexokinase I in the outer envelope membrane.

Other hexokinases can also be used in the context of the invention, sequences suitable for creating suitable expression vectors can be found in the gene database, for example, under the keyword “hexokinase” (for example, at the filing date, there were over 1300 nucleic acid sequences for hexokinases recorded in the NCBI library) partially overlap).

In addition to the hexokinase mentioned in the accompanying examples, the person skilled in the art is familiar with further suitable anchoring proteins or parts of proteins which are used as

Anchorage domain can be used, known. Various proteins that are anchored in the outer mitochondrial membrane are listed below. The person skilled in the art can, using the references below, use conventional cloning methods to prepare suitable fusion proteins and use them to generate the desired mitochondrial conglomerates.

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Nature 388: 195-200 (1997). - OM06_YEAST, Accession No. P33448: Kassenbrock C.K., Cao W., Douglas M.G. "Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex."; EMBO J. 12: 3023-3034 (1993

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- OM07_SCHPO, Accession No. 042999

- OM07_YEAST, Accession No. P53507: Hoenlinger A., Boemer U., Alconada A., Eckerskorn C, Lottsspeich F., Dietmeier K., Pfanner N .;

"Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import." EMBO J. 15: 2125-2137 (1996)

- OM20_CAEEL, Accession No. Q 19766 - OM20_HUMAN, Accession No. Q15388: Seki N., Moczko M., Nagase T., Zufall N., Ehmann B., Dietmeier K., Schaefer E., Nomura N., Pfanner N.

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- OM20_NEUCR, Accession No. P35848: Mayer A., Nargans F.E., Neupert W., LiU R.

"MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19." EMBO J. 14: 4204-4211 (1995)

- OM20_RAT, Accession No. Q62760; Accession No. Q63804; Accession No. O08517

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- OM40_CAEEL, Accession No. Q 18090

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"The genome sequence of Drosophila melano gaster." Science 287: 2185-2195 (2000)

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Ashworth L.K., Van Bockxmeer F.M., Dawkins R.L.

"Sequencing of 42kb of the APO E-C2 gene cluster reveals a new gene: PEREC1." DNA Seq. 9: 89-101 (1998)

- OM40_MOUSE, Accession No. Q9QYA2; Accession No. Q9Z2N1

- OM40_NEUCR, Accession No. P24391: Kiebler M., Pfaller R., Soellner T., Griffiths G., Horstmann H.,

RA Pfanner N., Neupert W. "Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins." Nature 348: 610-616 (1990)

- OM40_SCHPO, Accession No. 013656; Accession No. 013655

- OM40_YEAST, Accession No. P23644: Baker K.P., Schaniel A., Vestweber D., Schatz G.

"A yeast mitochondrial outer membrane protein essential for protein import and cell viability."

Nature 348: 605-609 (1990) - OM45_YEAST, Accession No. P16547: Yaffe MP, Jensen RE, Guido EC "The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function."

J. Biol. Chem. 264: 21091-21096 (1989) - OM70_NEUCR, Accession No. P23231: Steger H.F., Soellner T., Kiebler M.,

Dietmeier K.A., Pfaller R.,

RA Truelzsch K.S., Tropschug M., Neupert W., Pfanner N.

"Import of ADP / ATP carrier into mitochondria: two receptors act in parallel."

J. Cell Biol. 111: 2353-2363 (1990) - OM70_YEAST, Accession No. P07213: de Antoni A., D Angelo M., Dal Pero F.,

Sartorello F., Pandolfo D.,

RA Pallavicini A., Lanfranchi G., Valle G.

"The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames."; Yeast 13: 261-266 (1997)

- PORl_HUMAN, Accession No. P21796; Accession No. Q9UIQ5; Accession No. Q9UPL0: Blachly-Dyson E.G., Zambronicz E.B., Yu W.H., Adams V., McCabe E.R., Adelman J.P., Colombini M., Forte M .;

"Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel."; J. Biol. Chem. 268: 1835-1841 (1993)

- PORl_MOUSE, Accession No. Q60932: Sampson M.J., Lovell R.S., Craigen W.J. "Isolation, characterization, and mapping of two mouse mitochondrial voltage-dependent anion channel isoforms." Genomics 33: 283-288 (1996)

- PORl_RABIT, Accession No. Q9TT15

- PORl_RAT, Accession No. Q9Z2L0: Anflous K., Blondel O., Bernard A., Khrestchatisky M., Ventura-Clapier R. "Characterization of rat porin isoforms: cloning of a cardiac type-3 variant encoding an additional methionine at its putative N-terminal region." Biochim. Biophys. Acta 1399: 47-50 (1998)

- PORl_WHEAT, Accession No. P46274 - PORl_YEAST, Accession No. P04840: Mihara K., Sato R.

"Molecular cloning and sequencing of cDNA for yeast porin, an outer mitochondrial membrane protein: a search for targeting signal in the primary structure." EMBO J. 4: 769-774 (1985)

- POR2_HUMAN, Accession No. P45880; Accession No. Q9Y5I6: Ha H., Hajek P., Bedwell D.M., Burrows P.D.

"A mitochondrial porin cDNA predicts the existence of multiple human porins." J. Biol. Chem. 268: 12143-12149 (1993)

- POR2_MELGA, Accession No. P82013

- POR2 MOUSE, Accession No. Q60930: Sampson M.J., Lovell R.S., Craigen W.J. "Isolation, characterization, and mapping of two mouse mitochondrial voltage-dependent anion channel isoforms."

Genomics 33: 283-288 (1996)

- POR2_RABIT, Accession No. Q9TT14

- POR2_RAT, Accession No. P81155: Bureau M.H., Khrestchatisky M., Heeren M.A., Zambrowicz E.B., Kim H., Grisar T.M., Colombini M., Tobin A.J., Olsen

R.W.

"Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptides from mammalian brain." J. Biol. Chem. 267: 8679-8684 (1992) - POR2_XENLA, Accession No. P81004: Steinacker P., Awni LA, Becker S., Cole T., Reymann S., Hesse D., Kratzin HD, Morris-Wortmann C, Schwarzer C, Thinnes FP, Hilschmann N. "The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels."

Int. J. Biochem. Cell Biol. 32: 225-234 (2000)

- POR2_YEAST, Accession No. P40478: Blachly-Dyson A., Song J., Colombini M., Forte M.

"Multicopy suppressors of phenotypes resulting frorn the absence of yeast NDAC encode a VDAC-like protein." Mol. Cell. Biol. 17: 5727-5738 (1997)

- POR3_HUMAΝ, Accession No. Q9Y277; Accession No. Q9UIS0: Mao M., Fu G., Wu J.-S., Zhang Q.-H., Zhou J., Kan L.-X., Huang Q.-H., He K.-L., Gu B.-W., Han

Z.-G., Shen Y., Gu J., Yu Y.-P., Xu S.-H., Wang Y.-X., Chen S.-J., Chen Z.

"Identification of genes expressed in human CD34 (+) hematopoietic stem / progenitor cells by expressed sequence tags and efficient full-length cDNA cloning."

Proc. Natl. Acad. Be. U.S.A. 95: 8175-8180 (1998) - POR3_MOUSE, Accession No. Q60931: Sampson M.J., Lovell R.S., Davison

D.B., Craigen W.J.

"A novel mouse mitochondrial voltage-dependent anion channel gene localizes to chromosome 8."

Genomics 36: 192-196 (1996) - POR3_PIG, Accession No. Q29380: Winteroe A.K., Fredholm M., Davies W.

"Evaluation and characterization of a porcine small intestine cDNA library: analysis of839 clones."

Mom. Genome 7: 509-517 (1996)

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"Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum." J. Biol. Chem. 267: 21072-21079 (1992)

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"Porins from plants. Molecular cloning and functional characterization of two new members of the porin family."

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"Porins from plants. Molecular cloning and functional characterization of two new members of the porin family."

J. Biol. Chem. 269: 25754-25760 (1994)

The person skilled in the art can determine, by means of routine methods, which proteins or parts of proteins are capable of being anchored in the outside by producing suitable gene constructs and transferring them to plant cells

Mitochondrial membrane and which proteins due to their amino acid sequence or because of their insufficient length, especially with parts of proteins, can not mediate anchoring in the outer mitochondrial membrane.

The person skilled in the art can also use the proteins described in the prior art, which have a cytoplasmic localization with anchoring in the outer mitochondrial membrane, in particular hexokinases and porins, to find other suitable proteins, for example by PCR using oligonucleotides that are of known nucleic acid sequences are derived, or by hybridization of cDNA or genomic libraries with gene probes which correspond to known nucleic acid sequences. The second component of the protein, the expression of which in plant cells leads to the desired formation of mitochondrial conglomerates, is the so-called shielding component, which causes steric shielding of determinants on the mitochondrial surface.

In a preferred embodiment, it is the green fluorescent protein (GFP), which glows green under UV radiation and is frequently used as a reporter gene or as a selection marker. In principle, however, any protein or any part of a protein which is capable of sterically shielding determinants on the surface of the mitochondria, in particular of receptors, is suitable.

In the case of this shielding or masking component, too

Experts can use routine experiments to easily determine the suitability of a particular protein or part of a protein by expressing it in

Check plant cells. It is important that the part protruding into the cytosol is capable of sterically shielding the relevant determinants on the surface of the mitochondria. It is believed that the

Shielding component should have a size that corresponds to a molecular weight of about 20 kDa, preferably 25 kDa and particularly preferably at least 28 kDa.

The anchoring component and the steric components are preferably expressed in the form of a fusion protein in the plant cell, i.e. the fusion protein is encoded by a chimeric gene construct that is an artificial one

Merging of the DNA sequences coding for the anchoring component and the steric component. In principle, however, it is not excluded that the expression of a naturally occurring protein for the process according to the invention for producing mitochondrial conglomerates is suitable. In addition, it is assumed that the expression of two interacting proteins, one of which acts as a shielding component and the other protein takes over the function of the anchoring component, also effects the desired conglomeration of the mitochondria.

The production of suitable gene constructs which code for fusion proteins, comprising the anchoring component and the steric component, is known to the person skilled in the art and is possible using standard molecular biological methods, see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

The various methods of transferring gene constructs and plasmids to plant cells are also well known to those skilled in the art. The same applies to regulatory sequences that are suitable for the expression of genes in plants, such as. B. constitutive promoters, tissue and development-specific promoters, inducible promoters, enhancer sequences and other regulatory sequences that control the transcription and translation of a coding operatively linked coding DNA sequence in transformed plant cells.

In a preferred embodiment, the promoter controlling the expression of the fusion protein is a constitutive promoter, such as the 35S RNA promoter from CaMV, or a leaf-specific promoter.

A large number of cloning vectors are available to prepare for the introduction of foreign genes into higher plants or their cells Replication signal for E. coli and a marker gene for the selection of transformed bacterial cells included. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence, in this case the fusion of (i) sequences which code for the anchoring component with (ii) sequences which are for the steric Coding shielding component can be introduced into the vector at a suitable restriction interface. The plasmid obtained is then used for the transformation of E. cob ^' cells. Transformed E. cob ^' cells are grown in an appropriate medium and then harvested and lysed, and the plasmid is recovered. As an analysis method to characterize the obtained

In general, plasmid DNA is used for restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.

A large number of techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation medium, the fusion of

Proplastics, injection, electroporation, direct gene transfer of isolated DNA into protoplasts, the introduction of DNA using the biolistic method and other options that have been well established for several years and are part of the usual repertoire of the specialist in plant molecular biology or plant biotechnology .

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies for direct gene transfer. Simple plasmids such as e.g. B. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is recommended. The usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.

Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. Are z. B. for the transformation of the plant cell uses the Ti or Ri plasmid, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti or Ri plasmid must be connected as a flank region with the genes to be introduced become. If Agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells. The use of T-DNA for the transformation of Plant cells have been intensively examined and have been adequately described in well-known overview articles and manuals on plant transformation. For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.

Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which shows the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and the like. a. mediated. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA. Alternative markers are also suitable for this, such as nutritive markers and screening markers. Of course, selection markers can also be completely dispensed with, but this is associated with a fairly high need for screening. If marker-free transgenic plants are desired, strategies are available to the person skilled in the art which permit subsequent removal of the marker gene, e.g. B. cotransformation, sequence-specific recombinases.

The regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using known nutrient media. The plants thus obtained can then be prepared using conventional methods, including molecular biological methods, such as PCR, blot analyzes, for the presence of the introduced nucleic acid, which codes for the fusion protein, consisting of anchoring component and shielding component, are examined.

The transgenic plant or the transgenic plant cells can be any monocot or dicot plant or its plant cell. They are preferably useful plants or cells of useful plants. It is particularly preferably corn, rice, wheat, barley, oats, rye, soybean, rapeseed, potato, tomato, tobacco, sugar beet, pea, banana, pineapple, paprika, yams, cassava, cotton.

The plant cells transformed with the fusion gene construct coding for the fusion protein show the inventive phenomenon of the formation of mitochondrial conglomerates, due to an inhibition of the naturally occurring mitochondrial division. This inhibition is mediated by anchoring the fusion protein in the outer mitochondrial membrane, caused by the anchoring component of the fusion protein, and shielding determinants on the mitochondrial surface, caused by the steric component of the fusion protein.

The mitochondrial conglomerates of the transformed plant cells can be genetically modified due to their size using known transformation methods.

Particle bombardment or microinjection is particularly suitable as a transformation method, but other methods are also conceivable if they are suitable for the transformation of plastids or protoplasts, for example, or have proven them for these objects.

The method of "particle bombardment" was published in 1987 (Klein et al., Nature 327: 70-73) and has meanwhile found its way into the relevant specialist books as a standard method. The method has also been used to mitochondria of Saccharomyces cerevisiae and Chlamydomonas reinhardtii to transform (see, e.g., Johnston et al. (1988) Science 240: 1538-1541; Boynton et al. (1996) Methods Enzymol. 264: 279-296; Butow and Fox (1990) Trends Biochem. Sci. 15: 465- 468).

Animal mitochondria have already been successfully transformed by electroporation (Collombet et al. (1997) J. Biol. Chem. 272: 5342-5347).

An alternative selection for transgenic mitochondria was by Ortega et al. (Ortega et al. (2000) Curr. Genet. 37: 315-321). This is based on mutations in the mitochondrially coded gene cob, through which resistance to the antibiotics Antimycin A (AA) or Myxothiazol (Myx) could be achieved. Both antibiotics reduced the growth of a tobacco cell culture to 11.4 ± 10% (AA) or 1.7 + 4% (Myx) at a concentration of 100 μM. 1 μM AA in combination with 1 mM SHAM (salicylhydroxamic acid) was found to be fatal.

Mutations in highly conserved amino acids are known in mammals, fungi and algae, which confer resistance to AA and Myx (Eposti et al. (1993) Biochim Biophys Acta 1143: 243-271). Similarly, in plants the replacement of amino acid glycine-43 by valine gives resistance to AA and the replacement of amino acid phenylalanine-135 by leucine gives resistance to myx. The inhibitory effect of myxothiazole or antimycin A on the mitochondrial respiratory chain thus lends itself as a selection marker for mitochondrial transformation (see also the examples below). The invention thus also relates to the use of cob genes as selection markers, as is explained in detail in the examples below.

Known for years as a means of nuclear transformation (Crossway et al. (1986) Mol. Gen. Genet. 202: 79-85), the method of injecting DNA using a fine capillary has now been refined to such an extent that chloroplasts are also transformed can. (Knoblauch et al. (1999) Nat. Biotechnol. 17: 906-909.) The mitochondrial conglomerates according to the invention partly exceed the size of chloroplasts, so that their transformation by microinjection should also be possible.

After successful transformation of the mitochondria, they have to be selected by backcrossing, analogous to transgenic chloroplasts. Back-crossing of their plants with transformed mitochondria is necessary to eliminate the phenotype of the mitochondrial conglomerates. Backcrossing can be facilitated by cotransforming the gene leading to the conglomerates with a negative selection marker. This negative selection marker kills all plants transformed in the nucleus under suitable conditions. Together with conditions which enable a positive selection of transformed mitochondria, only plants which have transformed mitochondria and whose gene leading to conglomerates together with the negative selection marker have been crossed out can survive. Examples of negative selection markers are, for example, codA (cytosine deaminase; Stougaard (1993) Plant J. 3: 755-761) and dhlA (haloalkane dehalogenase; Naested et al. (1999) Plant J. 18: 571-576).

The invention thus also relates to plant cells and plants which have mitochondria which have been transformed by the method according to the invention, and to propagation material and harvest products of these plant cells or plants, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.

The present invention is illustrated in the following examples, which are only the

Serve to illustrate the invention and are in no way to be understood as a limitation.

Examples

General cloning procedures

Cloning methods such as restriction cleavage, DNA isolation, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. cob ^' cells, cultivation of bacteria, recombinant sequence analysis DNA, were according to Sambrook et al. (1989, vide supra).

The transformation of Agrobacterium tumefaciens was carried out according to the

Höfgen and Willmitzer method (Nucl. Acids Res. (1988) 16, 9877). The agrobacteria were grown in YEB medium (Vervliet et al. (1975) J. Gen. Virol. 26, 33). Bacterial strains and plasmids

E. coli (XL-1 Blue) bacteria were purchased from Stratagene (La Jolla, California, USA). The Agrobacterium strain used for the plant transformation (C58C1 with the plasmid pGV 3850kan) was developed by Debleare et al. (1985, Nucl. Acids Res. 13, 4777). The vectors pCR-Blunt (Invitrogen, Carlsbad, California, USA), pBluescript SK- (Stratagene) and pBinAR (Höfgen and Willmitze (1990) Plant Sei. 66: 221-230) were used for the cloning.

Tobacco transformation

For the tobacco transformation of tobacco plants (Nicotiana tabacum L.cv. Samsun NN), 10 ml of an overnight culture of Agrobacterium tumefaciens grown under selection were centrifuged off, the supernatant was discarded, and the bacteria were resuspended in the same volume of antibiotic-free medium. Leaf disks of sterile plants (diameter approx. 1 cm) were bathed in this bacterial solution in a sterile petri dish. The leaf disks were then placed in Petri dishes on MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473) with 2% sucrose and 0.85 Bacto agar. After 2 days of incubation in the dark at 25 ° C, the leaf disks were washed on MS medium with 100 mg / 1 kanamycin, 500 mg / 1 claforan, 1 mg / 1 benzylaminopurine (BAP), 0.2 mg / 1 naphthylacetic acid ( NAA), 1.6% glucose and 0.85 Bacto agar and the cultivation (16 h light / 8 h dark) continued. Growing sprouts were transferred to hormone-free MS medium with 2% sucrose, 215 mg / 1 claforan and 0.8% Bacto agar.

Cloning of the chimeric transgenes

Two fusion constructs of hexokinase 1 (Hxkl, see above) were generated with the Green Fluorescent Protein (GFP), in which the Hxkl content was different. The Hxkl and GFP fusion constructs are shown in Fig. 1. In the shorter construct HxklN:: GFP (N stands for N-terminus), only the N-terminus of Hxkl, comprising the putative signal peptide, was fused with GFP. In the larger Hxkl :: GFP construct, the entire sequence coding for Hxkl was combined with that for GFP.

The fusion partners GFP and Hxkl and HxklN were created with the help of

Polymerase chain reaction (PCR) cloned. The polymerization steps were carried out in an automated T3 thermal cycler (Biometra, Göttingen, Germany) according to the programs given below. The PCR constructs were propagated in the vector pCR blunt (Invitrogen). The Hxkl subfragments were then labeled as BamHI / EcoRI

Restriction fragments ligated into the vector pBluescript SK- (Stragagene). The GFP fragment was connected as an EcoRI / Sall restriction fragment. The entire construct was then cut out as a BamHI / Sall fragment and ligated into the binary vector pBinAR. The following oligonucleotides and templates were used for the PCR amplifications.

HK9GFP3: GAA TTC GGA CTT ATC TTC AAG GTA HK9GFP5: GGA TCC C AA CTT TTA GCC AAC CTC C

HK9LGFP3: GAA TTC ACG AAG AAT AGC CAT AGC

K75: AT GAA TTC AGT AAA GGA GAA GAA CTT

K76: AT GTC GAC TTA TTT GTA TAG TTC ATC CAT GC

(The restriction sites for the enzymes EcoRI, BamHI and Sall are highlighted).

A HKI cDNA clone was used as a template for Hxkl and was isolated from a cDNA library which is identical to the one from which the one described by Wiese et al. published clone Hxkl was isolated (Wiese et al. (1999) FEBS Lett. 461: 13-8.).

The vector pBin-mGFP5-ER was used as the template for GFP (Siemering K.R. et al. (1996), Curr. Biol. 6: 1653-1663).

The composition of the PCR reaction batches and the PCR program used in each case are given below.

For HxklN:

Program:

For Hxkl

Program:

For GFP

Program:

The nucleic acid sequences of the fusion constructs described above are given below, along with the amino acid sequences of the fusion proteins encoded by the fusion constructs. Nucleic acid sequence of the HxklN :: GFP fusion construct

GGATCCCAACTTTTAGCCAACCTCCAATTCCTCTGCCGTGACAAAAAGAAAG GATGAAGAAAGCGACGGTGGGAGCCGCCGTAATTGGCGCCGCTACGGTATGT GCAGTGGCGGCATTAATAGTGAACCACCGTATGCGCAAATCTAGCAAATGGG CACGTGCTATGGCTATTCTTCGTGAAΓTCAGTAAAGGAGAAGAACTTTTCAC TGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAA TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCC TTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGT CACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATG AAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGA GGACCATCTTCTTCAAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAA GTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTC AAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCC ACAACGTATACATCATGGCCGACAAGCAAAAGAACGGCATCAAAGCCAACTT CAAGACCCGCCACAACATCGAAGACGGCGGCGTGCAACTCGCTGATCATTAT CAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATT ACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCA CATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGAT GAACTATACAAATAAGTCGAC

The first 25 bases correspond to the sequence of the PCR primer HK9GFP5, including the BamHI restriction site (italic). The Hxkl ATG start codon is highlighted. The EcoRI restriction interface, via which the GFP fragment was connected to the Hxkl fragment, is also shown in italics. The last 29 bases result from the PCR primer K76 used (counter strand, including Sall restriction site).

Amino acid sequence of the fusion protein HxklN:: GFP

MKKATVGAAVIGAATVCAVAALIVNHRMRKSSKWARAAILREFSKGEE FT GWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP PTLV TTFSYGVQCFSRYPDH KRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANF KTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH MVLLEFVTAAGITHGMDELYK

The EcoRI fusion results in amino acids 43-44, EF. The EcoRI interface GAATTC is already in the GFP template pBin-mGFP5-ER, as a result of which an endoplasmic reticulum signal sequence (pBin-mGFP5-ER) was connected upstream. However, this signal sequence was not used in the context of the invention and the EcoRI nucleotides are not necessary for the function of GFP (cf. access numbers U87974 with and U87973 without this restriction site). The sequence of Hxkl at the relevant point is GAATTT, which is also translated to EF. The amino acids EF can therefore still be included in the hexokinase sequence.

Nucleic acid sequence of the fusion construct Hxkl:: GFP

GGATCCCAACTTTTAGCCAACCTCCAATTCCTCTGCCGTGACAAAAAGAAAG GATGAAGAAAGCGACGGTGGGAGCCGCCGTAATTGGCGCCGCTACGGTATGT GCAGTGGCGGCATTAATAGTGAACCACCGTATGCGCAAATCTAGCAAATGGG CACGTGCTATGGCTATTCTTCGTGAATTTGAGGAAAAGTGTGGGACCCCTGA TGCTAAGCTCAAGCAAGTCGCTGATGCTATGACCGTCGAGATGCACGCTGGA CTTGCCTCCGAAGGTGGTAGCAAGCTCAAGATGCTTATCACTTACGTCGATA ATCTCCCCACCGGTGATGAAGCTGGCGTCTTTTATGCGTTGGATCTTGGTGG AACAAATTTTCGAGTATTGCGAGTGCAACTTGGTGGAAAAGATGGTGGTATT GTTCATCAGGAATTTGCGGAGGCATCAATTCCTCCAAATTTGATGGTTGGGA CTTCAGAAGCACTTTTTGATTATATTGCGGCAGAACTTGCAAAATTTGTCAA CGAGGAAGGGGAAAAGTTTCAACAACCTCCTGGTAAGCAGAGAGAACTAGGT TTCACCTTCTCATTCCCGGTAATGCAGACTTCAATCAACTCTGGGACTATTA TGAGGTGGACAAAGGGCTTCTCCATTGATGATGCGGTTGGCCAAGATGTTGT TGGAGAACTCGCAAAAGCTATGAAAAGAAAAGGAGTTGATATGCGGGTCTCA GCTTTGGTGAATGATACTGTTGGGACGTTGGCTGGTGGTAAATATACACACA ACGACGTAGCTGTTGCTGTTATCTTAGGTACAGGGACCAATGCAGCCTATGT GGAACGGGTGCAGGCGATTCCAAAGTGGCATGGTCCTGTGCCAAAATCTGGT GAAATGGTTATCAACATGGAATGGGGTAATTTTAGGTCATCCCATCTTCCCT TGACACAGTATGATCATGCGTTGGATACTAATAGTTTGAATCCTGGTGATCA GATATTTGAGAAGATGACTTCTGGCATGTACTTGGGAGAAATTTTACGCAGA GTTCTACTCAGGGTGGCCGAAGAAGCTGGCATTTTTGGTGATGAGG TCCCTC CAAAGCTCAAGAGTCCATTTGTATTGAGGACACCTGATATGTCTGCGATGCA TCATGACGCATCCTCTGATCTGAGAGTGGTTGGTGACAAGCTGAAGGATATT TTAGAGATATCTAATACCTCCTTGAAGACAAGGAGATTAGTCATTGAGCTGT GCAACATCGTTGCGACACGTGGGGCAAGGCTTGCAGCTGCGGGTGTATTGGG CATCTTGAAAAAGATGGGAAGGGATACTCCTCGGCAAGGTGGTCTAGAGAAG ACGGTTGTAGCCATGGATGGCGGATTGTACGAGCACTATACAGAATACAGGA TGTGCTTAGAGAACACTTTGAAGGAATTGCTTGGAGATGAATTGGCGACAAG CATTGTTTTCGAGCACTCCAATGATGGTTCTGGCATTGGTGCAGCTCTTCTT GCTGCCTCTAACTCAATGTACCTTGAAGATAAGTCCGAATTCAGTAAAGGAG AAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGT TAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCAT GGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATA CCCAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGA TACGTGCAGGAGAGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACAC GTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAA GGGAATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATAC AACTACAACTCCCACAACGTATACATCATGGCCGACAAGCAAAAGAACGGCA TCAAAGCCAACTTCAAGACCCGCCACAACATCGAAGACGGCGGCGTGCAACT CGCTGATCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTA CCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACG AAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTAC ACATGGCATGGATGAACTATACAAATAAGTCGAC

At Hxkl :: GFP the Hxk stop codon TAG was mutated by EcoRI in GAA. The amino acids EF are located at positions 498-499.

Amino acid sequence of the fusion protein Hxkl :: GFP

MKKATVGAAVIGAATVCAVAALI NHRMRKSSKWARAMAILREFEEKCGTPD AKLKQVADA TVEMHAGLASEGGSKLKMLITYNDNLPTGDEAGVFYALDLGG

TNFRVLRVQLGGKDGGIVHQEFAEASIPPNLMVGTSEALFDYIAAELAKFVN

EEGEKFQQPPGKQRELGFTFSFPVMQTSINSGTIMRWTKGFSIDDAVGQDW GELAKA KRKGVDMRVSALVNDTVGTLAGGKYTHNDVAVAVILGTGTNAAYV ERVQAIPKWHGPVPKSGE VINMEWGNFRSSHLPLTQYDHALDTNSLNPGDQ IFEKMTSGMYLGEILRRVLLRVAEEAGIFGDEVPPKLKSPFVLRTPDMSAMH HDASSDLRWGDKLKDILEISNTSLKTRR VIELCNIVATRGARLAAAGVLG ILKKMGRDTPRQGGLEKTWAMDGGLYEHYTEYRMCLENTLKELLGDELATS IVFEHSNDGSGIGAALLAASNS YLEDKSEFSKGEELFTGWPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRY PDH KRHDFFKSA PEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYI ADKQKNGIKANFKTRHNIEDGGVQL ADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDELYK

Confocal laser scanning microscopy

Epidermis was peeled off from the underside of a sheet and irradiated with light of the wavelengths 488 and 543 nm on the CLSM 410 from Zeiss (Jena, Germany). The emissions were limited to 510 to 525 nm by a bandpass filter. GFP protein became visible in light green.

Transfer and expression of the fusion proteins Hxkl :: GFP and HxklN :: GFP in tobacco

The fusion constructs were transferred to tobacco cells as indicated above and transgenic plants were regenerated. Epidermis samples of the transgenic plants showed green glowing signals of the GFP in the fluorescence microscope. What was surprising at first, however, was that the two constructs had different distribution patterns from GFP showed. While the small construct caused several small glowing GFP signals, the larger construct marked significantly larger glowing areas, comparable in size to the cell nucleus. The electron microscopic examination of the plant tissue then showed that the large GFP-marked areas were accumulations of mitochondria that were aggregated to varying degrees. Peroxisomes were also associated with these aggregates. In the plants transformed with the smaller construct, HxklN:: GFP, the mitochondria also showed an affinity for one another, but to a lesser extent than with Hxkl :: GFP.

The localization of GFP on the outer mitochondrial membrane led to the aggregation of the mitochondria. The degree of aggregation was dependent on the size of the GFP fusion protein. The chimeric protein Hxkl :: GFP resulted in large conglomerates of mitochondria and peroxisomes, while the smaller fusion protein HxklN:: GFP only generated an affinity for both organelle types. While the effect of the larger construct was already detectable in the light microscope, the small effect of HxklN :: GFP was only visible under the electron microscope.

It is assumed that the formation of the mitochondrial conglomerates is due to the fact that the large amount of foreign proteins anchored in the outer mitochondrial membrane interferes with the complete division of the organelles, and the presence of the fusion protein thus limits the division of the mitochondria by constriction - or prevented. The electron micrographs shown in Figure 3 also support this. Transformation of the mitochondrial conglomerates

In the first published transformation of yeast mitochondria, a mutation in the mitochondrial gene oxi3, coding for the largest subunit of cytochrome oxidase (COXI), was corrected by homologous recombination with the WT gene. Because the cells regained the ability to breathe, they could be screened. The transformation of the mitochondrial conglomerates according to the invention described here is based on an oligomycin resistance mediated by mitochondrial transformation.

Oligomycin specifically inhibits the mitochondrial FoFj ATPase (hence the name FO for oligomycin-sensitive; this name was then retained for the chloroplastid ATPase, although it is not sensitive). In the mitochondrial genome oligomycin-resistant yeast and hamster cells, 6 mutations were found in the gene for the subunit, which are probably for the

Resistance are responsible. (Breen et al. (1986) J. Biol. Chem. 261: 11680-11685; Holmans et al. (1987) Somat. Cell. Mol. Genet. 13: 347-353; John and Nagley (1986) FEBS Lett. 207: 79-83). A mutation in the mitochondrial gene for subunit 9 was made responsible for a further form of resistance to oligomycin, or venturicidin and ossamycin in yeast. (Galanis M et al. (1989) FEBS Lett. 249: 333-336; Nagley et al. (1986) FEBS Lett. 195: 159-163). Both genes are also widespread in plant mitochondria, so that a transformation protocol can be built up on resistance imparted thereby.

The first step towards a reproducible protocol is a mutated one

Form of one of the two named genes, which confers resistance to oligomycin, to be introduced into a mitochondrion. Be it particle bombardment or microinjection. Both processes become analogous to plastid transformation used. The screening for oligomycin resistance provides proof of a successful transformation.

The second step is the production of a gene which contains a) oligomycin resistance as a marker and b) the biotechnologically or physiologically interesting gene under the control of a mitochondrial promoter (for the mitochondrial genome of Arabidopsis thaliana see, for example, Unseld et al. (1997) Nat. Genet. 15: 57-61). The chimeric gene from oligomycin-resistant ATPase subunit, promoter and transgene is provided with mitochondrial DNA at both ends, which enables it to be homologously recombined into the target genome. Larger areas of the target genome may be exchanged. Here, the expert has to decide which areas of the original mitochondrial genome are unnecessary. There are two strategies: a) The original gene for ATPase subunit is replaced by the marker gene. Important genes may be replaced by the transgene that also integrates at this point. b) The chimeric gene integrates at an unimportant location in the mitochondrial genome, which ensures the survival of the original genes. The marker gene for oligomycin resistance does not replace the original gene for the ATPase subunit, but this also remains. Thus, the transformed mitochondrial genome would have two genes for the ATPase subunit, with only one conferring oligomycin resistance. The disadvantage here would be reduced resistance.

Thorsness and Fox succeeded in integrating a foreign gene (ura3) into the mitochondrial genome of yeast. This, however, without a promoter, since it was only examined whether this gene can get from the mitochondrial to the nuclear genome. (Thorsness and Fox (1990) Nature 346: 376-379). Analog is after that described above Protocol and the transformation methods also mentioned above, the integration of foreign DNA also possible in plant mitochondria.

Use of GUS (ß-glucuronidase) as a conglomeration component

Using the techniques and expression vectors described or cited above, transgenic tobacco plants were also produced which expressed a fusion protein consisting of Hxkl and GUS (β-glucuronidase).

For this purpose, the fusion construct Hxkl :: GUS outlined below was first produced.

EcoRI BamHI Spei Hind]

Using the oligonucleotides GUS_SalI_5 and NOS_HindIII_3, the plasmid pCambia 1304 (Cambia, Canberra) was amplified from the plasmid GUS and NOS terminator and ligated into the vector pBinAR via Sall / Hindlll (the sequences for GUS and NOS, or another terminator, of course also from other common

Sources can be obtained). Here, the OCS terminator of the BinAR vector was replaced by the NOS termination sequences of the Cambia plasmid. The vector's multiple cloning site has been expanded to include a Spel interface. The new vector created in this way was named pGUS-AR. Hxkl was amplified from the corresponding cDNA using the oligonucleotides HK9GFP5 and HK9-GFP-GUS_3 and ligated into the new vector pGUS-AR via BamHI / Spei.

The PCR was carried out under standard conditions using the following primers:

Oligonucleotides used:

The Hxkl :: GUS construct was introduced into tobacco plants of the SNN variety by means of Agrobacterium-mediated transformation. Selection was carried out on medium containing kanamycin. Resistant plants were screened using qualitative GUS staining (see e.g. Jefferson (1987) Plant Mol. Biol. Rep. 5: 387-405; Jefferson et al. (1987) EMBO J. 6: 3901-3907). 21 GUS-positive plants were identified and analyzed for the presence of mitochondrial conglomerates.

By staining with the dye specific for mitochondria CM-H ₂ TMROS (MitoTracker® from Molecular Probes), different degrees of conglomerates were detected, which overall showed clearly that GUS for

Generation of the desired mergers of mitochondria is generally suitable. In addition to conglomeration components other than GFP, such as GUS, anchoring components other than Hexokinase 1 were also tested. Another plant protein, namely Hexokinase 3, was expressed in fusion with GFP in transgenic tobacco plants. The proteins Hexokinase 1 and Hexokinase 3 are 87.9% identical. Also in the case of the fusion protein Hxk3 :: GFP, the phenomenon of mitochondrial conglomeration observed for Hxkl :: GFP was evident.

In addition to tobacco, Arabidopsis thaliana was also transformed with expression vectors which code for fusion proteins according to the invention. The desired mitochondrial conglomeration was also observed in transgenic Arabidopsis pu zes.

Both the transgenic tobacco and the transgenic Arabidops w plants show no visible phenotype.

Selection strategy for mitochondrial transformation

The above-mentioned attempts by Ortega et al. (2000, Curr. Genet. 37: 315-321) with tobacco cell cultures were carried out with leaf disks on callus induction medium. It was found that the callus growth in 16 h light was only slightly inhibited by the antibiotics every day. With only 30 minutes of light per day, the antibiotics had a clearer effect, but without killing.

Unlike by Ortega et al. described, SHAM also had an impact on callus growth alone. A clear effect was seen at more than 100 μM. At AA and Myx were already visible at 50 μM concentration inhibition, whereby Myx was somewhat stronger.

Based on this data, a selection for antimycin and myxothiazole, possibly in combination with SHAM, should be possible in the dark. The concentration of SHAM should not exceed 100 μM at least in the initial stage. The concentrations of AA and Myx should be at least 50 μM.

On the basis of the published tobacco cob sequence (access number U67396), oligonucleotides were derived, with the aid of which the complete ORF of the cob gene was amplified from tobacco DNA. After ligation into the vector pCR blunt (Invitrogen), those mutations were introduced by means of PCR which should lead to resistance to AA or Myx. The mutation of nucleotide gl28 to t leads to the amino acid exchange glycine-43 → valine. Mutation of nucleotide t405 to a leads to the amino acid exchange phenylalanine-135 -> leucine. In addition, nucleotide t423 was mutated to a in both plasmids, creating an Ncol cleavage site without changing the amino acid sequence. The resulting plasmids were cobG43V + NcoIbzw. called cobF135L + NcoI. The construction of the plasmids used for the selection of transgenic mitochondria is shown in Figure 5.

Generation of transgenic tobacco plants with mitochondrial conglomerates

Tobacco plants of the Havana variety were transformed by means of Agrobacterium-mediated gene transfer with Hxkl :: GFP. The positive plants selected on kanamycin showed the well-known phenomenon of large mitochondrial conglomerates. The Havana variety was used because it has proven itself in plastid transformation.

Implementation of the biolistic transformation

Leaf discs were taken from the transgenic tobacco plants with mitochondrial conglomerates, as well as from wild types. Using a particle gun, these were washed one to three times with the plasmid cobG43V + NcoIbzw. bombarded cobF135L + NcoI.

Screening of calli growing on selective medium

Leaf disks bombarded with cobG43 V + Ncol were designed for 50 μM AA after two days of rest, leaf disks bombarded with cobF135L + NcoI for 50 μM Myx. After one week, the concentration was increased to 100 μM each. After two weeks at 100 μM plus 50 μM SHAM. After three weeks, the promising callus approaches were transferred to 200 μM AA or Myx plus 100 μM SHAM. After five weeks, SHAM was also increased to 200 μM.

The selection marker could be further improved by introducing a further mutation into the plasmids used, whereby a Sphl interface present in the native cob gene is switched off, since this enables a PCR reaction to be used to distinguish between native cob and mutated cob. The DNA isolated from the regenerated callus can then be digested completely with SphI. The transgenic cob is retained and can still be amplified using PCR. An exchange of nucleotide T264 for C would open this possibility. characters

FIG. 1 shows the fusion constructs of hexokinase 1 (Hxkl) with the green fluorescent protein (GFP), in which the Hxkl content was of different sizes. In the shorter construct HxklN:: GFP, only the N-terminus of Hxkl, comprising the putative signal peptide, was fused to GFP. In the larger Hxkl :: GFP construct, the entire sequence coding for Hxkl was combined with that for GFP. In Figure 1, HK1 stands for Hxkl and HK1N for HxklN.

Figure 2 shows an electron micrograph of Hxkl :: GFP organelles. In the left picture, aggregated mitochondria can be seen in the middle, peroxisomes in the upper right corner. In the right picture are aggregated mitochondria and

To see chloroplasts. The black dots are gold-labeled antibodies against GFP.

FIGS. 3 a and b (different magnifications) likewise show electron micrographs of Hxkl :: GFP organelles, it being clearly evident that the formation of the mitochondrial conglomerates appears to be due to a disturbed mitochondrial division.

Figure 4 shows EM images of mitochondrial conglomerates. The left complex has a size of at least 7 μm, the right one of at least 10 μm. FIG. 5 shows the construction of the plasmids used for the selection of transgenic mitochondria.

Claims

1. A method for producing mitochondrial conglomerates comprising the expression of a first protein component capable of being anchored in the outer mitochondrial membrane and a second protein component which sterically shielding determinants on the surface of mitochondria in is able to plant cells.

2. The method of claim 1, wherein the first and second

Protein component in the form of a fusion protein can be expressed in the plant cell.

3. The method according to claim 1 or 2, wherein the protein component capable of being anchored in the outer mitochondrial membrane is the enzyme hexokinase or a porin or parts of this enzyme or a porin which is for anchoring in the outer mitochondria Membrane are capable.

4. The method according to any one of the preceding claims, wherein the protein component capable of sterically shielding determinants on the surface of mitochondria is the green fluorescent protein or parts thereof which are used for sterically shielding determinants on the surface of mitochondria in the Location.

5. The method according to any one of the preceding claims, wherein the protein component capable of sterically shielding determinants on the surface of mitochondria has a size which has a molecular weight of corresponds to at least 20 kDa, preferably at least 25 kDa and particularly preferably at least 28 kDa.

6. A process for the production of transgenic plants, comprising the production of mitochondrial conglomerates by the process according to one of the claims

I to 5.

7. A method for producing transgenic plants by means of mitochondrial transformation, comprising the steps of a) generating mitochondrial conglomerates by a method according to one of the preceding claims, b) transforming the mitochondria with a nucleic acid molecule and, if appropriate, c) crossing out the phenotype of the conglomerated mitochondria ,

8. The method according to claim 7, wherein the transformation in step b) is carried out by means of biolistic methods, in particular particle bombardment, by means of microinjection or by means of electroporation.

9. Use of mitochondrial conglomerates, produced by a method according to one of claims 1 to 5 for the transformation of mitochondria.

1 O. Trans gene plant produced by a method according to any one of claims 6 to 8.

11. Mitochondrial conglomerate with a size of at least 4 μm, preferably at least 7 μm and particularly preferably of at least 10 μm.

12. Use of the mitochondrial conglomerates according to claim 11 for the mitochondrial transformation.