Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-17
2001-10-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C536S023100, C536S024100
Reexamination Certificate
active
06303345
ABSTRACT:
It is generally known that genetic engineering techniques allow individual genes to be transferred into the genome of organisms, such as microorganisms, yeasts or plants, in a targeted manner. This technique, which is known as transformation or, in the case of higher cells, also as transfection, is carried out routinely by various routes, for example by particle gun bombardment (cf. M. E. Fromm, F. Morrish, C. Armstrong, R. Williams, J. Thomas and T. M. Klein: “Inheritance and expression of chimeric genes in the progeny of transgenic maize plants”, Bio/Technology 8: 833-839, 1990), naked DNA transfer (cf. P. Meyer, I. Heidmann, G. Forkmann and H. Saedler: “A new petunia flower colour generated by transformation of a mutant with a maize gene”, Nature 330: 677-678, 1987) or by Agrobacterium-mediated stable integration of genes or gene segments into the genome of a recipient plant. As an alternative for the chromosomal integration of foreign genes, it is possible, for example, to use extrachromosomally replicating vectors in order to express foreign genes in a desired organism without integration. Examples of extrachromosomally replicating vectors which are available for plants are those developed from plant viruses (cf., for example, J. W. Davies and J. Stanley: “Geminivirus genes and vectors”, Trends Genet. 5: 77-81, 1989). To do this, the foreign genes to be expressed in the chosen organisms must be brought under the control of regulatory signals (promoter, terminator) which are suitable for this organism and which ensure constitutive or inducible and/or optionally tissue- and/or development-specific transcription. Moreover, it is desirable to provoke an increased mRNA synthesis of the foreign gene by using a strong promoter.
It is known that there are great differences between the regulation signals of the transcription of genes to be expressed which are found in bacteria and eukaryotes (cf., for example, Mitra et al., 1994, Biochem. Biophys. Res. Commun. 204: 187-194; Pobjecky et al., 1990, Mol. Gen. Genet. 220: 314-316). Although, with regard to promoters, eukaryotes also contain a TATA or Hogness box which is very similar and corresponds to the -10-sequence of the prokaryotes (TATAAT), this box is further away from the transcription start in the case of eukaryotes. Additional elements exist in the eukaryotes which are vital for the promoter activity. This is why promoters derived from eukaryotes are, as a rule, unsuitable for regulating the transcription of genes in prokaryotes and vice versa. Also, differences between the promoter structures exist within the eukaryotes, for example between plants and fungi, and also, for example, with regard to the specificity of a promoter for specific cells within one and the same eukaryotic organism.
It would therefore be exceedingly useful for industry and research to have available promoters which are equally active in prokaryotes and eukaryotes, for example in bacteria, fungi and plants. For example, this would dispense with complicated recloning work when it is intended to employ, at the same time, prokaryotic and eukaryotic recipient organisms for the expression of foreign genes.
Such a promoter is the 35S RNA promoter of the cauliflower mosaic virus (CaMV). This promoter meets the requirements made on a strong constitutive promoter in plant cells and is employed predominantly for the transformation of plants, (cf., R. Walden: “Genetic Transformation in Plants”, Open University Press, Milton Keynes, 1988). However, it is known that the CaMV 35S promoter is also active in bacteria (Assaad and Signer, Molecular and General Genetics 223: 517-520, 1990).
Accordingly, it is an object of the invention to provide further promoters derived from a plant specific virus which are active in bacteria and/or fungi. Advantageously, they are active both in plants and, in particular, equally in plants and/or fungi, and bacteria so that they can be employed not only in eukaryotes, but also in prokaryotes. In addition, they advantageously also have a higher promoter activity in plants, fungi and/or bacteria compared with the CaMV 35S promoter.
The German patent DE 43 06 832 of the Max-Planck-Gesellschaft zur Förderung der Wissenschaften and Rohde et al., Plant Molecular Biology 27: 623-628, 1995 have described the use of a DNA which is derived from the CFDV virus (coconut foliar decay virus), which attacks the coconut palm
Cocos nucifera,
and whose structure is shown in
FIGS. 1
,
3
A and
3
B of the Patent Specification as a viral phloem-specific promoter for the tissue-specific expression of genes in transgenic plants.
The CFDV virus is located in the vascular system of the plant (cf. J. W. Randles et al.: “Localization of coconut foliar decay virus in coconut palm”, Ann. Appl. Biology 1992, 601-617). A DNA associated with the disease symptoms and the occurrence of viral particles has already been cloned, sequenced and its structure determined at an earlier point in time (cf. W. Rohde et al.: “Nucleotide sequence of a circular single-stranded DNA associated with coconut foliar decay virus”, Virology 176: 648-651, 1990). CFDV is a viral phytopathogen with a genome consisting of covalently closed-circular simplex DNA. Rohde et al., Virology 176: 648-651, 1990 described a DNA molecule of CFDV with a size of 1291 nucleotides (SEQ ID NO:1) and deletion mutants thereof. CFDV is not a representative of the geminivirus group, but probably constitutes the prototype of the DNA virus group of the “circoviruses”.
Surprisingly, it has now been found that the CFDV-DNA and fragments of the CFDV-DNA are also active as promoters in bacteria and fungi. Thus, for example, the promoter activity in
E. coli
is markedly higher than that of the CaMV 35S promoter which is also active in bacteria (Assaad and Signer, Molecular and General Genetics 223: 517-520, 1990); the CFDV constructs pRT CF4 and pRT CF9 even show an activity in
E. coli
which is up to 60 times higher than that of the CaMV 35S promoter; in tobacco protoplasts too, the CDFV fragment promoter which is contained in the construct pRT CF4 shows a slightly higher promoter activity than the CaMV 35S promoter. It is because of this activity that the CFDV promoters are suitable, in particular, for use in bacterial systems and fungal systems, for example for the generation of pharmacologically active proteins or peptides.
Accordingly, the object of the invention is the use, characterized in the claims, of the CFDV-DNA and of CFDV-DNA fragments as bacterial promoters and promoters in the fungi, in particular in yeasts.
To generate the CFDV-DNA fragments which are suitable for the use according to the invention, techniques which are well known to the skilled worker are used, such as, for example, suitable cleavage sites of the restriction endonucleases on the CFDV-DNA, or the polymerase chain reaction technique, which allows, starting from a full-length CFDV-DNA construct, CFDV-DNA fragments of the desired length to be amplified using a specific primer. To this end, the primers which suit the CFDV fragment are synthesized in a manner known per se using the nucleotide sequence of the CFDV virus described by W. Rohde et al. in Virology 176: 648-651, 1990 and, more specifically, the nucleotide sequences in the region of the 5′ or 3′ ends of the desired fragment.
CFDV DNA fragments which are preferred for the use according to the invention are the DNA fragments with the nucleotides 211 to 991, 409 to 991, 611 to 991, 711 to 991, 211 to 962, 409 to 962, 611 to 962 and 711 to 962 and the XhoI/StyI fragment of the CFDV DNA with the nucleotides 1 to 1157 of SEQ ID NO:1.
CFDV DNA fragments which are very particularly preferred for the use according to the invention are DNA fragments which only encompass the sequence section of the CFDV DNA including the repeated sequence (RPT), the 52 bp sequence and the TATAA box, without encompassing the sequence section up to the end of the open reading frame ORF1 and all of the nucleotides required for constructing the so-called stem-loop structure. Accordingly, very p
Becker Dieter
Hehn Alain
Randles John W.
Rohde Wolfgang
Salamini Francesco
Davis Katharine F
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
Needle & Rosenberg P.C.
Schwartzman Robert A.
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