Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-07-28
2001-03-06
Park, Hankvel (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S173300, C435S242000, C435S252300, C800S281000
Reexamination Certificate
active
06197542
ABSTRACT:
The invention relates to the field of genetic engineering by means of DNA recombinant techniques, more particularly to the field of the genetic engineering of eukaryotic organisms, such as yeasts, fungi and particularly plants.
The invention is particularly directed to genetic manipulations which lead to resistance of the host organism against one or more RNA viruses, and to genetic manipulations which render the host organism capable of an inducible or tissue-specific production of foreign proteins/peptides or RNAs.
For such genetic manipulations recombinant DNA is used comprising one or more sequences derived from RNA virus, or, more particularly, at least one nucleotide sequence which is derived from RNA virus which for its replication is dependent on a viral RNA/RNA polymerase (i.e. an RNA-dependent RNA polymerase, which is sometimes referred to as “replicase”). These RNA viruses may belong to the group of double-stranded RNA viruses (i.e. the genome of the virus consists of double-stranded RNA), to the group of positive-strand RNA viruses (i.e. the genome of the virus consists of “sense” or messenger single-stranded RNA), or to the group of negative-strand RNA viruses (i.e. the genome of the virus consists of “antisense” single-stranded RNA).
Not all RNA viruses, however, are dependent on a viral RNA/RNA polymerase for their replication. This holds in particular for the viruses belonging to the family of Retroviridae, which for their multiplication are dependent on DNA replication after the genomic RNA is transcribed into DNA by means of reverse transcriptase.
Examples of RNA viruses which for their replication are dependent on a viral RNA/RNA polymerase, are double-stranded viruses from the families of Reoviridae and Birnaviridae, negative-strand viruses from the families of Arenaviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae, and positive-strand viruses from the families of Togaviridae, Flaviviridae, Coronaviridae, Nodaviridae, Picornaviridae and Caliciviridae. Concrete examples of positive-strand viruses with a plant host are Tobacco Mosaic Virus, Tobacco Necrosis Virus, Brome Mosaic Virus, Cucumber Mosaic Virus, Tobacco Streak Virus, Tobacco Rattle Virus, Cowpea Mosaic Virus, Tomato Black Ring Virus, Potato Y Virus, Turnip Yellow Mosaic Virus, Tomato Bushy Stunt Virus, Southern Bean Mosaic Virus, Barley Yellow Dwarf Virus, Potatovirus X, Sugar Beet Yellows Virus, Carnation Latent Virus, Carnation Ringspot Virus, Barley Stripe Mosaic Virus, Alfalfa Mosaic Virus, Pea Enation Mosaic Virus and Tomato Spotted Wilt Virus.
Subject to the above-mentioned limitation to RNA viruses which for their replication are dependent on a viral RNA/RNA polymerase, the term “RNA virus” as used herein includes viruses and virusoids which for their replication are dependent on the help of another (helper) virus. As is well known, RNA virus infections may be accompanied by coinfections of, for instance, satellite viruses with a coat protein of their own, satellite RNA which is packed in mixed particles, and virusoids (a small circular RNA genome, packed in mixed particles). The term “RNA virus” as used herein also includes viroids, i.e. autonomous small bare RNA molecules. The further explanation of the invention in the experimental section to follow will be given with reference to an RNA satellite virus, viz. the Satellite Tobacco Necrosis Virus (STNV), a small plant virus (1.85×10
3
kD), which for its replication is entirely dependent on the presence of the helper Tobacco Necrosis Virus (TNV). The RNA genome of STNV contains 1239 nucleotides and codes for a coat protein of 195 amino acids.
The invention comprises incorporating genetic information into the genome of eukaryotes by means of genetic engineering, which information does not as such, nor through transcription products derived therefrom, constitute a burden to the host, and yet accomplishes a very effective protection of the host against viral infections, or enables an inducible or tissue-specific, very efficient production of foreign proteins (or peptides) or RNAs. The genetic information to be incorporated according to the invention comprises an expression-cassette for the host to be transformed containing two, 12-250 base pair long, inverted repeat nucleotide sequences with therebetween at least one nucleotide sequence which is derived from RNA virus which for its replication is dependent on a viral RNA/RNA polymerase, said RNA virus derived sequence comprising at least cis elements for replication, but no gene that codes for viral RNA/RNA polvmerase and no gene that codes for viral coat protein.
The genome of RNA viruses which for their replication are dependent on a viral RNA/RNA polymerase comprises various cis elements, i.e. elements or functions which function only for the nucleic acid by which they are encoded, such as structure elements of the nucleic acid. In addition to cis elements for replication (including in any case the binding site for an RNA/RNA polymerase) the genome of RNA viruses mostly also includes cis elements for transport (the binding site of transport proteins), cis elements for packing the nucleic acid in phage envelopes to form virus particles, and cis elements for translation of messenger RNA into protein, in particular coat protein. Examples of trans elements of the genome of RNA viruses are the genes which code for coat protein, transport protein and RNA/RNA polymerase.
An essential element of the invention is that the expression cassette incorporated into the genome of the host leads to transcription of the sequence derived from RNA virus to form a messenger RNA molecule with a panhandle structure. No strict requirements are set to the elements of the expression cassette which regulate this transcription, such as in particular the transcription promoter. The promoter may be, and in many cases will even preferably be, a relatively weak promoter so that the host will be virtually unburdened by this transcription and the transcription products formed in the process. Suitable promoters are known for many organisms. Naturally the expression cassette should also comprise a suitable polyadenylation site, while the expression cassette is flanked at both ends by so-called integration sites enabling integration into the genome of the intended host.
Various experiments have demonstrated that a successful expression in the host of the viral genetic information incorporated into the genome requires the presence of two, 12-250 base pair long, inverted repeat nucleotide sequences flanking the DNA in-between. These inverted repeat nucleotide sequences may for instance consist of dG-dC base pairs or dC-dG base pairs. The fact that the presence of inverted repeat nucleotide sequences leads to both replication and expression in infected cells of the host, is ascribed to the formation of RNA molecules with a stabilizing panhandle structure (see Van Emmelo et al., Virology 157, 1987, 480-487). For this purpose it is necessary that the inverted repeat nucleotide sequences have a length of at least 12 base pairs. Preferably, however, the inverted repeat nucleotide sequences have a length of at least 15 base pairs. Inverted repeat nucleotide sequences of a length of more than 250 base pairs are not very practical. Preferably, they are not longer than about 50 base pairs.
A further essential feature is the absence of RNA/RNA polymerase (or replicase), or the gene coding therefor, so that the amount of RNA virus-specific messenger RNA present in the cells of the host due to transcription is not further increased. When the RNA virus derived sequence to be incorporated is derived from a satellite virus, such as STNV, this requirement is automatically satisfied because the STNV genome does not contain an RNA/RNA polymerase gene (which explains why the satellite virus depends for its replication on the helper virus, which provides the required RNA/RNA polymerase).
According to the invention it is further of great importance that no viral coat protein is produced and that the nucleotide sequen
Ameloot Paul
De Lafonteyne Jean
Fiers Walter
Haute Eddie Van
Aveve N.V.
Hoffman & Baron LLP
Park Hankvel
LandOfFree
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