Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
2000-04-03
2002-05-21
Priebe, Scott D. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C800S306000, C435S091400, C435S091320, C435S468000, C536S023720
Reexamination Certificate
active
06392124
ABSTRACT:
SCOPE OF THE INVENTION
The invention refers to infective clones of the turnip mosaic virus (TuMV) and to plant infectious viral vectors based on said infectious clones.
BACKGROUND OF THE INVENTION
Infectious viral clones comprise a DNA molecule and some transcription regulating mechanisms, capable of synthesising a transcript which can infect plants of plant cells (protoplasts). Infectious clones, in themselves, constitute a very valuable tool in basic research in Virology and, besides, are the necessary starting element for the preparation of plant viral vectors.
Plant viruses show different types of genoma: single stranded DNA (for example, geminivirus), double stranded DNA (for example, pararetrovirus), positive polarity RNA, i.e., messenger direction (for example potyvirus), negative polarity RNA (for instance rabdovirus), double polarity RNA (for instance tospovirus) or double stranded RNA (for instance reovirus). Recombinant DNA technology initially allowed the development of infectious clones of plant viruses with DNA genoma (geminivirus and pararetrovirus). Afterwards, thanks to the development of reverse transcriptases infectious clones have been obtained from numerous plant virus groups with positive polarity RNA genoma [Boyer, J. C. & Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses.
Virology
, 198, 415-426]. Infectious clones from the following potyviruses have been described:
Sharka virus (PPV) [Maiss E., Timple U., Brisske Rode A., Lesemann D. E. & Casper R. (1992). Infectious in vivo transcripts of a plum pox potyvirus full length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter.
Journal of General Virology
, 73, 709-712; Riechmann J. L., Lain S. & Garcia J. A. (1990). Infectious in vitro transcripts from a plum box potyvirus cDNA clone.
Virology
, 177, 710-126];
Tobacco vein mottling virus (TVMV) [Domier L. L., Franklin K. M., Hunt A. G., Thoads R. E. & Shaw J. G. (1989). Infectious in vitro transcripts from cloned cDNA of a potyvirus, tobacco vein mottling virus.
Proceedings of the National Academy of Sciences USA
, 86, 3509-3513];
Tobacco engraving virus (TEV) [Doija V. V., McBride H. J. & Carrington J. C. (1992). Tagging of plant potyvirus replication and movement by insertion of □-glucuronidase into the viral polyprotein.
Proceedings of the National Academy of Sciences USA
, 89, 10208-10212];
Pea seedborne mosaic virus (PSbMV) [Johansen I. E., Dougherty W. G., Keller K. E., Wang D. & Hampton R. O. (1996). Multiple viral determinants affect seed transmission of pea seedborne mosaic virus in Pisum sativum.
Journal of General Virology
, 77, 3149-3154];
Peanut stripe virus (PStV) [Flasinki S., Gunasinghe U. B., Gonzales R. A. & Cassidy B. G. (1996). The cDNA sequence and infectious transcripts of peanut stripe virus.
Gene
, 171, 299-300];
Zucchini yellow mosaic virus (ZYMV) [Gal-On A., Antignus Y., Rosner A. & Raccah B. (1991). Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus,
Journal of General Virology
, 72, 2639-2643; Galon A., Meiri E., Huet H., Hua W. J., Raccah B. & Gaba V. (1995). Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus,
Journal of General Virology
, 76, 3223-3227];
Papaya ringspot potyvirus (PRSV) [Chiang C. H. & Yeh S. D. (1995). Construction of infectious in vitro transcripts of papaya ringspot potyvirus. 6
th
International Plant Virus Epidemiology Symposium, Ma'ale Hachamisha, Jerusalem, Israel, page 52]; and
Potato A virus (PVA) [Puurand V., Valkonen J., Makinen K., Rabenstein F. & Saarma M. (1996). Infectious in vitro transcripts form cloned cDNA of the potato A potyvirus.
Virus Research
, 40, 135-140.
The genoma of most of the plant viruses comprises one or several positive polarity RNA molecules. The infectious clones of plant viruses with RNA genoma basically comprise a complementary DNA (cDNA) to the viral genomic RNA and a promoter sequence linked to said cDNA. This promoter sequence can either be that of a bacteriophage, which allows the obtaining of viral RNA by in vitro transcription by the corresponding RNA polymerase, or else that of A plant functional gene, which allows, after the introduction of the DNA into the plants, in vivo production of the corresponding effective viral RNA.
The obtaining of an full length infectious clone from an RNA template (the viral genoma of an RNA virus) implies, in the first place, the use of a reverse transcriptase in order to make a first cDNA chain, in the second place, the synthesis of double stranded cDNA by means of a DNA polymerase, and, in the third place, the linking of different cDNA restriction fragments, although this third stage is not always carried out because some full length infectious clones have been prepared by means of the polymerase (RT-PCR) reserve transcription—chain reaction. The obtaining of a full length infectious clone from an RNA template is a complicated procedure which shows many difficulties since (i) a reading error in the polymerases may take place during either of the first two stages, and (ii) errors may also occur in the linking of the restriction fragments either by the presence of exonucleases or else by the presence of restriction fragments, small and difficult to detect, for the enzyme being used, entailing both kinds of errors the obtaining of full length clones which are not infectious [Boyer, J. C. & Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses.
Virology
, 198, 415-426]. Another factor which also has an influence is the precision of the linking of the promoter to the cDNA. The infectivity is very much decreased, even with 5′ extensions of 1 or 2 nucleotides, however, the 3′ extensions do not have such a big influence in the biological activity [Boyer, J. C. & Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA viruses.
Virology
, 198, 415-426]. On the other hand, the handling of full length viruses is very complex due to the unstableness problems which they show.
One of the main applications for plant infectious viral clones is their use in the construction of viral vectors. A plant infectious viral vector consists, in general, of a infectious viral clone modified in such a way that it contains a heterologous nucleic acid sequence which is expressed after inoculating plants or plant cells with said vector.
Plant viral vectors are used to research molecular biology processes in plants and are useful as tools in order to study plant viral infections processes. Besides, plant viral vectors have been developed in order to substitute and/or insert genes and to show epitopes. Among the commercial applications of known plant viral vectors are the expression of the interpheron alphaD gene, the expression of alpha-tricosanthine (inhibitor of viral replication in the human immunodeffiency virus [HIV]), the expression of the peptide inhibiting the enzyme which converts angiotensine I, the presentation of epitopes of the flu virus, of HIV-1, of malaria epitopes, and the expression of antigenic peptides of the glosopeda virus and human rhinovirus 14 (Scholthof, H. B., Scholthof, K. B. G. & Jackson, A. O. (1996). Plant virus gene vectors for transient expression of foreign proteins in plants.
Annual Review of Phytopathology
, 34, 299-323]. On the other hand, plant infectious vectors represent an alternative to the obtaining of transgenic plants and show numerous advantages, among which are, for instance, high expression in a short period of time, the expression of genes which can make more difficult the regeneration or the growth of transgenic plants and the possibility of a transient expression.
Plant viruses have a limited and specific range of hosts. Most of the plant species are not infected by most viruses, i.e., each virus is capable of infecting a unique combination of plant species &lsqb
Martinez Herrera David
Ponz Ascaso Fernando
Sanchez Sanchez Florentina
Torres Pascual Vicente
Institute Nacional de Investigacion Y Tecnologia Agraria Y Alime
Ostrolenk Faber Gerb & Soffen, LLP
Priebe Scott D.
Sorbello Eleanor
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