AP415A - Recombinant tospovirus DNA constructs and plants comprising such constructs. - Google Patents
Recombinant tospovirus DNA constructs and plants comprising such constructs. Download PDFInfo
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- AP415A AP415A APAP/P/1993/000496A AP9300496A AP415A AP 415 A AP415 A AP 415A AP 9300496 A AP9300496 A AP 9300496A AP 415 A AP415 A AP 415A
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Abstract
Recombinant Impatiens Necrotic Spot Virus (INSV) DNA constructs
Description
-' IMPROVEMENTS IN OR RELATING TO ORGANIC COMPOUNDS o
The present invention relates to. plants having reduced susceptibility to infection from tospoviruses, genetic material capable of generating tolerance to tospoviruses , probes suitable for isolating and diagnosing , and processes for obtaining such plants and genetic material and probes .
Viral infections in plants are frequently responsible for detrimental effects in growth, undesirable morphological changes , decreased yield and the like. Such infections often result in a higher susceptibility to infection in infected plants to other plant pathogens and plant pests.
' Transmission of plant viruses generally occurs via insect or fungal carriers or may occur through mechanical means.
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Plant breeders continuously look to develop varieties of crop plant species tolerant to or resistant to specific virus strains. In the past , virus resistance conferring genes have been transferred from wild types related to commercial plants into commercial varieties through breeding. The transfer of an existing resistance in the wild from the wild type gene pool to a cultivar is a tedious process in which the resistance conferring gene(s) must first be identified in a source (donor) plant species and then combined into the gene pool of a commercial variety. Resistance or tolerance generated in this way is typically active only against one or at best a few strains of the virus in question . One disadvantage of breeding cultivars
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AP Ο Ο Ο 4 Ο 3 for resistance to a particular virus species is that there is often a lack of a gene source suitable for conferring disease resistance within the crop species.
Other approaches to limit the effect of virus induced disease on plants include the use of chemicals such as insecticides, fungicides and the like which act against virus carriers , and/or rely on the employment of preventative methods such as efficient phytosanitary working conditions . However, the use of chemicals to combat virus disease by killing the carrier is' subject to increasingly tougher governmental regulations which present growers with a decreasing scala of permitted chemical plant-protectants.
In an alternative , a system referred to as crossprotection may be employed . Cross-protection is a phenomenon in which infection of a plant with one strain of a virus protects that plant against superinfection with a second related virus strain. The cross-protection method preferentially involves the use of avirulent virus strains to infect plants , which act to inhibit a secondary infection with a virulent strain of the same virus. However, the use of a natural cross-protection system can have several disadvantages. The method is very labour intensive because it requires inoculation of every plant crop , and carries the risk that an avirulent strain may mutate to a virulent strain, thus becoming a causal agent for crop disease in itself. A further possible hazard is that an avirulent virus strain in one plant species can act as a virulent strain in another plant species.
Several studies have indicated that the viral coat protein of the protecting virus plays an important role in crossprotection and that protection occurs when the resident virus and the challenging virus have the same or closely related coat protein structures.
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Recent developments in gene manipulation and plant transformation techniques have given rise to new methods for generating virus resistance in plants. Genetically engineered cross-protection is a form of virus resistance which phenotypically resembles natural cross-protection , but is achieved through the expression of genetic ( information of a viral coat protein from the genome of a genetically manipulated plant . Generation of virus resistance via genetic engineering has been described in for instance , EP 223 452 and reported by Abel et al [(1986) Science 232:738-743] . It was shown that expression of the tobacco mosaic virus strain Ul (TMV-Ul) coat protein gene from the genome of a transgenic plant resulted in a delay of symptom development after infection with any TMV strain. Similar results with respect to coat protein-mediated protection have also been obtained for alfalfa mosaic virus (AMV), potato virus X (PVX) and cucumber mosaic virus (CMV).
Although TMV, CMV, AMV and PVX belong to different virus
C groups, they share a common architecture: in all such viruses the viral RNA is a positive strand RNA encapsidated ( by a viral coat consisting of many individual but identical viral coat proteins.
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However , tospoviruses are essentially different from the plant viruses mentioned above. The genus tospovirus belongs to the family Bunyaviridae. All tospoviruses are transmitted by thrips. The virus particles are spherical in shape (80120 nm in diameter) and contain internal nucleocapsids surrounded by a lipid envelope studded with glycoprotein surface projections. The multipartite genome consists of linear single stranded RNA molecules of negative or ambisense polarity. The terminal nucleotides of these RNA molecules are characterised by a consensus sequence as follows: 5' AGAGCAAUX....................GAUUGCUCU 3', wherein X is C or U . Members of the tospovirus group include tomato spotted wilt virus (TSWV), Impatiens necrotic
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spot virus (INSV), and tomato chlorotic spot virus (TCSV), also known as tomato mottled spot virus (TMSV) or TSWV-like isolate BR-O3. A general description of a tospovirus, using TSWV as a representative of the genus tospoviruses can be found in our co-pending application EP 426 195 herein incorporated by reference .
The tospovirus particle contains at least 4 distinct structural proteins: an internal nucleocapsid protein N of 29 kd and two membrane glycoproteins: Gl, approximately 78 kd, and G2 approximately 58 kd. In addition, minor amounts of a large protein, L, approximately 260 kd have been detected in virus particles. Tospoviral genomes consist of three linear single stranded RNA molecules of about 2900 nucleotides (nt) (S RNA) , about 5000 nt , (M RNA) and about 8900 nt (L RNA) , each tightly associated with nucleocapsid proteins and a few copies of the L> protein to form circular nucleocapsids. A schematic structure outlining most properties of an INSV is given in Figure 1. Based on the above and other properties , INSV (like TSWV) has been classified as a member of the tospovirus genus.
Circumstantial evidence has been presented which suggests that an M RNA encoded gene is directly or indirectly involved in the synthesis of the Gl membrane glycoprotein [Verkleij and Peters ,(1983) J. Gen. Virol. 64:677-686] .
As mentioned above, tospoviruses such as TSWV , INSV and the like are transmitted by certain species of thrips. These tospovirus carriers belong to the family Tripidae and include tobacco thrips (Frankliniella fusca (Hinds.),, western flower thrips (F. occidentalis (Pergande)), common blossom thrips (F. Schultzei (Trybom)), chilli thrips (Scirtothrips dorsalis (Hood)), Thrips setosus (Moulton), onion thrips (T. tabaci (Lindeman, ), F. intonsa and melon thrips (T. palmi (Karny)). The tospovirus is acquired by
AP Ο Ο Ο 4 Ο 3 the virus before they pupate but adults more commonly transmit the virus. Adult thrips can remain infective throughout their lives .
Tospoviruses are widespread in temperate, subtropical and tropical climate zones throughout the world . The current distribution of tospoviruses covers all continents and makes them one of the most widely distributed of groups of plant viruses. At least 370 plant species representing 50 plant * J families, both monocotyledons and dicotyledons , are
-—i naturally infected by tospoviruses of the Bunvaviridae.
Tospoviruses seriously affect the production of food and ornamental crops . Symptoms of tospovirus infection in plants include stunting, ringspots, dark purple-brown sunken spots, stem browning, flower breaking, necrotic and pigmental lesions and patterns, yellows and non-necrotic mottle, mosaic in greens or even total plant death. Most plant hosts display only a few of these symptoms, however, the wide range of symptoms produced by tospovirus infection has complicated diagnosis of the disease and has led to individual diseases being given several different names . A further complication is that tospovirus symptoms within the same plant species may vary depending on the age of the plant, time of infection during the life-cycle of the plant , nutritional levels , environmental conditions, such as temperature , and the like.
Although TSWV has been known for many years, is widely distributed, and is the causal agent of a disease which leads to significant loss in yield in crops and ornamentals, limited progress has been made in identifying sources of genes capable of conferring resistance to TSWV or other tospoviruses . A monogenic TSWV tolerance has been identified in Lycopersicon peruvianum, but this trait has not been transferred to cultivated tomatoes so far , nor has a resistance source been identified for other crop species . The use of natural cross-protection systems to
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AP Ο Ο Ο 4 0 J decrease the invasive effects by tospovirus strains capable of causing damage is not well documented. Limited positive results have been reported for tomato and lettuce.
The introduction of genetic information capable of conferring resistance or tolerance to tospoviruses into plant gene pools by means of genetic manipulation provides the breeder and grower alike with a new method for combatting tospovirus induced disease. In particular, it has been found that genetic manipulation techniques may be employed to confer resistance to INSV related disease in plants .
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Detailed Description
According to the present invention there is provided a recombinant INSV DNA construct comprising a DNA sequence coding for transcription into
a) an RNA sequence of an INSV or an RNA sequence homologous thereto ;
b) an RNA sequence of an INSV or an RNA sequence homologous thereto capable of encoding for an INSV protein or a part thereof , in which one or more codons have been replaced by synonyms , or an RNA sequence homologous thereto ; or
c) an RNA sequence complementary to an RNA sequence according to a) or b) , which INSV DNA is under expression control of a promoter capable of functioning in plants and includes a terminator
V ' capable of functioning in plants.
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The DNA sequences defined under a), b) and c) above, for the purposes of the present invention will be referred to as INSV Related DNA Sequences hereinafter. An INSV Related DNA Sequence according to the invention may be modified as appropriate to create mutants or modified sequences homologous to such INSV Related DNA Sequences from which they are derived, using methods known to those skilled in the art such as site-directed mutagenesis and the like. Such mutants or modified coding sequences are embraced within the spirit and scope of the invention.
The term RNA sequence of an INSV may refer to a sequence of the S, M or L RNA strand, preferably an S or M
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The term Η RNA sequence homologous to an RNA sequence of an INSV refers to an RNA sequence of an INSV wherein a number of nucleotides have been deleted and/or added but which is still capable of hybridization to a nucleotide sequence complementary to an RNA sequence of an INSV under appropriate hybridization conditions. For the purposes of the present invention appropriate hybridization conditions may include but are not limited to , for example , an incubation for about 16 hours at 42’C , in a buffer system comprising 5 x standard saline citrate (SSC) , 0.5% sodium dodecylsulphate (SDS) , 5 x Denhardt ’ s solution , 50% formamide and 100 gg/ml carrier DNA· (hereinafter the buffer
Sx- system) , followed by washing 3x in buffer comprising 1 x
SSC and 0.1% SDS at 65'C for approximately an hour each time .
Preferably , hybridization conditions employed in the present invention may involve incubation in a buffer system for about 16 hours at 49'C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 55-C for about an hour each time . More preferably , hybridization conditions may involve incubation in a buffer system for about 16 hours at 55‘C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 65’C for approximately an hour each time .
The length of the INSV Related DNA Sequence will i.a. depend on the particular strategy to be followed , as will become apparent from the description hereinafter . In general , the INSV Related DNA Sequence may comprise at least 20 , and suitably 50 or more nucleotides .
The term promoter refers to the nucleotide sequence upstream from the transcriptional start site and which contains all the regulatory regions required for transcription, including the region coding for the leader sequence of mRNA (which leader sequence comprises the
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AP Ο Ο Ο 4 Ο 3 ribosomal binding site and initiates translation at the AUG start codon) .
Examples of promoters suitable for use in DNA constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant cells. The promoter may express the DNA constitutively or differentially . Suitable examples of promoters differentially regulating DNA expression are promoters inducible by disease carriers, such as thrips, e.g. so-called wound-inducible promoters. It will be j-- appreciated that the promoter employed should give rise to the expression of an INSV Related DNA Sequence at a rate sufficient to produce the amount of RNA necessary to decrease INSV susceptibility in a transformed plant. The required amount of RNA to be transcribed may vary with the type of plant. Particularly preferred promoters include the cauliflower mosaic virus 35S (CaMV 35S) promoter, derivatives thereof, and a promoter inducible after wounding by a disease carrier such as thrips, e.g. a wound inducible promoter. Examples of further suitable promoters include nopaline synthase, octopine synthase and the like.
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The term terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription . Terminators are DNA 3 ‘ -non-translated sequences that contain a polyadenylation signal, that causes the addition of polyadenylate sequences to the 3 1 end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants. Examples of terminators particularly suitable for use in the DNA constructs of the invention include the nopaline synthase terminator of A. tumefaciens, the 35S terminator of CaMV and the zein terminator from Zea mays.
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In accordance with the present invention , an RNA sequence is complementary to another RNA sequence if it is able to form a hydrogen-bonded complex therewith , according to rules of base pairing under appropriate hybridization conditions (as described hereinabove) .
The present invention also provides a vector capable of introducing the DNA construct of the invention into plants and methods of producing such vectors.
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The term vector as employed herein refers to a vehicle with which DNA constructs of INSV or fragments thereof may be incorporated into the cells of a host organism.
The term plants refers to differentiated plants as well as undifferentiated plant material such as protoplasts, plant cells, including cybrids and hybrids, seeds, plantlets and the like which under appropriate conditions can develop into mature plants , progeny thereof and parts thereof such as cuttings , fruits of such plants and the like.
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The invention further provides plants comprising in their C genome a DNA construct of the invention, and methods of producing such plants. Such methods include plant breeding, plantlets derived from protoplast fusion and the like.
The plants according to the invention have reduced susceptibility to diseases induced by INSV or diseases related to INSV infection and suffer from substantially fewer or none of the disadvantages and limitations of plants obtained by classical methods as mentioned hereinabove.
Many types of plants are susceptible to INSV infection however only in some types is INSV infection known to give
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Such types of plants include the ornamental or flowering plants. Examples of such plants include but are not limited to Ageratum , Amaranthus , Anthirrhinum , Aquilegia , 3egonia , Chrysanthemum , Cineraria , clover. Cosmos , cowpea , Cyclamen , Dahlia , Datura , Delphinium ,
Gerbera , Gladiolus , Gloxinia , Hippeastrum , Impatiens , Mesembryanthemum , petunia , Primula , Saint Paulia , Salpiglossis , Tagetes , Verbena , Viola , Vinca ,
Zinnia , Pelargonium and the like .
Other types of plants may be susceptible to INSV infection but these plants may not present disease symptoms directly associated with INSV infection, however such plants may present symptoms of a disease as a result of a secondary infection by a different organism made possible as a result of an initial infection by INSV. Such plants may therefore be viewed as being the subject of an INSV infection related disease and may include plants selected from a wider group of plant types. Further examples of this group of plant types may include vegetable and other crops . Such crop types include alfalfa , aubergine , beet , broad bean , broccoli , brussels sprouts , cabbage , cauliflower , celery , chicory , cow pea , cucumber , endive , gourd , groundnut , lettuce , melon , onion , papaya , pea , peanut , pepper , pineapple , potato , safflower , snap bean , soybean , spinach , squash , sugarbeet , sunflower , tobacco , tomato , water melon and the like .
The invention relates in particular to ornamental plants and preferably to those listed ornamental plants comprising in their plant genome a DNA construct of the invention.
The particular features of tospoviruses including those of INSV are illustrated hereinafter.
The S, M and L RNA are single stranded RNA molecules. The S RNA of INSV is about 3000 nucleotides long( SEQ. ID No.l ;
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SEQ ID No. 2 ) and comprises two genes, one (SEQ ID No.3) encoding a non-structural protein (NSs) in viral sense, the other one (SEQ ID No.11) encoding the nucleocapsid protein (N) in viral complementary sense. The intergenic region between the NSs- and N-gene can be folded into a secondary structure (Seq ID No. 7 and SEQ ID No.8) . The 5'- and 3‘terminal sequences of the S RNA are capable of hybridizing to each other such that the first nucleotide is opposite (and complementary) to the last nucleotide of said S RNA strand . For the purposes of the description the doublestranded structure obtained by hybridizing both RNA termini (' will be referred to as a pan-handle (SEQ ID No.5 and SEQ
ID NO. 6) hereinafter.
The M RNA strand of INSV comprises about 5000 nucleotides (SEQ ID No. 14) . It contains at least two open reading frames, one encoding a non-structural protein (NSm) in viral sense ( SEQ ID No.15), and another open reading frame (SEQ ID No.21) in viral complementary sense. This open reading frame is translated on polysomes located on the endoplasmic reticulum where the nascent polypeptide chain is cleaved co-translationally to form the spike proteins G1 and G2 respectively. As with S RNA, the termini of the M RNA strand are complementary to each other and may likewise
C hybridize to form a pan-handle ( SEQ ID No.13 and SEQ
ID No.19).
The L RNA strand of INSV comprises about 8900 nucleotides. It contains complementary 3' and 5' ends for a length of from about 50 to about 80 nucleotides. The RNA has a negative polarity, with one open reading frame (ORF) located as the viral complementary strand. This ORF corresponds to a primary translation product of about 2875 amino acids in length with an anticipated Mw of between about 300,000 to about 350,000. Comparison with the polymerase proteins of other negative strand viruses indicates that this protein probably represents a viral
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Ο polymerase. In some mutant strains, shortened L RNA molecules have been found in addition to the wild type, full length L RNA. These shortened L RNAs however are observed to possess the characteristic terminal nucleotide sequences and thus are capable of forming “pan handle* structures. They are also encapsidated with nucleocapsid protein and are included in virus particles. Their presence suppresses symptom development resulting in less severe detrimental effect . Thus , these shortened L RNA molecules
L- can be regarded as defective interfering (DI) RNAs . A ς defective interfering RNA is one which is capable of interfering in replication by competing with other genomic RNAs for polymerases and therefore is capable of being replicated , and by so doing inhibits the replication and/or expression of other genomic RNAs with which it is competing .Thus , a DI RNA may comprise any RNA sequence which is capable of being replicated and may be an L , S , or M RNA within the context of the present invention . Such DI RNA sequences may comprise RNA sequences which have had nucleotides either deleted from or added thereto provided that they are capable of competing for polymerases and of £ replicating .
( A preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV RNA sequences of a pan-handle (SEQ ID No.5 , SEQ ID NO. 6 ; SEQ ID No. 18 , SEQ ID No. 19), or into INSV RNA sequences homologous thereto.
Another preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV-RNA sequences of an open reading frame in viral complementary sense i.e. having negative polarity , or into corresponding RNA sequences in which one or more codons have been replaced by their synonyms, or into RNA sequences
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A further preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV-RNA sequences of a hairpin ( SEQ ID No.7 , SEQ ID No. 8 ; SEQ ID No. 13 , SEQ ID No. 16 ) , or into RNA sequences homologous thereto.
Preferably, the INSV-RNA sequence referred to hereinabove has at least 20 nucleotides. Preferably, the INSV-RNA sequence has at least 50 nucleotides.
Examples of DNA constructs suitable for use according to the invention include INSV-Related DNA Sequences coding for transcription into (reference is made to the sequence listing );
i) the viral S RNA nucleotide sequence from 1 to 3017 (SEQ. ID NO.1) ii) the viral S RNA nucleotide sequence from position 25 to 3017 (SEQ. ID No.2);
iii) the viral S RNA nucleotide sequence from 87 to 1436 (SEQ. ID NO.3);
iv) the viral S RNA nucleotide sequence from 2030 to
2868 (SEQ. ID No.4);
v) the viral S RNA pan-handle ’ structure comprising;
a) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral S RNA
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b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral S RNA
vi) | the viral 2079; (SEQ | S ID | RNA No. | nucleotide 7) | sequence | from | 1437 | to |
vii) | the viral | s | RNA | nucleotide | sequence | from | 1440 | to |
2041; (SEQ | ID | No. | 8) |
Q viii) the viral complementary S RNA nucleotide sequence from 1 to about 3017; (SEQ ID No.9) ix) the viral complementary S RNA nucleotide sequence from 1 to 2993; (SEQ ID No.10)
x) the viral complementary S RNA nucleotide sequence from 150 to 933; (SEQ ID No.11) xi) the S RNA nucleotide sequence from 1581 to 2930 of the viral complementary S RNA strand; (SEQ ID No.12);
c xii) the viral complementary S RNA secondary structure having a nucleotide sequence of 642 nucleotides from 939 to 1580; (SEQ ID No.13) xiii) S RNA nucleotide sequence from 87 to 1436 in which one or more codons have been replaced by their synonyms;
xiv) S RNA nucleotide sequence from 2080 to 2868 in which one or more codons have been replaced by their synonyms;
xv) the M RNA nucleotide sequence from 1 to 4970 (SEQ ID No.14);
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AP Ο Ο Ο 4 Ο 3 xvi) the Μ RNA sequence from 86 to 997 (SEQ ID No.15);
xvii) the M RNA sequence of the intergenic region from 998 to 1470 (SEQ ID No.16);
xviii) | the M RNA 17) | sequence from 1471 to 4884; (SEQ ID No. | |
xix) | the M RNA pan-handle structure comprising ; a) a first nucleotide sequence of from about 30 to about |
nucleotides in length from the 5' end of the viral M RNA and
b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral M RNA xx) the complementary viral M RNA sequence from 1 to 4970; (SEQ ID No.20)
O xxi) the complementary viral M RNA sequence from position 87 to position 3500 of the complementary viral M RNA sequence; ( SEQ ID No.21) xxii) the complementary viral M RNA sequence from position 3974 to 4885 (SEQ ID No.22) xxiii) rna sequences homologous to the nucleotide sequences defined under i) to xii) and xv, to xxii) hereinabove.
xxiv) fragments of sequences defined under i) to xxii)
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Preferred INSV-Related DNA Sequences code for transcription into the RNA sequences according to sequences iv) to xii) and xv) to xxii) as defined above, or into RNA sequences homologous thereto, or into fragments thereof comprising at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 50 nucleotides.
According to another preferred embodiment of the invention the DNA constructs of the invention comprise INSV Related θ DNA Sequences coding for transcription into a combination of the 5' and 3' terminal sequences (ie pan-handles) of
C viral S, M or L RNA respectively, more preferably of S or M
RNA, and most preferably of S RNA . Examples of S RNA and M RNA terminal sequences include
i) a first nucleotide sequence 36 nucleotides in length from the 5' end of the viral S RNA :
' AGAGCAATNN NNNNNNNNNN NNNNGAACAAC CCAAGC 3 ' (SEQ ID No.5 ie nucleotides from position 1 to 36 of SEQ ID No.l , where N stands for A,T,G,or
C C) c
and a second nucleotide sequence 36 nucleotides in length from the 3‘ end of the viral S RNA:
5' GATTATATG ATGTTATATT CGTGACACAA TTGCTCT 3' (SEQ ID No.6 ie nucleotides from position 2981 to 3017 of SEQ ID No.l) ii) a first nucleotide sequence of 36 nucleotides in length
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51 AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAA 31 (SEQ ID No.18 ie nucleotides from position 1 to 36 of SEQ ID NO.13) and
b) a second nucleotide sequence 36 nucleotides in length from the 3' end of the viral M RNA
5' TGTTGTATGT AGAGATTTTG TTTGCACTGA TTGCTC T 3' (SEQ ID No.19 ie nucleotides from position 4941 to 4970 Of SEQ ID No. 13)
In the case of the terminus at the 5' end of the S RNA it is not known whether or not there are sixteen or seventeen nucleotides in the unknown region demarked by a series of N s , however the exact number of nucleotides in this region is not considered to be critical to the formation of pan-handle structures so long as the 5' end of the S RNA is capable of complementing the 3‘ end of the S RNA thus enabling the formation of a pan-handle structure .
The invention further provides probes suitable for use as diagnostic tools for the diagnosis of disease in plants suspected of being infected with INSV tospoviruses. Such probes comprise a labeled oligonucleotide (RNA or DNA) sequence complementary to an RNA sequence of an INSV tospovirus. The desired length of the sequence and appropriate method for diagnostic use of probes are known by those skilled in the art. A suitable probe may comprise a nucleotide sequence of at least 12 to about 800 nucleotides, preferably at least 15, more preferably more than 30 nucleotides, and most preferably from about 400 to bad ORIGINAL
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600 nucleotides complementary to an RNA sequence of an INSV tospovirus .
Probes according to the invention are helpful in identifying INSV tospovirus RNA or parts thereof in infected plant material i.a. for diagnostic purposes prior to full presentation of disease symptoms in plants .
The invention accordingly also provides a diagnostic method of determining INSV tospovirus infection in plants which comprises detecting INSV tospovirus replicative forms employing the probes of the invention in dot-blot type assays .
Probes according to the invention are useful in the construction of and use of chimeric genes comprising a DNA sequence corresponding to an RNA sequence of an INSV tospovirus.
The DNA constructs of the invention may be obtained by insertion of an INSV Related DNA Sequence in an appropriate expression vector, such that the sequence is brought under expression control of a promoter capable of functioning in plants and its transcription is terminated by a terminator capable of functioning in plants.
The term “appropriate expression vector as used herein refers to a vector containing a promoter region and a terminator region which are capable of functioning in plant cells.
The insertion of an INSV Related DNA Sequence into an appropriate expression vector may be carried out in a manner known per se. Suitable procedures are illustrated in
Likewise the construction of an appropriate expression vector may be carried out in a manner known per se.
AP000403
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Plants according to the invention may be obtained by
a) inserting into the genome of a plant cell a DNA construct as hereinbefore defined;
b) obtaining transformed cells; and
c) regenerating from the transformed cells genetically transformed plants.
DNA vectors of the present invention may be inserted into the plant genome of plants susceptible to INSV infection. Such plant transformation may be carried out employing techniques known per se for the transformation of plants, such as plant transformation techniques involving Ti plasmids derived from Agrobacterium tumefaciens, A. rhizogenes or modifications thereof, naked DNA transformation or electroporation of isolated plant cells or organized plant structures, the use of micro-projectiles to deliver DNA, the use of laser systems, liposomes, or viruses or pollen as transformation vectors and the like.
Plants of the invention may be monitored for expression of an INSV-Related DNA Sequence by mechods known in the art, including Northern analysis, Southern analysis, PCR techniques and/or immunological techniques and the like. The plants of the invention show decreased susceptibility to INSV infection as demonstrated by tests whereby the plants are exposed to INSV preferentially at a concentration in the range at which the rate of disease symptoms correlates linearly with INSV concentration in the inoculum.
Methods suitable for INSV inoculation are known in the art and include mechanical inoculation, and in particular, the use of appropriate vectors.
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Plants of the invention may also be obtained by the crossing of a plant obtained according to the methods of the invention with another plant to produce plants having in their plant genome a DNA construct of the invention.
The invention is illustrated by the following non-limiting examples and accompanying figures.
Figure 1: Schematic representation of an INSV particle .
Figure 2; Sequence strategy for INSV viral S RNA.
Figure 3: Open reading frame analysis of the INSV S RNA, full bars represent translational stop codons (TAA, TAG, TGA), half size bars indicate start codons (ATG).
Figure 4: Schematic review of the construction of a suitable expression vector (pZU-B).
Figure 5 suitable sequence. | : Schematic review of the | construction of a V N protein-coding | |||
plasmid | comprising | the | INS’ | ||
Figure 6 | : Schematic review | of | the | construction of a | |
suitable | plasmid | comprising | the | INSV | NSs protein-coding |
sequence.
Figure 7: Schematic review of the construction of a suitable plasmid comprising the INSV NSm protein-coding sequence.
Figure 8: Schematic review of the construction of a suitable plasmid comprising the INSV G1/G2 glycoprotein precursor-coding sequence.
Figure 9: Schematic review of the construction of a INSV N gene-containing plant transformation vector.
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Figure 10: Schematic review of the construction of a INSV NSs gene-containing plant transformation vector.
Figure 11: Schematic review of the construction of a INSV G1/G2 glycoprotein precursor gene-containing plant transformation vector.
Figure 12: Schematic review of the construction of a INSV NSm gene-containing plant transformation vector.
Figure 13: The secondary structure located at the intergenic region of INSV S RNA.
Suitable examples of preferred INSV Related DNA Sequences coding for transcription into a sequence of the secondary structure of the intergenic region of S RNA or of RNA sequences homologous thereto are sequences coding for the 1437 to 2079 nucleotide sequence of S RNA or for a sequence homologous to such sequences.
Other advantageous features of the present invention will
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MATERIAL AND METHODS
All INSV RNA-derived sequences presented here are depicted as DNA sequences for the sole purpose of uniformity. It will be appreciated that this is done for convenience.
Cultivars of Nicotiana t a ba cum and Petunia hybrida, used in plant transformation studies, are grown under standard greenhouse conditions. Axenic explant material is grown on θ standard MS media [Muras’nige and Skoog, (1962) Physiol Plant
15: 473-497] containing appropriate phytohormones and sucrose concentrations.
E. coli bacteria are grown on rotary shakers at 37°C in standard LB-medium. Agrobacterium tumefaciens strains are grown at 28°C in MinA medium supplemented with 0.1 % glucose [Ausubel et al., (1987) Current Protocols in Molecular Biology , Green Publishing Associates and Wiley Intersciences , New York , Chichester , Brisbane , Toronto , and Singapore] .
In all cloning procedures the Ξ. coli strain JM83, (F~, ,—
V. A(lac-pro), ara, rpsL, 080, dlacZMl5) is used as a recipient for recombinant plasmids.
(
Binary vectors are conjugated to Agrobacterium tumefaciens strain LBA 4404, a strain containing the Ti-plasmid vir region, (Hoekema et al. , (1983) Nature 303:179-180] in standard triparental matings using the E. coli HB101, containing the plasmid pRK2013 as a helper strain. [Figurski and Helinski, (1979) Proc. Natl. Acad. Sci.USA 76:1648-1652] Appropriate Agrobacterium tumefaciens recipients are selected on media containing rifampicin (50 gg/ml) and kanamycine (50 μg/ml) .
Cloning of fragments in the vectors pUCl9 [Yanish-Perron
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ΡΒΙΝ19 [Bevan et al.,(1984) Nucl Acids Res. 12:8711-8721] or derivatives, restriction enzyme analysis of DNA, transformation to E. coli recipient strains, isolation of plasmid DNA on small as well as large scale, nicktranslation, in vitro transcription, DNA sequencing, Southern blotting and DNA gel electrophoresis are performed according to standard procedures [Maniatis et al., (1982) Molecular Cloning , a Laboratory Manual . Cold Spring Harbor Laboratory , New York ; Ausubel et al.supra, (1987)].
DNA amplification using the polymerase chain reaction (PCR) were performed as recommended by the supplier of the Tag polymerase (Perkin Elmer Cetus ).
Amplifications of RNA by reverse transcription of the target RNA followed by standard DNA amplification were performed using the Gene Amp RNA PCR Kit as recommended by the supplier (Perkin Elmer Cetus).
Examples
Example 1; Isolation of INSV particles and genetic material therein
INSV isolate NL-07, an isolate from Impatiens, is maintained on Impatiens by grafting. Virus is purified from systemically infected Nicotiana rustica leaves, after mechanical inoculation essentially as described by Tas et al. [(1977) J. Gen. Virol. 36:81-91 ] . All material used in the isolation procedure should be maintained at a temperatue of 4 °C . Twelve days after inoculation 100 grams of infected leaves are harvested and ground for 5-10 seconds at a low speed setting in 5 volumes extraction buffer (0.1 M NaH2PO4, 0.01 M Na2SO3, pH 7) in a Waring blender. The suspension is filtered through cheesecloth and the filtrate is centrifuged for 10 minutes at 16,000 x g. The resulting
AP Ο Ο Ο 4 Ο 3 pellet is resuspended in three volumes resuspension buffer (0.01 M NaH2PO4, 0.01 M Na2SO3, pH 7). The pellet is dissolved by stirring carefully at 4°C. After centrifuging for 10 minutes at 12,500 x g the pellet is discarded and the supernatant centrifuged again for 20 minutes at 50,000 x g. The pellet is resuspended in 0.2 volume of resuspension buffer (0.01 M NaH2PO4, 0.01 M Na2SO3, pH 7) and kept on ice for 30 minutes. Anti-serum raised in rabbits against material from non-infected Nicociana rustica is added to the solution and carefully stirred for 1 hour . Non-viral complexes are pelleted after 10 minutes centrifuging at 16,000 x g. The cleared supernatant is loaded on a linear 5% - 40 % sucrose gradient in resuspension buffer(0.01 M
NaH2PO4, 0.01 M Na2SO3, pH 7) , and spun for 45 minutes at 95,000 x g. The opalescent band containing INSV particles is carefully collected with a syringe and diluted 4 times with resuspension buffer. Washed viruses are pelleted by centrifugation for 1.5 hours at 21,000 x g and resuspended in one volume of resuspension buffer. Generally, 100 grams of leaf material yields approximately 0,5 mg of INSV viruses. INSV RNA is recovered preferentially from purified ζ virus preparations by SDS-phenol extractions followed by ethanol precipitation . From 1 mg INSV , 1-5 μg of RNA is
C extracted. The isolated RNA molecules are analysed for intactness by electrophoresis on an agarose gel. Three distinct RNA molecules are identified with apparent sizes of about 3000 nucleotides (S RNA), about 4900 nucleotides (M RNA) and about 8900 nucleotides (L RNA) respectively.
Example 2: Sequence determination of the 3'-termini of the INSV viral RNAs
In order to perform direct RNA sequencing , INSV RNA is extracted from purified nucleocapsids essentially according to Verkleij et al. (1983 ) supra . Twelve days after inoculation 100 grams of infected leaves are harvested and
AP Ο Ο Ο 4 Ο 3 volumes of TAS-E buffer (O.O1 M EDTA, 0.01 M NajSCb, 0.1 % cysteine, 0.1 M TRIS pH 8.0) in a Waring blender. The suspension is filtered through cheesecloth and centrifuged for 10 minutes at 1,100 x g. Nucleocapsids are recovered from the supernatant after 30 minutes of centrifuging at 66,000 x g. The pellet is carefully resuspended in one volume of TAS-R buffer (1 % Nonidet NP-40, 0.01 M EDTA, 0.01 M Na2SO3, 0.1 % cysteine, 0.01 M glycine, 0.01 M TRIS , pH 7.9). The pellet is dissolved by stirring carefully for 30 minutes at 4 °C. The supernatant is cleared by centrifuging for 10 minutes at 16,000 x g. Crude nucleocapsids are collected from the cleared supernatant by sedimentation through a 30 % sucrose cushion for 1 hour at 105,000 x g. The nucleocapsid pellet is resuspended in 400 μΐ 0.01 M Na-citrate pH 6.5, layered on a 20 - 40 % sucrose (in 0.01 M Na-citrate pH 6.5) and spun for 2 hours at 280,000 x g. The three different opalescent bands, respectively L, M and S nucleocapsid, are collected separately. INSV RNA is recovered preferentially from purified nucleocapsid preparations by SDS-phenol extractions followed by ethanol precipitation. Generally , 100 μg of RNA are obtained from 100 grams of infected leaves. The 3'-ends of the separate INSV RNAs are labeled using RNA ligase and 5'-[3:P]pCp. The end-labeled RNA molecules are separated on a low gelling temperature agarose gel [Wieslander, (1979) Anal Biochem 98: 305-309] . The enzymatic approach described by Clerx-Van Haaster and Bishop [(1980) Virology 105:564-574] and Clerx-Van Haaster et al. [(1982) J Gen Virol 61:289-292] is used to determine the 30 terminal nucleotides of the 3'and 5'-ends of both S and M RNA.
Synthetic oligonucleotides complementary to the 3'-termini are synthesized using a commercially available system (Applied Biosystems) and used for dideoxy-sequencing with reverse transcriptase.
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Example 3: cDNA cloning of INSV genetic material
Oligonucleotides complementary to the 3' -end of the S RNA are used for priming first strand cDNA synthesis. With these primers, double stranded DNA to INSV RNA is synthesized according to Gubler and Hoffman ((1983) Gene 25:263-269].
Two different approaches are used to generate cDNA clones to the INSV viral RNAs. A first series of clones is obtained by random priming of the INSV RNA using fragmented single stranded calf thymus DNA, followed by first and second strand cDNA synthesis. cDNA is made blunt-ended using T4-DNA polymerase and ligated with T4 ligase into the Smal site of pUC19.
A second series of INSV cDNA clones is obtained by priming first strand DNA synthesis with the oligonucleotides complementary to the 20 terminal nucleotides at the 3 *-ends of the INSV RNAs. Blunt ended cDNA fragments are cloned into the Sma I site of pUCl9.
cDNA clones from both series containing viral inserts are ί selected via colony hybridization, essentially according to the method of Grunstein and Hogness [(1975) Proc. Natl.
I Acad. Sci. USA 72:3961-3965] using [32]p-labeled, randomly primed first strand cDNA as a probe. Sets of overlapping cDNA clones are selected by Southern analysis followed by plasmid walking, in order to construct a restriction map, based on cDNA derived sequences of the S RNA (Figure 2J.
Example 4: Sequence determination of the INSV S RNA
In order to determine the sequence of the S RNA 5 selected cDNA clones are subcloned into pBluescript, resulting in the plasmids pINSV-S2, pINSV-S15, pINSV-S61, plNSV-S60 and pINSV-S39 , (Figure 2). The clones are sequenced in both directions using the protocol of Zhang et al. ((1988) Nucl.
AP Ο Ο Ο 4 Ο 3 of the S RNA is determined by primer extension of the synthetic oligonucleotide INSV-S60 (5' d (AGAGCAATTGTGTCAJ. which is complementary to the 15 nucleotides of the 3'terminus. Sequence data from the INSV S RNA (3017 nt) is summarized in the sequence listing (SEQ ID No.l to SEQ ID No.12).
Computer simulated translation of the 6 different reading frames on the viral strand and viral complementary strand reveals the presence of two putative open reading frames (Figure 3). On the viral strand an open reading frame is found starting at position 87 and terminating at a UAA stopcodon at position 1436 encoding a protein of 449 amino acids with a predicted molecular mass of about 51.2 kd. This protein is a non-structural protein, tentatively designated NSs (Figure 3/ SEQ ID No. 26). The other open reading frame is located on the viral complementary strand from position 2080 to 2868 (SEQ ID No. 11) , encoding a 262 amino acid long polypeptide with a predicted molecular mass of about 28.7 kd. This open reading frame encodes the viral nucleocapsid protein N (Figure 3/ SEQ ID No 25). Thus Figure 3 shows the coding capacities of the viral and the viral complementary strand of INSV S RNA, indicating the NSs and N protein genes are expressed from subgenomic mRNAs (SEQ ID No.3 , SEQ ID No.11 respectively) . Thus, the situation occurs that a plant virus RNA has an ambisense gene arrangement. Other important features of this S RNA sequence is the existence of complementary terminal repeats capable of forming so-called pan-handle structures. These structures play an important role in replication and transcription of viral RNA. Another putative regulatory element is the secondary structure in the intergenic region of the S RNA, which most likely contains the transcription termination signals for both subgenomic mRNAs, encoding respectively the N and NSs-protein._ bad original
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The nucleotide sequence of the INSV M and L RNA is elucidated employing similar strategies and methods as used to determine the nucleotide sequence of the S RNA.
Example 5: Construction of an expression vector pZU-B
The recombinant plasmid pZO347 is a derivative of pBluescript carrying a 496 bp BamHI-Smal fragment containing a 426 bp 35S promoter fragment (Hindi fragment) of CaMV strain Cabb-S, linked to a 67 bp fragment of the nontranslated leader region, the so-called Ω-region, of the f tobacco mosaic virus. This results in a chimeric promoter with a complete transcriptional fusion between the promoter of CaMV to the untranslated leader of TMV. By using in vitro mutagenesis the original position of the TMV ATG startcodon is mutated to a Sinai site.
The plasmid pZO008 carries the nopaline synthase (NOS) terminator as a 260 bp Pstl-Hindlll fragment. This PstlHindlll fragment is excised from pZO008 and ligated using T4 ligase into Pstl-Hindlll linearized pZO347. The resulting recombinant plasmid pZU-B is another plant expression vector. The sequence of this 35S-Q promoter as used in the plant expression vector pZU-B is shown as SEQ ID No.23 .The resulting recombinant plasmid pZU-3 contains the 35S HincIITMV Ω fusion (35S-Ω) , unique Smal and Pstl sites and the NOS terminator (Figure 4 ). This expression vector is preferentially used in constructing translational fusions of the gene for expression downstream of the chimaeric promoter 353-Ω.
Example 6; Subcloning of the INSV N protein gene
The INSV N protein coding sequence is obtained by fusion of the cDNA clones plNSV-S60 and plNSV-S39 ( Figure 5) . The cDNA clone pINSV-S60 is subjected to Spel digestion and the fragment containing the 31-end of the INSV N protein gene is
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AP Ο Ο Ο 4 Ο 3 separated electrcphoretically and purified from the gel using a DEAE membrane (NA-45, Schleicher and Schall) and cloned in the largest Spel fragment of pINSV-S39 linearized resulting in the recombinant plasmid pINSV-N. Primers are designed homologous to the translational start and stop codon. Primer INSV-066 d(GCAGATATCATGAACAAAGC) creates an EcoRV site just proximal to the start codon.
Primer INSV-070 d(GCAACCTGCAGCTCAAATCTCTT) creates a Pstl site just distal to the stop codon. These primers are used in standard PCR experiments in which pINSV-N is used as the template. The resulting PCR fragment is isolated from the gel using a DEAE membrane (NA-45, Schleicher and Schall) and cloned in the Smal linearized pBluescript to generate plasmid pINSV-N2 . The added restriction sites, EcoRV and Pstl, facilitate the construction of further plasmids. (Alternatively, one may choose to add the sites in different ways such as but not limited to site-directed mutagenesis or by ligation of other synthetic oligonucleotide linkers. Such methods are all known to a person skilled in the art.)
Example 7 : Subcloning of the INSV non-etructural protein genes (NSs gene) of INSV S RNA
The sequence of the gene corresponding to the non-structural protein NSs is isolated using RNA based PCR on isolated INSV S RNA. Two primers are designed which are homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon , the stop codon primer has a Pstl site just distal thereto . Purified INSV S RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid plNSV-NSs (Figure 6) .
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Example 8: Subcloning of the INSV non-structural protein gene (NSm gene) of the INSV M RNA
The sequence of the gene corresponding to the non-structural protein NSm is isolated using RNA based PCR on isolated INSV M RNA. Two primers are designed which are homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon, the stop codon primer has a Pstl site just distal thereto . Purified INSV S RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid pINSV-NSm (Figure 7).
Example 9: Subcloning of the INSV G1/G2 glycoprotein gene (G1/G2 gene) of the INSV M RNA
The sequence of the gene corresponding to the G1/G2 glycoprotein precursor is isolated using RNA based PCR on isolated INSV M RNA. Two primers are designed homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon, the stop codon primer has a Pstl site just distal thereto . Purified INSV M RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid pINSV-Gl/G2 (Figure 8).
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Example 10 : Construction of plant transformation vectors containing INSV sequences
Example 10A: N protein constructions in pZU-B
In order to make a fusion in which the ATG start codon from the N protein gene is fused directly to the 3'-end of the TMV untranslated leader of the 35S-Q promoter the start codon of the N gene has to be mutated using the PCR approach as hereinbefore described. The N protein gene is excised from the plasmid pINSV-N2 via an EcoRV-Pstl digestion. The fragment is isolated and inserted into the Smal-Pstl linearised pZU-B, resulting in recombinant plasmid pINSV-NB. The chimeric cassette containing the 35S-Q promoter, the N gene and the NOS terminator is excised from the plasmid pINSV-NB via a BamHI/Xbal digestion. The isolated chimaeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NBB. The resulting plasmid pINSV-NBB (Figure 9) is used in plant transformation experiments using methods well known to a person skilled in the art.
Example 10B: NSs protein gene constructions in pZU-B
In order to create a fusion in which the ATG start ccdon from the NSs protein is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the start codon of the NSs gene is mutated, using the PCR approach. The plasmid pINSVNs is digested with EcoRV and Pstl and the NSs containing fragment is isolated from the gel and inserted into Smal/ Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-NSsB. The chimaeric cassette containing the 35S-Q promoter, the mutated NSs protein gene and the NOS terminator is excised from the plasmid pINSV-NSsB via a BamHI/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NSsBB. The
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AP Ο 00 4 Ο 3 resulting plasmid pINSV-NSsBB (Figure 10) is used in plant transformation experiments using methods well known to a person skilled in the art.
Example 10C: Gl/62 glycoprotein gene constructions in pZU-B
In order to create a fusion in which the ATG start codon from the G1/G2 glycoproteinprecursor is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the start codon of the G1/G2 gene is mutated, using the PCR approach. The plasmid pINSV-Gl/G2 is digested with EcoRV and Pstl and the G1/G2 containing fragment is isolated from the gel and inserted into Smal/Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-Gl/G2B. The chimeric cassette containing the 35S-Q promoter, the mutated G1/G2 glycoprotein gene and the NOS terminator is excised from the plasmid pINSV-Gl/G2B via a BamHI/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBlNl9 to create the binary f transformation vector pINSV-Gl/G2BB. The resulting plasmid pINSV-Gl/G2BB (Figure 11) is used in plant transformation experiments using methods well known to a person skilled in the art.
Example 10D: NSm protein gene construct iona in pZU-B
In order to create a fusion in which the ATG start codon from the NSm protein is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the startcodon of the NSm gene is mutated, using the PCR approach. The plasmid plNSVNSm is digested with EcoRV and Pstl and the NSm-containing fragment is isolated from the gel and inserted into Smal. Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-NSmB. The chimeric cassette containing the 35S-Q promoter, the mutated NSm protein gene and the NOS terminator is excised from the plasmid pINSV-NSmB via a
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BamHi/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NSmBB. The resulting plasmid pINSV-NSmBB (Figure 12) is used in plant transformation experiments using methods well known to a person skilled in the art.
Example 10E: 5'- and 3'-termini pan-handle f constructions in pZU-B
A DNA analysis programme is used to. locate the pan-handle element of the loop in the viral INSV S RNA. The strongest pan-handle structure that is detected includes about the first 24-25 nucleotides at the 5‘-end (1 to 24 or 25) of the viral S RNA and about the last 36 nucleotides at the 31-end
of the viral S RNA length of the pan-ha nucleotides long. | (SEQ .ndle | ID Nos 5 element | and 6 of the | respectively).The loop is about 36 |
These regions are | synthesized | on a | commercial | DNA |
v synthesizer and appropriate linker sequences are added.
Construction of the pan-handle vectors of S and M RNA
Z results in respectively: pINSV-termS and pINSV-termM. Using appropriate restriction enzyme combination these fragments are inserted between the 35S-Q promoter and the NOS terminator of pZU-B yielding the chimeric cassettes: pINSVtermSA , pINSV-termMA , pINSV-termSB and pINSV-termMB. These cassettes are then transferred into the binary transformation vector pBINl9 using appropriate enzyme combinations yielding the following plasmids: pINSV-termSAB, plNSV-termMAB, pINSV-termSBB and pINSV-termMBB. Alternatively, it is possible to design “pan-handle constructs including the 3'- and 5'-end termini that are larger than indicated above, or separated by any other DNA sequence in order to enhance the stability of the transcripts produced from these recombinant genes in plants.
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All pan-handle constructs resemble shortened tospovirus RNA molecules , specifically INSV RNA molecules and therefore can be regarded as defective interfering RNAs.
Example_1 OF-·._Construction_containing INSV 5 RNA g_eoondarY_structure region in dzu-b.
A DNA.analysis programme is used to locate a secondary structure in the viral INSV S RNA. The strongest secondary structure detectable starts at nucleotide 1440 and ends at nucleotide 2041 of SEQ ID No.l , (SEQ id No 8).
The DNA fragment carrying the secondary structure region is isolated from plNSV-S61 using a PCR approach similar to that described earlier. The two primers used contain the sequences 1440-1460 and 2021-2041 of SEQ ID No.l. The PCR fragment is excised from an agarose gel and subsequently treated with T4 polymerase to create blunt ends and is subsequently cloned into the Smal site of the expression vector pZU-B, resulting in the recombinant plasmid pINSVHpS3. The plasmid plNSV-HpSB is digested with Hindlll and the fragment containing the chimeric gene is excised from an agarose gel and ligated into Xbal linearized pBIN19, resulting in the transformation vector pINSV-HpSBB.
(It is clear to a person skilled in the art that other fragments can be isolated from the cDNA clones of the INSV S RNA containing the hairpin region as described above without interference to function. Also, a fragment containing the hairpin region may be synthesized using a DNA-synthesizer.)
Example 1 1 ; Transformation of binary vectors to tobacco plant material
Methods to transfer binary vectors to plant material are well established and known to a person skilled in the art.
Variations in procedures exist due to for instance differences in used Agrobacterium strains , different
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AP Ο Ο Ο 4 Ο 3 sources of explant material , differences in regeneration systems depending on as well the cultivar as the plant species used .
The binary plant transformation vectors as described above are used in plant transformation experiments according to the following procedures. The constructed binary vector is transferred by tri-parental mating to an acceptor Agrobacterium tumefaciens strain, followed by southern analysis of the ex-conjugants for verification of proper transfer of the construct to the acceptor strain, inoculation and cocultivation of axenic explant material with the Agrobacterium tumefaciens strain of choice, selective killing of the Agrobacterium tumefaciens strain used with appropriate antibiotics, selection of transformed cells by growing on selective media containing kanamycine, transfer of tissue to shoot - inducing media, transfer of selected shoots to root inducing media, transfer of plantlets to soil, assaying for intactness of the construct by southern analyses of isolated total DNA from the transgenic plant, assaying for proper function of the inserted chimeric gene by northern analysis and/or enzyme assays and western blot analysis of proteins.
Example 12 ; Expression of INSV 3 RNA sequences in tobacco plant cells
RNA is extracted from leaves of regenerated plants using the following protocol. Grind 200 mg leaf material to a fine powder in liquid nitrogen. Add 800 μΐ RNA extraction buffer (100 mM Tris-HCl (pH 8,0), 500 mM NaCl, 2 mM EDTA, 200 mM SMercapto-ethanol, 0,4% SDS) and extract the homogenate with phenol, collect the nucleic acids by alcohol precipitation. Resuspend the nucleic acids in 0,5 ml 10 mM Tris-HCl (pH 8,0), 1 mM EDTA, add LiCl to a final concentration of 2 M, leave on ice for maximal 4 hours and collect the RNA by centrifugation. Resuspend in 400 μΐ 10 mM Tris-HCl (pH 8,0),_ bad ORIGINAL
APO 00 4 Ο 3 mM EDTA and precipitate with alcohol, finally resuspend in 50 μΐ 10 mM Tris-HCl (pH 8,0), 1 mM EDTA. RNAs are separated on glyoxal/agarose gels and blotted to Genescreen as described by van Grinsven et al. [(1986) Theor Appl Gen 73:94-101]. INSV S RNA sequences are detected using DNA or RNA probes labeled with [32P] , [35S] or by using nonradioactive labeling techniques. Based on northern analysis, it is determined to what extent the regenerated plants express chimaeric INSV S RNA sequences.
Plants transformed with chimaeric constructs containing an INSV N protein-encoding sequence are also subjected to western blot analysis. Proteins are extracted from leaves of transformed plants by grinding in sample buffer according to the method of Laemmli [(1970) Nature 244: 29-30] . A 50 gg portion of protein is subjected to electrophoresis in a 12,5 % SDS-polyacrylamide gel essentially as described by Laemmli (1970) supra . Separated proteins are transferred to nitrocellulose electrop’noretically as described by Towbin et al. [(1979) Proc. Natl. Acad. Sci. USA 76:4350-4354]. Transferred proteins are reacted with antiserum raised against purified INSV structural or non-structural proteins (Towbin et al.(1979) supra. Based on the results of the western analysis, it is determined that transformed plants do contain INSV N proteins encoded by the inserted chimaeric
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Example 13: Resistance of plants against INSV infection
Transformed plants are grown in the greenhouse under standard quarantine conditions in order to prevent any infections by pathogens. The transformants are selfpollinated and the seeds harvested. Progeny plants are analyzed for segregation of the inserted gene and subsequently infected with INSV by mechanical inoculation. Tissue from plants systemically infected with INSV is ground in 5 volumes of ice-cold inoculation buffer (10 mM phosphate buffer supplemented with 1% Na2SO3) and rubbed in the presence of carborundum powder on the first two fully extended leafs of approximately 5 weeks old seedlings. Inoculated plants are monitored for symptom development during 3 weeks after inoculation.
Plants containing INSV Related DNA Sequences show reduced susceptibility to INSV infection as exemplified by a delay in symptom development, whereas untransformed control plants show severe systemic INSV symptoms within 7 days after inoculat ion.
Example 14: Use of synthetic oligonucleotides for diagnostic purposes
RNA is extracted from leaves of suspected plants using the following protocol: grind 1 gram of leaf material, preferentially showing disease symptoms, in 3 ml 100 mM Tris-HCl, 50 mM EDTA, 1.5 M NaCl and 2% CTAB (pH 8.0). After grinding, 1 ml of the homogenate is subjected to chloroform extraction and incubated at 65 °C for 10 minutes. The inorganic phase is then collected and extracted with phenol/chloroform (1:1), followed by a last extraction with chloroform. The ribonucleic acids are isolated from the inorganic phase, containing the total nucleic acids, by adding LiCl to a final concentration of 2 M. The preparation is incubated at 4°C for 1 hour, after which the ribonucleic
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APO 00 4 0 3 acids are collected by centrifugation. The ribonucleic acid pellet is resuspended in 25 μΐ 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). The ribonucleic acids are recovered by standard alcohol precipitation. The ribonucleic acid pellet is resuspended in 25 μΐ 10 mM Tris-HCl, 1 mM EDTA (pH 8.0).
μΐ of the purified ribonucleic acids is spotted on a nylon blotting membrane (e.g. Hybond-N, Amersham UK). The presence of INSV in the plant is detected by standard hybridization, using any part or parts of the sequence isolated from virions or preferentially by designing synthetic oligomers on the basis of disclosed sequence information as a probe. (Alternatively, in vitro transcripts of regions of the INSV genome are used to detect INSV Related RNA Sequences in diseased plants.) A diseased plant is diagnosed by the occurrence of hybridization at the dot containing RNA material from the diseased plant.
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SEQUENCE LISTING
SEQ. ID No. 1
Sequence type: Nucleotide Sequence length: 3017 nucleotides Strandedness: single stranded
Molecule type: Viral S RNA nucleotide sequence from position 1 to position 3017
Original Source Organism: | Impat iens | Necrotic Spot Virus | ||||
AGAGCAATNN | NNNNNNNNNN | NNNNGAACAA | CCAAGCTACA | ACAAATCTTA | CAATATTGTC | 60 |
AATTACATTA | CTACTTCCAT | TTTAACATGT | CTAGTGCAAT | GTATGAAACA | ATTATCAAAT | 120 |
CGAAGTCCTC | AATCTGGGGA | ACAACATCTT | CGGGTAAAGC | AGTAGTAGAT | AGTTATTGGA | 180 |
TTCATGATCA | ATCTTCCGGA | AAGAAGTTGG | TCGAAGCTCA | ACTCTATTCT | GACTCCAGGA | 240 |
GCAAGACCAG | TTTCTGTTAC | ACTGGTAAAG | TTGGCTTTCT | CCCAACAGAA | GAAAAAGAAA | 300 |
TTATAGTGAG | ATGTTTTGTG | CCTATTTTTG | ATGACATTGA | TCTGAATTTC | TCCTTTTCAG | 360 |
GGAATGTTGT | CGAAATTCTG | GTCAGATCTA | ACACAACAAA | CACAAACGGT | GTTAAACATC | 420 |
AAGGTCATCT | CAAAGTGTTA | TCCTCTCAGT | TGCTCAGAAT | GCTTGAAGAG | CAAATAGCAG | 480 |
TGCCTGAAAT | TACTTCAAGA | TTCGGTCTGA | AAGAATCTGA | CATCTTCCCT | CCAAATAATT | 540 |
TCATTGAAGC | TGCAAATAAA | GGATCATTGT | CTTGTGTCAA | AGAAGTCCTT | TTTGATGTCA | 600 |
AGTATTCAAA | CAACCAATCC | ATGGGCAAAG | TCAGTGTTCT | TTCTCCTACC | AGAAGTGTTC | 660 |
ATGAATGGCT | GTACACACTT | AAGCCTGTTT | TTAACCAATC | CCAGACCAAC | AACAGGACAG | 720 |
TAAACACTTT | GGCTGTAAAA | TCACTGGCAA | TGTCTGCAAC | TTCTGATTTA | ATGTCAGATA | 780 |
CTCATTCGTT | TGTCAGGCTC | AATAATAACA | AGCCTTTTAA | AATCAGCCTT | TGGATGCGCA | 340 |
TCCCTAAAAT | AATGAAATCA | AACACATACA | GCCGGTTCTT | CACCCTGTCT | GATGAATCTT | 900 |
CTCCTAAAGA | GTATTATATA | AGCATTCAAT | GTCTTCCGAA | TCACAACAAT | GTTGAAACAG | 960 |
TCATTGAATA | TAACTTTGAT | CAGTCAAACC | TCTTCTTGAA | TCAACTCCTT | CTAGCAGTGA | 1020 |
TTCATAAAAT | TGAGATGAAT | TTTTCTGATC | TAAAAGAACC | TTACAATGTT | ATCCATGATA | 1CS0 |
TGTCGTATCC | TCAAAGAATT | gttcattcac | TTCTTGAAAT | CCACACAGAA | CTTGCTCAAA | 1140 |
CTGTCTGTGA | CAGTGTTCAG | CAAGACATGA | TTGTCTTCAC | TATAAATGAG | CCAGATCTAA | 1200 |
AGCCAAAAAA | GTTTGAGCTA | GGGAAAAAGA | CTTTAAATTA | TTCAGAAGAT | GGTTATGGGA | 1260 |
GAAAATATTT | CCTTTCTCAG | ACCTTGAAAA | GTCTTCCGAG | AAACTCACAA | ACAATGTCTT | 1320 |
ATTTGGATAG | CATCCAGATG | CCCGATTGGA | AATTTGACTA | TGCTGCAGGT | GAAATAAAAA | 1380 |
BAD ORIGINAL L
AP Ο Ο Ο 4 Ο 3
TTTCTCCTAG | ATCAGAGGAT | GTTTTGAAAG | CTATTTCTAA | ATTAGATTTA | AATTAACCTT | 1440 |
GGTTAAACTT | GTCCCTAAGT | AAAGTTTGTT | TACATGCATT | TAGATCAGAT | TAAACAAATC | 1500 |
TAATAACAGA | TAAACCAAAA | ACAATCATAT | GAAATAAATA | AATAAACATA | AAATATATAA | 1560 |
AAAATACAAA | AAAAATCATA | AAATAAATAA | AAACCAAAAA | AGGATGGCCT | TCGGGCACAA | 1620 |
TTTGGTTGCT | TTAATAATGC | TTTAAAATGA | ATGTATTAGT | AAATTATAAA | CTTTAAATCC | 1680 |
AATCTACTCA | CAAATTGGCC | AAAAATTTGT | ATTTGTTTTT | GTTTTTGTTT | TTTGTTTTTT | 1740 |
GTTTTTGTTT | TGTTTTATTT | GTTTTTTATT | TTGTTTTTTG | TTTTTTGTTT | TTTATTTTAT | 1800 |
TTATATATAT | ATATATATAT | ATTTTGTAGT | ggtttttatt | gtttttatta | TTTTTTGTAG | 1860 |
CTTTTTTACT | TGTTTATTTC | ACACGCAAAC | ACACTTTCAA | GTTTATATAT | TAAAACACAC | 1920 * |
ATTAAACTTA | TTTCAAATAA | TTTATAAAAG | CACACTTAAT | ACACTCAAAC | AATAATTAAT | 1980 |
TATTTTATTT | TTTATTTTAT | TTTTTATTTT | TATTATTTTT | ATTTTTATTT | ATTTAAATGC | 2040 |
ATTTAACACA | ACACAAAGCA | AACCAAGCTC | AAATCTCTTT | TAAATAGAAT | catttttccc | 2100 |
AAAATCAATA | GTAGCATTAA | ACATGCTGTA | AATGGATGTA | AGCCCTTCTT | TGTAGTGGTC | 2160 |
CATTGCAGCA | AGTCCTTTAG | CTTTCGGACT | ACAAGCCTTT | AGTATATCTG | CATATTGTTT | 2220 |
AGCCTTGCCA | ATTTCAACAG | AGTTCATGCT | ATATCCTTTG | CTTTTTAGAA | CTGTGCACAC | 2230 |
TTTCCCAACT | gcctctttag | TGCTAAACTT | AGACATGTCA | ATTCCAAGCT | CAACATGTTT | 2340 |
AGCATCTTGA | TAAATAGCCG | GAACTAGTGC | AGCTATTTCA | AAATTCAGTA | CAGATGCTAT | 2400 |
CAGAGGAAGA | CTTCCTCCTA | AGAGAACACC | CAAGACACAG | GATTTCAAAT | CTGTGGTTGC | 2460 |
AAGACCATAT | GAGGCAATCA | GAGGGTGACT | TGGAAGGCTA | TTTATAGCTT | CAGTCAGAGC | 2520 |
AGATCCATTG | TCCTTTATCA | TTCCAACAAG | ATGAACTCTC | ACCATTGCAT | CAAGTCTTCG | 2530 |
GAAAGTCATA | TCATTGACCC | CAACTCTTTC | TGAATTGTTT | CTAGTTTTCT | TAATTGTGAC | 2640 |
TGATCCAAAA | GTGAAGTCAG | CACTCTTAAT | GACTCTCATT | ATAGATTGCC | TATTCTTGAG | 2700 |
GAAGGATAGG | CAGGATGCAG | TAGTCATGTT | CTGAATCTTT | TCACGGTTGT | TGGTAAAGAA | 2760 |
GTCAGTGAAA | TTGAAAGACC | CTTCATTTTG | AGTTTCCTCA | AATTCTAAGG | AATCAGATTG | 2820 |
AGTCAAAAGC | TTGACTATGT | TCTCCTTGGT | AATCTTTGCT | TTGTTCATCT | TGATCTGCTG | 2880 |
ACTTTACTAA | CTTTAAAGCT | TAAAGTGTTC | AAATTACTAA | ATAGTACTTG | CGGTTAAAGT | 2940 |
AGTATTTGGT | AAAATTTGTA | ATTTTTCAGT | TTCTAGCTTT | GGATTATATG | ATGTTATATT | 3000 |
CGTGACACAA | TTGCTCT | 3 017 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
SEQ ID NO: 2
Sequence type: nucleotides
Sequence length: 2993 nucleotides
Strandedness: single stranded
Molecule type: Viral S RNA nucleotide sequence from position 25 to position 3017 of SEQ ID NO.l
Original Source Organism: Impatiens Necrotic Spot Virus
GAACAACCAA | GCTACAACAA | ATCTTACAAT | ATTGTCAATT | ACATTACTAC | TTCCATTTTA | 60 |
ACATGTCTAG | TGCAATGTAT | GAAACAATTA | TCAAATCGAA | GTCCTCAATC | TGGGGAACAA | 120 |
CATCTTCGGG | TAAAGCAGTA | GTAGATAGTT | ATTGGATTCA | TGATCAATCT | TCCGGAAAGA | 180 |
AGTTGGTCGA | AGCTCAACTC | TATTCTGACT | CCAGGAGCAA | GACCAGTTTC | TGTTACACTG | 240 |
GTAAAGTTGG | CTTTCTCCCA | ACAGAAGAAA | AAGAAATfAT | AGTGAGATGT | TTTGTGCCTA | 300 |
TTTTTGATGA | CATTGATCTG | AATTTCTCCT | TTTCAGGGAA | TGTTGTCGAA | ATTCTGGTCA | 360 |
GATCTAACAC | AACAAACACA | AACGGTGTTA | AACATCAAGG | TCATCTCAAA | GTGTTATCCT | 420 |
CTGAGTTGCT | CAGAATGCTT | GAAGAGCAAA | TAGCAGTGCC | TGAAATTACT | TCAAGATTCG | 480 |
GTCTGAAAGA | ATCTGACATC | TTCCCTCCAA | ATAATTTCAT | TGAAGCTGCA | AATAAAGGAT | 540 |
CATTGTCTTG | TGTCAAAGAA | GTCCTTTTTG | ATGTCAAGTA | TTCAAACAAC | CAATCCATGG | 600 |
GCAAAGTCAG | TGTTCTTTCT | CCTACCAGAA | GTGTTCATGA | ATGGCTGTAC | ACACTTAAGC | 660 |
ctgtttttaa | CCAATCCCAG | ACCAACAACA | GGACAGTAAA | CACTTTGGCT | GTAAAATCAC | 720 |
TGGCAATGTC | TGCAACTTCT | GATTTAATGT | CAGATACTCA | TTCGTTTGTC | AGGCTCAATA | 780 |
ATAACAAGCC | TTTTAAAATC | AGCCTTTGGA | TGCGCATCCC | TAAAATAATG | AAATCAAACA | 840 |
CATACAGCCG | GTTCTTCACC | CTGTCTGATG | AATCTTCTCC | TAAAGAGTAT | TATATAAGCA | 900 |
TTCAATGTCT | TCCGAATCAC | AACAATGTTG | AAACAGTCAT | TGAATATAAC | TTTGATCAGT | 960 |
CAAACCTCTT | CTTGAATCAA | CTCCTTCTAG | CAGTGATTCA | TAAAATTGAG | ATGAATTTTT | 1020 |
CTGATCTAAA | AGAACCTTAC | AATGTTATCC | ATGATATGTC | GTATCCTCAA | AGAATTGTTC | 1080 |
ATTCACTTCT | TGAAATCCAC | ACAGAACTTG | CTCAAACTGT | CTGTGACAGT | GTTCAGCAAG | 1140 |
ACATGATTGT | CTTCACTATA | AATGAGCCAG | ATCTAAAGCC | AAAAAAGTTT | GAGCTAGGGA | 1200 |
AAAAGACTTT | AAATTATTCA | GAAGATGGTT | ATGGGAGAAA | ATATTTCCTT | TCTCAGACCT | 1260 |
TGAAAAGTCT | TCCGAGAAAC | TCACAAACAA | TGTCTTATTT | GGATAGCATC | CAGATGCCCG | 1320 |
ATTGGAAATT | TGACTATGCT | GCAGGTGAAA | TAAAAATTTC | TCCTAGATCA | GAGGATGTTT | 1380 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
TGAAAGCTAT TTCTAAATTA GATTTAAATT AACCTTGGTT AAACTTGTCC CTAAGTAAAG 1440
TTTGTTTACA TGCATTTAGA TCAGATTAAA CAAATCTAAT AACAGATAAA CCAAAAACAA 1500
TCATATGAAA TAAATAAATA AACATAAAAT ATATAAAAAA TACAAAAAAA ATCATAAAAT 1560
AAATAAAAAC CAAAAAAGGA TGGCCTTCGG GCACAATTTG GTTGCTTTAA TAATGCTTTA 1620
AAATGAATGT ATTAGTAAAT TATAAACTTT AAATCCAATC TACTCACAAA TTGGCCAAAA 1680
ATTTGTATTT GTTTTTGTTT TTGTTTTTTG TTTTTTGTTT TTGTTTTGTT TTATTTGTTT 1740
TTTATTTTGT TTTTTGTTTT TTGTTTTTTA ΤΤΤΤΑΤΤΤΑΤ ΑΤΑΤΑΤΑΤΑΤ ΑΤΑΤΑΤΑΤΤΤ 1800
TGTAGTGGTT TTTATTGTTT ΤΤΑΤΤΑΤΤΤΤ TTGTAGCTTT TTTACTTGTT TATTTCACAC 1860 ί'’', ' GCAAACACAC TTTCAAGTTT ATATATTAAA ACACACATTA AACTTATTTC ΑΑΑΤΑΑΤΤΤΑ 1920 ( TAAAAGCACA CTTAATACAC TCAAACAATA ΑΤΤΑΑΤΤΑΤΤ ΤΤΑΤΤΤΤΤΤΑ ΤΤΤΤΑΤΤΤΤΤ 1980
ΤΑΤΤΤΤΤΑΤΤ ΑΤΤΤΤΤΑΤΤΤ ΤΤΑΤΤΤΑΤΤΤ AAATGCATTT AACACAACAC AAAGCAAACC 2040
AAGCTCAAAT CTCTTTTAAA TAGAATCATT TTTCCCAAAA TCAATAGTAG CATTAAACAT 2100
GCTGTAAATG GATGTAAGCC CTTCTTTGTA GTGGTCCATT GCAGCAAGTC CTTTAGCTTT 2160
CGGACTACAA GCCTTTAGTA TATCTGCATA TTGTTTAGCC TTGCCAATTT CAACAGAGTT 2220
CATGCTATAT CCTTTGCTTT TTAGAACTGT GCACACTTTC CCAACTGCCT CTTTAGTGCT 2280
AAACTTAGAC ATGTCAATTC CAAGCTCAAC ATGTTTAGCA TCTTGATAAA TAGCCGGAAC 2340
TAGTGCAGCT ATTTCAAAAT TCAGTACAGA TGCTATCAGA GGAAGACTTC CTCCTAAGAG 2400
AACACCCAAG ACACAGGATT TCAAATCTGT GGTTGCAAGA CCATATGAGG CAATCAGAGG 2460 θ GTGACTTGGA AGGCTATTTA TAGCTTCAGT CAGAGCAGAT CCATTGTCCT TTATCATTCC 2520 ζ- . AACAAGATGA ACTCTCACCA TTGCATCAAG TCTTCGGAAA GTCATATCAT TGACCCCAAC 2580
TCTTTCTGAA TTGTTTCTAG TTTTCTTAAT TGTGACTGAT CCAAAAGTGA AGTCAGCACT 2640
CTTAATGACT CTCATTATAG ATTGCCTATT CTTGAGGAAG GATAGGCAGG ATGCAGTAGT 2700
CATGTTCTGA ATCTTTTCAC GGTTGTTGGT AAAGAAGTCA GTGAAATTGA AAGACCCTTC 2760
ATTTTGAGTT TCCTCAAATT CTAAGGAATC AGATTGAGTC AAAAGCTTGA CTATGTTCTC 2320
CTTGGTAATC TTTGCTTTGT TCATCTTGAT CTGCTGACTT TACTAACTTT AAAGCTTAAA 2830
GTGTTCAAAT TACTAAATAG TACTTGCGGT TAAAGTAGTA TTTGGTAAAA TTTGTAATTT 2940
AP Ο Ο Ο 4 Ο 3
SZQ ID NO. 3
Sequence type: Nucleotide
Sequence length: 1350 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA nucleotide sequence coding for the non-structural (NSs) protein from position 87 to position 1435 of SEQ ID NO :1
Original source organism: Impatiens Necrotic Spot Virus
ATGTCTAGTG | CAATGTATGA | AACAATTATC | AAATCGAAGT | CCTCAATCTG | GGGAACAACA | 60 |
TCTTCGGGTA | AAGCAGTAGT | AGATAGTTAT | TGGATTCATG | ATCAATCTTC | CGGAAAGAAG | 120 |
TTGGTCGAAG | CTCAACTCTA | TTCTGACTCC | AGGAGCAAGA | CCAGTTTCTG | TTACACTGGT | 180 |
AAAGTTGGCT | TTCTCCCAAC | AGAAGAAAAA | GAAATTATAG | TGAGATGTTT | TGTGCCTATT | 240 |
TTTGATGACA | TTGATCTGAA | TTTCTCCTTT | TCAGGGAATG | TTGTCGAAAT | TCTGGTCAGA | 300 |
TCTAACACAA | CAAACACAAA | CGGTGTTAAA | CATCAAGGTC | ATCTCAAAGT | GTTATCCTCT | 350 |
CAGTTGCTCA | GAATGCTTGA | AGAGCAAATA | GCAGTGCCTG | AAATTACTTC | AAGATTCGGT | 420 |
CTGAAAGAAT | CTGACATCTT | CCCTCCAAAT | AATTTCATTG | AAGCTGCAAA | TAAAGGATCA | 480 |
TTGTCTTGTG | TCAAAGAAGT | CCTTTTTGAT | GTCAAGTATT | CAAACAACCA | ATCCATGGGC | 540 |
AAAGTCAGTG | TTCTTTCTCC | TACCAGAAGT | GTTCATGAAT | GGCTGTACAC | ACTTAAGCCT | 500 |
GTTTTTAACC | AATCCCAGAC | CAACAACAGG | ACAGTAAACA | CTTTGGCTGT | AAAATCACTG | 550 |
GCAATGTCTG | CAACTTCTGA | TTTAATGTCA | GATACTCATT | CGTTTGTCAG | GCTCAATAAT | 720 |
AAC zirtGC C TT | TTAAAATCAG | CCTTTGGATG | CGCATCCCTA | AAATAATGAA | ATCAAACACA | 730 |
TACAGCCGGT | TCTTCACCCT | GTCTGATGAA | TCTTCTCCTA | AAGAGTATTA | TATAAGCATT | 840 |
CAATGTCTTC | CGAATCACAA | CAATGTTGAA | ACAGTCATTG | AATATAACTT | TGATCAGTCA | 900 |
aacctcttct | TGAATCAACT | CCTTCTAGCA | GTGATTCATA | AAATTGAGAT | GAATTTTTCT | 950 |
GATCTAAAAG | AACCTTACAA | TGTTATCCAT | GATATGTCGT | ATCCTCAAAG | AATTGTTCAT | 1020 |
TCACTTCTTG | AAATCCACAC | AGAACTTGCT | CAAACTGTCT | GTGACAGTGT | TCAGCAAGAC | 1080 |
ATGATTGTCT | TCACTATAAA | TGAGCCAGAT | CTAAAGCCAA | AAAAGTTTGA | GCTAGGGAAA | 1140 |
AAGACTTTAA | ATTATTCAGA | AGATGGTTAT | GGGAGAAAAT | ATTTCCTTTC | TCAGACCTTG | 1200 |
AAAAGTCTTC | CGAGAAACTC | ACAAACAATG | TCTTATTTGG | ATAGCATCCA | GATGCCCGAT | 1260 |
TGGAAATTTG | ACTATGCTGC | AGGTGAAATA | AAAATTTCTC | CTAGATCAGA | GGATGTTTTG | 1320 |
AAAGCTATTT | CTAAATTAGA | TTTAAATTAA | 1350 |
bad original
AP Ο Ο Ο 4 Ο 3
SEQ ID NO: 4
Sequence type: Nucleotide
Sequence length: 789 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA nucleotide sequence from 2080 to 2868 of SEQ ID NO: 1
Original Source Organism: Impatiens Necrotic Spot Virus < TTAAATAGAA TCATTTTTCC CAAAATCAAT AGTAGCATTA AACATGCTGT AAATGGATGT 60
AAGCCCTTCT TTGTAGTGGT CCATTGCAGC AAGTCCTTTA GCTTTCGGAC TACAAGCCTT 120
TAGTATATCT GCATATTGTT TAGCCTTGCC AATTTCAACA GAGTTCATGC TATATCCTTT 180
GCTTTTTAGA ACTGTGCACA CTTTCCCAAC TGCCTCTTTA GTGCTAAACT TAGACATGTC 240
AATTCCAAGC TCAACATGTT TAGCATCTTG ATAAATAGCC GGAACTAGTG CAGCTATTTC 300
AAAATTCAGT ACAGATGCTA TCAGAGGAAG ACTTCCTCCT AAGAGAACAC CCAAGACACA 360
GGATTTCAAA TCTGTGGTTG CAAGACCATA TGAGGCAATC AGAGGGTGAC TTGGAAGGCT 420
ATTTATAGCT TCAGTCAGAG CAGATCCATT GTCCTTTATC ATTCCAACAA GATGAACTCT 480
CACCATTGCA TCAAGTCTTC GGAAAGTCAT ATCATTGACC CCAACTCTTT CTGAATTGTT 540 θ TCTAGTTTTC TTAATTGTGA CTGATCCAAA AGTGAAGTCA GCACTCTTAA TGACTCTCAT 600
TATAGATTGC CTATTCTTGA GGAAGGATAG GCAGGATGCA GTAGTCATGT TCTGAATCTT 660
TTCACGGTTG TTGGTAAAGA AGTCAGTGAA ATTGAAAGAC CCTTCATTTT GAGTTTCCTC 720
AAATTCTAAG GAATCAGATT GAGTCAAAAG CTTGACTATG TTCTCCTTGG TAATCTTTGC 780
APO00403
SEQ ID KO i 5
Sequence Type: Nucleotide
Sequence length: 36 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA nucleotide sequence from the 5' end of the viral S RNA sequence
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATNN NNNNNNNNNN NNNNGAACAAC CCAAGC
SEQ ID NO :6
Sequence Type: Nucleotide
Sequence length: 36 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA nucleotide sequence from the 3' end of the viral S RNA sequence
Original Source Organism: Impatiens Necrotic Spot Virus
GATTATATGA TGTTATATTC GTGACACAAT TGCTCT
SEQ ID Ho. 7
Sequence type: Nucleotide
Sequence length:643 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA
Original Source Organism: Impatiens Necrotic Spot Virus
CCTTGGTTAA | acttgtccct | AAGTAAAGTT | tgtttacatg | CATTTAGATC | AGATTAAACA |
AATCTAATAA | CAGATAAACC | AAAAACAATC | atatgaaata | AATAAATAAA | CATAAAATAT |
ATAAAAAATA | CAAAAAAAAT | CATAAAATAA | ataaaaacca | AAAAAGGATG | GCCTTCGGGC |
ACAATTTGGT | TGCTTTAATA | ATGCTTTAAA | ATGAATGTAT | TAGTAAATTA | TAAACT7TAA |
ATCCAATCTA | CTCACAAATT | GGCCAAAAAT | TTGTATTTGT | TTTTGTTTTT | GTTTTTTGTT |
T-tttgtTTTT | GTTTTGTTTT | atttgttttt | TATTTTGTTT | TTTGTTTTTT | GTTTTTTATT |
BAD ORIGINAL
AP Ο Ο Ο 4 0 j
ΤΤΑΤΤΤΑΤΑΤ ATATΑΤΑΤΑΤ ATATATTTTG TAGTGGTTTT TATTGTTTTT ΑΤΤΑΤΤΤΤΤΤ 420
GTAGCTTTTT TACTTGTTTA TTTCACACGC AAACACACTT TCAAGTTTAT ATATTAAAAC 480
ACACATTAAA CTTATTTCAA ATAATTTATA AAAGCACACT TAATACACTC AAACAATAAT 540
ΤΑΑΤΤΑΤΤΤΤ ΑΤΤΊΤΊΤΑΤΤ ΤΤΑΤΤΤΤΤΤΑ ΊΤΤΤΤΑΤΤΑΤ ΤΤΤΤΑΤΤΤΤΤ ΑΤΤΤΑΤΤΤΑΑ 600
ATGCATTTAA CACAACACAA AGCAAACCAA GCTCAAATCT CTT 643
SSQ ID No. 9
Sequence type: Nucleotide
Sequence length: 602 nucleotides
Strandedness: Single stranded
Molecule type: Viral S RNA from 1440 to 2041 of SEQ ID No.l Original Source Organism: Impatiens Necrotic Spot Virus
TGGTTAAACT | TGTCCCTAAG | |
CTAATAACAG | ATAAACCAAA | |
AAAAATACAA | AAAAAATCAT | |
ATTTGGTTGC | TTTAATAATG | |
CAATCTACTC | ACAAATTGGC | |
C- | TGTTTTTGTT | TTGTTTTATT |
( | TTTATATATA | TATATATATA |
V | GCTTTTTTAC | TTGTTTATTT |
CATTAAACTT | ATTTCAAATA | |
TTATTTTATT | TTTTATTTTA | |
CA |
TAAAGTTTGT | TTACATGCAT | TTAGATCAGA | TTAAACAAAT | 60 |
AACAATCATA | TGAAATAAAT | AAATAAACAT | AAAATATATA | 120 |
AAAATAAATA | AAAACCAAAA | AAGGATGGCC | TTCGGGCACA | 180 |
CTTTAAAATG | AATGTATTAG | TAAATTATAA | ACTTTAAATC | 240 |
CAAAAATTTG | TATTTGTTTT | TGTTTTTGTT | TTTTGTTTTT | 300 |
TGTTTTTTAT | TTTGTTTTTT | gttttttgtt | TTTTATTTTA | 360 |
TATTTTGTAG | TGGTTTTTAT | tgtttttatt | ATTTTTTGTA | 420 |
CACACGCAAA | CACACTTTCA | agtttatata | TTAAAACACA | 480 |
ATTTATAAAA | GCACACTTAA | TACACTCAAA | CAATAATTAA | 540 |
TTTTTTATTT | TTATTATTTT | ΤΑΤΤΤΤΤΑΤΤ | TATTTAAATG | 600 |
602
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
SEQ ID HO: 9
Sequence type: Nucleotide
Sequence length: 3017 nucleotides
Strandedness: single stranded
Molecule type: Complementary viral S RNA nucleotide sequence from 1 to about 3017
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATTG | TGTCACGAAT | ATAACATCAT | ATAATCCAAA | GCTAGAAACT | GAAAAATTAC | 60 |
AAATTTTACC | AAATACTACT | TTAACCGCAA | GTACTATTTA | GTAATTTGAA | CACTTTAAGC | 120 |
TTTAAAGTTA | GTAAAGTCAG | CAGATCAAGA | TGAACAAAGC | AAAGATTACC | AAGGAGAACA | 180 |
TAGTCAAGCT | TTTGACTCAA | TCTGATTCCT | TAGAATTTGA | GGAAACTCAA | AATGAAGGGT | 240 |
CTTTCAATTT | CACTGACTTC | TTTACCAACA | ACCGTGAAAA | GATTCAGAAC | ATGACTACTG | 300 |
CATCCTGCCT | ATCCTTCCTC | AAGAATAGGC | AATCTATAAT | GAGAGTCATT | AAGAGTGCTG | 360 |
ACTTCACTTT | TGGATCAGTC | ACAATTAAGA | AAACTAGAAA | CAATTCAGAA | AGAGTTGGGG | 420 |
TCAATGATAT | GACTTTCCGA | AGACTTGATG | CAATGGTGAG | AGTTCATCTT | GTTGGAATGA | 430 |
TAAAGGACAA | TGGATCTGCT | CTGACTGAAG | CTATAAATAG | CCTTCCAAGT | CACCCTCTGA | 540 |
TTGCCTCATA | TGGTCTTGCA | ACCACAGATT | TGAAATCCTG | TGTCTTGGGT | GTTCTCTTAG | 600 |
GAGGAAGTCT | TCCTCTGATA | GCATCTGTAC | tgaattttga | AATAGCTGCA | CTAGTTCCGG | 660 |
CTATTTATCA | AGATGCTAAA | CATGTTGAGC | TTGGAATTGA | CATGTCTAAG | TTTAGCACTA | 720 |
AAGAGGCAGT | TGGGAAAGTG | TGCACAGTTC | TAAAAAGCAA | AGGATATAGC | ATGAACTCTG | 730 |
TTGAAATTGG | CAAGGCTAAA | CAATATGCAG | ATATACTAAA | GGCTTGTAGT | CCGAAAGCTA | 840 |
AAGGACTTGC | TGCAATGGAC | CACTACAAAG | AAGGGCTTAC | ATCCATTTAC | AGCATGTTTA | 9C0 |
ATGCTACTAT | TGATTTTGGG | AAAAATGATT | CTATTTAAAA | GAGATTTGAG | CTTGGTTTGC | 960 |
TTTGTGTTGT | GTTAAATGCA | TTTAAATAAA | TAAAAATAAA | AATAATAAAA | ATAAAAAATA | 1020 |
AAATAAAAAA | TAAAATAATT | AATTATTGTT | TGAGTGTATT | AAGTGTGCTT | TTATAAATTA | 1080 |
TTTGAAATAA | GTTTAATGTG | TGTTTTAATA | TATAAACTTG | AAAGTGTGTT | TGCGTGTGAA | 1140 |
ATAAACAAGT | AAAAAAGCTA | CAAAAAATAA | TAAAAACAAT | AAAAACCACT | ACAAAATATA | 1200 |
TATATATATA | TATATAAATA | AAATAAAAAA | CAAAAAACAA | AAAACAAAAT | AAAAAACAAA | 1260 |
TAAAACAAAA | CAAAAACAAA | AAACAAAAAA | CAAAAACAAA | AACAAATACA | AATTTTTGGC | 1320 |
CAATTTGTGA | GTAGATTGGA | TTTAAAGTTT | ATAATTTACT | AATACATTCT | TTTAAAGCAT | 1330 |
TATTAAAGCA | ACCAAATTGT | GCCCGAAGGC | CATCCTTTTT | TGGTTTTTAT | TTATTTTATG | 1440 |
attttttttg | TATTTTTTAT | ATATTTTATG | TTTATTTATT | TATTTCATAT | GATTGTTTTT | 1500 |
GGTTTATCTG | TTATTAGATT | TGTTTAATCT | GATCTAAATG | CATGTAAACA | AACTTTACTT | 1560 |
AGGGACAAGT | TTAACCAAGG | TTAATTTAAA | TCTAATTTAG | AAATAGCTTT | CAAAACATCC | 1620 |
TCTGATCTAG | GAGAAATTTT | TATTTCACCT | GCAGCATAGT | CAAATTTCCA | ATCGGGCATC | 1630 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
TGGATGCTAT | CCAAATAAGA | CATTGTTTGT | GAGTTTCTCG | GAAGACTTTT | CAAGGTCTGA | 1740 |
GAAAGGAAAT | ATTTTCTCCC | ATAACCATCT | TCTGAATAAT | TTAAAGTCTT | TTTCCCTAGC | 1800 |
TCAAACTTTT | TTGGCTTTAG | ATCTGGCTCA | TTTATAGTGA | AGACAATCAT | GTCTTGCTGA | 1860 |
ACACTGTCAC | AGACAGTTTG | AGCAAGTTCT | GTGTGGATTT | CAAGAAGTGA | ATGAACAATT | 1920 |
CTTTGAGGAT | ACGACATATC | ATGGATAACA | TTGTAAGGTT | CTTTTAGATC | AGAAAAATTC | 1980 |
ATCTCAATTT | TATGAATCAC | TGCTAGAAGG | AGTTGATTCA | AGAAGAGGTT | TGACTGATCA | 2040 |
AAGTTATATT | CAATGACTGT | TTCAACATTG | TTGTGATTCG | GAAGACATTG | AATGCTTATA | 2100 |
TAATACTCTT | TAGGAGAAGA | TTCATCAGAC | AGGGTGAAGA | ACCGGCTGTA | TGTGTTTGAT | 2160 |
TTCATTATTT | TAGGGATGCG | CATCCAAAGG | CTGATTTTAA | AAGGCTTGTT | ATTATTGAGC | 2220 |
CTGACAAACG | AATGAGTATC | TGACATTAAA | TCAGAAGTTG | CAGACATTGC | CAGTGATTTT | 2280 |
ACAGCCAAAG | TGTTTACTGT | CCTGTTGTTG | GTCTGGGATT | GGTTAAAAAC | AGGCTTAAGT | 2340 |
GTGTACAGCC | ATTCATGAAC | ACTTCTGGTA | GGAGAAAGAA | CACTGACTTT | GCCCATGGAT | 2400 |
TGGTTGTTTG | AATACTTGAC | ATCAAAAAGG | ACTTCTTTGA | CACAAGACAA | TGATCCTTTA | 2460 |
TTTGCAGCTT | CAATGAAATT | ATTTGGAGGG | AAGATGTCAG | ATTCTTTCAG | ACCGAATCTT | 2520 |
GAAGTAATTT | CAGGCACTGC | TATTTGCTCT | TCAAGCATTC | TGAGCAACTG | AGAGGATAAC | 2580 |
ACTTTGAGAT | GACCTTGATG | TTTAACACCG | TTTGTGTTTG | TTGTGTTAGA | TCTGACCAGA | 2640 |
ATTTCGACAA | CATTCCCTGA | AAAGGAGAAA | TTCAGATCAA | TGTCATCAAA | AATAGGCACA | 2700 |
AAACATCTCA | CTATAATTTC | TTTTTCTTCT | GTTGGGAGAA | AGCCAACTTT | ACCAGTGTAA | 2760 |
CAGAAACTGG | TCTTGCTCCT | GGAGTCAGAA | TAGAGTTGAG | CTTCGACCAA | CTTCTTTCCG | 2320 |
GAAGATTGAT | CATGAATCCA | ATAACTATCT | ACTACTGCTT | TACCCGAAGA | TGTTGTTCCC | 2330 |
CAGATTGAGG | ACTTCGATTT | GATAATTGTT | TCATACATTG | CACTAGACAT | GTTAAAATGG | 2940 |
AAGTAGTAAT | GTAATTGACA | ATATTGTAAG | ATTTGTTGTA | GCTTGGTTGT | TCNNNNNNNN | 3000 |
NNNNNNNNNA | TTGCTCT | 3 017 |
SEQ ID NO: 10
Sequence Type: Nucleotide
Sequence length: 2993
Strandedness: Single stranded
Molecule type: Complementary S RNA nucleotide sequence from 1 to 2993 zt SEQ ID NO :9
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATTG TGTCACGAAT ATAACATCAT ATAATCCAAA GCTAGAAACT GAAAAATTAC 60
AAATTTTACC AAATACTACT TTAACCGCAA GTACTATTTA GTAATTTGAA CACTTTAAGC '.20
TTTAAAGTTA GTAAAGTCAG CAGATCAAGA TGAACAAAGC AAAGATTACC AAGGAGAACA '.30 bad original O
AP Ο Ο Ο 4 Ο 3
TAGTCAAGCT TTTGACTCAA TCTGATTCCT TAGAATTTGA GGAAACTCAA AATGAAGGGT 240 CTTTCAATTT CACTGACTTC TTTACCAACA ACCGTGAAAA GATTCAGAAC ATGACTACTG 300
CATCCTGCCT ATCCTTCCTC AAGAATAGGC AATCTATAAT GAGAGTCATT AAGAGTGCTG 360
ACTTCACTTT TGGATCAGTC ACAATTAAGA AAACTAGAAA CAATTCAGAA AGAGTTGGGG 420 TCAATGATAT GACTTTCCGA AGACTTGATG CAATGGTGAG AGTTCATCTT GTTGGAATGA 480 TAAAGGACAA TGGATCTGCT CTGACTGAAG CTATAAATAG CCTTCCAAGT CACCCTCTGA 540 TTGCCTCATA TGGTCTTGCA ACCACAGATT TGAAATCCTG TGTCTTGGGT GTTCTCTTAG 600 GAGGAAGTCT TCCTCTGATA GCATCTGTAC TGAATTTTGA AATAGCTGCA CTAGTTCCGG 660 CTATTTATCA AGATGCTAAA CATGTTGAGC TTGGAATTGA CATGTCTAAG TTTAGCACTA 720 AAGAGGCAGT TGGGAAAGTG TGCACAGTTC TAAAAAGCAA AGGATATAGC ATGAACTCTG 780
TTGAAATTGG CAAGGCTAAA CAATATGCAG ATATACTAAA GGCTTGTAGT CCGAAAGCTA 840 *
AAGGACTTGC TGCAATGGAC CACTACAAAG AAGGGCTTAC ATCCATTTAC AGCATGTTTA 900
ATGCTACTAT TGATTTTGGG AAAAATGATT CTATTTAAAA GAGATTTGAG CTTGGTTTGC 960
TTTGTGTTGT GTTAAATGCA ΤΤΤΑΑΑΤΑΑΑ ΤΑΑΑΑΑΤΑΑΑ ΑΑΤΑΑΤΑΑΑΑ ΑΤΑΑΑΑΑΑΤΑ 1020
ΑΑΑΤΑΑΑΑΑΑ ΤΑΑΑΑΤΑΑΤΤ AATTATTGTT TGAGTGTATT AAGTGTGCTT ΤΤΑΤΑΑΑΤΤΑ 1080
TTTGAAATAA GTTTAATGTG TGTTTTAATA TATAAACTTG AAAGTGTGTT TGCGTGTGAA 1140
ATAAACAAGT AAAAAAGCTA CAAAAAATAA TAAAAACAAT AAAAACCACT ACAAAATATA 1200
ΤΑΤΑΤΑΤΑΤΑ ΤΑΤΑΤΑΑΑΤΑ ΑΑΑΤΑΑΑΑΑΑ CAAAAAACAA AAAACAAAAT AAAAAACAAA 1260
TAAAACAAAA CAAAAACAAA AAACAAAAAA CAAAAACAAA AACAAATACA AATTTTTGGC 1320 CAATTTGTGA GTAGATTGGA TTTAAAGTTT ATAATTTACT AATACATTCA TTTTAAAGCA 13 80
TTATTAAAGC AACCAAATTG TGCCCGAAGG CCATCCTTTT TTGGTTTTTA ΤΤΤΑΤΤΤΤΑΤ 1440
GATTTTTTTT GTATTTTTTA ΤΑΤΑΤΤΤΤΑΤ GTTTATTTAT TTATTTCATA TGATTGTTTT 1500 TGGTTTATCT GTTATTAGAT TTGTTTAATC TGATCTAAAT GCATGTAAAC AAACTTTACT 1560
TAGGGACAAG TTTAACCAAG GTTAATTTAA ATCTAATTTA GAAATAGCTT TCAAAACATC 1620
CTCTGATCTA GGAGAAATTT TTATTTCACC TGCAGCATAG TCAAATTTCC AATCGGGCAT 1680 CTGGATGCTA TCCAAATAAG ACATTGTTTG TGAGTTTCTC GGAAGACTTT TCAAGGTCTG 1740 AGAAAGGAAA TATTTTCTCC CATAACCATC TTCTGAATAA TTTAAAGTCT TTTTCCCTAG 1800 CTCAAACTTT TTTGGCTTTA GATCTGGCTC ATTTATAGTG AAGACAATCA TGTCTTGCTG 1860 AACACTGTCA CAGACAGTTT GAGCAAGTTC TGTGTGGATT TCAAGAAGTG AATGAACAAT 1920 TCTTTGAGGA TACGACATAT CATGGATAAC ATTGTAAGGT TCTTTTAGAT CAGAAAAATT 1980 CATCTCAATT TTATGAATCA CTGCTAGAAG GAGTTGATTC AAGAAGAGGT TTGACTGATC 2040 AAAGTTATAT TCAATGACTG TTTCAACATT GTTGTGATTC GGAAGACATT GAATGCTTAT 2100 ATAATACTCT TTAGGAGAAG ATTCATCAGA CAGGGTGAAG AACCGGCTGT ATGTGTTTGA 2160 TTTCATTATT TTAGGGATGC GCATCCAAAG GCTGATTTTA AAAGGCTTGT TATTATTGAG 2220 CCTGACAAAC GAATGAGTAT CTGACATTAA ATCAGAAGTT GCAGACATTG CCAGTGATTT 2280 TACAGCCAAA GTGTTTACTG TCCTGTTGTT GGTCTGGGAT TGGTTAAAAA CAGGCTTAAG 2340 TGTGTACAGC CATTCATGAA CACTTCTGGT AGGAGAAAGA ACACTGACTT TGCCCATGGA 2400
AP Ο Ο Ο 4 Ο 3
TTGGTTGTTT GAATACTTGA CATCAAAAAG GACTTCTTTG ACACAAGACA ATGATCCTTT 2460
ATTTGCAGCT TCAATGAAAT TATTTGGAGG GAAGATGTCA GATTCTTTCA GACCGAATCT 2520 TGAAGTAATT TCAGGCACTG CTATTTGCTC TTCAAGCATT CTGAGCAACT GAGAGGATAA 2580 CACTTTGAGA TGACCTTGAT GTTTAACACC GTTTGTGTTT GTTGTGTTAG ATCTGACCAG 2640 AATTTCGACA ACATTCCCTG AAAAGGAGAA ATTCAGATCA ATGTCATCAA AAATAGGCAC 2700 AAAACATCTC ACTATAATTT CTTTTTCTTC TGTTGGGAGA AAGCCAACTT TACCAGTGTA 2760 ACAGAAACTG GTCTTGCTCC TGGAGTCAGA ATAGAGTTGA GCTTCGACCA ACTTCTTTCC 2820 GGAAGATTGA TCATGAATCC AATAACTATC TACTACTGCT TTACCCGAAG ATGTTGTTCC 2880 CCAGATTGAG GACTTCGATT TGATAATTGT TTCATACATT GCACTAGACA TGTTAAAATG 2940 GAAGTAGTAA TGTAATTGAC AATATTGTAA GATTTGTTGT AGCTTGGTTG TTC 2993
SEQ ID NO: 11
Sequence type: Nucleotide
Sequence length: 789 nucleotides
Strandedness: Single stranded
Molecule type: Viral complementary S RNA nucleotide sequence coding for the nucleocapsid (N) protein from position 150 to position 938 of
SEQ ID NO:9
Original Source Organism: Impatiens Necrotic Spot Virus
ATGAACAAAG | CAAAGATTAC | CAAGGAGAAC | ATAGTCAAGC | TTTTGACTCA | ATCTGATTCC | 60 |
TTAGAATTTG | AGGAAACTCA | AAATGAAGGG | TCTTTCAATT | TCACTGACTT | CTTTACCAAC | 120 |
AACCGTGAAA | AGATTCAGAA | CATGACTACT | GCATCCTGCC | TATCCTTCCT | CAAGAATAGG | 180 |
CAATCTATAA | TGAGAGTCAT | TAAGAGTGCT | GACTTCACTT | TTGGATCAGT | CACAATTAAG | 240 |
AAAACTAGAA | ACAATTCAGA | AAGAGTTGGG | GTCAATGATA | TGACTTTCCG | AAGACTTGAT | 300 |
GCAATGGTGA | GAGTTCATCT | TGTTGGAATG | ATAAAGGACA | ATGGATCTGC | TCTGACTGAA | 360 |
GCTATAAATA | GCCTTCCAAG | TCACCCTCTG | ATTGCCTCAT | ATGGTCTTGC | AACCACAGAT | 420 |
TTGAAATCCT | GTGTCTTGGG | TGTTCTCTTA | GGAGGAAGTC | TTCCTCTGAT | AGCATCTGTA | 480 |
CTGAATTTTG | AAATAGCTGC | ACTAGTTCCG | GCTATTTATC | AAGATGCTAA | ACATGTTGAG | 540 |
CTTGGAATTG | ACATGTCTAA | GTTTAGCACT | AAAGAGGCAG | TTGGGAAAGT | GTGCACAGTT | 600 |
CTAAAAAGCA | AAGGATATAG | CATGAACTCT | GTTGAAATTG | GCAAGGCTAA | ACAATATGCA | 660 |
GATATACTAA | AGGCTTGTAG | TCCGAAAGCT | AAAGGACTTG | CTGCAATGGA | CCACTACAAA | 720 |
GAAGGGCTTA | CATCCATTTA | CAGCATGTTT | AATGCTACTA | TTGATTTTGG | GAAAAATGAT | 780 |
AP Ο Ο Ο 4 Ο 3
SEQ ID NO: 12
Sequence type: Nucleotide
Sequence length: 1350 nucleotides
Strandedness: Single stranded ·
Molecule type: Complementary S RNA nucleotide sequence (to the viral S RNA nucleotide sequence) from position 1581 to position 2930 of SEQ ID NO:9
Original Source Organism: Impatiens Necrotic Spot Virus
TTAATTTAAA | TCTAATTTAG | AAATAGCTTT | CAAAACATCC | TCTGATCTAG | GAGAAATTTT | 60 |
TATTTCACCT | GCAGCATAGT | CAAATTTCCA | ATCGGGCATC | TGGATGCTAT | CCAAATAAGA | 120 |
CATTGTTTGT | GAGTTTCTCG | GAAGACTTTT | CAAGGTCTGA | GAAAGGAAAT | ATTTTCTCCC | 180 |
ATAACCATCT | TCTGAATAAT | TTAAAGTCTT | TTTCCCTAGC | TCAAACTTTT | TTGGCTTTAG | 240 |
ATCTGGCTCA | TTTATAGTGA | AGACAATCAT | gtcttgctga | ACACTGTCAC | AGACAGTTTG | 300 |
AGCAAGTTCT | GTGTGGATTT | CAAGAAGTGA | ATGAACAATT | CTTTGAGGAT | ACGACATATC | 360 |
ATGGATAACA | TTGTAAGGTT | CTTTTAGATC | AGAAAAATTC | ATCTCAATTT | TATGAATCAC | 420 |
TGCTAGAAGG | AGTTGATTCA | AGAAGAGGTT | TGACTGATCA | AAGTTATATT | CAATGACTGT | 480 |
TTCAACATTG | TTGTGATTCG | GAAGACATTG | AATGCTTATA | TAATACTCTT | TAGGAGAAGA | 540 |
TTCATCAGAC | AGGGTGAAGA | ACCGGCTGTA | TGTGTTTGAT | TTCATTATTT | TAGGGATGCG | 600 |
CATCCAAAGG | CTGATTTTAA | AAGGCTTGTT | ATTATTGAGC | CTGACAAACG | AATGAGTATC | 660 |
TGACATTAAA | TCAGAAGTTG | CAGACATTGC | CAGTGATTTT | ACAGCCAAAG | TGTTTACTGT | 720 |
CCTGTTGTTG | GTCTGGGATT | GGTTAAAAAC | AGGCTTAAGT | GTGTACAGCC | ATTCATGAAC | 780 |
ACTTCTGGTA | GGAGAAAGAA | CACTGACTTT | GCCCATGGAT | TGGTTGTTTG | AATACTTGAC | 840 |
ATCAAAAAGG | ACTTCTTTGA | CACAAGACAA | TGATCCTTTA | TTTGCAGCTT | CAATGAAATT | 900 |
ATTTGGAGGG | AAGATGTCAG | ATTCTTTCAG | ACCGAATCTT | GAAGTAATTT | CAGGCACTGC | 960 |
TATTTGCTCT | TCAAGCATTC | TGAGCAACTG | AGAGGATAAC | actttgagat | GACCTTGATG | 1020 |
TTTAACACCG | TTTGTGTTTG | TTGTGTTAGA | TCTGACCAGA | ATTTCGACAA | CATTCCCTGA | 1080 |
AAAGGAGAAA | TTCAGATCAA | TGTCATCAAA | AATAGGCACA | AAACATCTCA | CTATAATTTC | 1140 |
TTTTTCTTCT | GTTGGGAGAA | AGCCAACTTT | ACCAGTGTAA | CAGAAACTGG | TCTTGCTCCT | 1200 |
GGAGTCAGAA | TAGAGTTGAG | CTTCGACCAA | CTTCTTTCCG | GAAGATTGAT | CATGAATCCA | 1260 |
ATAACTATCT | ACTACTGCTT | TACCCGAAGA | TGTTGTTCCC | CAGATTGAGG | ACTTCGATTT | 1320 |
GATAATTGTT | TCATACATTG | CACTAGACAT | 1350 |
BAD ORIGINAL
APO00403
SEQ ID NO :13
Sequence type: Nucleotide
Sequence length: 642 nucleotides
Strandedness: single stranded
Molecule type: Viral complementary S RNA strand of the intergenic region from position 939 to position 1580 of SEQ ID NO:9
Original Source Organism:Impatiens Necrotic Spot Virus
AAGAGATTTG | AGCTTGGTTT | GCTTTGTGTT | GTGTTAAATG | CATTTAAATA | AATAAAAATA | 60 |
AAAATAATAA | AAATAAAAAA | TAAAATAAAA | AATAAAATAA | TTAATTATTG | TTTGAGTGTA | 120 |
TTAAGTGTGC | TTTTATAAAT | TATTTGAAAT | AAGTTTAATG | TGTGTTTTAA | TATATAAACT | 180 |
TGAAAGTGTG | TTTGCGTGTG | AAATAAACAA | GTAAAAAAGC | TACAAAAAAT | AATAAAAACA | 240 |
ATAAAAACCA | CTACAAAATA | TATATATATA | TATATATAAA | TAAAATAAAA | AACAAAAAAC | 300 |
AAAAAACAAA | ATAAAAAACA | AATAAAACAA | AACAAAAACA | AAAAACAAAA | AACAAAAACA | 360 |
AAAACAAATA | CAAATTTTTG | GCCAATTTGT | GAGTAGATTG | GATTTAAAGT | TTATAATTTA | 420 |
CTAATACATT | CTTTTAAAGC | ATTATTAAAG | CAACCAAATT | GTGCCCGAAG | GCCATCCTTT | 480 |
TTTGGTTTTT | ΑΤΤΤΑΤΊΤΤΑ | TGATTTTTTT | tgtatttttt | ATATATTTTA | TGTTTATTTA | 540 |
TTTATTTCAT | ATGATTGTTT | ttggtttatc | TGTTATTAGA | TTTGTTTAAT | CTGATCTAAA | 600 |
TGCATGTAAA | CAAACTTTAC | TTAGGGACAA | GTTTAACCAA | GG | 642 |
SEQ ID NO :14 ( Sequence type:Nucleotide
Sequence length: 4970 nucleotides Strandedness: single stranded Molecule type: Viral M RNA
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAATTCA ATCATTATCT TCTCAATATT 60 TTAATTCTTA ATCTACCGTC CAGAGATGAA TAGTTTTTTC AAATCACTCA GATCATCTAG 120 CAGCAGGGAG CTAGATCACC CTAGGGTTAC AACTACCCTC TCTAAACAAG GAGCAGACAT 180 TGTTGTACAC AATCCTTCTG CTAATCACAA CAACAAGGAA GTTCTCCAAA GAGCCATGGA 240 TAGCTCTAAA GGGAAGATTT TGATGAACAA TACAGGCACC TCATCACTAG GCACATATGA 300 GTCTGACCAG ATATCTGAAT CAGAGTCTTA TGATCTTTCT GCTAGAATGA TTGTTGATAC 360 AAATCATCAT ATCTCCAGCT GGAAAAATGA TCTTTTTGTA GGTAATGGTG ATAAAGCTGC 420 AACCAAGATA ATTAAGATAC ATCCAACCTG GGATAGCAGA AAACAATACA TGATGATCTC 480
AP Ο 00 4 Ο 3
TTTAATTGAT | CCTAACAAGA | GTGTTAATGC | CAGAACTGTT | TTGAAAGGGC | AAGGAAGCAT | 600 |
TAAAGATCCT | ATATGTTTTG | TTTTTTATCT | AAATTGGTCC | ATTCCAAAAG | TTAACAACAC | 660 |
TTCAGAGAAT | TGTGTTCAGC | TTCATTTATT | ATGTGATCAA | GTTTACAAGA | AAGATGTTTC | 720 |
TTTTGCTAGT | GTCATGTATT | CTTGGACAAA | AGAATTCTGT | GATTCACCAA | GAGCAGATCT | 780 |
GGATAAAAGC | TGCATGATAA | TACCCATCAA | TAGGGCTATT | AGAGCCAAAT | CGCAAGCCTT | 840 |
CATTGAAGCC | TGCAAGTTAA | TCATACCTAA | AGGCAATTCT | GAAAAGCAAA | TTAGAAGACA | 900 |
ACTTGCAGAG | CTAAGTGCTA | ATTTAGAGAA | ATCTGTTGAA | GAAGAGGAGA | ATGTTACTGA | 960 |
TAACAAGATA | GAGATATCAT | TTGATAATGA | AATCTAAATA | TGTTTTCATT | TAATAATAAA | 1020 |
TAATATATAT | TGTTCATAAT | ATTTTGAATG | TTTAAGTAAA | AAATAAAGCA | AGATAAAAAA | 1080 |
CTATATATAT | ATATATATAT | AGAAGTATAA | AATATATATG | TATTTGTGTT | TAAAAACAAA | 1140 |
TCAAAAACCA | AAAAAGAAAA | AAGAAAAAAT | AAACAAAAAA | CAAAAACAAA | AACAAAAACA | 1200 |
AACAAAAAGC | AAAAAATAGA | AAAAAGTTGA | AAAAAACCAA | AAAAATTTTT | TTTGTAAATA | 1260 |
AATAAGGCTC | CGGCCAGATT | TGGTCTAAGA | CCTTTTTATT | TGTTTTTATA | CATTTTATTT | 1320 |
GTTTTTGTTG | ATTTTTATTT | TTATTATTTT | ΤΑΤΑΤΤΤΓΤΤ | ATATAGTTTG | CTTATTTAAC | 1380 |
ACTTATTTAG | ACAAATTAAA | TTTATTTGAT | TACAATCATT | CTGCCTTATT | TAATTTAAAA | 1440 |
CACATTTGGT | GTATATTCCA | ATGAATTTAA | TCATATACCG | CTGAAGTCTA | GAGGAGGTCT | 1500 |
TCTTCTAGTG | ATGGTGTCTT | TACCAGAAGA | CGTGGAAACC | AAAGAATAAT | CATTAGTGTC | 1560 |
TTCAATATAT | TTTGTCTTGT | AAGACTTGTT | TCTAACATAG | CCTCTACACA | TTGTGGCAAC | 1620 |
AATAGAGCAG | AGGTAAGCAA | GAGCAAATAC | AAAGAGTATG | AGCAATACTA | CTCTGACTGT | 1680 |
ATCAAAGAAG | GATCCAAAGT | GGCTTGCTAT | AAAGTTAAAA | GGGCTTTTAA | CATAGTCCCA | 17 4 0 |
AAAGCTCCAA | ACTGATGTGT | CAGAATTATA | TTGCTGTTCC | TCGTGTGCAT | GTTGGTCATT | 1800 |
TTGATCAATT | ATGTTTTCTG | GTTCCAGCAC | AGCAACAGAA | TCTACAAGTG | CCTCAACTGA | 1860 |
GTATGATTTG | TCTCCTTCTG | GTTCTATAAT | CATTTTTTGT | TTTTCTGGGT | TAGAAGTGCA | 1920 |
GAACATTGTC | AAGTTATACT | TATTAGCACC | TTTCTTTACT | GCTATCTGGT | ATGTTGACAA | 1980 |
TGAACATTGT | TTCATGGTTA | ACCTTGCAGA | AAAAGTTATG | TCTGATATAA | ATGAGGCAGC | 2040 |
ACACCTCAGC | CCTTGGCTAC | ATAAGAAACA | TCCCTTACAG | CTTAAAGAGA | CAGAACTCAA | 2100 |
TATAGGCTTT | TTTGGTACAG | TTTTAAACAA | TTCAGAAGGT | AGATCCAAAA | CAATTTTAAG | 2160 |
CTTACCTAGA | CTAAAGATCT | TTTCCATATA | AAAACTATTC | TGGTCAGTAA | ACTGAACTGG | 2220 |
AATGTCCGAT | atttggttca | AACCTGTTTT | AAATCTGTAT | GTGTCATAAC | CACATGATTT | 2280 |
TATCGTAATT | GTTTTTTTAC | CAATTGCTGA | ACAATCCCAG | GACAGATCGT | TTGTATCTAA | 2340 |
TGTTTTCTTA | GAGAAAATGG | GATCACCTTG | GTGTGAAAGT | TGAGGATGAC | CAAACATTTT | 2400 |
TGATGGATTA | TTTAATCTAG | CTATGTTTCC | CGCATATACG | TGACTATCAG | GTCCATGAGC | 2460 |
TATCAGCTGG | CCTATTGTTA | AGCCATCATT | ATGGAAATCC | GCTAATATAT | CAGCCTGGAA | 2520 |
ATATCCTGAT | TCAGATGGGA | CTTCCTCAGA | TACAGTGAAA | CACTTTGCTC | CCACAAATCC | 2580 |
AGATATACAT | ACTTCAGACT | TGATTGTTGA | TTTAATAACA | GAATAAATCC | TGAAAGATTG | 2640 |
ATCCATATCA | TACACATTTC | TACAAAACCC | ACAAGTGGCT | CCTTCATTGA | TAGCCAAACA | 2700 |
CCAAACCTCT | TCACAACCCC | AGTAAGATGT | TGGTGTTATG | CAGAAATCTT | GATACCCAGT | 2760 |
AP000403 )
TATCGGTTGT TCTTTTCTGC AATCTGAGCA TTTACCTGTG CATGTTGAAA AGAAATCAGT 2820 GTGGGTGCTT TGTATAGGAG CTGTAGTGTA TTGTTCAGAA ACATCATACT GTATTCTAAC 2880 ΤΤΤΤΤΤΑΑΤΑ TAAACAACAA ACTTCTGAGC AGTGCTAGAA CTTTTGTCAT TAAGAGAGAA 2940 AACTGTGCCC CCACCTGATA ATAAAGATTC TTCTATCATG TATCTATATT TTCCATCTAT 3000
CACCGAGTCA AATATGAGAG ATTTTCTTGG AAAAATGCTT TCAGGTATGT CTGATTCATT 3060 AGATTTAAGT GCATCTCCAG AAATGTATCC ATATTTTTCA GTTTTATTGT AGAAATCAAT 3120 TATACCATTC CTAAGCCTTT TCATGAAGTG TAGATTCACA GCATTCAATC CCAATGTGTC 3180 ACCAGAATAT TCTAAGAACC CATTATCTAA AGGCTTGCTT TGGAAAATAG AGGCATACTC 3240 ACAACCAAAT CTGCATTTGA CAAAAGTTAC TAAAGCATTT TCAGTTATCC TGCCTTTGCA 3300 TTCTTGATAA GGTATACAAT CCATAGGACC TTCTGTCACA ACATTGGTTA GAAAGTTAGA 3360 TTCTACAATA GAATTTTCTT TAATAGCACA GAAGCATTGG TCTTTTTCAG GACATTTGTC 3420 *' ATATCTGTTT GTAACAAAGC GGTCACAACC AGGGACATAA TAACAGCTAT CCAAACACTG 3480 AGCAGTTTGA GCCATAGACA TAGGCATCTG TGACAAAATC AGAAATCCTA TCAAAGTTTC 3540 TGTGACTGCT TTTAGGAAAG AGAGGCCTAT TTTTGTATTA ACTATCAAAT GGAACCATTC 3600 AATGCTAGCC CAGTTGTATT TTTTATTCTT CTCTGCTGTT CTAGTTATTA TAGGACATTC 3660 TTCTGAGTGT TCTTCAGAGG CTTTGTTTTT GTTACAAATG CATAATTTTG AGCATTCATG 3720 GGTTACCAAA CATAAATTTC CACAGACCTT ACATTTCAAG GGAAAATAAG ACCATAAATA 3780 ATTTATCAGT AGTAGTATAG GATACGTTAT CAATCCCAGA AGATCATACC CATAGAACAG 3840 TGTTTTAGAT GTTTTGTTTA CCAAGTACCT TATAGGGAAA TAGACAATCA GAGCAATCAT 3900
GATCAATCTA AACCATGAGA AGTTGATGCA AGCAGTTTGT TTGTAAATAT TTTTGGAGTA 3960 CTTTATAATA CAATCTCTAA CTCTTTTGTC CACTAAAGGA ACTTTAGAAG ACTTGTCACC 4020
GCACAATAGG TTATGCTTAC CATCCATATT TTCTTCTGTG AAAGTCAAAC TAACTGAGCC 4080
AGAGAAGCTT ATTATGGAAT GGCTCATGTC ACTTCCTTCT CTTTTGACGA CGTAACCCAT 4140 GATTTTCTCA GGTGTAGTTA ATGAAACTGT ATAAGAATTA ACTATGTTTG TTTTTGATAT 4200
TTTACAATCA CCTGAGAATT TCACACTCTG GAGAGAGACT GTGCCATTAG TTGGTCTAGA 4260
ATTGTACATG ATTGGATAAT TGTAATTCTC CAAACTTTCA ATTATATAGA ATTTAGTTCC 4320
TATAGATAAT TTCCTTTTGT TATCGATTTT TGTTATTGGT ACAACTGGAA CAGTTTCAAA 4380 GCTTCTTGGC AATTCAGAAG ATCCTTCACA GTTTCCCAAT TTAGTTATAG TGTCACTGAT 4440 ACATGAATAT ATAACACCAT TGCTTTCTAC TTGGTAATAA ACATTGAATG TTGAAACTCC 4500 TTTAATGCTA CAAGTCAAAC TTGAAGCATT TAGGCATGGA TTTGGTAAAT CCATAACTGA 4560 TATAGTTGTT GGTGTAGAAG ACAATCCACT TGGAGATTGA GGTACCTCAT TATTGGCAAG 4620 AACAGTTTGA GTATCTCGTG TTGGTCTAAG GGTTTTACCT GTTGCATTCT GGAGCATTTC 4680 AGCCAAAGTA TCTAGAATTT CATTTTTATG ATCTACAGAA CGGTCATAAT AAGCTTCATC 4740 ATAAATTTCT GGATGATCGC CCCTTTCAAC ATGAATCTTT GCATCTGTCT CCTTTAATGC 4800 CATAAAGGAT AAGATAACAG AAGTAACAAC TAGTGTACAT ACACTAATTT TAACAAGTAA 4860 CTCGCACATC TTTAGAATTT TCATTCTAAA AAGTCGAATA ACACTAGTTC TAAAATTGCT 4910 TTATGAGTTT GATCTGTTGT ATGTAGAGTT TTGTTTGCAC TGATTGCTCT 4970
BAD ORIGINAL
AP 0 00 4 0 3
SEQ ID NO» 15
Sequence type: Nucleotide
Sequence length: 912 nucleotides
Strandedness: single strand
Molecule type: Viral M RNA nucleotide sequence coding for the NSm protein gene from position 86 to position 997 of SEQ ID NO:14
Original Source Organism: Impatiens Necrotic Spot Virus
ATGAATAGTT | TTTTCAAATC | ACTCAGATCA | TCTAGCAGCA | GGGAGCTAGA | TCACCCTAGG | 60 |
GTTACAACTA | CCCTCTCTAA | ACAAGGAGCA | GACATTGTTG | TACACAATCC | TTCTGCTAAT | 120 |
CACAACAACA | AGGAAGTTCT | CCAAAGAGCC | ATGGATAGCT | CTAAAGGGAA | GATTTTGATG | 180 |
AACAATACAG | GCACCTCATC | ACTAGGCACA | TATGAGTCTG | ACCAGATATC | TGAATCAGAG | 240 |
TCTTATGATC | TTTCTGCTAG | AATGATTGTT | GATACAAATC | ATCATATCTC | CAGCTGGAAA | 300 |
AATGATCTTT | TTGTAGGTAA | TGGTGATAAA | GOTGCAACCA | AGATAATTAA | GATACATCCA | 360 |
ACCTGGGATA | GCAGAAAACA | ATACATGATG | ATCTCAAGGA | TAGTTATCTG | GATATGCCCT | 420 |
ACTATAGCTG | ATCCTGATGG | GAAATTGGCT | GTAGCTTTAA | TTGATCCTAA | CAAGAGTGTT | 480 |
AATGCCAGAA | CTGTTTTGAA | AGGGCAAGGA | AGCATTAAAG | ATCCTATATG | TTTTGTTTTT | 540 |
TATCTAAATT | GGTCCATTCC | AAAAGTTAAC | AACACTTCAG | AGAATTGTGT | TCAGCTTCAT | 600 |
TTATTATGTG | ATCAAGTTTA | CAAGAAAGAT | GTTTCTTTTG | CTAGTGTCAT | GTATTCTTGG | 6 6 0 |
ACAAAAGAAT | TCTGTGATTC | ACCAAGAGCA | GATCTGGATA | AAAGCTGCAT | GATAATACCC | |
ATCAATAGGG | CTATTAGAGC | CAAATCGCAA | GCCTTCATTG | AAGCCTGCAA | GTTAATCATA | ~30 |
CCTAAAGGCA | attctgaaaa | GCAAATTAGA | AGACAACTTG | CAGAGCTAAG | TGCTAATTTA | 34 0 |
GAGAAATCTG | TTGAAGAAGA | GGAGAATGTT | ACTGATAACA | AGATAGAGAT | ATCATTTGAT | 9 0 0 |
AATGAAATCT | AA | ? 1 2 |
SEQ ID No »16
Sequence type: nucleotide
Sequence length: 473 nucleotides
Strandedness: single stranded
Molecule type: Intergenic region of viral M RNA from position 998 to position 1470 of SEQ ID NO:14
Original Source Organism: Impatiens necrotic spot virus
ATATGTTTTC ATTTAATAAT AAATAATATA TATTGTTCAT AATATTTTGA ATGTTTAAGT -?
AAAAAATAAA GCAAGATAAA AAACTATATA TATATATATA TATAGAAGTA TAAAATATAT 120
ATGTATTTGT GTTTAAAAAC AAATCAAAAA CCAAAAAAGA AAAAAGAAAA AATAAACA-A I 60
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
AAACAAAAAC AAAAACAAAA ACAAACAAAA AGCAAAAAAT AGAAAAAAGT TGAAAAAAAC 240 CAAAAAAATT TTTTTTGTAA ATAAATAAGG CTCCGGCCAG ATTTGGTCTA AGACCTTTTT 300 ATTTGTTTTT ATACATTTTA TTTGTTTTTG TTGATTTTTA ΤΤΙΤΤΑΤΤΑΤ ΊΤΤΤΑΤΑΤΤΤ 360 TTTATATAGT TTGCTTATTT AACACTTATT TAGACAAATT ΑΑΑΤΤΤΑΤΤΤ GATTACAATC 420 ATTCTGCCTT ΑΤΤΤΑΑΤΤΤΑ AAACACATTT GGTGTATATT CCAATGAATT ΤΑΑ 473
SEQ ID Νο. 17
Sequence type: Nucleotide
Sequence length: 3414 nucleotides
Strandedness: single stranded
Molecule Type: Viral M RNA from 1471 - 4884
Original Source Organism: Impatiens Necrotic Spot Virus
TCATATACCG | CTGAAGTCTA | GAGGAGGTCT | TCTTCTAGTG | ATGGTGTCTT | TACCAGAAGA | 60 |
CGTGGAAACC | AAAGAATAAT | CATTAGTGTC | TTCAATATAT | TTTGTCTTGT | AAGACTTGTT | 120 |
TCTAACATAG | CCTCTACACA | TTGTGGCAAC | AATAGAGCAG | AGGTAAGCAA | GAGCAAATAC | 180 |
AAAGAGTATG | AGCAATACTA | CTCTGACTGT | ATCAAAGAAG | GATCCAAAGT | GGCTTGCTAT | 240 |
AAAGTTAAAA | GGGCTTTTAA | CATAGTCCCA | AAAGCTCCAA | ACTGATGTGT | CAGAATTATA | 300 |
TTGCTGTTCC | TCGTGTGCAT | GTTGGTCATT | TTGATCAATT | ATGTTTTCTG | GTTCCAGCAC | 360 |
AGCAACAGAA | TCTACAAGTG | CCTCAACTGA | GTATGATTTG | TCTCCTTCTG | GTTCTATAAT | 420 |
CATTTTTTGT | TTTTCTGGGT | TAGAAGTGCA | GAACATTGTC | AAGTTATACT | TATTAGCACC | 480 |
TTTCTTTACT | GCTATCTGGT | ATGTTGACAA | TGAACATTGT | TTCATGGTTA | ACCTTGCAGA | 540 |
AAAAGTTATG | TCTGATATAA | ATGAGGCAGC | ACACCTCAGC | CCTTGGCTAC | ATAAGAAACA | 600 |
TCCCTTACAG | CTTAAAGAGA | CAGAACTCAA | TATAGGCTTT | TTTGGTACAG | TTTTAAACAA | 660 |
TTCAGAAGGT | AGATCCAAAA | CAATTTTAAG | CTTACCTAGA | CTAAAGATCT | TTTCCATATA | 720 |
AAAACTATTC | TGGTCAGTAA | ACTGAACTGG | AATGTCCGAT | ATTTGGTTCA | AACCTGTTTT | 780 |
AAATCTGTAT | GTGTCATAAC | CACATGATTT | TATCGTAATT | GTTTTTTTAC | CAATTGCTGA | 840 |
ACAATCCCAG | GACAGATCGT | TTGTATCTAA | TGTTTTCTTA | GAGAAAATGG | GATCACCTTG | 900 |
GTGTGAAAGT | TGAGGATGAC | CAAACATTTT | TGATGGATTA | TTTAATCTAG | CTATGTTTCC | 960 |
CGCATATACG | TGACTATCAG | GTCCATGAGC | TATCAGCTGG | CCTATTGTTA | AGCCATCATT | 1020 |
ATGGAAATCC | GCTAATATAT | CAGCCTGGAA | ATATCCTGAT | TCAGATGGGA | CTTCCTCAGA | 1080 |
TACAGTGAAA | CACTTTGCTC | CCACAAATCC | AGATATACAT | ACTTCAGACT | TGATTGTTGA | 1140 |
TTTAATAACA | GAATAAATCC | TGAAAGATTG | ATCCATATCA | TACACATTTC | TACAAAACCC | 1200 |
ACAAGTGGCT | CCTTCATTGA | TAGCCAAACA | CCAAACCTCT | TCACAACCCC | AGTAAGATGT | 1260 |
TGGTGTTATG | CAGAAATCTT | GATACCCAGT | TATCGGTTGT | TCTTTTCTGC | AATCTGAGCA | 1320 |
TTTACCTGTG | CATGTTGAAA | AGAAATCAGT | GTGGGTGCTT | TGTATAGGAG | CTGTAGTGTA | 1380 |
BAD ORIGINAL
APO00403
TTGTTCAGAA ACATCATACT GTATTCTAAC TTTTTTAATA TAAACAACAA ACTTCTGAGC 1440 AGTGCTAGAA CTTTTGTCAT TAAGAGAGAA AACTGTGCCC CCACCTGATA ATAAAGATTC 1500 TTCTATCATG TATCTATATT TTCCATCTAT CACCGAGTCA AATATGAGAG ATTTTCTTGG 1560 AAAAATGCTT TCAGGTATGT CTGATTCATT AGATTTAAGT GCATCTCCAG AAATGTATCC 1620 ATATTTTTCA GTTTTATTGT AGAAATCAAT TATACCATTC CTAAGCCTTT TCATGAAGTG 1680 TAGATTCACA GCATTCAATC CCAATGTGTC ACCAGAATAT TCTAAGAACC CATTATCTAA 1740 AGGCTTGCTT TGGAAAATAG AGGCATACTC ACAACCAAAT CTGCATTTGA CAAAAGTTAC 1800 TAAAGCATTT TCAGTTATCC TGCCTTTGCA TTCTTGATAA GGTATACAAT CCATAGGACC 1860 TTCTGTCACA ACATTGGTTA GAAAGTTAGA TTCTACAATA GAATTTTCTT TAATAGCACA 1920 GAAGCATTGG TCTTTTTCAG GACATTTGTC ATATCTGTTT GTAACAAAGC GGTCACAACC 1980 AGGGACATAA TAACAGCTAT CCAAACACTG AGCAGTTTGA GCCATAGACA TAGGCATCTG 2040’ TGACAAAATC AGAAATCCTA TCAAAGTTTC TGTGACTGCT TTTAGGAAAG AGAGGCCTAT 2100 TTTTGTATTA ACTATCAAAT GGAACCATTC AATGCTAGCC CAGTTGTATT TTTTATTCTT 2160
CTCTGCTGTT CTAGTTATTA TAGGACATTC TTCTGAGTGT TCTTCAGAGG CTTTGTTTTT 2220
GTTACAAATG CATAATTTTG AGCATTCATG GGTTACCAAA CATAAATTTC CACAGACCTT 2280 ACATTTCAAG GGAAAATAAG ACCATAAATA ATTTATCAGT AGTAGTATAG GATACGTTAT 2340
CAATCCCAGA AGATCATACC CATAGAACAG TGTTTTAGAT GTTTTGTTTA CCAAGTACCT 2400
TATAGGGAAA TAGACAATCA GAGCAATCAT GATCAATCTA AACCATGAGA AGTTGATGCA 2460
AGCAGTTTGT TTGTAAATAT TTTTGGAGTA CTTTATAATA CAATCTCTAA CTCTTTTGTC 2520 CACTAAAGGA ACTTTAGAAG ACTTGTCACC GCACAATAGG TTATGCTTAC CATCCATATT 2580
TTCTTCTGTG AAAGTCAAAC TAACTGAGCC AGAGAAGCTT ATTATGGAAT GGCTCATGTC 2540
ACTTCCTTCT CTTTTGACGA CGTAACCCAT GATTTTCTCA GGTGTAGTTA ATGAAACTGT 2700
ATAAGAATTA ACTATGTTTG TTTTTGATAT TTTACAATCA CCTGAGAATT TCACACTCTG 2760 GAGAGAGACT GTGCCATTAG TTGGTCTAGA ATTGTACATG ATTGGATAAT TGTAATTCTC 2820
CAAACTTTCA ATTATATAGA ATTTAGTTCC TATAGATAAT TTCCTTTTGT TATCGATTTT 2880 TGTTATTGGT ACAACTGGAA CAGTTTCAAA GCTTCTTGGC AATTCAGAAG ATCCTTCACA 2940 GTTTCCCAAT TTAGTTATAG TGTCACTGAT ACATGAATAT ATAACACCAT TGCTTTCTAC 3000 TTGGTAATAA ACATTGAATG TTGAAACTCC TTTAATGCTA CAAGTCAAAC TTGAAGCATT 3060 TAGGCATGGA TTTGGTAAAT CCATAACTGA TATAGTTGTT GGTGTAGAAG ACAATCCACT 3120 TGGAGATTGA GGTACCTCAT TATTGGCAAG AACAGTTTGA GTATCTCGTG TTGGTCTAAG 3180 GGTTTTACCT GTTGCATTCT GGAGCATTTC AGCCAAAGTA TCTAGAATTT CATTTTTATG 3240 ATCTACAGAA CGGTCATAAT AAGCTTCATC ATAAATTTCT GGATGATCGC CCCTTTCAAC 3300 ATGAATCTTT GCATCTGTCT CCTTTAATGC CATAAAGGAT AAGATAACAG AAGTAACAAC 3360
AP Ο Ο Ο 4 Ο 3
SEQ ID NO: 18
Sequence type: nucleotide
Sequence length: 36 nucleotides
Strandedness: single stranded
Molecule type: 5' end of M RNA pan-handle
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAA 36
SEQ ID NO: 19
Sequence type: Nucleotide
Sequence length: 36 nucleotides
Strandedness: single stranded
Molecule type: 3* end of M RNA pan-handle '
Original Source Organism: Impatiens Necrotic Spot Virus
CTGTTGTATG TAGAGTTTTG TTTGCACTGA TTGCTC 36
SEQ ID NO: 20
Sequence type: Nucleotide
Sequence length: 4970 nucleotides
Strandedness: single stranded
Molecule type: Complementary M RNA
Original Source Organism: Impatiens Necrotic Spot Virus
AGAGCAATCA | GTGCAAACAA | AACTCTACAT | ACAACAGATC | AAACTCATAA | AGCAATTTTA | 60 |
GAACTAGTGT | TATTCGACTT | TTTAGAATGA | AAATTCTAAA | GATGTGCGAG | TTACTTGTTA | 120 |
AAATTAGTGT | ATGTACACTA | GTTGTTACTT | CTGTTATCTT | atcctttatg | GCATTAAAGG | 180 |
AGACAGATGC | AAAGATTCAT | GTTGAAAGGG | GCGATCATCC | AGAAATTTAT | GATGAAGCTT | 240 |
ATTATGACCG | TTCTGTAGAT | CATAAAAATG | AAATTCTAGA | TACTTTGGCT | GAAATGCTCC | 300 |
AGAATGCAAC | AGGTAAAACC | CTTAGACCAA | CACGAGATAC | TCAAACTGTT | CTTGCCAATA | 360 |
ATGAGGTACC | TCAATCTCCA | AGTGGATTGT | CTTCTACACC | AACAACTATA | TCAGTTATGG | 420 |
ATTTACCAAA | TCCATGCCTA | AATGCTTCAA | GTTTGACTTG | TAGCATTAAA | GGAGTTTCAA | 480 |
CATTCAATGT | TTATTACCAA | GTAGAAAGCA | ATGGTGTTAT | ATATTCATGT | ATCAGTGACA | 540 |
CTATAACTAA | ATTGGGAAAC | TGTGAAGGAT | CTTCTGAATT | GCCAAGAAGC | TTTGAAACTG | 600 |
BAD ORIGINAL &
AP Ο Ο Ο 4 Ο 3 ι
TTCCAGTTGT | ACCAATAACA | AAAATCGATA | ACAAAAGGAA | ATTATCTATA | GGAACTAAAT | 660 |
TCTATATAAT | TGAAAGTTTG | GAGAATTACA | ATTATCCAAT | CATGTACAAT | TCTAGACCAA | 720 |
CTAATGGCAC | AGTCTCTCTC | CAGAGTGTGA | AATTCTCAGG | TGATTGTAAA | ATATCAAAAA | 780 |
CAAACATAGT | TAATTCTTAT | ACAGTTTCAT | TAACTACACC | TGAGAAAATC | ATGGGTTACG | 840 |
TCGTCAAAAG | AGAAGGAAGT | GACATGAGCC | ATTCCATAAT | AAGCTTCTCT | GGCTCAGTTA | 900 |
GTTTGACTTT | CACAGAAGAA | AATATGGATG | GTAAGCATAA | CCTATTGTGC | GGTGACAAGT | 960 |
CTTCTAAAGT | TCCTTTAGTG | GACAAAAGAG | TTAGAGATTG | TATTATAAAG | TACTCCAAAA | 1020 |
ATATTTACAA | ACAAACTGCT | TGCATCAACT | TCTCATGGTT | TAGATTGATC | ATGATTGCTC | 1080 |
TGATTGTCTA | TTTCCCTATA | AGGTACTTGG | TAAACAAAAC | ATCTAAAACA | CTGTTCTATG | 1140 |
GGTATGATCT | TCTGGGATTG | ATAACGTATC | CTATACTACT | ACTGATAAAT | TATTTATGGT | 1200 |
CTTATTTTCC | CTTGAAATGT | AAGGTCTGTG | G.AAATTTATG | TTTGGTAACC | CATGAATGCT | 1260 |
CAAAATTATG | CATTTGTAAC | AAAAACAAAG | CCTCTGAAGA | ACACTCAGAA | GAATGTCCTA | 1320 |
TAATAACTAG | AACAGCAGAG | AAGAATAAAA | AATACAACTG | GGCTAGCATT | GAATGGTTCC | 1380 |
ATTTGATAGT | TAATACAAAA | ATAGGCCTCT | CTTTCCTAAA | AGCAGTCACA | GAAACTTTGA | 1440 |
TAGGATTTCT | GATTTTGTCA | CAGATGCCTA | TGTCTATGGC | TCAAACTGCT | CAGTGTTTGG | 1500 |
ATAGCTGTTA | TTATGTCCCT | GGTTGTGACC | GCTTTGTTAC | AAACAGATAT | GACAAATGTC | 1560 |
CTGAAAAAGA | CCAATGCTTC | TGTGCTATTA | AAGAAAATTC | TATTGTAGAA | TCTAACTTTC | 1620 |
TAACCAATGT | TGTGACAGAA | GGTCCTATGG | ATTGTATACC | TTATCAAGAA | TGCAAAGGCA | 1680 |
GGATAACTGA | AAATGCTTTA | GTAACTTTTG | TCAAATGCAG | ATTTGGTTGT | GAGTATGCCT | 1740 |
CTATTTTCCA | AAGCAAGCCT | TTAGATAATG | GGTTCTTAGA | ATATTCTGGT | GACACATTGG | 1800 |
GATTGAATGC | TGTGAATCTA | CACTTCATGA | AAAGGCTTAG | GAATGGTATA | ATTGATTTCT | 1860 |
ACAATAAAAC | TGAAAAATAT | GGATACATTT | CTGGAGATGC | ACTTAAATCT | AATGAATCAG | 1920 |
ACATACCTGA | AAGCATTTTT | CCAAGAAAAT | CTCTCATATT | TGACTCGGTG | ATAGATGGAA | 1980 |
AATATAGATA | CATGATAGAA | GAATCTTTAT | TATCAGGTGG | GGGCACAGTT | TTCTCTCTTA | 2040 |
ATGACAAAAG | TTCTAGCACT | GCTCAGAAGT | TTGTTGTTTA | TATTAAAAAA | GTTAGAATAC | 2100 |
AGTATGATGT | TTCTGAACAA | TACACTACAG | CTCCTATACA | AAGCACCCAC | ACTGATTTCT | 2160 |
TTTCAACATG | CACAGGTAAA | TGCTCAGATT | GCAGAAAAGA | ACAACCGATA | ACTGGGTATC | 2220 |
AAGATTTCTG | CATAACACCA | ACATCTTACT | GGGGTTGTGA | AGAGGTTTGG | TGTTTGGCTA | 2280 |
TCAATGAAGG | AGCCACTTGT | GGGTTTTGTA | GAAATGTGTA | TGATATGGAT | CAATCTTTCA | 2340 |
GGATTTATTC | TGTTATTAAA | TCAACAATCA | AGTCTGAAGT | ATGTATATCT | GGATTTGTGG | 2400 |
GAGCAAAGTG | TTTCACTGTA | TCTGAGGAAG | TCCCATCTGA | ATCAGGATAT | TTCCAGGCTG | 2460 |
ATATATTAGC | GGATTTCCAT | AATGATGGCT | TAACAATAGG | CCAGCTGATA | GCTCATGGAC | 2520 |
CTGATAGTCA | CGTATATGCG | GGAAACATAG | CTAGATTAAA | TAATCCATCA | AAAATGTTTG | 2580 |
GTCATCCTCA | ACTTTCACAC | CAAGGTGATC | CCATTTTCTC | TAAGAAAACA | TTAGATACAA | 2640 |
ACGATCTGTC | CTGGGATTGT | TCAGCAATTG | GTAAAAAAAC | AATTACGATA | AAATCATGTG | 2700 |
GTTATGACAC | ATACAGATTT | AAAACAGGTT | TGAACCAAAT | ATCGGACATT | CCAGTTCAGT | 2760 |
TTACTGACCA | GAATAGTTTT | TATATGGAAA | AGATCTTTAG | TCTAGGTAAG | CTTAAAATTG | 2820 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
TTTTGGATCT | ACCTTCTGAA | TTGTTTAAAA | CTGTACCAAA | AAAGCCTATA | TTGAGTTCTG | 2880 | |
TCTCTTTAAG | CTGTAAGGGA | TGTTTCTTAT | GTAGCCAAGG | GCTGAGGTGT | GCTGCCTCAT | 2940 | |
TTATATCAGA | CATAACTTTT | TCTGCAAGGT | TAACCATGAA | ACAATGTTCA | TTGTCAACAT | 3000 | |
ACCAGATAGC | AGTAAAGAAA | GGTGCTAATA | AGTATAACTT | GACAATGTTC | TGCACTTCTA | 3060 | |
ACCCAGAAAA | ACAAAAAATG | ATTATAGAAC | CAGAAGGAGA | CAAATCATAC | TCAGTTGAGG | 3120 | |
CACTTGTAGA | TTCTGTTGCT | GTGCTGGAAC | CAGAAAACAT | AATTGATCAA | AATGACCAAC | 3180 | |
ATGCACACGA | GGAACAGCAA | TATAATTCTG | ACACATCAGT | TTGGAGCTTT | TGGGACTATG | 3240 | |
TTAAAAGCCC | TTTTAACTTT | ATAGCAAGCC | ACTTTGGATC | CTTCTTTGAT | ACAGTCAGAG | 3300 | |
TAGTATTGCT | CATACTCTTT | GTATTTGCTC | TTGCTTACCT | CTGCTCTATT | GTTGCCACAA | 3360 | |
) | TGTGTAGAGG | CTATGTTAGA | AACAAGTCTT | ACAAGACAAA | ATATATTGAA | GACACTAATG | 3420 |
ATTATTCTTT | GGTTTCCACG | TCTTCTGGTA | AAGACACCAT | CACTAGAAGA | AGACCTCCTC | 3480 *' | |
TAGACTTCAG | CGGTATATGA | TTAAATTCAT | TGGAATATAC | ACCAAATGTG | TTTTAAATTA | 3540 | |
AATAAGGCAG | AATGATTGTA | ATCAAATAAA | TTTAATTTGT | CTAAATAAGT | GTTAAATAAG | 3600 | |
CAAACTATAT | AAAAAATATA | AAAATAATAA | AAATAAAAAT | CAACAAAAAC | AAATAAAATG | 3660 | |
TATAAAAACA | AATAAAAAGG | TCTTAGACCA | AATCTGGCCG | GAGCCTTATT | TATTTACAAA | 3720 | |
AAAAATTTTT | TTGGTTTTTT | TCAACTTTTT | TCTATTTTTT | GCTTTTTGTT | TGTTTTTGTT | 3730 | |
TTTGTTTTTG | TTTTTTGTTT | ATTTTTTCTT | TTTTCTTTTT | TGGTTTTTGA | TTTGTTTTTA | 3840 | |
AAC AC AAAT A | CATATATATT | TTATACTTCT | ATATATATAT | ATATATATAG | TTTTTTATCT | 3900 | |
TGCTTTATTT | TTTACTTAAA | CATTCAAAAT | ATTATGAACA | ATATATATTA | TTTATTATTA | 3960 | |
AATGAAAACA | TATTTAGATT | TCATTATCAA | ATGATATCTC | TATCTTGTTA | TCAGTAACAT | 4020 | |
TCTCCTCTTC | TTCAACAGAT | TTCTCTAAAT | TAGCACTTAG | CTCTGCAAGT | TGTCTTCTAA | 4090 | |
TTTGCTTTTC | AGAATTGCCT | TTAGGTATGA | TTAACTTGCA | GGCTTCAATG | AAGGCTTGCG | 4140 | |
ATTTGGCTCT | AATAGCCCTA | TTGATGGGTA | TTATCATGCA | GCTTTTATCC | AGATCTGCTC | 4200 | |
TTGGTGAATC | ACAGAATTCT | TTTGTCCAAG | AATACATGAC | ACTAGCAAAA | GAAACATCTT | 4250 | |
TCTTGTAAAC | TTGATCACAT | AATAAATGAA | GCTGAACACA | ATTCTCTGAA | GTGTTGTTAA | 4320 | |
CTTTTGGAAT | GGACCAATTT | AGATAAAAAA | CAAAACATAT | AGGATCTTTA | ATGCTTCCTT | 4330 | |
GCCCTTTCAA | AACAGTTCTG | GCATTAACAC | TCTTGTTAGG | ATCAATTAAA | GCTACAGCCA | 4440 | |
ATTTCCCATC | AGGATCAGCT | ATAGTAGGGC | ATATCCAGAT | AACTATCCTT | GAGATCATCA | 4500 | |
TGTATTGTTT | TCTGCTATCC | CAGGTTGGAT | GTATCTTAAT | TATCTTGGTT | GCAGCTTTAT | 4550 | |
CACCATTACC | TACAAAAAGA | TCATTTTTCC | AGCTGGAGAT | ATGATGATTT | GTATCAACAA | 4520 | |
TCATTCTAGC | AGAAAGATCA | TAAGACTCTG | ATTCAGATAT | CTGGTCAGAC | TCATATGTGC | 4530 | |
CTAGTGATGA | GGTGCCTGTA | TTGTTCATCA | AAATCTTCCC | TTTAGAGCTA | TCCATGGCTC | 4^40 | |
TTTGGAGAAC | TTCCTTGTTG | TTGTGATTAG | CAGAAGGATT | GTGTACAACA | ATGTCTGCTC | 4300 | |
CTTGTTTAGA | GAGGGTAGTT | GTAACCCTAG | GGTGATCTAG | CTCCCTGCTG | CTAGATGATC | 4 s50 | |
TGAGTGATTT | GAAAAAACTA | TTCATCTCTG | GACGGTAGAT | TAAGAATTAA | AATATTGAGA | 4'-20 | |
AGATAATGAT | TGAATTCGGC | TAGATATAAT | TTTGATGCAC | TGATTGCTCT | 4-70 |
bad ORIGINAL
AP Ο Ο Ο 4 Ο 3
SEQ ID NO: 21
Sequence type: Nucleotide
Sequence length: 3414 nucleotides
Strandedness: Single stranded
Molecule type: Complementary M RNA coding for the G1/G2 precursor proteins from position 87 to position 3500 of SEQ ID NO: 20
Original Source Organism:
ATGAAAATTC | TAAAGATGTG | CGAGTTACTT | GTTAAAATTA | GTGTATGTAC | ACTAGTTGTT | 60 |
ACTTCTGTTA | TCTTATCCTT | TATGGCATTA | AAGGAGACAG | ATGCAAAGAT | TCATGTTGAA | 120 |
AGGGGCGATC | ATCCAGAAAT | TTATGATGAA | GCTTATTATG | ACCGTTCTGT | AGATCATAAA | 180 |
AATGAAATTC | TAGATACTTT | GGCTGAAATG | CTCCAGAATG | CAACAGGTAA | AACCCTTAGA | 240 |
CCAACACGAG | ATACTCAAAC | TGTTCTTGCC | AATAATGAGG | TACCTCAATC | TCCAAGTGGA | 300 |
TTGTCTTCTA | CACCAACAAC | TATATCAGTT | ATGGATTTAC | CAAATCCATG | CCTAAATGCT | 360 |
TCAAGTTTGA | CTTGTAGCAT | TAAAGGAGTT | TCAACATTCA | ATGTTTATTA | CCAAGTAGAA | 420 |
AGCAATGGTG | TTATATATTC | atgtatcagt | GACACTATAA | CTAAATTGGG | AAACTGTGAA | 480 |
GGATCTTCTG | AATTGCCAAG | AAGCTTTGAA | ACTGTTCCAG | TTGTACCAAT | AACAAAAATC | 540 |
GATAACAAAA | GGAAATTATC | TATAGGAACT | aaattctata | TAATTGAAAG | TTTGGAGAAT | 600 |
TACAATTATC | CAATCATGTA | CAATTCTAGA | CCAACTAATG | GCACAGTCTC | TCTCCAGAGT | 660 |
GTGAAATTCT | CAGGTGATTG | TAAAATATCA | AAAACAAACA | TAGTTAATTC | TTATACAGTT | 720 |
TCATTAACTA | CACCTGAGAA | AATCATGGGT | TACGTCGTCA | AAAGAGAAGG | AAGTGACATG | 780 |
AGCCATTCCA | TAATAAGCTT | CTCTGGCTCA | GTTAGTTTGA | CTTTCACAGA | AGAAAATATG | 840 |
GATGGTAAGC | ATAACCTATT | GTGCGGTGAC | AAGTCTTCTA | AAGTTCCTTT | AGTGGACAAA | 900 |
AG AGTTAGAG | ATTGTATTAT | AAAGxAClCC | AAAAATATTT | ACAAACAAAC | TGCTTGCATC | 960 |
AACTTCTCAT | GGTTTAGATT | GATCATGATT | GCTCTGATTG | TCTATTTCCC | TATAAGGTAC | 1020 |
TTGGTAAACA | AAACATCTAA | AACACTGTTC | TATGGGTATG | ATCTTCTGGG | ATTGATAACG | 1080 |
TATCCTATAC | TACTACTGAT | AAATTATTTA | TGGTCTTATT | TTCCCTTGAA | ATGTAAGGTC | 1140 |
TGTGGAAATT | TATGTTTGGT | AACCCATGAA | TGCTCAAAAT | TATGCATTTG | TAACAAAAAC | 1200 |
AAAGCCTCTG | AAGAACACTC | AGAAGAATGT | CCTATAATAA | CTAGAACAGC | AGAGAAGAAT | 1260 |
AAAAAATACA | ACTGGGCTAG | CATTGAATGG | TTCCATTTGA | TAGTTAATAC | AAAAATAGGC | 1320 |
CTCTCTTTCC | TAAAAGCAGT | CACAGAAACT | TTGATAGGAT | TTCTGATTTT | GTCACAGATG | 1380 |
CCTATGTCTA | TGGCTCAAAC | TGCTCAGTGT | TTGGATAGCT | GTTATTATGT | CCCTGGTTGT | 1440 |
GACCGCTTTG | TTACAAACAG | ATATGACAAA | TGTCCTGAAA | AAGACCAATG | CTTCTGTGCT | 1500 |
ATTAAAGAAA | ATTCTATTGT | AGAATCTAAC | TTTCTAACCA | ATGTTGTGAC | AGAAGGTCCT | 1560 |
ATGGATTGTA | TACCTTATCA | AGAATGCAAA | GGCAGGATAA | CTGAAAATGC | TTTAGTAACT | 1620 |
TTTGTCAAAT | GCAGATTTGG | TTGTGAGTAT | GCCTCTATTT | TCCAAAGCAA | GCCTTTAGAT | 1680 |
APO0040 3
C'·
AATGGGTTCT | TAGAATATTC | TGGTGACACA | TTGGGATTGA | ATGCTGTGAA | TCTACACTTC | 1740 | |
ATGAAAAGGC | TTAGGAATGG | TATAATTGAT | TTCTACAATA | AAACTGAAAA | ATATGGATAC | 1800 | |
ATTTCTGGAG | ATGCACTTAA | ATCTAATGAA | TCAGACATAC | CTGAAAGCAT | TTTTCCAAGA | 1860 | |
AAATCTCTCA | TATTTGACTC | GGTGATAGAT | GGAAAATATA | GATACATGAT | AGAAGAATCT | 1920 | |
TTATTATCAG | GTGGGGGCAC | AGTTTTCTCT | CTTAATGACA | AAAGTTCTAG | CACTGCTCAG | 1980 | |
AAGTTTGTTG | TTTATATTAA | AAAAGTTAGA | ATACAGTATG | ATGTTTCTGA | ACAATACACT | 2040 | |
ACAGCTCCTA | TACAAAGCAC | CCACACTGAT | TTCTTTTCAA | CATGCACAGG | TAAATGCTCA | 2100 | |
GATTGCAGAA | AAGAACAACC | GATAACTGGG | TATCAAGATT | TCTGCATAAC | ACCAACATCT | 2160 | |
TACTGGGGTT | GTGAAGAGGT | TTGGTGTTTG | GCTATCAATG | AAGGAGCCAC | TTGTGGGTTT | 2220 | |
Θ | TGTAGAAATG | TGTATGATAT | GGATCAATCT | TTCAGGATTT | ATTCTGTTAT | TAAATCAACA | 2280 |
ATCAAGTCTG | AAGTATGTAT | ATCTGGATTT | GTGGGAGCAA | AGTGTTTCAC | TGTATCTGAG | 2340 ’ | |
GAAGTCCCAT | CTGAATCAGG | ATATTTCCAG | GCTGATATAT | TAGCGGATTT | CCATAATGAT | 2400 | |
GGCTTAACAA | TAGGCCAGCT | GATAGCTCAT | GGACCTGATA | GTCACGTATA | TGCGGGAAAC | 2460 | |
ATAGCTAGAT | TAAATAATCC | ATCAAAAATG | TTTGGTCATC | CTCAACTTTC | ACACCAAGGT | 2520 | |
GATCCCATTT | TCTCTAAGAA | AACATTAGAT | ACAAACGATC | TGTCCTGGGA | TTGTTCAGCA | 2580 | |
ATTGGTAAAA | AAACAATTAC | GATAAAATCA | TGTGGTTATG | ACACATACAG | ATTTAAAACA | 2640 | |
GGTTTGAACC | AAATATCGGA | CATTCCAGTT | CAGTTTACTG | ACCAGAATAG | TTTTTATATG | 2700 | |
GAAAAGATCT | TTAGTCTAGG | TAAGCTTAAA | ATTGTTTTGG | ATCTACCTTC | TGAATTGTTT | 2760 | |
AAAACTGTAC | CAAAAAAGCC | TATATTGAGT | TCTGTCTCTT | TAAGCTGTAA | GGGATGTTTC | 2820 | |
TTATGTAGCC | AAGGGCTGAG | GTGTGCTGCC | TCATTTATAT | CAGACATAAC | TTTTTCTGCA | 2880 | |
AGGTTAACCA | TGAAACAATG | TTCATTGTCA | ACATACCAGA | TAGCAGTAAA | GAAAGGTGCT | 2940 | |
) | AATAAGTATA | ACTTGACAAT | gttctgcact | TCTAACCCAG | AAAAACAAAA | AATGATTATA | 3000 |
a > t - . | GAACCAGAAG | GAGACAAATC | ATACTCAGTT | GAGGCACTTG | TAGATTCTGT | TGCTGTGCTG | 3060 |
( ’ | GAACCAGAAA | ACATAATTG ATCAAAATGAC | CAACATGCAC | ACGAGGAACA | GCAATATAAT | 3120 | |
TCTGACACAT | CAGTTTGGAG | CTTTTGGGAC | TATGTTAAAA | GCCCTTTTAA | CTTTATAGCA | 3180 | |
AGCCACTTTG | GATCCTTCTT | TGATACAGTC | AGAGTAGTAT | TGCTCATACT | CTTTGTATTT | 3240 | |
GCTCTTGCTT | acctctgctc | TATTGTTGCC | ACAATGTGTA | GAGGCTATGT | TAGAAACAAG | 3300 | |
TCTTACAAGA | CAAAATATAT | TGAAGACACT | AATGATTATT | CTTTGGTTTC | CACGTCTTCT | 3360 | |
GGTAAAGACA | CCATCACTAG | AAGAAGACCT | CCTCTAGACT | TCAGCGGTAT | ATGA | 3414 |
SEQ ID No. 22
Sequence types Nucleotide
Sequence length: 912 nucleotides
Strandedness: single stranded
Molecule type: Complementary viral M RNA from 3974 to 4885 of SEQ ID No. 20
AP Ο Ο Ο 4 Ο 3
I
TTAGATTTCA | TTATCAAATG | ATATCTCTAT | CTTGTTATCA | GTAACATTCT | CCTCTTCTTC | 60 |
AACAGATTTC | TCTAAATTAG | CACTTAGCTC | TGCAAGTTGT | CTTCTAATTT | GCTTTTCAGA | 120 |
ATTGCCTTTA | GGTATGATTA | ACTTGCAGGC | TTCAATGAAG | GCTTGCGATT | TGGCTCTAAT | 180 |
AGCCCTATTG | ATGGGTATTA | TCATGCAGCT | TTTATCCAGA | TCTGCTCTTG | GTGAATCACA | 240 |
GAATTCTTTT | GTCCAAGAAT | ACATGACACT | AGCAAAAGAA | ACATCTTTCT | TGTAAACTTG | 300 |
ATCACATAAT | AAATGAAGCT | GAACACAATT | CTCTGAAGTG | TTGTTAACTT | TTGGAATGGA | 360 |
CCAATTTAGA | TAAAAAACAA | AACATATAGG | atctttaatg | CTTCCTTGCC | CTTTCAAAAC | 420 |
AGTTCTGGCA | TTAACACTCT | TGTTAGGATC | AATTAAAGCT | ACAGCCAATT | TCCCATCAGG | 480 |
ATCAGCTATA | GTAGGGCATA | TCCAGATAAC | TATCCTTGAG | ATCATCATGT | ATTGTTTTCT | 540 |
GCTATCCCAG | GTTGGATGTA | TCTTAATTAT | CTTGGTTGCA | GCTTTATCAC | CATTACCTAC | 600 |
AAAAAGATCA | TTTTTCCAGC | TGGAGATATG | ATGATTTGTA | TCAACAATCA | TTCTAGCAGA | 660 |
AAGATCATAA | GACTCTGATT | CAGATATCTG | GTCAGACTCA | TATGTGCCTA | GTGATGAGGT | 720 |
GCCTGTATTG | TTCATCAAAA | TCTTCCCTTT | AGAGCTATCC | ATGGCTCTTT | GGAGAACTTC | 780 |
CTTGTTGTTG | TGATTAGCAG | AAGGATTGTG | TACAACAATG | TCTGCTCCTT | GTTTAGAGAG | 840 |
GGTAGTTGTA | ACCCTAGGGT | GATCTAGCTC | CCTGCTGCTA | GATGATCTGA | GTGATTTGAA | 900 |
AAAACTATTC | AT | 912 |
SEQ ID No. 23
Sequence type: Nucleotide
Sequence length: 446 nucleotides
Strandedness: Single stranded
Molecule type: CaMV 35S' promoter and Ω sequence of tobacco mosaic virus Original Source Organism:
GGATCCGGAA CATGGTGGAG CACGACACGC TTGTCTACTC CAAAAATATC 50 AAAGATACAG TCTCAGAAGA CCAAAGGGCA ATTGAGACTT TTCAACAAAG 100 TTATTGTGAA GATAGTGGAA AAGGAAGGTG GCTCCTACAA ATGCCATCAT 150 TGCGATAAAG GAAAGGCCAT CGTTGAAGAT GCCTCTGCCG ACAGTGGTCC 200 CAAAGATGGA CCCCCACCCA CGAGGAGCAT CGTGGAAAAA GAAGACGTTC 250 CAACCACGTC TTCAAAGCAA GTGGATTGAT GTGATATCTC CACTGACGTA 300 AGGGATGACG CACAATCCCA CTATCCTTCG CAAGACCCTT CCTCTATATA 350 AGGAAGTTCA TTTCATTTGG AGAGGACTTT TTACAACAAT TACCAACAAC 400
AP Ο Ο Ο 4 Ο 3
SEQ ID NO :24
Sequence type:Amino acid
Sequence length: 303 amino acids (NSm protein)
Original Source Organism: Impatiens Necrotic Spot Virus c
Met | Asn | Ser | Phe | Phe 5 | Lys | Ser | Leu | Arg | Ser 10 | Ser | Ser | Ser | Arg | Glu 15 | 15 |
Leu | Asp | His | Pro | Arg 20 | Val | Thr | Thr | Thr | Leu 25 | Ser | Lys | Gin | Gly | Ala 30 | 30 |
Asp | lie | Val | Val | His 35 | Asn | Pro | Ser | Ala | Asn 40 | His | Asn | Asn | Lys | Glu 45 | 45 |
Val | Leu | Gin | Arg | Ala 50 | Met | Asp | Ser | Ser | Lys 55 | Gly | Lys | He | Leu | Met 60 | 60 |
Asn | Asn | Thr | Gly | Thr 65 | Ser | Ser | Leu | Gly | Thr 70 | Tyr | Glu | Ser | Asp | Gin 75 | 75 |
lie | Ser | Glu | Ser | Glu 80 | Ser | Tyr | Asp | Leu | Ser 85 | Ala | Arg | Met | lie | Val 90 | 90 |
Asp | Thr | Asn | His | His 95 | lie | Ser | Ser | Trp | Lys 100 | Asn | Asp | Leu | Phe | Val 105 | 105 |
BAD ORIGINAL £
AP Ο Ο Ο 4 Ο 3
Gly Asn Gly Asp Lys | Ala Ala Thr Lys | lie 115 | lie | Lys | He | His | Pro 120 | 120 | |||||||
110 | |||||||||||||||
Thr | Trp | Asp | Ser | Arg | Lys | Gin | Tyr | Met | Met | lie | Ser | Arg | He | Val | 135 |
125 | 130 | 135 | |||||||||||||
lie | Trp | lie | Cys | Pro | Thr | lie | Ala | Asp | Pro | Asp | Gly | Lys | Leu | Ala | 150 |
140 | 145 | 150 | |||||||||||||
Val | Ala | Leu | lie | Asp | Pro | Asn | Lys | Ser | Val | Asn | Ala | Arg | Thr | Val | 165 |
155 | 160 | 165 | |||||||||||||
Leu | Lys | Gly | Gin | Gly | Ser | He | Lys | Asp | Pro | He | Cys | Phe | Val | Phe | 180 |
170 | 175 | 180 | |||||||||||||
Tyr | Leu | Asn | Trp | Ser | He | Pro | Lys | Val | Asn | Asn | Thr | Ser | Glu | Asn | 195 |
185 | 190 | 195 | |||||||||||||
Cys | Val | Gin | Leu | His | Leu | Leu | Cys | Asp | Gin | Val | Tyr | Lys | Lys | Asp | 210 |
200 | 205 | 210 | |||||||||||||
Val | Ser | Phe | Ala | Ser | Val | Met | Tyr | Ser | Trp | Thr | Lys | Glu | Phe | Cys | 225 |
215 | 220 | . | 225 | ||||||||||||
Asp | Ser | Pro | Arg | Ala | Asp | Leu | Asp | Lys | Ser | Cys | Met | He | He | Pro | 240 |
230 | 235 | 240 | |||||||||||||
He | Asn | Arg | Ala | He | Arg | Ala | Lys | Ser | Gin | Ala | Phe | He | Glu | Ala | 255 |
245 | 250 | 255 | |||||||||||||
Cys | Lys | Leu | He | He | Pro | Lys | Gly | Asn | Ser | Glu | Lys | Gin | lie | Arg | 270 |
260 | 265 | 270 | |||||||||||||
Arg | Gin | Leu | Ala | Glu | Leu | Ser | Ala | Asn | Leu | Glu | Lys | Ser | Val | Glu | 285 |
275 | 280 | 285 | |||||||||||||
Glu | Glu | Glu | Asn | Val | Thr | Asp | Asn | Lys | lie | Glu | lie | Ser | Phe | Asp | 300 |
290 | 295 | 300 | |||||||||||||
Asn | Glu | lie | 3 03 |
SEQ ID NO :25
Sequence types Amino acids
Sequence length: 262 amino acids (N protein)
Original Source Organism: Impatiens Necrotic Spot Virus
Met | Asn | Lys | Ala | Lys 5 | He | Thr | Lys | Glu | Asn 10 | lie | Val | Lys | Leu | Leu 15 | 15 |
Thr | Gin | Ser | Asp | Ser 20 | Leu | Glu | Phe | Glu | Glu 25 | Thr | Gin | Asn | Glu | Gly 30 | 3 0 |
Ser | Phe | Asn | Phe | Thr 35 | Asp | Phe | Phe | Thr | Asn 40 | Asn | Arg | Glu | Lys | lie 45 | 45 |
Gin | Asn | Met | Thr | Thr 50 | Ala | Ser | Cys | Leu | Ser 55 | Phe | Leu | Lys | Asn | Arg 60 | 6 ·? |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3 οο
Gin | Ser | He | Met | Arg 65 | Val | He | Lys | Ser | Ala 70 | Asp | Phe | Thr | Phe | Gly 75 | 75 |
Ser | Val | Thr | He | Lys 80 | Lys | Thr | Arg | Asn | Asn 85 | Ser | Glu | Arg | Val | Gly 90 | 90 |
Val | Asn | Asp | Met | Thr 95 | Phe | Arg | Arg | Leu | Asp 100 | Ala | Met | Val | Arg | Val 105 | 105 |
His | Leu | Val | Gly | Met 110 | lie | Lys | Asp | Asn | Gly 115 | Ser | Ala | Leu | Thr | Glu 120 | 120 |
Ala | lie | Asn | Ser | Leu 125 | Pro | Ser | His | Pro | Leu 130 | lie | Ala | Ser | Tyr | Gly 135 | 135 |
Leu | Ala | Thr | Thr | Asp 140 | Leu | Lys | Ser | Cys | Val 145 | Leu | Gly | Val | Leu | Leu 150 | 150 |
Gly | Gly | Ser | Leu | Pro 155 | Leu | He | Ala | Ser | Val 160 | Leu | Asn | Phe | Glu | He 165 | 165 |
Ala | Ala | Leu | Val | Pro 170 | Ala | He | Tyr | Gin | Asp 175 | Ala | Lys | His | Val | Glu 180 | 180 |
Leu | Gly | He | Asp | Met 185 | Ser | Lys | Phe | Ser | Thr 190 | Lys | Glu | Ala | Val | Gly 195 | 195 |
Lys | Val | Cys | Thr | Val | Leu | Lys | Ser | Lys | Gly | Tyr | Ser | Met | Asn | Ser | 210 |
200 | 205 | 210 | |||||||||||||
Val | Glu | He | Gly | Lys | Ala | Lys | Gin | Tyr | Ala | Asp | lie | Leu | Lys | Ala | 225 |
215 | 220 | 225 | |||||||||||||
Cys | Ser | Pro | Lys | Ala | Lys | Gly | Leu | Ala | Ala | Met | Asp | His | Tyr | Lys | 240 |
230 | 235 | 240 | |||||||||||||
Glu | Gly | Leu | Thr | Ser | lie | Tyr | Ser | Met | Phe | Asn | Ala | Thr | lie | Asp | 255 |
245 | 250 | 255 | |||||||||||||
Phe | Gly | Lys | Asn | Asp | Ser | lie | 262 |
260
SEQ ΙΟ NO: 26
Sequence type: Amino acid
Sequence length: 449 amino acids
Molecule type: NSs protein
Original Source Organism: Impatiens Necrotic Spot Virus
Met Ser Ser Ala Met lyr Glu Thr lie He Lys Ser Lys Ser Ser 15 5 10 15 lie Trp Gly Thr Thr Ser Ser Gly Lys Ala Val Val Asp Ser Tyr 30
AP Ο Ο Ο 4 Ο 3 (?)
Trp | Ile | His | Asp | Gin 35 | Ser | Ser | Gly | |
Leu | Tyr | Ser | Asp | Ser 50 | Arg | Ser | Lys | |
Lys | Val | Gly | Phe | Leu 65 | Pro | Thr | Glu | |
Cys | Phe | Val | Pro | Ile 80 | Phe | Asp | Asp | |
& | Ser | Gly | Asn | Val | Val 95 | Glu | Ile | Leu |
C | Thr | Asn | Gly | Val | Lys 110 | His | Gin | Gly |
Gin | Leu | Leu | Arg | Met 125 | Leu | Glu | Glu | |
Thr | Ser | Arg | Phe | Gly 140 | Leu | Lys | Glu | |
Asn | Phe | Ile | Glu | Ala 155 | Ala | Asn | Lys | |
Glu | Val | Leu | Phe | Asp 170 | Val | Lys | Tyr | |
Lys | Val | Ser | Val | Leu 185 | Ser | Pro | Thr | |
4 | Tyr | Thr | Leu | Lys | Pro 200 | Val | Phe | Asn |
( | Thr | Val | Asn | Thr | Leu 215 | Ala | Val | Lys |
Ser | Asp | Leu | Met | Ser 230 | Asp | Thr | His | |
Asn | Lys | Pro | Phe | Lys 245 | Ile | Ser | Leu | |
Met | Lys | Ser | Asn | Thr 260 | Tyr | Ser | Arg | |
Ser | Ser | Pro | Lys | Glu 275 | Tyr | Tyr | Ile | |
His | Asn | Asn | Val | Glu 290 | Thr | Val | Ile | |
Asn | Leu | Phe | Leu | Asn 305 | Gin | Leu | Leu | |
Glu | Met | Asn | Phe | Ser 320 | Asp | Leu | Lys |
Lys | Lys 40 | Leu | Val | Glu | Ala | Gin 45 | 45 |
Thr | Ser 55 | Phe | Cys | Tyr | Thr | Gly 60 | 60 |
Glu | Lys 70 | Glu | Ile | Ile | Val | Arg 75 | 75 |
Ile | Asp 85 | Leu | Asn | Phe | Ser | Phe 90 | 90 |
Val | Arg 100 | Ser | Asn | Thr | Thr | Asn 105 | 105 |
His | Leu 115 | Lys | Val | Leu | Ser | Ser 120 | 120 |
Gin | Ile 130 | Ala | Val | Pro | Glu | Ile 135 | 135 |
Ser | Asp 145 | Ile | Phe | Pro | Pro | Asn 150 | 150 |
Gly | Ser 160 | Leu | Ser | Cys | Val | Lys 165 | 165 |
Ser | Asn 175 | Asn | Gin | Ser | Met | Gly 180 | 180 |
Arg | Ser 190 | Val | His | Glu | Trp | Leu 195 | 195 |
Gin | Ser 205 | Gin | Thr | Asn | Asn | Arg 210 | 210 |
Ser | Leu 220 | Ala | Met | Ser | Ala | Thr 225 | 225 |
Ser | Phe 235 | Val | Arg | Leu | Asn | Asn 240 | 240 |
Trp | Met 250 | Arg | Ile | Pro | Lys | Ile 255 | 255 |
Phe | Phe 265 | Thr | Leu | Ser | Asp | Glu 270 | 270 |
Ser | Ile 280 | Gin | Cys | Leu | Pro | Asn 285 | 285 |
Glu | Tyr 295 | Asn | Phe | Asp | Gin | Ser 300 | 300 |
Leu | Ala 310 | Val | He | His | Lys | Ile 315 | 315 |
Glu | Pro 325 | Tyr | Asn | Val | Ile | His 330 | 330 |
AP Ο Ο Ο 4 Ο 3
Asp Met | Ser | Tyr Pro 335 | Gin | Arg | Ila | Val His 340 | Ser Leu Leu | Glu lie 345 | 345 |
His Thr | Glu | Leu Ala 350 | Gin | Thr | Val | Cys Asp 355 | Ser Val Gin | Gin Asp 360 | 360 |
Met He | Val | Phe Thr 365 | He | Asn | Glu | Pro Asp 3.70 | Leu Lys Pro | Lys Lys 375 | 375 |
Phe Glu | Leu | Gly Lys 380 | Lys | Thr | Leu | Asn Tyr 385 | Ser Glu Asp | Gly Tyr 390 | 390 |
Gly Arg | Lys | Tyr Phe 395 | Leu | Ser | Gin | Thr Leu 400 | Lys Ser Leu | Pro Arg 405 | 405 |
Asn Ser | Gin | Thr Met 410 | Ser | Tyr | Leu | Asp Ser 415 | lie Gin Met | Pro Asp 420 | 420 |
Trp Lys | Phe | Asp Tyr 425 | Ala | Ala | Gly | Glu lie 430 | Lys lie Ser | Pro Arg 435 | 435 |
Ser Glu Asp Val Leu Lys Ala lie Ser Lys Leu Asp Leu 440 445 . SEQ ID NO :27 Sequence type: Amino acid Sequence length: 1137 eunino acids (G1G2 precursor protein) Original Source Organism: Impatiens Necrotic Spot Virus | Asn 1 | 449 | |||||||
Met Lys | lie | Leu Lys 5 | Met | Cys | Glu | Leu Leu 10 | Val Lys lie | Ser Val 15 | 15 |
Cys Thr | Leu | Val Val 20 | Thr | Ser | Val | lie Leu 25 | Ser Phe Met | Ala Leu 30 | 30 |
Lys Glu | Thr | Asp Ala 35 | Lys | lie | His | Val Glu 40 | Arg Gly Asp | His Pro 45 | 45 |
Glu lie | Tyr | Asp Glu 50 | Ala | Tyr | Tyr | Asp Arg 55 | Ser Val Asp | His Lys 60 | 60 |
Asn Glu | lie | Leu Asp 65 | Thr | Leu | Ala | Glu Met 70 | Leu Gin Asn | Ala Thr 75 | 75 |
Gly Lys | Thr | Leu Arg 80 | Pro | Thr | Arg | Asp Thr 85 | Gin Thr Val | Leu Ala 90 | 90 |
Asn Asn | Glu | Val Pro 95 | Gin | Ser | Pro | Ser Gly 100 | Leu Ser Ser | Thr Pro 105 | 105 |
Thr Thr | lie | Ser Val 110 | Met | Asp | Leu | Pro Asn 115 | Pro Cys Leu | Asn Ala 120 | 120 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
Ser | Ser | Leu | Thr | Cys 125 | Ser | He | Lys | Gly Val 130 | Ser | Thr | Phe | Asn | Val 135 | 135 |
Tyr | Tyr | Gin | Val | Glu 140 | Ser | Asn | Gly | Val lie 145 | Tyr | Ser | Cys | He | Ser 150 | 150 |
Asp | Thr | He | Thr | Lys 155 | Leu | Gly | Asn | Cys Glu 160 | Gly | Ser | Ser | Glu | Leu 165 | 165 |
Pro | Arg | Ser | Phe | Glu 170 | Thr | Val | Pro | Val Val 175 | Pro | lie | Thr | Lys | lie 180 | 180 |
Asp | Asn | Lys | Arg | Lys 185 | Leu | Ser | lie | Gly Thr 190 | Lys | Phe | Tyr | lie | He 195 | 195 |
Glu | Ser | Leu | Glu | Asn 200 | Tyr | Asn | Tyr | Pro He 205 | Met | Tyr | Asn | Ser | Arg 210 | 210 |
Pro | Thr | Asn | Gly | Thr 215 | Val | Ser | Leu | Gin Ser 220 | Val | Lys | Phe | Ser | Gly 225 | 225 |
Asp | Cys | Lys | He | Ser 230 | Lys | Thr | Asn | lie Val 235 | ^sn | Ser | Tyr | Thr | Val 240 | 240 |
Ser | Leu | Thr | Thr | Pro 245 | Glu | Lys | lie | Met Gly 250 | Tyr | Val | Val | Lys | Arg 255 | 255 |
Glu | Gly | Ser | Asp | Met 260 | Ser | His | Ser | lie lie 265 | Ser | Phe | Ser | Gly | Ser 270 | 270 |
Val | Ser | Leu | Thr | Phe 275 | Thr | Glu | Glu | Asn Met 280 | Asp | Gly | Lys | His | Asn 285 | 285 |
Leu | Leu | Cys | Gly | Asp 290 | Lys | Ser | Ser | Lys Val 295 | Pro | Leu | Val | Asp | Lys 300 | 300 |
Arg | Val | Arg | Asp | Cys 305 | lie | lie | Lys | Tyr Ser 310 | Lys | Asn | He | Tyr | Lys 315 | 315 |
Gin | Thr | Ala | Cys | He 320 | Asn | Phe | Ser | Trp Phe 325 | Arg | Leu | He | Met | lie 330 | 330 |
Ala | Leu | lie | Val | Tyr 335 | Phe | Pro | lie | Arg Tyr 340 | Leu | Val | Asn | Lys | Thr 345 | 345 |
Ser | Lys | Thr | Leu | Phe 350 | Tyr | Gly | Tyr | Asp Leu 355 | Leu | Gly | Leu | lie | Thr 360 | 360 |
Tyr | Pro | lie | Leu | Leu 365 | Leu | He | Asn | Tyr Leu 370 | Trp | Ser | Tyr | Phe | Pro 375 | 375 |
Leu | Lys | Cys | Lys | Val 380 | Cys | Gly | Asn | Leu Cye 385 | Leu | Val | Thr | His | Glu 390 | 390 |
Cys | Ser | Lys | Leu | Cys 395 | lie | Cys | Asn | Lys Asn 400 | Lys | Ala | Ser | Glu | Glu 405 | 405 |
His | Ser | Glu | Glu | Cys 410 | Pro | He | He | Thr Arg 415 | Thr | Ala | Glu | Lys | Asn 420 | 420 |
BAD ORIGINAL &
AP Ο Ο Ο 4 Ο 3 ) 72
Lys | Lys | Tyr | Asn | Trp 425 | Ala | Ser | lie | Glu | Trp 430 | Phe | His | Leu | lie | Val 435 | 435 | |
As η | Thr | Lys | He | Gly 440 | Leu | Ser | Phe | Leu | Lys 445 | Ala | Val | Thr | Glu | Thr 450 | 450 | |
Leu | He | Gly | Phe | Leu 455 | lie | Leu | Ser | Gin | Met 460 | Pro | Met | Ser | Met | Ala 465 | 465 | |
Gin | Thr | Ala | Gin | Cys 470 | Leu | Asp | Ser | Cys | Tyr 475 | Tyr | Val | Pro | Gly | Cys 480 | 480 | |
Asp | Arg | Phe | Val | Thr 485 | Asn | Arg | Tyr | Asp | Lys 490 | Cys | Pro | Glu | Lys | Asp 495 | 495 | |
'\ | Gin | Cys | Phe | Cys | Ala 500 | lie | Lys | Glu | Asn | Ser 505 | He | Val | Glu | Ser | Asn 510 | 510 |
<· | Phe | Leu | Thr | Asn | Val 515 | Val | Thr | Glu | Gly | Pro 520 | Met | Asp | Cys | lie | Pro 525 | 525 |
Tyr | Gin | Glu | Cys | Lys 530 | Gly | Arg | lie | Thr | Glu 535 | Asn | Ala | Leu | Val | Thr 540 | 540 | |
Phe | Val | Lys | Cys | Arg 545 | Phe | Gly | Cys | Glu | Tyr 550 | Ala | Ser | lie | Phe | Gin 555 | 555 | |
Ser | Lys | Pro | Leu | Asp 560 | Asn | Gly | Phe | Leu | Glu 565 | Tyr | Ser | Gly | Asp | Thr 570 | 570 | |
Leu | Gly | Leu | Asn | Ala 575 | Val | Asn | Leu | His | Phe 580 | Met | Lys | Arg | Leu | Arg 585 | 585 | |
Asn | Gly | lie | He | Asp 590 | Phe | Tyr | Asn | Lys | Thr 595 | Glu | Lys | Tyr | Gly | Tyr 600 | 600 | |
C | lie | Ser | Gly | Asp | Ala 605 | Leu | Lys | Ser | Asn | Glu 610 | Ser | Asp | He | Pro | Glu 615 | 615 |
< | Ser | lie | Phe | Pro | Arg 620 | Lys | Ser | Leu | lie | Phe 625 | Asp | Ser | Val | He | Asp 630 | 630 |
Gly | Lys | Tyr | Arg | Tyr 635 | Met | He | Glu | Glu | Ser 640 | Leu | Leu | Ser | Gly | Gly 645 | 645 | |
Gly | Thr | Val | Phe | Ser 650 | Leu | Asn | Asp | Lys | Ser 655 | Ser | Ser | Thr | Ala | Gin 660 | 660 | |
Lys | Phe | Val | Val | Tyr 665 | lie | Lys | Lys | Val | Arg 670 | Ila | Gin | Tyr | Asp | Val 675 | 675 | |
Ser | Glu | Gin | Tyr | Thr 680 | Thr | Ala | Pro | He | Gin 685 | Ser | Thr | His | Thr | Asp 690 | 590 | |
Phe | Phe | Ser | Thr | Cys 695 | Thr | Gly | Lys | Cys | Ser 700 | Asp | Cys | Arg | Lys | Glu 705 | 7 05 | |
Gin | Pro | lie | Thr | Gly 710 | Tyr | Gin | Asp | Phe | Cys 715 | lie | Thr | Pro | Thr | Ser 720 | <» 1 1 4. V |
BAD ORIGINAL &
AP Ο Ο Ο 4 Ο 3
Ο ™
Tyr Trp Gly Cys Glu Glu Val Trp Cys Leu Ala lie Asn Glu Gly 735 725 730 735
Ala Thr Cys Gly Phe Cys Arg Asn Val Tyr Asp Met Asp Gin Ser 7 50 740 745 750
Phe Arg lie Tyr Ser Val lie Lys Ser Thr lie Lys Ser Glu Val 765 755 760 765
Cys He Ser Gly Phe Val Gly Ala Lys Cys Phe Thr Val Ser Glu 780 770 775 780
Glu Val Pro Ser Glu Ser Gly Tyr Phe Gin Ala Asp He Leu Ala 795 785 790 795
Asp Phe His Asn Asp Gly Leu Thr lie Gly Gin Leu lie Ala His 810 800 805 810 /', Gly Pro Asp Ser His Val Tyr Ala Gly Asn lie Ala Arg Leu Asn 825
815 820 825
Asn Pro Ser Lys Met Phe Gly His Pro Gin Leu Ser His Gin Gly 840 830 835 840
Asp Pro He Phe Ser Lys Lys Thr Leu Asp Thr Asn Asp Leu Ser 855 845 850 855
Trp Asp Cys Ser Ala He Gly Lys Lys Thr He Thr He Lys Ser 870 860 865 870
Cys Gly Tyr Asp Thr Tyr Arg Phe Lys Thr Gly Leu Asn Gin He 385 875 880 885
Ser Asp lie Pro Val Gin Phe Thr Asp Gin Asn Ser Phe Tyr Met 900 890 895 900
Glu Lys He Phe Ser Leu Gly Lys Leu Lys He Val Leu Asp Leu 915 905 910 915 ( Pro Ser Glu Leu Phe Lys Thr Val Pro Lys Lys Pro He Leu Ser 930
920 925 930
Ser Val Ser Leu Ser Cys Lys Gly Cys Phe Leu Cys Ser Gin Gly 945 935 940 945
Leu Arg Cys Ala Ala Ser Phe He Ser Asp He Thr Phe Ser Ala 960 950 955 960
Arg Leu Thr Met Lys Gin Cys Ser Leu Ser Thr Tyr Gin He Ala 975 965 970 975
Val Lys Lys Gly Ala Asn Lys Tyr Asn Leu Thr Met Phe Cys Thr 990 980 985 990
Ser Asn Pro Glu Lys Gin Lys Met He He Glu Pro Glu Gly Asp 1005 995 1000 1005
Lys Ser Tyr Ser Val Glu Ala Leu Val Asp Ser Val Ala Val Leu 1020
AP Ο Ο Ο 4 Ο 3
Glu | Pro Glu Asn lie 1025 | He Asp Gin Asn Asp Gin His Ala His Glu 1035 | |||||||||||
1030 | 1035 | ||||||||||||
Glu | Gin | Gin | Tyr Asn | Ser | Asp | Thr | Ser Val | Trp | Ser | Phe | Trp Asp | 1050 | |
1040 | 1045 | 1050 | |||||||||||
Tyr | Val | Lys | Ser Pro | Phe | Asn | Phe | lie Ala | Ser | His | Phe | Gly Ser | 1065 | |
1055 | 1060 | 1065 | |||||||||||
Phe | Phe | Asp | Thr Val | Arg | Val | Val | Leu Leu | He | Leu | Phe | Val Phe | 1080 | |
1070 | 1075 | 1080 | |||||||||||
Ala | Leu | Ala | Tyr Leu | Cys | Ser | lie | Val Ala | Thr | Met | Cys | Arg Gly | 1095 | |
1085 | 1090 | 1095 | |||||||||||
Tyr | Val | Arg | Asn Lys | Ser | Tyr | Lys | Thr Lys | Tyr | He | Glu | Asp Thr | 1110 | |
1100 | 1105 | 1110 | |||||||||||
ρ | |||||||||||||
Asn | Asp | Tyr | Ser Leu | Val | Ser | Thr | Ser Ser | Gly | Lys | Asp | Thr He | 1125 | |
1115 | 1120 | 1125 | |||||||||||
Thr | Arg | Arg | Arg Pro | Pro | Leu | Asp | Phe Ser | Gly | lie | 1137 | |||
1130 | 1135 |
BAD ORIGINAL
AP Ο Ο Ο 4 Ο 3
HAVING NOW PARTICULARIV DFSCRI8FD AND ASCFR?*·—rf ·\ ΛΗΑΤ MA.V.·.» Ή· :... · ·,
ΤΟ βν ri.nl OrtiVlEL;. I, Μ DlClARL lhAI rthAi * Λ, Ci-,· .· ·,
Claims (6)
- CLAIMS: 1. Recombinant INSV DNA constructs comprising a DNA sequence coding for transcription intoa) an RNA sequence of an INSV or an RNA sequence homologous thereto ;b) an RNA sequence of an INSV or an RNA sequence homologous thereto capable of encoding for an INSV protein or a part thereof , in which one or more codons have been replaced by synonyms , or an RNA sequence homologous thereto ; orc) an RNA sequence complementary to an RNA sequence according to a) or b), which INSV DNA is under expression control of a promoter and a terminator capable of functioning in plants.
- 2. A DNA construct according to Claim 1, wherein the INSV DNA sequences code for transcription into:i) the viral S RNA nucleotide sequence from 1 to 3017 (SEQ. ID No.l) ii) the viral S RNA nucleotide sequence from position 25 to 3017 (SEQ. ID No.2);iii) the viral S RNA nucleotide sequence from 87 to 1436 (SEQ. ID No.3);iv) the viral S RNA nucleotide sequence from 2080 to 2868 (SEQ. ID No.4);BAD ORIGINALAPΟ Ο ο 4 Ο 3ν) the viral S RNA * pan-handle · structure comprisinga) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral S RNA andb) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral S RNA vi) the viral S RNA nucleotide sequence from 1437 to 2079; (SEQ ID No. 7) vii) the viral S RNA nucleotide sequence from 1440 to 2041; (SEQ ID No.8) viii) the viral complementary S RNA nucleotide sequence from 1 to about 3017; (SEQ ID No.9) ix) the viral complementary S RNA nucleotide sequence from 1 to 2993; (SEQ ID No.10)x) the viral complementary S RNA nucleotide sequence from 150 to 938; (SEQ ID No.11) xi) the S RNA nucleotide sequence from 1581 to 2930 of the viral complementary S RNA strand; (SEQ ID No.12);xii) the viral complementary S RNA secondary structure having a nucleotide sequence of 642 nucleotides from 939 to 1580; (SEQ ID No.13)BAD ORIGINAL ftAP Ο Ο Ο 4 Ο 3Ο xiii) xiv) χν) xvi) xvii) xviii) xix) txx) xxi)S RNA nucleotide sequence from 87 to 1436 in which one or more codons have been replaced by their synonyms;S RNA nucleotide sequence from 2080 to 2868 in which one or more codons have been replaced by their synonyms;the M RNA nucleotide sequence from 1 to 4970 (SEQ ID NO.14);the M RNA sequence from 86 to 997 (SEQ ID No.15);the M RNA sequence of the intergenic region from 993 to 1470 (SEQ ID No.16);the M RNA sequence from 1471 to 4884; (SEQ ID No. 17) the M RNA pan-handle structure comprising : a) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral M RNA andb) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral M RNA;the complementary viral M RNA sequence from 1 to 4970; (SEQ ID No.20) the complementary viral M RNA sequence from position 87 to position 3500 of the complementary viral M RNA sequence; ( SEQ ID No.21)BAD ORIGINALAP Ο Ο Ο 4 Ο 3 xxii) the complementary viral M RNA sequence from position 3974 to 4385 (SEQ ID No.22) xxiii) RNA sequences homologous to the nucleotide sequences defined under i) to xii) and xv) to xxii) hereinabove.xxiv) fragments of sequences defined under i) to xxii) hereinabove.
- 3. A DNA construct according to Claim 1, wherein the DNA sequence codes for transcription into INSV-RNA sequences of a pan-handle, or into RNA sequences homologous thereto.
- 4. A DNA construct according to Claim 1 wherein the DNA sequence codes for transcription into INSV-RNA sequences of a pan-handle wherein the pan-handle structure comprises two complementary strands comprising 36 nucleotides in length.
- 5. A DNA construct according to Claim 1, wherein the DNA sequence codes for transcription into INSV RNA sequences of an open reading frame in viral complementary sense, or into corresponding RNA sequences in which one or more codons have been replaced by synonyms thereof, or into RNA sequences homologous thereto.
- 6. A DNA construct according to Claim 1, wherein the the DNA sequence codes for transcription into INSVRNA sequences of a secondary structure, or into RNA
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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GB929206016A GB9206016D0 (en) | 1992-03-19 | 1992-03-19 | Improvements in or relating to organic compounds |
Publications (2)
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AP9300496A0 AP9300496A0 (en) | 1993-04-30 |
AP415A true AP415A (en) | 1995-09-29 |
Family
ID=10712460
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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APAP/P/1993/000496A AP415A (en) | 1992-03-19 | 1993-03-18 | Recombinant tospovirus DNA constructs and plants comprising such constructs. |
Country Status (10)
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US (1) | US5773700A (en) |
EP (1) | EP0566525A3 (en) |
JP (1) | JPH067170A (en) |
AP (1) | AP415A (en) |
AU (1) | AU664241B2 (en) |
CA (1) | CA2091806A1 (en) |
GB (1) | GB9206016D0 (en) |
IL (1) | IL105086A0 (en) |
MX (1) | MX9301522A (en) |
ZA (1) | ZA931985B (en) |
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AU685428B2 (en) | 1993-01-29 | 1998-01-22 | Cornell Research Foundation Inc. | Tomato spotted wilt virus |
GB9505907D0 (en) * | 1995-03-23 | 1995-05-10 | Sandoz Ltd | Improvements in or relating to organic compounds |
BR9807587A (en) * | 1997-02-19 | 2000-03-21 | Cornell Res Foundation Inc | DNA construction, dna expression vector, host cell, transgenic plant, process to give plants a trace, transgenic plant seed, and process to give plants a trace |
US6121511A (en) * | 1997-09-12 | 2000-09-19 | Ball Horticultural Company | Production of transgenic impatiens |
US6528703B1 (en) | 1998-09-11 | 2003-03-04 | Ball Horticultural Company | Production of transgenic impatiens |
US6818804B1 (en) | 1999-10-22 | 2004-11-16 | Cornell Research Foundation, Inc. | Tospovirus resistance in plants |
MXPA03009627A (en) | 2001-04-24 | 2004-01-29 | Cornell Res Foundation Inc | Synthetic nucleic acid molecule for imparting multiple traits. |
US9441244B2 (en) | 2003-06-30 | 2016-09-13 | The Regents Of The University Of California | Mutant adeno-associated virus virions and methods of use thereof |
US9233131B2 (en) | 2003-06-30 | 2016-01-12 | The Regents Of The University Of California | Mutant adeno-associated virus virions and methods of use thereof |
CN101124321B (en) | 2004-11-05 | 2012-02-29 | 恰根北美控股有限公司 | Compositions and methods for purifying nucleic acids from stabilization reagents |
ITUD20060280A1 (en) * | 2006-12-29 | 2008-06-30 | Univ Degli Studi Udine | ARTIFICIAL SEQUENCE OF DNA EVENT FUNCTION OF LEADER IN 5 '(5'-UTR) OPTIMIZED FOR THE OVEREXPRESSION OF HETEROLOGICAL PLANT PROTEINS |
US8669413B2 (en) * | 2009-11-25 | 2014-03-11 | Hm.Clause, Inc. | Breeding and selection for resistance to melon severe mosaic virus (MeSMV) |
US8663624B2 (en) | 2010-10-06 | 2014-03-04 | The Regents Of The University Of California | Adeno-associated virus virions with variant capsid and methods of use thereof |
CN103561774B (en) | 2011-04-22 | 2016-12-14 | 加利福尼亚大学董事会 | There is adeno-associated virus virion and the using method thereof of variant capsids |
US9738879B2 (en) | 2012-04-27 | 2017-08-22 | Duke University | Genetic correction of mutated genes |
EP3597741A1 (en) | 2012-04-27 | 2020-01-22 | Duke University | Genetic correction of mutated genes |
ITUD20130002A1 (en) * | 2013-01-16 | 2014-07-17 | Transactiva S R L | ARTIFICIAL SEQUENCE OF DNA HAVING LEADER FUNCTION IN 5 '(5'-UTR) OPTIMIZED FOR THE OVER-EXPRESSION OF RECOMBINANT PLANTS AND PLANT FOR THE PRODUCTION OF RECOMBINANT PLANT PROTEINS |
US9828582B2 (en) | 2013-03-19 | 2017-11-28 | Duke University | Compositions and methods for the induction and tuning of gene expression |
CN105247044B (en) | 2013-05-31 | 2021-05-07 | 加利福尼亚大学董事会 | Adeno-associated virus variants and methods of use thereof |
KR102551324B1 (en) | 2013-06-05 | 2023-07-05 | 듀크 유니버시티 | Rna-guided gene editing and gene regulation |
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WO2016081924A1 (en) | 2014-11-20 | 2016-05-26 | Duke University | Compositions, systems and methods for cell therapy |
JP6929791B2 (en) | 2015-02-09 | 2021-09-01 | デューク ユニバーシティ | Compositions and methods for epigenome editing |
CN114480502A (en) | 2015-03-02 | 2022-05-13 | 阿德夫拉姆生物技术股份有限公司 | Compositions and methods for intravitreal delivery of polynucleotides to retinal cones |
WO2016154344A1 (en) | 2015-03-24 | 2016-09-29 | The Regents Of The University Of California | Adeno-associated virus variants and methods of use thereof |
WO2017015637A1 (en) | 2015-07-22 | 2017-01-26 | Duke University | High-throughput screening of regulatory element function with epigenome editing technologies |
KR20180038558A (en) | 2015-08-25 | 2018-04-16 | 듀크 유니버시티 | Compositions and methods for improving specificity in genomic manipulation using RNA-guided endonuclease |
EP4089175A1 (en) | 2015-10-13 | 2022-11-16 | Duke University | Genome engineering with type i crispr systems in eukaryotic cells |
CN114672516A (en) | 2016-07-29 | 2022-06-28 | 加利福尼亚大学董事会 | Adeno-associated virus virions with variant capsids and methods of use thereof |
AU2017345470B2 (en) | 2016-10-19 | 2023-08-03 | Adverum Biotechnologies, Inc. | Modified AAV capsids and uses thereof |
AU2018324477A1 (en) | 2017-08-28 | 2019-10-17 | The Regents Of The University Of California | Adeno-associated virus capsid variants and methods of use thereof |
CN118059211A (en) * | 2024-01-09 | 2024-05-24 | 珠海市藤栢医药有限公司 | Polypeptide-containing pharmaceutical composition and application thereof |
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EP0426195A1 (en) * | 1989-11-03 | 1991-05-08 | S&G Seeds B.V. | Improvements in or relating to organic compounds |
-
1992
- 1992-03-19 GB GB929206016A patent/GB9206016D0/en active Pending
-
1993
- 1993-03-16 EP EP19930810190 patent/EP0566525A3/en not_active Withdrawn
- 1993-03-17 CA CA002091806A patent/CA2091806A1/en not_active Abandoned
- 1993-03-17 AU AU35237/93A patent/AU664241B2/en not_active Ceased
- 1993-03-17 IL IL105086A patent/IL105086A0/en unknown
- 1993-03-18 MX MX9301522A patent/MX9301522A/en unknown
- 1993-03-18 JP JP5058584A patent/JPH067170A/en not_active Withdrawn
- 1993-03-18 AP APAP/P/1993/000496A patent/AP415A/en active
- 1993-03-19 ZA ZA931985A patent/ZA931985B/en unknown
-
1996
- 1996-12-06 US US08/764,100 patent/US5773700A/en not_active Expired - Fee Related
Patent Citations (1)
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EP0426195A1 (en) * | 1989-11-03 | 1991-05-08 | S&G Seeds B.V. | Improvements in or relating to organic compounds |
Also Published As
Publication number | Publication date |
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IL105086A0 (en) | 1993-07-08 |
EP0566525A3 (en) | 1993-12-08 |
AU3523793A (en) | 1993-09-23 |
GB9206016D0 (en) | 1992-04-29 |
US5773700A (en) | 1998-06-30 |
JPH067170A (en) | 1994-01-18 |
EP0566525A2 (en) | 1993-10-20 |
AU664241B2 (en) | 1995-11-09 |
CA2091806A1 (en) | 1993-09-20 |
AP9300496A0 (en) | 1993-04-30 |
MX9301522A (en) | 1994-07-29 |
ZA931985B (en) | 1994-09-19 |
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