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  • 1
    ISSN: 1432-203X
    Keywords: Key words Banana bunchy top virus ; Promoter ; Vascular expression ; Green fluorescent protein ; Intron-mediated enhancement
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants.
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  • 2
    ISSN: 1432-203X
    Keywords: Key words Musa ; Banana ; Transformation ; Microprojectile bombardment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into the banana genome.
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  • 3
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Four cDNA clones were generated from the genomic dsRNA of an Australian isolate of pangola stunt Fijivirus (PaSV). Each clone hybridized with nucleic acid extracts from PaSV infected plants but not healthy plants. Further, each clone hybridized with more than one segment of the PaSV dsRNA genome. One clone was used to demonstrate that homology existed between the Australian isolate of PaSV and a South American isolate of PaSV although the isolates differed in the sizes of the genomic dsRNAs and in the vector species. The clone also hybridized with some segments of the maize rough dwarf Fijivirus genome.
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  • 4
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Banana bract mosaic (BBMV) is a relatively new, non-persistently aphid transmitted disease of bananas in the Philippines. Partially purified preparations from infected plants contained low numbers of flexuous virions, 660 to 760 nm in length, and a 38kDa protein, possibly the coat protein, which reacted with a general potyvirus antiserum in western blots. There were insufficient virions for conventional antiserum production or cDNA synthesis. Therefore, DNA was amplified using potyvirus-specific degenerate primers and reverse transcriptase PCR. The PCR products were cloned, sequenced and analysed and contained a 5′ open reading frame of up to 150 amino acids and a 3′ untranslated region of up to 190 nucleotides which were clearly related to the C-terminal half of the coat proteins and the 3′ untranslated regions, respectively of potyviruses. The BBMV open reading frame amino acid sequence was most similar to the C-terminal half of the maize dwarf mosaic potyvirus coat protein (71.3% similarity) and the BBMV 3′ untranslated region was most similar to that of ornithogalum mosaic potyvirus (39.6% similarity). Our results show that BBMV is a distinct potyvirus and also demonstrate the application of virus group specific primers in the characterisation of previously undescribed viruses.
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  • 5
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.
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  • 6
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary.  We have sequenced the coat protein (CP) coding region of 11 field isolates of SCMV from Australia, USA and South Africa. The differences between the nucleotide sequences of the isolates was 0.2 to 4.1% and the encoded amino acid sequences differed by 0.0 to 3.5%. Phylogenetic analysis of the CP coding sequences of the SCMV isolates and the related potyviruses SCMV-MDB, JGMV, SrMV, MDMV-A and PVY showed that the SCMV isolates formed a tightly clustered group, with SCMV-MDB forming a separate branch. This indicated that (i) the SCMV isolates are of one strain (SCMV-A) and not geographically distinct species and (ii) SCMV-MDB is clearly distinct, and may represent another potyvirus species.
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  • 7
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A 93 nucleotide sequence was found to be strongly conserved between two ssDNA genomic components of banana bunchy top virus (BBTV). Two outwardly extending degenerate primers were designed from this sequence and used in a polymerase chain reaction (PCR) with DNA extracted from purified BBTV virions. PCR amplified products consisting of at least seven distinct bands all approximately 1 kb and possibly representing full-length BBTV dsDNA were resolved. The PCR amplified products were cloned and the clones screened by restriction enzyme analysis. Four distinct restriction analysis groups were identified. These results confirm that the genome of BBTV contains at least five components and that it belongs to a previously undescribed group of plant viruses which may also contain subterranean clover stunt virus.
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  • 8
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary A high titre (1:10000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed inE. coli. The fusion protein consisted of theMalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3′ end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting theNcoI restriction site (C/CATGG) and creating a uniqueEco47III site (AGC/GCT). Endonuclease restriction withEco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 µg of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed inE. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.
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  • 9
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The coat protein (CP) of strain SC of sugarcane mosaic virus (SCMV-SC) was expressed transiently in sugarcane protoplasts after electroporation with one of two plasmids encoding the CP gene. The CP gene was fused with either the cauliflower mosaic virus 35S promoter or the synthetic monocotyledon promoter “Emu”. The coat protein gene was also inducibly expressed inEscherichia coli when fused to the trc promoter. The protein expressed in both systems had the same electrophoretic mobility and antigenic specificity as purified SCMV-SC coat protein. Transient expression of the 35S-CP gene in protoplasts could only be demonstrated in Western blots developed with the chemiluminescence enzyme substrate luminol.
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  • 10
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary.  We have sequenced the entire coat protein (CP)-coding region and 5′ 162 nucleotides of the 3′ untranslated region (UTR) of nine different isolates of banana bract mosaic virus (BBrMV) from five different countries. Further, we have sequenced the 3′ 621 nucleotides of the NIb-coding region of a Philippines isolate. This is the first report of BBrMV in Thailand, Vietnam and Western Samoa. When the sequences of the CP-coding region and 3′ UTR were compared to each other, variability of between 0.3% and 5.6%, and 0.3% and 4.3%, was observed at the nucleotide and amino acid levels, respectively. Phylogenetic analysis of the BBrMV isolates did not reveal any relationship between the geographic location of the isolates. The BBrMV CP was expressed in Escherichia coli as a fusion protein and the purified recombinant protein was used to produce a high titre BBrMV-specific polyclonal antiserum. This antiserum was used to develop a F(ab′)2 indirect double antibody sandwich ELISA and compared with immuno-capture PCR (IC-PCR) and reverse transcription PCR (RT-PCR) assays for BBrMV detection. RT-PCR was shown to be the most sensitive test followed by ELISA and IC-PCR.
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