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  • 1
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. We have used a new method for binning minisatellite alleles (semi-automated allele aggregation) and report the extent of population diversity detectable by eleven minisatellite loci in 2689 individuals from 19 human populations distributed widely throughout the world. Whereas population relationships are consistent with those found in other studies, our estimate of genetic differentiation (Fst) between populations is less than 8%, which is lower than comparative estimates of between 10%–15% obtained by using other sources of polymorphism data. We infer that mutational processes are involved in reducing Fst estimates from minisatellite data because, first, the lowest Fst estimates are found at loci showing autocorrelated frequencies among alleles of similar size and, second, Fst declines with heterozygosity but by more than predicted assuming simple models of mutation. These conclusions are consistent with the view that minisatellites are subject to selective or mutational constraints in addition to those expected under simple stepwise mutation models.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Archives of virology 144 (1999), S. 89-105 
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary.  Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3′ RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3′ untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequences together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.
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  • 3
    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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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Archives of virology 142 (1997), S. 1673-1680 
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary. Banana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense. A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein. The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6. This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV. The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit. The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.
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  • 5
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary.  Complete nucleotide sequences of the coat protein gene (DNA-3) of banana bunchy top virus (BBTV) were obtained from five geographical isolates by PCR. Analysis of these sequences revealed two distinct groups of BBTV isolates with those from the Philippines, Taiwan and Vietnam forming the Asian group while the South Pacific/African group consisted of isolates from Australia, Burundi and Fiji. At the nucleotide level, the sequences of DNA-3 were more similar between isolates from the same group (maximum 5.86%) than between members of the two different groups (maximum 13.05%). At the amino acid level, the BBTV coat protein remained highly conserved, with a maximum of 〈3% sequence variation between all isolates in this study. There was a significantly higher degree of divergence between the Asian isolates, which may indicate that BBTV has been present in this region for an extended period of time or that there have been multiple introductions of BBTV into bananas. The high level of conservation in the BBTV coat protein suggests that any of the DNA-3 sequences presented in this study would probably be equally effective as transgene in attempts to generate transgenic banana plants with resistance to both groups of BBTV isolates.
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  • 6
    ISSN: 1432-8798
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary.  The 5′ and 3′ terminal sequences of the plus strand of Fiji disease fijivirus (FDV) segments 2, 3, 9 and 10 possess the conserved terminal sequences, 5′AAGUUUUU… . .CAGCAGAUGUC 3′. The 5′ sequence is identical to that of maize rough dwarf fijivirus (MRDV) and rice black-streaked dwarf fijivirus (RBSDV), whereas the FDV 3′ sequence shares the consensus, CAGCNNNNGUC, with MRDV and RBSDV. The FDV terminal sequences, and the amino acid sequences from FDV segment 9, are more closely related to those from MRDV and RBSDV than to those from oat sterile dwarf fijivirus (OSDV) and Nilaparvata lugens reovirus (NLRV; a putative Fijivirus).
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  • 7
    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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  • 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 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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  • 10
    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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