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ABSTRACT: Optimization of oocyte culture conditions is a crucial aspect of reproductive biology and technology. In the present study, maturation of germinal vesicle-stage marmoset oocytes were evaluated in the following media: Waymouth medium, Waymouth medium containing porcine follicular fluid (pFF) (Waymouth-pFF medium), and porcine oocyte medium (POM). Oocytes cultured in Waymouth-pFF medium had higher maturation rates to the metaphase II stage than those cultured in Waymouth medium (36.1% vs. 24.8%, respectively, P < 0.05), indicating the suitability of this medium for culturing marmoset oocytes. Hence, maturation of marmoset oocytes cultured in POM was subsequently evaluated. The rate of maturation to the metaphase I stage was significantly higher and degradation rates were significantly lower in oocytes cultured in POM than those cultured in Waymouth medium. In addition, three offspring were successfully obtained after transfer of embryos matured in chemically defined medium. Therefore, we concluded that POM was suitable for marmoset oocyte culture. Furthermore, this was apparently the first report of marmoset offspring derived from oocytes cultured in chemically defined medium.
Theriogenology 08/2012; 78(7):1487-93. · 1.96 Impact Factor
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ABSTRACT: Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.
Archives of Virology 02/2007; 152(10):1839-49. · 2.11 Impact Factor
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ABSTRACT: The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.
Journal of General Virology 09/2000; 81(Pt 8):2085-93. · 3.36 Impact Factor
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ABSTRACT: A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1.41 and 1.43 g/cm(3) in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3' poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa ('23K'; ORF1) and 235 kDa ('235K'; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa ('108K'). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the COMOVIRIDAE: The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.
Journal of General Virology 03/2000; 81(Pt 2):541-7. · 3.36 Impact Factor
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ABSTRACT: We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.
Archives of Virology 02/1999; 144(12):2475-83. · 2.11 Impact Factor
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Welding International 01/1998; 12(5):360-365.
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ABSTRACT: Biological, morphological and serological properties of grapevine berry inner necrosis virus (GINV), the casual virus of grapevine berry inner necrosis disease occurring in Japan, were compared with those of several known trichoviruses. Host range and particle length of GINV were quite similar to those of apple chlorotic leaf spot trichovirus (ACLSV). In ultrathin sections of the infected tissues, GINV particles existed in aggregated masses in the cytoplasm of vascular parenchyma and mesophyll cells. No virus specific inclusion bodies, such as pinwheels, viroplasmas or vesicles were observed. Serological relationships were not found between GINV and ACLSV, grapevine virus A or grapevine virus B. The cDNAs of the 3'-terminal region of the GINV genome were synthesized from poly (A)+RNAs extracted from infected tissues by PCR-selected cDNA subtraction and 3'-RACE PCR. The sequence of the 3'-terminal 2469 nucleotides contained three open reading frames (ORF) encoding a protein with the conserved motifs of RNA polymerase (ORF1), a 39 kDa putative movement protein (ORF2) and a 22 kDa protein (ORF3). The 22 kDa protein expressed in Escherichia coli reacted with antiserum against GINV, indicating that it is the coat protein of GINV. Polymerase and coat protein amino acid sequence comparisons and phylogenetic analyses with nine species of the genera Trichovirus and Capillovirus indicated that GINV is a new trichovirus relatively close to ACLSV.
Archives of Virology 02/1997; 142(7):1351-63. · 2.11 Impact Factor
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ABSTRACT: A putative movement protein of molecular mass 50 kDa encoded by the ORF2 of the apple chlorotic leaf spot virus (ACLSV) genome was expressed in Escherichia coli using an expression vector and was then used to produce an antiserum. Immunoblot analysis using an antiserum raised against this protein showed that the ORF2 protein of ACLSV was detected in both cell wall and cell membrane fractions prepared from infected Chenopodium quinoa tissues. The ORF2 protein from infected tissues had a molecular mass of 52 kDa, larger than that of the full-length ORF2 protein (50 kDa protein) expressed in E. coli. Incubation of the 52 kDa protein with alkaline phosphatase resulted in a decrease in its apparent molecular mass from 52 kDa to 50 kDa, strongly suggesting that the ORF2 protein of ACLSV is phosphorylated in infected plant tissues.
Journal of General Virology 07/1995; 76 ( Pt 6):1503-7. · 3.36 Impact Factor
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ABSTRACT: The complete nucleotide sequence of the genome of an apple isolate of apple chlorotic leaf spot virus (ACLSVA) was determined. The genome is 7552 nucleotides excluding the poly(A) tail and contains three open reading frames (ORFs 1, 2 and 3), encoding proteins with M(r) values of 216,503 (216.5K), 50,453 (50.4K) and 21,394 (21.4K), respectively. Nucleotide sequence comparisons between ACLSV-A and the previously sequenced ACLSV from plum (ACLSV-P) showed that the sequence identity at the nucleotide level was 79.8%. Amino acid sequence identities of ORFs 1 and 2 between both isolates were 88.4% and 79.9%, respectively. The 21.4K protein encoded by ORF 3 of ACLSV-A had an amino acid sequence identity of 88.6% with the 28.3K protein encoded by ORF 3 of ACLSV-P. Immunoblot analysis of the 21.4K protein expressed in Escherichia coli showed that this protein is the coat protein of ACLSV-A.
Journal of General Virology 10/1993; 74 ( Pt 9):1927-31. · 3.36 Impact Factor
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ABSTRACT: The complete nucleotide sequence of apple stem grooving virus (ASGV) genome has been determined. The genome is 6496 nucleotides in length excluding a 3'-terminal poly(A) tail and contains two overlapping open reading frames (ORFs). ORF1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at position 4788 and can encode a 36-kDa protein. The 241-kDa protein contains two consensus sequences associated with the RNA-dependent RNA polymerase and the NTP-binding helicase. Comparisons of amino acid sequences around these conserved motifs with other RNA viruses revealed that ASGV has extensive similarities with apple chlorotic leaf spot, tymo-, carla-, and potexviruses, and is a member of the sindbis-like supergroup. ASGV coat protein is found to be located in the C-terminal region of the 241-kDa polyprotein. The 36-kDa protein encoded by ORF2 contains the consensus sequence Gly-Asp-Ser-Gly found in the active site of several cellular and viral serine proteases.
Virology 12/1992; 191(1):98-105. · 3.35 Impact Factor
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ABSTRACT: Apple stem grooving virus (ASGV) RNA was translated in a rabbit reticulocyte lysate system and shown to direct the synthesis of several polypeptides of Mr ranging from 200K to 43K. A polypeptide of 200K was a major product, but no polypeptide with electrophoretic mobility the same as that of the ASGV coat protein was synthesized. Immunoprecipitation experiments showed that a polypeptide of 200K was selectively precipitated by antiserum against purified ASGV. These results indicate that ASGV coat protein is translated as part of a 200K polyprotein.
Journal of General Virology 06/1992; 73 ( Pt 5):1313-5. · 3.36 Impact Factor
