Tained SDS-PAGE gels, as well as Western blot 1-phenyl-4-(4,4,5,5-tetr…
Writer Marita Thorpe Thorpe
Date 24-11-21 08:32
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- Country : Italy
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- Business Section : K4-eco
3955527643
- Email : maritathorpe@aol.com
- Phone : 3955527643
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As shown in Figure 2A, we were able to obtain WT as well as mutant CPs with satisfactory yields. An equal amount of purified WT and mutant BBSV CPs were then separated on an SDS-PAGE gel, transferred to nitrocellulose membrane, and renatured prior to being exposed to the digoxigenin-labeled BBSV genomic RNA, with the similarly purified GFP as a negative control. Figure 2B shows the result of one of the representative North-western experiments. As shown in lane 8, The GFP protein failed to immobilize any BBSV RNA, thus confirming that the RNA-binding is a specific property of WT BBSV CP (lane 1). Significantly, the RNA-binding activity of BM3, 4, 5, and 7 were only slightly reduced (Figure 2B, lanes 4?), thus revealing less significant participation of the basic residues after amino acid position 6 of BBSV CP (see Figure 1A for detailed changes in the respective mutants). It is particularly noteworthy that BM5 CP (Figure 2B, lane 6), despite the 2-Bromo-1,3-difluoro-4-nitrobenzene replacement ofFigure 2 Delineation of the amino acid residues within the N-terminus of BBSV CP involved in RNA binding with Northwestern blot analysis. (A) Confirmation of the quantity and purity of wild type (WT) and mutated BBSV CPs prepared with an E. coli expression system using SDS AGE (top) and Western blot (bottom). GFP was used as a control. (B) RNA binding capability of WT and mutant CPs were assayed using non-radioactively labeled BBSV RNA (see Materials and 4-(4 Methods for details). The experiment was repeated three times with similar results.Zhang et al. Virology Journal 2013, 10:200 http://www.virologyj.com/content/10/1/Page 4 ofall amino acid residues between positions 7 and 13 with A, was still able to bind BBSV RNA with affinity levels approaching that of WT CP. By contrast, BM1 and BM2 CPs, which shared the 4KR5 AA changes, were able to bind only trace amounts of BBSV RNA (Figure 2B, lanes 2 and 3). Together, these results clearly illustrated that these two basic residues are indispensible for the RNAbinding PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/4155310 activity of BBSV CP. Therefore, the reduced accumulation of BM1 and BM2 CPs in infected protoplasts, in which these two residues were replaced by alanines, was most likely caused by a lack of RNA-binding activity, and hence an inability to assemble stable virions.Virus assembly was compromised by the N-terminal mutations to varying degreesHaving revealed the critical role of 4KR5 in the interaction between CP and viral RNA of BBSV, we next wanted to determine whether substituting these two amino acid residues indeed impaired BBSV virion assembly. We hence tried to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13867361 purify virions from the inoculated leaves of the N. benthamiana plants infected with the mutant transcripts, at 10 days post inoculation (dpi. See Methods section for details). The purified virions were first examined with electron microscopy (EM).Surprisingly, only BM3 and BM4 produced virions at sufficiently high concentrations to permit the detection by EM (Figure 3A, right two panels). In an attempt to detect lower levels of virion assembly by other mutants, we then subjected the virion preparations to Western blot with a BBSV CP antibody. As shown in Figure 3B (top panel), a BBSV CP-specific band was detected in BM3, 4, 5, and 7 samples, with decreasing intensities, but not in BM1 and BM2 samples. It should be noted that the extensive purification procedure would have removed most other proteins not associated with assembled virions. Indeed, the.
- Item Name :
- Business Section : K4-eco
3955527643
- Email : maritathorpe@aol.com
- Phone : 3955527643
- Message :
As shown in Figure 2A, we were able to obtain WT as well as mutant CPs with satisfactory yields. An equal amount of purified WT and mutant BBSV CPs were then separated on an SDS-PAGE gel, transferred to nitrocellulose membrane, and renatured prior to being exposed to the digoxigenin-labeled BBSV genomic RNA, with the similarly purified GFP as a negative control. Figure 2B shows the result of one of the representative North-western experiments. As shown in lane 8, The GFP protein failed to immobilize any BBSV RNA, thus confirming that the RNA-binding is a specific property of WT BBSV CP (lane 1). Significantly, the RNA-binding activity of BM3, 4, 5, and 7 were only slightly reduced (Figure 2B, lanes 4?), thus revealing less significant participation of the basic residues after amino acid position 6 of BBSV CP (see Figure 1A for detailed changes in the respective mutants). It is particularly noteworthy that BM5 CP (Figure 2B, lane 6), despite the 2-Bromo-1,3-difluoro-4-nitrobenzene replacement ofFigure 2 Delineation of the amino acid residues within the N-terminus of BBSV CP involved in RNA binding with Northwestern blot analysis. (A) Confirmation of the quantity and purity of wild type (WT) and mutated BBSV CPs prepared with an E. coli expression system using SDS AGE (top) and Western blot (bottom). GFP was used as a control. (B) RNA binding capability of WT and mutant CPs were assayed using non-radioactively labeled BBSV RNA (see Materials and 4-(4 Methods for details). The experiment was repeated three times with similar results.Zhang et al. Virology Journal 2013, 10:200 http://www.virologyj.com/content/10/1/Page 4 ofall amino acid residues between positions 7 and 13 with A, was still able to bind BBSV RNA with affinity levels approaching that of WT CP. By contrast, BM1 and BM2 CPs, which shared the 4KR5 AA changes, were able to bind only trace amounts of BBSV RNA (Figure 2B, lanes 2 and 3). Together, these results clearly illustrated that these two basic residues are indispensible for the RNAbinding PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/4155310 activity of BBSV CP. Therefore, the reduced accumulation of BM1 and BM2 CPs in infected protoplasts, in which these two residues were replaced by alanines, was most likely caused by a lack of RNA-binding activity, and hence an inability to assemble stable virions.Virus assembly was compromised by the N-terminal mutations to varying degreesHaving revealed the critical role of 4KR5 in the interaction between CP and viral RNA of BBSV, we next wanted to determine whether substituting these two amino acid residues indeed impaired BBSV virion assembly. We hence tried to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13867361 purify virions from the inoculated leaves of the N. benthamiana plants infected with the mutant transcripts, at 10 days post inoculation (dpi. See Methods section for details). The purified virions were first examined with electron microscopy (EM).Surprisingly, only BM3 and BM4 produced virions at sufficiently high concentrations to permit the detection by EM (Figure 3A, right two panels). In an attempt to detect lower levels of virion assembly by other mutants, we then subjected the virion preparations to Western blot with a BBSV CP antibody. As shown in Figure 3B (top panel), a BBSV CP-specific band was detected in BM3, 4, 5, and 7 samples, with decreasing intensities, but not in BM1 and BM2 samples. It should be noted that the extensive purification procedure would have removed most other proteins not associated with assembled virions. Indeed, the.
