My Annotations
| Status | Page | Date/Time | GO Term (Aspect) | Reference | Evidence | Notes | Links |
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| updatedbyinstructor | EBVB9:BRRF1 | 2013-03-30 22:25:41 CDT | GO:0019046 release from viral latency (P) | PMID:21325409 | ECO:0000315 mutant phenotype evidence used in manual assertion | Figure 1 A-C: Three different cell lines were transfected with plasmids for the over-expression of Na (which is the product of gene BRRF1) as well as control plasmids with no over-expression of Na. To see if Na initiated lysis in the cells in which Na was overproduced, immunoassays were performed to search for lytic cell products. The proteins searched for include BMRF1, Z protein, R protein, Na (through FLAG), and β-actin. In all three types of cells with plasmids over-expressing BRRF1, these lytic proteins were found in a greater amount than in the cells without the over-expression plasmids. Expression of BRRF1 leads to a product that affects maintenance of viral latency.
The evidence code is IMP because there is over-expression of the gene. Presence of the gene product resulted in an increase in specific lytic proteins measured by an immunoblot assay.
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| updatedbyinstructor | HCMVM:AN | 2013-04-14 14:47:28 CDT | GO:0004529 exodeoxyribonuclease activity (F) | PMID:21900421 | ECO:0000314 direct assay evidence used in manual assertion | Figure 2 shows the results demonstrating UL98's exonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled. They were then combined with 14C-labelled DNA. The amount of cleaved DNA was measured over time. Figure 2a shows the significantly different cleavage rates between the WT gene and the R164A mutant, whose function was greatly impaired. Figure 2b show the total cleavage done by the WT and all the mutants over the course of an hour. The WT gene product exhibited significantly more exonuclease activity than the UL98-impaired mutants.
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| updatedbyinstructor | HCMVM:AN | 2013-04-14 14:47:28 CDT | GO:0004520 endodeoxyribonuclease activity (F) | PMID:21900421 | ECO:0000314 direct assay evidence used in manual assertion | Figure 3 shows the results demonstrating UL98's endonuclease activity. There is a wild type (WT) UL98 strain and five mutants in which alanine was used to substitute another residue within the gene. The expressed proteins were purified and labelled.
In figure 3a, the WT and mutants were incubated with closed-circular plasmid DNA to see if the gene products would convert it to open-circular and linear forms using endonuclease activity. Following incubation, gel electrophoresis measured the leftover DNA products. The WT and one of the mutants were able to degrade the entire DNA substrate by nicking the supercoiled substrate and processing the products into smaller fragments not visible on the gel. Other mutants were only able to nick the supercoiled DNA, but not digest anything else.
In figure 3b, the WT and mutants were incubated with a ssDNA substrate that had a 3' quencher and a 5' fluorophore. Endonucleolytic cleavage would result in detectable fluorescence. Over time, more protein was added to the reaction in increments. The WT and one of the mutants produced significantly more fluorescence than the rest of the mutants. This indicates that those mutants suffered from impaired endonuclease function. The R164A mutant had a slightly reduced endonuclease activity compaired to the WT.
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| requireschanges | HCMVM:DEN | 2013-04-14 15:37:50 CDT | GO:0004843 ubiquitin-specific protease activity (F) | PMID:2786740 | ECO:0000315 mutant phenotype evidence used in manual assertion | Figure 1 shows that HCMV UL48 has deubiquitinase activity. A UL48 wild type was isolated as well as two point mutants that had amino acid substitutions in the presumed active site. They also generated several fusion proteins. All of these had GST at their amino ends and were tagged. One of these fusion proteins contained ubiquitin (Ub) . Figure 1C shows that when expressed UL48 was incubated with the fusion proteins, it cleaved GST-Ub-HA to produce GST-Ub, but it did not cleave any of the other proteins. This was measured using gel electrophoresis. When the two active-site UL48 mutatns were tested in figure 1D, not activity was detected, showing that their ubiquitinase activity was impaired. Protein staining in figure 1E showed that there were comparable amounts of the WT and mutant UL48 proteins in each assay, so the differences in activity can't be attributed in differences in amount of protein present.
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