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Post by jontrip247 on Oct 9, 2015 10:18:25 GMT -6
Thread created by Gabriela & Jon
Genus: Begomovirus
Two species examples: Tomato yellow leaf curl virus (YLCV), Bean dwarf mosaic virus (BDMV)
> Key characteristics of the genus translation
- Leaky scanning
~ A phenomenon in which a weak initiation codon triplet on mRNA is sometimes skipped by ribosome in translation initiation
~ The weak initiation codon can be an ACG, or an ATG
- Reinitiation
~ The 40S ribosomal subunit continues scanning to further initiation codon, so mRNA can encode for several different proteins
***This is done so that the virus can reach downstream major ORFs***
- Studies on leaky scanning mechanism, using Begomovirus, were preformed to study cellular stress of eukaryotic cells in which EIF2alpha is phosphorylated, like in the case of dsRNA/PKR activation. Additionally, geminivirus genomes contain few target sites for eukaryotic DNA methylation. This reflects evolutionary selection for undermethylated DNA, because geminivirus promoters and origins are both negatively influenced by DNA methylation.
> Other factors to consider
- TATA box
~ This, like in eukaryotic cells, acts as a promoter in some of the viruses of this genus
~ Indicating that the plant basal transcription apparatus is necessary for geminivirus expression
- G-box element
~ Essential for activity
~ Found in the complementary-sense promoter for Rep expression of the viruses
~ Also found in a variety of plant promoters
~ Several plant proteins specifically bind the G-box motifs and activate transcription
~ The G-box conserves its location at the base of the hairpin in geminivirus genomes
***The corresponding G-box factor represents the first example of a plant transcription factor that also acts as a replication factor. Initiation of DNA replication and transcription both require the recruitment and assembly of large protein complexes on DNA. These common requirements have resulted in proteins that function in both processes, and many systems achieve these goals through similar mechanisms***
***Studies have also reflected the usefulness of geminiviruses as episomal vectors in reverse genetic experiments for eukaryotes***
> What about the genome?
- We observe bipartite or monopartite.
~ Bipartite begomoviruses, an A component encodes five or six proteins:
~ On the v-sense:
o Coat protein
~ On the c-sense:
o REP, AC4, transcriptional activator (TrAP/AL2), replication enhancer (REn), and, in some strains an optional protein AV2
~ Monopartite begomoviruses six proteins are encoded:
o Rep, TrAP, CP, REn, C4, and V2
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Post by Admin on Oct 10, 2015 6:53:02 GMT -6
well done
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Post by omararias on Oct 17, 2015 10:33:57 GMT -6
Thanks for the information shared, please I have some questions:
- In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised?
- Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?.
Thank you so much.
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Post by gabriela on Oct 19, 2015 14:02:58 GMT -6
Thanks for the information shared, please I have some questions: - In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised? - Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?. Thank you so much. Hey Omar..  thanks so much for your questions. Here are some answers:
In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised?
For the genus that we choose (Begamovirus) just EIF2alpha was reported to be involved as translation elongation factor described during cellular stress. However, we were searching some information for another ssDNA virus and several other factors have been studied and observed to be participating during translation. One example of those and that we considered is quite important is the Eukaryotic initiation factor 4A (eIF4A). This factor is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' end. The idea of this initiator factor is that its proximal secondary structure facilitates the attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors ( which are called as eIF4E and eIF4G). As well, in ssDNA virus this initiator factor has two isoforms eIF4AI and eIF4AII, which are encoded by two different genes. Recently another member has being include to the eIF4A, which is called eIF4AIII, however its function in translation has not been well characterized. Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?
It was actually complex. In summary they designed that they use was described as a methylation sensitive extension assay. Where global DNA methylation status was evaluated using a cytosine extension assay. Briefly, 1 µg of genomic DNA was digested overnight with a 10-fold excess of MspI, leaving an overhang at non-methylated sites. A second DNA aliquot from mock inoculated samples was incubated without restriction enzyme and served as a background control. Single-nucleotide-extension reactions with α32P-dCTP and Taq DNA polymerase were then performed.
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Post by dulanjani on Oct 21, 2015 13:37:26 GMT -6
Can you explain more about Giminivirus preventing Eukaryotic DNA methylation Sites? I have read in literature that Giminivirus can prevent methylation-mediated transcriptional gene silencing by interfering the function of plant methylation cycle. Apparently they found a novel counter defense strategy used by many Giminivirus such as Replication associated protein (Rep)which can suppress post transcriptional gene silencing by down regulating Methyltransferese1 and Chromomethylase3.
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Post by gabriela on Oct 21, 2015 16:15:09 GMT -6
Can you explain more about Giminivirus preventing Eukaryotic DNA methylation Sites? I have read in literature that Giminivirus can prevent methylation-mediated transcriptional gene silencing by interfering the function of plant methylation cycle. Apparently they found a novel counter defense strategy used by many Giminivirus such as Replication associated protein (Rep)which can suppress post transcriptional gene silencing by down regulating Methyltransferese1 and Chromomethylase3. Some recently data have suggested that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Which has been actually considered as a novel counter - defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases specifically methyltransferase1 and crhomomethylase3, both of them related with local and systemic defense reaction of infected tissues "preventing plant recover or resist against the virus". Some studies have been done in Arabidopsis thaliana with the incorporation of a transgene, where they observed that Rep (replication associated protein) suppressed TGS. Same results, were confirmed by bisulfite sequencing, which showed that the expression of Rep causes the reduction of levels of DNA methylation and in consequences, it suggested that Rep (viral protein) is essential for virus replication and blocking of plant defense systems.. pretty cool. please if you need some papers let me know.. |
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Post by omararias on Oct 21, 2015 16:38:39 GMT -6
Thanks for the information shared, please I have some questions: - In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised? - Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?. Thank you so much. Hey Omar.. thanks so much for your questions. Here are some answers:
In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised?
For the genus that we choose (Begamovirus) just EIF2alpha was reported to be involved as translation elongation factor described during cellular stress. However, we were searching some information for another ssDNA virus and several other factors have been studied and observed to be participating during translation. One example of those and that we considered is quite important is the Eukaryotic initiation factor 4A (eIF4A). This factor is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' end. The idea of this initiator factor is that its proximal secondary structure facilitates the attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors ( which are called as eIF4E and eIF4G). As well, in ssDNA virus this initiator factor has two isoforms eIF4AI and eIF4AII, which are encoded by two different genes. Recently another member has being include to the eIF4A, which is called eIF4AIII, however its function in translation has not been well characterized. Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?
It was actually complex. In summary they designed that they use was described as a methylation sensitive extension assay. Where global DNA methylation status was evaluated using a cytosine extension assay. Briefly, 1 µg of genomic DNA was digested overnight with a 10-fold excess of MspI , leaving an overhang at non-methylated sites. A second DNA aliquot from mock inoculated samples was incubated without restriction enzyme and served as a background control. Single-nucleotide-extension reactions with α32P-dCTP and Taq DNA polymerase were then performed .
I just have one extra curisoty regarding the two isoforms eIF4AI and eIF4AII encoded by two different genes that you mention. From what you have read, is there any slight difference in the funtion of those initiation factors. |
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Post by omararias on Oct 21, 2015 16:57:22 GMT -6
Can you explain more about Giminivirus preventing Eukaryotic DNA methylation Sites? I have read in literature that Giminivirus can prevent methylation-mediated transcriptional gene silencing by interfering the function of plant methylation cycle. Apparently they found a novel counter defense strategy used by many Giminivirus such as Replication associated protein (Rep)which can suppress post transcriptional gene silencing by down regulating Methyltransferese1 and Chromomethylase3. Some recently data have suggested that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Which has been actually considered as a novel counter - defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases specifically methyltransferase1 and crhomomethylase3, both of them related with local and systemic defense reaction of infected tissues "preventing plant recover or resist against the virus". Some studies have been done in Arabidopsis thaliana with the incorporation of a transgene, where they observed that Rep (replication associated protein) suppressed TGS. Same results, were confirmed by bisulfite sequencing, which showed that the expression of Rep causes the reduction of levels of DNA methylation and in consequences, it suggested that Rep (viral protein) is essential for virus replication and blocking of plant defense systems.. pretty cool. please if you need some papers let me know.. Great information, please could you help me with one question, according to the studies that you mention there is kind of a total suppression of the TGS. But do you think that is possible that besides of the plant gene TGS, there is another gene that could be involved in the plant defense methylation mechanism?, in which case there would be a partial suppression by the viral protein. |
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Post by gabriela on Oct 23, 2015 15:47:55 GMT -6
Some recently data have suggested that the largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle. Which has been actually considered as a novel counter - defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases specifically methyltransferase1 and crhomomethylase3, both of them related with local and systemic defense reaction of infected tissues "preventing plant recover or resist against the virus". Some studies have been done in Arabidopsis thaliana with the incorporation of a transgene, where they observed that Rep (replication associated protein) suppressed TGS. Same results, were confirmed by bisulfite sequencing, which showed that the expression of Rep causes the reduction of levels of DNA methylation and in consequences, it suggested that Rep (viral protein) is essential for virus replication and blocking of plant defense systems.. pretty cool. please if you need some papers let me know.. Great information, please could you help me with one question, according to the studies that you mention there is kind of a total suppression of the TGS. But do you think that is possible that besides of the plant gene TGS, there is another gene that could be involved in the plant defense methylation mechanism?, in which case there would be a partial suppression by the viral protein. I am pretty sure that there will be more genes involved, however I searched for extra information and, so far there is no more genes that have been reported as clear as the TGS, however related with the Geminiviridae family, from where our genus was chosen. |
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Post by gabriela on Oct 23, 2015 15:54:47 GMT -6
Hey Omar.. thanks so much for your questions. Here are some answers:
In the studies of cellular stress that you guys described besides of the EIF2alpha is there any factor that was compromised?
For the genus that we choose (Begamovirus) just EIF2alpha was reported to be involved as translation elongation factor described during cellular stress. However, we were searching some information for another ssDNA virus and several other factors have been studied and observed to be participating during translation. One example of those and that we considered is quite important is the Eukaryotic initiation factor 4A (eIF4A). This factor is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' end. The idea of this initiator factor is that its proximal secondary structure facilitates the attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors ( which are called as eIF4E and eIF4G). As well, in ssDNA virus this initiator factor has two isoforms eIF4AI and eIF4AII, which are encoded by two different genes. Recently another member has being include to the eIF4A, which is called eIF4AIII, however its function in translation has not been well characterized. Which is the experimental designed that was carried out to localize the target sites for DNA methylation regulation?
It was actually complex. In summary they designed that they use was described as a methylation sensitive extension assay. Where global DNA methylation status was evaluated using a cytosine extension assay. Briefly, 1 µg of genomic DNA was digested overnight with a 10-fold excess of MspI , leaving an overhang at non-methylated sites. A second DNA aliquot from mock inoculated samples was incubated without restriction enzyme and served as a background control. Single-nucleotide-extension reactions with α32P-dCTP and Taq DNA polymerase were then performed .
I just have one extra curisoty regarding the two isoforms eIF4AI and eIF4AII encoded by two different genes that you mention. From what you have read, is there any slight difference in the funtion of those initiation factors. The eIF4A isoforms are highly conserved, are thought to be functionally interchangeable, and are directed to the 5' m(7)GpppN cap structure of mRNAs during translation initiation. After some experiments they have found that the respective roles of eIF4AI and eIF4AII is also related with translation, however suppression of eIF4AI increases transcription of the eIF4AII gene, leading to elevated eIF4AII mRNA and protein levels. Inhibition of eIF4AI suppresses protein synthesis, and although eIF4AII protein levels increase above and beyond it is sufficient to compensate for the decrease in eIF4AI levels. So basically they are correlated somehow, to up and down regulation, but eIF4A has a really important funtion during the formation of cap structure. |
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Post by omararias on Oct 25, 2015 12:21:09 GMT -6
Great information, please could you help me with one question, according to the studies that you mention there is kind of a total suppression of the TGS. But do you think that is possible that besides of the plant gene TGS, there is another gene that could be involved in the plant defense methylation mechanism?, in which case there would be a partial suppression by the viral protein. I am pretty sure that there will be more genes involved, however I searched for extra information and, so far there is no more genes that have been reported as clear as the TGS, however related with the Geminiviridae family, from where our genus was chosen. Thanks for the updated information. |
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Post by omararias on Oct 25, 2015 12:24:24 GMT -6
Thank you so much, for the information.
I just have one extra curisoty regarding the two isoforms eIF4AI and eIF4AII encoded by two different genes that you mention. From what you have read, is there any slight difference in the funtion of those initiation factors. The eIF4A isoforms are highly conserved, are thought to be functionally interchangeable, and are directed to the 5' m(7)GpppN cap structure of mRNAs during translation initiation. After some experiments they have found that the respective roles of eIF4AI and eIF4AII is also related with translation, however suppression of eIF4AI increases transcription of the eIF4AII gene, leading to elevated eIF4AII mRNA and protein levels. Inhibition of eIF4AI suppresses protein synthesis, and although eIF4AII protein levels increase above and beyond it is sufficient to compensate for the decrease in eIF4AI levels. So basically they are correlated somehow, to up and down regulation, but eIF4A has a really important funtion during the formation of cap structure. Thanks for the information. It was very helpful to know about the isoforms role in regulation in transcription and translation. |
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Post by cuvozidoke on May 15, 2019 13:07:06 GMT -6
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thanks so much for your questions. Here are some answers:
