SEA-PHAGES | All posts created by kbutela

Link to this post \| posted 07 May, 2019 18:52
kbutela	I'm currently QCing Sekhmet, a Cluster CZ4 Gordonia phage. Phages in this cluster have the tail assembly chaperone programmed translational frameshift occurring as GT to GD with a slippery sequence of GGGGACT. The G part of Sekhmet doesn't contain any of the verified slippery sequences from the slippery sequence paper, but it does contain GGGAAAC, one base off from the verified GGGAAAT GN to GK slippery sequence yielding the same GN to GK frameshift. I've attached a screenshot of the six-frame translation with the original frameshift boxed in red and the GGGAAAC frameshift boxed in blue. Was there some particular reason that the GT to GD frameshift was chosen instead of GN to GK? 89Kb

Link to this post | posted 07 May, 2019 18:52

I'm currently QCing Sekhmet, a Cluster CZ4 Gordonia phage. Phages in this cluster have the tail assembly chaperone programmed translational frameshift occurring as GT to GD with a slippery sequence of GGGGACT. The G part of Sekhmet doesn't contain any of the verified slippery sequences from the slippery sequence paper, but it does contain GGGAAAC, one base off from the verified GGGAAAT GN to GK slippery sequence yielding the same GN to GK frameshift. I've attached a screenshot of the six-frame translation with the original frameshift boxed in red and the GGGAAAC frameshift boxed in blue. Was there some particular reason that the GT to GD frameshift was chosen instead of GN to GK?

Posted in: Frameshifts and Introns → Cluster CZ4 Frameshift

Link to this post \| posted 19 Mar, 2019 14:46
kbutela	Thanks Welkin and Veronique!

Posted in: Request a new function on the SEA-PHAGES official list → Ribonuclease toxin BrnT

Link to this post \| posted 18 Mar, 2019 19:51
kbutela	I have encountered this same pattern of HHPred hits for PhrostedPhlake gp39; no matches to RelE are showing up in this HHPred search. gp38 (reverse, so the "downstream" gene) has no hits to BrnA but does contain a ribbon-helix-helix DNA binding domain similar to BrnA (antitoxin). Clustal Omega alignments to other BrnT toxins are poor (found in some Mycobacterium and Rhodococcus sp, along with numerous gram negatives), and the conserved residues present in the gram-negatives tested in the Heaton et al paper that Veronique posted are also not present in the PhrostedPhlake gp39 and other BrnT genes annotated in some Actinobacteria. Should we just call this as NKF since no consensus was reached on the call of BrnT?

Link to this post | posted 18 Mar, 2019 19:51

kbutela

I have encountered this same pattern of HHPred hits for PhrostedPhlake gp39; no matches to RelE are showing up in this HHPred search. gp38 (reverse, so the "downstream" gene) has no hits to BrnA but does contain a ribbon-helix-helix DNA binding domain similar to BrnA (antitoxin). Clustal Omega alignments to other BrnT toxins are poor (found in some Mycobacterium and Rhodococcus sp, along with numerous gram negatives), and the conserved residues present in the gram-negatives tested in the Heaton et al paper that Veronique posted are also not present in the PhrostedPhlake gp39 and other BrnT genes annotated in some Actinobacteria.

Should we just call this as NKF since no consensus was reached on the call of BrnT?

Posted in: Request a new function on the SEA-PHAGES official list → Ribonuclease toxin BrnT

Link to this post \| posted 10 Aug, 2018 13:58
kbutela	I'm also on 10.13.6. This is good information to know so that we can encourage students to upgrade their OS!

Posted in: DNA Master → Copy and paste in DNA Master

Link to this post \| posted 08 Aug, 2018 13:15
kbutela	So strange! I am using a generic wireless mouse with my MacBook Pro. I have always found it easier to use with DNA Master than the Apple mouse.

Posted in: DNA Master → Copy and paste in DNA Master

Link to this post \| posted 08 Aug, 2018 01:01
kbutela	I also cannot get WINE DNA Master to copy/paste from documentation to documentation directly and am not sure why this happens, but I found a reliable work-around. Copy the text from the documentation, then go to Tools –> Text Editor. A Text Editor window will open; paste your text here. You can use either the edit menu or the shortcut button within the Text Editor window. Select your text in the Text Editor window and copy it again, either using the edit menu function or the copy shortcut button in Text Editor. This text can now be copied directly into a documentation.

Link to this post | posted 08 Aug, 2018 01:01

kbutela

I also cannot get WINE DNA Master to copy/paste from documentation to documentation directly and am not sure why this happens, but I found a reliable work-around. Copy the text from the documentation, then go to Tools –> Text Editor. A Text Editor window will open; paste your text here. You can use either the edit menu or the shortcut button within the Text Editor window. Select your text in the Text Editor window and copy it again, either using the edit menu function or the copy shortcut button in Text Editor. This text can now be copied directly into a documentation.

Posted in: DNA Master → Copy and paste in DNA Master

Link to this post \| posted 08 Jul, 2018 23:33
kbutela	I am QCing Skysand, a Cluster CR3 Gordonia phage. The only annotated member of this cluster is Patio. Skysand technically contains three possible slippery sequences (from the Xu et al paper 2004): L5 GGGGGAA GE to GG TM4 GGGAAAA GK to GK HK97 GGAAAAG EK to GK The only possible amino acid switch that is found in the three FORWARD frames in Skysand is GK to GK, so I'm operating under the assumption that the frameshift follows the one found in Mycobacteriophage TM4 (-1). Other phages (Milly) with a TM4-like slippery sequence call the frameshift presumably when the A and P sites are occupied by GGA-AAA respectively, with the repeated nucleotide called as the third nucleotide in GGA. This frameshift seems to use the G in the first frame and the K in the next frame. In the six-frame translation for Patio (screenshot attached) the frameshift is called at nucleotide 23746, which corresponds to GGG-GAA initial ribosome occupancy (GG to GG frameshift; original frame is GG to GK with the frameshift occurring between GG in the first frame. The slippery sequence in Patio (and Skysand) seems to be "extra slippy" (CCCCGGGGGAAAA), so it's not entirely clear if the repeated nucleotide would occur as in TM4 or instead like the annotation in Patio. Given the nature of this "extra slippy" sequence and the location of the frameshift in the Patio annotation, is it safe to call the frameshift occurring between GG or should it follow the TM4 model where the frameshift occurs between G and K? Do members of CR have a different slippery sequence that's not listed in the Xu paper? Edited 08 Jul, 2018 23:34 69Kb

Link to this post | posted 08 Jul, 2018 23:33

kbutela

I am QCing Skysand, a Cluster CR3 Gordonia phage. The only annotated member of this cluster is Patio.

Skysand technically contains three possible slippery sequences (from the Xu et al paper 2004):

L5 GGGGGAA GE to GG
TM4 GGGAAAA GK to GK
HK97 GGAAAAG EK to GK

The only possible amino acid switch that is found in the three FORWARD frames in Skysand is GK to GK, so I'm operating under the assumption that the frameshift follows the one found in Mycobacteriophage TM4 (-1).

Other phages (Milly) with a TM4-like slippery sequence call the frameshift presumably when the A and P sites are occupied by GGA-AAA respectively, with the repeated nucleotide called as the third nucleotide in GGA. This frameshift seems to use the G in the first frame and the K in the next frame.

In the six-frame translation for Patio (screenshot attached) the frameshift is called at nucleotide 23746, which corresponds to GGG-GAA initial ribosome occupancy (GG to GG frameshift; original frame is GG to GK with the frameshift occurring between GG in the first frame. The slippery sequence in Patio (and Skysand) seems to be "extra slippy" (CCCCGGGGGAAAA), so it's not entirely clear if the repeated nucleotide would occur as in TM4 or instead like the annotation in Patio. Given the nature of this "extra slippy" sequence and the location of the frameshift in the Patio annotation, is it safe to call the frameshift occurring between GG or should it follow the TM4 model where the frameshift occurs between G and K? Do members of CR have a different slippery sequence that's not listed in the Xu paper?

Edited 08 Jul, 2018 23:34

Posted in: Frameshifts and Introns → Annotating the CR3 Frameshift

Link to this post \| posted 05 Jul, 2018 20:23
kbutela	Thanks Jackie and Welkin! Should this be added to the "Cluster Specific Annotation Tips" forum for Cluster A phages?

Posted in: Functional Annotation → Cluster A1 terminase, small subunit

Link to this post \| posted 02 Jul, 2018 20:37
kbutela	NEHalo gp3 (http://phagesdb.org/genes/NEHALO_DRAFT_3/) currently Pham 37118 ) is called in several phages as "terminase, small subunit" or "tail protein" in four other phages. The sole HHPred match at 94% probability is to PF05119.11 Terminase_4, phage terminase, small subunit but is not linked to a crystal structure in the PDB. This match has an E-value of 0.17 with coverage of 47%. All other matches show low probability scores of 40% or below. COILS did not reveal the coil-coiled domain that is typically found in many small subunit terminase sequences in gp3. I ran the sequence through Phyre2 (output attached) and got low confidence matches to the regulatory domain of mycobacterial adenylyl cyclase rv1264 (80; 12% id) and a phage tail protein (40.6, 22% id). No other evidence of the call for terminase, small subunit was found other than Blast matches to previously annotated phages and that one HHPred hit with low e-value. Should this call of "terminase, small subunit" be changed to NKF? Gp10 is annotated as "terminase, large subunit" and has well supported HHPred matches to other large terminases. There are a few NKF genes located in between the putative "terminase, small subunit" gp3 and gp10 from a synteny perspective. Edited 02 Jul, 2018 20:39 282Kb

Link to this post | posted 02 Jul, 2018 20:37

kbutela

NEHalo gp3 (http://phagesdb.org/genes/NEHALO_DRAFT_3/) currently Pham 37118 ) is called in several phages as "terminase, small subunit" or "tail protein" in four other phages.

The sole HHPred match at 94% probability is to PF05119.11 Terminase_4, phage terminase, small subunit but is not linked to a crystal structure in the PDB. This match has an E-value of 0.17 with coverage of 47%. All other matches show low probability scores of 40% or below.

COILS did not reveal the coil-coiled domain that is typically found in many small subunit terminase sequences in gp3. I ran the sequence through Phyre2 (output attached) and got low confidence matches to the regulatory domain of mycobacterial adenylyl cyclase rv1264 (80; 12% id) and a phage tail protein (40.6, 22% id). No other evidence of the call for terminase, small subunit was found other than Blast matches to previously annotated phages and that one HHPred hit with low e-value.

Should this call of "terminase, small subunit" be changed to NKF? Gp10 is annotated as "terminase, large subunit" and has well supported HHPred matches to other large terminases. There are a few NKF genes located in between the putative "terminase, small subunit" gp3 and gp10 from a synteny perspective.

Edited 02 Jul, 2018 20:39

Posted in: Functional Annotation → Cluster A1 terminase, small subunit

Link to this post \| posted 10 May, 2018 14:34
kbutela	I've seen this happen a few times. I have my students set up a DNA Master Archive folder somewhere on their Mac, then I have them set this folder to default in DNA Master Preferences. I discourage my students from using the WINE "DNA Master Archive" folder since sometimes files in there seem to get lost or corrupted more easily. Are the students opening files from the DNA Master "open" menu? In my experience, there is usually a brief hang time for opening files. If the program still isn't opening files, then I have the students uninstall and reinstall the program which seems to take care of the problem. Randall DeJong This will hopefully be a silly, easy-to-answer question! We don't typically use DNA Master for Mac in our course, but we have some students who wish to continue unfinished annotations into the summer. They have downloaded and installed the Wine Bottle version posted by Baylor (thanks SEA colleagues!), and it appears to run fine. However, some run into trouble in that they cannot open files - they just hang. My suspicion is that the files need to be in the DNA Master archives folder, only those files aren't 'seen' when in the Mac Finder. Is my suspicion correct or is something else? Any solutions? Thanks!

Link to this post | posted 10 May, 2018 14:34

kbutela

I've seen this happen a few times. I have my students set up a DNA Master Archive folder somewhere on their Mac, then I have them set this folder to default in DNA Master Preferences. I discourage my students from using the WINE "DNA Master Archive" folder since sometimes files in there seem to get lost or corrupted more easily. Are the students opening files from the DNA Master "open" menu? In my experience, there is usually a brief hang time for opening files. If the program still isn't opening files, then I have the students uninstall and reinstall the program which seems to take care of the problem.

Randall DeJong
This will hopefully be a silly, easy-to-answer question!
We don't typically use DNA Master for Mac in our course, but we have some students who wish to continue unfinished annotations into the summer. They have downloaded and installed the Wine Bottle version posted by Baylor (thanks SEA colleagues!), and it appears to run fine. However, some run into trouble in that they cannot open files - they just hang. My suspicion is that the files need to be in the DNA Master archives folder, only those files aren't 'seen' when in the Mac Finder. Is my suspicion correct or is something else? Any solutions?

Thanks!

Posted in: DNA Master → Running DNA Master on a Mac using Wine

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