Link to this post | posted 30 Jul, 2023 22:55
kcornely	Is it possible for a phage to have two WhiB family transcription factors? We think we've identified two in phage Maravista.

Link to this post | posted 30 Jul, 2023 22:09
kcornely	Greetings, all! We are getting ready to submit our annotation for Maravista and we haven't received a reply to this query, so I hope someone can help us out. To clarify, it's a reverse gene and it's gp 41 on phagesdb and we are going with the GeneMark start at 32125. We're fairly certain it's a transposase but want to be sure. Thanks!

Link to this post | posted 23 Jun, 2023 18:47

kcornely

Greetings, all, Is anyone else having trouble updating DNA Master? We get an error message when we attempt to update. I've attached a screenshot. We've tried computers on campus and off campus, laptops and desk tops, and nothing is working. The update reaches 33%, and then DNA Master either freezes, or we get an FTP error. It appears that DNA Master is having difficulty connecting to the server. Any advice to solve this problem would be appreciated and thanks in advance! 71Kb

Link to this post | posted 22 Jun, 2023 16:47
kcornely	We are annotating a cluster F1 phage (Maravista)and have identified a transposase (gp 41) and we need to find the corresponding transposon. We haven't done this before. What tools would be effective to assist us in identifying a transposon? Thanks in advance for the help!

Link to this post | posted 09 Jun, 2023 18:15
kcornely	Talking to Debbie in person! We clarified that the start site is the second methionine in the sequence, which means that the translated protein has a single initial methionine and not two Mets, irrespective of the direction of translation. Edited 09 Jun, 2023 19:25

Link to this post | posted 02 May, 2023 02:26

kcornely

Greetings, all! We are annotating a cluster A3 mycobacteriophage and we see that the lysin A and lysin B proteins do not follow the expected synteny in which lysin A, then holin, and then lysin B follow the minor tail proteins expressed downstream of the tape measure protein. Instead we see lysin A and lysin B near the 5' end of the genome, expressed upstream of terminase. And we aren't able to find a protein we can assign as a holin. Is synteny different in cluster A3 phages? Thanks in advance for your help!

Link to this post | posted 05 Apr, 2023 19:23

kcornely

We are annotating a cluster A3 phage LBerry and I found this discussion to be really helpful, because I didn't expect to find minor tail proteins on the left hand side of the genome. We have three genes, gp3, gp4, and gp5, that are candidate minor tail proteins. The gp4 and gp5 proteins seem promising because we get HHPred hits to collagen-type proteins, and BLAST hits to phage tail fiber proteins. The sizes of gp4 and gp5 also seem to be about right (945 bp and 636 bp, respectively). We're not sure about gp3. The size is smaller (321 bp), but we get some nice HHPred hits to tail fibers for L5 and the E. coli phage T5 (for example, the L subunit of phage T4; PDB code = 7QG9). Is gp3 still too short to be a minor tail protein? Or could we still call the function as a minor tail protein because gp3 might be a subunit that is part of a larger assembly?

Link to this post | posted 08 Aug, 2022 22:54

kcornely

Thanks, Debbie and Karen! I met with my students today, and I also consulted with a biochemistry colleague, and we investigated some of the other genes in pham 37230. There are 266 members of this pham, and I looked on phagesdb and these genes are all about 800-1000 bp in length. I looked at some of the other pham members and I used the domain function in Phamerator (and I also did a BLASTp on NCBI to get the domain information for Langerak_46 and BiteSize_54) and from what I can see, these proteins all have hits to exonuclease domains that cover the length of the protein, and no alignments with helicases. And the encoded proteins don't appear to be large enough to have both exonuclease and helicase functionalities. It is possible that the earlier annotations of the P1 cluster phages of this pham as RecB-like exonuclease/helicase were incorrect? I'm also curious about RedRock_72 as an example of a RecB-like exonuclease/helicase. I looked on phagesdb and found that gp72 in RedRock belongs to pham 24. Virtually all members of pham 24 are assigned a Cas4 exonuclease function (as Karen says in her post), and again, these genes are about the same size (800-900 base pairs)indicating that the proteins aren't large enough to have both helicase and nuclease functionalities. Maybe we don't have an example of a phage protein that has both functionalities because one doesn't exist, as least as far as we know? Thanks to both of you to responding to my posts–my next task is to look over a QC'd genome that we recently received, and we got quite a few of the functional assignment wrong. As a biochemist, I ought to be good at this, and it's important for me to get this right so that I can properly mentor my students. Thanks again, Kathleen

Link to this post | posted 05 Aug, 2022 14:56
kcornely	Karen, this is very helpful, thank you. An example of a RecB-like protein would be very helpful. I understand your thought process and your logic as identifying this protein as a Cas4 endonuclease. But why do so many other P1 cluster phages assign the function as RecB-like exonuclease/helicase? Shouldn't proteins in the same pham all have the same function? Thanks again for your reply, Kathleen

Link to this post | posted 03 Aug, 2022 18:48

kcornely

Greetings, all! I'd like to come back to my previous post regarding Starterator. We are encountering quite a few genes in Frankenweenie in which the suggested Starterator start is not the same as the most annotated start. I give an example above; here's another example: For gene 30, the original Glimmer call @bp 15143 has strength 3.13 ** not called by GeneMark; ST: start @15143 is the suggested start, but start @15146 has five most annotated. If we choose the start at 15146, which we believe is correct, we obtain q1:s1 data for all five BM cluster phages, with an alignment of 98.8%. In addition, the 15146 start produces a -1 bp overlap and expresses a gene length of 249 bp. All of the other five BM cluster phages that express the gene of this pham (7493) are also 249 bp. The Glimmer/Starterator start at 15143 provides a -4 bp overlap. So this gene has both -4 and -1 bp overlap options and the advice we got from the forum is to go with the -1 bp overlap. So we believe that the most annotated start, 15146, is correct. But we don't understand why Starterator didn't call the most annotated start. As always, thanks for helping me to understand Starterator better! Best wishes, Kathleen