Link to this post | posted 23 Jun, 2020 06:27

anders

Hi, I am QCing the cluster F phage Coco12, which has an HNH endonuclease inserted into lysin A, and am looking for guidance on annotating the lysin A bits. The insertion seems to split the gene between domains. HHPred easily identifies the amidase domain in the downstream half, but it does not provide any evidence for a peptidase domain in the upstream half. BLAST does show that the upstream half has the same sequence as lysin As in Payne and Hatfull that contain the "N3" putative peptidase domain, as in Tweety gp30 (Payne and Hatfull, 2012). Cluster F phage Frankie has nearly the same situation with an HNH endonuclease inserted in nearly the same position. To my surprise, the lysin A gene in Frankie is annotated as if it has an intron – in two sections that are connected together to make one lysin A polypeptide. The GenBank entry reads gene 26909..28665 /locus_tag="SEA_FRANKIE_33" CDS join(26909..27370,27946..28665) I have attached phamerator screenshots of the Coco12 and Frankie lysin As aligned with intact lysin As. Should Coco12 lysin A be annotated with an intron, as in Frankie? (I am thinking that bench evidence ought to support an annotation like this.) Or, should each lysin A part be annotated with a domain function appended? We have good HHPred evidence supporting the amidase domain for the downstream section, but we lack HHPRed or CDD evidence for a peptidase function in the upstream bit. BLASTp shows that the upstream section is nearly identical to Tweety gp30, which is the canonical lysin A listed in Payne and Hatfull that carries the N3 hydrolase domain (M23B peptidase in phi29 gp13 tail protein). Or is there another option that is the best in this case? Thanks! Kirk ################################################# Addendum 6/26/20: I said above that the first half of Coco12's lysin A did not hit anything on HHPRed. I have never fiddled with HHPred settings before, but I changed the MSA generation method from the default HHBlits=>UniRef30 to PSI-BLAST=>nr70 and this now hits a group of proteins that does include phi29 gp13. The region that matches phi29 gp13 is not to its m23 metallo-peptidase-like domain, but to its lysozyme domain. I've attached a screenshot and link to the HHPred output. Edited 26 Jun, 2020 16:49 150Kb 101Kb

Link to this post | posted 26 Jun, 2020 17:09
welkin	Hi Kirk, Can you stitch the lysin back together in silico and get really good BLASTN matches to other lysins? That would be the most compelling evidence for an intron, other than the HNH (the HNH in the gap is already pretty compelling).

Link to this post | posted 26 Jun, 2020 18:47

anders

Hi Welkin, Yes, when I remove the inserted HNH and the 6 bp duplication that was created, a lysin A is made that is identical to others such as MilleniumForce lysin A. I've attached my notes and sequences. Thanks, Kirk ###################################################### addendum: I have also attached documentation of the final decision we made for the splice junctions. Edited 08 May, 2021 00:54 522Kb 154Kb

Link to this post | posted 07 May, 2021 14:29

andrea

Hi, We found this in JalFarm20 as well. We tried to change this in PECAAN by deleting the original 2 Lysin A genes (see below) and adding a gene that spanned the entire length of the protein. The pop-up allowed us to enter the proper stop coordinate (28,603) but there was no place to enter the proper start coordinate (26,866), and it did not appear amongst the gene candidates, even when we deleted genes 32, 33, and 34 or just 33+34 first: Gene 32: 26,866-27,360 Forward (Lysin A) Gene 33: 27,847-27,341 Reverse (HNH Endonuclease) Gene 34: 27,884-28,603 Forward (Lysin A) Does anyone know of a way to do this in PECAAN? We were not sure how to use the advanced Join Position for adding a gene, even after reading the PECAAN guide. Or should we delete gene 34 in PECAAN and manually change this in DNA Master? Thank you, Andrea

Link to this post | posted 07 May, 2021 20:03
debbie	Hi Andrea, i do not know how to do this in PECAAN, but in following Kirk's explanation for Coco12, i would annotate it the same way he did. debbie

Link to this post | posted 07 May, 2021 20:27
andrea	Hi Debbie, Thanks for your speedy reply. I was in touch with Kirk to verify our annotation of the Lysin A gene. I made the change in DNA Master to reflect this. Have a great weekend! Andrea

Link to this post | posted 07 May, 2021 20:42
debbie	You too! Terrific that you contacted Kirk! Thanks, debbie