SEA-PHAGES | All posts created by ClaireRinehart

Link to this post \| posted 14 Feb, 2019 09:28
ClaireRinehart	First let me point out that we have added two new fields to the Phagesdb BLAST table, Cluster and Pham. We added these two fields because when considering the functions in Phagesdb BLAST we often found ourselves asking, is this phage from the same sub cluster or is this a gene function that has been acquired from another group of phages? Once we added these fields we found it much easier to sort based on cluster and pham. While clicking on the headers to sort and using the search tool is a wonderful way to explore the Phageseb BLAST information, we found ourselves thinking, wouldn't it be nice if we just had a summary count of the functions along with the cluster and pham information. That is why the summary table was created. The count just shows the number of hits for each function/cluster/pham while the frequency shows the fraction of the total number of phages with an assigned function. When checking genes with no assigned function, NKF, I find it very useful to be able to check the Phagesdb Summary and if there are no function/cluster/pham lines then I know it is not really useful to sort and search the Phagesdb BLAST table. Another interesting feature of this summary table is that it shows the variety of names for the same function. This may be useful to point out to students as a reason for using the "approved function list" represented in the drop down window that appears as functions are typed in. Finally, the Phagesdb Summary is useful to see which clusters may have exchanged this gene and you can use the Pham to make sure that they are fairly close relatives. Hope that this helps and that the new features are useful to you and your students. Thanks, Claire

Link to this post | posted 14 Feb, 2019 09:28

First let me point out that we have added two new fields to the Phagesdb BLAST table, Cluster and Pham. We added these two fields because when considering the functions in Phagesdb BLAST we often found ourselves asking, is this phage from the same sub cluster or is this a gene function that has been acquired from another group of phages? Once we added these fields we found it much easier to sort based on cluster and pham. While clicking on the headers to sort and using the search tool is a wonderful way to explore the Phageseb BLAST information, we found ourselves thinking, wouldn't it be nice if we just had a summary count of the functions along with the cluster and pham information. That is why the summary table was created. The count just shows the number of hits for each function/cluster/pham while the frequency shows the fraction of the total number of phages with an assigned function.
When checking genes with no assigned function, NKF, I find it very useful to be able to check the Phagesdb Summary and if there are no function/cluster/pham lines then I know it is not really useful to sort and search the Phagesdb BLAST table.
Another interesting feature of this summary table is that it shows the variety of names for the same function. This may be useful to point out to students as a reason for using the "approved function list" represented in the drop down window that appears as functions are typed in.
Finally, the Phagesdb Summary is useful to see which clusters may have exchanged this gene and you can use the Pham to make sure that they are fairly close relatives.
Hope that this helps and that the new features are useful to you and your students.
Thanks,
Claire

Posted in: PECAAN → New Features in PECAAN

Link to this post \| posted 17 Oct, 2018 21:10
ClaireRinehart	I have a question on what to list as the function for Kalb97 gene 76, which has good matches to the Immunity Repressor genes in other phages. An alignment of Kalb97 with one of it's closest relatives, Marius, show that Kalb97 has suffered a 630 bp deletion at base 44554. This deletion removes about 266 bp from the 5' end of gene 79 of Marius (Kalb97 gene 76), which is the immunity repressor, and about 10 bp from the 3' end of gene 80 in Marius (Kalb97 gene 77). Therefore the termination site is deleted in Kalb97 gene 77, thus allowing the reading frame to continue. The space for a promoter/regulation and the translation start site for Kalb97 gene 76 as well as about half the N-termal end of the immunity repressor coding region is missing and therefore it is doubtful if this phage has any immunity regulation. The Phagesdb entry for Kalb97 lists it as a Temperate phage but the plaque pictures look to be clear plaques. The deletion is also evident in the NCBI BLAST results where the T:Q From is 90:1 for Marius. My question is, what do I list as the function? Do I still put Immunity repressor and then try to put a note in the gene that indicates that this gene carries a clear plaque deletion? Or do I simply label it as NKF and ignore the discovery of the deletion?

Link to this post | posted 17 Oct, 2018 21:10

ClaireRinehart

I have a question on what to list as the function for Kalb97 gene 76, which has good matches to the Immunity Repressor genes in other phages. An alignment of Kalb97 with one of it's closest relatives, Marius, show that Kalb97 has suffered a 630 bp deletion at base 44554. This deletion removes about 266 bp from the 5' end of gene 79 of Marius (Kalb97 gene 76), which is the immunity repressor, and about 10 bp from the 3' end of gene 80 in Marius (Kalb97 gene 77). Therefore the termination site is deleted in Kalb97 gene 77, thus allowing the reading frame to continue. The space for a promoter/regulation and the translation start site for Kalb97 gene 76 as well as about half the N-termal end of the immunity repressor coding region is missing and therefore it is doubtful if this phage has any immunity regulation. The Phagesdb entry for Kalb97 lists it as a Temperate phage but the plaque pictures look to be clear plaques. The deletion is also evident in the NCBI BLAST results where the T:Q From is 90:1 for Marius.

My question is, what do I list as the function? Do I still put Immunity repressor and then try to put a note in the gene that indicates that this gene carries a clear plaque deletion? Or do I simply label it as NKF and ignore the discovery of the deletion?

Posted in: Functional Annotation → Truncated Immunity Repressor

Link to this post \| posted 24 Jul, 2018 17:00
ClaireRinehart	Tammy, We currently do not place the function into the function box in our DNA Master Full Annotation export. Welkin just pointed out to me that the online guide tells how to generate minimal files from the complete notes when functions are recorded in the function field. We will work on adding this feature to the complete file export. Meanwhile, you can use the Export CDS Function button on the export page of PECAAN to export a file that can be copy/pasted into the DNA Master Documentation page which will parse the functions into the notes field. These minimal functions can then be copied en masse into the product or function fields in DNA Master by clicking on the right hand triangle in the Notes field. Thanks, Claire

Link to this post | posted 24 Jul, 2018 17:00

ClaireRinehart

Tammy,
We currently do not place the function into the function box in our DNA Master Full Annotation export. Welkin just pointed out to me that the online guide tells how to generate minimal files from the complete notes when functions are recorded in the function field. We will work on adding this feature to the complete file export. Meanwhile, you can use the Export CDS Function button on the export page of PECAAN to export a file that can be copy/pasted into the DNA Master Documentation page which will parse the functions into the notes field. These minimal functions can then be copied en masse into the product or function fields in DNA Master by clicking on the right hand triangle in the Notes field.
Thanks,
Claire

Posted in: PECAAN → New Features in PECAAN

Link to this post \| posted 23 May, 2018 17:29
ClaireRinehart	Ruth Plymale recently asked how to annotate a frameshift in PECAAN. Since this is a new feature in PECAAN, I thought that I would post the answer here also. Since I am currently working on AugsMagnumOpus I will demo how to find and record frameshifts with this phage. First a quick look at the GeneMark coding capacity plot shows the coding capacity for the two parts of the longer frameshifted gene: 15175 – 15573 and 15520-15971. (Fig. 1) Notice that the left half is in the top forward reading frame and overlaps with the right half that is coded for in the bottom forward reading frame. If you go to the Sequence menu in PECAAN and scroll down you will see the six-frame translation. The top three are forward translation and the bottom three are reverse. All potential Start (green) and Stop (red) codons are displayed in the Top and Bottom DNA strands. The amino acid in each translation is oriented over the first base of the triplet codon that codes for it. If you type 15520 into the Seek position: box in the Sequence window and press return, the left side of the window will scroll 15520 (Fig 2). Notice that the green gene highlight is in the bottom forward translation and corresponds to the left gene. Recall that in GeneMark, this was in the top forward frame. So, the correlation in frames between PECAAN Six Frame and GeneMark is as follows: PECAAN Six Frame……….GeneMark Forward Top…………..Forward Bottom Forward Middle………..Forward Middle Forward Bottom………..Forward Top Therefore, we will expect the frame shift to move from the bottom reading frame in the PECAAN Six Frame figure to the top reading frame since in GeneMark the left half was in the top reading frame and the right half in the bottom reading frame. If we look for a potentially slippery spot in the Forward DNA strand we see in the middle of Figure 2 there is a stretch of five Gs that correspond to PGE in the bottom translation and AGGS in the top frame translation. During the frameshift the tRNA with the Glycine under label 1 shifts back one base to correspond with the Glycine under label 2 in Figure 2. Therefore, the Guanine under label 3 in Figure 2 is both the last base used in translation of the bottom reading frame and becomes the first base in continuing translation of the top reading frame. To accurately find the position of the G under label 3 in Figure 2, take and drag the figure to the left until the G, under label 3, is positioned as the first base at the left of the window and then use the number under Start pos as its proper location which is 15549. Using the Mouse pos can sometimes be inaccurate and should be used to get general locations. Therefore the two parts of the frameshifted gene are then defined by the ranges 15175 .. 15549 and 15549 .. 15971. Now, return to the Genes window by clicking on Genes in the top menu. Click on the top Add Gene button and the Add Gene window will pop-up. Enter the Gene Stop as 15971 and ensure that the Forward Orientation is displayed. Click on the Advanced button and another field will appear for you to enter the beginning of the right half of the frameshifted gene. Enter 15549 and press the blue + button. Another field will appear that will allow you to enter the right end of the left half of the frameshifted gene which is also 15549 in this case. Again, click the blue + button and you will see the complete entry shown in Figure 3. At this point click the Add Gene button at the bottom of the window and the gene will be added and you will see join(start..15549,15549..15971) as the gene range for the new frame shifted gene. One last thing. You will need to go to the original gene representing the right half of the frame shifted gene and uncheck the Gene Included box so that you don’t have it included in the genome. You should see it designated as (Not Included) when you look at the gene selection drop-down menu. Edited 23 May, 2018 17:38 163Kb

Link to this post | posted 23 May, 2018 17:29

ClaireRinehart

Ruth Plymale recently asked how to annotate a frameshift in PECAAN.

Since this is a new feature in PECAAN, I thought that I would post the answer here also.

Since I am currently working on AugsMagnumOpus I will demo how to find and record frameshifts with this phage.
First a quick look at the GeneMark coding capacity plot shows the coding capacity for the two parts of the longer frameshifted gene: 15175 – 15573 and 15520-15971. (Fig. 1) Notice that the left half is in the top forward reading frame and overlaps with the right half that is coded for in the bottom forward reading frame.
If you go to the Sequence menu in PECAAN and scroll down you will see the six-frame translation. The top three are forward translation and the bottom three are reverse. All potential Start (green) and Stop (red) codons are displayed in the Top and Bottom DNA strands. The amino acid in each translation is oriented over the first base of the triplet codon that codes for it. If you type 15520 into the Seek position: box in the Sequence window and press return, the left side of the window will scroll 15520 (Fig 2). Notice that the green gene highlight is in the bottom forward translation and corresponds to the left gene. Recall that in GeneMark, this was in the top forward frame. So, the correlation in frames between PECAAN Six Frame and GeneMark is as follows:
PECAAN Six Frame……….GeneMark
Forward Top…………..Forward Bottom
Forward Middle………..Forward Middle
Forward Bottom………..Forward Top

Therefore, we will expect the frame shift to move from the bottom reading frame in the PECAAN Six Frame figure to the top reading frame since in GeneMark the left half was in the top reading frame and the right half in the bottom reading frame. If we look for a potentially slippery spot in the Forward DNA strand we see in the middle of Figure 2 there is a stretch of five Gs that correspond to PGE in the bottom translation and AGGS in the top frame translation. During the frameshift the tRNA with the Glycine under label 1 shifts back one base to correspond with the Glycine under label 2 in Figure 2. Therefore, the Guanine under label 3 in Figure 2 is both the last base used in translation of the bottom reading frame and becomes the first base in continuing translation of the top reading frame. To accurately find the position of the G under label 3 in Figure 2, take and drag the figure to the left until the G, under label 3, is positioned as the first base at the left of the window and then use the number under Start pos as its proper location which is 15549. Using the Mouse pos can sometimes be inaccurate and should be used to get general locations. Therefore the two parts of the frameshifted gene are then defined by the ranges 15175 .. 15549 and 15549 .. 15971.

Now, return to the Genes window by clicking on Genes in the top menu.
Click on the top Add Gene button and the Add Gene window will pop-up. Enter the Gene Stop as 15971 and ensure that the Forward Orientation is displayed. Click on the Advanced button and another field will appear for you to enter the beginning of the right half of the frameshifted gene. Enter 15549 and press the blue + button. Another field will appear that will allow you to enter the right end of the left half of the frameshifted gene which is also 15549 in this case. Again, click the blue + button and you will see the complete entry shown in Figure 3. At this point click the Add Gene button at the bottom of the window and the gene will be added and you will see join(start..15549,15549..15971) as the gene range for the new frame shifted gene.

One last thing. You will need to go to the original gene representing the right half of the frame shifted gene and uncheck the Gene Included box so that you don’t have it included in the genome. You should see it designated as (Not Included) when you look at the gene selection drop-down menu.

Edited 23 May, 2018 17:38

Posted in: PECAAN → New Features in PECAAN

Link to this post \| posted 16 Apr, 2018 17:13
ClaireRinehart	Welkin, So, you are implying that we should not call any gene as a Tail Assembly Chaperone at this point in the B1 Cluster? If we can call genes that match tail assembly chaperones in other B cluster phages, how many can we call, I see some with one, some with two, and some with none? Edited 16 Apr, 2018 17:15

Posted in: Cluster B Annotation Tips → Tail assembly chaperones?

Link to this post \| posted 25 Jan, 2018 14:28
ClaireRinehart	I am working with gene 72 from Niza, which is an A1 Mycobacteriophage. None of the phagesdb genes show a function but the top HHPred match is to pfam 14373, which is an Immunity Superfection protein based on the E.coli T4 gene described in J. Virol 1989 63:3472-8. Abstract below: "The immunity (imm) gene of the Escherichia coli bacteriophage T4 effects exclusion of phage superinfecting cells already infected with T4. A candidate for this gene was placed under the control of the lac regulatory elements in a pUC plasmid. DNA sequencing revealed the presence of an open reading frame encoding a very lipophilic 83-residue (or 73-residue, depending on the unknown site of translation initiation) polypeptide which most likely represents a plasma membrane protein. This gene could be identified as the imm gene because expression from the plasmid caused exclusion of T4 and because interruption of the gene in the phage genome resulted in a phage no longer effecting superinfection immunity. It was found that the fraction of phage which was excluded upon infection of cells possessing the plasmid-encoded Imm protein ejected only about one-half of their DNA. Therefore, the Imm protein inhibited, directly or indirectly, DNA ejection." The pfam14373 description is: "This family includes the E. coli bacteriophage T4 superinfection immunity (imm) protein. When E. coli is sequentially infected with two T-even type bacteriophage the DNA of the superinfecting phage is excluded from the host, into the periplasmic space. The immunity protein plays a role in this process." Looking at Niza-72, the TMHMM predicts three transmembrane domains that covers 71 residues. The total length of the T4 protein is 76 amino acids and Niza-72 is 104 residues. Finally, NCBI has chosen to recently add the pfam14373 region note to all of the top BLAST hits to Niza-72. A decision for inclusion or exclusion of "superinfection immunity protein" in the accepted function list needs to be made since all future annotators of this Cluster A1 protein will want to include this as a function. It looks like Acinetobacter Baumannii (WP_062937363) has already included it as a function. I didn't attach a DNA Master file since it is readily available on PECAAN. Thanks, Claire

Link to this post | posted 25 Jan, 2018 14:28

ClaireRinehart

I am working with gene 72 from Niza, which is an A1 Mycobacteriophage. None of the phagesdb genes show a function but the top HHPred match is to pfam 14373, which is an Immunity Superfection protein based on the E.coli T4 gene described in J. Virol 1989 63:3472-8. Abstract below:
"The immunity (imm) gene of the Escherichia coli bacteriophage T4 effects exclusion of phage superinfecting cells already infected with T4. A candidate for this gene was placed under the control of the lac regulatory elements in a pUC plasmid. DNA sequencing revealed the presence of an open reading frame encoding a very lipophilic 83-residue (or 73-residue, depending on the unknown site of translation initiation) polypeptide which most likely represents a plasma membrane protein. This gene could be identified as the imm gene because expression from the plasmid caused exclusion of T4 and because interruption of the gene in the phage genome resulted in a phage no longer effecting superinfection immunity. It was found that the fraction of phage which was excluded upon infection of cells possessing the plasmid-encoded Imm protein ejected only about one-half of their DNA. Therefore, the Imm protein inhibited, directly or indirectly, DNA ejection."

The pfam14373 description is: "This family includes the E. coli bacteriophage T4 superinfection immunity (imm) protein. When E. coli is sequentially infected with two T-even type bacteriophage the DNA of the superinfecting phage is excluded from the host, into the periplasmic space. The immunity protein plays a role in this process."

Looking at Niza-72, the TMHMM predicts three transmembrane domains that covers 71 residues. The total length of the T4 protein is 76 amino acids and Niza-72 is 104 residues.

Finally, NCBI has chosen to recently add the pfam14373 region note to all of the top BLAST hits to Niza-72.

A decision for inclusion or exclusion of "superinfection immunity protein" in the accepted function list needs to be made since all future annotators of this Cluster A1 protein will want to include this as a function. It looks like Acinetobacter Baumannii (WP_062937363) has already included it as a function.

I didn't attach a DNA Master file since it is readily available on PECAAN.

Thanks,
Claire

Posted in: Request a new function on the SEA-PHAGES official list → Superinfection Immunity Protein

Link to this post \| posted 22 Jan, 2018 18:17
ClaireRinehart	Sally, I assume that you are referring to Sansa, Aminay, Steamy, Acolyte, and LastResort. I checked on the web Phamerator and could not find them therefore I wonder if they have been Phamerated. PECAAN talks to PITT and St. Louis to pull Phamerator and Starterator information respectively. If a phage hasn't been Phamerated then I don't think that it will show up in the Pitt database. Thanks, Claire

Link to this post | posted 22 Jan, 2018 18:17

ClaireRinehart

Sally,
I assume that you are referring to Sansa, Aminay, Steamy, Acolyte, and LastResort. I checked on the web Phamerator and could not find them therefore I wonder if they have been Phamerated. PECAAN talks to PITT and St. Louis to pull Phamerator and Starterator information respectively. If a phage hasn't been Phamerated then I don't think that it will show up in the Pitt database.

Thanks,
Claire

Posted in: PECAAN → New Features in PECAAN

Link to this post \| posted 18 Jan, 2018 02:30
ClaireRinehart	Excellent! Thank you both. I just updated PECAAN today to match the list. -Claire

Posted in: Request a new function on the SEA-PHAGES official list → Approved list

Link to this post \| posted 17 Jan, 2018 17:39
ClaireRinehart	Dan, I try regularly to update our function list in PECAAN so that it matches the Official Function List here at SEA PHAGES (https://seaphages.org/blog/2017/10/30/official-function-list/). I noticed that you have a line at the top of the page: Written by Dan Russell on Oct. 30, 2017 . My question is this, does this date change whenever you update the list? If not, then could you put a "Last Updated" cell in the sheet itself that will give me something to monitor? -Claire

Link to this post | posted 17 Jan, 2018 17:39

ClaireRinehart

Dan, I try regularly to update our function list in PECAAN so that it matches the Official Function List here at SEA PHAGES (https://seaphages.org/blog/2017/10/30/official-function-list/). I noticed that you have a line at the top of the page:

Written by Dan Russell on Oct. 30, 2017 .

My question is this, does this date change whenever you update the list? If not, then could you put a "Last Updated" cell in the sheet itself that will give me something to monitor?

-Claire

Posted in: Request a new function on the SEA-PHAGES official list → Approved list

Link to this post \| posted 11 Aug, 2017 13:55
ClaireRinehart	In the annotation of Biglebops_31 (27784-28920) we found multiple hits to endonucleases (Hammy_34, Vendetta_66, Splinter_66) and to exonucleases LindNT_35, Marcoliusprime_34), all with e-values of 0. HHPRED also had several hits to endo- and exo-nucleases as well as hits to MRE11 which has both activities and is a DNA double-strand break repair protein (99.5% probability, 60-70% coverage and e-values 10^-12 to 10^-14) that pairs with RAD50, an ATPase. We have no accepted annotation that would best fit this situation. Could we add a "DNA double strand break repair, Mre11-like protein" annotation function to our accepted list? I have included below, an associated paper abstract for the top hit 1ii7_A that explains the relationship of Mre11 and Rad50. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Hopfner, K.P., Karcher, A., Craig, L., Woo, T.T., Carney, J.P., Tainer, J.A. (2001) Cell(Cambridge,Mass.) 105: 473-485 PubMed: 11371344 Search on PubMed Primary Citation of Related Structures: 1II7 1II8 PubMed Abstract: To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker. Edited 11 Aug, 2017 13:59

Link to this post | posted 11 Aug, 2017 13:55

ClaireRinehart

In the annotation of Biglebops_31 (27784-28920) we found multiple hits to endonucleases (Hammy_34, Vendetta_66, Splinter_66) and to exonucleases LindNT_35, Marcoliusprime_34), all with e-values of 0. HHPRED also had several hits to endo- and exo-nucleases as well as hits to MRE11 which has both activities and is a DNA double-strand break repair protein (99.5% probability, 60-70% coverage and e-values 10^-12 to 10^-14) that pairs with RAD50, an ATPase. We have no accepted annotation that would best fit this situation. Could we add a "DNA double strand break repair, Mre11-like protein" annotation function to our accepted list? I have included below, an associated paper abstract for the top hit 1ii7_A that explains the relationship of Mre11 and Rad50.

Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.

Hopfner, K.P., Karcher, A., Craig, L., Woo, T.T., Carney, J.P., Tainer, J.A.

(2001) Cell(Cambridge,Mass.) 105: 473-485

PubMed: 11371344 Search on PubMed
Primary Citation of Related Structures: 1II7 1II8
PubMed Abstract:
To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Edited 11 Aug, 2017 13:59

Posted in: Request a new function on the SEA-PHAGES official list → DNA ds break repair Mre11

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