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All posts created by Pollenz

| posted 25 Feb, 2020 12:58
The short duplicated areas in the DJ phages appear to have sequence similarity to the sequences in the Cluster BI1 phages even though they come from different species.

The DJ phages have eight of these directly upstream of start codons:

CGCAtatATTACAtGCCtTATAATGAGACCCCTAcGAAAGG(xxxxxxx 6-9bp)ATG
RS Pollenz
Posted in: Choosing Start SitesSD scoring matrix
| posted 24 Feb, 2020 15:22
Yes, very interesting. There is one sequence of ~44bp that is replicated PRIOR to 5 consecutive genes…..
RS Pollenz
Posted in: Choosing Start SitesSD scoring matrix
| posted 24 Feb, 2020 14:03
We are annotating a Gordonia DJ phage Secretariat. It appears there is an region across most of these DJ genomes that begins about 32,000bp and contains ~15 small genes that are divergent across the different phages and all the genes are separated by 20-100bp gaps between them (no 1-4bp overlaps or small <10bp gaps). The issue is that the called start for many of these in NOT the LO that in many cases will significantly reduce the gaps between these genes. It appears that the RBS data is very poor (example, Z value 0.446, spacer 8, final score -8, compared to the "called" start of 1.77/11/-5.3) for many of these. All of the genes are hypothetical, so no evidence of how these genes are related (an opernon??). The Coding Potential may drop off at the LO on some of these, but its not that different from many genes that have been called. We know that there are many aspects that impact translation initiation beyond the RBS/SD such as RNA structure, operons, translation of previous genes, etc. What are your thoughts on selecting the LO and reducing gaps when the RBS data is so poor?
RS Pollenz
Posted in: Choosing Start SitesSD scoring matrix
| posted 29 Jan, 2020 20:34
We installed the virula machine and windows10 on a new Macbook Air and the window size (screen) is very small when you launch the VM. How do you change the size to make it bigger. The MAC controls the OS screen, not the VM screen. Thank you
RS Pollenz
Posted in: Using WINE to run DNA Master on a MacHelp with WINE
| posted 14 Jun, 2019 15:31
hello

I have a new 2019 DELL laptop and have been trying to install DNA master. I do not get all the files in the download (no library or ssleay32 files) and I do not see the DNAMas updated version showing up like I saw when I did this on an older machine I borrowed prior to the conference last week (running windows 7). Thus, the program that is downloaded will not update from the 2012 version. I tried form both Phages DB links and seaphages.org. I have uninstalled and tried several times but get the same results. I noticed that when I initially download the file and open in the downloads folder, the DNAmas.ex will not initially launch and gives an error saying that it will not run on a PC, but eventually the DNAmas.ex does turn into the DNA master icon so I can launch the installer……but only seems to contain the minimal files listed above.
RS Pollenz
Posted in: DNA MasterDNAS master install on 2019 PC
| posted 23 May, 2019 17:32
We have worked in Gordonia r. and M. foliorum the past few years. Typically, the uncut DNA from isolated that have high concentration (> 200ng/ul) runs with a high mol mass band, but does show some amount of smear (see enclosed example). The gels sometimes show a double band or gap in the high mol mass DNA. If we have low yield samples (<50ng/ul), they sometimes do not run as a high mol mass band and appear smeared but its hard to tell if that's because the concentration is already low and was degraded during the isolation.
RS Pollenz
Posted in: Phage BiologyDNA Smear
| posted 05 Feb, 2019 19:49
On further review I was thinking Emperor could be a prophage…….this is posted on the Emperor Phages DB: Emperor was found as a prophage of Gordonia westfalica NRRL-B-24152. Liquid Gordonia westfalica was spotted on Gordonia terrae 3612, and the resulting phage release was picked and amplified, and called Emperor. So, now it make sense and perhaps since the Emperor genes are redundant to the Gordonia genome genes, they do not hot when doing a BLASTP.
RS Pollenz
Posted in: AnnotationPhage hits not showing in NCBI but 100% to Gordonia wetsfalica
| posted 05 Feb, 2019 14:31
We are currently annotating a DM phage, Ashton. This cluster has few members and Ashton is from M. foliorum while the others are from Gordonia terrea. Ashton has some nucleotide similarity to SallySpecial and Emperor and there are high and low areas as you would expect. In the BLASTP analysis, we are not consistently seeing the genes from Emperor as hits but getting hits to proteins from Gordonia westfalica as the top match when we expect to see products from Emperor. When I do a BLASTP on the products from Emperor, they all hit with 100% alignment and 100% identity to Gordonia westfalica (the genes from Emperor are NOT showing as hits), even Emperor gene #9 listed as an orpham (see screen shot) hits to the westfalica! I can also manually align Ashton and Emperor products to show alignments that NCBI is not showing me vs giving me the Gordonia westfalica hits. Emperor is in Genbank, so I can find the sequences and accession numbers. Am I missing something here? Why is Emperor phage 100% matching to an annotated bacteria genome from a different Gordonia strain? We do see hits to the SallySpecial products even though they have much reduced identity vs. Emperor in some instances.
RS Pollenz
Posted in: AnnotationPhage hits not showing in NCBI but 100% to Gordonia wetsfalica
| posted 04 Feb, 2019 20:03
OK great. Here is another interesting situation. We have a double start codon GTGATG where the GTGA creates the 4 bp overhang to to the previous gene. I know from Welkin that we typically choose the 2nd start codon if there are two consecutive……Thoughts since choosing the ATG is a one bp overlap to the TGA stop. The RBS data show identical values for the GTG and ATG.
RS Pollenz
Posted in: Choosing Start SitesGTGA overlaps
| posted 22 Jan, 2019 17:14
I see mention of the ATGA 4bp overlap across 2 genes as a preferred configuration and can assist in selecting a correct start site. What is the situation with a GTGA 4bp overlap? We have several genes in our EE phage that show this GTGA configuration. Is this also a preferred orientation that should be noted in the annotation notes file?
RS Pollenz
Posted in: Choosing Start SitesGTGA overlaps