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In this paper, the authors cloned a GPD promoter for use in driving a GFP gene from Lentinula edodes using a set of provided primers. The primers were derived from the following accession GQ457137.1 containing the GPD gene for this organism.

In fact, there are three accessions in GenBank containing gpd promoter sequences for Lentinula edodes, each of varied size and homology to the aforementioned sequence used in the paper.

I am working on defining a transformation protocol for Psilocybe cubensis (of which there are no published transformations) and I would like to design some primers as done in the paper above in order to clone the GPD promoter for this organism from its DNA; however, just as with L. edodes, there are multiple accessions of GPD in psilocybe cubensis: KM273235.1 and KT067727.1

In my case; however, there is no literature referencing either of these accessions and so I don't know which of these accession I could or should use to design my primers.

I have the following questions:

  1. Why does the same promoter within the same organism have such varied sequences?
  2. If the promoter sequence varies even within the same organism, the how can anyone extract the promoter with certainty since the organism may carry a different sequence for the promoter?
  3. Generally, I'm trying to find the GPD promoter for P. cubensis (or any strong promoter) and clone it for use in my plasmid for transformation. I'm stuck as to how to go about finding a suitable candidate.

Thank you.

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Why does the same promoter within the same organism have such varied sequences?

While there may be other reasons, the fact that the same species has several genes encoding GPD proteins is an indication that this enzymatic activity is important for different mechanisms, times or conditions. For example, I mentioned in a comment to one of your previous questions that the three GPD-encoding genes in budding yeast (TDH1, TDH2 and TDH3) are expressed during different growth phases. Therefore, although the proteins encoded by these genes might share both sequence homology and function, their promoter regions most likely will not. We even expect their promoters to be different, to allow for such differential expression. Put differently: if the three TDH promoters were all highly similar, we would expect them to also be active at the same times, effectively rendering the three genes redundant.

There is therefore no reason for assuming that promoters driving the expression of two different genes will share any homology, just because the encoded proteins happen to share function.

If the promoter sequence varies even within the same organism, the how can anyone extract the promoter with certainty since the organism may carry a different sequence for the promoter?

The only way of knowing exactly what region upstream of a gene that regulates it's expression (i.e., what functional elements that defines its promoter) is to characterize the promoter region. This is way beyond the scope of your work. What we tend to do in budding yeast when we clone an uncharacterized promoter, is to simply take 1000 bp upstream of a gene's start codon, and assume that this includes all elements necessary for proper regulation of the gene in question (e.g. possible transcription factor binding sites (TFBSs), transcriptional start site (TSS), etc). 1000 bp is a relatively safe length in budding yeast, since the distance between genes rarely exceeds this, and we tend to assume that promoter regions don't overlap neighboring genes' coding regions. But this might be different in your species. Also, if you know the location your gene's TSS, this would be a safer reference point than the start codon.

Generally, I'm trying to find the GPD promoter for P. cubensis (or any strong promoter) and clone it for use in my plasmid for transformation. I'm stuck as to how to go about finding a suitable candidate.

My guess is that the authors that cloned the GPD promoter from Lentinula edodes either already knew that this specific gene is highly expressed and therefore selected its promoter; alternatively they might have cloned all GPD promoter variants, and then simply published the one that they saw worked best.

If you don't have any information about the expression of the GPD-encoding genes from your species, any decision as to which promoter to choose will inevitably be more or less guesswork. It seems to me that the evolutionary distance between Lentinula edodes and Psilocybe cubensis is way too large to make any direct inferences. Nevertheless, with the lack of better options here is what I suggest:

  1. Try to identify which of Psilocybe cubensis's GPD proteins is most similar (in terms of sequence homology) to the protein the authors chose for their Lentinula edodes GPD promoter. This might indicate that these are direct orthologs, and with a bit of luck may also share similar relative expression patterns and/or levels. You could also do a similar matching with the Tdh3 protein from budding yeast, since TDH3 is indeed a very strong promoter (article behind paywall, but easy to find elsewhere). If you still don't know which of the promoters to choose, proceed with both candidates and see which one works best (although that means doubled cost and effort).

  2. Once you have identified your candidate GPD gene, you define an appropriate region upstream of your gene’s coding region as the promoter region. This can be arbitrarily picking 1000 bp, or the entire intergenic region, as @Cholpisit suggests.

  3. Design your primers for cloning this region into your target vector, upstream of GFP.

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  • $\begingroup$ I really appreciate your effort to help, but I can't help but feel like you aren't quite understanding what I'm asking even though I've taken a best effort to elucidate it as clearly as possible. Furthermore, I'm asking these questions specifically to learn and reconcile my ignorance before actually designing the primers or running the experiment - you're first sentence seems to presume otherwise. This conversation format doesn't really accommodates a proper discussion where some of the miscommunication could probably be resolved in a few quick back/forth in a chat. $\endgroup$
    – doremi
    Apr 16 at 19:52
  • $\begingroup$ Anywho, I will digest the info further and see if I can think of a way to progress the conversation forward. I am sincerely grateful for your efforts. $\endgroup$
    – doremi
    Apr 16 at 19:54
  • $\begingroup$ If you don't feel my response answer your question(s) you can wait with accepting it and see if someone else addresses it better :) $\endgroup$
    – gaspanic
    Apr 16 at 20:51
  • $\begingroup$ My intent was not to insinuate that you are not trying to understand, I just think that if you don't understand why homology between promoters of paralogous genes is unlikely, you will probably want to understand that before you go about with your endeavor. But explaining why this is the case is a completely different question. In any case, I will edit that comment out from my answer. Also, if you can point out exactly what parts you didn't get answered, you might get better answer from me or from others. $\endgroup$
    – gaspanic
    Apr 16 at 21:02
  • $\begingroup$ I accepted it to ensure my comments didn't take away from the effort you put into the response! Duly noted re: the other parts. $\endgroup$
    – doremi
    Apr 16 at 22:21
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I'm not well-verse in fungal transcriptional system but I did some digging in bacteria before, so not entirely sure this perspective will help.

The promoter region of any gene should be upstream of the transcriptional unit and usually remains in the intergenic region, not being part of the CDS of interest and the adjacent gene. If we're digging a promoter of an essential gene, probably constitutively express with decent strength, ones might be able to chop the whole intergenic region and the part of the adjacent genes (in case they contribute to the transcriptional control). A well-annotated genome would be required for this approach.

Considering the different promoter/gene annotations available in the database. It might be hard to pinpoint if they're the same strain/substrain or not and that may affect the genetic variation to a certain level.

  • The doubting 3 GPD promoters of Lentinula edodes you mentioned are from unpublished sources from 2 teams. Do we know if they're exactly the same strain? As for those two sequences from the same team, they share sequence similarity to a certain level, GQ457137 is part of GQ457138 according to the alignment.
  • As for the P. cubensis GPD promoter, you can try chopping the intergenic sequence upstream of the coding sequence as gaspanic mentioned above. The GPD accessions KM273235 and KT067727 might be referring to different strains as well. Depending on the strain you're working on, you may prefer the sequence submitted as that strain. However, you'll have to look for more information since those deposited sequences refer to partial CDS, not the promoter itself like in the case of Lentinula edodes.
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