1
$\begingroup$

I'm reading a paper on genetic transformation of a fungi and the plasmid used in the paper uses two forms of the same GPD (glyceraldehyde3-phosphate dehydrogenase) promoter to drive a GFP gene, one from Agaricus bisporus and one from Lentinula edodes (GenBank: GQ457137.1).

However, I noticed that the sequences for the aforementioned GPD promoters do not match the reference sequence in GenBank (NC_007251.2) which itself is derived from another organism.

Why are there different sequences for the same promoter? Furthermore, how would I identify the GPD gene in another organism if I'm unable to compare it to a known sequence?

The organism I wish to transform has had it's complete genome sequenced and my transformation would be much more effective if I could use a native promoter like GPD.

$\endgroup$
4
  • $\begingroup$ I think in general you are looking for a more general answer about finding homologous sequences or analogous sequences. There is a lot of discussion about how you define a homolog/analog and whether it is a sequence homolog or a functional homolog/analog. This question gives a good answer about potential tools to use. You might also be inte $\endgroup$ Apr 13 at 0:39
  • $\begingroup$ Nice synthetic biology question; want to support the SynBio SE proposal? area51.stackexchange.com/proposals/125068/… $\endgroup$
    – jakebeal
    Apr 13 at 2:19
  • 1
    $\begingroup$ @jakebeal Supported! $\endgroup$
    – doremi
    Apr 13 at 23:40
  • $\begingroup$ @doremi Thank you! $\endgroup$
    – jakebeal
    Apr 14 at 0:12
1
$\begingroup$

I might be misunderstanding you here, but I take from your links above that you want to match the GPD promoter regions from two (distantly related) fungi Agaricus bisporus and Lentinula edodes, to the GPD promoter of Leishmania major, which belongs to a completely different Kingdom!

Promoter regions tend to be relatively poorly conserved between species compared with protein coding regions, even for closely related species. Given the evolutionary distance between the species you are mentioning, the chance you will find any homology between their promoter regions is probably zero.

Furthermore, how would I identify the GPD gene in another organism if I'm unable to compare it to a known sequence?

What I would do is to take the translated GPD protein sequence, which for Lentinula edodes would be GenBank BAA83550.1. I would then use that to search for protein matches using blastp, specifically subsetting for Leishmania major; and use the result to locate the coding gene in the genome. You can also do this in one single step with tblastn, which looks for matches in a translated nucleotide database (see this tblastn example query).

You can then simply take the 1000 bp or so upstream of the coding region to represent your GPD promoter.

$\endgroup$
8
  • $\begingroup$ This might be an ignorant question, but how can completely different sequences produce the same GPD dehydrogenase? In the blastp, for example, even the translated proteins only have 40% homology in some cases. $\endgroup$
    – doremi
    Apr 13 at 23:14
  • $\begingroup$ As an added example, the GPD protein sequences for KT067727.1 and KM273235.1 are both from the same species (the one I'm transforming), but they vary wildly. How am I to know which one I can use? $\endgroup$
    – doremi
    Apr 13 at 23:45
  • $\begingroup$ This section is not a good place to answer your first question, but in short: not all residues are equally important for an enzyme's activity. See this Wikipedia article on active sites for some intuition. $\endgroup$
    – gaspanic
    Apr 14 at 0:44
  • $\begingroup$ The two proteins are probably paralogs. Which promoter to choose depends on your experiment and what you might know about each gene's expression. Compare for example the GPD equivalents in budding yeast: TDH1, TDH2, and TDH3. The latter two are expressed in exponentially growing cells, whereas TDH1 is apparently only found in stationary phase. $\endgroup$
    – gaspanic
    Apr 14 at 1:02
  • $\begingroup$ Related to this question, I want to replace the gpdA promoter from that plasmid with the GPD promoter from KT067727.1 or KM273235.1 (my target organism). If I have the organism in-hand, how am I supposed to design a primer to clone the promoter if the seq varies even within the same species? If I just had it synthesized, how would I decide which accession to use? $\endgroup$
    – doremi
    Apr 14 at 16:45

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.