Closed MPiovesana closed 9 months ago
No problem identifying Uniprot ID; cyp51A gene of Aspergillus fumigatus (Q4WNT5).
NOTE: THESE NAMES HAVE BEEN MODIFIED AFTER DISCUSSION IN TICKET #157; SEE LATEST COMMENT BELOW!
In this paper, CYP51A alleles are amplified from a wt (ND158) and two mutant (MS6 and R7-1) strains of Aspergillus fumigatus, and cloned under the control of the constitutive gpdA promoter from Aspergillus nidulans in an integrative plasmid. A modified ND158 strain of A. fumigatus (harbouring a small deletion in pyrG locus to allow for auxotrophic selection) is then transformed with plasmids. Important to note that endogenous CYP51A is NOT replaced in recipient strain. As mutant alleles are derived from strains other than the recipient, and the plasmids are integrated ectopically in the genome, I have chosen "transformant" to describe these genotypes.
I have added the small disruption in pyrG locus as background info (pyrG-) to all genotypes, and I have named the alleles as follows:
gpdA::CYP51Awt(CYP51A-aaG54) --> for mutant harbouring wt plasmid-encoded cyp51A
gpdA::MS6-CYP51A(CYP51A-aaG54R) --> for mutant harbouring CYP51A-aaG54R derived from strain MS6;
gpdA::R7-1-CYP51A(CYP51A-aaG54W) --> for mutant harbouring CYP51A-aaG54W derived from strain R7-1.
Must I add something else to the allele description to indicate the presence of the endogenous wt copy of CYP51A?
Authors quantify expression levels of CYP51A in all mutant lines (Fig 2), and observe increased expression; however, given that this increased expression is a result of the introduction of extra copies of the gene in the transformant lines, I selected "ectopic" as expression level. Is that correct?
AC: I think 'ectopic' is correct here, as it seems that the altered phenotype is due to the AA substitution in Cyp51A rather than overexpression of the gene.
Phenotype annotation is quite straightforward (Table 2); with "normal growth on posaconazole" and "resistant to posaconazole" terms used for respective genotypes.
@CuzickA Another example for discussion - allele type "transformant".
gpdA::CYP51Awt(CYP51A-aaG54) --> for mutant harbouring wt plasmid-encoded cyp51A
gpdA::MS6-CYP51A(CYP51A-aaG54R) --> for mutant harbouring CYP51A-aaG54R derived from strain MS6;
gpdA::R7-1-CYP51A(CYP51A-aaG54W) --> for mutant harbouring CYP51A-aaG54W derived from strain R7-1.
Genotypes names modified according to recent discussion of transformant allele type:
CYP51A transformant(ND158-CYP51A(G54))[Ectopic]
CYP51A transformant(MS6-CYP51A(G54R))[Ectopic]
CYP51A transformant(R7-1-CYP51A(G54W))[Ectopic]
Disruption in pyrG locus and presence of endogenous CYP51A gene were recorded as background info (-pyrG / endogenous CYP51A present)
Maybe it is better to create genotypes and annotations based on the spontaneous mutations in Cyp51A identified by culturing pathogen with posaconazole. Only one AA change was identified within the Cyp51A gene compared to the WT (Table 1 data). However, as it is is possible that other spontaneous mutations have occurred throughout the genotype, maybe it is also worth recording the transformant genotypes as well, which confirm that this AA change in Cyp51A is responsible for the phenotype (Table 2 data).
I have added the following annotations to capture the spontaneous mutant data in Table 1.
Current transformant genotype annotations
These seem okay, but I will delete the top one as this is really a control and behaves in the same way as the untransformed WT in Table 2
Now looks like this
AE alteration_in _archetype
G54R; Cyp51A; ASPEFU G54W; Cyp51A; ASPEFU
Questions for Nichola
Are these correct? I could not find info on Cyp51A in your S file. I found the above information from Table 2 in Mair et al 2016.
Session will be approved after archetype check. Closing ticket.
G54R; Cyp51A; ASPEFU G54W; Cyp51A; ASPEFU
This is correct
AE alteration_in _archetype checked by Nichola. Session now approved.
Curation by @MPiovesana https://canto.phi-base.org/curs/b6da8569a501db3e