A recent PLoS One article “A Genetic Code Alteration Is a Phenotype Diversity Generator in the Human Pathogen Candida albicans” finds some pretty dramatic changes in gene expression and phenotypes by replacing the tRNAs for CUG back to Leucine (Leu; in the standard genetic code) from their meaning of Serine (Ser) in these Candida species. The CUG codon transition in some Candida spp has been of interest since it is an example of a recent change in the genetic code and provides a comparative system to study the mechanism and genome changes of how a genetic code shift is manifested.
Few organisms are as well understood at the genetic level as Saccharomyces cerevisiae. Given that there are more yeast geneticists than yeast genes and exemplary resources for the community (largely a result of their size), this comes as no surprise. What is curious is the large number of yeast genes for which we’ve been unable to characterize. Of the ~6000 genes currently identified in the yeast genome, 1253 have no verified function (for the uninclined, this is roughly 21% of the yeast proteome). Egads! If we can’t figure this out in yeast, what hope do we have in non-model organisms?Lourdes Peña-Castillo and Timothy R. Hughes discuss this curious observation and its cause in their report in Genetics.
A quick link to a Neurospora paper in Genetics today entitled “Alternative Splicing Gives Rise to Different Isoforms of the Neurospora crassa Tob55 Protein That Vary in Their Ability to Insert ÃŸ-Barrel Proteins Into the Outer Mitochondrial Membrane”. The authors investigated alternative splicing of a gene found in the TOB complex on the outside of the mitochondria. They found reduced growth rate when a strain expressed only the the longest form of three isoforms and confirmed the protein expression of the three isoforms with mass spec.
A recent paper “Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence”, from the Nosanchuk lab at Yeshiva University, shows the role of secreted lipases in virulence of this pathogen. C. parapsilosis is second only to the evolutionarily closely related commensal Candida albicans as worldwide cause of invasive candidiasis. This paper demonstrates a knockout system using selectable marker which confers resistance to the drug Nourseothricin. The authors sought to delete the adjacent and convergently-transcribed lipase genes CpLIP1 and CpLIP2 and characterize the phenotype of the lipase deficient mutants as blood-borne C. parapsilosis infections are in a lipid rich environment.
Through a series of experiments testing growth in rich media, media with olive-oil, and in infection models they showed that the importance of lipase activity. The knockout strain was unable to grow efficiently on YNB media+olive oil indicating that these two genes are the only ones capable of lipase activity. The murine infection experiments indicated that the knockout could be cleared in 4 days while the WT and reconstituted were cleared in 7. The authors acknowledge some limitations in the infection model in that it does not fully recapitulate an invasive candidiasis because mice were infected intravenously so the role of endothelial cell invasion was tested in vivo.
This is not the first paper on targeted gene knockouts in this fungus. A paper from earlier this summer, “Development of a gene knockout system in Candida parapsilosis reveals a conserved role for BCR1 in biofilm formation”, from Geraldine Butler’s group at University College group, who work on both evolutionary and pathogenesis questions in Candida species, developed a knockout system using the same drug marker. The Butler laboratory also showed that the C. parapsilosis MAT locus, part of the sexual reproduction machinery of fungi, has degraded, consistent with the observed asexuality of these species.
The improving genetic tools for targeted disruption of loci in additional species is permitting experiments that get at the heart of what makes some fungi pathogenic. With the genome sequence of many of the relatives of the pathogens we can systematically dissect what genetic differences have a role in virulence. It will be interesting to reconstruct whether the ancestor of many of these Candida spp always had the potential for virulence or if it co-evolved with its human or other mammalian commensal lifestyle.
Back from ISMB/ECCB and a mountain of things left undone that somehow still need doing … including a quick entry about what was interesting at the conference.
I heard many good talks and only a few bad ones – maybe I guessed properly in darting between the multiple sessions. The meeting itsself was much better than past ones I had attended. The combination of Special Interest Groups meeting (BOSC, AFP, and Microbial Comparative Genomics being the ones I spent my time in). I got to give my talks and tutorial during the first few days and was able to just try and soak up the rest of the meeting (when my brain wasn’t melting from the heat). Particularly good was Carole Goble’s presentation on 7-deadly sins of bioinformatics software development.
Some general evolutionary talks that I found really interesting (some of these are probably biased since I count many of the presenters as friends):
- Mike Eisen’s keynote on evolution of regulatory sequences in Drosophila and other flies. Was a wild ride with a couple of different points he has made in the past, but was great to see it all come together.
- Alan Moses presented his work on predicting CDK targets through similar methodology that he pioneered in Eisen lab on cis-regulatory binding site clustering.
- Alex Hartemink gave two incredibly lucid presentations in that I really understood what he was trying to accomplish and how they did it. I wasn’t lost in the algorithm complexities but still understood the approach they were taking. One on Neuronal Information Flow using songbird brain profiling data and a second talk on their work in the epigenetics session on predicting imprinted genes.
- Pilpel Yitzhak presented a summary of published work on selection on translational efficiency and found some very interesting patterns in codon biases in aerobic and anaerobic fungi.
- Ilan Wapinski talked about his approach to building orthologous groups of genes that seems to be quite robust among the ascomycete fungi he used as benchmarked against the YGOB. I’m excited to work with him to apply it to larger sample of fungi as well as other particular clades of fungi.
- Ines Hellmann from Rasmus Nielsen’s group talked about some very cool work to look at population genetic analyses based on whole genome tiling data.
I’ll write a quick post on the BoF session on open source and data sharing as well.
When first discovered, the gene LaeA was thought to be a master switch for silencing of several NRPS secondary metabolite gene clusters in Aspergillus. NRPS and PKS are important genes in filamentous fungi as they produce many compounds that likely help fungi compete in the ecological niche mycotoxins (e.g. aflatoxin, gliotoxin), plant hormone (e.g. Gibberellin), and a potential wealth of additional undiscovered activities.
A recent paper from Nancy Keller’s lab entitled Transcriptional Regulation of Chemical Diversity in Aspergillus fumigatus by LaeA has followed up previous studies with whole genome expression profiling of a LaeA knockout strain to explore the breadth of the genome that is regulated by this transcriptional regulator. Continue reading Exploring a global regulator of gene expression in Aspergillus
A exciting research paper “Control of alternative RNA splicing and gene expression by eukaryotic riboswitches” published in Nature details the mechanism of how riboswitches work in Neurospora crassa. While riboswitches have been found and studied in bacteria there has not been extensive work showing how they work in fungi. In bacteria the riboswitch acts as the direct interacting sensor that switches gene expression off through a structural change in the RNA and fit in nicely with the RNA world view.
Using N. crassa, the authors show that alternative splicing is directly regulated through the thiamine metabolism genes which contains previously identified riboswitches. As also highlighted in the accompanying commentary this is also an interesting examples of direct RNA regulation of alternative splicing rather than through peptides like SR proteins.
A nice evolutionary analysis of peroxin genes entitled PEX Genes in Fungal Genomes: Common, Rare, or Redundant in the journal “Traffic” from Kiel et al out of the University of Groningen in The Netherlands. Within a species, the genes in the PEX family are not necessarily phylogenetically related to each other, but instead are all named as to how they were discovered in mutant screens, most of which were done in S. cerevisiae.
Peroxisomes are interesting because they are necessary for some biochemical reactions (fatty acid metabolism). In filamentous fungi there are additionally specialized peroxisomes called Woronin bodies that plug the septal pore that separates individuals cells in a hyphae. These are specific to filamentous fungi so it is interesting to contrast the numbers and types of genes in the PEX family that are present as determined from the genome sequences. To relate this to human biology, the authors suggest that understanding the complex phenotypes of human peroxisome biogenesis disorders (PBD) will be helped through the study of the disruptions of PEX genes in various filamentous fungi. Interestingly, they find that nearly all PEX genes are present in all fungi, yeast and filamentous alike, although there may be additional genes unidentified.
Woronin bodies in A. nidulans from Momany et al, Mycologia 2002
From Genetics this week a review discusses Why are there still 1000 Uncharacterized Yeast genes? Poor Yeast – so many more genes have no known function, while S. pombe has nearly 100% coverage in functional annotation. I’ll also point out that the 1000 genes refers to protein-coding genes, not ncRNA genes which may mean that there is alot more that is unknown.
I think this sentence from the abstract hits the nail on the head.
Notably,the uncharacterized gene set is highly enriched for genes whose only homologs are in other fungi. Achieving a full catalog of yeast gene functions may require a greater focus on the life of yeast outside the laboratory.
I’ve never worked with Magnaporthe grisea, the fungus responsible for rice blast, one of the most devastating crop diseases, but I do know that its life cycle is complicated and that knocking out roughly 61% of the genes in the genome and evaluating the mutant phenotype to infer gene function is not trivial. In their recent letter to Nature, Jeon et al did what many of us have dreamed of doing in our fungus of interest: manipulate every gene to find those that contribute to a phenotype of interest.
In their study, the authors looked for pathogenecity genes. Interestingly, the defects in appressorium formation and condiation had the strongest correlation with defects pathogenicity, suggesting that these two developmental stages are crucial for virulence. Ultimately, the authors identify 203 loci involved in pathogenecity, the majority of which have no homologous hits in the sequence databases and have no clear enriched GO functions. Impressively, this constitutes the largest, unbiased list of pathogenecity genes identified for a single species (though so of us, I’m sure, may have a problem with the term “unbiased”).
If you’d like to play with their data, the authors have made it available in their ATMT Database.