Category Archives: cell biology

Nuclear Pore Complex studied in thermophilic fungus

ResearchBlogging.orgSometimes choosing a hot lover can make all the difference. In this case, choosing a thermophilic fungus was the right eukaryote for the job to purify stable proteins from the nuclear pore complex and test their interactions. Since high temperatures (60C as compared to what its relative Chaetomium globosum prefers, 24C) will denature proteins, this fungus has evolved the ability to still fold up proteins nicely at those high temperatures. Thus at more standard laboratory room temperature or below, these proteins should be really stable and easier to work with.  This manuscript (not OpenAccess sadly) includes the genome sequence of Chaetomium thermophilum sequenced with 454 FLX and XLR at 24X and assembled into 20 scaffolds – (8 chromosomes expected so they say – and I agree – this is quite good).The used the Celera assembler to make this final assembly for those of you taking notes at home on how to assemble your fungal genomes. The genome is available for download at the authors’s site or at GenBank. Their assembly is quite a bit smaller (28.3 Mb) than the related C. globosum (34.9) or Neurospora crassa genome (41Mb – though the authors use the old version and report 39.2; they also say “*based on the published genome (Galagan et al., 2003), although there is a newer assembly of N. crassa available from Broad, the newer assembly is not annotated for protein coding genes yet.” which is kind of weird because there is an annotated version here). I do wonder if the tendency of repeated elements to be collapsed in the assembly process resulted in a smaller assembly or if this really does have a smaller genome and less genes (~7k genes while Neurospora has ~10k).  Also worth noting that several other thermophilic fungi have been public for a while at the JGI too – Thielavia terrestris and Sporotrichum thermophile and our lab and others are investigating the genome content and how some genome properties like transposons have evolved in these lineages.

The thermophilic adaptation of this fungus has lead to stable proteins which can be studied more easily than mesophilic fungi. They have been able to determine how the nucleoporins (Nups) interact because of the biochemical and structural assays that are possible with the more stable protein complexes. This highlights the value of targeting an experimental system that has the properties needed and the simple and straightforward tactics needed to generate and use the genome sequence (the genome is but a minor note in the findings of this paper).  I can only wonder why none of my de novo genome assemblies go together as nicely as this one, but I’m excited to see this work present new insights into the biology of nuclear pore complexes.

Amlacher, S., Sarges, P., Flemming, D., van Noort, V., Kunze, R., Devos, D., Arumugam, M., Bork, P., & Hurt, E. (2011). Insight into Structure and Assembly of the Nuclear Pore Complex by Utilizing the Genome of a Eukaryotic Thermophile Cell, 146 (2), 277-289 DOI: 10.1016/j.cell.2011.06.039

Methylation to the max!

A new paper from the Zilberman lab at UC Berkeley shows the application of high throughput sequencing to the study of DNA methylation in eukaryotes.  They generate an huge data set of whole genome methylation patterns in several plants, animals, and five fungi including early diverging Zygomycete.

The work was performed using Bisulfite sequencing (Illumina) to capture methylated DNA, RNA-Seq of mRNA. The also performed some ChIP-Seq of H2A.Z on pufferfish to look at the nucleosome positioning in that species. For aligning the reads, they used BowTie to align the bisulfite sequences (though I’d be curious how a new aligner, BRAT, designed for Bisulfite seq reads would perform) to the genome.  They also sequenced mRNA via RNA-Seq to assay gene expression for some of the species.

They find several interesting patterns in animal and fungal genomes.  I’ll highlight one in the fungi. They find an unexpected pattern in U. reesii of reduced CGs in repeats, which shows signatures of a RIP-like process, are also methylated.  This finding is also consistent with observations in Coccidioides (Sharpton et al, Genome Res 2009) that showed depleted CGs pairs in repeats.  Since the phenomenon is also found in Coccidioides genomes this methylation of some repeats is likely not unique to U. reesii but may be important in recent evolution of the Onygenales fungi or the larger Eurotiales fungi.  There are several other interesting findings with the first such study that shows methylation data for Zygomycete fungi and a basidiomycete close to my heart, Coprinopsis.  It will be interesting is to dig deeper into this data and see how the patterns of methylation compare to other genomic features and the mechanisms regulating methylation process.

Zemach, A., McDaniel, I., Silva, P., & Zilberman, D. (2010). Genome-Wide Evolutionary Analysis of Eukaryotic DNA Methylation Science DOI: 10.1126/science.1186366

Hey there fluffy

ResearchBlogging.orgI spy a picture of Neurospora growing on the cover of Genetics this month.  The cover highlights the results from the work of the lab of Luis Corrochano who works on  light regulation in a variety of systems like Neurospora and Phycomyces.  This work describes their work on the fluffy gene which regulates conidiation (production of conidia or asexual spores). They show that an important interplay between an inducer of light response, the White Collar Complex (WCC), and the FLD protein on fluffy.  The data from indicate hat FLD represses fluffy as a response to dark but that this repression is removed in response to light through the action of WCC.

Olmedo, M., Ruger-Herreros, C., & Corrochano, L. (2009). Regulation by Blue Light of the fluffy Gene Encoding a Major Regulator of Conidiation in Neurospora crassa Genetics, 184 (3), 651-658 DOI: 10.1534/genetics.109.109975

Gene prediction without training?

A new paper in Genome Research from Borodovsky lab at Georgia Tech provides an improved ab initio gene prediction building on their previous program GeneMark called GeneMark.hmm ES.  This application doesn’t require a training set when building models for gene prediction in fungal genomes and reports to have as good or better sensitivity and specificity than most of the commonly used ab initio programs. They are picking up on proviously described insights about fungal gene structures and introns which is the lack of a necessary branch site and varying degrees of conservation of splice-sites in most intron rich fungi (Schwartz et al, 2008) and that these intron sizes remain short across the fungi (Stajich et al. 2007).

In practice it should simplify the initial genome annotation protocols used and could really streamline the procedures. It doesn’t replace the need to gathering EST sequence data that can also be used generate a training set in an automated fashion.  EST and transcriptional evidence is still very important for identification of UTR and alternative splicing isoforms.

Hopefully these data from the predictions will integrate into the Cryptococcus and Coprinus genome annotations that are undergoing an update at the Broad.  We’ll see how well this performs on a couple of the Chytrid genome sequences we are working on as well.

Aspergillus comparative transcriptional profiling

Researchers from Technical University of Denmark published some interesting results from comparing expression across the very distinct Aspergillus species.

Kudos also goes to making it Open Access. I am posting a few key figures below the fold because I can! They grew the fungi in bioreactors fermenting glucose or xylose. After calibrating the growth curves they were able to sample the appropriate time points for comparison of gene expression across these three species. They found a set of genes commonly expressed.

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S.pombe telomerase RNA identified

Blogging on Peer-Reviewed Research Webb, C.J., Zakian, V.A. (2008). Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA. Nature Structural & Molecular Biology, 15(1), 34-42. DOI: 10.1038/nsmb1354

Leonardi, J., Box, J.A., Bunch, J.T., Baumann, P. (2008). TER1, the RNA subunit of fission yeast telomerase. Nature Structural & Molecular Biology, 15(1), 26-33. DOI: 10.1038/nsmb1343

Two papers in Nature Structural & Molecular Biology identify the telomerase RNA in Schizosaccharomyces pombe. Telomerase is a multi-unit enzyme that has both protein and RNA components. While the protein subunit is highly conserved and identifiable through sequence comparisons of eukaryotes, the RNA subunit has a variable size and sequence making identification through comparative means more difficult. The S. pombe telomerase RNA subunit, or TER1, was discovered by two labs applying similar biochemical approaches to identify the locus.

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Caffeine induced alternative splicing

A study shows how Caffeine regulates alternative splicing in a subset cancer-associated genes including the transcription factor and tumor suppressor KLF6 through the splicing factor SC35.  There is a necessary “caffeine response element” in the intron of KLF6 which plays a role in the splice-site choice, although caffeine induces up-regulation of SC35 and over-expression of SC35 is sufficient to mimic the caffeine response.

Candida White-Opaque switching

Blogging on Peer-Reviewed ResearchA paper in PLoS Biology from Sandy Johnson’s lab entitled “Interlocking Transcriptional Feedback Loops Control White-Opaque Switching in Candida albicans discusses phenotype switching in the human pathogenic fungus Candida albicans. Why is the important?

“White-opaque switching is an epigenetic phenomenon, where genetically identical cells can exist in two distinctive cell types, white and opaque. Each cell type is stably inherited for many generations, and switching between the two types of cells occurs stochastically and rarely—roughly one switch in 10^4 cell divisions”

white-opaque coloniesThere is also a review by Kira O’Day to discuss the implications of the findings. Understanding this sort of developmental and epigenetic signaling is important to better know how fungi adjust and interact with their environment. However, the authors do conclude that White-Opaque switching is exclusive to Candida albicans so aspects of this research only directly applicable to studies in this system. Phenotype switching is an active area of research for Candida biologists – some nice micrographs and SEM of the different cell morphologies can be seen at Prof. Joachim Morschhäuser’s page (and linked to the right).

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