A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications

Models of <i>KPTN</i>-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.</p

The olfactory transcriptomes of mice.

The olfactory (OR) and vomeronasal receptor (VR) repertoires are collectively encoded by 1700 genes and pseudogenes in the mouse genome. Most OR and VR genes were identified by comparative genomic techniques and therefore, in many of those cases, only their protein coding sequences are defined. Some also lack experimental support, due in part to the similarity between them and their monogenic, cell-specific expression in olfactory tissues. Here we use deep RNA sequencing, expression microarray and quantitative RT-PCR in both the vomeronasal organ and whole olfactory mucosa to quantify their full transcriptomes in multiple male and female mice. We find evidence of expression for all VR, and almost all OR genes that are annotated as functional in the reference genome, and use the data to generate over 1100 new, multi-exonic, significantly extended receptor gene annotations. We find that OR and VR genes are neither equally nor randomly expressed, but have reproducible distributions of abundance in both tissues. The olfactory transcriptomes are only minimally different between males and females, suggesting altered gene expression at the periphery is unlikely to underpin the striking sexual dimorphism in olfactory-mediated behavior. Finally, we present evidence that hundreds of novel, putatively protein-coding genes are expressed in these highly specialized olfactory tissues, and carry out a proof-of-principle validation. Taken together, these data provide a comprehensive, quantitative catalog of the genes that mediate olfactory perception and pheromone-evoked behavior at the periphery

Limited sexual dimorphism in the olfactory system.

<p>The mean gene expression is plotted against their log₂ fold change between male and female samples for the VNO (A) and OM (B). Genes with ±infinite fold changes were assigned to ±13 to ease visualization. Triangles depict genes located on the sex chromosomes. Genes significantly differentially expressed in one tissue (FDR 5%) are red while the 11 genes that are significantly differentially expressed in both tissues are blue. The genes in the VNO plotted in green are eight lipocalins that are highly variable between replicates. Dotted lines indicate a log₂ fold change of ±2.</p

Putative novel genes.

<p>Putative novel genes.</p

Expression of the complete OR repertoire in the OM.

<p>The mean FPKM expression values for all OR and trace amine-associated receptors (TAAR) genes in the OM. Genes are ordered by their chromosomal location and chromosomes are annotated in the boxes at the bottom. Class I OR genes are blue, class II OR genes are red and TAAR genes are green. Black shading below each bar indicates the gene is annotated as a functional receptor, and grey indicates an annotated pseudogene. Error bars represent the standard error of the mean from the six biological replicates. <i>Olfr1507</i> is the highest expressed OR gene and is indicated with an asterisk.</p

Novel genes are expressed in olfactory organs.

<p>(A) Chromosome 2 is schematized on the top and the locus where two previously unidentified genes were found is amplified below. In black are <i>Lcn16</i> and <i>Lcn17</i> gene models, where boxes correspond to the exons. The mapped RNAseq reads are below: each read is drawn in grey and blue lines join read fragments that span exon junctions. Black segments within the reads indicate indels. (B) <i>In situ</i> hybridization reveals <i>Lcn16</i> is expressed in glandular tissue of the VNO and (C) <i>Lcn17</i> is expressed in cells within the main olfactory epithelium. Scale bars: (B) 100 µm, (C) 50 µm.</p

RNAseq provides comprehensive gene models for ORs and VRs.

<p>(A–B) An example of new gene models generated for <i>Olfr168</i> (A) and <i>Vmn1r34</i> (B) are shown in black. Boxes correspond to exons and arrowheads indicate the direction of the gene. The existing Ensembl annotations for the genes are shown in red with their UTRs in grey. New 5′ exons and extended 3′UTRs were identified for both. The mapped RNAseq reads that support the models are below. Each read is drawn in grey and blue lines join read fragments that span exon junctions. Black segments within the reads indicate indels. (C) Boxplots of the transcript length as annotated in Ensembl (pink) or as obtained from the reconstructed models from our RNAseq data (blue) for the V1R, V2R and OR genes. The increase in transcript length is highly significant (*** P<0.0001, two-tailed Mann-Whitney test). (D) As above, but quantifying the proportion of unique sequence for probe design (*** P<0.0001 and *P<0.01, two-tailed Mann-Whitney test). The <i>uniqueness</i> corresponds to the proportion of all 100 nucleotide long windows within the transcript that map uniquely to the genome. In all boxplots, outliers are defined as data points that fall outside 1.5 of the inter-quartile range, and are plotted as open circles.</p

Expression of the complete VR repertoire in the VNO.

<p>The mean FPKM expression values are shown for all the VR and formyl peptide receptor (FPR) genes in the VNO. Genes are ordered by their chromosomal location and chromosomes are annotated in the boxes at the bottom. V1R genes are blue, V2R genes are red and FPR genes are green. Black shading below each bar indicates the gene is annotated as a functional receptor, and grey indicates an annotated pseudogene. Error bars represent the standard error of the mean from the six biological replicates. <i>Vmn2r89</i> is the highest VR gene expressed and is indicated with an asterisk.</p