Corneal dystrophies are phenotypically and genetically heterogeneous, often resulting in visual impairment caused by corneal opacification. We investigated the genetic cause of an autosomal dominant corneal stromal dystrophy in a pedigree with eight affected individuals in three generations. Affected individuals had diffuse central stromal opacity, with reduced visual acuity in older family members. Histopathology of affected cornea tissue removed during surgery revealed mild stromal textural alterations with alcianophilic deposits. Whole genome sequence data were generated for four affected individuals. No rare variants (MAF < 0.001) were identified in established corneal dystrophy genes. However, a novel heterozygous missense variant in exon 4 of SPARCL1, NM_004684: c.334G > A; p.(Glu112Lys), which is predicted to be damaging, segregated with disease. SPARC-like protein 1 (SPARCL1) is a secreted matricellular protein involved in cell migration, cell adhesion, tissue repair, and remodelling. Interestingly, SPARCL1 has been shown to regulate decorin. Heterozygous variants in DCN, encoding decorin, cause autosomal dominant congenital stromal corneal dystrophy, suggesting a common pathogenic pathway. Therefore, we performed immunohistochemistry to compare SPARCL1 and decorin localisation in corneal tissue from an affected family member and an unaffected control. Strikingly, the level of decorin was significantly decreased in the corneal stroma of the affected tissue, and SPARCL1 appeared to be retained in the epithelium. In summary, we describe a novel autosomal dominant corneal stromal dystrophy associated with a missense variant in SPARCL1, extending the phenotypic and genetic heterogeneity of inherited corneal disease.

• MeSH
• dědičné dystrofie rohovky * genetika   patologie   MeSH
• dekorin genetika   metabolismus   MeSH
• dospělí MeSH
• extracelulární matrix - proteiny * genetika   MeSH
• heterozygot MeSH
• lidé středního věku MeSH
• lidé MeSH
• missense mutace * MeSH
• proteiny vázající vápník * genetika   MeSH
• rodokmen * MeSH
• senioři MeSH
• stroma rohovky patologie   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dekorin MeSH
• extracelulární matrix - proteiny * MeSH
• proteiny vázající vápník * MeSH
• SPARCL1 protein, human MeSH Prohlížeč

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.

• Klíčová slova
• PABP, RNA processing, eIF4E, eIF4G, protein synthesis, translation initiation, trypanosome,
• Publikační typ
• časopisecké články MeSH

Invertebrate-microbial associations are widespread in the biosphere and are often related to the function of novel genes, fitness advantages, and even speciation events. Despite ~ 13,000 species of millipedes identified across the world, millipedes and their gut microbiota are markedly understudied compared to other arthropods. Exploring the contribution of individual host-associated microbes is often challenging as many are uncultivable. In this study, we conducted metatranscriptomic profiling of different body segments of a millipede at the holobiont level. This is the first reported transcriptome assembly of a tropical millipede Telodeinopus aoutii (Demange, 1971), as well as the first study on any Myriapoda holobiont. High-throughput RNA sequencing revealed that Telodeinopus aoutii contained > 90% of the core Arthropoda genes. Proteobacteria, Bacteroidetes, Firmicutes, and Euryarchaeota represented dominant and functionally active phyla in the millipede gut, among which 97% of Bacteroidetes and 98% of Firmicutes were present exclusively in the hindgut. A total of 37,831 predicted protein-coding genes of millipede holobiont belonged to six enzyme classes. Around 35% of these proteins were produced by microbiota in the hindgut and 21% by the host in the midgut. Our results indicated that although major metabolic pathways operate at the holobiont level, the involvement of some host and microbial genes are mutually exclusive and microbes predominantly contribute to essential amino acid biosynthesis, short-chain fatty acid metabolism, and fermentation.

• MeSH
• Bacteroidetes MeSH
• členovci * genetika   MeSH
• esenciální aminokyseliny MeSH
• kyseliny mastné těkavé MeSH
• střevní mikroflóra * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• esenciální aminokyseliny MeSH
• kyseliny mastné těkavé MeSH

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.

• Klíčová slova
• RNA-seq, RT-PCR, VUS, aberrant splicing, blood RNA,
• MeSH
• ABC transportéry genetika   MeSH
• alternativní sestřih * MeSH
• lidé MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• reverzní transkripce MeSH
• RNA * genetika   MeSH
• sestřih RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportéry MeSH
• ABCA4 protein, human MeSH Prohlížeč
• RNA * MeSH

In the venom of spiders, linear peptides (LPs), also called cytolytical or antimicrobial peptides, represent a largely neglected group of mostly membrane active substances that contribute in some spider species considerably to the killing power of spider venom. By next-generation sequencing venom gland transcriptome analysis, we investigated 48 spider species from 23 spider families and detected LPs in 20 species, belonging to five spider families (Ctenidae, Lycosidae, Oxyopidae, Pisauridae, and Zodariidae). The structural diversity is extraordinary high in some species: the lynx spider Oxyopes heterophthalmus contains 62 and the lycosid Pardosa palustris 60 different LPs. In total, we identified 524 linear peptide structures and some of them are in lycosids identical on amino acid level. LPs are mainly encoded in complex precursor structures in which, after the signal peptide and propeptide, 13 or more LPs (Hogna radiata) are connected by linkers. Besides Cupiennius species, also in Oxyopidae, posttranslational modifications of some precursor structures result in the formation of two-chain peptides. It is obvious that complex precursor structures represent a very suitable and fast method to produce a high number and a high diversity of bioactive LPs as economically as possible. At least in Lycosidae, Oxyopidae, and in the genus Cupiennius, LPs reach very high Transcripts Per Kilobase Million values, indicating functional importance within the envenomation process.

• Klíčová slova
• NGS spider venom transcriptome analysis, complex precursors, cytolytical peptides, linear peptides, lycosins, oxyopinins, tachykinin-like peptides, venom protease,
• Publikační typ
• časopisecké články MeSH

Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology (GO) enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of C. beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however, their validation is required.

• Klíčová slova
• Clostridium, HKG, biofuel, housekeeping genes, non-model organisms,
• Publikační typ
• časopisecké články MeSH

In many species, sexual differentiation is a vital prelude to reproduction, and disruption of this process can have severe fitness effects, including sterility. It is thus interesting that genetic systems governing sexual differentiation vary among-and even within-species. To understand these systems more, we investigated a rare example of a frog with three sex chromosomes: the Western clawed frog, Xenopus tropicalis. We demonstrate that natural populations from the western and eastern edges of Ghana have a young Y chromosome, and that a male-determining factor on this Y chromosome is in a very similar genomic location as a previously known female-determining factor on the W chromosome. Nucleotide polymorphism of expressed transcripts suggests genetic degeneration on the W chromosome, emergence of a new Y chromosome from an ancestral Z chromosome, and natural co-mingling of the W, Z, and Y chromosomes in the same population. Compared to the rest of the genome, a small sex-associated portion of the sex chromosomes has a 50-fold enrichment of transcripts with male-biased expression during early gonadal differentiation. Additionally, X. tropicalis has sex-differences in the rates and genomic locations of recombination events during gametogenesis that are similar to at least two other Xenopus species, which suggests that sex differences in recombination are genus-wide. These findings are consistent with theoretical expectations associated with recombination suppression on sex chromosomes, demonstrate that several characteristics of old and established sex chromosomes (e.g., nucleotide divergence, sex biased expression) can arise well before sex chromosomes become cytogenetically distinguished, and show how these characteristics can have lingering consequences that are carried forward through sex chromosome turnovers.

• MeSH
• genetická zdatnost MeSH
• pohlavní chromozomy genetika   MeSH
• procesy určující pohlaví genetika   MeSH
• rekombinace genetická MeSH
• sexuální diferenciace genetika   MeSH
• Xenopus genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Geografické názvy
• Ghana MeSH

BACKGROUND: Gynodioecious species exist in two sexes - male-sterile females and hermaphrodites. Male sterility in higher plants often results from mitonuclear interaction between the CMS (cytoplasmic male sterility) gene(s) encoded by mitochondrial genome and by nuclear-encoded restorer genes. Mitochondrial and nuclear-encoded transcriptomes in females and hermaphrodites are intensively studied, but little is known about sex-specific gene expression in plastids. We have compared plastid transcriptomes between females and hermaphrodites in two haplotypes of a gynodioecious species Silene vulgaris with known CMS candidate genes. RESULTS: We generated complete plastid genome sequences from five haplotypes S. vulgaris including the haplotypes KRA and KOV, for which complete mitochondrial genome sequences were already published. We constructed a phylogenetic tree based on plastid sequences of S. vulgaris. Whereas lowland S. vulgaris haplotypes including KRA and KOV clustered together, the accessions from high European mountains diverged early in the phylogram. S. vulgaris belongs among Silene species with slowly evolving plastid genomes, but we still detected 212 substitutions and 112 indels between two accessions of this species. We estimated elevated Ka/Ks in the ndhF gene, which may reflect the adaptation of S. vulgaris to high altitudes, or relaxed selection. We compared depth of coverage and editing rates between female and hermaphrodite plastid transcriptomes and found no significant differences between the two sexes. We identified 51 unique C to U editing sites in the plastid genomes of S. vulgaris, 38 of them in protein coding regions, 2 in introns, and 11 in intergenic regions. The editing site in the psbZ gene was edited only in one of two plastid genomes under study. CONCLUSIONS: We revealed no significant differences between the sexes in plastid transcriptomes of two haplotypes of S. vulgaris. It suggests that gene expression of plastid genes is not affected by CMS in flower buds of S. vulgaris, although both sexes may still differ in plastid gene expression in specific tissues. We revealed the difference between the plastid transcriptomes of two S. vulgaris haplotypes in editing rate and in the coverage of several antisense transcripts. Our results document the variation in plastid genomes and transcriptomes in S. vulgaris.

• MeSH
• genom plastidový genetika   MeSH
• Silene genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Silene vulgaris (bladder campion) is a gynodioecious species existing as two genders - male-sterile females and hermaphrodites. Cytoplasmic male sterility (CMS) is generally encoded by mitochondrial genes, which interact with nuclear fertility restorer genes. Mitochondrial genomes of this species vary in DNA sequence, gene order and gene content. Multiple CMS genes are expected to exist in S. vulgaris, but little is known about their molecular identity. RESULTS: We assembled the complete mitochondrial genome from the haplotype KRA of S. vulgaris. It consists of five chromosomes, two of which recombine with each other. Two small non-recombining chromosomes exist in linear, supercoiled and relaxed circle forms. We compared the mitochondrial transcriptomes from females and hermaphrodites and confirmed the differentially expressed chimeric gene bobt as the strongest CMS candidate gene in S. vulgaris KRA. The chimeric gene bobt is co-transcribed with the Cytochrome b (cob) gene in some genomic configurations. The co-transcription of a CMS factor with an essential gene may constrain transcription inhibition as a mechanism for fertility restoration because of the need to maintain appropriate production of the necessary protein. Homologous recombination places the gene cob outside the control of bobt, which allows for the suppression of the CMS gene by the fertility restorer genes. We found the loss of three editing sites in the KRA mitochondrial genome and identified four sites with highly distinct editing rates between KRA and another S. vulgaris haplotypes (KOV). Three of these highly differentially edited sites were located in the transport membrane protein B (mttB) gene. They resulted in differences in MttB protein sequences between haplotypes. CONCLUSIONS: Frequent homologous recombination events that are widespread in plant mitochondrial genomes may change chromosomal configurations and also the control of gene transcription including CMS gene expression. Posttranscriptional processes, e.g. RNA editing shall be evaluated in evolutionary and co-evolutionary studies of mitochondrial genes, because they may change protein composition despite the sequence identity of the respective genes. The investigation of natural populations of wild species such as S. vulgaris are necessary to reveal important aspects of CMS missed in domesticated crops, the traditional focus of the CMS studies.

• MeSH
• cytochromy b genetika   metabolismus   MeSH
• editace RNA MeSH
• genom mitochondriální * MeSH
• haplotypy MeSH
• homologní rekombinace * MeSH
• membránové glykoproteiny genetika   MeSH
• mitochondriální protonové ATPasy genetika   MeSH
• mitochondrie genetika   MeSH
• neplodnost rostlin genetika   MeSH
• otevřené čtecí rámce genetika   MeSH
• rostlinné proteiny genetika   MeSH
• Silene genetika   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochromy b MeSH
• membránové glykoproteiny MeSH
• mitochondriální protonové ATPasy MeSH
• rostlinné proteiny MeSH

Cytoplasmic male sterility (CMS) is a widespread phenomenon in flowering plants caused by mitochondrial (mt) genes. CMS genes typically encode novel proteins that interfere with mt functions and can be silenced by nuclear fertility-restorer genes. Although the molecular basis of CMS is well established in a number of crop systems, our understanding of it in natural populations is far more limited. To identify CMS genes in a gynodioecious plant, Silene vulgaris, we constructed mt transcriptomes and compared transcript levels and RNA editing patterns in floral bud tissue from female and hermaphrodite full siblings. The transcriptomes from female and hermaphrodite individuals were very similar overall with respect to variation in levels of transcript abundance across the genome, the extent of RNA editing, and the order in which RNA editing and intron splicing events occurred. We found only a single genomic region that was highly overexpressed and differentially edited in females relative to hermaphrodites. This region is not located near any other transcribed elements and lacks an open-reading frame (ORF) of even moderate size. To our knowledge, this transcript would represent the first non-coding mt RNA associated with CMS in plants and is, therefore, an important target for future functional validation studies.

• Klíčová slova
• Cytoplasmic male sterility, Silene vulgaris, editing, mitochondrion, non-coding RNA, splicing, transcriptome.,
• MeSH
• editace RNA MeSH
• květy genetika   růst a vývoj   MeSH
• mitochondriální geny * MeSH
• nekódující RNA * MeSH
• neplodnost rostlin * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Silene genetika   fyziologie   MeSH
• transkriptom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• nekódující RNA * MeSH
• rostlinné proteiny MeSH