Differences in transcription between free-living and CO_2-activated third-stage larvae of Haemonchus contortus

Background: The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results: Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma-secreted proteins (ASPs). Using an in silico-subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion: The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention

A Novel null homozygous mutation confirms <i>CACNA2D2</i> as a gene mutated in epileptic encephalopathy

Contribution to epileptic encephalopathy (EE) of mutations in CACNA2D2, encoding α2δ-2 subunit of Voltage Dependent Calcium Channels, is unclear. To date only one CACNA2D2 mutation altering channel functionality has been identified in a single family. In the same family, a rare CELSR3 polymorphism also segregated with disease. Involvement of CACNA2D2 in EE is therefore not confirmed, while that of CELSR3 is questionable. In a patient with epilepsy, dyskinesia, cerebellar atrophy, psychomotor delay and dysmorphic features, offspring to consanguineous parents, we performed whole exome sequencing (WES) for homozygosity mapping and mutation detection. WES identified extended autozygosity on chromosome 3, containing two novel homozygous candidate mutations: c.1295delA (p.Asn432fs) in CACNA2D2 and c.G6407A (p.Gly2136Asp) in CELSR3. Gene prioritization pointed to CACNA2D2 as the most prominent candidate gene. The WES finding in CACNA2D2 resulted to be statistically significant (p = 0.032), unlike that in CELSR3. CACNA2D2 homozygous c.1295delA essentially abolished α2δ-2 expression. In summary, we identified a novel null CACNA2D2 mutation associated to a clinical phenotype strikingly similar to the Cacna2d2 null mouse model. Molecular and statistical analyses together argued in favor of a causal contribution of CACNA2D2 mutations to EE, while suggested that finding in CELSR3, although potentially damaging, is likely incidental

EST analysis of gene expression in early cleavage-stage sea urchin embryos

A set of 956 expressed sequence tags derived from 7-hour (mid-cleavage) sea urchin embryos was analyzed to assess biosynthetic functions and to illuminate the structure of the message population at this stage. About a quarter of the expressed sequence tags represented repetitive sequence transcripts typical of early embryos, or ribosomal and mitochondrial RNAs, while a majority of the remainder contained significant open reading frames. A total of 232 sequences, including 153 different proteins, produced significant matches when compared against GenBank. The majority of these identified sequences represented ‘housekeeping’ proteins, i.e., cytoskeletal proteins, metabolic enzymes, transporters and proteins involved in cell division. The most interesting finds were components of signaling systems and transcription factors not previously reported in early sea urchin embryos, including components of Notch and TGF signal transduction pathways. As expected from earlier kinetic analyses of the embryo mRNA populations, no very prevalent protein-coding species were encountered; the most highly represented such sequences were cDNAs encoding cyclins A and B. The frequency of occurrence of all sequences within the database was used to construct a sequence prevalence distribution. The result, confirming earlier mRNA population analyses, indicated that the poly(A) RNA of the early embryo consists mainly of a very complex set of low-copy-number transcripts

Characterisation of novel transcripts in the 3’ region of L-type calcium channel genes in human brain

The alpha-1 subunit (CACNA1 family) of L-type voltage-gated calcium channels (LTCCs) play significant roles in brain function and neuropsychiatric disorders. Transcription of LTCCs is complex, with each alpha1 gene producing multiple isoforms. CACNA1S and CACNA1F were reported to have short-length transcripts from their 3’ region in the human brain which were not well characterized. Moreover, whether other CACNA1 genes also produce the 3’ transcripts have not been studied. This thesis aims to identify and understand the short-length transcripts arising from the 3’ region of LTCC genes in human brain, especially CACNA1S and CACNA1F, using a series of technical approaches and analyses. Firstly, using PCR amplification and long-read nanopore sequencing, multiple 3’ transcripts with exon-skipping possibilities were identified. Secondly, 5’ Rapid Amplification of cDNA Ends (5’ RACE) identified the 3’ transcripts as being complete transcripts, arising from alternative transcription start sites (TSS). These findings were corroborated by exon expression level findings from Genotype-Tissue Expression (GTEx) data. Analyses using PhyloP showed that the 5’-untranslated region (5’-UTR) of the 3’ transcripts are more conserved than the intronic regions. Finally, the translation and localization of the protein encoded by each 3’ transcript was investigated in transfected HEK-293T and SH-SY5Y cell lines. All proteins were detectable by western blot. C-CaV1.3 (encoded by CACNA1D) localized to the nucleus in both cell types; C-CaV1.2 (encoded by CACNA1C) translocated to the nucleus in the excitable (SH-SY5Y) cells but not the non-excitable (HEK-293T) cells. C-CaV1.1 and C-CaV1.4 (encoded by CACNA1S and CACNA1F respectively) were not localized to the nucleus. This thesis shows that LTCC alpha1 subunit genes produce 3’ transcripts using alternative TSS in human brain. Their potential functionality is supported by the evolutionary conservation of their 5’-UTRs and by their translation in vitro. However, further studies are required to identify the significance of these transcripts for brain function

New Perspectives on Host-Parasite Interplay by Comparative Transcriptomic and Proteomic Analyses of Schistosoma japonicum

Schistosomiasis remains a serious public health problem with an estimated 200 million people infected in 76 countries. Here we isolated ~ 8,400 potential protein-encoding cDNA contigs from Schistosoma japonicum after sequencing circa 84,000 expressed sequence tags. In tandem, we undertook a high-throughput proteomics approach to characterize the protein expression profiles of a number of developmental stages (cercariae, hepatic schistosomula, female and male adults, eggs, and miracidia) and tissues at the host-parasite interface (eggshell and tegument) by interrogating the protein database deduced from the contigs. Comparative analysis of these transcriptomic and proteomic data, the latter including 3,260 proteins with putative identities, revealed differential expression of genes among the various developmental stages and sexes of S. japonicum and localization of putative secretory and membrane antigens, enzymes, and other gene products on the adult tegument and eggshell, many of which displayed genetic polymorphisms. Numerous S. japonicum genes exhibited high levels of identity with those of their mammalian hosts, whereas many others appeared to be conserved only across the genus Schistosoma or Phylum Platyhelminthes. These findings are expected to provide new insights into the pathophysiology of schistosomiasis and for the development of improved interventions for disease control and will facilitate a more fundamental understanding of schistosome biology, evolution, and the host-parasite interplay

Characterization of PRL1 and its paralogue PRL2 in Arabidopsis thaliana

Das Arabidopsis-Gen PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) kodiert für ein Protein, das ein Mitglied einer konservierten WDR-Protein-Familie in Eukaryoten ist. PRL1-Mutationen führen zu Zucker-Überempfindlichkeit, verursachen zahlreiche pleiotrope Veränderungen bei der Wurzel-, Blatt- und Blüten-entwicklung und lösen Antworten auf abiotische und biotische Stress-Reize aus, indem sie die Pflanzen-hormon-Homöostase verändern. Im Gegensatz zu anderen Eukaryoten enthält das Arabidopsis-Genom mit PRL2 ein PRL1-Paralog, das mit PRL1 eine hochkonservierte C-terminale WDR-Domäne teilt. Verglichen mit PRL1 besitzt PRL2 jedoch unterschiedliche N-terminale Sequenzen. Nichtsdestotrotz weisen die N-terminalen Bereiche von PRL1 und PRL2 eine für die Evolution im Pflanzenreich außergewöhnlich hochkonservierte Struktur auf. Untersuchungen an Hefen und Säugetieren zeigten, dass PRL1 eine Schlüsselrolle bei der Aktivierung des Spliceosoms spielt. Dennoch impliziert die Divergenz der N-terminalen PRL1-Region in Arabidopsis pflanzenspezifische Funktionen beim Spleißen. Ein Ziel dieser Arbeit war es, Einblicke in regulatorische Funktionen von PRL1 zu ermöglichen. Affymetrix ATH1-Arrays und Tiling-Arrays bestätigten zusammen mit der RNA-Seq-Analyse die im gesamten Genom vorhandenen Veränderungen der prl1 Mutante. Darüber hinaus legt die Hochregulierung der Transposons zusammen mit der Hochregulierung von vielen weiteren Genen nahe, dass PRL1 ebenfalls an der Kontrolle des Gen-Silencing beteiligt ist. Zudem konnte durch die Verwendung von veränderten PRL1-Konstrukten gezeigt werden, dass PRL1-Transkription und PRL1-Stabilität stressabhängig sind und dass PRL1 nicht in die Proteasom-Aktivität eingreift. Ein weiteres Ziel war, das Ausmaß der funktionellen Übereinstimmung von PRL1 und PRL2 zu beurteilen. Ein Transkriptionsvergleich von PRL1 und PRL2 ergab, dass PRL2 in den meisten Gewebeteilen auf einem signifikant niedrigeren Niveau transkribiert wird. So wird PRL2 in Blüten speziell im männlichen Reproduktionsgewebe, in Pollenkörnern, im Endosperm und in der Zygote exprimiert, wohingegen PRL1 in sich entwickelnden Samenanlagen und im Integument der sich entwickelnden Samen aktiv ist. Die prl2-Mutationen sind embryoletal, während somatische prl2-Mosaike einen prl1-ähnlichen Phänotyp zeigen. Durch den Austausch der unterschiedlichen N-terminalen Domänen zwischen PRL1 und PRL2 konnte die Subfunktionalisation von PRL1 und PRL2 gezeigt werden. Die N-terminale Domäne von PRL2 komplementiert den prl1-Wurzelphänotyp nur teilweise. Sie kann jedoch die von prl2 verursachte embryonale Letalität wiederherstellen. Zudem ersetzt die N-terminale PRL1-Domäne zusammen mit der C-terminalen PRL2-Domäne den prl1-Phänotyp, aber nicht den embryoletalen prl2-Phänotyp. Zusätzlich zu der unterschiedlichen Transkription, die man bei PRL1 und PRL2 beobachtet , hat die Diversifikation der N-terminalen Domänen zu der Subfunktionalisation von PRL1 und PRL2 in Arabidopsis beigetragen. Die in dieser Arbeit vorgelegten Ergebnisse weisen zudem darauf hin, dass PRL1 und PRL2 eine spezielle Funktion bei der Kontrolle des Gen-Silencing in Arabidopsis spielen

Alternative translation initiation in rat brain yields K2P2.1 potassium channels permeable to sodium.

K(2P) channels mediate potassium background currents essential to central nervous system function, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Here, we show that alternative translation initiation (ATI) regulates function of K(2P)2.1 (TREK-1) via an unexpected strategy. Full-length K(2P)2.1 and an isoform lacking the first 56 residues of the intracellular N terminus (K(2P)2.1Delta1-56) are produced differentially in a regional and developmental manner in the rat central nervous system, the latter passing sodium under physiological conditions leading to membrane depolarization. Control of ion selectivity via ATI is proposed to be a natural, epigenetic mechanism for spatial and temporal regulation of neuronal excitability