Curation of a high-quality gene set is the critical first step in genome research, enabling subsequent analyses such as ortholog assignment, cis-regulatory element finding, and synteny detection. In this project, we have reannotated the genome of Caenorhabditis briggsae, the best studied sister species of the model organism Caenorhabditis elegans. First, we applied a homology-based gene predictor genBlastG to annotate the C. briggsae genome. We then validated and further improved the C. briggsae gene annotation through RNA-seq analysis of the C. briggsae transcriptome, which resulted in the first validated C. briggsae gene set (23,159 genes), among which 7347 genes (33.9% of all genes with introns) have all of their introns confirmed. Most genes (14,812, or 68.3%) have at least one intron validated, compared with only 3.9% in the most recent WormBase release (WS228). Of all introns in the revised gene set (103,083), 61,503 (60.1%) have been confirmed. Additionally, we have identified numerous trans-splicing leaders (SL1 and SL2 variants) in C. briggsae, leading to the first genome-wide annotation of operons in C. briggsae (1105 operons). The majority of the annotated operons (564, or 51.0%) are perfectly conserved in C. elegans, with an additional 345 operons (or 31.2%) somewhat divergent. Additionally, RNAseq analysis revealed over 10 thousand small-size assembly errors in the current C. briggsae reference genome that can be readily corrected. The revised C. briggsae genome annotation represents a solid platform for comparative genomics analysis and evolutionary studies of Caenorhabditis species.
Voltage gating of hyperpolarization-activated cation (HCN) channels is potentiated by direct binding of cAMP to a cytoplasmic cAMP-sensing domain (CSD). When unliganded, the CSD inhibits hyperpolarization-dependent opening of the HCN channel gate; cAMP binding relieves this autoinhibition so that opening becomes more favorable thermodynamically. This autoinhibition-relief mechanism is conserved with that of several other cyclic nucleotide receptors using the same ligand-binding fold. Besides its thermodynamic effect, cAMP also modulates the depolarization-dependent deactivation rate by kinetically that is formed by mode shift after prolonged hyperpolarization activation. This hysteretic activation-deactivation cycle is preserved by CSD substitution, but the change in deactivation kinetics of the liganded channel resulting from CSD substitution is not correlated with the change in autoinhibition properties. Thus the liganded and the unliganded forms of the CSD respectively provide the structural determinants for open-state trapping and autoinhibition, such that two distinct mechanisms for cAMP regulation can operate in one receptor. HCN channels are tetramers of homologous subunits; each subunit has six membrane-spanning helices (S1-S6) homologous to those of voltage-gated potassium channels, with a voltagesensing domain in S1-S4 (4). Hyperpolarization drives inward movement of the positively charged S4 (5), which is coupled to opening of the pore gate formed by the cytoplasmic C-terminal ends of the four S6 helices in the tetramer (6). S6 in each subunit is followed by an 80-residue "C-linker" with an unusual multihelix fold (7), then the cAMP-binding fold and a poorly conserved "extreme C-terminal" region. The C-linker and cAMP-binding fold together can be viewed as one "cAMP-sensing domain" (CSD; see Fig. 1A, Top), based on domain stability (7) and the C-linker's functional importance (8).Cyclic AMP binding potentiates hyperpolarization-dependent activation of HCN channels (9). That is, cAMP increases thermodynamic stability of the open channel relative to the closed channel, so that the voltage needed for half-maximal activation (V 1∕2 ) becomes less negative. A similar V 1∕2 shift can be produced by deleting the CSD through enzymatic or genetic truncation (10, 11). This establishes a classical autoinhibition mechanism (12) as the basis for cAMP regulation of HCN channels, in analogy with PKA (13) and EPAC (14). Thus the unliganded ("apo") CSD inhibits an intrinsic activity of the channel, and the liganded ("holo") form of the CSD has weakened or nonexistent capability for this inhibition, so that cAMP binding mimics CSD deletion. Because the autoinhibition-relief model attributes specific regulatory function to the apo CSD rather than the holo CSD, we introduce the designation "apo-driven" for this mechanism type. In a "holo-driven" mechanism, by contrast, the presence of the CSD with bound ligand would be required for a particular HCN channel structure that achieves optimal activation. A holodriven mechanis...
BackgroundWhole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G4) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase.ResultsTo identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F170 and F470 strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F470 (JNC170).ConclusionsHere, we identified variants that accumulated in C. elegans’ genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G4-forming potential, are vulnerable to deletion-formation in this genetic background.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1402-y) contains supplementary material, which is available to authorized users.
Keywords: Cyclin B3 (cyb-3), dynein heavy chain (dhc-1), mdf-1/MAD1, genome stability, spindle assembly checkpoint (SAC)Abbreviations: APC/C, anaphase promoting complex/cyclosome; CIN, chromosome instability; EMS, ethyl methanesulfonate; Him, high incidence of males; oaCGH, oligonucleotide array Comparative Genomic Hybridization; SAC, spindle assembly checkpoint; WGS, whole genome sequencing.Spindle assembly checkpoint (SAC) ensures genome stability by delaying anaphase onset until all the chromosomes have achieved proper spindle attachment. Once correct attachment has been achieved, SAC must be silenced. In the absence of mdf-1/MAD1, an essential SAC component, Caenorhabditis elegans cannot propagate beyond 3 generations. Previously, in a dog-1(gk10)/FANCJ mutator background, we isolated a suppressor of mdf-1(gk2) sterility (such-4) which allowed indefinite propagation in the absence of MDF-1. We showed that such-4 is a Cyclin B3 (cyb-3) duplication. Here we analyze mdf-1 such-4; dog-1, which we propagated for 470 generations, with freezing of samples for long time storage at F 170 and F 270 . Phenotypic analysis of this strain revealed additional suppression of sterility in the absence of MDF-1, beyond the effects of such-4. We applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) and whole genome sequencing (WGS) and identified a further amplification of cyb-3 (triplication) and a new missense mutation in dynein heavy chain (dhc-1). We show that dhc-1(dot168) suppresses the mdf-1(gk2), and is the second cloned suppressor, next to cyb-3 duplication, that does not cause a delay in anaphase onset. We also show that amplification of cyb-3 and dhc-1(dot168) cooperate to increase fitness in the absence of MDF-1.
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