bluuebunny/crossref_metadata_2025 huggingface dataset:
Marsupial X chromosome inactivation: past, present and future
Authors: Deakin Janine E.
Published: March 2013

Marsupial and eutherian mammals inactivate one X chromosome in female somatic cells in what is thought to be a means of compensating for the unbalanced X chromosome dosage between XX females and XY males. The hypothesis of X chromosome inactivation (XCI) was first published by Mary Lyon just over 50 years ago, with the discovery of XCI in marsupials occurring a decade later. However, we are still piecing together the evolutionary origins of this fascinating epigenetic mechanism. From the very first studies on marsupial X inactivation, it was apparent that, although there were some similarities between marsupial and eutherian XCI, there were also some striking differences. For instance, the paternally derived X was found to be preferentially silenced in marsupials, although the silencing was often incomplete, which was in contrast to the random and more tightly controlled inactivation of the X chromosome in eutherians. Many of these earlier studies used isozymes to study the activity of just a few genes in marsupials. The sequencing of several marsupial genomes and the advent of molecular cytogenetic techniques have facilitated more in-depth studies into marsupial X chromosome inactivation and allowed more detailed comparisons of the features of XCI to be made. Several important findings have come from such comparisons, among which is the absence of the XIST gene in marsupials, a non-coding RNA gene with a critical role in eutherian XCI, and the discovery of the marsupial RSX gene, which appears to perform a similar role to XIST. Here I review the history of marsupial XCI studies, the latest advances that have been made and the impact they have had towards unravelling the evolution of XCI in mammals.
Evolution of genomic imprinting as a coordinator of coadapted gene expression
Authors: Wolf Jason B.
Published: March 2013

Genomic imprinting is an epigenetic phenomenon in which the expression of a gene copy inherited from the mother differs from that of the copy inherited from the father. Many imprinted genes appear to be highly interconnected through interactions mediated by proteins, RNA, and DNA. These kinds of interactions often favor the evolution of genetic coadaptation, where beneficially interacting alleles evolve to become coinherited. Here I demonstrate theoretically that the presence of gene interactions that favor coadaptation can also favor the evolution of genomic imprinting. Selection favors genomic imprinting because it coordinates the coexpression of positively interacting alleles at different loci. Evolution is expected to proceed through a scenario where selection builds associations between beneficial combinations of alleles and, if one locus evolves to become imprinted, it leads to selection for its interacting partners to match its pattern of imprinting. This process should favor the evolution of physical linkage between interacting genes and therefore may help explain why imprinted genes tend to be found in clusters. The model suggests that, whereas some genes are expected to evolve their imprinting status because selection directly favors a specific pattern of parent-of-origin-dependent expression, other genes may evolve imprinting as a coevolutionary response to match the expression pattern of their interacting partners. As a result, some genes will show phenotypic effects consistent with the predictions of models for the evolution of genomic imprinting (e.g., conflict models), but other genes may not, having simply evolved imprinting to follow the lead of their interacting partners.
Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device
Authors: Marie Rodolphe , Pedersen Jonas N. , Bauer David L. V. , Rasmussen Kristian H. , Yusuf Mohammed , Volpi Emanuela , Flyvbjerg Henrik , Kristensen Anders , Mir Kalim U.
Published: March 2013

We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.
Role of ELOVL4 and very long-chain polyunsaturated fatty acids in mouse models of Stargardt type 3 retinal degeneration
Authors: Barabas Peter , Liu Aihua , Xing Wei , Chen Ching-Kang , Tong Zongzhong , Watt Carl B. , Jones Bryan W. , Bernstein Paul S. , Križaj David
Published: March 2013

Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the
ELOVL4
(
Elongation of very long chain fatty acids 4
) gene. Its protein product, ELOVL4, is an elongase required for the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs). It is unclear whether photoreceptor degeneration in STGD3 is caused by loss of VLC-PUFAs or by mutated ELOVL4 protein trafficking/aggregation. We therefore generated conditional knockout (cKO) mice with
Elovl4
ablated in rods or cones and compared their phenotypes to transgenic (TG) animals that express the human STGD3-causing
ELOVL4

STGD3

allele. Gas chromatography–mass spectrometry was used to assess C
30
–C
34
VLC-PUFA and
N
-retinylidene-
N
-retinylethanolamine content; electroretinography was used to measure phototransduction and outer retinal function; electron microscopy was used for retinal ultrastructure; and the optomotor tracking response was used to test scotopic and photopic visual performance.
Elovl4
transcription and biosynthesis of C
30
–C
34
VLC-PUFAs in rod cKO and TG retinas were reduced up to 98%, whereas the content of docosahexaenoic acid was diminished in TG, but not rod cKO, retinas. Despite the near-total loss of the retinal VLC-PUFA content, rod and cone cKO animals exhibited no electrophysiological or behavioral deficits, whereas the typical rod–cone dystrophic pattern was observed in TG animals. Our data suggest that photoreceptor-specific VLC-PUFA depletion is not sufficient to induce the STGD3 phenotype, because depletion alone had little effect on photoreceptor survival, phototransduction, synaptic transmission, and visual behavior.
Inhibition of TFG function causes hereditary axon degeneration by impairing endoplasmic reticulum structure
Authors: Beetz Christian , Johnson Adam , Schuh Amber L. , Thakur Seema , Varga Rita-Eva , Fothergill Thomas , Hertel Nicole , Bomba-Warczak Ewa , Thiele Holger , Nürnberg Gudrun , Altmüller Janine , Saxena Renu , Chapman Edwin R. , Dent Erik W. , Nürnberg Peter , Audhya Anjon
Published: March 2013

Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of gait disorders. Their pathological hallmark is a length-dependent distal axonopathy of nerve fibers in the corticospinal tract. Involvement of other neurons can cause additional neurological symptoms, which define a diverse set of complex hereditary spastic paraplegias. We present two siblings who have the unusual combination of early-onset spastic paraplegia, optic atrophy, and neuropathy. Genome-wide SNP-typing, linkage analysis, and exome sequencing revealed a homozygous c.316C>T (p.R106C) variant in the Trk-fused gene (
TFG
) as the only plausible mutation. Biochemical characterization of the mutant protein demonstrated a defect in its ability to self-assemble into an oligomeric complex, which is critical for normal TFG function. In cell lines, TFG inhibition slows protein secretion from the endoplasmic reticulum (ER) and alters ER morphology, disrupting organization of peripheral ER tubules and causing collapse of the ER network onto the underlying microtubule cytoskeleton. The present study provides a unique link between altered ER architecture and neurodegeneration.
Dpp-induced Egfr signaling triggers postembryonic wing development in
Drosophila
Authors: Paul Litty , Wang Shu-Huei , Manivannan Sathiya N. , Bonanno Liana , Lewis Sarah , Austin Christina L. , Simcox Amanda
Published: March 2013

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The
Drosophila melanogaster
wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early
Drosophila
wing disc.
vn
is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating
vn
expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated,
vn
expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.
State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels
Authors: Miranda Pablo , Contreras Jorge E. , Plested Andrew J. R. , Sigworth Fred J. , Holmgren Miguel , Giraldez Teresa
Published: March 2013

Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca
2+
binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca
2+
binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.
Is junk DNA bunk? A critique of ENCODE
Authors: Doolittle W. Ford
Published: March 2013

Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete? Here, I review older arguments for junk grounded in the C-value paradox and propose a thought experiment to challenge ENCODE’s ontology. Specifically, what would we expect for the number of functional elements (as ENCODE defines them) in genomes much larger than our own genome? If the number were to stay more or less constant, it would seem sensible to consider the rest of the DNA of larger genomes to be junk or, at least, assign it a different sort of role (structural rather than informational). If, however, the number of functional elements were to rise significantly with C-value then, (
i
) organisms with genomes larger than our genome are more complex phenotypically than we are, (
ii
) ENCODE’s definition of functional element identifies many sites that would not be considered functional or phenotype-determining by standard uses in biology, or (
iii
) the same phenotypic functions are often determined in a more diffuse fashion in larger-genomed organisms. Good cases can be made for propositions
ii
and
iii
. A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed.
Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products
Authors: Defenouillère Quentin , Yao Yanhua , Mouaikel John , Namane Abdelkader , Galopier Aurélie , Decourty Laurence , Doyen Antonia , Malabat Christophe , Saveanu Cosmin , Jacquier Alain , Fromont-Racine Micheline
Published: March 2013

Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with
SKI
and
LTN1
mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from
Non-Stop
mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.
Intracellular transport of insulin granules is a subordinated random walk
Authors: Tabei S. M. Ali , Burov Stanislav , Kim Hee Y. , Kuznetsov Andrey , Huynh Toan , Jureller Justin , Philipson Louis H. , Dinner Aaron R. , Scherer Norbert F.
Published: March 2013

We quantitatively analyzed particle tracking data on insulin granules expressing fluorescent fusion proteins in MIN6 cells to better understand the motions contributing to intracellular transport and, more generally, the means for characterizing systems far from equilibrium. Care was taken to ensure that the statistics reflected intrinsic features of the individual granules rather than details of the measurement and overall cell state. We find anomalous diffusion. Interpreting such data conventionally requires assuming that a process is either ergodic with particles working against fluctuating obstacles (fractional Brownian motion) or nonergodic with a broad distribution of dwell times for traps (continuous-time random walk). However, we find that statistical tests based on these two models give conflicting results. We resolve this issue by introducing a subordinated scheme in which particles in cages with random dwell times undergo correlated motions owing to interactions with a fluctuating environment. We relate this picture to the underlying microtubule structure by imaging in the presence of vinblastine. Our results provide a simple physical picture for how diverse pools of insulin granules and, in turn, biphasic secretion could arise.
B and T lymphocyte attenuator inhibits LPS-induced endotoxic shock by suppressing Toll-like receptor 4 signaling in innate immune cells
Authors: Kobayashi Yoshihisa , Iwata Arifumi , Suzuki Kotaro , Suto Akira , Kawashima Saki , Saito Yukari , Owada Takayoshi , Kobayashi Midori , Watanabe Norihiko , Nakajima Hiroshi
Published: March 2013

Although innate immune responses are necessary for the initiation of acquired immune responses and the subsequent successful elimination of pathogens, excessive responses occasionally result in lethal endotoxic shock accompanied by a cytokine storm. B and T lymphocyte attenuator (BTLA), a coinhibitory receptor with similarities to cytotoxic T-lymphocyte antigen (CTLA)-4 and programmed death (PD)-1, is expressed in not only B and T cells but also dendritic cells (DCs) and macrophages (Mϕs). Recently, several studies have reported that BTLA-deficient (BTLA
−/−
) mice show enhanced pathogen clearance compared with WT mice in early phase of infections. However, the roles of BTLA expressed on innate cells in overwhelming and uncontrolled immune responses remain unclear. Here, we found that BTLA
−/−
mice were highly susceptible to LPS-induced endotoxic shock. LPS-induced TNF-α and IL-12 production in DCs and Mϕs was significantly enhanced in BTLA
−/−
mice. BTLA
−/−
DCs also produced high levels of TNF-α on stimulation with Pam3CSK4 but not poly(I:C) or CpG, suggesting that BTLA functions as an inhibitory molecule on Toll-like receptor signaling at cell surface but not endosome. Moreover, BTLA
−/−
DCs showed enhanced MyD88- and toll/IL-1R domain-containing adaptor inducing IFN (TRIF)-dependent signaling on LPS stimulation, which is associated with impaired accumulation of Src homology 2-containing protein tyrosine phosphatase in lipid rafts. Finally, we found that an agonistic anti-BTLA antibody rescued mice from LPS-induced endotoxic shock, even if the antibody was given to mice that had developed a sign of endotoxic shock. These results suggest that BTLA directly inhibits LPS responses in DCs and Mϕs and that agonistic agents for BTLA might have therapeutic potential for LPS-induced endotoxic shock.
Binding of
Drosophila
Polo kinase to its regulator Matrimony is noncanonical and involves two separate functional domains
Authors: Bonner Amanda M. , Hughes Stacie E. , Chisholm Jennifer A. , Smith S. Kendall , Slaughter Brian D. , Unruh Jay R. , Collins Kimberly A. , Friederichs Jennifer M. , Florens Laurence , Swanson Selene K. , Pelot Marissa C. , Miller Danny E. , Washburn Michael P. , Jaspersen Sue L. , Hawley R. Scott
Published: March 2013

Significance

Polo kinase regulates many processes during cell division and is upregulated in many cancers. During
Drosophila
female meiosis, the protein Matrimony inhibits Polo kinase using a noncanonical mechanism of Polo binding. Complete loss of Matrimony leads to meiotic catastrophe, and partial loss leads to chromosome missegregation. Proper Matrimony–Polo binding is required to prevent these defects, indicating that preventing Polo from phosphorylating targets is necessary for proper completion of meiosis. This finding is in contrast to mitosis where phosphorylation by Polo is usually required for cell division. This work provides important insight into developing anticancer therapeutic agents targeting Polo kinase.
Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle
Authors: Hirose Yuu , Rockwell Nathan C. , Nishiyama Kaori , Narikawa Rei , Ukaji Yutaka , Inomata Katsuhiko , Lagarias J. Clark , Ikeuchi Masahiko
Published: March 2013

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330–680 nm). Two CBCR subfamilies detect near-UV to blue light (330–450 nm) via a “two-Cys photocycle” that couples bilin
15Z/15E
photoisomerization with formation or elimination of a second bilin–cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing
15Z
state (

15Z

P
g
) and a red-absorbing
15E
state (

15E

P
r
). We demonstrate that RcaE from
Fremyella diplosiphon
undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as

15Z

P
g
but protonated as

15E

P
r
. This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a “protochromic photocycle,” in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.
Ramshackle (Brwd3) promotes light-induced ubiquitylation of
Drosophila
Cryptochrome by DDB1-CUL4-ROC1 E3 ligase complex
Authors: Ozturk Nuri , VanVickle-Chavez Sarah J. , Akileswaran Lakshmi , Van Gelder Russell N. , Sancar Aziz
Published: March 2013

Cryptochrome (CRY) is the primary circadian photoreceptor in
Drosophila
. It resets the circadian clock by promoting light-induced degradation of the clock proteins Timeless and Period, as well as its own proteolysis. The E3 ligases that ubiquitylate Timeless and Period before degradation are known and it is known that
Drosophila
(d) CRY is degraded by the ubiquitin-proteasome system as well. To identify the E3 ligase for dCRY we screened candidates in S2 cells by RNAi. Knockdown of each of the 25 putative F-box proteins identified by bioinformatics did not attenuate the light-induced degradation of dCRY. However, knockdown of a WD40 protein, Bromodomain and WD repeat domain containing 3 (Brwd3) (CG31132/Ramshackle) caused strong attenuation of dCRY degradation following light exposure. We found that BRWD3 functions as a Damage-specific DNA binding protein 1 (DDB1)- and CULLIN (CUL)4-associated factor in a Cullin4-RING Finger E3 Ligase (CRL4) that mediates light-dependent binding of dCRY to CUL4-ROC1-DDB1-BRWD3, inducing ubiquitylation of dCRY and its light-induced degradation. Thus, this study identifies a light-activated E3 ligase complex essential for light-mediated CRY degradation in
Drosophila
cells.
Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles
Authors: Morgan Anthony J. , Davis Lianne C. , Wagner Siegfried K.T.Y. , Lewis Alexander M. , Parrington John , Churchill Grant C. , Galione Antony
Published: March 2013

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca2+ stores that release Ca2+ in response to the second messengers IP3 and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca2+ released from acidic organelles by NAADP subsequently recruits IP3 or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca2+ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca2+ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca2+-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca2+ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca2+ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca2+ oscillations and waves.
Rer1p maintains ciliary length and signaling by regulating γ-secretase activity and Foxj1a levels
Authors: Jurisch-Yaksi Nathalie , Rose Applonia J. , Lu Huiqi , Raemaekers Tim , Munck Sebastian , Baatsen Pieter , Baert Veerle , Vermeire Wendy , Scales Suzie J. , Verleyen Daphne , Vandepoel Roel , Tylzanowski Przemko , Yaksi Emre , de Ravel Thomy , Yost H. Joseph , Froyen Guy , Arrington Cammon B. , Annaert Wim
Published: March 2013

Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi–localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left–right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.
Functional characterization of a ClC transporter by solid-supported membrane electrophysiology
Authors: Garcia-Celma Juan , Szydelko Adrian , Dutzler Raimund
Published: March 2013

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.
Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels
Authors: Wang Zhuren , Dou Ying , Goodchild Samuel J. , Es-Salah-Lamoureux Zeineb , Fedida David
Published: March 2013

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.
A temporal switch model for estimating transcriptional activity in gene expression
Authors: Jenkins Dafyd J. , Finkenstädt Bärbel , Rand David A.
Published: March 2013

Motivation: The analysis and mechanistic modelling of time series gene expression data provided by techniques such as microarrays, NanoString, reverse transcription–polymerase chain reaction and advanced sequencing are invaluable for developing an understanding of the variation in key biological processes. We address this by proposing the estimation of a flexible dynamic model, which decouples temporal synthesis and degradation of mRNA and, hence, allows for transcriptional activity to switch between different states.

Results: The model is flexible enough to capture a variety of observed transcriptional dynamics, including oscillatory behaviour, in a way that is compatible with the demands imposed by the quality, time-resolution and quantity of the data. We show that the timing and number of switch events in transcriptional activity can be estimated alongside individual gene mRNA stability with the help of a Bayesian reversible jump Markov chain Monte Carlo algorithm. To demonstrate the methodology, we focus on modelling the wild-type behaviour of a selection of 200 circadian genes of the model plant Arabidopsis thaliana. The results support the idea that using a mechanistic model to identify transcriptional switch points is likely to strongly contribute to efforts in elucidating and understanding key biological processes, such as transcription and degradation.

Contact:  B.F.Finkenstadt@Warwick.ac.uk

Supplementary information:  Supplementary data are available at Bioinformatics online.
Galaxy LIMS for next-generation sequencing
Authors: Scholtalbers Jelle , Rößler Jasmin , Sorn Patrick , de Graaf Jos , Boisguérin Valesca , Castle John , Sahin Ugur
Published: March 2013

Summary: We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard and customizable sample information forms, barcoded submission forms, tracking of input sample quality, multiplex-capable automatic flow cell design and automatically generated sample sheets to aid physical flow cell preparation. In addition, the platform provides the researcher with a user-friendly interface to create a request, submit accompanying samples, upload sample quality measurements and access to the sequencing results. As the LIMS is within the Galaxy platform, the researcher has access to all Galaxy analysis tools and workflows. The system reports requests and associated information to a message queuing system, such that information can be posted and stored in external systems, such as a wiki. Through an API, raw sequencing results can be automatically pre-processed and uploaded to the appropriate request folder. Developed for the Illumina HiSeq 2000 instrument, many features are directly applicable to other instruments.

Availability and implementation: The code and documentation are available at http://tron-mainz.de/tron-facilities/computational-medicine/galaxy-lims/

Contact:  jelle.scholtalbers@tron-mainz.de
Distribution of dehalogenation activity in subseafloor sediments of the Nankai Trough subduction zone
Authors: Futagami Taiki , Morono Yuki , Terada Takeshi , Kaksonen Anna H. , Inagaki Fumio
Published: March 2013

Halogenated organic matter buried in marine subsurface sediment may serve as a source of electron acceptors for anaerobic respiration of subseafloor microbes. Detection of a diverse array of reductive dehalogenase-homologous (
rdhA
) genes suggests that subseafloor organohalide-respiring microbial communities may play significant ecological roles in the biogeochemical carbon and halogen cycle in the subseafloor biosphere. We report here the spatial distribution of dehalogenation activity in the Nankai Trough plate-subduction zone of the northwest Pacific off the Kii Peninsula of Japan. Incubation experiments with slurries of sediment collected at various depths and locations showed that degradation of several organohalides tested only occurred in the shallow sedimentary basin, down to 4.7 metres below the seafloor, despite detection of
rdhA
in the deeper sediments. We studied the phylogenetic diversity of the metabolically active microbes in positive enrichment cultures by extracting RNA, and found that Desulfuromonadales bacteria predominate. In addition, for the isolation of genes involved in the dehalogenation reaction, we performed a substrate-induced gene expression screening on DNA extracted from the enrichment cultures. Diverse DNA fragments were obtained and some of them showed best BLAST hit to known organohalide respirers such as
Dehalococcoides
, whereas no functionally known dehalogenation-related genes such as
rdhA
were found, indicating the need to improve the molecular approach to assess functional genes for organohalide respiration.
Organic cofactors in the metabolism of
Dehalococcoides mccartyi
strains
Authors: Schipp Christian J. , Marco-Urrea Ernest , Kublik Anja , Seifert Jana , Adrian Lorenz
Published: March 2013

Dehalococcoides mccartyi
strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published
Dehalococcoides
genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of
Dehalococcoides
physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in
Dehalococcoides
strain CBDB1.
Dehalococcoides
genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B
12
), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole.
Dehalococcoides
genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate,
S
-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.
Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases
Authors: Hug Laura A. , Maphosa Farai , Leys David , Löffler Frank E. , Smidt Hauke , Edwards Elizabeth A. , Adrian Lorenz
Published: March 2013

Organohalide respiration is an anaerobic bacterial respiratory process that uses halogenated hydrocarbons as terminal electron acceptors during electron transport-based energy conservation. This dechlorination process has triggered considerable interest for detoxification of anthropogenic groundwater contaminants. Organohalide-respiring bacteria have been identified from multiple bacterial phyla, and can be categorized as obligate and non-obligate organohalide respirers. The majority of the currently known organohalide-respiring bacteria carry multiple reductive dehalogenase genes. Analysis of a curated set of reductive dehalogenases reveals that sequence similarity and substrate specificity are generally not correlated, making functional prediction from sequence information difficult. In this article, an orthologue-based classification system for the reductive dehalogenases is proposed to aid integration of new sequencing data and to unify terminology.
The restricted metabolism of the obligate organohalide respiring bacterium
Dehalobacter restrictus:
lessons from tiered functional genomics
Authors: Rupakula Aamani , Kruse Thomas , Boeren Sjef , Holliger Christof , Smidt Hauke , Maillard Julien
Published: March 2013

Dehalobacter restrictus
strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It grows only via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor.
Dehalobacter restrictus
represents the paradigmatic member of the genus
Dehalobacter
, which in recent years has turned out to be a major player in the bioremediation of an increasing number of organohalides, both
in situ
and in laboratory studies. The recent elucidation of the
D. restrictus
genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood–Ljungdahl (WL) pathway for CO
2
fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one,
pceA
, has been characterized on genetic and biochemical levels. This study describes a multi-level functional genomics approach on
D. restrictus
across three different growth phases. A global proteomic analysis allowed consideration of general metabolic pathways relevant to organohalide respiration, whereas the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenases.
The origin, evolution, and functional impact of short insertion–deletion variants identified in 179 human genomes
Authors: Montgomery Stephen B. , Goode David L. , Kvikstad Erika , Albers Cornelis A. , Zhang Zhengdong D. , Mu Xinmeng Jasmine , Ananda Guruprasad , Howie Bryan , Karczewski Konrad J. , Smith Kevin S. , Anaya Vanessa , Richardson Rhea , Davis Joe ,   The 1000 Genomes Project Consortium, MacArthur Daniel G. , Sidow Arend , Duret Laurent , Gerstein Mark , Makova Kateryna D. , Marchini Jonathan , McVean Gil , Lunter Gerton
Published: March 2013

Short insertions and deletions (indels) are the second most abundant form of human genetic variation, but our understanding of their origins and functional effects lags behind that of other types of variants. Using population-scale sequencing, we have identified a high-quality set of 1.6 million indels from 179 individuals representing three diverse human populations. We show that rates of indel mutagenesis are highly heterogeneous, with 43%–48% of indels occurring in 4.03% of the genome, whereas in the remaining 96% their prevalence is 16 times lower than SNPs. Polymerase slippage can explain upwards of three-fourths of all indels, with the remainder being mostly simple deletions in complex sequence. However, insertions do occur and are significantly associated with pseudo-palindromic sequence features compatible with the fork stalling and template switching (FoSTeS) mechanism more commonly associated with large structural variations. We introduce a quantitative model of polymerase slippage, which enables us to identify indel-hypermutagenic protein-coding genes, some of which are associated with recurrent mutations leading to disease. Accounting for mutational rate heterogeneity due to sequence context, we find that indels across functional sequence are generally subject to stronger purifying selection than SNPs. We find that indel length modulates selection strength, and that indels affecting multiple functionally constrained nucleotides undergo stronger purifying selection. We further find that indels are enriched in associations with gene expression and find evidence for a contribution of nonsense-mediated decay. Finally, we show that indels can be integrated in existing genome-wide association studies (GWAS); although we do not find direct evidence that potentially causal protein-coding indels are enriched with associations to known disease-associated SNPs, our findings suggest that the causal variant underlying some of these associations may be indels.
Targeted chromosomal deletions and inversions in zebrafish
Authors: Gupta Ankit , Hall Victoria L. , Kok Fatma O. , Shin Masahiro , McNulty Joseph C. , Lawson Nathan D. , Wolfe Scot A.
Published: March 2013

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%–15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.
The Differential Transcription Network between Embryo and Endosperm in the Early Developing Maize Seed
Authors: Lu Xiaoduo , Chen Dijun , Shu Defeng , Zhang Zhao , Wang Weixuan , Klukas Christian , Chen Ling-ling , Fan Yunliu , Chen Ming , Zhang Chunyi
Published: March 2013

Transcriptome analysis of early-developing maize (Zea mays) seed was conducted using Illumina sequencing. We mapped 11,074,508 and 11,495,788 paired-end reads from endosperm and embryo, respectively, at 9 d after pollination to define gene structure and alternative splicing events as well as transcriptional regulators of gene expression to quantify transcript abundance in both embryo and endosperm. We identified a large number of novel transcribed regions that did not fall within maize annotated regions, and many of the novel transcribed regions were tissue-specifically expressed. We found that 50.7% (8,556 of 16,878) of multiexonic genes were alternatively spliced, and some transcript isoforms were specifically expressed either in endosperm or in embryo. In addition, a total of 46 trans-splicing events, with nine intrachromosomal events and 37 interchromosomal events, were found in our data set. Many metabolic activities were specifically assigned to endosperm and embryo, such as starch biosynthesis in endosperm and lipid biosynthesis in embryo. Finally, a number of transcription factors and imprinting genes were found to be specifically expressed in embryo or endosperm. This data set will aid in understanding how embryo/endosperm development in maize is differentially regulated.
Seedling establishment in a dynamic sedimentary environment: a conceptual framework using mangroves
Authors: Balke Thorsten , Webb Edward L. , van den Elzen Eva , Galli Demis , Herman Peter M. J. , Bouma Tjeerd J.
Published: March 2013

Vegetated biogeomorphic systems (e.g. mangroves, salt marshes, dunes, riparian vegetation) have been intensively studied for the impact of the biota on sediment transport processes and the resulting self‐organization of such landscapes. However, there is a lack of understanding of physical disturbance mechanisms that limit primary colonization in active sedimentary environments.

This study elucidates the effect of sediment disturbance during the seedling stage of pioneer vegetation, using mangroves as a model system. We performed mesocosm experiments that mimicked sediment disturbance as (i) accretion/burial of plants and (ii) erosion/excavation of plants of different magnitudes and temporal distribution in combination with water movement and inundation stress.

Cumulative sediment disturbance reduced seedling survival, with the faster‐growing Avicennia alba showing less mortality than the slower‐growing Sonneratia alba. The presence of the additional stressors (inundation and water movement) predominantly reduced the survival of S. alba.

Non‐lethal accretion treatments increased shoot biomass of the seedlings, whereas non‐lethal erosion treatments increased root biomass allocation. This morphological plasticity in combination with the abiotic disturbance history determined how much maximum erosion the seedlings were able to withstand.

Synthesis and applications. Seedling survival in dynamic sedimentary environments is determined by the frequency and magnitude of sediment accretion or erosion events, with non‐lethal events causing feedbacks to seedling stability. Managers attempting restoration of mangroves, salt marshes, dunes and riparian vegetation should recognize sediment dynamics as a main bottleneck to primary colonization. The temporal distribution of erosion and accretion events has to be evaluated against the ability of the seedlings to outgrow or adjust to disturbances. Our results suggest that selecting fast‐growing pioneer species and measures to enhance seedling growth or temporary reduction in sediment dynamics at the restoration site can aid restoration success for vegetated biogeomorphic ecosystems.