BackgroundThe mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.ResultsWe undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.ConclusionsComparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

A neurotransmitter transporter can potentially mediate uptake or release of substrate, and its stoichiometry is a key factor that controls the driving force and thus the neurotransmitter flux direction. We have used a combination of electrophysiology and radio-tracing techniques to evaluate the stoichiometries of two glycine transporters involved in glycinergic or glutamatergic transmission. We show that GlyT2a, a transporter present in glycinergic boutons, has a stoichiometry of 3 Na+/Cl-/glycine, which predicts effective glycine accumulation in all physiological conditions. GlyT1b, a glial transporter, has a stoichiometry of 2 Na+/Cl-/ glycine, which predicts that glycine can be exported or imported, depending on physiological conditions. GlyT1b may thus modulate glutamatergic synapses by increasing or decreasing the glycine concentration around N-methyl-D-aspartate receptors (NMDARs).

Two closely related ␤ subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus ␤ antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca 2؉ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian ␣ 1 cRNAs (␣ 1C and ␣ 1E ); (ii) coinjection of a Xenopus ␤ antisense oligonucleotide and excess rat ␤ cRNA rescued expression of ␣ 1 Ca 2؉ channel currents; and (iii) coinjection of mammalian ␣ 1 cRNA with cRNA encoding either of the two Xenopus ␤ subunits facilitated both activation and inactivation of Ca 2؉ channel currents by voltage, as happens with most mammalian ␤ subunits. The Xenopus ␤ subunit cDNAs (␤3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 ␤ subunit. We propose that ''␣ 1 alone'' channels are in fact tightly associated ␣ 1 ␤3xo channels, and that effects of exogenous ␤ subunits are due to formation of higher-order [␣ 1 ␤]␤ n complexes with an unknown contribution of ␤3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition ␣ 1 ␤, are [␣ 1 ␤]␤ n complexes that associate with ␣ 2 ␦ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of ␣ 1E have a ␤ subunit binding domain. This ␤ binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel ␣ 1 subunits.Xenopus oocytes translate exogenously injected mRNAs and cRNAs with relatively high efficiency. This has made them systems of choice for the functional expression and characterization of many cloned molecules, such as neuronal ligandgated ion channels, G protein-coupled receptors, and many voltage-gated ion channels, including voltage-dependent Ca 2ϩ channels. Voltage-dependent Ca 2ϩ channels are formed of an ␣ 1 pore-forming and voltage-sensing subunit and ␤ and ␣ 2 ␦ regulatory subunits. Functional expression in Xenopus oocytes has not only been used to define structure-function relations of voltage-gated calcium channels by assessing the effects of specific mutations of the ␣ 1 channel protein, but also to define identity and roles of the regulatory subunits in promoting ␣ 1 expression or modifying the properties of the expressed ␣ 1 subunit. Several nonallelic genes encoding ␣ 1 subunits, termed ␣ 1S and ␣ 1A -␣ 1E , have been identified by molecular cloning (1-4). Of these, all except ␣ 1S have been functionally expressed in the Xenopus oocyte. However, while certain variants of ␣ 1C and ␣ 1E can be expressed without coinjection of other subunit cRNAs (e.g., refs. 5 and 6), others, particularly ␣ 1A , yield only minimal currents in the absence of additional subunits, notably a ␤ subunit (7,8). The reasons for these differences are not understood.The interpreta...

Background:Head and neck squamous cell carcinoma (HNSCC) is associated with poor survival. To identify prognostic and diagnostic markers and therapeutic targets, we studied ANO1, a recently identified calcium-activated chloride channel (CaCC).Methods:High-resolution genomic and transcriptomic microarray analysis and functional studies using HNSCC cell line and CaCC inhibitors.Results:Amplification and overexpression of genes within the 11q13 amplicon are associated with the propensity for future distance metastasis of HPV-negative HNSCC. ANO1 was selected for functional studies based on high correlations, cell surface expression and CaCC activity. ANO1 overexpression in cells that express low endogenous levels stimulates cell movement, whereas downregulation in cells with high endogenous levels has the opposite effect. ANO1 overexpression also stimulates attachment, spreading, detachment and invasion, which could account for its effects on migration. CaCC inhibitors decrease movement, suggesting that channel activity is required for the effects of ANO1. In contrast, ANO1 overexpression does not affect cell proliferation.Interpretation:ANO1 amplification and expression could be markers for distant metastasis in HNSCC. ANO1 overexpression affects cell properties linked to metastasis. Inhibitors of CaCCs could be used to inhibit the tumourigenic properties of ANO1, whereas activators developed to increase CaCC activity could have adverse effects.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems.

The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.

Membrane neurotransmitter transporters control the concentration of their substrate in the synaptic clefts, through the thermodynamic coupling of uptake to the movement of Na + and other ions. In addition, excitatory amino acid transporters (EAAT) have a Cl − conductance which is gated by the joint binding of Na + and glutamate, but thermodynamically uncoupled to the flux of glutamate. This conductance is particularly large in the retina-specific EAAT5 isoform. In the mouse retina, we located EAAT5 in both cone and rod photoreceptor terminals and in axon terminals of rod bipolar cells. In these later cells, application of glutamate on the axon terminal evoked a current that reversed at E Cl , was insensitive to bicuculline, TPMPA, strychnine, DL-AP5, CNQX and MCPG, but blocked by the glutamate transporter inhibitor DL-tBOA. Furthermore, short depolarizations of the bipolar cells evoked a DL-tBOA and Cd 2+ -sensitive current whose amplitude was comparable to the glutamate-evoked current. Its kinetics indicated that EAAT5 was located close to the glutamate release site. For 2 ms depolarizations evoking maximal responses, the EAAT5-mediated current carried between 2 and 8 times more charge as an average inhibitory GABA or glycine postsynaptic current received spontaneously from amacrine cells, with 10 mM or 0.5 mM intracellular EGTA, respectively. In conditions for which reciprocal inhibition could be monitored, the charge carried by the EAAT5 current was 1.5 times larger than the one carried by the inhibitory postsynaptic currents received from amacrine cells. These results indicate that EAAT5 acts as a major inhibitory presynaptic receptor at mammalian rod bipolar cell axon terminals. This feedback mechanism could control glutamate release at the ribbon synapses of a non-spiking neuron and increase the temporal contrast in the rod photoreceptor pathway.

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through speci¢c transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters di¡er at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steadystate kinetics. A 3 Na + /1 Cl^/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine £ux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na + and Cl^concentra-tions. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the re¢lling of synaptic vesicles by the low a⁄nity, low speci¢city vesicular transporter VGAT/VIAAT. In contrast, the 2 Na + /1 Cl^/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors e¡ectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.Key words: Transporter; Glycine ; Stoichiometry; Driving force; Uptake; Synaptic transmission 1. Transporters of recapture in slow and fast transmission have di¡erent constraintsIn the central nervous system of Vertebrates, di¡usion and uptake of neurotransmitters by speci¢c transporters terminate synaptic transmission at the notable exception of acetylcholine which is hydrolyzed by acetylcholinesterase. Transporters of recapture are located in glial cells and/or neurons and uptake regulates the basal extracellular concentration and spillover of neurotransmitters, thus limiting synaptic cross talk. Though recapture is their principal mode of operation, transporters are bi-directional molecular machines and may also behave as a Ca 2þ -independent source of neurotransmitters, depending on the direction of the driving force.Transporters belong to two families of secondary active transporters (see reviews in [1,2]) with a distinct membrane topology and an ionic requirement for Cl 3 and K þ ions [3]. The larg...