A high-resolution single nucleotide polymorphism genetic map of the mouse genome

High-resolution genetic maps are required for mapping complex traits and for the study of recombination. We report the highest density genetic map yet created for any organism, except humans. Using more than 10,000 single nucleotide polymorphisms evenly spaced across the mouse genome, we have constructed genetic maps for both outbred and inbred mice, and separately for males and females. Recombination rates are highly correlated in outbred and inbred mice, but show relatively low correlation between males and females. Differences between male and female recombination maps and the sequence features associated with recombination are strikingly similar to those observed in humans. Genetic maps are available from http://gscan.well.ox.ac.uk/#genetic_map and as supporting information to this publication.     

Animal models of psychiatric disease

Animal models of psychiatric diseases are useful tools for screening new drugs and for investigating the mechanisms of those disorders. Despite the difficulties inherent in modelling human psychiatric phenotypes in animals, there has been recent success identifying mutations in mice that give rise to some of the characteristic features of anxiety, depression, schizophrenia, autism, obsessive-compulsive disorder and bipolar disorder. In some cases these models have the additional strength that drugs used to treat the human condition alleviate the symptoms in mice. Robust genetic evidence of the involvement of multiple susceptibility genes in psychiatric disease will enable future studies to move from single-gene models to models with multiple modified loci, with the promise of better representing the complexity of the human diseases.     

Computational design of an integrin I domain stabilized in the open high affinity conformation

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.     

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.     

GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28

Membrane proteins located on vesicles (v-SNAREs) and on the target membrane (t-SNAREs) mediate specific recognition and, possibly, fusion between a transport vesicle and its target membrane. The activity of SNARE molecules is regulated by several soluble cytosolic proteins. We have cloned a bovine brain cDNA encoding a conserved 117 amino acid polypeptide, denoted Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), that functions as a soluble transport factor. GATE-16 interacts with N-ethylmaleimidesensitive factor (NSF) and significantly stimulates its ATPase activity. It also interacts with the Golgi v-SNARE GOS-28 in an NSF-dependent manner. We propose that GATE-16 modulates intra-Golgi transport through coupling between NSF activity and SNAREs activation.     

Properties of monovalent nickel complexes with tetraaza-macrocyclic ligands in aqueous solutions

The effects of N-alkylation on the redox potential of the couples NiLⁱ²⁺/NiLⁱ⁺, L = tetraaza-14-membered-macrocyclic ligands, and on the properties of the monovalent nickel complexes in aqueous solutions are reported for 14 complexes. The spectra and lifetimes of the NiLⁱ⁺ complexes are reported. The self-exchange rates for the couples NiLⁱ²⁺/NiLⁱ⁺ were determined. Two of the ligands were synthesized for the first time for this study. Cyclic voltammetry and pulse radiolysis were used. The results point out that: (i) N-alkylation always shifts the redox potential to a less cathodic one; this effect stems to a large degree from the decrease in the solvation energy of the complex caused by the N-alkylation of the ligand. (ii). The lifetime of the monovalent complexes is not linearly related to the redox potential of the NiLⁱ²⁺/NiLⁱ⁺ couples. (iii) The NiLⁱ⁺ complexes exist in several isomeric forms; the rate of the isomerization depends on the structure of the ligand. (iv) Different isomers of NiLⁱ⁺ may be formed when the complex NiLⁱ²⁺ is reduced by different reagents; therefore, the pulse-radiolytically formed NiLⁱ⁺ complexes might have different properties than those formed electrochemically.     

Mouse inbred strain sequence information and yin-yang crosses for quantitative trait locus fine mapping

The shared ancestry of mouse inbred strains, together with the availability of sequence and phenotype information, is a resource that can be used to map quantitative trait loci (QTL). The difficulty in using only sequence information lies in the fact that in most instances the allelic state of the QTL cannot be unambiguously determined in a given strain. To overcome this difficulty, the performance of multiple crosses between various inbred strains has been proposed. Here we suggest and evaluate a general approach, which consists of crossing the two strains used initially to map the QTL and any new strain. We have termed these crosses "yin-yang," because they are complementary in nature as shown by the fact that the QTL will necessarily segregate in only one of the crosses. We used the publicly available SNP database of chromosome 16 to evaluate the mapping resolution achievable through this approach. Although on average the improvement of mapping resolution using only four inbred strains was relatively small (i.e., reduction of the QTL-containing interval by half at most), we found a great degree of variability among different regions of chromosome 16 with regard to mapping resolution. This suggests that with a large number of strains in hand, selecting a small number of strains may provide a significant contribution to the fine mapping of QTL.     

Cold spots are universal in protein–protein interactions

Proteins interact with each other through binding interfaces that differ greatly in size and physico‐chemical properties. Within the binding interface, a few residues called hot spots contribute the majority of the binding free energy and are hence irreplaceable. In contrast, cold spots are occupied by suboptimal amino acids, providing possibility for affinity enhancement through mutations. In this study, we identify cold spots due to cavities and unfavorable charge interactions in multiple protein–protein interactions (PPIs). For our cold spot analysis, we first use a small affinity database of PPIs with known structures and affinities and then expand our search to nearly 4000 homo‐ and heterodimers in the Protein Data Bank (PDB). We observe that cold spots due to cavities are present in nearly all PPIs unrelated to their binding affinity, while unfavorable charge interactions are relatively rare. We also find that most cold spots are located in the periphery of the binding interface, with high‐affinity complexes showing fewer centrally located colds spots than low‐affinity complexes. A larger number of cold spots is also found in non‐cognate interactions compared to their cognate counterparts. Furthermore, our analysis reveals that cold spots are more frequent in homo‐dimeric complexes compared to hetero‐complexes, likely due to symmetry constraints imposed on sequences of homodimers. Finally, we find that glycines, glutamates, and arginines are the most frequent amino acids appearing at cold spot positions. Our analysis emphasizes the importance of cold spot positions to protein evolution and facilitates protein engineering studies directed at enhancing binding affinity and specificity in a wide range of applications.     

Effect of hypothermia on reuptake of neuromediators by synaptosomes

Cranio-cerebral hypothermia (temperature of the body 32-30 degrees C, of the brain 29-27 degrees C) was studied for its effect on the reuptake of neuromediators (3H-noradrenaline and [14C]GABA) by the cortex and hypothalamus synaptosomes of the rat brain. It was found that the reuptake of [3H]noradrenaline by the cortex synaptosomes under narcosis and cranio-cerebral hypothermia was inhibited much stronger than that by the hypothalamus synaptosomes. At the same time GABA-ergic synapses of the cortex and hypothalamus were not sensitive to narcosis. Cranio-cerebral hypothermia essentially inhibited the reuptake of [14C] GABA by synaptosomes and hypothalamus.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.