SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN↓NGGCC, where N is any base and ↓ is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SfiI, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SfiI cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SfiI cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for kcat and Km were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SfiI activity is thus highly dependent on conformational variations in the spacer DNA.

Spatial heterogeneity of inhibitory surrounds in the middle temporal visual area.

A recurrent theme in the organization of vertebrate visual cortex is that of receptive fields with an associated "silent" opponency component. In the middle temporal area (area MT), a cortical visual area involved in the analysis of retinal motion in primates, this opponency appears in the form of a region outside the classical receptive field (CRF) that in itself gives no response but suppresses responses to motion evoked within the CRF. This antagonistic motion surround has been described as very large and symmetrically arrayed around the CRF. On the basis of this view, the primary function of the surround has long been thought to consist of simple figure-ground segregation based on movement. We have made use of small stimulus patches to map the form and extent of the surround and find evidence that the surround inhibition of many MT cells is in fact confined to restricted regions on one side or on opposite sides of the CRF. Such regions endow MT cells with the ability to make local-to-local motion comparisons, capable of extracting more complex features from the visual environment, and as such, may be better viewed as intrinsic parts of the receptive field, rather than as separate entities responsible for local-to-global comparisons.

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Circuitry for color coding in the primate retina.

Human color vision starts with the signals from three cone photoreceptor types, maximally sensitive to long (L-cone), middle (M-cone), and short (S-cone) wavelengths. Within the retina these signals combine in an antagonistic way to form red-green and blue-yellow spectral opponent pathways. In the classical model this antagonism is thought to arise from the convergence of cone type-specific excitatory and inhibitory inputs to retinal ganglion cells. The circuitry for spectral opponency is now being investigated using an in vitro preparation of the macaque monkey retina. Intracellular recording and staining has shown that blue-ON/yellow-OFF opponent responses arise from a distinctive bistratified ganglion cell type. Surprisingly, this cone opponency appears to arise by dual excitatory cone bipolar cell inputs: an ON bipolar cell that contacts only S-cones and an OFF bipolar cell that contacts L- and M-cones. Red-green spectral opponency has long been linked to the midget ganglion cells, but an underlying mechanism remains unclear. For example, receptive field mapping argues for segregation of L-and M-cone signals to the midget cell center and surround, but horizontal cell interneurons, believed to generate the inhibitory surround, lack opponency and cannot contribute selective L- or M-cone input to the midget cell surround. The solution to this color puzzle no doubt lies in the great diversity of cell types in the primate retina that still await discovery and analysis.

Mouse strain differences determine severity of iron accumulation in Hfe knockout model of hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/−), and knockout (Hfe −/−) mice from each strain. Although the Hfe −/− mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe −/− AKR mice that were 2.5 or 3.6 times higher than those of Hfe −/− C3H or Hfe −/− C57BL/6 mice, respectively. The Hfe −/− mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.