Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

The IRP1-HIF-2α Axis Coordinates Iron and Oxygen Sensing with Erythropoiesis and Iron Absorption

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Highlights? Derepression of HIF-2α mRNA in Irp1^?/? mice causes age-dependent polycythemia ? HIF-2α hyperactivity is observed in multiple tissues of Irp1^?/? mice ? The mRNA regulons of IRP1 and IRP2 are separable in vivo ? The IRP1-HIF-2α axis is a therapeutic target for hematologic or oncologic disorders     

Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at,or Prior to,Establishment of Double-Strand Breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.     

Haplotype probabilities for multiple-strain recombinant inbred lines

Recombinant inbred lines (RIL) derived from multiple inbred strains can serve as a powerful resource for the genetic dissection of complex traits. The use of such multiple-strain RIL requires a detailed knowledge of the haplotype structure in such lines. Broman (2005) derived the two- and three-point haplotype probabilities for 2(n)-way RIL; the former required hefty computation to infer the symbolic results, and the latter were strictly numerical. We describe a simpler approach for the calculation of these probabilities, which allowed us to derive the symbolic form of the three-point haplotype probabilities. We also extend the two-point results for the case of additional generations of intermating, including the case of 2(n)-way intermated recombinant inbred populations (IRIP).     

Long homozygous chromosomal segments in reference families from the centre d'Etude du polymorphisme humain

Using genotypes from nearly 8,000 short tandem-repeat polymorphisms typed in eight of the reference families from the Centre d'Etude du Polymorphisme Humain (CEPH), we identified numerous long chromosomal segments of marker homozygosity in many CEPH individuals. These segments are likely to represent autozygosity, the result of the mating of related individuals. Confidence that the complete segment is homozygous is gained only with markers of high density. The longest segment in the eight families spanned 77 cM and included 118 homozygous markers. All individuals in family 884 showed at least one segment of homozygosity: the father and mother were homozygous in 8 and 10 segments with an average length of 13 and 16 cM, respectively, and covering a total of 105 and 160 cM, respectively. The progeny in family 884 were homozygous over 5-16 segments with average length 11 cM. The progeny in family 102 were homozygous over 4-12 segments with average length 19 cM. Of the 100 individuals in the other six families, 1 had especially long homozygous segments, and 19 had short but significant homozygous segments. Our results indicate that long homozygous segments are common in humans and that these segments could have a substantial impact on gene mapping and health.     

Efficient Imputation of Missing Markers in Low-Coverage Genotyping-by-Sequencing Data from Multiparental Crosses

We consider genomic imputation for low-coverage genotyping-by-sequencing data with high levels of missing data. We compensate for this loss of information by utilizing family relationships in multiparental experimental crosses. This nearly quadruples the number of usable markers when applied to a large rice Multiparent Advanced Generation InterCross (MAGIC) study.