Rapid Genotyping of Mice with Hemoglobinopathies and Globin Transgenes

The hematology of the laboratory mouse has beenwell characterized. Normal genetic differences at thealpha- and beta-globin gene loci serve as useful markersfor a wide variety of types of experimental studies. There are a number of naturallyoccurring or induced mutations that disrupt globinexpression and produce thalassemic phenotypes. Inaddition, much has been learned of the workings of theglobin locus control region from studies of transgenicmice, including those with mutations induced by targetedsite-specific modifications. After a new mutation ortransgene has been created, it must be maintained in living mice, and the genotypes of theoffspring must be ascertained. While it is possible todetermine genotypes by DNA analyses, such assays aretime consuming and relatively expensive. An osmoticchallenge test -- originally developed for thegenotyping of large-deletion alpha-thalassemia mutationsin mice -- has proven useful in detecting bothsevere and milder alpha- and beta-thalassemias, as wellas some transgenic genotypes in mice carrying human globin genes.Reliable genotyping can, in some cases, be completedwithin a few minutes with minimal expense.Quantification of red cell fragility for a variety ofthalassemic and transgenic mice is described here, alongwith a simplified test suitable for rapid, routinegenotyping. The osmotic challenge test is perfectlyreliable for distinguishing genotypes that causesignificantly decreased release of hemoglobin from the redcells, but it is also useful for some of the conditionsin which overall erythrocyte osmotic fragility isessentially normal.     

Effects of habitat size and quality on equilibrium density and extinction time of Sorex araneus populations

1. The effects of changes in habitat size and quality on the expected population density and the expected time to extinction of Sorex araneus are studied by means of mathematical models that incorporate demographic stochasticity.
2. Habitat size is characterized by the number of territories, while habitat quality is represented by the expected number of offspring produced during the lifetime of an individual.
3. The expected population density of S. araneus is shown to be mainly influenced by the habitat size. The expected time to extinction of S. araneus populations due to demographic stochasticity, on the other hand, is much more affected by the habitat quality.
4. In a more general setting we demonstrate that, irrespective of the actual species under consideration, the likelihood of extinction as a consequence of demographic stochasticity is more effectively countered by increasing the reproductive success and survival of individuals then by increasing total population size.     

Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

Responses of juvenile rainbow trout, under food limitation, to chronic low pH and elevated summer temperatures, alone and in combination

Rainbow trout were exposed (90 days) in synthetic soft water to sublethal low pH (5.2) and a simulated climate warming scenario (+2°C above the control summer temperature range of 16.5–21° C), alone and in combination, under conditions of limited food (∼4% dry body weight day⁻¹). Weight specific oxygen consumption rates ( M o₂) were ∼55% of M o_2(max), in contrast to ∼75% of M o_2(max) found in trout fed an unlimited ration. This is likely due to a reduction in food quantity and thus feeding activity. However, the trout exposed to low pH at control temperatures exhibited higher conversion efficiencies and increased growth. In contrast, trout exposed to +2°C had reduced growth rates. No ionoregulatory disturbance occurred in any treatment, suggesting that this ration was sufficient to provide a replacement salt load in the diet. Energy budgets indicated that the limited ration resulted in a lowered optimum temperature for growth, with a greater proportion of the energy intake dissipated for metabolic expenditure, resulting in reduced conversion efficiencies. A fourfold reduction in faecal and unaccounted energy losses indicated higher absorption efficiencies than in satiation-fed trout.     

Accumulation Pattern of IgG Antibodies and Fab Fragments in Transgenic Arabidopsis thaliana Plants

For the further optimization of antibody expression in plants,it is essential to determine the final accumulation sites ofplant-made antibodies. Previously, we have shown that, uponsecretion, IgG antibodies and F_ab fragments can be detectedin the intercellular spaces of leaf mesophyil cells of transgenicArabidopsis thaliana plants. However, immunofluorescence microscopyshowed that this is probably not their final accumulation site.In leaves, IgG and F_abfragments accumulate also at the interiorside of the epidermal cell layers and in xylem vessels. Theseaccumulation sites correspond with the leaf regions where waterof the transpiration stream is entering a space impermeableto the proteins or where water is evaporating. In roots, plant-madeF_ab fragments accumulate in intercellular spaces of cortex cells,in the cytoplasm of pericycle and, to a lesser extent, endodermiscells, and in cells of the vascular cylinder. In other words,antibody accumulation occurs at the sites where water passeson its radial pathway towards and within the vascular bundle.Taken together, our results suggest that, upon secretion ofplant-made antibodies or F_ab fragments, a large proportion ofthese proteins are transported in the apoplast of A. thaliana,possibly by the water flow in the transpiration stream. ⁴Corresponding author. Fax 32-9-2645349; e-mail: anpic{at}gengenp.rug.ac.be     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.