IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

Influence of plant domestication on plant-pollinator interactions: Floral attributes and floral visitor communities in wild and cultivated squash plants

Premise

Domestication of plant species results in phenotypic modifications and changes in biotic interactions. Most studies have compared antagonistic plant-herbivore interactions of domesticated plants and their wild relatives, but little attention has been given to how domestication influences plant-pollinator interactions. Floral attributes and interactions of floral visitors were compared between sister taxa of the genus Cucurbita (Cucurbitaceae), the domesticated C. moschata, C. argyrosperma ssp. argyrosperma and its wild progenitor C. argyrosperma ssp. sororia in the place of origin.

Methods

We conducted univariate and multivariate analyses to compare floral morphological traits and analyzed floral reward (nectar and pollen) quantity and quality between flowers of wild and domesticated Cucurbita taxa. Staminate and pistillate flowers of all three taxa were video recorded, and visitation and behavior of floral visitors were registered and analyzed.

Results

Most floral morphological characteristics of flowers of domesticated taxa were larger in both staminate and pistillate flowers. Staminate and pistillate flowers presented distinct correlations between floral traits and integration indices between domesticated and wild species. Additionally, pollen quantity and protein to lipid ratio were greater in domesticated species. Cucurbit pollen specialists, Eucera spp., had the highest probability of visit for all Cucurbita taxa.

Conclusions

We provide evidence that floral traits of domesticated and wild Cucurbita species experienced different selection pressures. Domesticated Cucurbita species may have more resources invested towards floral traits, thereby increasing attractiveness to pollinators and potentially plant reproductive success. Wild ancestor plant populations should be conserved in their centers of origin to preserve plant-pollinator interactions.     

Allozymic characterization and evolutionary relationships in the BrazilianAkodon cursor species group (Rodentia-Cricetidae)

The present study involved an electrophoretic survey of 22 protein loci in 269 individuals belonging to three species of the genusAkodon, A. aff.cursor (2n=16),A. cursor (2n=14/15), andA. montensis (2n=24/25/26), collected in Eastern Brazil. The joint results of gene diversity, genetic distances, phenetic analyses, and phylogenetic trees suggested thatA. aff.cursor has recently separated fromA. cursor and that the three species have experienced a recent chromosomal divergence followed by low allozyme differentiation. These data are in agreement with their classification as sibling species.     

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12–p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 trans-activator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent—human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to threedifferent chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12–p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.     

The biosynthesis of glucose containing insect lipid linked oligosaccharide and its possible role in glycoprotein assembly

Summary Microsome enriched Ceratitis capitata extracts synthesized a glucosylated lipid linked oligosaccharide. Its properties were closely related to those of the previously described insect mannosylated dolichyl diphosphate oligosaccharides and almost the same as those of the rat liver dolichyl-diphosphate-(GlcNAc)₂-(Man)₉-(Glc)_1–3. The saccharide moiety of, the latter was transferred to an unknown endogenous protein-like acceptor by the fly extracts. These represent the first evidence of a protein glycosylation in a pluricellular invertebrate through dolichyl derivatives.Abbreviations Dol-P dolichyl phosphate - Dol-P-P dolichyl diphosphate     

Phospholipid dependence of rat liver microsomal acyl: CoA synthetase and acyl-CoA : 1-Acyl-sn-glycero-3-phosphocholine O-acyltransferase

Summary Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used—palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes—1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A₂ which both participate in the deacylation-reacylation cycle.     

The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3

The common cytokine receptor gamma chain (gamma c), an essential component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. Recently, a novel lymphokine (IL-21) and its receptor (IL-21R alpha) were described which profoundly affect the growth and activation state of B, T, and NK cells in concert with other lymphokines or stimuli [Parrish-Novak, J., et al. (2000) Nature 408, 57-63]. In this report, we show that gamma c is also a required signaling component of the IL-21 receptor (IL-21R) using the gamma c-deficient X-linked severe combined immunodeficiency (XSCID) lymphoblastoid cell line JT, and JT cells reconstituted with gamma c (JT/gamma c). Moreover, we demonstrate a functional requirement for both gamma c and the gamma c-associated Janus family tyrosine kinase 3 (JAK3) in IL-21-induced proliferation of pro-B-lymphoid cells engineered to express human IL-21R alpha (BaF3/IL-21R alpha). Retroviral-mediated transduction of wild-type gamma c into XSCID JT cells restored function to the IL-21R, as shown by IL-21-induced tyrosine phosphorylation of JAK1 and JAK3, and downstream activation of STAT5, in JT/gamma c cells as well as BaF3/IL-21R alpha and primary splenic B cells. In contrast, IL-21 failed to activate the JAK-STAT pathway in nonreconstituted JT cells. Monoclonal antibodies specific for the gamma c chain effectively inhibited IL-21-induced growth of BaF3/IL-21R alpha cells, supporting a functional role for this molecule in the IL-21R complex. In addition, the specific JAK3 tyrosine kinase inhibitor WHI-P131 significantly reduced IL-21-induced proliferation of BaF3/IL-21R alpha cells. Taken together, these results definitively demonstrate that IL-21-mediated signaling requires the gamma c chain, and indicate that JAK3 is an essential transducer of gamma c-dependent survival and/or mitogenic signals induced by this cytokine.