A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.     

Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.     

Mechanical properties of Xenopus egg cytoplasmic extracts

Cytoplasmic extracts prepared from Xenopus laevis eggs are used for the reconstitution of a wide range of processes in cell biology, and offer a unique environment in which to investigate the role of cytoplasmic mechanics without the complication of preorganized cellular structures. As a step toward understanding the mechanical properties of this system, we have characterized the rheology of crude interphase extracts. At macroscopic length scales, the extract forms a soft viscoelastic solid. Using a conventional mechanical rheometer, we measure the elastic modulus to be in the range of 2-10 Pa, and loss modulus in the range of 0.5-5 Pa. Using pharmacological and immunological disruption methods, we establish that actin filaments and microtubules cooperate to give mechanical strength, whereas the intermediate filament cytokeratin does not contribute to viscoelasticity. At microscopic length scales smaller than the average network mesh size, the response is predominantly viscous. We use multiple particle tracking methods to measure the thermal fluctuations of 1 microm embedded tracer particles, and measure the viscosity to be approximately 20 mPa-s. We explore the impact of rheology on actin-dependent cytoplasmic contraction, and find that although microtubules modulate contractile forces in vitro, their interactions are not purely mechanical.     

An empirical model of carbon flow through marine viruses and microzooplankton grazers

Viruses and microzooplankton grazers represent major sources of mortality for marine phytoplankton and bacteria, redirecting the flow of organic material throughout the world's oceans. Here, we investigate the use of nonlinear population models of interactions between phytoplankton, viruses and grazers as a means to quantitatively constrain the flow of carbon through marine microbial ecosystems. We augment population models with a synthesis of laboratory-based estimates of prey, predator and viral life history traits that constrain transfer efficiencies. We then apply the model framework to estimate loss rates in the California Current Ecosystem (CCE). With our empirically parameterized model, we estimate that, of the total losses mediated by viruses and microzooplankton grazing at the focal CCE site, 22 ± 3%, 46 ± 27%, 3 ± 2% and 29 ± 20% were directed to grazers, sloppy feeding (as well as excretion and respiration), viruses and viral lysate respectively. We identify opportunities to leverage ecosystem models and conventional mortality assays to further constrain the quantitative rates of critical ecosystem processes.     

Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.     

Combined association,segregation and aggregation analysis on case-control family data

Recently genetic epidemiologists have begun using case-control family study designs to investigate the role of genetic and environmental risk factors in disease etiology. The objective of these studies is to assess the association of environmental factors with the disease trait; to characterize the disease genes using segregation analysis; and to quantify the residual familial aggregation after controlling for environmental and genetic factors. Typically these objectives are achieved by conducting separate studies and analysis. This paper describes an estimating equation based approach for a combined association, segregation and aggregation analysis on data from case-control family studies. Simulations indicate that the method performs well in a variety of settings. The method is illustrated using simulated family history data made available to participants in a recent Genetic Analysis Workshop.