High-throughput trapping of secretory pathway genes in mouse embryonic stem cells

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.     

Gastrointestinal and renal responses to water intake in the green-backed firecrown (Sephanoides sephanoides), a South American hummingbird

To maintain water balance, nectar-feeding vertebrates oscillate between meeting the challenges of avoiding overhydration and preventing dehydration. To understand how green-backed firecrowns (Sephanoides sephanoides) accomplish this, we examined the response of water-handling processes in the gastrointestinal tract (GIT) and kidney to different rates of water intake during the evening, night, and morning. Fractional water absorption in the GIT was independent of water intake rate (evening: 0.91 +/- 0.08; morning: 0.88 +/- 0.04). Consistent with this nonregulated water absorption, we found linear increases in water flux, fractional turnover of body water, and the rate of renal water loading as water intake rate increased during both the evening and morning. Despite these relationships, glomerular filtration rate (GFR) was insensitive to water loading (evening: 2.08 +/- 0.56 ml/h; morning: 1.84 +/- 0.68 ml/h) and less than the allometric expectation (2.92 ml/h). During the evening, fractional renal water reabsorption decreased linearly as the rate of water intake increased. At night, a period of natural fasting for hummingbirds, mean GFR was not different from zero (0.00 +/- 0.05 ml/h). These findings indicate that green-backed firecrowns eliminate excess ingested water by decreasing water reabsorption in the kidney; to conserve water, it appears that hummingbirds arrest whole kidney GFR, effectively preventing urinary water losses. After discounting evaporative water losses, our results show that hummingbirds rely principally on their renal system to resolve the osmoregulatory quandary posed by nectarivory.     

Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.     

Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela

The mosquito Aedes aegypti is the main vector of dengue in Venezuela. The genetic structure of this vector was investigated in 24 samples collected from eight geographic regions separated by up to 1160 km. We examined the distribution of a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among 1144 Ae. aegypti from eight collections. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Seven haplotypes were detected throughout Venezuela and these were sorted into two clades. Significant differentiation was detected among collections and these were genetically isolated by distance.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

Immunochemical characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50B), enterocin P, and enterocin Q by Enterococcus faecium L50

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47 degrees C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32 degrees C, but production becomes negligible when the growth temperature is above 37 degrees C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47 degrees C. Maximum EntL50A and EntL50B production was detected at 25 degrees C, while EntP and EntQ are maximally produced at 37 and 47 degrees C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.