Transformation by Complementation of an Adenine Auxotroph of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Swollen basidiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per μg of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basidiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and other auxotrophic strains yielded Ade⁻ progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene.     

Nutritive value of the nuña popping bean

Nuñas (Thaseolus vulgaris, Fabaceae), commonly called popping beans, are traditionally grown in the Andean highlands of South America, and are consumed as a snack food after a quick toasting process. Proximate analysis of their nutritive value revealed that nunas have a higher content of starch, amylose, and copper than four dry bean varieties and a lower mean content of protein, phosphorous, iron, and boron. The unique texture and taste of nuñas may be related to their high starch content. Antinutritional factors such as lectins were higher in raw and boiled nuña samples than in toasted nuñas, while tannin levels did not change from raw to toasted treatments. Overall in-vitro digestibility was slightly lower for toasted nunas than boiled dry bean.     

Comparative value of four arcelin variants in the development of dry bean lines resistant to the Mexican bean weevil

Wild Phaseolus vulgaris L. accessions containing arcelin codominant alleles 1 through 5 were reconfirmed and characterized for resistance to the Mexican bean weevil, Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae). Accession G 02771 (arcelin 5) had the highest level of antibiosis resistance, followed by G 12952 (arcelin 4), G 12882 (arcelin 1) and G 12866 (arcelin 2). Arcelin 3 accessions conferred the lowest levels of resistance. As the presence of arcelin is inherited as a single dominant gene, a backcross breeding program has been used to transfer resistance to the Mexican bean weevil from wild beans to bean cultivars using serological techniques to detect the presence of arcelin and replicated insect feeding tests to measure resistance levels. Progeny containing arcelin 1 showed resistance equal or superior to that of the resistant check. Arcelin 2-deerived lines had intermediate levels of resistance while no resistant progenies were obtained from crosses with arcelin 3 and 4 sources. Results are discussed in relation to the deployment of arcelin alleles in bean cultivars.

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Valeurs comparées de 5 types d'arcéline dans l'obtention de lignées de Phaseolus vulgaris résistantes à Zabrotes subfasciatus

Résumé La résistance à Zabrotes subfasciatus est associée à la présence d'arcéline, une nouvelle protéine des graines, découverte chez quelques populations de Phaseolus vulgaris. 5 types d'arcéline, hérités comme allèles codominants ont été décrits dans la littérature. Nous avons reprécisé les différentes populations contenant différents types d'arcéline et caractérisé leurs résistances à Z. subfasciatus. La population G 02771, correspondant à l'arcéline 5, présente la résistance la plus élevée par antibiose, suivie de G 12952 (arcéline 4), G 12882 (arcéline 1) et G 12866 (arcéline 2). Les populations contenant l'arcéline 3 présentent le moins de résistance à Z. subfasciatus.Un programme de croisements en retour associé à des tests sérologiques pour déceler la présence d'arcéline chez les descendants jeunes et des expériences répétées d'alimentation par les insectes vec BC2F₃ a été réalisé pour transférer la résistance de populations naturelles à des cultivars de haricots. Les lignées, provenant de croisements avec des populations sauvages avec de l'arcéline 1, ont été fortement résistantes à Z. subfasciatus. Les lignées contenant de l'arcéline 2 ont été considérées comme ayant une résistance intermédiaire. Les lignées avec arcélines 3 et 4 étaient sensibles. Les raisons de l'échec du transfert de la résistance élevée des parents contenant de l'arcéline 4, sont inconnues. On a constaté que la concentration de l'arcéline dans les lignées contenant cet allèle était très faible, tandis que la concentration en arcéline 1 restait remarquablement élevée. Les recherches sont poursuivies pour déterminer les raisons de l'absence de transfert de l'arcéline 4 chez les descendants contenant cet allèle. Quoi qu'il en soit, les caractéristiques agronomiques et les qualités des lignées résistantes (codées RAZ) ont été évaluées en vue d'une diffusion pour les programmes nationaux de recherche des pays de basses altitudes intertropicaux ou Zabrotes subfasciatus fait des dégâts importants.

     

Improved template preparation for PCR-based assays for detection of food-borne bacterial pathogens

Shigella flexneri, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were applied to FTA filters, and the filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 and 200 CFU for the gram-negative enterics and Listeria, respectively. Different numbers of S. flexneri cells were used in controlled contamination experiments with several different foods (produce, beef, and apple cider). Aliquots from concentrated food washes subsequently spotted onto FTA filters and assayed by PCR gave consistently positive results and detection limits similar to those observed with pure-culture dilutions. This universal method for PCR template preparation from bacterial cells is rapid and highly sensitive and reduces interference from food-associated inhibitors of PCR. In addition, its broad applicability eliminates the need for multiple methods for analysis of food matrices.     

Improving our understanding of demographic monitoring: avian breeding productivity in a tropical dry forest

The ratio of juvenile to adult birds in mist‐net samples is used to monitor avian productivity, but whether it is a “true” estimate of per capita productivity or an index proportional to productivity depends on whether capture probability is not age‐dependent (true estimate) or age difference in capture probability is consistent among years (index). Better understanding of the processes affecting age‐ and year‐specific capture probabilities is needed to advance the application of constant‐effort mist‐netting for monitoring and conservation, particularly in many tropical settings where capture rates are often low. We ranked members of the avian community by capture frequencies, determined if temporary emigration influenced the availability of birds to be captured, and assessed the distribution of birds relative to mist‐nets and the parity between capture‐based productivity estimates and number of fledglings in nest plots in a tropical dry forest in Puerto Rico in 2009 and 2010. Few captures characterized the community of 25 resident species and, when estimable, capture probabilities were low, particularly for juveniles (typically < 0.1). Negative trends in capture probability, temporary emigration, and the distribution of birds suggest that avoidance of mist‐nets influenced capture rates in our study. Increasing mist‐net coverage or moving mist‐nets between sampling periods could increase capture rates. The number of fledglings observed in nest plots (25 ha/plot) did not correlate well with capture‐derived estimates (20 ha/net stations), suggesting the presence of immigrants or failure to find all nests. Our results suggest that indices of breeding productivity from mist‐netting data may track temporal changes in productivity, but such data likely do not reflect “true” productivity in most cases unless age‐specific differences in capture probability are incorporated into estimates. Pilot studies should be conducted to evaluate capture rates and the spatial extent sampled by mist‐nets to improve sampling design and inferences before informing decisions.     

Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy

Mammalian Genome - Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of...     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

Protein digestibility of hydrolyzed hog hair meal for pigs

Thirty-six crossbred castrated male and female pigs, initially averaging 76.2 kg, were used in a nitrogen balance study to determine apparent digestible protein, apparent net protein utilization and apparent biological values of hydrolyzed hog hair (HHH) meal for pigs. The HHH meal contained 93.4% crude protein and 22.6 MJ per kg of gross energy. Digestibility of protein was linearly decreased (P < 0.01) as HHH meal was substituted for 25 and 50% of the maize and soya bean meal protein in a basal diet fortified with minerals and vitamins and containing 14% crude protein. There was a trend for apparent net protein utilization values to decrease and apparent biological values to increase as HHH meal was substituted. Assuming linearity, nitrogen utilization values (%) calculated by difference for HHH meal were: apparent digestible protein, 74.7 ± 1.6; apparent net protein utilization, 44.0 ± 3.4; apparent biological value, 57.2 ± 3.7.     

Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.     

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.