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.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

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.     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

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...     

Verminous vasculitis, pneumonia and pulmonary infarction in a cynomolgus monkey after treatment with ivermectin

An apparently healthy cynomolgus monkey (Macaca fascicularis) died 2 hours after routine inhalation anesthesia and implantation of a femoral catheter. Gross necropsy findings included patchy raised areas of severe pulmonary hemorrhage and consolidation. Filarioid nematodes (Edesonfilaria malayensis) were located in pulmonary blood vessels and in numerous 0.1-2 cm fibrous cysts on the pleural surfaces of the lungs, pericardium, diaphragm, retroperitoneum, and in the urinary bladder wall. Microscopic lesions included verminous vasculitis, pulmonary infarcts and pneumonia. Many of the nematodes were more necrotic than the surrounding host tissue. During quarantine, 17 days before surgery, the monkey had been given a single dose of ivermectin (200 micrograms/Kg, intramuscular) as an anthelminthic for gastrointestinal nematodes. It is postulated that many of the filarioid nematodes were killed by this treatment. These parasitic emboli caused pulmonary infarction and the severe inflammatory reaction. The resulting pulmonary disease compromised pulmonary function and contributed to death after anesthesia. This complication should be considered if monkeys possibly harboring filarioid nematodes are treated with ivermectin.     

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.