Resolution of a common RNA sequencing ambiguity by terminal deoxynucleotidyl transferase

One of the more common ambiguities which arise when using reverse transcriptase and dideoxynucleotide-chain termination to sequence RNA is a radioactive band of cDNA that extends over all four lanes on a sequencing gel. The adjacent sequences both above and below the band are not affected. Assuming then, that these ambiguities are caused by the termination of the DNA polymerase activity of reverse transcriptase for reasons other than the insertion of a dideoxynucleotide in the growing cDNA chain, terminal deoxynucleotidyl transferase should be able to continue to add deoxynucleotides to these products after the sequencing reaction is complete. It does, clearing the improperly terminated cDNA from these pileup sites, revealing the correct sequence. This technique can also be used to identify the template RNA's 5'-terminal base, although far more units of terminal deoxynucleotidyl transferase are required.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26058/1/0000132.pd

Identification of sequence changes in the cold-adapted, live attenuated influenzavaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the 13132, P131, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27049/1/0000039.pd

Sequence comparison of wild-type and cold-adapted B/Ann Arbor/1/66 influenza virus genes

Consensus sequences for both wt and ca B/Ann Arbor/1 /66 viral PB2, PB1, PA, NP, M, and NS genes were directly determined from vRNA using a combination of chemical and chain-termination sequencing methods. There were 105 sites of difference between the wt and ca sets of these six RNA genes. The differences resulted in 26 amino acid substitutions distributed over the six proteins. The sequence changes were compared to the sequences of other known influenza type B wt viruses to pinpoint those changes that were unique to the ca B/Ann Arbor/1/66 virus. Of the 26 amino acid differences, only 11 were unique to the cold-adapted virus. These unique sites were distributed among five of the six genes. The NS protein had no amino acid substitutions. The sequence changes are discussed in terms of their probable mode of origin and selection, and in terms of their importance to the cold-adapted, temperature-sensitive, and attenuation phenotypes of ca B/AA/1 /66 virus. The sequence and organization of the PB2 gene and predicted protein are also given. The PB2 gene was 2396 nucleotides long, and it encoded a predicted protein of 770 amino acids with a molecular weight of 88,035 Da for the wt virus and 88,072 Da for the ca virus. Both proteins were predominantly hydrophilic, and each had an overall charge of +24.5 at pH 7.0.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27362/1/0000387.pd

Nitrogen and Sulfur Fertilization in Soybean: Impact on Seed Yield and Quality

Over time, plant breeding efforts for improving soybean [Glycine max (L.) Merr.] yield was prioritized and effects on seed nutritional quality were overlooked, decreasing protein concentration. This research aims to explore the effect of nitrogen (N) and sulfur (S) fertilization on soybean seed yield, seed protein and sulfur amino acids concentration. In 2018, ten field trials were conducted across the main US soybean producing region. The treatments were fertilization at 1) planting (NSP); during 2) vegetative growth (NSV); and 3) reproductive growth (NSR) and 4) unfertilized (Control). Nitrogen fertilization was applied at the rate of 40 lb/a utilizing urea ammo­nium nitrate (UAN), and S at 9 lb/a via ammonium sulfate (AMS). A meta-analysis was performed to consider small variations among experimental designs. A summary of the effect sizes did not show effects for seed yield. However, fertilization at planting (NSP) increased seed protein by 1% more than the control across all sites. Overall, sulfur amino acid concentration increased by 1.5% relative to the control, but the most consistent benefit came from fertilization during the reproductive growth (NSR), increasing sulfur amino acids by 1.9%. Although N and S fertilization did not affect seed yields, applying N and S in different stages of the crop growth can increase protein concentration and improve protein composition, providing the opportunity to open new US soybean markets

Influenza in Migratory Birds and Evidence of Limited Intercontinental Virus Exchange

Migratory waterfowl of the world are the natural reservoirs of influenza viruses of all known subtypes. However, it is unknown whether these waterfowl perpetuate highly pathogenic (HP) H5 and H7 avian influenza viruses. Here we report influenza virus surveillance from 2001 to 2006 in wild ducks in Alberta, Canada, and in shorebirds and gulls at Delaware Bay (New Jersey), United States, and examine the frequency of exchange of influenza viruses between the Eurasian and American virus clades, or superfamilies. Influenza viruses belonging to each of the subtypes H1 through H13 and N1 through N9 were detected in these waterfowl, but H14 and H15 were not found. Viruses of the HP Asian H5N1 subtypes were not detected, and serologic studies in adult mallard ducks provided no evidence of their circulation. The recently described H16 subtype of influenza viruses was detected in American shorebirds and gulls but not in ducks. We also found an unusual cluster of H7N3 influenza viruses in shorebirds and gulls that was able to replicate well in chickens and kill chicken embryos. Genetic analysis of 6,767 avian influenza gene segments and 248 complete avian influenza viruses supported the notion that the exchange of entire influenza viruses between the Eurasian and American clades does not occur frequently. Overall, the available evidence does not support the perpetuation of HP H5N1 influenza in migratory birds and suggests that the introduction of HP Asian H5N1 to the Americas by migratory birds is likely to be a rare event

Incorporating lessons from high-input research into a low-margin year

Increased soybean commodity prices in recent years have generated interest in developing high-input systems to increase yield. However, little information exists about the effects of input-intensive, high-yield management on soybean yield and profitability, as well as interactions with basic agronomic practices

High-Input Management Systems Effect on Soybean Seed Yield, Yield Components, and Economic Break-Even Probabilities

Elevated soybean [Glycine max (L.) Merr.] prices have spurred interest in maximizing soybean seed yield and has led growers to increase the number of inputs in their production systems. However, little information exists about the effects of high-input management on soybean yield and profitability. The purpose of this study was to investigate the effects of individual inputs, as well as combinations of inputs marketed to protect or increase soybean seed yield, yield components, and economic break-even probabilities. Studies were established in nine states and three soybean growing regions (North, Central, and South) between 2012 and 2014. In each site-year both individual inputs and combination high-input (SOYA) management systems were tested. When averaged between 2012 and 2014, regional results showed no seed yield responses in the South region, but multiple inputs affected seed yield in the North region. In general, the combination SOYA inputs resulted in the greatest yield increases (up to 12%) compared to standard management, but Bayesian economic analysis indicated SOYA had low break-even probabilities. Foliar insecticide had the greatest break-even probabilities across all environments, although insect pressure was generally low across all site-years. Soybean producers in North region are likely to realize a greater response from increased inputs, but producers across all regions should carefully evaluate adding inputs to their soybean management systems and ensure that they continue to follow the principles of integrated pest management

Characterizing Genotype X Management Interactions on Soybean Seed Yield

Increased soybean [Glycine max (L.) Merr.] commodity prices in recent years have generated interest in high-input systems to increase yield. The objective of this study was to evaluate the effects of current, high-yielding cultivars under high- and low-input systems on soybean yield and yield components. Research trials were conducted at 19 locations spanning nine states from 2012 to 2014. At each location, six high-yielding cultivars were grown under three input systems: (i) standard practice (SP, current recommended practices), (ii) high-input treatment consisting of a seed treatment fungicide, insecticide, nematistat, inoculant, and lipo-chitooligosaccharide (LCO); soil-applied N fertilizer; foliar LCO, fertilizer, antioxidant, fungicide and insecticide (SOYA), and (iii) SOYA minus foliar fungicide (SOYA-FF). An individual site-year yield analysis found only 3 of 53 (5.7%) site-years examined had a significant cultivar × input system interaction, suggesting cultivar selection and input system decisions can remain independent. Across all site-years, the SOYA and SOYA-FF treatments yielded 231 (5.5%) and 147 kg ha–1 (3.5%) more than the SP, and input system differences were found among maturity groups. Yield component measurements (seeds m–2, seed mass, early-season and final plant stand, pods plant–1, and seeds pod–1) indicated positive yield responses were due to increased seeds m–2 and seed mass. While both high-input systems increased yield on average, grower return on investment (ROI) would be negative given today’s commodity prices. These results further support the use of integrated pest management principles for making input decisions instead of using prophylactic applications to maximize soybean yield and profitability

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections