Christopher A. Bottoms

Genetic Properties of the Maize Nested Association Mapping Population

Codifying Maize Modifications Maize, one of our most important crop species, has been the target of genetic investigation and experimentation for more than 100 years. Crossing two inbred lines tends to result in “better” offspring, in a process known as heterosis. Attempts to map the genetic loci that control traits important for farming have been made, but few have been successful (see the Perspective by Mackay). Buckler et al. (p. 714) and McMullen et al. (p. 737) produced a genomic map of maize that relates recombination to genome structure. Even tremendous adaptations in very diverse species were produced by numerous, small additive steps. Differences in flowering time in maize among inbred lines were not caused by a few genes with large effects, but by the cumulative effects of numerous quantitative trait loci—each of which has only a small impact on the trait. Outcrossing vigor in maize is most likely due to retained variability in regions around the centromeres. Maize genetic diversity has been used to understand the molecular basis of phenotypic variation and to improve agricultural efficiency and sustainability. We crossed 25 diverse inbred maize lines to the B73 reference line, capturing a total of 136,000 recombination events. Variation for recombination frequencies was observed among families, influenced by local (cis) genetic variation. We identified evidence for numerous minor single-locus effects but little two-locus linkage disequilibrium or segregation distortion, which indicated a limited role for genes with large effects and epistatic interactions on fitness. We observed excess residual heterozygosity in pericentromeric regions, which suggested that selection in inbred lines has been less efficient in these regions because of reduced recombination frequency. This implies that pericentromeric regions may contribute disproportionally to heterosis.

A structurally conserved water molecule in Rossmann dinucleotide-binding domains.

A computational comparison of 102 high‐resolution (≤1.90 Å) enzyme‐dinucleotide (NAD, NADP, FAD) complexes was performed to investigate the role of solvent in dinucleotide recognition by Rossmann fold domains. The typical binding site contains about 9–12 water molecules, and about 30% of the hydrogen bonds between the protein and the dinucleotide are water mediated. Detailed inspection of the structures reveals a structurally conserved water molecule bridging dinucleotides with the well‐known glycine‐rich phosphate‐binding loop. This water molecule displays a conserved hydrogen‐bonding pattern. It forms hydrogen bonds to the dinucleotide pyrophosphate, two of the three conserved glycine residues of the phosphate‐binding loop, and a residue at the C‐terminus of strand four of the Rossmann fold. The conserved water molecule is also present in high‐resolution structures of apo enzymes. However, the water molecule is not present in structures displaying significant deviations from the classic Rossmann fold motif, such as having nonstandard topology, containing a very short phosphate‐binding loop, or having α‐helix “A” oriented perpendicular to the β‐sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide‐binding domain.

High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase

Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

Exploring structurally conserved solvent sites in protein families

Protein‐bound water molecules are important components of protein structure, and therefore, protein function and energetics. Although structural conservation of solvent has been studied in a few protein families, a lack of suitable computational tools has hindered more comprehensive analyses. Herein we present a semiautomated computational approach for identifying solvent sites that are conserved among proteins sharing a common three‐dimensional structure. This method is tested on six protein families: (1) monodomain cytochrome c, (2) fatty‐acid binding protein, (3) lactate/malate dehydrogenase, (4) parvalbumin, (5) phospholipase A2, and (6) serine protease. For each family, the method successfully identified previously known conserved solvent sites. Moreover, the method discovered 22 novel conserved solvent sites, some of which have higher degrees of conservation than the previously known sites. All six families studied had solvent sites with more than 90% conservation and these sites were invariably located in regions of the protein with very high sequence conservation. These results suggest that highly conserved solvent sites, by virtue of their proximity to conserved residues, should be considered as one of the defining three‐dimensional structural characteristics of protein families and folds. Proteins 2006.

Crystal structure of rat α-parvalbumin at 1.05 Å resolution

The crystal structure of rat α‐parvalbumin has been determined at 1.05 Å resolution, using synchrotron data collected at Advanced Photon Source beamline 19‐ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms, R = 0.132 and Rfree = 0.162. The average coordinate estimated standard deviations are 0.021 Å and 0.038 Å for backbone atoms and side‐chain atoms, respectively. Besides providing a more precise view of the α‐isoform than previously available, these data permit comparison with the 0.91 Å structure determined for pike β‐parvalbumin. Visualization of the anisotropic displacement parameters as thermal ellipsoids yields insight into the atomic motion within the Ca2+‐binding sites. The asymmetric unit includes three parvalbumin (PV) molecules. Interestingly, the EF site in one displays uncharacteristic flexibility. The ellipsoids for Asp‐92 are particularly large and non‐spherical, and the shape of the Ca2+ ellipsoid implies significant vibrational motion perpendicular to the plane defined by the four y and z ligands. The relative dearth of crystal‐packing interactions in this site suggests that the heightened flexibility may be the result of diminished intermolecular contacts. The implication is that, by impeding conformational mobility, crystal‐packing forces may cause serious overestimation of EF‐hand rigidity. The high quality of the data permitted 11 residues to be modeled in alternative side‐chain conformations, including the two core residues, Ile‐97 and Leu‐105. The discrete disorder observed for Ile‐97 may have functional ramifications, providing a mechanism for communicating binding status between the CD and EF binding loops and between the PV metal ion‐binding domain and the N‐terminal AB region.

Expanding Maize Genetic Resources with Predomestication Alleles: Maize–Teosinte Introgression Populations

Teosinte (Zea mays subsp. parviglumis H. H. Iltis & Doebley) has greater genetic diversity than maize inbreds and landraces (Z. mays subsp. mays). There are, however, limited genetic resources to efficiently evaluate and tap this diversity. To broaden resources for genetic diversity studies in maize, we developed and evaluated 928 near‐isogenic introgression lines (NILs) from 10 teosinte accessions in the B73 background. Joint linkage analysis of the 10 introgression populations identified several large‐effect quantitative trait loci (QTL) for days to anthesis (DTA), kernel row number (KRN), and 50‐kernel weight (Wt50k). Our results confirm prior reports of kernel domestication loci and identify previously uncharacterized QTL with a range of allelic effects enabling future research into the genetic basis of these traits. Additionally, we used a targeted set of NILs to validate the effects of a KRN QTL located on chromosome 2. These introgression populations offer novel tools for QTL discovery and validation as well as a platform for initiating fine mapping.

Computational Analysis of RNA-seq

Using High-Throughput DNA Sequencing (HTS) to examine gene expression is rapidly becoming a -viable choice and is typically referred to as RNA-seq. Often the depth and breadth of coverage of RNA-seq data can exceed what is achievable using microarrays. However, the strengths of RNA-seq are often its greatest weaknesses. Accurately and comprehensively mapping millions of relatively short reads to a reference genome sequence can require not only specialized software, but also more structured and automated procedures to manage, analyze, and visualize the data. Additionally, the computational hardware required to efficiently process and store the data can be a necessary and often-overlooked component of a research plan. We discuss several aspects of the computational analysis of RNA-seq, including file management and data quality control, analysis, and visualization. We provide a framework for a standard nomenclature -system that can facilitate automation and the ability to track data provenance. Finally, we provide a general workflow of the computational analysis of RNA-seq and a downloadable package of scripts to automate the processing.

Wanted: unique names for unique atom positions. PDB-wide analysis of diastereotopic atom names of small molecules containing diphosphate.

BackgroundBiological chemistry is very stereospecific. Nonetheless, the diastereotopic oxygen atoms of diphosphate-containing molecules in the Protein Data Bank (PDB) are often given names that do not uniquely distinguish them from each other due to the lack of standardization. This issue has largely not been addressed by the protein structure community.ResultsOf 472 diastereotopic atom pairs studied from the PDB, 118 were found to have names that are not uniquely assigned. Among the molecules identified with these inconsistencies were many cofactors of enzymatic processes such as mononucleotides (e.g. ADP, ATP, GTP), dinucleotide cofactors (e.g. FAD, NAD), and coenzyme A. There were no overall trends in naming conventions, though ligand-specific trends were prominent.ConclusionThe lack of standardized naming conventions for diastereotopic atoms of small molecules has left the ad hoc names assigned to many of these atoms non-unique, which may create problems in data-mining of the PDB. We suggest a naming convention to resolve this issue. The in-house software used in this study is available upon request.A version of the software used for the analyses described in this paper is available at our web site: http://digbio.missouri.edu/ddan/DDAN.htm.

IView: introgression library visualization and query tool

BackgroundAn introgression library is a family of near-isogenic lines in a common genetic background, each of which carries one or more genomic regions contributed by a donor genome. Near-isogenic lines are powerful genetic resources for the analysis of phenotypic variation and are important for map-base cloning genes underlying mutations and traits. With many thousands of distinct genotypes, querying introgression libraries for lines of interest is an issue.ResultsWe have created IView, a tool to graphically display and query near-isogenic line libraries for specific introgressions. This tool incorporates a web interface for displaying the location and extent of introgressions. Each genetic marker is associated with a position on a reference map. Users can search for introgressions using marker names, or chromosome number and map positions. This search results in a display of lines carrying an introgression at the specified position. Upon selecting one of the lines, color-coded introgressions on all chromosomes of the line are displayed graphically.The source code for IView can be downloaded from http://xrl.us/iview.ConclusionsIView will be useful for those wanting to make introgression data from their stock of germplasm searchable.