Change in Biotypic Diversity of Russian Wheat Aphid (Hemiptera: Aphididae) Populations in the United States.

A key component of Russian wheat aphid, Diuraphis noxia (Kurdjumov), management has been through planting resistant wheat cultivars. A new biotype, RWA2, appeared in 2003 which caused widespread damage to wheat cultivars containing the Dn4 gene. Biotypic diversity in Russian wheat aphid populations has not been addressed since 2005 when RWA2 dominated the biotype complex. Our objectives were to determine the biotypic diversity in the Central Great Plains and Colorado Plateau at regional (2010, 2011, 2013) and local (2012) levels and detect the presence of new Russian wheat aphid biotypes. Regional and within-field aphid collections were screened against Russian wheat aphid-resistant wheat genotypes containing genes Dn3, Dn4, Dn6, Dn7, Dn9, CI2401; and resistant barley STARS 9301B. In 2010, all aphid collections from Texas were avirulent to the Dn4 resistance gene in wheat. Regional results revealed Dn4 avirulent RWA6 was widespread (55-84%) in populations infesting wheat in both regions. Biotypes RWA1, 2, and 3/7 were equally represented with percentages<20% each while RWA8 was rarely detected. Combining percentages of RWA1, 6, and 8 across regions to estimate avirulence to Dn4 gene revealed high percentages for both 2011 (64-80%) and 2013 (69-90%). In contrast, the biotype structure at the local level differed where biotype percentages varied up to ≥2-fold between fields. No new biotypes were detected; therefore, Dn7, CI2401, and STARS9301B remained resistant to all known Russian wheat aphid biotypes. This study documents a shift to Dn4 avirulent biotypes and serves as a valuable baseline for biotypic diversity in Russian wheat aphid populations prior to the deployment of new Russian wheat aphid-resistant wheat cultivars.

Computational methods for protein sequence analysis.

This unit is presented as a guide to addressing the issue of what to do with a protein sequence once it is obtained. A theoretical background for protein sequence analysis is provided first, followed by a discussion of matrix methods for sequence comparison (Matrix Methods for Sequence Comparison: Dot Plots). Sequence similarity searching is then presented, including the BLAST and FASTA databases. Other aspects of protein sequence analysis covered here are alignment methods, scoring matrices, multiple alignments, cluster methods and trees, and identification of functional sites.

Sensitivity of biological models to errors in parameter estimates.

Since A. M. Turing's paper proposing a mathematical basis for pattern formation in developing organisms many mathematical approaches have been proposed to model biological phenomenon. Continued laboratory study and recent improvements in measurement capabilities have provided an immense quantity of raw gene expression data. The level of data now available demands the development of well-characterized and tested computational tools. Thus, we have examined one mathematical model's sensitivity to errors in estimating its' parameters. Errors in parameter estimation can arise from noise in the laboratory measurements and recasting of laboratory data. We elected to examine the rule-based mathematical model of Mjolsness et al for its' sensitivity to errors in estimated parameters. We have used the technique of sensitivity equations as generally applied in nonlinear systems analysis.

The identification of markers segregating with resistance to Schistosoma mansoni infection in the snail Biomphalaria glabrata.

Both snail and parasite genes determine the susceptibility of the snail Biomphalaria glabrata to infection with the trematode Schistosoma mansoni. To identify molecular markers associated with resistance to the parasite in the snail host, we performed genetic crosses between parasite-resistant and -susceptible isogenic snails. Because resistance to infection in adult snails is controlled by a single locus, DNA samples from individual F2 and F1 backcross progeny, segregating for either the resistant or susceptible phenotypes, were pooled (bulked segregant). Genotypes for both parents were determined with 205 arbitrary decamer primers by random amplified polymorphic DNA-PCR. Of the 205 primers, 144 were informative, and the relative allele frequencies between the pools for these primers were determined. Two primers, OPM-04 and OPZ-11, produced fragments in the resistant parent of one cross that were inherited in a dominant fashion in the resistant F2 and backcross-bulked segregant progeny. Subsequent typing of DNA samples of individual progeny snails showed that the 1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced by primer OPZ-11 segregated in the same dominant fashion with the resistant phenotype. Sequence analysis of the 1.2-kb marker showed that it corresponds to a repetitive sequence in the snail genome with no homology to existing DNA sequences in the public databases. Analysis of the 1. 0-kb marker showed that it also corresponds to a repetitive sequence in the B. glabrata genome that contains an imperfect ORF, with homology to retrovirus-related group-specific antigens (gag) polyprotein.

Cluster analysis and data visualization of large-scale gene expression data.

The discovery of any new gene requires an analysis of the expression context for that gene. Now that the cDNA and genomic sequencing projects are progressing at such a rapid rate, high throughput gene expression screening approaches are beginning to appear to take advantage of that data. We present a strategy for the analysis for large-scale quantitative gene expression measurement data from time course experiments. Our approach takes advantage of cluster analysis and graphical visualization methods to reveal correlated patterns of gene expression from time series data. The coherence of these patterns suggests an order that conforms to a notion of shared pathways and control processes that can be experimentally verified.

Large-scale temporal gene expression mapping of central nervous system development.

We used reverse transcription-coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. The data provide a temporal gene expression "fingerprint" of spinal cord development based on major families of inter- and intracellular signaling genes. By using distance matrices for the pair-wise comparison of these 112 temporal gene expression patterns as the basis for a cluster analysis, we found five basic "waves" of expression that characterize distinct phases of development. The results suggest functional relationships among the genes fluctuating in parallel. We found that genes belonging to distinct functional classes and gene families clearly map to particular expression profiles. The concepts and data analysis discussed herein may be useful in objectively identifying coherent patterns and sequences of events in the complex genetic signaling network of development. Functional genomics approaches such as this may have applications in the elucidation of complex developmental and degenerative disorders.

Effect of prosthetic superstructure accuracy on the osteointegrated implant bone interface.

OBJECTIVE: This study evaluated the biologic result of forces induced by a misfitting prosthetic superstructure on implants placed in a New Zealand white rabbit tibia model. STUDY DESIGN: Nine rabbits had two dental implants placed in both right and left proximal tibias. After 6 weeks, one animal was sacrificed for baseline integration data, and the remaining animals had fitting or misfitting prosthetic superstructures attached to the implants for 12 weeks. Implants were evaluated clinically, radiographically, and histomorphometrically at the scanning electron microscopic level. RESULTS: No clinical, radiographic, or histomorphometric evidence exists of integration failure with implants subjected to superstructure strain, although bone remodeling is noted. CONCLUSIONS: Given the limitations of sample size, animal model used, duration of prosthetic superstructure attachment, and loading confounders possible, the study of prosthetic framework misfit must be evaluated with another animal model, such as an intraoral primate model, to determine the relationship between clinical performance and histologic findings.

Intraphagosomal oxygen in stimulated macrophages.

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO2 in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (d-Cat1)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 +/- 3.4 microM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen.

Nucleic acid sequences coding for internal antisense peptides: are there implications for protein folding and evolution?

We have asked whether coding segments of nucleic acids generate amino acid sequences which have an antisense relationship to other amino acid sequences in the same chain (i.e. 'Internal Antisense'), and if so, could the internal antisense content be related to the structure of the encoded protein? Computer searches were conducted with the coding sequences for 132 proteins. The result for each search of a specific sequence was compared to the mean result obtained from 1000 randomly assembled nucleic acid chains whose length and base composition were identical to that of the native sequences. The study was conducted in all three reading frames. The normal reading frame (frame one) was found to be contain lower amounts of internal antisense than the randomly assembled chains, whereas the frame two results were much higher. The internal antisense content in frame three was not significantly different from that in the random chains. The amount of internal antisense in frames two and three was correlated with the GC content at the center position of the codons in that frame, but this correlation was absent in frame one. No correlation with chain length was found. Qualitatively similar results were obtained when the random model was limited to retain the same purine/pyrimidine ratio as the native chains at each position in the codons, but in this case the internal antisense in frame three was also significantly greater than the computer-generated sequences. The results suggest that the internal antisense content in the correct reading frame has a qualitatively different origin from that in the other two frames. The high amount in frames two and three is apparently an artifact resulting from the asymmetric distribution of G and C in the codons, while the low amount in frame one may suggest evolutionary selection against internal antisense. Thus, the results do not support a relationship between internal antisense and protein structure.

Comparison of the resistance of miniplates and microplates to various in vitro forces.

A comparison of the Luhr Mini System (Howmedica, Inc, Rutherford, NJ) and the Luhr Micro System (Howmedica, Inc) was undertaken to determine resistance to various forces using a biomechanical model. Miniplates and microplates were first tested to determine their resistance to forces of displacement on flat bend, edge bend, tension, and compression generated by a materials testing system machine. Then, miniplates and microplates were attached to fresh porcine ribs, fixed to a custom-made jig, and subjected to the same forces of displacement. The load was applied to the bone plate to permanent deformation in all tests. The mini and microplate systems resisted 14.50 and 1.14 kg, respectively, on edgewise bending, 2.65 and 1.10 kg, respectively, on flat bending, 92.03 and 16.44 kg, respectively, on tension, and 127.9 and 27.02 kg, respectively, on compression. The mini and microsystem biomechanical model resisted 1.89 and 0.94 kg, respectively, on edgewise bending, 5.20 and 0.85 kg, respectively, on flat bending, 37.60 and 15.72 kg, respectively, on tension, and 53.55 and 16.0 kg, respectively, on compression. The results suggest that the Luhr Mini Fixation System provides a significant amount of resistance to tensile and compressive forces, but is weakest when large forces are applied at 90 degrees to the flat portion of the plate. The system showed decreased force resistance in the biomechanical model except on flat bending. The Luhr Micro Fixation System has significantly less resistance to deformation, but shows no decrease in ability to resist forces of displacement in the biomechanical model.

Reliability of a facebow transfer procedure.

The three-dimensional orientation of a maxillary cast mounting from a simulated-kinematic facebow transfer was evaluated in multiple trials among three operators on a single subject. The anterior and posterior anatomic facial reference points were marked on the subject. Each operator performed a separate series of trials to reset the anterior facebow component, the two posterior facebow components, and a control series with no resetting of any facebow components relative to the subject. The x, y, and z coordinates of three reference points on the maxillary cast were determined with a machinist microscope relative to a fixed reference after each facebow transfer. A range of differences between mountings of the maxillary cast were found between trials with all three methods used. These mounting errors were due to setting of the instrument and would be expected in routine clinical use of this instrument.

Inhibition of SV40 gene expression by microinjected small antisense RNA and DNA molecules.

We tested the impact of antisense RNA and DNA molecules on SV40 gene expression by microinjection into TC7 cells. Short antisense stretches, complementary to either hairpin or loop structures on the T antigen mRNA, inhibited T antigen synthesis. In contrast, full-length antisense RNA and DNA molecules did not effect T antigen synthesis.

Mechanism of mRNA binding to bovine mitochondrial ribosomes.

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.

Accumulation and decay of DG42 gene products follow a gradient pattern during Xenopus embryogenesis.

The DG42 gene is expressed during a short window during embryogenesis of Xenopus laevis. The mRNA for this gene can be first detected just after midblastula, peaks at late gastrula, and decays by the end of neurulation. The sequence of the DG42 cDNA and genomic DNA predicts a 70,000-Da protein that is not related to any other known protein. Antibodies prepared against portions of the DG42 open reading frame that had been expressed in bacteria detected a 70,000-Da protein in the embryo with a temporal course of appearance and decay that follows that of the RNA by several hours. Localization of the mRNA in dissected embryos and immunohistochemical detection of the protein showed that DG42 expression moves as a wave or gradient through the embryo. The RNA is first detected in the animal region of the blastula, and by early gastrula is found everywhere except in the outer layer of the dorsal blastopore lip. By midgastrula DG42 protein is present in the inner ectodermal layer and the endoderm; it disappears from dorsal ectoderm as the neural plate is induced and later decays in a dorsoventral direction. The last remnants of DG42 protein are seen in ventral regions of the gut at the tailbud stage.

Cloning, nucleotide sequence and expression of the pyrBI operon of Salmonella typhimurium LT2.

The pyrB-pyrI region of the Salmonella typhimurium LT2 chromosome has been cloned and sequenced. The two genes were found to constitute an operon, with pyrI being the distal gene and separated from pyrB by a 15-bp intercistronic region. Sequence analysis revealed the presence of two potential promoters; transcription initiated from the promoter proximal to pyrB would produce a transcript which could direct the synthesis of a 33-amino-acid leader peptide. The leader sequence possesses the requisite features of a rho-independent transcriptional terminator (attenuator) which is positioned 22 bp upstream from the pyrB structural gene. A regulatory mutation imparting a 30-fold elevated expression of pyrBI was identified as a two-base-pair deletion in the track of A X T base pairs located on the 3' side of the region of dyad symmetry of the attenuator. The leader sequence also has an additional region of dyad symmetry (putative transcriptional pause site) located 33 nucleotides upstream from the start of the proposed attenuator. The intervening sequence between the putative pause site and the indicated attenuator is characterized by encoding a high content of uracil residues in the transcript. Construction and analysis of transcriptional and translational fusions provided evidence that the leader region has the necessary features to mediate polypeptide synthesis in vivo, the removal of the region corresponding to the pause site and attenuator results in constitutive expression and the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from Escherichia coli K-12 but notable differences are observed.