Gene families: the taxonomy of protein paralogs and chimeras.

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. Duplications can be tandem or dispersed and can involve entire coding regions or modules that correspond to folded protein domains. As a result, gene products may acquire new specificities, altered recognition properties, or modified functions. Extreme proliferation of some families within an organism, perhaps at the expense of other families, may correspond to functional innovations during evolution. The underlying processes are still at work, and the large fraction of human and other genomes consisting of transposable elements may be a manifestation of the evolutionary benefits of genomic flexibility.

Targeted screening for induced mutations.

With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.

Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene.

Databases of expressed sequence tags (EST) can be used to screen rapidly for potential polymorphisms in candidate proteins. As part of this study, we screened the gene for the enzyme thymidylate synthase (TS). TS is important physiologically because it is essential for the synthesis of deoxythymidylate, a nucleotide required for DNA synthesis and repair. TS is also a major target for cancer chemotherapeutic drugs, especially the widely used 5-fluorouracil. Using sequence alignment of ESTs, we identified a candidate 6-bp variation at bp 1494 in the 3'-untranslated region of the TS mRNA. This sequence variation occurred in 21 of 34 aligned ESTs at this location, including ESTs from various tissue sources. The presence of this polymorphism was confirmed in a Caucasian population (n = 95) by polymerase chain restriction amplification/RFLP analysis. The allele frequency of the 6-bp deletion was found to be 0.29 (wildtype +6 bp/+6 bp, 48%; +6 bp/-6 bp, 44%; -6 bp/-6 bp, 7%). Although the function of this polymorphism has not yet been investigated, the 3'-untranslated region of a gene can play a role in mRNA stability and translation. This study illustrates an approach to polymorphism discovery in candidate enzymes of physiological interest by searches of publicly available sequence data, a rapid and inexpensive method. The potential functional relevance of the common 6-bp deletion in the TS gene needs to be investigated, because this enzyme is plausibly of major importance not only in cancer treatment but also in cancer prevention.

Growth factor regulation of bovine satellite cell growth in vitro.

This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on the proliferation and differentiation of primary bovine satellite cells (BSC) in vitro. Individually, these three factors had the following effects on satellite cells: bFGF stimulated proliferation (P less than .01) but inhibited differentiation (P less than .05); IGF-I had no effect on proliferation but stimulated differentiation (P less than .01); and TGF-beta inhibited both proliferation and differentiation (P less than .01). When combined, the following effects were observed: maximum stimulation of proliferation (P less than .01) occurred in the presence of bFGF and IGF-I and differentiation was not stimulated; TGF-beta and bFGF continued to inhibit differentiation (P less than .01), but in the presence of bFGF, TGF-beta stimulated proliferation (P less than .01). No stimulation was observed in the presence of TGF-beta and IGF-I. Bovine satellite cells respond to these three growth factors that have been shown to regulate the activity of other myogenic cells, and in most instances, the responses among cells from various species are similar. These experiments indicate that the interactions of growth factors may be critical in regulating bovine satellite cell activity.

Technical note: equine skeletal preservation techniques to enhance teaching effectiveness.

When the decision was made to euthanatize an acutely laminitic Thoroughbred broodmare, graduate students from the Department of Animal Sciences and Industry reconstructed the skeleton for use as a teaching tool. The reproductive and gastrointestinal tracts were removed and preserved in formalin. The hide, muscle, tendons, ligaments, and organs were removed, and the bones were boiled in water for > or = 48 h to remove all remaining tissue. After boiling, the bones were soaked in gasoline to remove fat from the marrow cavities and then soaked in a bleach/detergent mixture as a final cleaning step. The bones were allowed to dry for several weeks, then a semi-gloss clear lacquer was applied to aid in preservation. The bones were connected with 17-gauge wire and supported by two 1.91-cm galvanized steel rods on a mobile platform. The vertebral column was aligned on flexible copper tube with a 1.27-cm diameter. Additional support was provided for the head and neck by aluminum and steel rods extending from the front support. The final product is a complete, mobile skeleton that will be used as a teaching aid in equine classes. The skeleton serves a function for all levels of the cognitive learning domain. Examples of applications include memorization, identification, and location of bones; use in case studies for synthesis and demonstration of brainstorming efforts; and evaluation of joint ailments for more advanced levels of learning.

Biodegradation of C5+ hydrocarbons by a mixed bacterial consortium from a C(5+)-contaminated site.

C5+, a mixture of benzene, toluene, xylene, styrene, dicyclopentadiene (DCPD), naphthalene and other compounds, is a byproduct of polyethylene production and has been introduced into the environment via accidental release. The degradation of C5+ was studied using a defined consortium of 11 distinct bacterial strains isolated from C(5+)-contaminated soil. Vigorous growth of individual strains on C5+ was no prediction of dominance in the consortium, when the latter was grown under the same conditions. The defined consortium was able to degrade benzene, toluene, styrene and naphthalene, and to codegrade m-xylene in the presence of toluene or naphthalene. It was unable to degrade DCPD, which was inhibitory when degradation of pairs of C5+ components was examined. The complete C5+ mixture appeared to be the best substrate for the consortium.

Nutrient stimulation of sulfolane biodegradation in a contaminated soil from a sour natural gas plant and in a pristine soil.

Natural gas in western Canada can contain up to 35% H2S. The Sulfinol process for sour gas treatment makes use of sulfolane and an amine to remove H2S and other sour components from natural gas. Sulfolane has leached into groundwaters at sour gas treatment plant sites, and poses a risk for off-site contamination. Sulfolane biodegradation was monitored in shake-flask cultures and air-sparged microcosms inoculated with uncontaminated topsoil or with sulfolane contaminated soil obtained near a Sulfinol process building at a sour gas treatment facility in western Canada. For both soils, supplementation with a source of fixed nitrogen stimulated sulfolane biodegradation. Topsoil cultures and microcosms were only slightly affected by the addition of phosphate. Contaminated soil microcosms and cultures were stimulated by phosphate addition, but not to the same degree as by the addition of nitrogen. For these cultures and microcosms, amendment with both fixed nitrogen and phosphate produced an additive effect. It was possible to predict the nutrient requirements of air-sparged microcosms inoculated with each soil type using shake-flask cultures. Shake-flask cultures require less time and effort and fewer materials than the more complex air-sparged soil microcosms, and will be useful for large-scale experiments to predict the nutrient supplements required for bioremediation of sulfolane-contaminated sites.

Nutrition-associated problems facing elite level three-day eventing horses.

REASONS FOR PERFORMING STUDY: The main goal of feeding elite 3-day event horses is to deliver nutrients in optimal amounts to allow the horse to maximize its health and performance. However, improper nutritional management and/or physiological stressors related to intense training and competition may increase the risk of nutrition-associated disorders in these horses. An understanding of the nutrition-associated problems contributing to poor performance is critical to the health and welfare of the horse. OBJECTIVES: To characterize the nutrition-associated problems affecting top level 3-day event horses during 2008. METHODS: Contact information for riders competing in the 2 highest levels of 3-day eventing in 2008 was obtained from the United States Eventing Association. A survey containing 10 questions pertaining to participant demographics and nutrition-associated problems experienced by their horses was mailed and e-mailed to the 81 individuals fitting our criteria of living in USA and Canada. Data was collected in April and May 2009. RESULTS: Twenty-nine of 81 riders completed the survey (35.8%). Respondents rode a total of 45 horses in top level 3-day events in 2008. The top 5 nutrition-associated problems that horses faced at a significantly higher level than the other problems (P < 0.0001) were gastric ulcers (42.2%), joint problems (37.7%), decreased appetite (31.1%), weight loss (31.1%) and hyperexcitability (22.2%). There was no significant difference in frequency of problems between home and competition (P = 0.22). CONCLUSIONS: Horses competing at a high level of 3-day eventing in 2008 were at risk of reduced performance given the significant rate of gastric ulcers, decreased appetite and weight loss. Research addressing specific causes of and/or feeding management changes that would reduce the incidence of these problems in these horses is needed to ensure optimal health and performance.

Development and usage of eXtension's HorseQuest: an online resource.

eXtension (pronounced e-extension) is an online resource transforming how faculty can collaborate and deliver equine education. As the first Community of Practice launched from eXtension, HorseQuest (HQ) offers free, interactive, peer-reviewed, online resources on a variety of equine-related topics at http://www.extension.org. This group has adapted traditional educational content to the online environment to maximize search engine optimization, to be more discoverable and relevant in the online world. This means that HQ resources are consistently being found on the first page of search results. Also, by researching key words searched by Internet users, HQ has guided new content direction and determined potential webcast topics based on relevance and frequency of those searches. In addition to establishing good search engine optimization, HQ has been utilizing the viral networking aspect of YouTube by uploading clips of existing equine educational videos to YouTube. HorseQuest content appears in mainstream media, is passed on by the user, and helps HQ effectively reach their community of interest (horse enthusiasts). HorseQuest partners with My Horse University to produce webcasts that combine concise knowledge exchange via a scripted presentation with viewer chat and incoming questions. HorseQuest has produced and published content including 12 learning modules, 8 webchats, 21 webcasts, and 572 videos segments. After the official public launch, there was a steady increase in average number of visits/mo and average page views/mo over the 26-mo period. These regressions show a statistically significant increase in visits (P < 0.001) of approximately 450 visits per month and a significant increase in page views (P = 0.004) of about 373 page views per month. HorseQuest is a resource for several state 4-H advancement and competition programs and will continue to be incorporated into traditional extension programs, while reaching and affecting global audiences.

The Spo0A protein of Bacillus subtilis inhibits transcription of the abrB gene without preventing binding of the polymerase to the promoter.

Repression of transcription of the abrB gene is essential to expression of many of the postexponential genes in Bacillus. The repression is due to the activity of the response regulator protein Spo0A. We have used in vitro transcription and DNase I and hydroxyl radical footprinting to explore the mechanism of transcription inhibition. Spo0A binds to specific DNA sequences (0A boxes), and two such boxes are found downstream of the tandem promoters for the abrB gene. The data indicate that both RNA polymerase and Spo0A bind simultaneously to a DNA fragment containing the promoters and the 0A boxes. The Spo0A prevents the polymerase from inducing DNA strand denaturation at the promoter for the abrB gene.

RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human.

We have cloned a human cDNA that is related to the RNA polymerase I transcription factor Rrn3 of Saccharomyces cerevisiae. The recombinant human protein displays both sequence similarity and immunological crossreactivity to yeast Rrn3 and is capable of rescuing a yeast strain carrying a disruption of the RRN3 gene in vivo. Point mutation of an amino acid that is conserved between the yeast and human proteins compromises the function of each factor, confirming that the observed sequence similarity is functionally significant. Rrn3 is the first RNA polymerase I-specific transcription factor shown to be functionally conserved between yeast and mammals, suggesting that at least one mechanism that regulates ribosomal RNA synthesis is conserved among eukaryotes.

Sulfolane degradation by mixed cultures and a bacterial isolate identified as a Variovorax sp.

Sulfolane (tetrahydrothiophene-1,1-dioxide) is used in the Sulfinol process for natural gas sweetening. At many sour-gas processing plants spills, landfills and leakage from unlined surface storage ponds have contaminated groundwaters with sulfolane. Due to its high water solubility and mobility in aquifers, sulfolane poses a risk for off-site contamination. This study investigated the aerobic biodegradation of sulfolane by two mixed microbial enrichment cultures and by three bacterial isolates. Sulfolane served as the sole C, S and energy source for these cultures. In the two mixed cultures, 60% and 80% of the sulfolane C was recovered as CO2, whereas in cultures of the three isolates only 40-42% of the substrate C was recovered as CO,. In the mixed cultures, 81% and 97% of the sulfolane S was converted to sulfate, and in the pure isolates, 55-90% of the substrate S was converted to sulfate. Thus, the mixed cultures were capable of greater mineralization than the pure isolates. One isolate, strain WP1, was identified using a combination of 16S rRNA gene sequencing, physiological traits and cell morphology. WP1 was determined to be most similar to Varioivorax paradoxus.

Mutants of Agrobacterium VirA that activate vir gene expression in the absence of the inducer acetosyringone.

In the presence of inducer molecules produced by wounded plants, the VirA/VirG two-component positive regulatory system of Agrobacterium tumefaciens initiates transcription of virulence genes required for crown gall tumor formation. Exactly how this system enables the bacterium to respond to an environmental signal is not known, but phosphorylation of VirA and VirG plays a role. To analyze further the function of VirA, we chemically mutagenized the virA gene. Two mutants that activate vir transcription without the plant inducer acetosyringone were found; these mutants alter VirA function by distinct mechanisms. One mutant functions entirely independently of acetosyringone, whereas the activity of the second mutant is enhanced by acetosyringone. Both mutants function best at acid pH, but respond differently to specific monosaccharides that stimulate induction by wild-type VirA. Both mutant phenotypes are dominant over wild-type VirA, and both need the conserved histidine at the autophosphorylation site for strong inducer-independent vir transcription.

Composition of soil microbial communities enriched on a mixture of aromatic hydrocarbons.

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp. before convergence on a composition dominated by Alcaligenes spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.

Increased coverage of protein families with the blocks database servers.

The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBI's IMPALA program and 3D display of blocks on PDB structures.

XDOM, a graphical tool to analyse domain arrangements in any set of protein sequences.

MOTIVATION: To extract the maximum possible information from a set of protein sequences, its modular organization must be known and clearly displayed. This is important both for structural and functional analysis. RESULTS: This paper presents an algorithm and a graphical interface called XDOM which performs a systematic analysis of the modular organization of any set of protein sequences. The algorithm is an automatic method to identify putative domains from sequence comparisons. The graphical tool displays the proteins as a set of linked boxes, corresponding to its domains. The method has been tested on a family of bacterial proteins and on whole genomes. It is currently applied to the complete SWISS-PROT database to build the PRODOM database. AVAILABILITY: XDOM is available free of charge by anonymous ftp:¿¿ftp://ftp.toulouse.inra.fr/pub/xdom¿ ¿. The ProDom database can be consulted at ¿¿http://protein.toulouse.inra.fr/prodom.html¿¿.