The lack of an instructional definition of bioinformatics delays its effective integration into biology coursework. Using an iterative process, our team of biologists, a mathematician/computer scientist, and a bioinformatician together with an educational evaluation and assessment specialist, developed an instructional definition of the discipline: Bioinformatics is "an interdisciplinary field that is concerned with the development and application of algorithms that analyze biological data to investigate the structure and function of biological polymers and their relationships to living systems." The field is defined in terms of its two primary foundational disciplines, biology and computer science, and its interdisciplinary nature. At the same time, we also created a rubric for assessing open-ended responses to a prompt about what bioinformatics is and how it is used. The rubric has been shown to be reliable in successive rounds of testing using both common percent agreement (89.7%) and intraclass correlation coefficient (0.620) calculations. We offer the definition and rubric to life sciences instructors to help further integrate bioinformatics into biology instruction, as well as for fostering further educational research projects.

Birds, with their broad geographic ranges and close association with humans, have historically played an important role as carriers of human disease and as reservoirs for drug-resistant bacteria. Here, we examine scientific literature over a 15-year timespan to identify reported avian-bacterial associations and factors that may impact zoonotic disease emergence by classifying traits of bird species and their bacteria. We find that the majority of wild birds studied were migratory, in temperate habitats, and in the order Passeriformes. The highest diversity of bacteria was found on birds in natural habitats. The most frequently reported bacteria were Escherichia coli, Salmonella enterica, and Campylobacter jejuni. Of the bacteria species reported, 54% have shown pathogenicity toward humans. Percentage-wise, more pathogens were found in tropical (vs. temperate) habitats and natural (vs. suburban, urban, or agricultural) habitats. Yet, only 22% were tested for antibiotic resistance, and of those tested, 75% of bacteria species were resistant to at least one antibiotic. There were no significant patterns of antibiotic resistance in migratory versus non-migratory birds, temperate versus tropical areas, or different habitats. We discuss biases in detection and representation, and suggest a need for increased sampling in non-temperate zones and in a wider range of avian species.

Evolutionary theory is critical for a comprehensive understanding of biology, yet students often fail to grasp its underlying principles. This results partially from ineffective teaching; however, the use of interactive activities could alleviate this problem. In this guided investigation of evolutionary mechanisms, students use LEGO bricks to simulate how mutation, migration, genetic drift, and natural selection can affect the evolution of a population. This exercise was undertaken and assessed with college introductory biology students, but is also appropriate for advanced high school students.

Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5′ splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5′ exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

OBJECTIVES:Our goal was to determine the diversity and abundance of Staphylococcus bacteria on different components of a public transportation system in a mid-sized US city (Portland, Oregon) and to examine the level of drug resistance in these bacteria.METHODS:We collected 70 samples from 2 cm × 4 cm sections from seven different areas on buses and trains in Portland, USA, taking 10 samples from each area. We isolated a subset of 14 suspected Staphylococcus spp. colonies based on phenotype, and constructed a phylogeny from16S rRNA sequences to assist in identification. We used the Kirbye-Bauer disk diffusion method to determine resistance levels to six common antibiotics.RESULTS:We found a range of pathogenic Staphylococcus species. The mean bacterial colony counts were 97.1 on bus and train floors, 80.1 in cloth seats, 9.5 on handrails, 8.6 on seats and armrests at bus stops, 3.8 on the underside of seats, 2.2 on windows, and 1.8 on vinyl seats per 8 cm(2) sample area. These differences were significant (p < 0.001). Of the 14 isolates sequenced, 11 were staphylococci, and of these, five were resistant to penicillin and ampicillin, while only two displayed intermediate resistance to bacitracin. All 11 isolates were sensitive to trimethoprim-sulfamethoxazole, vancomycin, and tetracycline.CONCLUSIONS:We found six different strains of Staphylococcus, and while there were varying levels of drug resistance, we did not find extensive levels of multidrug-resistant bacteria, and no S. aureus was found. We found floors and cloth seats to be areas on buses and trains that showed particularly high levels of bacteria.

BACKGROUND:Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.RESULTS:The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.CONCLUSIONS:The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

Group II introns are self-splicing RNAs that are frequently assumed to be the ancestors of spliceosomal introns. They are widely distributed in bacteria and are also found in organelles of plants, fungi, and protists. In this study, we present a broadscale phylogenetic analysis of group II introns using sequence data from both the conserved RNA structure and the intron-encoded reverse transcriptase (RT). Two similar phylogenies are estimated for the RT open reading frame (ORF), based on either amino acid or nucleotide sequence, whereas one phylogeny is produced for the RNA. In making these estimates, we confronted nearly all the classic challenges to phylogenetic inference, including positional saturation, base composition heterogeneity, short internodes with low support, and sensitivity to taxon sampling. Although the major lineages are well-defined, robust resolution of topology is not possible between these lineages. The approximately unbiased (AU) and Shimodaira-Hasegawa topology tests indicated that the RT ORF and RNA ribozyme data sets are in significant conflict under a variety of models, revealing the possibility of imperfect coevolution between group II introns and their intron-encoded ORFs. The high level of sequence divergence, large timescale, and limited number of alignable characters in our study are representative of many RTs and group I introns, and our results suggest that phylogenetic analyses of any of these sequences could suffer from the same sources of error and instability identified in this study.

Retroelements are usually considered to be eukaryotic elements because of the large number and variety in eukaryotic genomes. By comparison, reverse transcriptases (RTs) are rare in bacteria, with only three characterized classes: retrons, group II introns and diversity-generating retroelements (DGRs). Here, we present the results of a bioinformatic survey that aims to define the landscape of RTs across eubacterial, archaeal and phage genomes. We identify and categorize 1021 RTs, of which the majority are group II introns (73%). Surprisingly, a plethora of novel RTs are found that do not belong to characterized classes. The RTs have 11 domain architectures and are classified into 20 groupings based on sequence similarity, phylogenetic analyses and open reading frame domain structures. Interestingly, group II introns are the only bacterial RTs to exhibit clear evidence for independent mobility, while five other groups have putative functions in defense against phage infection or promotion of phage infection. These examples suggest that additional beneficial functions will be discovered among uncharacterized RTs. The study lays the groundwork for experimental characterization of these highly diverse sequences and has implications for the evolution of retroelements.

Group II introns are a major class of ribozymes found in bacteria, mitochondria, and plastids. Many introns contain reverse transcriptase open reading frames (ORFs) that confer mobility to the introns and allow them to persist as selfish DNAs. Here, we report an updated compilation of group II introns in Eubacteria and Archaea comprising 234 introns. One new phylogenetic class is identified, as well as several specialized lineages. In addition, we undertake a detailed search for ORF-less group II introns in bacterial genomes in order to find undiscovered introns that either entirely lack an ORF or encode a novel ORF. Unlike organellar group II introns, we find only a handful of ORF-less introns in bacteria, suggesting that if a substantial number exist, they must be divergent from known introns. Together, these results highlight the retroelement character of bacterial group II introns, and suggest that their long-term survival is dependent upon retromobility.

Intron origin and evolution are of high interest, yet the rates of insertion and loss are unclear. To investigate their spread, we studied ribosomal (r)DNA introns from the closely related lichens Physcia aipolia and P. stellaris. Both taxa are replete with rDNA spliceosomal introns and autocatalytic group I introns, many of which show presence/absence polymorphism when screened with the PCR approach. This initially suggested that Physcia could be a model for studying intron retention and loss. However, during the course of a population-level analysis, we discovered widespread intron presence/absence heterogeneity within lichen thalli. To address this result, we sequenced multiple clones encoding nuclear rDNA and the single-copy elongation factor-1α (EF-1α) from individual thalli. These data showed extensive rDNA heterogeneity within individuals, rather than the presence of multiple fungi within a thallus. Our results suggest that considerable care must be taken when interpreting intron presence/absence in lichen rDNA, an observation that has general implications for the study of rDNA intron evolution.

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support Was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can Support the insertion and/or retention of group I introns. In contrast. we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene.

BACKGROUND:Group I introns have spread into over 90 different sites in nuclear ribosomal DNA (rDNA) with greater than 1700 introns reported in these genes. These ribozymes generally spread through endonuclease-mediated intron homing. Another putative pathway is reverse splicing whereby a free group I intron inserts into a homologous or heterologous RNA through complementary base-pairing between the intron and exon RNA. Reverse-transcription of the RNA followed by general recombination results in intron spread. Here we used phylogenetics to test for reverse splicing spread in a taxonomically broadly sampled data set of fungal group I introns including 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont Symbiotaphrina buchneri.RESULTS:Our analyses reveal a complex evolutionary history of the fungal introns with many cases of vertical inheritance (putatively for the 9 introns in S. buchneri) and intron lateral transfer. There are several examples in which introns, many of which are still present in S. buchneri, may have spread through reverse splicing into heterologous rDNA sites. If the S. buchneri introns are ancient as we postulate, then group I intron loss was widespread in fungal rDNA evolution.CONCLUSION:On the basis of these results, we suggest that the extensive distribution of fungal group I introns is at least partially explained by the reverse splicing movement of existing introns into ectopic rDNA sites.

There are four major classes of introns: self-splicing group I and group II introns, tRNA and/or archaeal introns and spliceosomal introns in nuclear pre-mRNA. Group I introns are widely distributed in protists, bacteria and bacteriophages. Group II introns are found in fungal and land plant mitochondria, algal plastids, bacteria and Archaea. Group II and spliceosomal introns share a common splicing pathway and might be related to each other. The tRNA and/or archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA, rRNA and mRNA. The mechanisms underlying the self-splicing and mobility of a few model group I introns are well understood. By contrast, the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified. The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion, splicing and spread that can be tested experimentally.

BACKGROUND:We have studied spliceosomal introns in the ribosomal (r)RNA of fungi to discover the forces that guide their insertion and fixation.RESULTS:Comparative analyses of flanking sequences at 49 different spliceosomal intron sites showed that the G - intron - G motif is the conserved flanking sequence at sites of intron insertion. Information analysis showed that these rRNA introns contain significant information in the flanking exons. Analysis of all rDNA introns in the three phylogenetic domains and two organelles showed that group I introns are usually located after the most conserved sites in rRNA, whereas spliceosomal introns occur at less conserved positions. The distribution of spliceosomal and group I introns in the primary structure of small and large subunit rRNAs was tested with simulations using the broken-stick model as the null hypothesis. This analysis suggested that the spliceosomal and group I intron distributions were not produced by a random process. Sequence upstream of rRNA spliceosomal introns was significantly enriched in G nucleotides. We speculate that these G-rich regions may function as exonic splicing enhancers that guide the spliceosome and facilitate splicing.CONCLUSIONS:Our results begin to define some of the rules that guide the distribution of rRNA spliceosomal introns and suggest that the exon context is of fundamental importance in intron fixation.

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis.

This paper identifies criteria seen as essential to feminist research. In light of these criteria, issues which have arisen during our current research on women and their experiences of midlife and menopause are discussed. Issues considered include the researchers' responsibilities to participants when exploring sensitive and highly personal issues relating to participants' life experiences, and less clear cut issues such as knowledge construction, power and control. In relation to the latter the balance of power in the research-participant relationship, and the role and responsibilities of the researcher in knowledge construction, are explored. Foucault's notions of knowledge construction and power and control and the feminist researcher's position, are considered in terms of rigour in feminist research and dissemination of research reports. Issues which are seen as problematic and worthy of further debate are: the relations between interviewer and interviewee; the intellectual (the researcher) as the bearer of universal values and as truth teller; and the level of critical activism possible in research studies of this nature.