Characterization of Gfat1 (zeppelin) and Gfat2, Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in Drosophila melanogaster.

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.

Effect of personal response systems on student perception and academic performance in courses in a health sciences curriculum.

To increase student engagement, active participation, and performance, personal response systems (clickers) were incorporated into six lecture-based sections of four required courses within the Health Sciences Department major curriculum: freshman-level Anatomy and Physiology I and II, junior-level Exercise Physiology, and senior-level Human Pathophysiology. Clickers were used to gather anonymous student responses to questions posed within the class period after individual thought and peer discussion. Students (n = 293, 88% of students completing the courses) completed a perceptual survey on clicker effectiveness inserted into the Student Assessment of Learning Gains online instrument. Across courses and years, students uniformly rated several dimensions of clicker use as providing good to great gain in engaging them in active learning, increasing participation and involvement during class, maintaining attention, applying material immediately, providing feedback concerning their understanding, and offering an anonymous format for participation. Within these four sections, quiz grades were compared between clicker and nonclicker years. Significant increases in pre- and posttest scores were seen in Exercise Physiology in clicker years and on some, but not all material, in Anatomy and Physiology I and II based on content quizzes. Human Pathophysiology results were unexpected, with higher quiz scores in the nonclicker year. The results support the hypothesis of increased engagement with clicker use. The hypothesis of increased student performance was not consistently supported. Increased performance was seen in Exercise Physiology. In Anatomy and Physiology I and II, performance improved on some content quizzes. In Human Pathophysiology, performance did not improve with clickers.

A multiyear approach to student-driven investigations in exercise physiology.

Many undergraduate institutions offer individual research opportunities for upper-level students in independent study courses and summer undergraduate research programs. These are necessarily limited to a small number of students. Greater numbers of students can benefit from incorporating student-directed investigative experiences into laboratories in standard courses. In human performance investigations, any single course may not offer sufficient numbers of subjects to adequately test hypotheses comparing population groups or to examine longitudinal trends. In this exercise physiology course, exercise testing was conducted in three areas: 1) techniques of body composition analysis, 2) field tests for the estimation of maximal oxygen consumption, and 3) maximal anaerobic and aerobic power. All students enrolled over a 10-yr period participated as subjects and as testers. Working in small research groups, students added their results to those from previous years, generated a variety of hypotheses (correlations between tests, subgroup differences, etc.), and tested them statistically using the complete data set of 217 subjects. They then engaged in collaborative writing and peer review to prepare formal papers on their results. The multiyear approach allowed students to situate their work within and contribute to the accumulation of a large database and to practice essential scientific skills of hypothesis formation, data collection and analysis, collaborative work, and scientific communication. In addition, due to the larger number of subjects available to analyze, students observed statistically significant differences between test groups in the multiyear database that they were unable to demonstrate when conducting analysis on a single course. Finally, the large number of subjects and statistical power offered by the use of the database provides distinct pedagogical advantages.

Genetic and Molecular Analysis of Essential Genes in Centromeric Heterochromatin of the Left Arm of Chromosome 3 in Drosophila melanogaster.

A large portion of the Drosophila melanogaster genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila, and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.

Heterochromatic genes in Drosophila: a comparative analysis of two genes.

Centromeric heterochromatin comprises approximately 30% of the Drosophila melanogaster genome, forming a transcriptionally repressive environment that silences euchromatic genes juxtaposed nearby. Surprisingly, there are genes naturally resident in heterochromatin, which appear to require this environment for optimal activity. Here we report an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D. melanogaster. DmDbp80 is typical of previously described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast, DmRpL15 is uncharacteristically small. The orthologs of these genes were examined in D. pseudoobscura and D. virilis. In situ hybridization and whole-genome assembly analysis show that these genes are adjacent, but not centromeric in the genome of D. pseudoobscura, while they are located on different chromosomal elements in D. virilis. Dbp80 gene organization differs dramatically among these species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species demonstrates active repositioning of these genes both within and between chromosomal elements. This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these genes also provides unique insight into the dynamic nature of genome evolution.

A genetic and molecular characterization of two proximal heterochromatic genes on chromosome 3 of Drosophila melanogaster.

Heterochromatin comprises a transcriptionally repressive chromosome compartment in the eukaryotic nucleus; this is exemplified by the silencing effect it has on euchromatic genes that have been relocated nearby, a phenomenon known as position-effect variegation (PEV), first demonstrated in Drosophila melanogaster. However, the expression of essential heterochromatic genes within these apparently repressive regions of the genome presents a paradox, an understanding of which could provide key insights into the effects of chromatin structure on gene expression. To date, very few of these resident heterochromatic genes have been characterized to any extent, and their expression and regulation remain poorly understood. Here we report the cloning and characterization of two proximal heterochromatic genes in D. melanogaster, located deep within the centric heterochromatin of the left arm of chromosome 3. One of these genes, RpL15, is uncharacteristically small, is highly expressed, and encodes an essential ribosomal protein. Its expression appears to be compromised in a genetic background deficient for heterochromatin protein 1 (HP1), a protein associated with gene silencing in these regions. The second gene in this study, Dbp80, is very large and also appears to show a transcriptional dependence upon HP1; however, it does not correspond to any known lethal complementation group and is likely to be a nonessential gene.

dSet1 is the main H3K4 di- and tri-methyltransferase throughout Drosophila development.

In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a "global" or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans.

An investigative laboratory course in human physiology using computer technology and collaborative writing.

Active investigative student-directed experiences in laboratory science are being encouraged by national science organizations. A growing body of evidence from classroom assessment supports their effectiveness. This study describes four years of implementation and assessment of an investigative laboratory course in human physiology for 65 second-year students in sports medicine and biology at a small private comprehensive college. The course builds on skills and abilities first introduced in an introductory investigations course and introduces additional higher-level skills and more complex human experimental models. In four multiweek experimental modules, involving neuromuscular, reflex, and cardiovascular physiology, by use of computerized hardware/software with a variety of transducers, students carry out self-designed experiments with human subjects and perform data collection and analysis, collaborative writing, and peer editing. In assessments, including standard course evaluations and the Salgains Web-based evaluation, student responses to this approach are enthusiastic, and gains in their skills and abilities are evident in their comments and in improved performance.