Michael J. Wolyniak

Implication of a novel multiprotein Dam1p complex in outer kinetochore function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

A Course-Based Research Experience: How Benefits Change with Increased Investment in Instructional Time

While course-based research in genomics can generate both knowledge gains and a greater appreciation for how science is done, a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. Nonetheless, this is a very cost-effective way to reach larger numbers of students.

A Central Support System Can Facilitate Implementation and Sustainability of a Classroom-Based Undergraduate Research Experience (CURE) in Genomics

There have been numerous calls to engage students in science as science is done. A survey of 90-plus faculty members explores barriers and incentives when developing a research-based genomics course. The results indicate that a central core supporting a national experiment can help overcome local obstacles.

Harnessing genomics to explore the processes and evolution of mRNA export.

The export of quality-controlled mRNAs across the nuclear membrane for translation is mediated by a set of core proteins that are conserved throughout eukaryotes and have been best-characterized in S. cerevisiae. The increased genomic complexity that arose during metazoan evolution, however, has resulted in increased systematic complexity that is reflected in the presence of additional metazoan mRNA export factors. In some cases, metazoans encode families of closely-related factors whereas in fungi, a single homolog is present. An exciting new study examines metazoan mRNA export from a global perspective through the use of a genome-wide RNA interference screen in Drosophila melanogaster. This screen identified several novel factors that contribute to mRNA export while reaffirming the strong evolutionary conservation that exists between Drosophila and yeast homologs that are essential for mRNA export. The study also showed that several factors were required for the export of an intron-containing transcript but not for one lacking an intron. Taken together, this study underscores the value of genomic approaches for understanding complex biological processes.

Improving medical students' knowledge of genetic disease: a review of current and emerging pedagogical practices

Genetics is an essential subject to be mastered by health professional students of all types. However, technological advances in genomics and recent pedagogical research have changed the way in which many medical training programs teach genetics to their students. These advances favor a more experience-based education focused primarily on developing student’s critical thinking skills. In this review, we examine the current state of genetics education at both the preclinical and clinical levels and the ways in which medical and pedagogical research have guided reforms to current and emerging teaching practices in genetics. We discover exciting trends taking place in which genetics is integrated with other scientific disciplines both horizontally and vertically across medical curricula to emphasize training in scientific critical thinking skills among students via the evaluation of clinical evidence and consultation of online databases. These trends will produce future health professionals with the skills and confidence necessary to embrace the new tools of medical practice that have emerged from scientific advances in genetics, genomics, and bioinformatics.

Extensive phenotypic variation among allelic T-DNA inserts in Arabidopsis thaliana.

T-DNA insertion mutants are a tool used widely in Arabidopsis thaliana to disrupt gene function. We phenotyped multiple homozygous T-DNA A. thaliana mutants at each of two loci (AT1G11060 and AT4G00210). We measured life history traits, including germination, size at reproduction and fruit production. Allelic T-DNA lines differed for most traits at AT1G11060 but not at AT4G00210. However, insertions in exons differed from other insertion positions in AT4G00210 but not in AT1G11060. We found evidence for additional insertions in approximately half of the lines, but found few phenotypic consequences. In general, our results suggest that a cautious interpretation of T-DNA phenotypes is warranted.

Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes.

The mechanisms that fungi use to co-regulate subsets of genes specifically associated with morphogenic states represent a basic unsolved problem in fungal biology. Candida albicans is an important model of fungal differentiation both for rapid interconversion between yeast and hyphal growth forms and for white/opaque switching mechanisms. The Sundstrom lab is interested in mechanisms regulating hypha-specific expression of adhesin genes that are critical for C. albicans hyphal growth phenotypes and pathogenicity. Early studies on hypha-specific genes such as HWP1 and ALS3 reported 5’ intergenic regions that are larger than those typically found in an average promoter and are associated with hypha-specific expression. In the case of HWP1, activation and repression involves a 368 bp region, denoted the HWP1 control region (HCR), located 1410 bp upstream of its transcription start site. In previous work we showed that HCR confers developmental regulation to a heterologous ENO1 promoter, indicating that HCR by itself contains sufficient information to couple gene expression to morphology. Here we show that the activation and repression mediated by HCR are localized to distinct HCR regions that are targeted by the transcription factors Nrg1p and Efg1p. The finding that Efg1p mediates both repression via HCR under yeast morphological conditions and activation conditions positions Efg1p as playing a central role in coupling HWP1 expression to morphogenesis through the HCR region. These localization studies revealed that the 120 terminal base pairs of HCR confer Efg1p-dependent repressive activity in addition to the Nrg1p repressive activity mediated by DNA upstream of this subregion. The 120 terminal base pair subregion of HCR also contained an initiation site for an HWP1 transcript that is specific to yeast growth conditions (HCR-Y) and may function in the repression of downstream DNA. The detection of an HWP1 mRNA isoform specific to hyphal growth conditions (HWP1-H) showed that morphology-specific mRNA isoforms occur under both yeast and hyphal growth conditions. Similar results were found at the ALS3 locus. Taken together, these results, suggest that the long 5’ intergenic regions upstream of hypha-specific genes function in generating mRNA isoforms that are important for morphology-specific gene expression. Additional complexity in the HWP1 promoter involving HCR-independent activation was discovered by creating a strain lacking HCR that exhibited variable HWP1 expression during hyphal growth conditions. These results show that while HCR is important for ensuring uniform HWP1 expression in cell populations, HCR independent expression also exists. Overall, these results elucidate HCR-dependent mechanisms for coupling HWP1-dependent gene expression to morphology uniformly in cell populations and prompt the hypothesis that mRNA isoforms may play a role in coupling gene expression to morphology in C. albicans.

Improved student linkage of Mendelian and molecular genetic concepts through a yeast-based laboratory module.

A study of modern genetics requires students to successfully unite the principles of Mendelian genetics with the functions of DNA. Traditional means of teaching genetics are often successful in teaching Mendelian and molecular ideas but not in allowing students to see how the two subjects relate. The laboratory module presented here attempts to present classical and molecular genetic concepts together as an inquiry‐based exploration appropriate for high school or introductory undergraduate students. Using the non‐essential APQ12 gene in the budding yeast Saccharomyces cerevisiae, students perform PCR, selective growth, and sporulation experiments to establish the ploidy and APQ12 zygosity of a series of unknown strains. Each experiment contributes data to characterize the unknown strains, but complete characterization is not possible without assimilating the data from all of the experiments. The module allows students to consider concepts normally introduced and emphasized in Mendelian genetics and explore them using molecular and experimental tools. Comparison of pre‐module and post‐module assessment surveys show an increase in student ability to link Mendelian concepts to experimental procedures relying on DNA analysis. The development of modules such as these provides students of all backgrounds with the tools to engage the complexities and issues that constitute modern principles of inheritance.