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  • Using Yeast to Determine the Functional Consequences of Mutations in the Human p53 Tumor Suppressor Gene: An Introductory Course-Based Undergraduate Research Experience in Molecular and Cell Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Hekmat-Scafe, D. S., Brownell, S. E., Seawell, P. C., Malladi, S., Imam, J. F., Singla, V., Bradon, N., Cyert, M. S., Stearns, T. 2017; 45 (2): 161-178

    Abstract

    The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course-based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students' high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA-binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):161-178, 2017.

    View details for DOI 10.1002/bmb.21024

    View details for Web of Science ID 000398045000010

  • A High-Enrollment Course-Based Undergraduate Research Experience Improves Student Conceptions of Scientific Thinking and Ability to Interpret Data CBE-LIFE SCIENCES EDUCATION Brownell, S. E., Hekmat-Scafe, D. S., Singla, V., Seawell, P. C., Imam, J. F., Eddy, S. L., Stearns, T., Cyert, M. S. 2015; 14 (2)

    Abstract

    We present an innovative course-based undergraduate research experience curriculum focused on the characterization of single point mutations in p53, a tumor suppressor gene that is mutated in more than 50% of human cancers. This course is required of all introductory biology students, so all biology majors engage in a research project as part of their training. Using a set of open-ended written prompts, we found that the course shifts student conceptions of what it means to think like a scientist from novice to more expert-like. Students at the end of the course identified experimental repetition, data analysis, and collaboration as important elements of thinking like a scientist. Course exams revealed that students showed gains in their ability to analyze and interpret data. These data indicate that this course-embedded research experience has a positive impact on the development of students' conceptions and practice of scientific thinking.

    View details for DOI 10.1187/cbe.14-05-0092

    View details for Web of Science ID 000355555900011

    View details for PubMedID 26033869

    View details for PubMedCentralID PMC4477737

  • Seizure Sensitivity Is Ameliorated by Targeted Expression of K+-Cl- Cotransporter Function in the Mushroom Body of the Drosophila Brain GENETICS Hekmat-Scafe, D. S., Mercado, A., Fajilan, A. A., Lee, A. W., Hsu, R., Mount, D. B., Tanouye, M. A. 2010; 184 (1): 171-183

    Abstract

    The kcc(DHS1) allele of kazachoc (kcc) was identified as a seizure-enhancer mutation exacerbating the bang-sensitive (BS) paralytic behavioral phenotypes of several seizure-sensitive Drosophila mutants. On their own, young kcc(DHS1) flies also display seizure-like behavior and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The product of kcc shows substantial homology to KCC2, the mammalian neuronal K(+)-Cl(-) cotransporter. The kcc(DHS1) allele is a hypomorph, and its seizure-like phenotype reflects reduced expression of the kcc gene. We report here that kcc functions as a K(+)-Cl(-) cotransporter when expressed heterologously in Xenopus laevis oocytes: under hypotonic conditions that induce oocyte swelling, oocytes that express Drosophila kcc display robust ion transport activity observed as a Cl(-)-dependent uptake of the K(+) congener (86)Rb(+). Ectopic, spatially restricted expression of a UAS-kcc(+) transgene was used to determine where cotransporter function is required in order to rescue the kcc(DHS1) BS paralytic phenotype. Interestingly, phenotypic rescue is largely accounted for by targeted, circumscribed expression in the mushroom bodies (MBs) and the ellipsoid body (EB) of the central complex. Intriguingly, we observed that MB induction of kcc(+) functioned as a general seizure suppressor in Drosophila. Drosophila MBs have generated considerable interest especially for their role as the neural substrate for olfactory learning and memory; they have not been previously implicated in seizure susceptibility. We show that kcc(DHS1) seizure sensitivity in MB neurons acts via a weakening of chemical synaptic inhibition by GABAergic transmission and suggest that this is due to disruption of intracellular Cl(-) gradients in MB neurons.

    View details for DOI 10.1534/genetics.109.109074

    View details for Web of Science ID 000281784000016

    View details for PubMedID 19884312

  • Mutations in the K+/Cl- cotransporter gene kazachoc (kcc) increase seizure susceptibility in Drosophila JOURNAL OF NEUROSCIENCE Hekmat-Scafe, D. S., Lundy, M. Y., Ranga, R., Tanouye, M. A. 2006; 26 (35): 8943-8954

    Abstract

    During a critical period in the developing mammalian brain, there is a major switch in the nature of GABAergic transmission from depolarizing and excitatory, the pattern of the neonatal brain, to hyperpolarizing and inhibitory, the pattern of the mature brain. This switch is believed to play a major role in determining neuronal connectivity via activity-dependent mechanisms. The GABAergic developmental switch may also be particularly vulnerable to dysfunction leading to seizure disorders. The developmental GABA switch is mediated primarily by KCC2, a neuronal K+/Cl- cotransporter that determines the intracellular concentration of Cl- and, hence, the reversal potential for GABA. Here, we report that kazachoc (kcc) mutations that reduce the level of the sole K+/Cl- cotransporter in the fruitfly Drosophila melanogaster render flies susceptible to epileptic-like seizures. Drosophila kcc protein is widely expressed in brain neuropil, and its level rises with developmental age. Young kcc mutant flies with low kcc levels display behavioral seizures and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The kcc mutation enhances a series of other Drosophila epilepsy mutations indicating functional interactions leading to seizure disorder. Both genetic and pharmacological experiments suggest that the increased seizure susceptibility of kcc flies occurs via excitatory GABAergic signaling. The kcc mutants provide an excellent model system in which to investigate how modulation of GABAergic signaling influences neuronal excitability and epileptogenesis.

    View details for DOI 10.1523/JNEUROSCI.4998-05.2006

    View details for Web of Science ID 000240151900010

    View details for PubMedID 16943550

  • Seizure suppression by gain-of-function escargot mutations GENETICS Hekmat-Scafe, D. S., Dang, K. N., Tanouye, M. A. 2005; 169 (3): 1477-1493

    Abstract

    Suppressor mutations provide potentially powerful tools for examining mechanisms underlying neurological disorders and identifying novel targets for pharmacological intervention. Here we describe mutations that suppress seizures in a Drosophila model of human epilepsy. A screen utilizing the Drosophila easily shocked (eas) "epilepsy" mutant identified dominant suppressors of seizure sensitivity. Among several mutations identified, neuronal escargot (esg) reduced eas seizures almost 90%. The esg gene encodes a member of the snail family of transcription factors. Whereas esg is normally expressed in a limited number of neurons during a defined period of nervous system development, here normal esg was expressed in all neurons and throughout development. This greatly ameliorated both the electrophysiological and the behavioral epilepsy phenotypes of eas. Neuronal esg appears to act as a general seizure suppressor in the Drosophila epilepsy model as it reduces the susceptibility of several seizure-prone mutants. We observed that esg must be ectopically expressed during nervous system development to reduce seizure susceptibility in adults. Furthermore, induction of esg in a small subset of neurons (interneurons) will reduce seizure susceptibility. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. We anticipate that some of these genes may ultimately serve as targets for novel antiepileptic drugs.

    View details for DOI 10.1534/genetics.104.036558

    View details for Web of Science ID 000228450800024

    View details for PubMedID 15654097

  • Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster GENOME RESEARCH Hekmat-Scafe, D. S., Scafe, C. R., McKinney, A. J., Tanouye, M. A. 2002; 12 (9): 1357-1369

    Abstract

    Olfaction is of considerable importance to many insects in behaviors critical for survival and reproduction, including location of food sources, selection of mates, recognition of colony con-specifics, and determination of oviposition sites. An ubiquitous, but poorly understood, component of the insect's olfactory system is a group of odorant-binding proteins (OBPs) that are present at high concentrations in the aqueous lymph surrounding the dendrites of olfactory receptor neurons. OBPs are believed to shuttle odorants from the environment to the underlying odorant receptors, for which they could potentially serve as odorant presenters. Here we show that the Drosophila genome carries 51 potential OBP genes, a number comparable to that of its odorant-receptor genes. We find that the majority (73%) of these OBP-like genes occur in clusters of as many as nine genes, in contrast to what has been observed for the Drosophila odorant-receptor genes. Two of the presumptive OBP gene clusters each carries an odorant-receptor gene. We also report an intriguing subfamily of 12 putative OBPs that share a unique C-terminal structure with three conserved cysteines and a conserved proline. Members of this subfamily have not previously been described for any insect. We have performed phylogenetic analyses of the OBP-related proteins in Drosophila as well as other insects, and we discuss the duplication and divergence of the genes for this large family. [The sequence data from this study have been submitted to FlyBase. Annotations for these sequences are available as supplementary material at http://www.genome.org.]

    View details for DOI 10.1101/gr.239402

    View details for Web of Science ID 000177964700007

    View details for PubMedID 12213773

  • Expression mosaic of odorant-binding proteins in Drosophila olfactory organs MICROSCOPY RESEARCH AND TECHNIQUE Shanbhag, S. R., Hekmat-Scafe, D., Kim, M. S., Park, S. K., Carlson, J. R., Pikielny, C., Smith, D. P., Steinbrecht, R. A. 2001; 55 (5): 297-306

    Abstract

    Deciphering the genome of the fruitfly, Drosophila melanogaster, has revealed 39 genes coding for putative odorant-binding proteins (OBPs), more than are known at present for any other insect species. Using specific antibodies, the expression mosaic of five such OBPs (OS-E, OS-F, LUSH, PBPRP2, PBPRP5) on the antenna and maxillary palp has been mapped in the electron microscope. It was found that (1) OBP expression does correlate with morphological sensillum types and subtypes, (2) several OBPs may be co-localized in the same sensillum, and (3) OBP localization is not restricted to olfactory sensilla. The expression of PBPRP2 in antennal epidermis sheds some light on the possible evolution of OBPs.

    View details for Web of Science ID 000172542700003

    View details for PubMedID 11754509

  • Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yager, J., Richards, S., Hekmat-Scafe, D. S., Hurd, D. D., Sundaresan, V., Caprette, D. R., Saxton, W. M., Carlson, J. R., Stern, M. 2001; 98 (18): 10445-10450

    Abstract

    Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Here we demonstrate with genetic methods that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. We show that growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn(2+)-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. Our results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis.

    View details for Web of Science ID 000170738000076

    View details for PubMedID 11517334

  • Olfactory coding in a compound nose - Coexpression of odorant-binding proteins in Drosophila International Symposium on Olfaction and Taste XII Hekmat-Scafe, D. S., Steinbrecht, R. A., Carlson, J. R. NEW YORK ACAD SCIENCES. 1998: 311–315

    Abstract

    Odorant-binding proteins (OBPs) are small, soluble proteins present in the aqueous medium surrounding olfactory receptor neurons. Their function in olfaction is unknown: they have been proposed to facilitate the transit of hydrophobic molecules to olfactory receptors, to deactivate the odorant stimulus, and/or to play a role in chemosensory coding. We have examined the genomic organization and expression patterns of two olfactory-specific genes (OS-E and OS-F) of Drosophila melanogaster, the products of which are members of a protein family in Drosophila sharing sequence similarity with moth OBPs. We found that the OS-E and OS-F transcription units are located < 1 kb apart. They are oriented in the same direction and display a similar intron-exon organization. Expression of both OS-E and OS-F proteins is spatially restricted to the ventrolateral region of the Drosophila antenna. Within this region, both OS-E and OS-F proteins are expressed within two different types of sensory hairs: in most, if not all, sensilla trichodea and in approximately 40% of the interspersed small sensilla basiconica. We consistently observe that OS-E and OS-F are coexpressed, indicating that an individual sensillum can contain more than one odorant-binding protein. This finding has potential implications for the roles of odorant-binding proteins in olfactory coding.

    View details for Web of Science ID 000078304300047

    View details for PubMedID 9929625

  • Coexpression of two odorant-binding protein homologs in Drosophila: Implications for olfactory coding JOURNAL OF NEUROSCIENCE HEKMATSCAFE, D. S., Steinbrecht, R. A., Carlson, J. R. 1997; 17 (5): 1616-1624

    Abstract

    Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurons. Their function in olfaction is still unknown: they have been proposed to facilitate the transit of hydrophobic molecules to olfactory receptors, to deactivate the odorant stimulus, and/or to play a role in chemosensory coding. In this study we examine the genomic organization and expression patterns of two olfactory-specific genes (OS-E and OS-F) of Drosophila melanogaster, the products of which are members of a protein family in Drosophila sharing sequence similarity with moth OBPs. We show that the OS-E and OS-F transcription units are located <1 kb apart. They are oriented in the same direction and display a similar intron-exon organization. Expression of both OS-E and OS-F proteins is restricted spatially to the ventrolateral region of the Drosophila antenna. Within this region both OS-E and OS-F proteins are expressed within two different types of sensory hairs: in most, if not all, sensilla trichodea and in approximately 40% of the interspersed small sensilla basiconica. We consistently observe that OS-E and OS-F are coexpressed, indicating that an individual sensillum can contain more than one odorant-binding protein. The functional significance of the observed expression pattern and its implications for olfactory coding are discussed.

    View details for Web of Science ID A1997WJ08700009

    View details for PubMedID 9030621

  • Genetic and molecular studies of olfaction in Drosophila Symposium on Olfaction in Mosquito-Host Interactions HEKMATSCAFE, D. S., Carlson, J. R. JOHN WILEY & SONS LTD. 1996: 285–301

    Abstract

    Drosophila melanogaster, an insect amenable to convenient molecular and genetic manipulation, has a highly sensitive olfactory system. A number of Drosophila olfactory mutants have been isolated and characterized. The smellblind mutant has a defect affecting a voltage-gated Na+ channel. The norpA mutant, defective in a phospholipase C, has a reduced response to odorants in one type of olfactory organ, providing genetic evidence for use of the inositol-1,4,5-trisphosphate signal transduction pathway in olfaction. Since the norpA gene is also required for phototransduction, this work demonstrates overlap in the molecular genetic basis of vision and olfaction. Interestingly, genetic analysis indicates that some olfactory information flows through a pathway which does not depend on norpA. Some mutants, such as ptg, acj6 and Sco, show odorant specificity, in the sense that some odorant responses are greatly reduced, whereas others are little affected, if at all. Some, but not all, mutations affect both larval and adult olfactory responses. Two tightly-linked Drosophila genes encode homologues of moth pheromone-binding proteins (PBPs). Genetic analysis may help determine whether PBPs facilitate transit of pheromones to or from olfactory receptor neurons. Information from Drosophila could be useful in designing means of controlling mosquitoes. It may also be possible to study olfactory genes, such as those encoding PBPs, from other insects by mutating them, introducing them into Drosophila and analysing their function in vivo.

    View details for Web of Science ID A1996BG20T00020

    View details for PubMedID 8894304

  • PUTATIVE DROSOPHILA PHEROMONE-BINDING PROTEINS EXPRESSED IN A SUBREGION OF THE OLFACTORY SYSTEM JOURNAL OF BIOLOGICAL CHEMISTRY McKenna, M. P., HEKMATSCAFE, D. S., Gaines, P., Carlson, J. R. 1994; 269 (23): 16340-16347

    Abstract

    Four genes expressed in the olfactory system of Drosophila melanogaster have been identified by subtractive hybridization. Two of these genes, OS-E and OS-F, are related to genes encoding moth pheromone-binding proteins. The OS-E and OS-F genes are tightly linked and are expressed in a subregion of the antenna (the primary olfactory organ). A protein sequence analysis suggests the possibility that pheromone-binding proteins are members of a larger class of proteins, extending beyond the olfactory system. The predicted product of a third gene, OS-D, shares features common to vertebrate odorant-binding proteins, but has a primary structure unlike odorant-binding proteins. The fourth gene, OS-C, encodes a novel 13-kDa protein that contains a putative nuclear import sequence and an acid-rich region. The expression patterns of these genes differ within the antenna; their transcript distributions support the notion of specialized roles for different olfactory sensilla. The functions of the OS gene products have not been demonstrated; however, the potential identification of pheromone-binding proteins in Drosophila, a species with well characterized genetics, may offer a means of analyzing the function of these molecules that is not available in other systems.

    View details for Web of Science ID A1994NQ72900060

    View details for PubMedID 8206941

  • MUTATIONS IN A CONSERVED REGION OF RNA POLYMERASE-II INFLUENCE THE ACCURACY OF MESSENGER-RNA START SITE SELECTION MOLECULAR AND CELLULAR BIOLOGY HEKMATPANAH, D. S., Young, R. A. 1991; 11 (11): 5781-5791

    Abstract

    A sensitive phenotypic assay has been used to identify mutations affecting transcription initiation in the genes encoding the two large subunits of Saccharomyces cerevisiae RNA polymerase II (RPB1 and RPB2). The rpb1 and rpb2 mutations alter the ratio of transcripts initiated at two adjacent start sites of a delta-insertion promoter. Of a large number of rpb1 and rpb2 mutations screened, only a few affect transcription initiation patterns at delta-insertion promoters, and these mutations are in close proximity to each other within both RPB1 and RPB2. The two rpb1 mutations alter amino acid residues within homology block G, a region conserved in the large subunits of all RNA polymerases. The three strong rpb2 mutations alter adjacent amino acids. At a wild-type promoter, the rpb1 mutations affect the accuracy of mRNA start site selection by producing a small but detectable increase in the 5'-end heterogeneity of transcripts. These RNA polymerase II mutations implicate specific portions of the enzyme in aspects of transcription initiation.

    View details for Web of Science ID A1991GL38200043

    View details for PubMedID 1922077