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[
International Worm Meeting,
2003]
Project Lab was developed and funded with an NSF CAREER grant designed to integrate research and education. Project Lab is an intensive new laboratory course in which undergraduate students participate in supervised research projects. These research projects are directly related to ongoing research in my laboratory. Project Lab has a class size of 7-12 students and, each year, it focuses on research activities appropriate for that particular stage of the research project. The addition of this course to our biology curriculum enhances the science education of undergraduate students at Santa Clara University by providing hands-on laboratory experience and exposure to real research problems. It also enhances the research program of the principal investigator by training and motivating students for research.I have taught Project Lab four times at SCU (in 1996, 1998, 1999, and 2000). We have been using a molecular approach to conduct a functional analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans. Project Lab classes have already made considerable progress, resulting in a peer-reviewed paper on which three Project Lab students were authors (Miller et al., Genetics 156: 1595, 2000). Results from the two more recent Project Lab classes will soon be submitted as a paper with four undergraduate authors.As part of the assessment goals for the NSF grant, each Project Lab class was asked to fill out four different surveys: an initial survey at the beginning of the course (given primarily for demographic purposes), an end-of-the-quarter survey during the last week of class, a 1-year-post survey approximately one year after finishing the course, and a 2-year-post survey approximately two years after finishing the course. The response rate for all surveys has been outstanding. There were 116 responses for 124 surveys, for a response rate of 94%! This is especially impressive given that half the surveys were sent to students 1 or 2 years after finishing the course, when most of them had graduated. The Project Lab surveys included questions about the impact of the course on skills, career goals, and confidence. Two main results were obtained from these surveys: (1) All students highly valued this research-intensive course and (2) student impression of course impact increased over time. In other words, students appreciated the course even more after graduation. Details of the surveys results will be presented.
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[
International Worm Meeting,
2003]
Eukaryotic genomes from diverse species contain distinct patterns of homologous genes ordered along their chromosomes. Chromosomal patterns change when rearrangements such as translocations, duplications, deletions, or inversions occur. These observations raise two important questions. Are genes randomly distributed in genomes? If not, do chromosomal rearrangements have adaptive value? We are beginning to address these questions through studies of a sperm-sensing mechanism that controls the rate and efficiency of reproduction in C. elegans. Sperm stimulate oocyte meiotic maturation, oocyte mitogen-activated protein kinase activation, somatic gonadal sheath contraction, and spermathecal dilation using the major sperm protein (MSP) as a signaling molecule. We show that genes in these signaling pathways are nonrandomly distributed in the genome and cluster with twenty-eight msp genes located at three loci on chromosomes II and IV. Other tightly linked genes have been implicated in regulating MSP transcription or trafficking during spermatogenesis. We also detected overlapping, nonrandom distributions of genes that control the onset of oogenesis (via the translational regulation of
fem-3), but not of genes in other delineated pathways. Therefore, we have defined clusters of reproductive genes that function in common pathways. To evaluate the magnitude of these clusters, we used a global microarray-based strategy to determine the chromosomal distributions of genes with upregulated germline transcriptional profiles. Large nonrandom aggregations of sperm-enriched and germline-enriched genes colocalize with the chromosomal regions we defined by functional studies. By contrast, muscle-enriched or dauer-enriched genes are proportionately represented in these regions. Our results strongly support the hypothesis that reproductive genes are nonrandomly distributed in the C. elegans genome. To investigate the significance of these reproductive clusters, we examined chromosomal distributions of related genes in C. briggsae and other metazoan genomes. Comparative mapping and phylogenetic studies suggest that these clusters were built by large numbers of chromosomal rearrangements occurring in two temporal phases. Because rearrangements generated nonrandom patterns within the C. elegans genome, selection is predicted to drive this (re)distribution process. Therefore, chromosomal rearrangements may have adaptive value. We will discuss reasons why clustering may confer a selective advantage and implications for genome evolution.
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[
East Coast Worm Meeting,
1998]
PKC3 is a C.elegans gene encoding a novel protein kinase C with strong sequence homologies to atypical PKC's in mammals (Wu et al., 1998). A PKC3::gfp construct has been used to demonstrate that the enzyme has restricted expression throughout late embryonic, larval and adult life in about 80-90 cells, particularly those along the alimentary canal, e.g. pharynx, intestine, the pharyngeal valve, the rectal valve, and the rectal valve muscles. There is also some expression associated with the vulva and spermatheca, and in the hyp cells of the tail tip. Similar staining of the alimentary canal was noted using immunofluorescence and confocal microscopy, using an affinity-purified polyclonal Ab raised against a PKC3 fusion protein including AA's 497-597 from the C terminus. A different pattern of patchy expression was noted by immunofluorescence in early embryoes, apparently involving membrane plaques between cell pairs. We have used postembedding immunoEM techniques (Hall, 1995) to localize the PKC3 protein more closely, using the same purified Ab. Binding of the anti-PKC3 Ab on thin sections of adult worms was similar to the pattern noted with the gfp construct, with highest labelling along the alimentary canal and in the extreme tail tip. Strongest label was found on the pharyngeal valve, and on the apical portions of the pharynx and the intestine, with appreciable labeling of the intestinal lumen. At the extreme tail tip, labeling was found both in the hypodermis and in the newly formed cuticle. Specific binding could be blocked by pre-soaking the Ab with antigen; curiously, the resulting Ab/antigen complex bound strongly to all nuclei. Localization of a protein kinase to extracellular space (intestinal lumen and tail cuticle) is highly unusual. The localization of PKC3 to the apical portions of cells along the alimentary canal is thought to be mediated by direct binding to PICK1 protein (cf. Staudinger et al, 1995, 1997), which probably associates with the cytoskeletal matrix underlying the plasma membrane.
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[
International C. elegans Meeting,
1989]
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[
International C. elegans Meeting,
2001]
I have used C. elegans in two different undergraduate lab courses at Santa Clara University. One is an upper division research-focused "Project Lab" course described below and the other is an upper division Genetics course that uses C. elegans for its laboratory component. The "Project Lab" course was developed with an NSF grant to integrate research and education by designing and implementing an intensive new laboratory course in which undergraduate students participate in supervised research projects. These research projects are directly related to ongoing research in my laboratory. Project Lab has a class size of 7-12 students and, each year, it focuses on research activities appropriate for that particular stage of the research project. The addition of this course to our biology curriculum enhances the science education of undergraduate students at Santa Clara University by providing hands-on laboratory experience and exposure to real research problems. It also enhances the research program of the principal investigator by training and motivating students for research. Specifically, we are using sequencing and site-directed mutagenesis to conduct a structure/function analysis of LIN-31, a member of the winged-helix family of transcription factors, which is required for the proper specification of vulval cell fates in C. elegans . The first two Project Lab classes made considerable progress, resulting in a paper on which three Project Lab students were authors (Miller et al. , Genetics 156: 1595, 2000). Results from the two most recent Project Lab class will be presented as a poster at this meeting (see Mora-Blanco et al. ). I have also successfully used C. elegans in my upper division genetics laboratory course. This is a 10-week course in which students use C. elegans to study basic genetic principles such as dominance, independent assortment, recombination, and complementation. In order to do this, they first find and then analyze their own genetic mutant. As the quarter progresses, they use their mutants to study the basic genetic principles listed above. The first lab introduces them to C. elegans and teaches them how to grow, manipulate, and sex the animals. In the second lab, they "find" their mutant by doing a mock mutant screen. In subsequent labs, they carry out genetic manipulations (crosses) to better understand their mutants. By the end of the quarter, they can answer the following questions about their mutant: Is the mutant allele dominant or recessive to the wild-type allele? What chromosome does it map to? Between what other genes does it map? Is it an allele of a known gene? In summary, C. elegans is ideally suited for an undergraduate laboratory course. With appropriate training, undergraduate students can quickly learn to manipulate nematodes for use in genetic and/or molecular experiments. Furthermore, the flexibility of the 3-, 4-, or 7-day life cycle makes C. elegans particularly appealing for institutions on the quarter system. Access to teaching materials for both courses will be available at the teaching poster session and on my web page (www-acc.scu.edu/~lmiller/homepage.html).
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Chemical signaling processes of the parasitic nematode Ascaris suum are central in creating a model to understand how nervous systems control behavior. Research is currently being done to isolate and sequence neuropeptides, including the FMRFamide-like (Phe-Met-Arg-Phe-NH2-like) peptides endogenous to A. suum (AF peptides). By associating neuropeptide localization and sequence information with observed behavioral effects, we hope to better explain the chemical signaling processes of the nematode nervous system. In this study, the effects of two groups of endogenous AF peptides were measured by monitoring the inhibition or potentiation of acetylcholine induced contraction on A. suum dorsal muscle strips. The AF peptides in each group have been isolated from the same cell and are encoded by the same transcript; their amino acid sequences are similar but differ by the presence or absence of P (Pro) preceding a stereotyped NFLRFa (Asn-Phe-Leu-Arg-Phe-NH2) ending. A comparison of the AF peptide groups was used to determine if subtle sequence differences function to amplify similar or generate diverse behavioral effects. Experiments demonstrate that peptides from both groups act to inhibit acetylcholine induced dorsal muscle contraction with varying efficacy and time course.
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[
International Worm Meeting,
2003]
GFP is widely utilized in C. elegans to visualize specific cells and labeled proteins. For co-localization studies, CFP (cyan) and YFP (yellow) variants can be used simultaneously to generate discrete two-color images. Conventional fluorescence filter sets, however, do not allow the use of GFP in combination with either CFP or YFP due to extensive overlap of these emission spectra. Here we describe new optical tools now available in the Zeiss LSM 510 META confocal microscope for simultaneous multicolor imaging with CFP, GFP and YFP. In the META, a diffraction grating disperses the sample fluorescence over a bank of detectors. This configuration subdivides the visible spectrum into 10.7 nm bandwidths and thereby effectively generates an emission spectrum for each pixel in the image plane. A linear unmixing algorithm utilizes reference spectra for each fluorophore (CFP, GFP, YFP) to calculate a best fit to the composite emission spectrum for each pixel. Pixels that exactly match a specific reference spectrum are assigned a single corresponding pseudocolor whereas pixels that register signal from more than one reporter are assigned the appropriate combination of pseudocolors and relative intensities for each contributing fluorophore. We used this strategy to generate images of transgenic animals expressing CFP, GFP, and YFP in specific motor neuron classes:
unc-25::CFP (DD, VD, blue);
unc-53::GFP (DA, AS, green);
acr-2::YFP (DA, VA, DB, VB, red). DA motor neurons were assigned a fourth color, yellow due to overlapping expression of green GFP and red YFP in these cells. An added benefit of the linear unmixing algorithm is that gut autofluorescence which does not match reporter reference spectra can be digitally removed without degrading the fidelity of the image.
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[
International C. elegans Meeting,
1999]
This NSF-sponsored project integrates research and education by developing an intensive new laboratory course in which undergraduate students participate in a supervised research project. This research project is directly related to ongoing research in the laboratory of the principal investigator. The course is called "Project Lab" and is taught for one quarter each year with a class size of 7-16 students. Each year, the course focuses on research activities appropriate for that particular stage of the research project. The addition of this course to our biology curriculum enhances the science education of undergraduate students at Santa Clara University by providing hands-on laboratory experience and exposure to real research problems. It also enhances the research program of the principal investigator by training and motivating students for research. Specifically, students isolate and study mutations in genes that are required for cell fate determination in the process of vulval development in the nematode, C. elegans . The identification and analysis of these genes will set the stage for future experiments in cell fate determination. C. elegans is ideally suited for an undergraduate laboratory course. With appropriate training, undergraduate students can quickly learn to manipulate nematodes for use in genetic and/or molecular experiments. The first two Project Lab classes have made considerable progress on two different projects. For the first project, Project Lab students isolated and characterized over 30 new mutations that represents genes involved in the proper choice of cell fate during C. elegans vulval development. For the second project, several Project Lab students have been involved in a structure/function analysis of a transcription factor required for proper patterning of cell fate in C. elegans vulval development. There is a manuscript in preparation for this project, which will be submitted for publication within the next year. Three Project Lab students will be authors on this paper. I have also successfully used C. elegans in my upper division genetics laboratory course. This is a 10-week course in which students use nematodes to study inheritance patterns and map genes (using STS mapping).
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[
International C. elegans Meeting,
2001]
Sophisticated regulatory systems have evolved to coordinate fertilization and oocyte meiotic cell cycle progression. However, the molecular interactions that govern critical oocyte cell cycle transitions and their conservation in the metazoa are not clear. C. elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of the smooth muscle-like gonadal sheath cells, which are necessary for ovulation. Using an in vivo bioassay, we have shown that the major sperm cytoskeletal protein (MSP) acts as a bipartite signal for both oocyte maturation and sheath contraction. During nematode sperm locomotion, MSP plays a role analogous to actin indicating that this 14 kDa sperm-specific protein has acquired extracellular signaling and intracellular cytoskeletal functions during evolution. Proteins with MSP-like domains have been found in plants, fungi, and other animals raising the possibility that MSP signaling functions may exist in other phyla. We are particularly interested in understanding how MSP promotes the resumption of meiosis in oocytes. A receptor for a maturation-promoting factor has not been identified in any animal. Thus, identifying and characterizing the MSP receptor(s) is a major goal. We are taking a genomic approach to identify receptor candidates. Using an in situ MSP binding assay, candidates can be quickly screened for binding following RNAi. In this assay, MSP-flouroscein specifically binds oocytes and sheath cells of the proximal gonad arm. MSP-flouroscein is active in promoting both oocyte maturation and sheath contraction in vivo . Binding is not observed in distal female gonads, male gonads, or following incubation with BSA-flouroscein. Preincubation with a 20-fold excess of unlabeled MSP completely abrogates MSP-flouroscein binding. Further, binding is not observed in
emo-1(
oz1) oocytes, which lack a functional secretory system due to a mutation in a Sec61p g homologue (Iwasaki et al., 1996). Using data from DNA microarrays (Reinke et al., 2000), we have identified three conserved transmembrane proteins whose transcripts are highly enriched in oocytes. MSP binding to oocytes is dramatically reduced in two loss of function backgrounds and enhanced in the other. Each gene is tightly linked to one of the two large MSP clusters in the genome--a finding of unknown significance. We currently have mutations in two genes and are doing detailed phenotypic analyses. We are also interested in examining protein localization in situ and evaluating MSP binding in a heterologous system. Our results will be discussed in the context of a new model for oocyte meiotic maturation. Iwasaki et al. (1996). J Cell Biol 134:699-714 Reinke et al. (2000). Mol Cell 6:605-616
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[
International Worm Meeting,
2009]
It has been recently demonstrated that hydrogen sulfide (H2S) can protect animals against damage and death associated with decreased O2 availability. For example, H2S protects mammals against otherwise lethal hypoxia and improves outcome in mammalian models of severe blood loss, myocardial infarction, aortic occlusion and hepatic ischemia/reperfusion. The mechanisms by which H2S exerts beneficial effects in mammals are unknown. We have developed a C. elegans model investigate the genetic factors that contribute to damage associated with ischemia in animals and to define the molecular mechanisms that mediate the beneficial effects of H2S. Here we show that, as in mammals, H2S protects against hypoxia in C. elegans. We have found that specific hypoxic conditions induce aggregation of polyglutamine-containing proteins. The range of O2 concentrations that cause aggregation is expanded by mutations in the highly conserved hypoxia-inducible transcription factor,
hif-1. These data suggest that perturbations in protein homeostasis occur when the cellular response to hypoxia is overwhelmed. Adaptation to H2S protects against hypoxia-induced disturbances of protein homeostasis. Even transient exposure to H2S early in development is sufficient to protect against hypoxia-induced aggregation of polyglutamine proteins. The H2S-induced changes that protect against hypoxia are distinct from those that cause increased lifespan and thermotolerance. These data show that exposure to H2S results in persistent physiological effects that can influence responses to changing environmental conditions.