Notre Dame NSF Biology REU 2003
Abstracts
GPCR Studies:
Characterization Of The Opsin
Gene Family In The Malarial Vector, Anopheles gambiae
Perciliz Tan, Catherine Hill,
G protein-coupled
receptors (GPCRs) are a large family of proteins with various functions such as
chemo- and photoreception, neurophysiological function, and hormone regulation.
Consequently GPCRs are important drug and insecticide targets. Among the
sensory and non-sensory GPCRs that we have identified in silico from the
genome of the principal malarial vector mosquito, Anopheles gambiae, are
a family of opsins thought to be involved in visual processes as part of
phototransduction signaling cascades in the mosquito eye.
In Anopheles
gambiae, crepuscular behaviors such as mating, feeding, host location, and
the selection of oviposition sites that are dependent upon light and spectral
conditions. At the present, the role of opsins in mosquito photoreception and
vision related behaviors are not known. The opsins are of interest as they have
undergone a gene expansion of the blue/green/violet sensitive receptors
relative to Drosophila melanogaster. Therefore we have undertaken a detailed
analysis of the opsin gene family and this gene expansion in A.
gambiae. We have used a combination of bioinformatic approaches together
with direct DNA sequencing, and quantitative RT-PCR to analyze opsin expression
in A. gambiae heads and bodies and in male and female mosquitoes
collected during the light and dusk photoperiods.
Preliminary results suggest that there
are different expression levels of opsin transcripts in male and female A.
gambiae at different times throughout the twenty-four hour photoperiod. The
observed differences may result from sexual dimorphisms, photoreceptor membrane
turnover, or other specialized roles that allow A. gambiae to be very
active at low light intensities. This is the first study of the A. gambiae
opsin gene family and is part of an ongoing effort to discover possible targets
that may lead to the establishment of attractants, repellants, or novel methods
for control of mosquitoes and other disease vectors.
Steuter, John, Reilly,Heather, Patel, Jigar, Deng, Bingbing, Ferdig, Michael
Biological
Sciences,
The disease malaria affects 300 to 500 million people and accounts for an estimated 2 million deaths a year. Of the malaria parasites, Plasmodium falciparum causes the most severe and deadly form of malaria. An important determining factor of the parasites effects on humans is its proliferative capacity in the erythrocytic cycle. Thus, the identification of factors that contribute to the overall growth rate of the parasite will result in a greater understanding of the disease. In this research we have looked at the relative differences in cycle time of the parasites, cell cycle synchronization, and overall growth rate. To score overall growth rate we used radiolabeled hypoxanthine (Hx) incorporation into proliferating progeny populations derived from a parental cross of HB3 and Dd2. The growth data was then analyzed using quantitative trait loci (QTL) mapping to identify chromosomal regions carrying genes controlling parasite growth. Cycle time and synchronization were measured using blood smear data from the parents HB3 and Dd2. Knowledge of genetic mechanisms of growth rate, cycle time, and cell cycle synchronization will lead to an enhanced biological understanding of the disease.
Erin C. Ward, Jennifer
Wietzke, and JoEllen Welsh. Department
of Biological Sciences,
Anopheles gambiae, or the African malaria mosquito, is characterized by profound
genetic plasticity, which is manifested as rapid specialization for humans and
human-manipulated environments. The
mosquito’s exploitation of agriculturally generated water pools and rice
fields resulted in its reproductive expansion into the dry season as well as
into perpetually arid regions of
A. gambiae has been sub-classified into M and S molecular forms by virtue of X-linked ribosomal DNA (rDNA) distinctions—an indicator of incipient speciation. The M and S forms of A. gambiae also exhibit a strong pre-mating barrier and discrete ecological niches. Two accounts of microsatellite allele frequency differences between the forms have previously been mapped to the X chromosome, and a fixed transposable element difference has been found in the vicinity of a pericentromeric X-linked gene. However, previous studies have not found variation on distal regions of the X chromosome or elsewhere in the genome. This information suggests the existence of differential selection in noncoding markers which should presumably evolve neutrally. We hypothesize this phenomenon is occurring because genes linked to these markers are being targeted for selection.
DNA was extracted from 480 male mosquitoes
captured in
The ultimate aim of this study is to reveal genes responsible for early stages of speciation between A. gambiae molecular forms. Recent sequencing and microsatellite data suggest these genes may be linked to the X chromosome, specifically in the pericentromeric region. Better comprehension of the mosquito adaptation process may provide information for the development of alternate approaches toward treatment and prevention of malaria.
Adam D. Will, Neil F. Lobo, and Frank H. Collins; Dept. of
Biological Sciences,
Mayetiola destructor, commonly known as the “Hessian” fly, is a major pest of wheat (Triticum sp.), but efforts to combat crop yield loss by the breeding of resistant strains of wheat are complicated by the virulence-avirulence interactions of Triticum and Mayetiola. Results of previous investigations suggest that a gene-for-gene relationship underlies Hessian fly avirulence to wheat virulence; however, the specific gene involved in the wheat-fly H13-vH13 interaction has not yet been identified. Markers specific to the vH13 locus have facilitated the screening of a Mayetiola destructor BAC library for genomic BAC8i18, which is associated with the avirulence locus. In this investigation, BAC8i18 was subcloned, sequenced, and assembled. Sequence analysis using available bioinformatics programs (FGENESH, GENSCAN, and geneid) revealed six putative genes, designated Mad1 through Mad6. NCBI BLASTp (nr) and Ensembl tBLASTn (A. gambiae masked genomic sequence; D. melanogaster genomic sequence) were used to reveal possible homology and synteny with insect genomic sequences. Mad1, Mad3, and Mad6, though predicted by all three gene-finding programs, do not have significant matches (each has an E-score>0.01) in the NCBI nr database or the Ensembl assemblies. Genes Mad2, Mad4, and Mad5 show significant sequence identity with D.melanogaster genes encoding a-catenin-related protein, Toll-6, and GCN2, respectively, and uncharacterized predicted transcripts in the A. gambiae assembly. Furthermore, Mad2, Mad4, and Mad5 all localize on the 3L chromosome of A. gambiae, suggesting evolutionary synteny by gene cluster conservation (but not through conservation of gene order); interestingly, this localization of Mad genes is not observed when D. melanogaster provides the reference genome. This initial analysis not only provides important clues to the identity of the gene responsible for the vH13 trait in Mayetiola destructor, but also raises important questions regarding the selective pressures that resulted in synteny between Mayetiola and Anopheles but not Drosophila.
Nick Russell*, Mehgan Schaffner*, Shannon Gray*, Daniela DeToni*,
Jennifer Brisson°, and
*Department of Biological Sciences,
Department of Biology,
Clare Boothe Luce Associate Professor,
Recently, scientists have been attempting
to find a genetic basis behind evolutionary changes and to understand how
changes at the molecular level can affect phenotypic diversity. This is currently a popular research area
because it can lead to a better understanding of natural selection pathways
resulting in the divergence of phenotypes among a single species. A major candidate gene that is thought to be
associated with this phenotypic divergence is the yellow gene. This project involves investigation of both
environmental and genetic effects on the phenotype of abdominal pigmentation in
Drosophila polymorpha. This
species is unique in that it exhibits a cline in abdominal pigmentation on
mainland
Characterization of
Apoptotic Pathways in a Murine Mammary Tumor Cell Line
Anna Acosta1,
Meggan Valrance2, and JoEllen Welsh2
1:
2:
Elitza Sevova and Dr.
Cloning of the HuC Promoter for Analysis of Retinal Repair in
Zebrafish
Zebrafish is a useful model system for studying neuronal regeneration from various types of retinal damage. It was shown that constant exposure to intense light results in photoreceptor apoptosis in the zebrafish retina, but that a return to normal light conditions allows photoreceptor regeneration. Recent findings present compelling evidence that Müller glial cells play a role in this regeneration process. A DNA construct utilizing the Cre recombinase and lox P sites was proposed to specifically label Müller glial cells and trace their fate during retinal regeneration. To test the feasibility of the Cre/lox system in the transgenic zebrafish retina, a preliminary construct involving the neuronal-specific HuC promoter was designed. Genomic zebrafish DNA was used to PCR amplify this promoter as either a 3.1 kb or 2.5 kb fragment that contains the necessary neuronal-specific sequences. These PCR products were ligated into the TOPO 2.1 vector and sequenced. Preliminary sequence data confirmed the cloning of the expected promoter elements. Both forms of the HuC promoter were ligated into the pT2KXIG poly transformation vector. The next step will involve ligating the Cre recombinase gene into the HuC promoter-containing pT2KXIG vector. Upon completion, the construct will be injected into zebrafish embryos, which will be crossed with wild-type fish upon maturation. The F1 progeny of that cross will then be analyzed for Cre expression by immunoblots. Those F1 fish that express Cre will then be crossed with fish containing a lox P-EGFP transgene to determine if the Cre/lox system can be used in future experiments with zebrafish. Ultimately, the Cre/lox system will be used to identify the role of Müller cells in retinal regeneration.
MECHANISMS OF
TRAIL-INDUCED APOPTOSIS IN OVARIAN CANCER CELL LINES
Chrissy Ratajczak &
Dr. Alan Johnson
Rab5 as a stimulatory molecule for the phagocytosis of mycobacterium
avium
Phagocytosis is an essential first step for the ingestion and subsequent killing of microbes by macrophages and other phagocytes. Based on its vital role in endocytotic fusion events, it is thought that the small molecular weight GTPase Rab5 may play an essential role in the phagocytotic process. Mycobacteria are known to undergo receptor-mediated phagocytosis by macrophages and have been shown to localize with Rab5 on their phagosomal membranes. To analyze the phagocytotic effects of Rab5, we overexpressed Rab5 in J774 cells and infected the cells with Mycobacterium avium. We found that overexpression of Rab5 caused a significant increase in complement opsinized M. avium uptake by the cells in comparison to cells transfected with an empty plasmid vector. Experiments also showed that overexpression of both constitutively active and dominant negative Rab5, Rab5(Q79L) and Rab5(S34N) respectively, increased uptake levels to a similar extent as compared to the wildtype Rab5. These findings indicate that Rab5 does not have an active function in complement mediated phagocytosis of mycobacteria, but the presence of Rab5 in the host cell does promotes uptake of M. avium, suggesting the possibility of Rab5 as a stimulatory molecule for phagocytosis.
Association of CBD1-4 SNP Polymorphism in AeIMUC1 gene with
Aedes agypti Susceptibility to Plasmodium gallinaceum
Mitosis and Cytokinesis in Acentrosomal BSC-1 Cells
Department
of Biology,
Centrosomes form the major component in the microtubule-organizing center (MTOC) of eukaryotic cells. However, it is not fully known what parts of mitosis the centrosomes and MTOC play a critical role in. The aim of this experiment was to determine what events during mitosis and cytokinesis involving spindle formation and organization of the various microtubules are dependent upon the centrosome. BSC-1 African green monkey kidney cells were transfected with a green fluorescent protein (GFP)-a tubulin construct and a cell line constitutively expressing GFP-a tubulin was established (BSC-1a). The BSC-1a cell line showed no significant difference in mitotic doubling time from the control BSC-1 cells. BSC-1a cells showed an average cell cycle timing of 12.5 hours from the entrance into M phase to subsequent M phase under both normal conditions and when exposed to fluorescent light. The microtubule network showed no observed defects in spindle formation due to the GFP labeled a-tubulin (Rusan, 2001). The centrosomes and resulting MTOC were microsurgically removed from BSC-1a cells during interphase. Cut BSC-1a cells healed the surgery point after an average of 2.5 hours (N=7). The acentrosomal BSC-1a cells were imaged under fluorescent microscopy from the cut point through mitosis and cytokinesis. The acentrosomal cells showed a non-bipolar spindle formation 75% of the time during M phase (N=4). This failure to produce a bipolar spindle most likely indicates that the centrosome has a role in proper spindle formation. In the non-bipolar spindles, astral microtubules appeared to be absent as the spindle showed sporadic movement inside the cell. The same 75% of the cells failed to have proper chromosomal segregation and generated multiple cleavage furrows during cytokinesis. The movement of the spindle within the cell during M phase confirms that astral microtubules nucleate from the MTOC. Midbody formation was also observed in 75% of the cells during cytokinesis (N=4). The presence of the midbody in the acentrosomal cells may show that the midzone microtubules may not nucleate from the MTOC like normal microtubule networks.
Elucidating the role of ARF6 in the Raf/MEK/ERK signaling pathway
during melanoma cell invasion.
Anne Marie Corgan, Vandhana Muralidharan and Crislyn D’Souza-Schorey, Department of Biological Sciences, University of Notre Dame
The incidence rate of melanoma is increasing faster than any other form of
cancer; it is rising at an epidemic rate of more than 4% annually. Melanomas are highly invasive. Understanding the mechanisms contributing to
cell invasion could potentially lead to new therapies and treatments for
melanomas. ERK
(Extracellular-signal-regulated kinase) is found to be upregulated in melanoma
cells, tissues, and organs. However, there still exists considerable
gaps in the understanding of molecular events that result in increased ERK
activation during melanoma tumor progression. Previous studies done by our
laboratory using invasive melanoma cell lines have suggested that ARF6
(ADP-ribosylation factor 6), a member of the Ras superfamily of small GTPases,
may regulate invasion through the MEK/ERK signaling pathway. We have shown that
activation of ARF6 is increased in response to physiological stimuli that
provoke ERK activation, whereas the inhibition of ARF6 activity blocks ERK
phosphorylation during melanoma cell invasion in vitro extracellular matrix
degradation assays. Our current efforts
are directed at testing the hypothesis that ARF6-GTP promotes ERK activation by
enhancing the activity of one or more kinases of the Raf-MEK-ERK pathway. Our studies on the relationship between ARF6
and Raf-1 will be discussed.