University of Notre Dame
Department of Biology
NSF Summer Undergraduate Research Program

Summer 2002 Symposium Abstracts

Analysis of Anopheles gambiae Y-chromosome to infer population
history and dynamics of an important malaria vector

Anthony Griswold
Dr. Nora Besansky

Understanding the population genetics of malaria vectors is key to the utilization of malaria control strategies that aim to replace vector with non-vector populations.  Genetic manipulations of vectors, which make them incapable of disease transmission, are of value when applied to natural populations, making the study of Anopheles gambiae populations imperative.  To help understand the population dynamics of this African vector, this study examined the Y-chromosome of species from the western and eastern coasts of Africa (Senegal and Kenya respectively).  As a haploid chromosome, the Y is believed to escape recombination while maintaining polymorphic markers, making it a useful tool to study population history and dynamics.  Based on PCR amplification, purification, and DNA sequencing of about 3kb from the Y-chromosome, populations across the African continent were found to have no DNA variation. Possible explanations of this lack of variation include lowered effective population size of the Y-chromosome, asymmetric mating in the populations, and natural selection on the chromosome.  However, the data from this experiment along with previous research tends overall to suggest that current An. gambiae population in Africa arose from a small founding group relatively recently in evolutionary time.

Resveratrol (3,5,4’-trihydroxystilbene) is a compound found at high concentrations in red wine which may play a protective role against the progression of breast cancer by mimicking some effects of estrogen. The aim of our study was to determine the effect of resveratrol on the estrogen receptor positive human breast cancer cell line T47D.  Western blot analysis indicated that low doses of resveratrol up-regulated the expression of the vitamin D receptor (VDR) protein. Through binding to the VDR, vitamin D is known to cause cell cycle arrest and apoptosis in breast cancer cells and therefore resveratrol-mediated up-regulation of VDR may sensitize the cells to vitamin D treatment.  In crystal violet analysis, vitamin D caused a dose-dependent decrease in breast cancer cell number compared to vehicle control.  Further analysis showed at combined treatment with resveratrol and vitamin D was more effective in reducing cell number than vitamin D alone.  Cell cycle analysis in progress will quantitate the percentage of cells undergoing cell cycle arrest and apoptosis following treatments. By comparing the effect of vitamin D alone to that of resveratrol in combination with vitamin D, we will determine if up-regulation of VDR by resveratrol sensitizes breast cancer cells to vitamin D treatment, which may increase expression of vitamin D target genes involved in cell cycle arrest and apoptosis.  Since analogs of vitamin D are currently in clinical trials to treat breast cancer, this study may show that dietary factors such as resveratrol could enhance a patient’s responsiveness to treatment.

Microsatellites have proven to be very useful as genetic markers, as they seem to be ubiquitous and randomly distributed throughout most eukaryote genomes.  However, our laboratory and others have determined that this paradigm does not necessarily apply to the mosquito Aedes aegypti.  Here we describe our efforts to date to identify and develop microsatellite markers for A. aegypti.  We developed three libraries specifically enriched for GA, AAT, and TAGA microsatellite sequences, respectively.  From these libraries we were able to identify a small number of PCR-amplifiable putative single-copy polymorphic microsatellites.  Despite this success, our results also provide some insights into microsatellites’ general lack of utility for this mosquito.  We obtained sequence data on 253 clones from the three libraries, and these were analyzed to determine whether they were 1) located within known mosquito repetitive elements, 2) situated in new, undescribed repetitive elements, or 3) represented a re-isolation of the same sequence.  Only 69 sequences passed this analysis, suggesting that microsatellites in A. aegypti, although relatively abundant and likely distributed throughout the genome, are most often associated with or within short interspersed repetitive elements, making them useless as genetic markers.

Mosquito-borne diseases have and still continue to cause significant problems in human medicine.  Methods to control these diseases have been difficult and extensive.  The most promising breakthroughs have been in the area of utilizing transposable elements to transform insects.  Because successful transformation of Drosophila melanogaster has been achieved, it is hoped that such techniques can be used to reach similar results in mosquitoes that carry malaria.  Our research focused on the initial stages of creating a plasmid vector that will insert a specific gene into the genome of Anopheles gambiae, one of the major carriers of malaria.  By using the transposable element, piggyBac, we hope to insert the green fluorescent protein (GFP) gene into the germ-line cells Anopheles gambiae mosquitoes.  Successful insertion would result in generations of mosquitoes that glow a fluorescent green color.  This preliminary achievement will open new doors to eventually producing genetically engineered mosquitoes that are resistant to carrying malaria and other diseases.

Understanding the genetic aberrations that lead to retinal degeneration is necessary for elucidating the essential processes in photoreceptor cells. In a previous experiment, two D. melanogaster mutants were created through irradiation of the second chromosome. Both mutants exhibit dominant retinal degeneration and are homozygous lethal. Deletion and recombination mapping were employed to identify the cytologic location of the genetic mutations in order to determine whether the mutant phenotypes result from mutations in the same gene. Preliminary results of 65 deficiency crosses localized the lethal mutation in both stocks to one of several chromosomal regions not covered by the deficiency kit.

 Prostate cancer is the second leading cause of cancer related death for men in the United States.  Anti-androgens such as Casodex are a widely used form of treatment.  Patients treated with anti-androgens ultimately become resistant to the drug and develop hormone refractory tumors.  Studies have shown a positive correlation between increased levels of Bcl-2, a protein involved in the regulation of the mitochondrial apoptotic pathway, and prostate cancer progression. Two sublines of LNCaP non-invasive human prostate cancer cells over expressing Bcl-2 were treated with varying concentrations of tumor necrosis factor a (TNF-a) and Casodex.  Crystal Violet assays were used to determine relative viability and MTT assays to determine the relative mitochondrial activity.  Western blot analyses were run to determine the androgen receptor status of the Bcl-2 over-expressing cells.  The Bcl-2 over-expressing cell lines treated with TNF-a had a significantly lower amount of death than the wild-type cell line.  Western blots showed androgen receptors were absent in the nuclei of the cells treated with Casodex.

Previous studies have shown that 1,25-dihydroxyvitamin D3, the active form of Vitamin D3, induces growth arrest and apoptosis in MCF-7 breast cancer cells.  1,25-dihydroxyvitamin D3 mediated apoptosis is characterized by cytochrome c release from the mitochondria and subcellular redistribution of Bax.  TPA, a phorbol ester, causes growth arrest in MCF-7 cells without triggering these apoptotic events.  To determine whether 1,25-dihydroxyvitamin D3 and TPA trigger independent pathways in breast cancer cells, MCF-7 cells were treated with 1,25-dihydroxyvitamin D3 in the presence or absence of TPA, and cell growth and apoptosis were assessed by Crystal Violet assay, Western Blotting, and immunofluorescence.  When added together to MCF-7 cells, TPA was found to potentiate the apoptotic effects of 1,25-dihydroxyvitamin D3.  The interaction of TPA and Vitamin D3 in a D3 resistant MCF-7 cell derivative was also studied, and the data indicate that while Vitamin D3 alone did not induce apoptosis in MCF-7DRes cells, TPA sensitized the cells to the apoptotic action of 1,25-dihydroxyvitamin D3 when the compounds were added simultaneously.  These studies indicate that TPA and 1,25-dihydroxyvitamin D3 trigger independent pathways leading to growth arrest and apoptosis in breast cancer cells, and that Vitamin D3 resistance might be overcome through activation of TPA dependent pathways.  These findings support the concept that combining Vitamin D3 analog therapy with agents that mimic TPA might be useful in treatment of breast cancer.

 RNAi (interference) is a phenomenon whereby when double-stranded RNA that corresponds to a gene of interest is introduced into an organism and interferes with expression of the endogenous gene.  Should we successfully develop such a system as a powerful tool for creating loss of function transgenic organisms, specific loss of function mutants can be used in the development of species-specific insecticides in efforts to control insect-borne diseases.  Using Drosophila melanogaster as a model insect and cinnabar (gene for brown eye pigment) as a model gene provides a non-lethal method of detecting RNAi in one of the most versatile organisms used in molecular genetic research.  In theory, by using the foldback cinnabar sequence, we will prevent the phenotypic expression of the ommochrome eye-pigment biosynthesis pathway with RNAi resulting in reduction or depletion of eye color in Drosophila.  This experiment requires microinjection of the pHermes vector containing the cinnabar foldback sequence along with the piggyBac transposable element into Drosophila embryos.  Piggybac will excise and insert the foldback transgene into the genome of Drosophila.  Double-stranded RNA will be produced by in-vivo transcription, thereby, interfering with cinnabar expression.

Single nucleotide polymorphisms (SNPs) are variations in genomic DNA, where alternative forms of a sequence exist in individuals of a population with a frequency of 1% or more.  SNPs are increasing in popularity as markers in genetic analysis because they are more prevalent in the genome, and if located in genes, SNPs can often directly affect protein structure or expression levels, and are much more stably inherited.  Presently our lab is focusing on trying to identify and characterize SNPs in laboratory strains of Aedes aegypti.  The strains of interest are Red, Moyo R, Liverpool, Formosa, Trinidad, Rockefeller, and Bronze.  The process used to identify SNPs is as follows:  PCR is first used to amplify genomic DNA in individuals representing each strain, then the PCR product is digested by a restriction enzyme, lastly, the digested DNA is analyzed by McSNP assay using the HYBAID DASH machine.  The individual mosquitoes were screened for the presence of previously identified SNPs at two genetic marker loci. Polymorphism was detected with both markers between and within strains.

 Anopheles funestus Giles and Anopheles gambiae Giles are the two most important malaria vectors in Africa. The activity of An. funestus spans the dry season, when An. gambiae is normally inactive. This overlapping characteristic of mosquito activity gives malaria a year-round deadliness in the Afrotropical region. Host feeding behaviors and vector competence have been shown to be under genetic control. Known genetic markers are necessary to localize these genes of interest and a physical map is vital for map-based cloning efforts. While the An. gambiae genome project has facilitated much genomic research within An. gambiae, An. funestus has received much less attention. Therefore, we have constructed and analyzed a cDNA library which loosely represents the transcriptome of An. funestus. Approximately 200 cDNA clones were sequenced, with the goal to sequence 1000. Then, the sequences were blasted against the NCBI non-redundant (nr) database to make predictions in protein function. Also, this library enabled us to conduct 3 types of comparative genomic studies between An. funestus and An. gambiae. Firstly, arm correspondence between species was investigated. Previous cytological studies indicate that arm 2L of An. funestus corresponds to arm 3R of An. gambiae and consequently arm 3R of An. funestus corresponds to arm 2L of An. gambiae. All other arms correspond (X=X, 2R=2R, 3L=3L). These results were confirmed using in situ hybridization of cDNA probes with both An. funestus and An. gambiae. However it is not yet known if the entire arm or a part of it was translocated during the evolutionary history of An. funestus. Secondly, gene order comparisons are currently being done. The next step we will take is to compare gene sequence homology between species. Of special note are the sequences of genes known to be involved in malaria transmission. Change in the sequence of these genes has important consequences in any control approach, whether the strategy involves drugs or a transformed mosquito line.

Cytoplasmic dynein (CD) is a microtubule motor protein responsible for multiple aspects of membrane transport and chromosomal segregation during mitosis.  The Vaughan Laboratory is interested in mechanisms that cytoplasmic dynein uses to bind cargo. Several subunits have been implicated in mediating organelle binding, including the intermediate chains (ICs), light intermediate chains (LICs) and light chains (LCs).  We have focused on the ICs because previous studies revealed that they interact with the p150Glued subunit of dynactin and that this interaction is regulated via IC phosphorylation at serine-84. To determine the impact of IC phosphorylation, site-directed mutants that mimic either  phosphorylated (S84D) or dephosphorylated (S84A) IC-2C were transfected into COS-7 cells and tested for membrane transport defects.  S84A mutants perturbed dynein-mediated transport by competing with intact dynein for organelle binding. In contrast, S84D mutants failed to compete with dynein for organelle binding or disrupt dynein-mediated transport.  Because previous studies had been performed using full-length ICs which can associate with the dynein LC subunits, we prepared truncated ICs which lack LC-binding sites and tested for organelle transport defects by transfection. The results using truncated ICs were different from results gathered using full-length ICs, with the S84A mutants displaying less disruption of dynein-mediated transport, and S84D mutants displaying more disruption. Although further work will be needed to clarify these observations, these findings suggest that the N-terminus of the ICs and interacting proteins could contribute to organelle binding.