Alternative splicing is a biological phenomenon found in eukaryotes that produces mRNAs for different protein isoforms from the same gene. To examine brain specific alternative splicing we are using the microarray data set generated by a recent collaboration between UCSC and Affymetrix. The microarray was specifically designed to probe for mouse alternative splicing events. The intensity value of each splice junction probe was compared to the overall expression level of the gene in which the splice junction resides in each tissue. These measurements were clustered to group tissues with similar splice junction profiles together. All of the brain tissues were found to be in a distinct cluster, indicating that the nervous system seems to produce distinct alternative mRNAs that may be important for neuron specific functions. Microarray data was validated with PCR analysis on a group of twenty candidate genes. We are now using the microarray data to discover splicing factors that regulate brain specific alternative splicing. A set of genes that code for RNA binding proteins is being studied to determine whether any show expression patterns that are correlated or anti-correlated with observed patterns of splice junction use. Once the splicing factors are known, biological pathways can be pieced together and further tests can be made to better understand the role of different protein isoforms in neuronal processes and how the failure of alternative splicing can lead to neurodegenerative disease.

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VALIDATION OF ALTERNATIVE SPLICING IN THE HUMAN GENOME. Jessica Bryant, Paul Perry, Aaron Miller, Talia Romano, Mike Smith, Manuel Ares. Hughes Undergraduate Research Lab and Department of Molecular, Cell, and Developmental Biology, UC Santa Cruz.

The enigma of how a relatively small number of genes (35,000) can code for the entire make up of the human body is one of the larger questions resulting from the near completion of the Human Genome. Alternative splicing is one mechanism, which may help explain this discrepancy. Varying translational biological products may be derived from the same gene by inclusion or exclusion of certain exons or introns in the primary transcript. The goal of our current research is to validate micro array assays using RT-PCR, which profile the various isoforms derived from suspected alternatively splicing genes. APLP2, NASP MYL6 are genes of particular interest due to their biological importance. APLP2 is an amyloid precursor protein that may cause Alzheimer's disease. NASP codes for a highly autoammunogenic protein responsible for histone binding. MYL6 codes for myosin alkali light chains, which are structural proteins found in various forms of smooth muscle and non-muscle cells. By comparing signals in alternative splicing micro arrays with measurements made by RT-PCR, we will be able to examine how well the arrays function as a tool for analyzing alternative splicing and where they need improvements.

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EXPRESSION OF CD36 POLYMORPHISMS ASSOCIATED WITH PLASMODIUM BINDING ABILITY IN VARIANT HEALTHY HUMAN TISSUE. Mariska Brady, Emily White, Oded Porat, Manuel Ares. Hughes Undergraduate Research Laboratory, Department of Molecular, Cell, and Developmental Biology, UC Santa Cruz.

Malaria is a major source of morbidity in the world. The causative agent of malaria is a mosquito-borne parasitic protozoan called Plasmodium. CD36 is a key human cell surface protein reported to be essential for binding of Plasmodium to human cells. It has been reported that there are two isoforms of CD36, one containing exons 4 and 5 and one lacking these two exons. The form lacking exons 4 and 5 encodes a shorter version of CD36 reported to have altered ability to bind the malarial parasite. Increased severity of cerebral malaria has been reported in individuals carrying polymorphisms that increase the skipping of CD36 exons 4 and 5. These reports raise questions concerning the role of CD36 alternative splicing in malaria infection. Our research goal is to explore the prospective alternative splicing of CD36, beginning with a survey of normal human tissues. In order to detect the two isoforms reported thus far, we are designing specific primers for PCR amplification of the region of the CD36 mRNA spanning exons 4 and 5. We will then use the primers to detect alternatively spliced CD36 mRNA in different human tissues. Understanding expression of CD36 may reveal whether this protein plays a major role in the course of cerebral or other courses of infection by Plasmodium.

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VALIDATING ALTERNATE SPLICING IN HUMAN GENES HMRT1L1 AND ITGA6. Lily Edmondson, Rebecca Loewen, Sean Fitzwater, Manuel Ares. Hughes Undergraduate Research Laboratory and Department of Molecular, Cell, and Developmental Biology, UC Santa Cruz.

Using the UCSC genome browser, the Hughes Undergraduate Research Lab created a database of alternate splicing predictions of a subset of human genes. We will test the alternate splicing predictions made about HRMT1L1, which codes for arginine N-methyltransferase 2, and the integrin gene ITGA6, encoding an extracellular matrix protein. Microarrays designed to detect predicted isoforms of these genes have been made. RNA was isolated from different cell lines and the cDNA was hybridized to the array. We will validate and further characterize the array data by performing RT-PCR designed to detect the given isoforms. We will reverse transcribe HRMT1L1 and ITGA6 mRNAs by using the proper primers, determined by analysis of the human genome. The cDNA obtained will be amplified by PCR and the products will be run on electrophoresis gel. We expect to see two different sizes of cDNA for each gene: the shorter segments will represent the mRNA in which an exon was excluded. We will compare our results to the array data to determine the accuracy of the array.

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RNA RECOGNITION AND CLEAVAGE BY RNASE III FROM THE THERMOPHILIC BACTERIA THERMATOGA MARITIMA. Laura Tomedi, Manuel Ares. Department of Molecular, Cell, and Developmental Biology, UC Santa Cruz.

RNase III enzymes are a large family of specific double stranded RNA (dsRNA) endonucleases. Members include Rnt1 from Saccharomyces cerevisiae, Dicer from Drosophila melanogaster, and RNase III from Escherichia coli. They are found in eubacteria and eukaryotes, but not in archae and process a variety of RNA in the cell, including snRNA, snoRNA, RNA for RNAi, and rRNA. In bacteria, this includes the processing of the pre-rRNA transcript into the 23S and 16S rRNA. RNase III of the thermophile Thermatoga maritima, was chosen because it has been found that enzymes from thermophiles usually crystallize more easily, and the enzyme's structure can be studied using X-ray crystallography. To characterize the function of the enzyme and map cleavage sites, we used Thermatoga RNase III protein and a synthetic dsRNA (mimicking the stem of the pre-23S rRNA). The RNase III open reading frame was cloned from Thermatoga genomic DNA into an E. coli expression vector, introducing a 6-his tag to the C terminus of the protein. The tagged protein was then over expressed in E. coli, and purified using a nickel chelate column. Christine Dunham chemically synthesized the RNA substrate in the Scott lab. Initial results show that the protein efficiently and specifically cleaves the substrate in a magnesium-dependent fashion at 65¡C. Characterizing the cleavage of this substrate by our protein will hopefully lead to a greater understanding of the specificity of the RNase III family.

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ALTERNATIVE SPLICING ANALYSIS OF THE MTR2 GENE IN S. CEREVISIAE. LaurenWilliams, Manuel Ares. Hughes Undergraduate Research Laboratory, and Department of Molecular, Cellular, Developmental Biology, Sinsheimer Laboratories, University of California Santa Cruz.

Compared to the 12,000 alternatively spliced genes in the human genome, only 2 S. cerevisiae genes are alternatively spliced. However, these few may hold a key to our understanding of the evolution of human molecular systems. MTR2 encodes a 21 KDa protein that couples with Mex67p to provide the essential function of mature mRNA export out of the nucleus. MTR2 is predicted to encode 6 splice variants, 5 of which have been confirmed through biochemical experiments. The unconfirmed variant is most interesting, as it would encode a protein with an extra 25 amino acids at its N terminus. The focus of my research has been to understand the structure and expression of MTR2, especially with respect to alternative splicing. When the intron region of this gene is aligned with the corresponding region from related yeasts, we find that the intron splice sites are not conserved. However, it is clear that this region of the S. cerevisiae genome is producing spliced RNA. Through biochemical experiments, we hope to understand the evolutionary context behind the presence of this intronic region in S. cerevisiae. Also, we hope to understand the in vivo expression of all variants, the regulation of this expression, and how the regulation of different variants affects mRNA export out of the nucleus.

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AN OLIGONUCLEOTIDE MICROARRAY TO PROFILE ALTERNATIVE SPLICING IN HUMANS AND ADENOVIRUS. Ross Centers, Manuel Ares. Hughes Undergraduate Research Laboratory, Department of Molecular, Cell, and Developmental Biology, and Department of Computer Science, Jack Baskin School of Engineering, UC Santa Cruz.

With the human genome estimated to contain only 35,000 genes, attention has focused on alternative pre-mRNA splicing as a mechanism to generate needed protein diversity from a relatively small genome. A pilot microarray was designed to profile alternative splicing across the adenovirus type 5 genome and in 65 human genes, selected for expression and characterized splicing. Oligonucleotide probes specific to alternative splice junctions were designed by combining sequence from two exons that are joined by splicing. In order to minimize "half-hybridization", where a probe binds a target containing only one of these two exons, splice junction probe sets were created which balance the contribution to probe-target melting temperature from each half junction. Multiple strategies were employed. "C" probes are centered on the junction with 20 nt from each exon. "S" probes are created by sliding a window of 40 nt across the junction until the difference in Tm of the sequences in either side is minimized. The "W" probes are created by allowing the sequence to grow from the junction until a target total Tm is reached, comprised of approximately equally stable halves. These sets were combined with probes specific to individual exons to fabricate a platform for the comprehensive observation of alternative splicing. Data from human tissue and cell lines could be evaluated to determine the efficacy of these strategies.

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PERL SCRIPTS FOR TRANSPORTING DATA FROM THE HUMAN GENOME BROWSER TO THE SPLICING MICROARRAY DATABASE. Bret Barnes, Manuel Ares. Hughes Undergraduate Research Laboratory, Department of Molecular, Cell, and Developmental Biology, and Department of Computer Science, Jack Baskin School of Engineering.

The human genome browser provides vast amounts of genomic information. We are interested in how the genome is interpreted by the machinery that carries out splicing. The browser contains tracks showing how spliced RNAs (mRNA and EST files) originate from the genes. In order to make microarrays that can detect and measure when and how splicing affects the expression of each gene, we need to access this information from the browser. Data contained in the browser data tables, such as exon genomic positions and exon and intron sequences are needed in order to design oligonucleotides for these microarrays. One of the fastest ways of executing this data retrieval process is to write simple perl scripts that extract the needed information directly from the browsers generated HTML pages. Perl is an excellent tool to use for this because of its extremely efficient parsing ability. Our lab has designed a script that extracts all sequence and position data of exons along with sequence of their neighboring introns. In the future, this script will allow us to rapidly, accurately, and automatically retrieve alternative splicing sequence data for thousands of genes quickly. This data will be used to design the oligonucleotides needed for our labs DNA array experiments.

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CORRELATION OF EXONS WITH PROTEINS OF KNOWN STRUCTURE: AN APPLICATION FOR VISUAL INTERPRETATION. Justin Hayes, Manuel Ares, Carol Rohl. Hughes Undergraduate Research Laboratory, Department of Molecular, Cell, and Developmental Biology, and Department of Computer Science, Baskin School of Engineering.

Do exons correlate with structural components within the proteins that they encode? I present a tool that allows researchers to map exons to proteins of known structure. The application, ExonPDB, provides an interface that allows one to retrieve a particular gene from an exon/intron database (EID1) and compare the gene to the PDB database via a BLAST alignment. Alternatively, a researcher may acquire EID data via a BLAST query against a known PDB sequence. Either way, the resulting alignment is used to produce a RasMol script, which coordinates the exon positions with their relative locations along the protein backbone. ExonPDB provides the researcher with both the RasMol script and the appropriate PDB file. The resulting image is displayed in a ribbon format with each exon's relative position on the protein depicted as a unique color. Through this means, a visual interpretation allows a researcher to formulate preliminary hypotheses as to the relationship between exons with a genome and the secondary structures within the proteins that they encode.

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