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Serial analysis of gene expression (SAGE) is an experimental technique used for quantitative, high throughput gene expression analysis (Velculescu et al., 1995). SAGE involves the isolation of short unique sequence tags from a specific location within each transcript. These sequence tags are concatenated, cloned, and sequenced. The frequency of particular transcripts within the starting cell population is reflected by the number of times the associated sequence tag is encountered within the sequence population. The SAGEmap database is a repository for some of this SAGE data, and tools that allow gene expression analysis are also available on the SAGEmap Web site (Lal et al., 1999). The virtual Northern predicts the SAGE tag in a user-supplied mRNA sequence and calculates the distribution of the tag in the SAGE libraries, thus providing a virtual picture of the expression pattern of the mRNA. In the SAGE xProfiler, the user selects two pools of libraries, such as colon cancer versus normal colon. The tool calculates which SAGE tags are more abundant in one pool or the other. The SAGE tags are mapped to UniGene clusters to provide biological context for the results.
High-density oligonucleotide and cDNA microarrays are a relatively new technique being used to monitor gene expression on a genome-wide scale. The technique uses the same principles of nucleic acid hybridization as do Northern and Southern blots but on a much larger scale. Thousands of gene-specific probes are arrayed on a small matrix, such as a glass slide or microchip, and this matrix is probed with labeled nucleic acid synthesized from a tissue type, developmental stage, or other condition of interest. The expression profiles of thousands of genes under that condition can thus be assayed simultaneously. The array probes can be derived from oligonucleotides or cDNAs. In many cases, the probes for cDNA arrays are 3’ ESTs (Duggan et al., 1999). For human expression analysis, UniGene clusters can be used as a source of additional information about the ESTs on the array. Microarray technologies and the bioinformatics challenges surrounding them are discussed in Chapter 16.
EXPRESSED SEQUENCE TAGS (ESTs)
INTERNET RESOURCES FOR TOPICS PRESENTED IN CHAPTER 12
dbEST home page http://www. ncbi.nlm.nih.gov/dbEST/
List of dbEST libraries http://www. ncbi.nlm.nih.gov/dbEST/libs—byorg.html
dbEST summary by http://www. ncbi.nlm.nih.gov/dbEST/dbEST_
How to submit ESTs to http://www. ncbi.nlm.nih.gov/dbEST/how—to—
EST Projects at http://genome.wustl.edu/gsc/est/navest.pl
The I.M.A.G.E. http://image.llnl.gov/
UniGene http://www. ncbi.nlm.nih.gov/UniGene/
The UniGene build http://www.ncbi.nlm.nih.gov/UniGene/build.html
UniGene query engine http://www. ncbi.nlm.nih.gov/UniGene/query.cgi
TIGR Gene Indices http://www. tigr. org/tdb/tgi.html
TIGR Orthologous Gene http://www. tigr. org/tdb/toga/toga.html
GeneMap ’ 98 http://www. ncbi.nlm.nih.gov/genemap/
dbSNP http://www. ncbi.nlm.nih.gov/SNP/
Cancer Genome Anatomy http://www. ncbi.nlm.nih.gov/ncicgap/
CGAP Digital Differential http://www.ncbi.nlm.nih.gov/CGAP/info/ddd.cgi
CGAP xProfiler http://www.ncbi.nlm.nih.gov/CGAP/hTGI/xprof/
Serial Analysis of Gene cgapxpsetup.cgi http://www. ncbi.nlm.nih.gov/SAGE/
SAGE virtual Northern http://www. ncbi.nlm.nih.gov/SAGE/sagevn.cgi
SAGE xProfiler http://www. ncbi.nlm.nih.gov/SAGE/sagexpsetup.cgi
You have been studying the histone deacetylase gene, RPD3, in the yeast Sacchar-omyces cerevisiae. You are moving to a lab that works on zebrafish, and you would like to continue your work on this gene. You wonder how difficult it will it be to clone the zebrafish ortholog of RPD3.
1. What is the GenBank accession number of the first listed RPD3 protein sequence from Saccharomyces cerevisiae?
2. Do the public sequence databases already contain any zebrafish proteins that are likely orthologs of RPD3?
a. What type of sequence comparison search should you perform?
b. To interpret the search results of your sequence comparison, you will need to know the scientific name for zebrafish. What is the scientific name?
c. Are there any zebrafish protein orthologs of yeast RPD3?
3. You remember that the EST database is an excellent source of sequence data.
a. What type of sequence comparison should you perform to find EST hits to the yeast protein sequence?
b. Are there any zebrafish EST hits to this yeast protein sequence?
4. Do the five top scoring ESTs belong to the same UniGene cluster?
5. What is the GenBank accession number of the human sequence that matches this UniGene cluster?
6. What cDNA clone does the top scoring EST hit come from?
7. Is this EST from the or 3' end of the cDNA clone?