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Gene Expression And Regulation Bioinformatics January 11, 2006 D. A. McClellan ([email protected]) Gene Expression • Expressed in the transcriptome • Every eukaryotic genome contains between 5000-60,000 protein-coding genes • Only a small subset of those genes are transcribed Gene expression is regulated in several basic ways • by region (e.g. brain versus kidney) • in development (e.g. fetal versus adult tissue) • in dynamic response to environmental signals (e.g. immediate-early response genes) • in disease states • by gene activity Page 157 Central Dogma of Molecular Biology DNA RNA protein phenotype cDNA Page 159 DNA RNA protein cDNA DNA RNA protein cDNA UniGene SAGE microarray Fig. 6.2 Page 159 Expression Databases & Analyses • UniGene: for the comparison of cDNA libraries – Goals: (1) create one unique entry for each gene, (2) collect all the ESTs associated with each gene • SAGE: Serial Analysis of Gene Expression library • DNA microarrays 5’ exon 1 3’ intron exon 2 3’ exon 3 5’ intron transcription 5’ 3’ RNA splicing (remove introns) 3’ 5’ polyadenylation 5’ AAAAA 3’ Export to cytoplasm Fig. 6.3 Page 161 Relationship of mRNA to genomic DNA for RBP4 Fig. 6.4 Page 162 Analysis of gene expression in cDNA libraries A fundamental approach to studying gene expression is through cDNA libraries. • Isolate RNA (always from a specific organism, region, and time point) insert • Convert RNA to complementary DNA • Subclone into a vector vector • Sequence the cDNA inserts. These are Expressed Sequence Tags Page 162-163 UniGene: unique genes via ESTs • Find UniGene at NCBI: www.ncbi.nlm.nih.gov/UniGene • UniGene clusters contain many ESTs • UniGene data come from many cDNA libraries. Thus, when you look up a gene in UniGene you get information on its abundance and its regional distribution. Page 164 Cluster sizes in UniGene This is a gene with 1 EST associated; the cluster size is 1 Page 164 & Fig. 2.3, Page 23 Cluster sizes in UniGene This is a gene with 10 ESTs associated; the cluster size is 10 Page 164 Cluster sizes in UniGene (human) Cluster size 1 2 3-4 5-8 9-16 17-32 Number of clusters 10,400 7,100 6,800 5,300 3,800 3,100 500-1000 2000-4000 8000-16,000 16,000-30,000 UniGene build 186, 9/05 1,500 130 12 3 Page 164 Ten largest human UniGene clusters Cluster size 22,925 22,320 16,562 16,309 16,231 11,076 10,517 10,087 9,973 8,966 UniGene build 186, 9/05 Gene eukary. translation EF (Hs. 522463) eukary. translation EF (Hs. 4395522) actin, gamma 1 (Hs.514581) GAPDH (Hs.169476) actin, beta (Hs.520640) ribosomal prot. L3 (Hs.119598) dehydrin (Hs.524390) enolase 1 (alpha)(Hs.517145) ferritin (Hs.433670) metastasis associated (Hs.187199) Table 6.2 Page 165 UniGene brain libraries UniGene lung libraries Fig. 6.7 Page 167 Brain Lung Fig. 6.7 Page 167 CamKII up-regulated in brain n-sec1 up-regulated in brain surfactant upregulated in lung Page 167 Fisher’s exact test provides a p value Digital differential display (DDD) results in UniGene are assessed for significance using Fisher’s exact test to generate a p value. p= NA! NB! c! C! (NA + NB)! g1A! g1B! (NA – g1A)!(NB – g1B)! The null hypothesis (that gene 1 is not differentially regulated in a comparison of two libraries) is rejected when p is < 0.05/G (where G = the number of UniGene clusters analyzed). Pages 165 Pitfalls in interpreting cDNA library data • bias in library construction • variable depth of sequencing • library normalization • error rate in sequencing • contamination (chimeric sequences) Pages 166-168 http://mgc.nci.nih.gov Fig. 6.8 p. 168-169 Serial analysis of gene expression (SAGE) • 9 to 11 base “tags” correspond to genes • measure of gene expression in different biological samples • SAGE tags can be compared electronically Page 169 SAGE tags are mapped to UniGene clusters Tag 1 Tag 1 Tag 2 Tag n Cluster 1 Cluster 2 Cluster 3 Cluster 1 Page 169