1. Gene Expression And Regulation Bioinformatics January 11, 2006 D. A. McClellan (david_mcclellan@byu.edu)

2. 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

3. 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 Gene expression is regulated in several basic ways Page 157

4. DNARNA cDNA phenotypeprotein Page 159 Central Dogma of Molecular Biology

5. DNARNA cDNA protein DNARNA cDNA protein UniGene SAGE microarray Fig. 6.2 Page 159

6. 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

7. Fig. 6.3 Page 161 exon 1exon 2exon 3intron transcription RNA splicing (remove introns) polyadenylation Export to cytoplasm AAAAA 3’5’ 3’ 5’3’

8. Relationship of mRNA to genomic DNA for RBP4 Fig. 6.4 Page 162

9. 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) Convert RNA to complementary DNA Subclone into a vector Sequence the cDNA inserts. These are Expressed Sequence Tags Page 162-163 vector insert

10. 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

11. Cluster sizes in UniGene This is a gene with 1 EST associated; the cluster size is 1 Page 164 & Fig. 2.3, Page 23

12. Cluster sizes in UniGene This is a gene with 10 ESTs associated; the cluster size is 10 Page 164

13. Cluster sizes in UniGene (human) Cluster sizeNumber of clusters 1  10,400 27,100 3-46,800 5-85,300 9-163,800 17-323,100  500-10001,500  2000-4000130  8000-16,00012  16,000-30,0003 UniGene build 186, 9/05 Page 164

14. Ten largest human UniGene clusters Cluster sizeGene 22,925eukary. translation EF ( Hs. 522463 ) 22,320eukary. translation EF ( Hs. 4395522 ) 16,562actin, gamma 1 ( Hs.514581 ) 16,309GAPDH ( Hs.169476 ) 16,231actin, beta ( Hs.520640 ) 11,076ribosomal prot. L3 ( Hs.119598 ) 10,517dehydrin (Hs.524390) 10,087enolase 1 (alpha) (Hs.517145) 9,973ferritin (Hs.433670) 8,966metastasis associated ( Hs.187199 ) UniGene build 186, 9/05 Table 6.2 Page 165

15. UniGene brain libraries

16. UniGene lung libraries

17. Fig. 6.7 Page 167

18. Fig. 6.7 Page 167 BrainLung

19. n-sec1 up-regulated in brain CamKII up-regulated in brain surfactant up- regulated in lung Page 167

20. 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 = 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 N A ! N B ! c! C! (N A + N B )! g1 A ! g1 B ! (N A – g1 A )!(N B – g1 B )!

21. 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

22. Fig. 6.8 p. 168-169 http://mgc.nci.nih.gov

23. 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

24. Tag 1 Tag 2 Tag n Cluster 1 Cluster 2 Cluster 3 Cluster 1 SAGE tags are mapped to UniGene clusters Page 169