1. Gene Sleuthing Lorraine Sartori Majid Masso Paul R. McCreary

2. Objectives: Search a data base to find a colon cancer gene Obtain information about the gene Use “Biology Workbench” to find the expressed gene’s protein sequence Determine the structure of the protein Use microarray to locate the normal gene

3. Search For a Colon Cancer Gene 1.Go to: http://bioquest.org/bedrock/http://bioquest.org/bedrock/ 2.Click on resources and then Other Resources 3.Click on “OMIM” 4.Search for –colon cancer 5.Select #114500

4. Obtain Information about the gene

5. Growth factor-induced protein phosphorylation plays a key role in the signal transduction that leads to mitogenic responses. Most growth factor receptors are transmembrane tyrosine kinases or are associated with cytoplasmic tyrosine kinases. However, another class of transmembrane receptors is predicted to function as serine-threonine kinases. The type II activin receptor (102581) and the types I (190181) and II TGF-beta receptors (Lin et al., 1992) belong to the serine-threonine kinase family. The many activities of TGF-beta in regulating cell proliferation and differentiation and extracellular matrix production are mediated through these receptors. With the use of cells overexpressing truncated type II receptors as dominant-negative mutants to block type II receptor signaling selectively, Chen et al. (1993) demonstrated the existence of 2 receptor pathways. The type II receptors, possibly in conjunction with type I receptors, mediate the induction of growth inhibition and hypophosphorylation of the retinoblastoma gene product. The type I receptors are responsible for effects on extracellular matrix, such as the induction of fibronectin and plasminogen activator inhibitor I, and for increased JUNB (165161) expression. Selective inactivation of the type II receptors alters the response to TGF-beta in a manner similar to the functional inactivation of the RB protein, suggesting a role for RB in the type II, but not the type I, receptor pathway.102581190181165161

6. “Biology Workbench” Use “Biology Workbench” to find the expressed gene’s protein sequence: Go to: http://bioquest.org/bedrock/http://bioquest.org/bedrock/ Click on resources and then Other Resources Click on Biology Workbench Log in

7. Choose Protein Choose Ndjinn Search for colon cancer gene 1ktz Choose the pdb finder Search for tgf beta type II receptor Import the Protein Sequence Use Swiss –PDB Viewer to study Protein Structure

8. PDB ID: 1KTZ Transforming Growth Factor II Receptor

9. Use microarray to locate the normal gene You have been given a microarray chip with spots of normal human DNA restriction fragments from chromosome 3 You have Cy3 labeled (green) normal cDNA You have Cy5 labeled (red) cDNA from the tumor cells You flood the microarray with the sample mixture containing both labeled cDNAs View the chip

10. Microarray Data Visualization

11. What do your detective skills tell you?

12. Future Work –DNA –Isolate the fragment from the DNA chip – Sequence the fragment –Look for similar fragments in other organisms –Do multiple alignments –Determine relatedness

13. Future Work –Protein –Look for similar protein sequences –Do multiple sequence alignments –Determine relatedness