1. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Transcriptional Control in Eukaryotes Background Information Microarrays

2. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Differential gene transcription Single most important mechanism of cell differentiation Different genes are expressed by cells at different stages Different genes are expressed by cells in different tissues Different genes are expressed by cells exposed to different stimuli

3. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E DNA microarrays (DNA chips) Allows the visualization of “portraits” of gene expression in time and space developed by Patrick Brown & Joe DeRisi (Stanford) thousands of genes to be monitored in single experiment

4. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Yeast cells at 2 different growth conditions DNA fragments representing individual genes to be studied are generated –different microplate wells contain different genes –spotted in ordered array on glass slide (robot) –thousands of genes in small area mRNAs used to make fluorescently labeled cDNAs –2 different cell populations used –one with green fluorescent dye & the other with red fluorescent dye –mixed & hybridized to slide slide reader records pattern of expression

5. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Example of microarray experiment... yeast genome (roughly 6200 protein-coding genes) –hybridized with a mixture of 2 different cDNA populations –green dye, high glucose: fermentation to ethanol –red dye, no glucose: oxidative phosphorylation –spots without color: genes not transcribed –yellow: genes transcribed under both conditions –concentrations of individual mRNAs can vary by >100- fold in yeast cells –mRNA can be detected at <1 copy per cell

6. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.30a

7. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 12.30a

8. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Classification of Leukemias Acute Lymphoblastic Leukemia (ALL) Acute Myloblastic Leukemia (AML) –Difficult to diagnose –Cytologically very similar Chemotherapy: –ALL: corticosteroids, vincristine, methotrexate, asparaginase –AML: daunorubicin and cytarabine

9. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Microarray Classification Discovery –38 bone marrow samples (27 ALL, 11 AML) –Affymetrix microarrays: 6817 human genes –1100 genes were more highly correlated with the AML-ALL class distinction than random Predicition –50-gene predictor derived –assigned 36 of 38 as AML or ALL; 2 uncertain –All 36 agreed with the patients' clinical diagnosis. –Predictors of 10 to 200 genes 100% accurate

10. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

11. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E

12. Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E HoxA9: bad news Clinical Outcome (remission after chemo) –not predicted by groups of genes –strongly correlated with HoxA9 expression –HoxA9 can transform myeloid cells in culture –HoxA9 causes leukemia in transgenic animals –HoxA9 is rearranged by a t(7;11)(p15;p15) chromosomal translocation in a rare subset of AML patients, who tend to have poor outcomes