“ The Discovery of Reverse Transcriptase” November 2, 2010 Biology 161 Dr. Hong Fei By: David Meyer Kylene Wolfe Ryan Schramer Adrian Bachman

History Genetic information was demonstrated through the transfer of information of the biological system during the years of: 1940's, 1950's & 1960's In 1970 more of a focus on RNA tumor viruses but left many unanswered questions.

Howard Temin Studied RNA tumor viruses most of his life His early days he spent time working on Rous sarcoma virus (RSV) Temin was the one that came up with the first hypothesis for the study of Reverse Transcriptase

Howard Temin Hypothesis “The RNA genome of RSV is converted into a DNA provirus.”

Howard Temin Proposal “RSV is sensitive to inhibitors of DNA synthesis and suggesting that DNA, complementary to the RSV genomic RNA, is present in transformed cells.”

David Baltimore Spent time studying virus replication He looked at RNA and DNA at a more biochemical view looking deep within individual virions He later focused on RNA tumor viruses and concluded with Rauscher murine leukemia virus (R-MLV)

Howard Temin and David Baltimore walked two different pathways

Experiment Time! They disrupted their pure stock viruses by using nonionic detergents “Can a retrovirus perform DNA synthesis?” Then “radiolabeled deoxthymidine triphosphate (dTTP) along with three deoxynucleotide triphosphates (dATP, dCTP, dGTP) to the virion preparation.”

Did these reactions contain any incorporation of radioactive dTTP into DNA? YES! Each experiment, dTTP turned into nucleic acid

They tested their experiment by adding enzymes reacted with RNA or DNA Results: Sensitive to Dnase

One more question? “What was the template?”

One more test! Baltimore and Temin both “preincubated the virions with RNase, which catalyzes the degradation of RNA into ribonucleotide monophosphates.”

Results: “ The longer the pretreatment of virions with RNase, the lower the amount of DNA-synthesizing activity, - thus an enzyme in the virion could catalyze RNA-dependent DNA synthesis.”

Results/Data Demonstration of RNA-dependent DNA Synthesis Radioactive Tymidine Incorporated into Nucleic Acid Experimental Treatment:R-MLVRSV Standard Conditions3.69 pmol9110 dpm Virions pretreated with RNase0.52 pmol2650 dpm Untreated Product1425 dpm8350 dpm After product is treated with RNase1361 dpm7200 dpm After Product is treated with Dnase125 dpm1520 dpm

This was reverse to what normally was happened in DNA and RNA giving it's name: “Reverse Transcriptase”

Conclusion: Temin believed that his hypothesis was true all along he just had to prove it to the world Baltimore believed a different theory but tested Temin theory by checking RNA- dependent and DNA polymerization.

What has this done to help the world? Concert mRNA to DNA help create cDNA libraries coloning and study of tissues study of many genes throughout biology Explosion of research in retroviruses/ RNA viruses

Temin/Baltimore Future Since HIV-AIDS is a retrovirus! This drove Temin and Baltimore to a Noble Prize in 1975

QUIZ TIME!!! 1. Why was the research done? a. Win a Noble prize b. Become president c. To test whether or not RNA genome of RSV is converted into a DNA provirus d. Test Michael Jackson's DNA

Question 2 2. What method did these men use? a. Newton's Law method b. Broke down a virus and started doing DNA/RNA test on it c. Dr. Fei Method d. Heartland College Education Method

Question 3 3. With the results was this experiment successful? a. YES! b. No c. Maybe d. Not sure

Question 4 4. What could you conclude from this experiment? a. It was the worlds greatest experiment b. The longer the pretreatment of virions with RNase, the lower the amount of DNA-synthesizing activity, - thus an enzyme in the virion could catalyze RNA-dependent DNA synthesis.” c. If water goes up, it must come back down d. DNA/RNA is the same in all people

Question 5 5. What is one way this research has helped the world? a. It helped two men win a Noble Prize b. It's going to save the world c. It gave a cure to AIDS d. Explosion of research in retroviruses/ RNA viruses

Bonus Question Who is the greatest Biology instructor in the who world? a. Dr. Hong Fei b. President Obama c. Rod Blagojevich d. Police Officer

The End!