“The Discovery of Reverse Transcriptase”

November 2, 2010

Biology 161Dr. Hong Fei

By: David MeyerKylene Wolfe

Ryan SchramerAdrian 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-MLV RSV

Standard Conditions 3.69 pmol 9110 dpm Virions pretreated with RNase 0.52 pmol 2650

dpm

Untreated Product 1425 dpm 8350 dpmAfter product is treated with RNase 1361 dpm 7200 dpmAfter Product is treated with Dnase 125 dpm 1520 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 tissuesstudy 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 prizeb. Become president

c. To test whether or not RNA genome of RSV is converted into a DNA provirusd. Test Michael Jackson's DNA

Question 2

2. What method did these men use?

a. Newton's Law methodb. Broke down a virus and started doing

DNA/RNA test on itc. Dr. Fei Method

d. Heartland College Education Method

Question 3

3. With the results was this experiment successful?

a. YES!b. No

c. Maybed. Not sure

Question 4

4. What could you conclude from this experiment?

a. It was the worlds greatest experimentb. 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 Prizeb. It's going to save the world

c. It gave a cure to AIDSd. Explosion of research in retroviruses/ RNA

viruses

Bonus Question

Who is the greatest Biology instructor in the who world?

a. Dr. Hong Feib. President Obamac. Rod Blagojevich

d. Police Officer

The End!