Instructional materials summary – Harvard SI 2012 Title of teachable tidbit: __Genes in Pieces___

1. Instructional materials summary – Harvard SI 2012Title of teachable tidbit: __Genes in Pieces___

2. Team 6 Genes in Pieces • Meg Kenna, Lehigh University • Mike Kuchka, Lehigh University • Rich Losick, Harvard University • Lynne Mullen, Harvard University • Carolyn Sealfon, Princeton University • Heather Thieringer, Princeton University • Facilitators: • Christov Roberson, Harvard University • Marvin O’Neal III, Stony Brook University

3. Team 6: Gene Expression Teachable Unit for an introductory cell and molecular biology course • Prior to the teachable tidbit, students • will already understand: • Central Dogma • RNA transcription • Translation • Prokaryotic gene expression • DNA/RNA hybridization • Gel electrophoresis • Electron microscopy

4. Learning Goals: • Understand how genetic information is organized in eukaryotes. • Interpret classical experimental data about eukaryotic gene organization.

5. Imagine it’s the early days of molecular biology…. For your research project you: 1. Obtain a piece of DNA containing an entire bacterial gene. 2. Obtain mRNA for the same gene. 3. Separate the DNA into two single strands. 4. Hybridize the mRNA to its complementary DNA strand. 5. Visualize the DNA-RNA hybrids by electron microscopy.

6. Draw what you expect to see on an electron micrograph when mRNA hybridizes to its complementary DNA strand! Note: Double stranded polynucleotides are thicker than single-stranded polynucleotides in electron micrographs Think-Pair-Share

7. Draw what you expect to see! Does it look something like this?

8. Draw what you expect to see! Does it look something like this?

9. Draw what you expect to see! Does it look something like this?

10. Now you repeat the experiment but this time the gene and its mRNA are from a eukaryote.

11. Here is what you now see! Single-stranded Double-stranded Think-Pair-Share

12. Here is what you now see! Single-stranded Double-stranded Why is this different than what you expected to see from a prokaryote?

13. What is the best explanation for this result? The gene contains a sequence that was removed from the transcript during the formation of the mRNA. The gene contains a sequence that was skipped over by RNA polymerase in transcribing the gene. The mRNA transcript acquired an insert after its synthesis that is absent from the gene. I can’t distinguish among A-C based on only this data. I have no idea; I am lost.

14. Genes are often in pieces. The coding bits are exons and the interruptions are introns. The introns are removed by “splicing” after the gene is transcribed into RNA. DNA Transcription pre-mRNA Splicing mRNA intron

15. What is the best explanation for this result? The gene contains a sequence that was removed from the transcript during the formation of the mRNA. The gene contains a sequence that was skipped over by RNA polymerase in transcribing the gene. The mRNA transcript acquired an insert after its synthesis that is absent from the gene. I can’t distinguish among A-C based on only this data. I have no idea; I am lost.

16. Single-stranded Double-stranded • Which strand is DNA? • Red • Blue • I don’t know

17. Single-stranded Double-stranded • Which strand is DNA? • Red • Blue • I don’t know

18. In the electron micrograph, the mRNA was alreadyspliced out. The loop you saw was DNA! DNA Transcription pre-mRNA Splicing mRNA intron

19. Recap Primary Transcript(pre-mRNA) exon intron exon 5’ 3’ a b c Splicing mRNA 5’ 3’ a c Hybridize to DNA mRNA: DNA hybrid 5’ 3’ 3’ 5’

20. Your next experiment: Use S1 nuclease to reveal the presence of introns Primary Transcript(pre-mRNA) exon intron exon 5’ 3’ a b c Splicing mRNA 5’ 3’ a c Hybridize to DNA mRNA: DNA hybrid 5’ 3’ 3’ 5’

21. a c 5’ 3’ 3’ 5’ a c b S1 nuclease: digests single stranded RNA and DNA What will remain after treatment with nuclease? Sketch a diagram and discuss with your neighbor.

22. a c 5’ 3’ 5’ 3’ a c Does your picture look like this? a c 5’ 3’ 3’ 5’ a c b S1 nuclease: digests single stranded RNA and DNA

23. a c 5’ 3’ 5’ 3’ a c non-denaturing conditions Imagine you are running a gel just like you did in lab earlier this semester a c 5’ 3’ 5’ 3’ a c b S1 nuclease: digests single stranded RNA and DNA How many bands of DNA are in the gel? A. Two bands of lengths a and c B. Single band of length a + c C. Three bands of lengths a, b, and c ?

24. a c 5’ 3’ 5’ 3’ a c Imagine you are running a gel just like you did in lab earlier this semester a c 5’ 3’ 5’ 3’ a c b S1 nuclease: digests single stranded RNA and DNA How many bands of DNA are in the gel? A. Two bands of lengths a and c B. Single band of length a + c C. Three bands of lengths a, b, and c non-denaturing conditions a + c

25. a c 5’ 3’ 5’ 3’ a c denaturing conditions Now you run a new kind of gel that denatures double-stranded nucleic acid a c 5’ 3’ 5’ 3’ a c b S1 nuclease: digests single stranded RNA and DNA How many bands of DNA are in the gel? A. Two bands of lengths a and c B. Single band of length a + c C. Three bands of lengths a, b, and c ?

26. a c 5’ 3’ 5’ 3’ a c Now you run a new kind of gel that denatures double stranded nucleic acid a c 5’ 3’ 5’ 3’ a c b S1 nuclease: digests single stranded RNA and DNA How many bands of DNA are in the gel? A. Two bands of lengths a and c B. Single band of length a + c C. Three bands of lengths a, b, and c denaturing conditions a c

27. This experiment demonstrates that the mRNA lacks a stretch of sequence that is present in the DNA. a c 3’ 5’ 3’ 5’ a c non-denaturing conditions denaturing conditions a c duplex single-stranded a c 5’ 3’ 3’ 5’ a c b S1 nuclease: digests single stranded RNA and DNA a + c

28. Introns vary enormously • Some yeast genes have one intron.

29. Introns vary enormously • Some yeast genes have one intron. • The dihydrofolate reductase gene in mammals has only five introns, but they account for 29 of the 31 kilobases (kb) of the gene.

30. Introns vary enormously • Some yeast genes have one intron. • The dihydrofolate reductase gene in mammals has only five introns, but they account for 29 of the 31 kilobases (kb) of the gene. • The champion is the human gene Titin with 363 introns.

31. Introns vary enormously • Some yeast genes have one intron. • The dihydrofolate reductase gene in mammals has only five introns, but they account for 29 of the 31 kilobases (kb) of the gene. • The champion is the human gene Titin with 363 introns. • Exons are usually ~150 bp but introns can be as long as 800 kb!

32. Mistakes in splicing can have major health effects • Breast Cancer (BRCA1) • Duchenne Muscular Dystrophy (DMD) • Neurofibromatosis (NF-1) • Thalassemias • Ocular albinism (OA-1)

33. Learning outcomes for ‘Genes in Pieces’You now are able to: • Recognize that introns are spliced out of pre-mRNA • Analyze electron micrograph data to show that genes are in pieces • Predict the gel electrophoresis patterns of nuclease protection assays • Know that genes vary greatly in the size and frequency of their introns, and that splicing is important in human health.

34. What’s coming next! Splicing is mediated by a molecular machine known as the spliceosome, the great discovery of Joan Steitz.