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Instructional materials summary – Harvard SI 2012 Title of teachable tidbit: __Genes in Pieces___ General Topic:gene expression Two sentence synopsis of.

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Presentation on theme: "Instructional materials summary – Harvard SI 2012 Title of teachable tidbit: __Genes in Pieces___ General Topic:gene expression Two sentence synopsis of."— Presentation transcript:

1 Instructional materials summary – Harvard SI 2012 Title of teachable tidbit: __Genes in Pieces___ General Topic:gene expression Two sentence synopsis of tidbit:Students learn to interpret experimental data that led to the discovery of splicing Type of activity (or activities):Think pair share, clickers, drawing, class discussion centered on data slides Designed for what level course and type of students? intro course for majors in molecular biology Materials required:Clickers, pad and pencil Comments on out of class preparation required by students and instructor: Students are expected to understand from earlier in the course: Central Dogma, transcription, translation, prokaryotic gene expression, DNA/RNA hybridization, gel electrophoresis, electron microscopy General comments:Data images are artificial representations for simplifying the presentation. Learning Goals for teachable unit are in separate Word document. List five keywords that would allow others to search for this activity in a database: Splicing, mRNA processing, electron microscopy, S1 nuclease mapping Names and institutions of group members: Meg Kenna, Lehigh University; Mike Kuchka, Lehigh University; Rich Losick, Harvard University Lynne Mullen, Harvard University; Carolyn Sealfon, Princeton University; Heather Thieringer, Princeton University Contact person for questions:Mike Kuchka Lynne Mullen

2 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 Team 6 Genes in Pieces

3 Prior to the teachable tidbit, students will already understand: 1.Central Dogma 2.RNA transcription 3.Translation 4.Prokaryotic gene expression 5.DNA/RNA hybridization 6.Gel electrophoresis 7.Electron microscopy Team 6: Gene Expression Teachable Unit for an introductory cell and molecular biology course

4 Learning Goals: 1.Understand how genetic information is organized in eukaryotes. 2.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 Single- stranded Double- stranded Here is what you now see! Why is this different than what you expected to see from a prokaryote?

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

14 DNA pre-mRNA Transcription Splicing mRNA intron 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.

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

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

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

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

19 abc exon intron ac 5’3’ Splicing 5’3’ Primary Transcript (pre-mRNA) Hybridize to DNA mRNA mRNA: DNA hybrid Recap 5’3’ 5’

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

21 5’3’ 5’ ac ac 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 5’3’ 5’ ac ac b S1 nuclease: digests single stranded RNA and DNA Does your picture look like this? 5’ 3’ 5’ ac ac

23 non-denaturing conditions ? 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 Imagine you are running a gel just like you did in lab earlier this semester 5’ 3’ 5’ ac ac 3’ 5’ ac ac b 3’ S1 nuclease: digests single stranded RNA and DNA

24 non-denaturing conditions 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 Imagine you are running a gel just like you did in lab earlier this semester 5’ 3’ 5’ ac ac 3’ 5’ ac ac b 3’ a + c S1 nuclease: digests single stranded RNA and DNA

25 denaturing conditions ? 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 Now you run a new kind of gel that denatures double-stranded nucleic acid 5’ 3’ 5’ ac ac 3’ 5’ ac ac b 3’ S1 nuclease: digests single stranded RNA and DNA

26 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 Now you run a new kind of gel that denatures double stranded nucleic acid 5’ 3’ 5’ ac ac 3’ 5’ ac ac b 3’ denaturing conditions acac S1 nuclease: digests single stranded RNA and DNA

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

28 Some yeast genes have one intron. Introns vary enormously

29 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.


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