Eukaryotic genes are often in pieces. The coding segments are ...

Eukaryotic genes are often in pieces. The coding segments are ...

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 ([email protected]) Lynne Mullen ([email protected]) 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 ONeal III, Stony Brook University Team 6: Gene Expression Teachable Unit for an introductory cell and molecular biology course Prior to the teachable tidbit, students will already understand: 1. 2. 3. 4. 5. 6. 7.

Central Dogma RNA transcription Translation Prokaryotic gene expression DNA/RNA hybridization Gel electrophoresis Electron microscopy Learning Goals: 1. Understand how genetic information is organized in eukaryotes. 2. Interpret classical experimental data about eukaryotic gene organization. Imagine its 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. Think-Pair-Share 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 singlestranded polynucleotides in electron micrographs Draw what you expect to see! Does it look something like this?

Draw what you expect to see! Does it look something like this? Draw what you expect to see! Does it look something like this? Now you repeat the experiment but this time the gene and its mRNA are from a eukaryote. Here is what you now see! Singlestranded Doublestranded Think-Pair-Share Here is what you now see!

Singlestranded Doublestranded Why is this different than what you expected to see from a prokaryote? 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. D. I cant distinguish among A-C based on only this data. E. I have no idea; I am lost. 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 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. D. I cant distinguish among A-C based on only this data. E. I have no idea; I am lost.

Singlestranded Doublestranded Which strand is DNA? A.Red B.Blue C.I dont know Singlestranded Doublestranded Which strand is DNA? A.Red B.Blue C.I dont know In the electron micrograph, the mRNA was already spliced out. The loop you saw was DNA! DNA

Transcription pre-mRNA Splicing mRNA intron Recap exon 5 intron a exon b

c 3 Primary Transcript (pre-mRNA) Splicing 5 a c 3 mRNA

Hybridize to DNA 3 5 3 5 mRNA: DNA hybrid Your next experiment: Use S1 nuclease to reveal the presence of introns exon 5 intron

a exon b c 3 Primary Transcript (pre-mRNA) Splicing 5 a

c 3 mRNA Hybridize to DNA 3 5 3 5 mRNA: DNA hybrid

a 3 5 c a c 3 5 b S1 nuclease: digests single stranded RNA

and DNA What will remain after treatment with nuclease? Sketch a diagram and discuss with your neighbor. Does your picture look like this? a c 5 3 3 a 5

c b S1 nuclease: digests single stranded RNA and DNA 5 a c 3 a c

3 5 Imagine you are running a gel just like you did in lab earlier this semester 5 3 a c 3 a 5 c b

5 a 3 a S1 nuclease: digests single stranded RNA and DNA c 3 c 5 How many bands of DNA are in the gel? non-denaturing conditions

? 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 a c 3 a

5 c b 5 a 3 a S1 nuclease: digests single stranded RNA and DNA c 3 c 5

How many bands of DNA are in the gel? non-denaturing conditions a+c 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 a c

a 3 5 c b 5 a 3 a S1 nuclease: digests single stranded RNA and DNA

c 3 c 5 How many bands of DNA are in the gel? denaturing conditions ? 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 a c a 3 5 c b 5 a

3 a S1 nuclease: digests single stranded RNA and DNA c 3 c 5 How many bands of DNA are in the gel? denaturing conditions a c A. Two bands of lengths a and c

B. Single band of length a + c C. Three bands of lengths a, b, and c This experiment demonstrates that the mRNA lacks a stretch of sequence that is present in the DNA. 3 5 a c a 3 5

c b 5 3 S1 nuclease: digests single stranded RNA and DNA c 3 a a c non-denaturing conditions

5 denaturing conditions a+c a c duplex single-stranded Introns vary enormously Some yeast genes have one intron. 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. 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. 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! Mistakes in splicing can have major health effects

Breast Cancer (BRCA1) Duchenne Muscular Dystrophy (DMD) Neurofibromatosis (NF-1) Thalassemias Ocular albinism (OA-1) 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. Whats coming next! Splicing is mediated by a molecular machine known as the spliceosome, the great discovery of Joan Steitz.

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