Download Genes in Pieces (PowerPoint) Northeast 2012

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 O’Neal 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 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.
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.
A. I can’t distinguish among A-C based on only this data.
A. 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.
A. I can’t distinguish among A-C based on only this data.
A. I have no idea; I am lost.
Singlestranded
Doublestranded
Which strand is DNA?
A.Red
B.Blue
C.I don’t know
Singlestranded
Doublestranded
Which strand is DNA?
A.Red
B.Blue
C.I don’t 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
5’
3’
c
a
c
3’
5’
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.
What’s coming next! Splicing is mediated by a
molecular machine known as the spliceosome,
the great discovery of Joan Steitz.