An introduction to molecular biology as a tool for studying neuroscience

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An introduction to molecular biology as a tool
for studying neuroscience

• Rich Reimer
• rjreimer_at_stanford.edu

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Outline
Eukaryotic gene structure nucleotides, DNA,
RNA introns and exons mRNA structure Manipulatin
g DNA recombinant technology plasmids and
endonucleases making cDNAs PCR heterologous
expression blocking expression - dominant
negative proteins, knockouts and RNAi
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DNA RNA - Nucleotide Bases
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DNA RNA Chains
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DNA base pairs
Genomic DNA for a given gene is made of two
strands - coding and non-coding
Chargaffs rule AT and GC
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Gene to protein - eukaryotic gene structure
Sense strand
heteronuclear RNA (hnRNA)
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Transcription
Promoters Gene specific sequences regulating
binding of RNA pol II - allow temporal, spatial
and environmental regulation of gene expression.
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mRNA maturation
Splicing Consensus sequences are recognized by
the spliceosome, a multisubunit complex which
clips out intronic sequences Variable splicing
can lead to expression of proteins with different
functional properties
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Structure and regulation of mRNA

• Translated and untranslated regions
• 5 end -cap site and translation initiation site
• 3 end polyA site
• Regulator sites for stabilization and degradation
  occur throughout mRNA molecules

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mRNA structure - coding regions
Translated regions form triplet codons for amino
acids
The code is degenerate - there are even 3 stop
codons, but only 1 Met Codons recruit tRNAs for
elongation polypeptide chain on the
ribosome Different species have preferences for
different codons
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Translation initiation sites
The first ATG is almost always the site of
transcription initiation. (GCC)n(A/G)CCATGG
(Kozak sequence) Most mammalian mRNAs have this
short recognition sequence at the site of
transcription initiation. It greatly facilitates
the initial binding of the small subunit of the
ribosome. In rare instances other codons can
serve as the site of transcription initiation
(ACG, CTG)
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RNA editing
enzymatic conversion of adenosine to
inosine Occurs in hnRNA inosine is translated as
guanosine The responsible enzyme ADAR is itself
edited regulates nervous system
proteins Inosine is relatively abundant in brain
mRNA edited AMPA and kainate receptors have
altered Ca permeability editing of Squid
neuronal proteins alters temp dependence of
kinetics
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RNA editing
R-gtG editing in the GluR2 glutamate receptor
subunit mRNA
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Recombinant DNA technology
Allows for the isolation and amplification of
specific segments of DNA. bacteria serve as
hosts for autonomously replicating segments of
DNA (plasmids) Plasmids small circular
double-stranded DNA replicate separately but
analogous to bacterial chromosomes
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Recombinant DNA technology
Cohen et al. Construction of Biologically
Functional Bacterial Plasmids In Vitro. Proc.
Nat. Acad. Sci. USA, November 1973
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pBluescript - a cloning vector
Key features Amp resistance origin of
replication LacZ gene mcs
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subcloning
EcoR1
Restriction enzymes recognize unique
sequences form sticky or blunt ends
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Manipulating DNA - cDNAs
cDNAs DNA sequences generated from mRNA.
First strand is the reverse complement of the mRNA
antisense strand
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Making cDNA
Once a ds cDNA molecule is made, linkers can be
attached with specific restriction enzyme
sites. ss cDNA can be used in PCR reactions
antisense strand
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PCR Basics
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Exponential amplification through PCR
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Modern cloning
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Site directed mutatgenesis

• uses PCR based system to introduce mutations in
  plasmids.
• Mutations are engineered into oligos (1-5 bases)
• takes advantage of the fact that PCR generated
  DNA is not methylated and insensitive to DpnI

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Heterologous expression
Generation of vectors with novel characteristics
allow for expression of genes in mammalian cells.
In certain cells (eg Xenopus oocytes) RNA can be
directly injected into the cell.
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pcDNA3 - mammalian expression vector
Key features amp resistance CMV promoter
polyA tail neo resistance mcs - cDNA must
be properly oriented
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Transfecting mammalian cells
Multiple techniques electroporation Ca/PO4--
cationic lipids Plasmids initially in the
cytoplasm and can be replicated in some
cells Can select for cells in which the DNA has
been stably integrated into the cells genomic
DNA with selective antibiotic (eg neomycin)
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Viral expression vectors
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Down regulating protein function - knockouts
Selectively inactivate a single gene by inserting
exogenous sequences that disrupts the normal gene
structure
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Down regulating protein function RNAi
siRNA molecule
microRNA pathway
ds RNA pathway
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RNAi
Mechanisms for using RNAi in mammalian cells
siRNAs
Long dsRNAs subjected to dicer
Vector based systems that express hairpinned dsRNA
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Online tools

• The Allen Brain Atlas
• http//www.brain-map.org/welcome.do
• Gensat
• http//www.gensat.org/makeconnection.jsp
• MMRC
• http//www.mmrrc.org/

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Using recombinant proteins to probe the nervous
system

• A bacterially produced glutamate biosensor to
  monitor neurotransmitter release
• A luciferase reporter gene for monitoring TGF-?
  signaling

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Sakiko Okumoto
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wavelength (nm)
Sakiko Okumoto
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GABAzine Wash in 30 sec after application
Chris Dulla
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GABAzine Wash in 50 sec after application
Chris Dulla
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GABAzine Wash in 70 sec after application
Chris Dulla
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Engineering of Reporter Mice for TGF-?
Signaling to Monitor Brain Injury and
Neurodegeneration
Wyss-Coray Lab
Amy Lin/Jian Luo
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Bioluminescence in Fireflies (Photinus pyralis)
Wyss-Coray Lab
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In vivo Bioluminescence Imaging
Luciferin
Wyss-Coray Lab
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In vivo Bioluminescence Imaging
Luciferin
Wyss-Coray Lab
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Kainate-Induced Excitotoxic Injury as a Model
of Neurodegeneration
Wyss-Coray Lab
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Imaging of Kainate-Induced Reporter Gene
Activation in Living Mice
Wyss-Coray Lab
Amy Lin
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Bioluminescence in Brain Slices from
SBE-luciferase Mice
Wyss-Coray Lab
Amy Lin