Flies and Worms:Genetic Studies of Touch and Taste

October 23, 2003

Overview

BLASTing genes

Real Time PCR

in situ hybridization

Expression of proteins in cell culture

Dominant-negative constructs

RNAi

Primary C. elegans cultures

FRET imaging in C. elegans

Notes on electrophysiology

BLAST

The Website:

www.ncbi.nlm.nih.gov/BLAST/

                   

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Info

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Nucleotide•Discontiguous megablast •Megablast •Nucleotide-nucleotide BLAST (blastn) •Search for short, nearly exact matches •Search trace archives with megablast or discontiguous megablast

Protein•Protein-protein BLAST (blastp) •PHI- and PSI-BLAST •Search for short, nearly exact matches •Search the conserved domain database (rpsblast) •Search by domain architecture (cdart)

Translated•Translated query vs. protein database (blastx) •Protein query vs. translated database (tblastn) •Translated query vs. translated database (tblastx)

Genomes•Human, mouse, rat •Fugu rubripes, zebrafish •Flies, nematodes, plants, yeasts, malaria •Microbial genomes, other eukaryotic genomes

Special•Align two sequences (bl2seq) •Screen for vector contamination (VecScreen) •Immunoglobin BLAST (IgBlast)

Meta•Retrieve results by RID •Get this page with javascript-free links

BLAST

What you can learn from sequence homology

I have this gene but I don’t know its function-- are there other genes that look like my gene? What do they do?

I think I have a gene that serves a particular function-- do other genes that serve similar function look like my gene?

There is a gene family that I want to study-- are there other genes in this family that have not been studied?

How similar are different genes in this family from my gene?

Are there certain regions that are very similar across this family?

What function does my gene serve in other species?

Real Time PCR

Quantifies changes in gene expression by means of fluorescent markers

Best for showing relative changes in gene expression

Uses reference sample and experimental samples

Relies on fluorescent reporter that binds specifically to dsDNA, emitting fluorescence only when bound

RT-PCR is performed on both samples

As PCR product increases, fluorescence increases (linear relationship)

Samples can be compared to determine relative quantities

Ambion Tech Notes www.ambion.com.techlib/tn/85/857.html and 81/813.html

In Situ Hybridization

Dioxygenin

Used as alternative to radioactive labeling

Probe is anti-sense RNA labeled with Dioxygenin

Labeled RNA binds to mRNA in sample

Apply antibody to dioxygenin

Apply stain for antibody

Determine location/absence of mRNA for your gene in sample

www.fruitfly.org/about/methods/cytogenetics.html

Dioxygenin

“Digoxigenin can be incorporated at either terminus, at both termini, and/or within the sequence. Eurogentec produces these oligonucleotides by using either a dT-amino modification or a C6-amino modifier to create an attachment site for the digoxigenin moeity”

http://usa.eurogentec.com/code/en/page_02.asp?Page=77

Cell Culture Studies

Basic Protocol:

Select cell line

Clone gene of interest into vector (includes tags, promotors)

Transfect gene into cells

Induce gene expression

(image to see fluorescent gene expression or immuonstain for specific proteins)

Lyse cells and spin to remove cell debris

Run over purification column to isolate protein

Remove from column

Run on gel-- use antibody for protein or tag to confirm

Creation of Dominant-Negatives

Basic Principle: Create mutant that expresses the wild-type gene, but this gene cannot perform its function due to specific

interference by another construct

Examples:

Binding by inactive protein domain

Overexpression of inactive subunit of complex

Blocking of active sites on protein (direct or indirect)

Caveats:

Theoretical construct does not always work as d-n

Must be sure that effect seen is due to interaction with your gene

RNAi

Usually potent and specific

Double-stranded more effective than sense or anti-sense single stranded

(Injected single strands probably rapidly degraded)

dsRNAi mimics loss of function phenotype

Injection produces decrease in endogenous mRNA

May act at level of chromatin structure or transcription

Crosses cellular boundries (injection in head of one animal can result in interference in progeny)

Fire et al. (1998) Nature 391: 806-811

RNAi

Limitations:

If members of a gene family share similar sequence, construct may interfere with multiple genes

If gene is expressed at low levels, not all genes or all cells will be influenced

Fire et al. (1998) Nature 391: 806-811

C. elegans Primary Cultures

Enables access to individual cell types for functional and molecular studies

Isolated cells differentiate into cells which would be found in L1 larva (as yet, no postembryonic development)

Uses strains with cell-type specific fluorescent markers to identify desired cells

Fluorescence Activated Cell Sorting (FACS)

Plated cells express cell-specific markers

Effective techniques for functional characterization:

Patch clamp

Whole cell recordings

dsRNA knock down of specific gene expression

Christensen et al. (2002) Neuron 33:503-514

C. elegans Primary Cultures

Christensen et al. (2002) Neuron 33:503-514

Figure 2. Micrographs of Cultured Embryonic Cells Expressing Cell-Specific GFP Reporters(A) myo-3::GFP expression in cultured body muscle cells. myo-3 encodes a myosin heavy chain isoform expressed in body muscles. The myo-3 reporter strain expresses two GFPs with peptide signals that target them to either the nucleus (arrows) or mitochondria (arrowheads). Inset: immunolocalization of UNC-54 myosin in body wall muscle cells. Cultures derived from myo-3::GFP-expressing worms were fixed and incubated with anti-UNC-54 and a Cy3-conjugated (red) secondary antibody. Yellow indicates overlap of GFP and Cy3 fluorescence.(B) Cultured mechanosensory neuron expressing mec-4::GFP. mec-4 encodes a degenerin-type ion channel subunit expressed largely in neurons that respond to gentle body touch.(C) Cultured cholinergic motor neurons expressing unc-4::GFP. unc-4 encodes a homeodomain transcription factor. Inset: immunolocalization of synaptotagmin in motor neurons. Cultures derived from unc-4::GFP-expressing worms were fixed and incubated with anti-synaptotagmin and a Cy3-conjugated (red) secondary antibody. unc-4::GFP, anti-SNT-1, and DAPI fluorescence are shown in green, red, and blue, respectively. Yellow indicates overlap of GFP and Cy3 fluorescence.(D) Cultured neuron expressing opt-3::GFP. OPT-3 is a H+/oligopeptide transporter expressed in glutamatergic neurons.

C. elegans Primary Cultures

Christensen et al. (2002) Neuron 33:503-514

Figure 2. Micrographs of Cultured Embryonic Cells Expressing Cell-Specific GFP Reporters(E) Combined DIC and fluorescence micrograph of an unc-119::GFP-expressing neuron. unc-119 encodes a novel protein that is expressed in all neurons and eight head muscle cells.(F) Combined DIC and fluorescence micrograph of an unc-4::GFP-expressing cholinergic motor neuron physically interacting with a body wall muscle cell.(G) Lateral view of a transgenic larval worm showing expression of unc-4::CFP (green) in A-type motor neurons and acr-5::YFP (red) in B-type motor neurons in the ventral nerve cord. Anterior is to left.(H) unc-4::CFP and acr-5::YFP are expressed in separate sets of neurons in vitro after 5 days in culture. All scale bars are 10 μm.

Chameleon

What is FRET?

Fluorescence Resonance Energy Transfer

Emission wavelength of one molecule is excitation wavelength for another molecule

When the 2 molecules are in very close proximity and the first molecule is excited, emission spectra from the second molecule

can be detected

If the molecules are not in close proximity, emission spectra from the first molecule will be detected

Chameleon

How does chameleon work as a calcium sensor?

Miyawaki et al. (1997) Nature 388:882-887

Chameleon

Advantages/Disadvantages

Advantages:

Precise targeting

Easy to image

Does not interfere with normal function of cell

Clean and specific

Able to detect a wide range of Ca++ concentration

Can be quantified with high spatiotemporal resolution

Disadvantages:

Nonzero baseline-- rare associations would be hard to detect

Miyawaki et al. (1997) Nature 388:882-887

Notes on Electrophysiology

Neurons maintain their resting potential near reversal potential for K+ (around -80 mV)

Na+/K+ pump results in high extracellular Na+ and low extracelluar K+

But, neurons are leaky

Adding large amounts of K+ externally upsets the ionic balance, changes the reversal potential for K+ and depolarizes

the cell

The measurement in cell recordings is the flow of Cl- ions on and off a silver-plated reference electrode

Additional Resources

National Center for Biotechnology Information http://ncbi.nlm.nih.gov

FlyBase http://flybase.bio.indiana.edu

WormBase http://www.wormbase.org

C. elegans genetics http://elegans.swmed.edu/genome.shtml

Worm Anatomy and Behavoir http://www.wormatlas.org