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/
PubMed Entrez BLAST OMIM Taxonomy Structure
24 October 2003 We will be removing the obsolete 'nph-blast_report' web service. Read more...
Disclaimer Privacy statement
Accessibility This page is valid XHTML 1.0.
Info
•FAQs •News •References •Credits Education
•Program selection guide
•Tutorial •URL API guide Download
•Executables •Databases •Source code Support
•Helpdesk •Mailing list
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
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