Download Advanced Techniques in Molecular Biology

Advanced Techniques in
Molecular Biology
Sonchita Bagchi
[email protected]
Some basic Molecular Biology techniques
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DNA cloning
Polymerase Chain Reaction
Gel electrophoresis
molecular hybridization
Mutagenesis
DNA and protein arrays
Model organism
Tissues attained and
processed – parafin
sections, cryo sections
National Human Genome Research Institute
(NHGRI) http://www.genome.gov/12514471
Ref: European Molecular
Biology Laboratory: Explore
Model organisms at EMBL
Detection techniques
•Fluorophore or fluorochrome
•3,3'-diaminobenzidine tetrahydrochloride (DAB)
•AminoEthyl Carbazole (AEC)
•Alkaline Phosphatase (AP)
http://www.immunohistochemistry.us/index.php?page=ihc-principle
Quantum dots
Quantum dots shine brighter, last longer, and are often less toxic than dyes previously
used. In addition, specific organic molecules can be attached to the quantum dots for
tracking.
Advantages over conventional fluorophores
•Photo stability
•Narrow spectra
Source: Kim Krieger,
Science 2004
Quantum dots are semiconductive
crystals that have been synthesized to be
as small as possible, normally from 2-30
nanometers wide. Quantum dots can be
synthesized so accurately that they can be
tuned to absorb and release specific
energy amounts.
Source:Ishikawa-Ankerhold et.al. Molecules 2012
Functional studies using Co-immunoprecipitation
Western Blot with antibody against the protein (if known!)
Advantages:
• Proteins that interact in a typical Co-IP are post-translationally modified and
conformationally natural.
• In Co-IP, proteins interact in a non-denaturing condition which is almost physiological.
Disadvantages:
• The signals of low-affinity of protein interactions might not be detected.
• There might be a third protein in certain protein-protein interaction.
• To choose an appropriate antibody, the target protein needs to be properly predicted. Or
there would not be a positive result in Co-IP.
• WB can be difficult to achieve.
Proximity Ligation Assay (PLA)
Source:Duolink
Fluorescence Resonance Energy Transfer
The energy is transferred from an excited donor fluorochrome to another molecule or
acceptor without emission of a photon. FRET relies on the close physical interaction of
the two molecules less than approximately 10 nm apart. Thus, FRET can be used to
determine molecular interaction/molecular proximity beyond the resolution limits of the
classical light microscope.
Source:Life Technologies
•Molecules must be <10nm apart.
•Donor emission must overlap Acceptor excitation
Fluorescence recovery after photobleaching (FRAP)
Source: University of Gothenburg, Centre for cellular imaging
RNA interference
Source: National Institute of General Medical Sciences
Overexpression of genes
•Constitutive promoters
•Inducible promoters
Source: Muller et. al. Cytokine, 2008
Fluorescence Microscopy
Confocal Microscopy
Electron Microscopy
Atomic force microscopy (AFM)
Source: Syddansk Universitet
Source: Bagchi et. al. Molecular Microbiology, 2008
Source: Lehenkari et. al. Ultramicroscopy, 2000
Questions??
Advanced Techniques in Molecular Biology
Part 2
Epigenetics
The term ‘Epigenetics’ was coined by Conrad H. Waddington in 1942, which was
derived from the Greek word “epigenesis” which originally described the
influence of genetic processes on development.
Genetic assimilation (1990) is a process by which a phenotype originally
produced
in
response
to
an
environmental
condition,
later
becomes genetically encoded via artificial selection or natural selection.
Today “Epigenetics” refers to covalent modification of DNA, protein, or RNA,
resulting in changes to the function and/or regulation of these molecules,
without altering their primary sequences. Epigenetics is the reason why a skin
cell looks different from a brain cell or a muscle cell. All three cells contain the
same DNA, but their genes are expressed differently, which creates the different
cell types.
Is it possible to pass epigenetic changes to future generations?
• DNA methylation
• Histone modification
• Non-coding RNA (ncRNA)-associated gene silencing
DNA methylation: the addition of a methyl group to part of the DNA molecule, which
prevents certain genes from being expressed.
Determination of differential methylation
with methylation-sensitive and
methylation-insensitive restriction
enzymes.
Genome-wide methods to measure DNA methylation
Histone Modifications
Histone modifications are proposed to affect chromosome function. The first
mechanism suggests modifications may alter the electrostatic charge of the histone
resulting in a structural change in histones or their binding to DNA. The second
mechanism proposes that these modifications are binding sites for protein recognition
modules that recognize acetylated or methylated amino acid.
Posttranslational modifications of histones create an epigenetic mechanism for the
regulation of a variety of normal and disease-related processes.
Purifying Histones
Non-coding RNA (ncRNA)-associated gene silencing
• Chromatin remodelling
• Transcriptional regulation
• Post-transcriptional regulation
Molecular phylogenetics
Phylogenetic systematics deals with identifying and understanding evolutionary
relationships among the many different kinds of life on earth, both living (extant)
and dead (extinct).
• Alignment—building the data model and extracting a dataset.
• Determining the substitution model—consider sequence variation.
• Tree building.
• Tree evaluation.
Protein Modeling or Molecular Modeling
• Identify the proteins with known three-dimensional structures that are
related to the target sequence.
• Align the related three-dimensional structures with the target sequence and
determine those structures that will be used as templates.
• Construct a model for the target sequence based on its alignment with the
template structure(s).
• Evaluate the model against a variety of criteria to determine if it is
satisfactory.
Prion - proteinaceous infectious particle
• Prion is an abnormal form of a normally harmless protein found in the brain
that is responsible for a variety of fatal neurodegenerative diseases of both
animals and humans.
• Prions can enter the brain through infection, or they can arise from mutations
in the gene that encodes the protein. Once present in the brain, prions
multiply by inducing benign proteins to refold into the abnormal shape.
• The normal protein structure is thought to consist of a number of flexible coils
called alpha helices. In the prion protein some of these helices are stretched
into flat structures called beta strands.
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The normal protein conformation can be degraded rather easily by cellular
enzymes called proteases, but the prion protein shape is more resistant to this
enzymatic activity. Thus, as prion proteins multiply they are not broken down
by proteases and instead accumulate within nerve cells, destroying them.
Glutamine-Glutamate cycle in depression, mood disorder and schizophrenia
Histological analysis of SLC38A6 (SNAT6) expression in
mouse brain shows selective expression in excitatory
neurons with high expression in the synapses.
Publications so far
Histological analysis of SLC38A6 (SNAT6) expression in mouse brain shows
selective expression in excitatory neurons with high expression in the synapses.
Bagchi S, Baomar HA, Al-Walai S, Al-Sadi S, Fredriksson R.
PLoS One. 2014 Apr; 9(4): e95438-e95438.
The evolutionary history and tissue mapping of amino acid transporters belonging
to solute carrier families SLC32, SLC36, and SLC38.
Sundberg BE, Wååg E, Jacobsson JA, Stephansson O, Rumaks J, Svirskis S, Alsiö J,
Roman E, Ebendal T, Klusa V, Fredriksson R.
J Mol Neurosci. 2008 Jun; 35(2):179-93.
SLC38A6 is widely expressed in the mouse brain
The evolutionary history and tissue mapping of amino acid transporters belonging to solute carrier families SLC32, SLC36, and
SLC38.
Sundberg BE, Wååg E, Jacobsson JA, Stephansson O, Rumaks J, Svirskis S, Alsiö J, Roman E, Ebendal T, Klusa V, Fredriksson R.
J Mol Neurosci. 2008 Jun; 35(2):179-93.
SNAT6 is selectively expressed in
excitatory neurons
SNAT6 has high expression in the synapses
Genes predicted to influence SNAT6:
According to the PCR results the top three genes interacting with SLC38A6 are as follows:
1. PAG (GLS1) (glutaminase) [already investigated]
2. CTPS2 (CTP Synthase) and it Catalyses the formation of CTP from UTP with the
concomitant deamination of glutamine to glutamate.
ATP+UTP+GLUTAMINE→ADP+Pi+CTP+GLUTAMATE
3. GRM2 (Glutamate Receptor, Metabotropic): It is a metabotropic glutamate receptor
that appears on excitatory neurons.
It is worth mentioning that according to the computational simulation, two other slc38
transporters seem to be interacting with A6 and they are SLC38A1 and SLC38A5.
The results shows Gls, Grm-2 ,ctps2 and Dner
had the biggest relative fold change. It is
interesting to note that GLS and Grm2 are
upregulated in the wild type, while Dner and
Ctps2 are upregulated in the siRNA treated
cells.
Functional basis: Speculations!
• Gls2 is an phosphate-activated amidohydrolase that catalyzes the hydrolysis of
glutamine to glutamate and ammonia. This protein (primarily expressed in the brain and
kidney) plays an essential role in generating energy for metabolism, synthesizing the
brain neurotransmitter glutamate and maintaining acid-base balance in the kidney. Thus
it makes sense that in case of decreasing the SNAT6( siRNA condition), the production of
the Gls protein decreases, as there is not enough glutamine to hydrolyse to glutamate.
• Grm-2 (Metabotropic glutamate receptor 2) exists on the surface of neurons and they
receive glutamate. As the glutamine percentage is decreasing in the treated cells, the
need for more glutamate decreases and thus they are down regulated.
• One of the main functions of Dner is clearing of glutamate, thus it seems very
reasonable to increase its production in order to compensate the loss of glutamine.
• ctps2 is a rate-limiting enzyme in the synthesis of cytosine nucleotides (ATP + UTP +
glutamine → ADP + Pi + CTP + glutamate). Hence, ctps2 is upregulated as the cell tries to
decrease the production of glutamate to conserve glutamine.
Direct Interaction between SNAT6 and CTPase2
• PLA (raised in same species)
• CoIP (too low Antibody conc.)
Questions??