Biology Reference
In-Depth Information
Box 5.1 Key Terms and Concepts
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Sensitized genetic background: A sensitized genetic back-
ground is a mutant state in which an allele of a gene leads to
a weak phenotype in the biological process under study (for
example eye development in
phenotype is manifested is called epistatic, while the gene
whose phenotype is modified as the result of the epistatic
gene is called hypostatic.
). Thus, a weak
allele for gene X may lead to fruit flies with abnormal/small
eyes, whereas a strong/null allele would produce flies with
no eyes. In the sensitized background carrying the weak
allele for gene X it is possible to screen for new mutants that
either enhance the phenotype (small eyes to no eyes) or
suppress the phenotype (small eyes to normal eyes). Such
sensitized genetic screens can lead to the identification of
genes that function in the same pathway or genes that act
redundantly in parallel pathways.
Drosophila
Synthetic lethality: Mutations in two genes are said to be
synthetically lethal when cells with either of the single
mutations are viable but cells with both mutations are
lethal. A synthetic lethal genetic screen, starts with a muta-
tion in gene X that does not kill the cell, but may confer
a weak phenotype (such as, slow growth). This genetic
background is then used to systematically test mutations in
other genes that may lead to lethality.
l
SDS-PAGE (sodium dodecyl-polyacrylamide gel electro-
phoresis): SDS-PAGE is a technique that is widely used in
molecular biology and biochemistry to separate proteins as
a function of their length and charge by application of an
electric field.
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Saturation screen: A saturation screen is a genetic screen
that is performed to discover all genes that are involved in
a particular biological process. One of
l
the first satura-
tion screens was performed in
by Christiane
N
ยจ
sslein-Volhard and Eric Wieschaus (1980) to uncover
genes that were associated with embryonic lethality and
changes in cuticle morphology. In such genetic screens,
a mutagen such as a chemical or radiation is used to
generate mutations in the organism's chromosomes. Indi-
viduals that exhibit the phenotype of interest are selected
and the mutant alleles are mapped and cloned to identify
every gene involved.
Drosophila
iTRAQ (isobaric tags for relative and absolute quantifica-
tion) and TMT (tandem mass tags): iTRAQ and TMT are
used to identify differentially phosphorylated proteins
between different samples. These amine-reactive molecules
enable multiplexing of up to 4
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8 samples: the small
molecules, identical in structure and mass, differ in the
isotopic substitution of atoms comprising their backbone.
This altered distribution permits the unambiguous spectral
identification of unique reporter ions generated from frag-
mentation of each tag during MS. The fragmentation of each
tag during MS results in the release of a signature reporter
ion that differs in mass from the other tags; the signature ions
released from the six tag set differ successively by 1 Dalton
and their relative levels can be considered to reflect differ-
ences in peptide levels between samples. iTRAQ and TMT
enable the concurrent analysis of multiple samples, and the
assessment of the relative levels of phosphopeptides.
e
Loss-of-function mutation: Changes in the DNA sequence
of a gene that leads to reduced or abolished function of the
gene product.
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Gain-of-function mutation: Changes in the DNA sequence
of a gene that confers a new and/or abnormal function to the
gene product.
l
Epistasis analysis: Epistasis is the interaction between two or
more genes where the effect of one gene on a particular
phenotype is modified by other gene(s). The gene whose
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Such properties have been instrumental in working out the
epistatic (see
Box 5.1
for definition) relationships between
genes and in ordering components of a given pathway into
linear, minimally branched cascades
[4]
. These pioneering
studies have helped to reduce complex biological and
developmental processes to a finite number of paradigms
and clarified the identity and relationship of key compo-
nents in evolutionary conserved pathways. Reflecting the
implied linearity and independence of these pathways from
each other, most are named after the activating ligand or
a central effector protein, for example the Wnt/Wingless,
Hedgehog (Hh), TGF-
b
, JAK/STAT, Toll, NF
k
B, Notch,
receptor tyrosine kinase/extracellular regulated kinase
(RTK/ERK), Akt/TOR, Jun Kinase (JNK), G protein-
coupled receptor (GPCR) and steroid hormone pathways
[5]
. The analysis of signal transduction pathways in
mammalian cells has presented a more complicated view,
hinting at significant bridges or
various signal transduction modules/pathways. Cross-talk
or interaction between pathways allows cells to respond in
a coordinate manner to the combined extracellular and
intracellular cues that they are exposed to in order to
produce the appropriate response. Cross-talk between
pathways accounts for many complex signaling behaviors,
including signal integration, the ability to generate a variety
of different responses to a signal, and/or to reuse proteins
between pathways. For example, the SMAD proteins,
which have been assigned to the TGF-
b
signaling cassette,
can be phosphorylated by MAPK, which functions in
a separate pathway in lower eukaryotes. Thus, rather than
following a simple path, a signal received by a mammalian
cell may be relayed through multiple channels. Similarly,
other proteins such as Ras, protein kinase C (PKC), and
protein kinase B (Akt) are also activated by multiple
extracellular ligands. However, much of this knowledge
has originated from studies in cell
'cross-talks' between
lines and in vitro
