Transcript Lecture 13 - Lectures For UG-5

PLANT BIOTECHNOLOGY & GENETIC
ENGINEERING
(3 CREDIT HOURS)
LECTURE 13
ANALYSIS OF THE TRANSCRIPTOME
ANALYSIS OF THE TRANSCRIPTOME
• Important insights into gene function can be gained by
expression profiling, i.e., determining where and when particular
genes are expressed. For example, some genes are switched on
(induced) or switched off (repressed) by external chemical
signals reaching the cell surface.
• In multicellular organisms, many genes are expressed in
particular cell types or at certain developmental stages.
• Furthermore, mutating one gene can alter the expression of
others.
• All this information helps to link genes into functional networks,
and genes can be used as markers to define particular cellular
states.
ANALYSIS OF THE TRANSCRIPTOME
• In the past, genes and their expression profiles have been
studied on an individual basis. Therefore, defining functional
networks in the cell has been rather like completing a large
and complex jigsaw puzzle.
• More recently, technological advances have made it possible
to study the expression profiles of thousands of genes
simultaneously, culminating in global expression profiling,
where every single gene in the genome is monitored in one
experiment.
• This can be carried out at the RNA level (by direct sequence
sampling or through the use of DNA arrays) or at the protein
level.
• Global expression profiling produces a holistic view of the
cell’s activity.
ANALYSIS OF THE TRANSCRIPTOME
• Complex aspects of biological change, including differentiation,
response to stress, and the onset of disease, can thus be
studied at the genomic level.
• Instead of defining cell states using single markers, it is now
possible to use clustering algorithms to group data obtained
over many different experiments and identify groups of coregulated genes.
• This produces a new way to define cellular phenotypes, which
can help to reveal novel drug targets and develop more
effective pharmaceuticals.
• Furthermore, anonymous genes can be functionally annotated
on the basis of their expression profiles, since two or more
genes that are co-expressed over a range of experimental
conditions are likely to be involved in the same general
function.
THE TRANSCRIPTOME IS THE COLLECTION OF ALL
MESSENGER RNAs IN THE CELL
• The full complement of mRNA molecules produced by the
genome has been termed the transcriptome, and the methods
for studying the transcriptome are grouped under the term
transcriptomics.
• Taking human beings as an example, it has been shown that
only 3% of the genome is represented by genes, suggesting
that the transcriptome is much simpler than the genome.
• This is not the case, however, because the transcriptome is
much more than just the transcribed portion of the genome.
• The complexity of the transcriptome is increased by processes
such as alternative splicing and RNA editing, so that each gene
can potentially give rise to many transcripts, each of which may
have a unique expression profile.
THE TRANSCRIPTOME IS THE COLLECTION
OF ALL MESSENGER RNAs IN THE CELL
• In extreme cases, where a gene has many introns and
undergoes extensive differential processing, one gene may
potentially produce thousands or even millions of distinct
transcripts.
• An example is the Drosophila gene Dscam (the homolog of the
human Down Syndrome cell adhesion molecule), which can be
alternatively spliced to generate nearly 40,000 different mature
transcripts (twice the number of genes in the Drosophila
genome).
• Each of these transcripts potentially encodes a distinct receptor
that may play a unique role in axon guidance.
THE TRANSCRIPTOME IS THE COLLECTION
OF ALL MESSENGER RNAs IN THE CELL
• Complex as the transcriptome is, it is never seen as a complete
system in vivo.
• This is because all genes are not expressed simultaneously, in
the same tissues, at the same levels.
• Cells transcribe a basic set of housekeeping genes whose
activity is required at all times for elementary functions, but
other luxury genes are expressed in a regulated manner, e.g., as
part of the developmental program or in response to an
external stimulus.
• Similarly, post-transcriptional events such as splicing are also
regulated processes.
• Researchers use phrases such as “human brain transcriptome”
or “yeast meiotic transcriptome” to emphasize this.
•
THE TRANSCRIPTOME IS THE COLLECTION
OF ALL MESSENGER RNAs IN THE CELL
• A typical human cell is thought to express, on average, about
15,000-20,000 different mRNAs, some of which have
housekeeping functions and some of which are more
specialized.
• A proportion of these will be splice variants of the same
primary transcript.
• Some of the mRNAs will be very abundant, some moderately
so, and others very rare.
• For a truly global perspective of RNA expression in the cell,
all of these transcripts must be quantified at the same time.
• This requires a highly parallel assay format which is both
sensitive and selective.
THE TRANSCRIPTOME IS THE COLLECTION
OF ALL MESSENGER RNAs IN THE CELL
There are two major types of strategy currently used for global RNA
expression analysis:
•The direct sampling of sequences from source RNA populations or
cDNA libraries, or from sequence databases derived therefrom.
•Hybridization analysis with comprehensive, non-redundant
collections of DNA sequences immobilized on a solid support. These
are known as DNA arrays.
Although such analysis is often called transcriptional profiling it is
important to emphasize that one is not really looking at the level of
transcription, but at the steady-state mRNA level, which also takes
into account the rate of RNA turnover. Furthermore, most of the
transcriptional profiling techniques do not measure absolute RNA
levels, but rather compare relative levels within and/or between
samples.
THE END