Title: PLANT BIOTECHNOLOGY
1PLANT BIOTECHNOLOGY GENETIC ENGINEERING(3
CREDIT HOURS)
- LECTURE 13
- ANALYSIS OF THE TRANSCRIPTOME
2ANALYSIS 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.
3ANALYSIS 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 cells activity.
4ANALYSIS 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
co-regulated 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.
5THE 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.
6THE 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.
7THE 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. -
8THE 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.
9THE 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.
10THE END