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PLANT BIOTECHNOLOGY

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Title: PLANT BIOTECHNOLOGY


1
PLANT BIOTECHNOLOGY GENETIC ENGINEERING(3
CREDIT HOURS)
  • LECTURE 13
  • ANALYSIS OF THE TRANSCRIPTOME

2
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.

3
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 cells activity.

4
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
    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.

5
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.

6
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.

7
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.

8
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.

9
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.

10
THE END
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