Biomedical Engineering Reference
In-Depth Information
Primary transcript
E1
I
E2
I
E3
Differential splicing
E1
E2
E1
E3
Translation
Polypeptide 1
Polypeptide 2
Figure 4.3 Differential splicing of mRNA can yield different polypeptide products. Transcription of a gene
sequence yields a 'primary transcript' RNA. This contains coding regions (exons) and non-coding regions (introns).
A major feature of the subsequent processing of the primary transcript is 'splicing', the process by which introns
are removed, leaving the exons in a contiguous sequence. Although most eukaryotic primary transcripts produce
only one mature mRNA (and hence code for a single polypeptide), some can be differentially spliced, yielding two
or more mature mRNAs. The latter can, therefore, code for two or more polypeptides. E: exon; I: intron
Additionally, the cellular location at which the resultant polypeptide will function often can-
not be predicted from RNA delection/sequences nor can detailed information regarding how the
polypeptide product's functional activity will be regulated (e.g. via post-translational mechanisms
such as phosphorylation, partial proteolysis, etc.). Therefore, protein-based drug leads/targets are
often more successfully identifi ed by direct examination of the expressed protein complement of
the cell, i.e. its proteome. Like the transcriptome (total cellular RNA content), and in contrast to
the genome, the proteome is not static, with changes in cellular conditions triggering changes in
cellular protein profi les/concentrations. This fi eld of study is termed proteomics.
Proteomics, therefore, is closely aligned to functional genomics and entails the systematic and
comprehensive analysis of the proteins expressed in the cell and their function. Classical proteomic
studies generally entailed initial extraction of the total protein content from the target cell/tissue,
followed by separation of the proteins therein using two-dimensional electrophoresis (Chapter 7).
Isolated protein 'spots' could then be eluted from the electrophoretic gel and subjected to further
analysis; mainly to Edman degradation, in order to generate partial amino acid sequence data.
The sequence data could then be used to interrogate protein sequence databanks in order to, for
example, assign putative function by sequence homology searches (Figure 4.4). Two-dimensional
electrophoresis, however, is generally capable of resolving no more than 2000 different proteins,
and proteins expressed at low levels may not be detected at all if their gel concentration is below
 
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