Gene Cloning

Genome Organization 11
both of which have arisen through the duplication of regions of the
genome during evolution. A duplicated region can either be on the same or
a separate chromosome.
Introns
Most eukaryotic genes are made up of stretches of DNA that code for
amino acids, interrupted by non-coding sequences. The coding and noncoding
sections are called exons and introns respectively. An example of
eukaryotic gene structure is shown in Figure 2.1, where the gene indicated
has three exons separated by two introns. Eukaryotic RNA polymerases
transcribe both the exons and introns to yield an RNA molecule called the
primary transcript; it requires further processing before it can be used as a
template for translation (Figure 2.2). This processing includes removal of
the introns in a process called splicing, which edits the exons into a contiguous
coding sequence that can be translated by the ribosome. Introns
make up 24–28% of the human genome, and the “average” human gene is
encoded by nine small exons of 145 bp each separated by large introns of
over 3 kb each.
The existence of introns has many implications for gene cloning and
analysis. Most genetic engineering requires the manipulation of a
sequence that is devoid of introns, i.e. is a single contiguous coding
sequence. As you can see from Figure 2.2a, the mature messenger RNA
contains an uninterrupted copy of the protein coding sequence. Therefore
the easiest way to obtain a contiguous coding sequence is to isolate messenger
RNA and to then create a DNA copy. A DNA copy of messenger RNA
is called cDNA, and the way in which this is produced and used will be discussed
in Section 4.18. Another issue that is raised by introns is the problem
of identifying genes within whole genome sequences. This is made
particularly difficult because of the fact that small exons are separated by
very large introns and predicting exon-intron boundaries is a difficult challenge.
The problem of identifying the coding sequences within genomic
kb
0 10 20 30 40 50 60 70 80 90 100
Genes
CpG
SSR
ITDR
Figure 2.1 Schematic representation of 100 kb of a human chromosome. The four rows
represent different classes of sequence found within the genome. This region of the genome
contains only one gene, the exons are shown as blocks and the introns as lines. The position of
simple sequence repeats (SSRs), interspersed transposon-derived repeats (ITDRs) and CpG islands
(CpG) are indicated by the blocks.