Gene transfer to animal cells - Principles of Gene Manipulation - page 200

Box 10.2 continued

example, the 5 ′ UTR may contain one or more AUG
codons upstream of the authentic translational start
site, and these are often detrimental to translational
initiation. The 3 ′ UTR may contain regulatory
elements that control mRNA stability (e.g. AU-rich
sequences that reduce stability have been identified
(see Shaw & Kamen 1986)). Furthermore, both the
5 ′ and 3 ′ UTRs may be rich in secondary structure,
which prevents efficient translation. In animal
systems, UTR sequences are generally removed from
transgene constructs to maximize expression.

The inclusion of a polyadenylation signal
Polyadenylation signals (terminators) are required in
eukaryotic genes to generate a defined 3 ′ end to the
mRNA. In most cases, this defined end is extended by
the addition of several hundred adenosine residues
to generate a poly(A) (polyadenylate) tail. This tail is
required for the export of mRNA into the cytoplasm,
and also increases its stability. In the absence of such
a site, the level of recombinant protein produced in
transformed cells can fall by as much as 90%
(Kaufman 1990). Poly(A) sites from the SV40 early
transcription unit or mouse b -globin gene are often
incorporated into mammalian expression vectors.

Optimization of the transgene for
translational efficiency
The sequence around the translational initiation
site should conform to Kozak's consensus, which is
defined as 5 ′ -CCRCCAUGG-3 ′ (Kozak 1986, 1999).
Of greatest importance is the purine at the − 3
position (identified as R) and the guanidine at
position + 4. The adenosine of the AUG initiation
codon (underlined) is defined as position + 1, and
the immediately preceding base is defined as

The removal of unnecessary
untranslated sequence
Eukaryotic mRNAs comprise a coding region (which
actually encodes the gene product) bracketed by
untranslated regions (UTRs) of variable lengths.
Both the 5 ′ and 3 ′ UTRs can influence gene
expression in a number of ways (Kozak 1999). For

T7 ATG Ig κ Leader myc epitope 6 x His TAA

pSecTag2

continued