as a binding site for the heatshock protein, HSP101, which is required for translational
enhancement. The efficiency of translation initiation is also affected by other mRNA struc-
tures, including the length of the leader—short leader sequences lead to reduced translation
efficiency. Secondary structures, both upstream and downstream of the AUG start codon,
can inhibit ribosome entry and again reduce translation efficiency. The consensus
nucleotide sequence surrounding the AUG start codon in dicots (dicotyledonous plants)
is aaA(A/C)aAUGGCu, while in monocots (monocotyledonous plants) it is c(a/c)(A/
G)(A/C)cAUGGCG). The presence of upstream AUG codons are particular features of
some genes that can reduce translational efficiency [for a review, see Kozak (2005)].
Foreign genes often contain nucleotide sequences that are not commonly used by plants
to encode amino acids. Unusual codon usage can affect mRNA stability. For example,
Bacillus thuringiensis(Bt) toxin genes are typically A/T-rich with an A or a T in the
third position of codons, which occurs only rarely in plants. Extensive modification of
the nucleotide sequence in the coding region of these genes can result in increased
expression so that enough Bt toxin would be produced to kill target insects that fed on
host plants. The plant species chosen for modification may also influence the design of
the transgene construct, since the codon bias in monocot genes tends to be more stringent
than it is in dicot genes.
Agrobacterium-mediated plant transformation has had a limited taxonomic host range,
with most successful reports of transformation among dicots. Modifications to plant trans-
formation protocols can, however, lead to the successful transfer of genes to plant species
once thought to be beyond the host range ofAgrobacterium, including a number of mono-
cots, such as rice and wheat. Despite these advances, monocots are most often transformed
using microparticle bombardment (Biolisticsw) (for a more detailed description of micro-
projectile bombardment-mediated transformation, see Chapter 10). Particle bombardment
does not require the use of plant binary vectors containing a T-DNA, since the DNA is
physically delivered into the cell by the force of the projected particle. In early plant trans-
formations using particle bombardment, entire plasmids were used, but more recently, only
the transgene cassette (promoter, gene, and terminator sequences) has been used. This
approach has reduced the transgene copy number and eliminated the insertion of unwanted
vector sequences.
7.3 Greater Demands Lead to Innovation
Recombinant DNA technology has become more sophisticated as new techniques have
emerged and greater demands have been made in the analysis of genes and the development
of biotechnological innovations. Today it would not be unusual, in the course of analyzing
a gene, to express the gene under a variety of promoters, make fusions with reporter genes
(Chapter 9) for subcellular localization studies, or make fusions with a purification tag for
biochemical analyses. All these types of analysis involve complex DNA manipulations so
that a gene and/or its promoter can be inserted into the appropriate vector. Such manipula-
tions have been facilitated by vectors that incorporate a series of restriction endonuclease
recognition sites in a sequence known as apolylinkerormultiple cloning siteso that
there is a convenient place in the vector to insert DNA. However, since vectors do not
always contain a standardized polylinker, DNA molecules are not easily exchanged
between vector types. In addition, genes and their promoters differ. Genes are rarely
flanked by convenient restriction sites for cloning and often contain internal restriction
170 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION