Download 4th Edition CHAPTER 16 1. The advantages of biological over

4th Edition
CHAPTER 16
1. The advantages of biological over chemical insecticides is that organophosphates and chlorinated
hydrocarbons have long-term and dramatic side effects on animals (i.e. low specificity). Biological
insecticides are usually highly specific but they have low potency.
3. Bt toxin is not toxic to humans because when the protoxin is ingested by an insect, the protein is
activated by the neutral pH of the insects' gut (pH 7.5-8.0) and by specific digestive proteases. The
active form is inserted into the membrane of the gut epithelial cells and creates an ion channel. The ion
channel results in the loss of ATP which halts cellular metabolism. Humans do not have the correct
stomach pH or the digestive enzymes, so they are not affected by the Bt toxin.
7. Creating a chimeric cry1C-cry1Ab protein is 3-34 times more affective than using just the cry1C
toxin, because the cry1Ab portion (even though it is cleaved off) increases the stability and therefore
the half-life of the protein. The protein degrades slower giving it more time to kill insects.
9. To lessen or avoid the development of insect resistance, one could stack the Cry genes (so that
multiple, different Cry proteins are produced in the same transgenic plant). It is unlikely that an insect
will become resistant to both genes simultaneously if the toxin binds to two different receptors. One
could plant Bt crops and still use a chemical insecticide or a different Bt toxin to kill off the insects that
do develop resistance to the transgenic Bt gene. One could fuse the Cry gene to a gene for another type
of biological toxin (e.g. chitinase), or one could plant the Bt crop alongside a buffer of the normal crop
species to keep up the population of insects that have not developed the resistance.
13. It is unlikely that any insects will develop resistance to the B. thuringiensis subsp. israelensis
because this subspecies produces at least three different Cry proteins along with Cyt1A (an endotoxin
with an entirely different mode of action that can act as a receptor for the Cry genes). It is extremely
unlikely that an insect will evolve resistance to the Cry and Cyt1A genes at the same time since each is
lethal in its own right.
15. One could improve the insecticidal properties of baculovirus by speeding up the killing of the
targets. Virulence can be enhanced by introducing a second insecticidal gene. Diuretic hormone,
juvenile hormone esterase, B. thuringiensis, scorpion, mite and wasp toxin have been used.
CHAPTER 18
1. The Ti plasmid from Agrobacterium tumefaciens has evolved to naturally insert the genes between
the left and right border sequences (T-DNA) into the chromosome of the host plant as part of the
bacterium's pathology. Scientists just had to replace the genes between the right and left borders with
their genes of interest (a few other changes have also been made to make cloning easier and more
efficient).
3. Reporter genes encode an activity that can be assayed. Many of them are dominant-marker selective
which allow scientists to isolate only transformed cells. Antibiotic resistance genes, for example, mean
that only transformed cells with the resistance gene can grow on media supplemented with the
antibiotic. All un-transformed cells die. Neomycin and hygromycin phosphotransferase are both
selectable markers and reporter genes. Beta-glucuronidase and green fluorescent protein are two
examples of reporter genes that are not selectable markers. These protein products can be detected in
situ and can be used in a variety of different ways to monitor transgenic plants.
4. Microprojectile bombardment is an alternative to the Ti plasmid method for introducing genes into
a plant. Tungsten or gold particles are coated with DNA (precipitated with CaCl2, spermidine or
polyethylene glycol). The particles are accelerated in a gene gun, propelled by high-pressure helium.
The projectiles enter into the plant cells and the DNA is released from the particles. The DNA
occasionally integrates into the host cell's chromosome. The transformed cells can be selected using a
selectable marker and then grown in sterile culture. A whole plant can be regenerated from a few
transformed cells.
5. A root specific promoter could be isolated by inserting a promoter-tagging vector with a two gene
selectable marker system (a constitutive promoter is placed in front of a selectable marker alongside a
promoterless reporter gene). The selectable marker that is constitutively expressed allows for isolation
of only the transformed cells. These cells can then be regenerated into full plants. The second gene, the
reporter gene, has no promoter so it will only be transcribed and translated if it was inserted into the
chromosome behind an endogenous promoter. To find a root specific promoter, use a marker gene like
GFP or GUS and then examine the plant for fluorescence or colored protein, respectively, that is visible
only in the root. The sequence of the promoter can then be determined by adaptor ligation, inverse
PCR, TAIL-PCR or some similar procedure, giving you the sequence of a root specific promoter.
10. The expression of a plant gene can be down-regulated by inserting an additional copy of the gene
(cosupression occurs, through RNA interference), addition of an antisense gene, use of ribozymes, or
virus-induced gene silencing. All of these techniques destroy the gene product at the mRNA stage,
preventing the production of the protein.
11. Introducing a gene into the chloroplast instead of the nuclear genome is advantageous because
chloroplast DNA is inherited maternally so there is no danger of spread of the transgene to the wild
population through pollen spread. High levels of expression can be achieved from multiple copies of
the gene in the chloroplast DNA, and because of operons and polycistronic mRNAs in the chloroplast it
would be easier to genetically engineer a metabolic pathway: one regulatory unit could produce
multiple genes needed for the pathway.