Molecular genetics of Drosophila development

Drosophila gene transfer techniques were worked out by Allan Spradling and Gerry Rubin to create fertile transgenic flies. This method, called P element transformation, has allowed Drosophila geneticists to unequivocally demonstrate which Drosophila genes are responsible for the host of developmental mutants that have intrigued biologists for decades.

P element-mediated transformation

Most Drosophila species contain transposons, the best characterized of which is the P element transposon. P element is a 2.9 kb DNA segment encoding a 87 kDa transposase enzyme flanked by an inverted repeat sequence of 31 bp. Drosophila in the wild contain about 30-50 genomic copies of randomly-integrated P element sequences.

P element transformation is a method to obtain germline DNA integration following co-injection of preblastoderm embryos with purified DNA; one plasmid encodes the transposase, and the other plasmid contains the inverted repeats flanking the experimental gene . Typically, the marker gene on the P element transformation plasmid encodes an eye color marker (e.g., the rosy gene, ry) that can be used to identify transgenes by virtue of their red eyes (ry+), against a background of non-transformed flies with white eyes (ry-).

In this example, the 5’ regulatory region of a developmentally-expressed gene is inserted upstream of the lacZ reporter gene. Second generation females (G1) with ry+ eyes contain the P element insertion in every cell and can be used to monitor expression of the lacZ reporter gene in early embryos.

Identification of strains with single P element insertions and genetic backcrossing is performed with G1 flies to obtain a homozygous isogenic strain. As shown in this example, the protein expression pattern of the endogenous target gene, and lacZ reporter gene, are overlapping in early embryos indicating that the cloned target gene regulatory region is fully functional.

Matings between transgenic lacZ females, and males with defects in genes encoding transcription factors required for embryo development, can be used to determine which transcription factors control cell-specific expression of the target gene.

P element "enhancer trap" vectors

Walter Gehring and his colleagues designed a special P element cloning vector that contained the lacZ gene fused in-frame with the second exon of the transposase. The weak promoter of the transposase-lacZ fusion gene is insufficient to allow significant transcription in the absence of a linked enhancer element.

A variation of the original enhancer trap strategy developed by Andrea Brand and Norbert Perrimon uses P element insertions to identify cell-specific enhancers that selectively direct expression of the yeast Gal4 gene contained on the enhancer trap vector. By crossing one of these Gal4 transgenes with a fly containing an integrated copy of a Gal4-responsive lacZ expression vector, it is possible to obtain cell-specific expression of beta-galactosidase activity.

What are the similarities and differences between P element transposition strategies in Drosophila, and retroviral expression systems in mammalian cells?


What explains the observation that the same transgenic female containing a developmental gene regulatory region fused to the lac Z, can produce embryos with different striping patterns depending on the transcripiton factor defect in the mated male, i.e., what does this tell you about the developmental gene regulatory region?


What are some of the advantages and disadvantages of the P element enhancer trap system?


What is the value of having cell-specific Gal4 expression in the Brand and Perrimon enhancer trap strategy? How is this different than Gehring's method of having the lacZ enhancer trap vector integrated into the same abdominal-specific gene region?

Construction of transgenic crop plants

Two approaches have traditionally been taken to increase the quality and quantity of crop yields.

1) Plant breeders have incorporated desirable traits into seed stocks using standard genetic manipulations.

2) Inhibit the deleterious effects of insects and weeds by spraying crops with pesticides and herbicides.

Plants have several unique characteristics that influence their suitability for transgenesis.

- Hormones and nutrients can be used in culture to permit whole plant regeneration from a de-differentiated tissue mass called a callus.

- Plants cannot move.

- Plants undergo self-fertilization.

- Produce a large number of progeny which are easy to harvest.

Two methods have been developed to generate transgenic plants:

1) Transformation of dicotyledon (dicots) plants such as soybean, squash, and tomato, by Agrobacterium-mediated bacterial conjugation.

2) Particle bombardment of monocotyledon (monocots) plants, represented by rice, corn and wheat, using biolistic devices.

Agrobacterium-mediated gene transfer

Agrobacterium were first identified as the pathogen responsible for the appearance of crown gall tumors in areas of plant wounding. The best characterized strain of Agrobacterium is A. tumefaciens, which transfers a ~25 kb segment of DNA, called T-DNA, into plant cells by a transfer mechanism that resembles conjugation and appears to involve a bacterial pilus structure.

Agrobacterium proteins recognize and cleave Ti plasmid DNA at short repeat sequence elements called the right border (RB) and left border (LB), which define the termini of T-DNA. Agrobacterium-mediated plant transformation has been exploited as an efficient gene transfer method in a variety of dicot plants.

Electroporation of the recombinant T-DNA plasmid into an Agrobacterium strain containing a Ti-plasmid encoding vir genes, but lacking LB and RB sequences, establishes a binary plasmid system in the presence of tetracycline. Exposure of wounded plant tissue to this Agrobacterium strain, promotes T-DNA transfer and random integration of the cloned genes into the plant genome. Plant regeneration in the presence of kanamycin, would produce a transgenic plant with root-specific AMG expression.

Transgenesis of rice and corn with biolistics

Monocot plants are often resistant to A. tumefaciens transformation due to inappropriate wound responses and hormonal differences between dicots and monocots.

John Sanford developed what can only be described as a "shotgun approach" to monocot transgenesis. This "Swat Team" DNA transfection strategy was first done by shooting DNA-coated metal projectiles out of a 22 caliber gun that was aimed directly at meristematic regions of whole plants. Biolistic devices have become much more sophisticated and are routinely used to create transgenic rice, corn and sorghum plants.

Schematic drawing of the Bio-Rad Biolistic PDS-1000/HE transformation system. This instrument uses an evacuated sample chamber that positions the target seedling directly in the path of DNA-coated tungsten or gold particles traveling at a velocity of 1,100 mph!.

A pressurized hand-held gene gun can also be used to shoot DNA-coated gold particles directly into the meristematic tissue of plants growing in experimental fields.

Agricultural impact of transgenic crop plants

It is inevitable that within the next decade, most all economically important plant crops will be derived from transgenic strains. Within the last 5 years alone, transgenic varieties of over 25 commercially grown plants have been approved for agricultural purposes by the US Department of Agriculture (USDA).

This rapid introduction of "foreign genes" into food producing plants has not been without controversy. The term Genetically Modified Organism (GMO) is being used to label transgenic plants and animals. The GMO controversy is catching headlines around the world and is pervasive on the Internet (don't believe everything your read, especially on the Internet!).

One of the first GMOs to hit the market was the Flavr Savr tomato developed by Calgene, a Californian biotechnology company that has since been bought by Monsanto. The idea behind this product was to produce a hearty tomato that could vine ripen and yet still withstand the bumps and bruises of shipping. What ever happened to this pioneering GMO?

What are the Major GMO crops and what is all the fuss about?

Round-Up Ready Soybean plants grown in the US will contain a herbicide-resistance gene that protects the crop from treatment with the broad-spectrum herbicide Round-Up™, a shikimic acid pathway inhibitor biochemically known as glyphosate. Transgenic plants containing the gene encoding Roundup®-tolerant enzyme (CP4-EPSPS) are resistant because this bypass enzyme avoids the block in amino acid biosynthesis caused by Round-Up. These GMO soybean crops can be sprayed repeatedly with Round-Up to eliminate weeds without harming the agricultural product. Round-Up Ready seed products are made by Monsanto and plenty of people are worried about it.

Bt-corn is a genetically modified corn plant that produces the Bt protein of the soil bacterium Bacillus thuringiensis. The Bt protein is toxic to the European corn borer, as well as other insects, including the Monarch butterfly. Read about the controversy; arguments against the use of Bt transgenic corn and other studies showing benefits outweigh the risks.

The protein structure of the endotoxin has been elucidated and biochemical analysis indicate that the toxin specifically binds to cell receptors in the insect intestine and forms pores resulting in loss of membrane integrity. Transgenic Bt-corn contain the toxin in the inactive form, which after ingestation by the larva, is specifically activated in the insect by protealytic cleavage. Current models of Bt toxin function suggest that multiple subunits form a complex that inserts into the membrane like an "umbrella". Concerns regarding human cosumption of Bt corn are related to possible allergic responses in humans to a bacterial toxin.

Bt-corn seed is produced by Novartis and it has provoked much controversy over the possibility that Bt may be an allergen in humans and therefore, Bt-corn is not approved for human food such as taco shells.

Bt cotton has beed developed for use in areas where the cotton bollworm causes crop damage. In 1998, bt cotton accounted for over a quarter of U.S. harvested cotton acreage. Bt cotton is big business in Arizona.

What are some of the concerns regarding introducing GMO crops into agriculture?


What is the difference between methods used to improve crop plants incrementally through classical genetics, and the use of transgenesis to create super crops, i.e, can they lead to the same GMO if given enough time?



What data supports the view that fields planted with Bt corn could lead to the demise of the Monarch butterfly? What data refutes this contention? How can this argument best be resolved?



What is the Flavr Savr tomato? What is the molecular genetic basis for its properties? Is the Flavr Savr tomato available in grocery stores?