Lecture 21: P elements Readings : Hartwell Reference D:

1. Lecture 21: P elements Readings: Hartwell Reference D: Drosophila melanogaster: Genetic Portrait of the Fruit Fly. Genetic Portrait of the fruit fly, esp. 818-826. Also 460-465, Transposable Genetic Elements Outline: General introduction -- hybrid dysgenesis Transposition, including regulation by splicing and delta2-3 Transformation -- Spradling and Rubin Mutagenesis -- the single P element gene disruption project Enhancer traps Mosaic analysis Gene replacement methods

2. General introduction -- hybrid dysgenesis Hybrid dysgenesis. Hybrid dysgenesis occurs only when P males mate with M females. The progeny of such crosses are semisterile. Male recombination is observed, and their progeny show mutations.

3. Events during a dysgenic cross. Eggs produced by M females have no repressor protein, so P elements brought into the embryo in sperm from P fathers will transpose rapidly.

4. Transposition, including regulation by splicing and delta2-3

6. P element anatomy (Engels, modified from Lindsley and Zimm 1992) : An autonomous P element is shown with some of its sequence features and a restriction map. Three examples of nonautonomous P elements, including the Type II repressor-making elements, KP and D50. Deletion of the germline specific intron (the D2-3 P element) boosts overall transposase activity and allows somatic activity

7. P element transformation

14. The enhancer-trap technique

15. Sensory mother cells in the wing imaginal disc. The sensory mother cells (bluish here) are easily revealed in this special strain of Drosophila, which contains an artificial lacZ reporter gene that, by chance, has inserted itself in the genome next to a control region that causes it to be expressed selectively in sensory mother cells. Animals such as this provide a way to detect and track down specific control regions in the genome - the so-called enhancer-traptechnique. The purple stain shows the expression pattern of the scute gene; this foreshadows the production of sensory mother cells and fades as the sensory mother cells successively develop. (From P. Cubas, J.-F. de Celis, S. Campuzano, and J. Modolell, Genes Dev. 5:996-1008, 1991.) -- legend from Molecular Biology of the Cell, 3rd edition.

18. Mosaic Analysis

23. Clones homozygous for the B52 null allele are seen as white patches (lacking the P[lacZ] transgene.

25. Oogenesis in Drosophila The oogonium divides by mitosis. Incomplete cytokinesis creates 16 cells interconnected by cytoplasmic bridges. The central cell will become the oocyte and the others will become nurse cells. The oocyte is surrounded by somatic cells known as follicle cells. It is the assymmetric distribution of materials from the follicle cells that is ultimately responsible for polarity in the embryo.

27. "Flip out" method

28. Effect of ectopic expression of Hedgehog on Drosophila wing development. (a) An interruption cassette construct can be used to express Hedgehog ectopically. This construct is introduced into Drosophila along with one containing an inducible promoter linked to the recombinase gene. Expression of the hedgehog gene (orange) is disrupted by the intervening yellow gene (blue). Brief exposure to heat shock induces expression of the recombinase enzyme, which catalyzes recombination at the FRT sites, leading to excision of the yellow gene and fusion of the constitutive promoter to the hedgehog gene. (b) In the developing wing of a normal fly, Hedgehog is expressed in the posterior (P) compartment (orange). Expression of Hedgehog in a localized sector in the anterior (A) compartment during development leads to mirror image duplication of anterior structures. This effect is similar to that resulting from ectopic expression of Sonic Hedgehog in the anterior region of the chick limb bud [See K. Basler and G. Struhl, 1994, Nature 368:208.] Legend from Molecular Cell Biology.

29. Gene Replacement

30. Targeted mutagenesis by homologous recombination in D. melanogaster. Rong et al. 2002. Genes Dev. 16: 1568-1581

31. Allelic substitution. In the first step, a standard ends-in targeting is used to carry a single point mutation into the target locus (see Fig. 1). In the second step (shown here), the target locus duplication is reduced to a single copy by HR between the repeated sequence elements. This event is stimulated by an I-CreI-generated DSB between the repeats.

35. Anterior-posterior specification in Drosophila. The syncytial blastoderm allows direct transcriptional regulation to generate spatially restricted patterns of expression for a number of genes, most significantly, the homeotic selector genes.