ESTRUCTURA DE UNA GUÍA DE UN ESTUDIO DE MERCADO

Four genomic sequence clones that map between Ac008344 and A c004307 were analysed for satellite sequences using the same method described previously. The eleven microsatellites chosen for analysis are shown in the list below:

Clone: Ac008096 Ac004299 Ac004311 Ac005148 Location: (deduced 56C6-10) (deduced 56D2-D11) 56D11-E1 56D15-E1 Size: unordered -87.6 kb -45.1 kb -95.2 kb No.satellites selected: 4 3 2 2

________________________________________________________________Chapter 4 1 o f these 11 microsatellites were examined by SSCP; only one showed a polymorphism between the two parental lines under the SSCP conditions used. This microsatellite (in clone Ac004299, at base pair position 28,821) was tested and found to represent a population polymorphism and was sequenced (Fig 4-8(A)). I genotyped the remaining 4 recombinant lines for this polymorphism, and found tight linkage between the lethal locus and the polymorphism (Table 4-4). This result maps the lethal locus to ±0.3 cM from this polymorphism, as it can be deduced that all 73 recombinants show complete linkage between the lethal locus and this polymorphism (see legend to Table 4-3). No further information could be inferred as to whether the lethal locus mapped to the left or right o f the polymorphism. In summary I have mapped the lethal locus on W049^ to ±0.3 cM from the microsatellite at base pair 28,821 o f Ac004299.

Discussion

When fra was identified as the lethal locus on W049,1 believed I had uncovered the single lethal locus on the right arm of chromosome W049 as an EMS mutagenesis, where the dose of mutagen produces more than one lethal mutation per line, is more likely to induce mutations that lie on different chromosomes or chromosome arms. The finding that another fr a allele also had the maternal segmentation phenotype strengthened my belief that I had identified the correct maternal segmentation gene. However, I subsequently found that there were two lethal loci on chromosome W049, and have shown that the maternal segmentation locus segregates away from fra. Chromosome FQ36 also carries two lethal loci: fra, and a weaker allele o f the maternal segmentation locus^. These two alleles can be used as part o f an allelic series to study the function of this locus.

^ If we estimate that the D rosophila genome has 12,000 genes (BDGP estimate), and that there are 4,800 genes on chromosome 2, then the likelihood of two fra mutant chromosomes (W049 and FQ36)

both containing a second mutation at an identical locus, when derived from independent EMS screens, is 1/4800.1 observed it in 1/9/ra mutant chromosomes tested, and the likelihood of this is 1/4800 x 1/9

________________________________________________________________Chapter 4 The maternal segmentation locus has been mapped using linkage to genome polymorphisms detected using SSCP. I observed a high frequency of polymorphism between the two parental lines as summarised below:

SNPs

No. tested

21 (SSCP and sequenced)

No. polymorphic

8

STSs containing SNPs 4 (SSCP only, not sequenced)

M icrosatellites 11 (SSCP only) 3 (checked

by sequencing)

This high frequency implies that it should be possible to easily detect and use genome polymorphisms between any two different populations for mapping loci by linkage. In one case, I also found a SNP in sequence near to a microsatellite, further indicating the abundance of polymorphisms between any two parental strains. Thus mapping using genome polymorphisms should be straightforward. I also found that using SSCP to detect the polymorphisms was very easy and effective when compared with the methods others have used, such as sequencing (e.g. Jakubowski and Komfeld, 1999).

In summary, this method o f mapping using linkage to genome polymorphisms mapped the locus to a finer resolution compared to mapping using linkage to visible markers, and it required a relatively small number o f recombinants. For example, I mapped the lethal locus to within ± 0.3 cM (-150-300 kb) using genome polymorphisms, whereas for the same number of recombinants (73) and using linkage to the visible marker c, one can only map the locus to a resolution of ± 1.5 map units (-750-1500 kb).

________________________________________________________________Chapter 4 The advantages of this method of mapping are:

(1) The frequency o f polymorphisms in any two populations is not likely to be limiting, whereas the number of visible markers available for linkage analysis is frequently limiting.

(2) Molecular detection of markers means that I required only a single round of recombination for this mapping experiment. Subsequent linkage to any new marker that is found can be demonstrated at a later date by PCR and SSCP on stored DNA samples of the recombinants. In other mapping schemes, such as those using visible markers, a new round of recombination is required every time a new marker is used. Thus mapping by the method I have used can be done within 6-8 weeks. I required 2 generations to generate the recombinants, and a further generation for the complementation test to the lethal locus. Subsequently all mapping is done molecularly, and this can proceed very rapidly as with each mapping step one can exclude many recombinants as they are found to lie outside useful regions for mapping. For example, my first SNP genotyping was done on 73 recombinants, but the next step was done with only 48 and the third microsatellite genotyping was performed on 10 lines only.

(3) One does not need to establish stocks o f all the recombinants. A single fly DNA preparation is sufficient for 25-50 individual PCR reactions, and thus can be used several times for genotyping with respect to different polymorphisms as mapping is refined further. This preparation can also be stored at 4°C for several months. (4) The SSCP analysis gives clear results and is easy to use. The PCR product can be

directly loaded onto the gel and I found that most polymorphisms were detected using one of two SSCP running conditions.

(5) Meiotic mapping and linkage analysis o f this type detects the presence o f multiple mutations on a chromosome as it can map all phenotypes. Thus it could have detected the presence of two lethal loci on chromosome W049.

In contrast, physical mapping using chromosomal deficiencies would not map a locus to such resolution. Male recombination (Chen et al, 1998) is an alternative approach to map a locus, but this technique requires more flies to be screened (~1000-2000) per P-element, and requires a separate round o f recombination for each P-element used

_____________________________________________________________ Chapter 4 for mapping. Additionally, this technique is limited by the density o f P-elements within the region o f interest, and some P-element insertions lines fail to generate recombinants (Chen et al, 1998).

The future work for this project involves the finer mapping and eventual cloning of the maternal segmentation locus, for which we have devised a scheme (described below), and the project will be continued by B. Jennings. Briefly, more informative recombinants within the region 56C1-D1 and 56D11-E6 are needed, and these can be obtained by using P-elements as dominant markers that flank this region, rather than the more distant c and p x markers. Thus, two P-element lines o f intermediate eye colour (containing the mini-w^ transgene) that map on either side o f this region (-500 kb apart) are selected, and a double recombinant line generated that carries both P- elements (P-P), and has a correspondingly stronger eye colour. From the progeny of P-P/W049 females, recombinant males with a single P-element can be selected which show a reversion to intermediate eye colour. These males are recombinants in the region of interest, and after successful mating to W049^ f\iQS to test for allelism, their DNA can be extracted and their polymorphisms genotyped as before. We estimate that obtaining about 100 recombinant males should map the locus down to about 10- 20 kb, after which direct DNA sequencing will allow the mutated gene to be identified. Its role in segmentation can then be examined.

_______________________________________________________________ Chapter 5

CHAPTER 5

MISEXPRESSION OF FRAZZLED DERIVATIVES DURING

TARGET SELECTION IN THE PERIPHERY

Introduction

In chapter 3 f r a was identified as a maternal segmentation gene, and two experimental approaches were initiated in parallel to address its function. One approach to study its functions in different developmental processes was to generate m utant forms o f f r a and misexpress them during segmentation and neural development. The other genetic approach (described in Chapter 4) however, showed that f r a did not encode a maternal segmentation function. Therefore the misexpression of mutant forms offra was examined only in neural development and not during segmentation. Fra transduces the attractive Netrin signal during pathfmding in neural development, but the mechanism by which this signal is transduced is unknown. In this chapter I present the results of experiments initiated to investigate Fra signal transduction during pathfmding at the midline and target selection in the periphery. The same signaling mechanism may be used in both processes, and the findings will have global implications as the Net-Fra ligand- receptor system is evolutionarily conserved (Chapter 1).

fra and net mutants have similar, partially penetrant, phenotypes in the CNS and periphery in Drosophila. In the CNS, the axon commissures are thinner and sometimes missing and the longitudinal axon tracts have occasional breaks. In the periphery, the ISN fails to recognise muscles 1 and 2 of its dorsal muscle targets and the ISNb fails to innervate its ventrolateral target muscles 6 and 7, however pathfmding by these motoneurons is unaffected (Chapter 1). Fra is expressed on all CNS and motoneuron axons; Net-A is expressed by midline cells and by dorsal muscles 1 and (more weakly) 2, and Net-B is expressed by midline cells and by dorsal muscle 2 and ventral muscles 6 and 7 (Figure 1-7). The CNS phenotypes suggest that the Netrins may be acting as long-range chemotropic signals from the

_______________________________________________________________ Chapter 5 midline, whereas the peripheral phenotypes described above are more indicative o f a local attractive effect at the level o f target selection by the motoneurons as they innervate the body wall muscles (Mitchell et al, 1996, Harris et al, 1996, Kolodziej et al, 1996).

Aside from the net mutant phenotype, the role o f the Netrins in target selection is also supported by genetic experiments where the Netrins are misexpressed on all or a subset o f muscles in a variety of genetic backgrounds (Winberg et al, 1998; see Chapter 1). Together with the partial penetrance of motoneuron defects seen in net and fr a mutants, these observations support the combinatorial model of target selection, and point to the existence of other signaling cues. These other cues may be detected if they enhance/suppress the penetrance of the ISN and ISNb phenotypes of net mutants. For example, Semall has an antagonistic effect to Netrin on RP3 innervation of muscles 6/7, as Sem all net double mutants suppress the net ISNb phenotype, and Sem all overexpression enhances the net ISNb phenotype. Thus Net and Semall may be acting together to target RP3 innervation of muscles 6 and 7. FasIII is also a candidate targeting cue, but neither FasIII net nor Fas III fra double mutants show altered penetrance of the net RP3 phenotype (Winberg et al, 1998).

Little is known about how Fra transduces the Netrin signal and the protein contains no identified signaling motifs. Fra has three conserved intracellular domains (Figure 5-1) which might serve as interaction interfaces with downstream mediators o f signaling. The only function ascribed so far to any o f the conserved intracellular domains o f Fra is in Netrin-dependent repulsion in vitro, mediated by an interaction o f the PI motif in DCC with UNC-5 (Hong et al, 1999). Additionally, as the same pair o f ligands and receptors (Netrin and Fra) interacts during axon guidance in the CNS and muscle target selection in the periphery, one might expect that the signal transduction pathways for both processes are also the same. These issues o f how Fra signals and whether the same signaling pathway operates in both midline pathfinding and target selection can be addressed by examining the expression of mutant forms of Fra to define domains required for its function. Other genetic approaches, e.g. to

Chapter 5

Figure 5-1: Sequence alignment of fra homologues

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