4.2.1 The lethal P element l(3)rG166 is an insertion within the CG1135 ORF
The CGI 135 locus was found to contain an uncharacterised lethal P element insertion, l(3)r0166. Sequencing of the insertion site had been performed (AQ026355, (Flybase 1998)) and was found to match sequences within the CGI 135 gene. Further analysis of this sequence found it to be in the last exon of the transcript within the ORF. Figure 4.12 shows a scheme of the genomic region of CGI 135 and the position of the P element. The insertion site is also marked in the CGI 135 sequence in figure 4.1. The P element is the ry^ lacZ enhancer trap P{PZ} (Rubin and Spradling 1983). The lacZ
transcription unit is oriented in the opposite direction to the orientation of the CGI 135 gene. Staining using a P-galactosidase antibody found that the enhancer trap is expressed in a restricted pattern in the eye and leg disc (figure 4.4), but not in the embryo (not shown). Due to the orientation of the P element and the fact that CGI 135 has been shown to be expressed ubiquitously, at least in the embryo, it is probable that the P element is not a CGI 135 enhancer trap. It may however represent the expression of the CGI 1359 gene which is transcribed in the same direction as the enhancer trap and starts just upstream of the beginning of the CGI 135 transcription unit (see figure 4.12). However, it is still formally possible that the enhancer trap does represent a subset of the CGI 135 expression domain which is controlled by elements 3’ of the transcription unit.
As the l(3)rG166 P element is inserted within the ORF of CGI 135, it assumed to be an allele of this gene. Insertion of the P element would in the least disruptive case cause the production of a truncated CGI 135 protein missing most of the FHA domain. This protein would be expected to be non-functional and could possibly represent a dominant
Chapter 4
negative version. However, the P element insertion would also lead to the production of a truncated mRNA lacking the 3’UTR and poly A signal. Therefore the allele may represent a protein null for CGI 135.
4.2.2 Investigation o f genetic interactions between l(3)rGI66 and mirror
Genetic interactions between l(3)rG166 and mirror were first assayed by crossing the P element line to various mirror alleles and looking at viability of progeny carrying both mutations. The mirror alleles mrr^\ m r /^ and have been described previously (chapter 3). As can be seen from table 4.1, the presence of the l(3)rG166 allele did not affect the survival of any of the mirror alleles. The percentage of surviving progeny carrying both mutations was increased compared to the expected mendelian ratios, but this is probably due to the deleterious effects of the balancer chromosomes which also cause reduced viability. In order to investigate a genetic interaction further, the l(3)rGI66 allele was recombined with the three mirror alleles. Double mutant chromosomes were confirmed to contain both the l(3)rGI66 and m irror mutations by backcrossing to the original stocks. The effect of the l(3)rG166 mutation on homozygous viability and the survival of m r / f m r /^ flies was then tested. The results in table 4.1 indicate that the presence of the CGI 135 allele does not drastically affect the survival of flies with reduced amounts of Mirror, although a slight decrease in viability was observed.
Chapter 4
Figure 4.4 Expression of p-galactosidase from the l(3)rG166 enhancer trap
Figure 4.4 Confocal images of an eye-antennal disc (A, anterior left, dorsal up) and a leg disc (B) from larvae of the l(3)rG166 enhancer trap line. The discs were stained with anti- P-galactosidase antibody (green). The enhancer trap shows expression posterior to the morphogenetic furrow (arrow) and in patches in the dorsal head region and the antennal disc. The enhancer trap is also expressed in distal parts of the leg disc.
Chapter 4
Table 4.1 Genetic interactions between CG1135 and mirror alleles
Alleles % viable progeny /mirror alleles
m r/^ mrr^^ mrf^^ l(3)rG166 46% (50%) 44% (33%) 36% (25%) l(3)rG166, m r/^ 37% (50%) 16% (33%) ND viable 33% (50%) ND l(3)rG166, m r/^ 25% (50%) lethal lethal m rr^ 33% (50%) lethal lethal
Table 4.1. Crosses were /fijrG7(56/TM3 x mri^V l(3)rG166/T'M3 x mrr^^/TM6;
l(3)rG166/TM3 x l(3)rG166, m r/^/TM 3 x m rr^^/m r/^\ l(3)rG166, m r/^
X m r / ’VTMô; l(3)rG166, mri^^ x m rr^^/ m rr^\ Viable progeny of the indicated
combination of alleles were counted (expected percentage of progeny in brackets). ND: not determined.
As described in the introduction, various combinations of mirror alleles which lead to reduced expression levels, have been shown to exhibit a mild Iroquois phenotype characterised by the loss of some lateral bristles and duplication in scutellar bristles. (Kehl
et a l 1998). These phenotypes are also associated with the mrr^V mrr^^ and mri^^/ m r/^
allelic combinations (I. Dahlsveen and H.McNeill, figure 4.5). It was found that the presence of the l(3)rG166 allele affects these phenotypes. Figure 4.5 shows the absence of bristles in m r /^ and m r /^ flies carrying the l(3)rG166 allele. Adults heterozygous for the CGI 135 mutation did not show any bristle defects (not shown). In order to quantify the effect, the presence of bristles in the notum of double mutant flies was recorded. The results are presented in table 4.2 and show that the CGI 135 mutation increases the loss of the posterior superalar and the anterior postalar bristles and increases the frequency of duplication of the anterior scutellar bristle.
Figure 4.5 Loss of lateral bristles in CGI 135, mirror double mutants
Figure 4.5 Adult thorax from flies carrying mirror and CGI 135 mutations. A) mirr^^l+;
B) mirr^^/mirr^^; C) l(3)rG166, mirr^^l mlrr^^\ D) l(3)rG166, mirr^^l mirr^^. The presence of the l(3)rG166 allele causes increased frequency of loss of the posterior super-alar (pSA, white arrow) and anterior post-alar (aPA, black arrow) as well as increased frequency of doubling of the anterior scutellar (aSC, red arrow) bristles. Please see table 4.2 for details.
Chapter 4
Table 4.2 The effect of the CGI 135 allele on the mirror bristle phenotype Allelic combination Frequency of presence Frequency of
duplication of aSC
N=
aSA pSA aPA pPA
mrr^^l mrr^^ 1 0.75 0.53 0.14 0.34 242
m r/^l l(3)rG166, mrr^^ 1 0.86 0.27 0.13 0.63 366
mrr^^l mrr^^ 0.99 0.59 0.12 0.05 0.06 332
m r/^l l(3)rG166, mrr^^ 0.99 0.15 0.08 0.07 0.25 142
mrr^^l l(3)rG166, mrr^^ I 0.22 0.06 0.05 0.24 221
Table 4.2. Frequency of the presence of bristles (aSA: anterior superalar; pSA: posterior superalar; aPA: anterior postalar; pFA: posterior postalar) per hemi-notum (N= number of hemi-nota scored) for various combinations of mirror and CG1135 alleles. Frequency of duplication of the aSC (anterior scutellar) bristle also included.
The above results indicate that m irror interacts genetically with the l(3)rG166
allele. It seems that reducing the amounts of CG1135 somehow impairs Mirror function. In chapter 3, genetic interactions were also investigated using two over-expression systems for Mirror. A deficiency covering CGI135 was found to enhance the phenotypes associated with ectopic Mirror expression. However, these effects could not be reproduced using the l(3)rG166 allele (not shown).