RESULTS

Morphological analysis of the Om(1J)Su34^R flie´s eyes. In the Om(1J)Su34 Om(1D)9 flies, the Om(1J)Su gene suppressed the Om(1D)9 phenotype reverting the mutant phenotype to the wild type and in the Om(1J)Su34^R Om(1D)9 flies the phenotype is Om(1D)9. In the Om(1J)Su34^R mutants as well as Om(1D)9 ones, there are fewer ommatidias than in the wild type, however, they have a normal cell constitution. In the Om(1J)Su34^R mutants it was observed a reduction in the compound eye size and an anterior indentation around which the ortogonal characteristic array of ommatidias is disturbed. The Om(1J)Su34^R mutants eyes also present an additional indentation in the posterior margen, however, as well as in Om(1D)9 flies, not always present (fig.1).

Cytological analysis of the revertants.Table 1 presents a resume of the cytological characteristics observed in the revertant´s chromosomes. In general, Om(1J)Su34^R chromosomes showed various chromosomes arrangements, all of them sharing the association of chromosomes arms (Fig.2). There were no visible arrangements in the X chromosome of control larvaes. These chromosomal association made difficult the chromosome preparation extending and the identification of the chromosomes involved in those association. Om(1J)Su34^R8 Om(1D)9 presented a terminal transposition of the 1A-6C segment of the X chromosome to the 24A region of 2L. In some individuals of this line, the 6C region of the XL was associated to the 24A region of the 2L, but it was not seeing any brakes nor transposition (Fig 2c and d). These data suggest that the revertion of the Om(1J)Su34 phenotype in Om(1J)Su34^R8 could be attributed to the arrangments observed in the X chromosome.

In the Om(1J)Su34^R7Om(1D)9 individuals, transposition events were also observed in the 6C region of the X chromosome (Table 1). Some of the flies presented the transposition of the terminal fragment of the X to chromosome 2 and also the association of chromosome 3 to the X-2 transposition site (Fig. 2).

InOm(1J)Su34^R9 Om(1D)9 there were no visible rearrangements in the X chromosome, eventhough there could be puntual mutations or microdeletions in the Om(1J)Su region that are not detected by our techniques. In general, all the revertant mutatants presented small insertions of about 2 bands in lenghts in different sites of the genome, that could be due to transposition events. Fig. 2 The in situ hybridization analysis of the Om(1J)Su34^R and control polytenic chromosomes with probe pBB1.9 (Fig 3) marked the position 6C of XL, the site assigned to the Om(1J) locus. However, probe pSB5.2 (Fig 3) showed multiple hybridization sites, specially in the X chromosome. The data obtained with this probe, pSB5.2 is not clear but it is possible that it contain repetitive sequences and that these results are due to the recognition of the multiple sites were this sequence is in the genome. The sequence analysis of this probe as well as the homologous regions will give evidence for this hypothesis. The revertants chromosomes were also analyzed with probe ptom28 containing the sequence of tom element (provided by Tsuchida). This probe recognize the 6C position and multiple other insertion sites in the genome of Om(1J)Su34 (control) and the Om(1J)Su34^R (revertants). However, this probe was not found to be associated to the small transpositions nor the rearrangments of the revertants (data not shown).

Genetic map of Om(1J)Su34^R.The Om(1J)Su34 flies does not have a visible phenotype by themselves. The can be indentified only by combination with another Om phenotype, as for example, Om(1D)9. We assigned the Om(1J)Su34^R to the X chromosome using the cis combination of two Om mutations: Om(1J) and Om(1D).The position of Om(1J)Su34^R in the X chromosome was found by analyzing the recombination value in the progeny from ct y Om(1J) Om(1D)9 and Om(1J)Su34^R Om(1D)9 males. The genetic markers were the yellow-Om(1J) mutations. Table 2 present the data obtained from the anlysis. These data allowed as to map the Om(1J)Su34^R alleles to 2.9 cM from the yellow locus (Fig 4), in concordance with the location of Om(1J) ⁽¹¹⁾.

Om(1J)Su34^R mutants genomic analysis The results obtained from the cytological analysis of the Om(1J)Su34^R mutants, suggested that the mutations are caused by rearrangements in the Om(1J) locus. We wanted to check if the reversion of the Om(1J)Su suppressor phenotype was associated to the lost of the tom element in the region. With this purpose, different fragments of the ca;px Om(1J) region (Fig 3), were used as probes for the analysis of the DNA of the mutants flies. The genomic DNA was digested with EcoRI and HindIII restriction enzymes, determining fragments of about 4 kb and 15.5 kb in the Om(1J) region. The Southern blot data (Fig 5) indicate that there are no differences in the Om(1J)) region between the Om(1J)Su34 and Om(1J)Su34^R flies. From the analysis of the region with the pBB1.9, pSB5.2 and pES5.2 probes it is concluded that all the Om(1J)Su34^R mutants have the tom element inserted in a Hind III-digested fragment of about 15.5 kb. The restriction map of this region showed to be similar in the Om(1J)Su34 Om(1D)9 and Om(1J)Su34^R Om(1D)9 flies. On the other hand, the hybridization with the pSB5.2 probe showed some differences (Fig 6) The pSB5.2 probe detected a deletion of about 2 Kb in a fragment of 12 Kb in the Hind III-digested DNA, moreover, there were unexpected bands of about 15 Kb and 23 Kb in the Om(1J)Su34^R mutants and control flies. Due to the length of these bands, it was not possible to assign them to the Om(1J) locus because they are not equivalent in number nor size to those expected after the available restriction map of this region done by Fujioka (1995). More evidence for this expectation is given by the fact that these fragments were not detected with the pES5.2 and pBB1.9 probes that partially connect to pSB5.2 (Fig 3). According to the in situ hybridization to the polytenic chromosomes with pSB5.2 probe, these unexpected bands could correspond to genomic fragments that contain repeated sequences.

We also checked for the presence of the tom element in the pSB5.2 probe. The results show that the tom element is not part of the sequence in this probe (data not shown). A posterior analysis of the probe showed that there is at least one extra HindIII restriction size 2 Kb away from the SalI 5´end of the probe, which was not considered in the original restriction map. Even though, these results do not explain the size of the obtained fragments. Due to the differences between our data and the one originally obtained by Fujioka on the restriction map of the Om(1J) region, it was necessary to analyze all the probes respect to the presence of undetected restriction sites and the construction of a new Om(1J) restriction map. These results did not let us assign a specific region as the cause of the reversion of the Om(1J)Su34 suppressor mutation.

DISCUSSION

In Drosophila, suppressor mutations have, in general, a spontaneous origin and some of them are associated with the insertion of a transposable element into the genes. One of these classes presents structural characteristic similar to the retroviral provirus of the vertebrates. Among these suppressor mutations, the Hairy-wing, su(Hw) locus has been intensively studied in D. melanogaster. Most of su(Hw) alleles are caused by the insertion of gypsy ^(14,15). This retrotransposon presents its own transcription initiation signal that interacts with the regulatory sequences of adjacent genes, affecting their expression and causing the mutant phenotype ⁽¹⁶⁾. The studies on Om mutations suggest that it mechanism of action could be similar to su(Hw). In 1988, Dr. Hinton proposed that the mechanism by which the tomelement cause the Om mutations in D. ananassae is by an over-expression of those genes where tom inserts. In concordance with this hypothesis, it has been shown that the tom element presents it own promoter for the expression of the Om genes when activated by tissue-specific signals ⁽¹⁷⁾.

The Om genes suppressor effect it is observed only when the suppressor Omgenes are expressed with other Om genes; the Om(1K) gene do not suppress other mutations that affect the eye structure, as Pu and Lo or sn^{g9 (11)}. The lack of information about the Drosophila suppressor genes make the Om genes and specially the Om(1J) region an interesting material for the study of the molecular mechanisms of suppressor mutations.

The analysis of Om(1J)Su34 showed that the mRNA detected in this region do not correspond to the suppressor gene. Other Om genes also present transcription domains near the tom insertion site that do not correspond to the mutated gene. The observations of Yoshida et al. ⁽⁵⁾ and Juni et al. ⁽⁶⁾ about the Om(2D) and Om(1E) genes, show that in Om(2D) there are at least four independent coding regions near the tom insertion site. One of them is the precursor gene for the OATtom near a gene do not ever promote it expression ⁽⁵⁾. In the Om(1E)53 allele, tom insertion site it is located 15 kb upstream respect to the Om(1E) transcription initiation site, and even though there are two transcripts in between this two regions, their expression do not result in a mutant phenotype ⁽⁶⁾. In Om(1A), Om(2D) and Om(1D), the site of tom insertion reside 70 Kb downstream the transcription initiation site ^(2,4,5).

The Om(1J) transcripts are expressed in both wild type and mutant individuals, without neither quantitative nor qualitative differences in the pattern of expression in the imaginal discs. With this data we are able to affirm that the transcripts detected in the Om(1J) region are not responsible for the suppressor effect, however their participation in the suppressor effect can not be discarded yet. The transformation of Om(1D) with a construction of hsp70 promoter and the messenger sequence, would give evidence of the function of these transcripts.

The cytological analysis of Om(1J)Su34 individuals gave additional information about the transcription domain of this gene. The brake point observed in the 6C region of the X chromosome in the Om(1J)Su34^R8 and Om(1J)Su34^R7 revertants reaffirm the location of the Om(1J) mutated locus to this site. This indicate that Om(1J)Su locus extends it transcription domain into this region, and that the tom element promoter effect over Om(1J)Su34^R has been suppressed, possibly, due to the chromosomal rearrangements involving this region, specially the transposition involving the 6C region. Then we suppose that in Om(1J)Su34^R the tom promoter effect over Om(1J)Su it is interrupted due to the separation of the Om(1J) transcription unit from the tom element. With the obtained data, it is not possible to assign the transcription unit 5´ or 3´ from the tom insertion site.

Respect to the complete reversion of the Om(1J)Su34 mutation, it is possible to propose that the revertants present a modification of the suppressor effect in the specific stage of the Om(1D)9 gene expression. There is a considerable expression of Om(1D) in the imaginal disc of the wild type and in the developing photoreceptors located posteriori to the morphogenetic furrow, where tom is also expressed ^(2,18). These results suggested to us a model of the mechanism by which the tom element conduce to obtain the Om(1D) mutants: early over-expression of the Om(1D) protein would cause the death of the undifferentiated cells in the region anterior to the morphogenetic furrow, without affecting the differentiating photoreceptors. It has been observed that this cellular effect it is suppressed when the Om(1D) mutation is combined with the Om(1K)Su mutations ⁽¹¹⁾.

In general, the Om phenotype seems to be caused by an excessive early transcription of the Om genes ^(3,7,4,5) under the control of the tom element promoter effect ⁽¹⁷⁾. The model proposed for the suppressor effect in D. ananassae it is illustrated in Fig 7 The suppressor genes of the Om system would be activated by the tom element insertion into the Om genes ⁽¹¹⁾. The early expression of the Om genes and the suppressor Om gene at the same time, would let both genes interact as in a normal development, avoiding the accumulation of the Om transcripts and allowing the eye development to continue in a normal way. The regulation of the suppressor effect would be dependent on the amount of the suppressor transcripts, in a feedback mechanism. This affirmation it is based on the experiments done by Hinton, where the Om(1J)Su suppressor effect showed to be dose-dependent: Su/Su > Su/+ > +/+ , and that the presence of extra copies of the suppressor transcript (Su/Su/Su) did not modify it effect. However, the concomitance expression of the Om suppressor genes and the Om mutations, in most of the cases do not result in a suppression of the mutant phenotype. It is supposed that the regulatory factors would act in a complementary way regulating the suppressor gene product and the tom element in the suppressed gene (Yoshida, personal communication). Even though the Om(1J)Su identification is still pendant, it is possible to assume that it gene product would be the result of the over-expression induced by the tom element promoter effect.

The analysis of the Om(1J)Su^R mutations it is important in the characterization of the suppressor mutations. The Southern blot assay showed almost no differences in the mutants Om(1J) region, the cytological analysis showed the presence of various chromosomal rearrangements, some of which were around the 6C region of the X chromosome, the site assigned to the Om(1J) locus ⁽¹²⁾.

The tom element transposition frequency has not been studied yet, however, Hinton experiments ⁽¹¹⁾ indicate that the frequency of Om mutants origin it is not reduced in the presence of Om suppressors. This indicate that the intrinsic functions necessary for the tom element transposition are not subject of suppression, at least in the primary oocytes, where the Om mutants originate. Then it is reasonable to think that the mobilization of these sequences in the genome is the result of transposition events.

The Om genes are normally involved in the eye development pathway, however, it similitude with D. melanogaster has been suggested just for two of them, the Om(1D) and Om(1A) genes. We do not doubt that these genes play a role in the eye developmental pathway, but it way of action and the specificity of the tom element insertion still need to be clarified.

BIBLIOGRAPHY

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Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [TABLES AND FIGURES] [TABLES AND FIGURES-2] [DISCUSSION] [BIBLIOGRAPHY] [Discussion Board]

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	[Cell Biology & Cytology]	[Genetics & Bioinformatics]

Monica Acosta, Kiyohito Yoshida
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Last update: 14/01/00