Monica Acosta⁽¹⁾, Kiyohito Yoshida^(2)
(1)(2)Hokkaido University - Sapporo. Japan

	[Cell Biology & Cytology]	[Genetics & Bioinformatics]

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 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