Cloning of the eyc gene was initiated from an eyc P-element allele (P1363), which failed to complement eyc1. Plasmid rescue of P1363 recovered ~16 kb genomic DNA flanking the P-element. A ~2 kb genomic probe (P3), 3' to the P-insertion site revealed a ~1.1 kb transcript which is elevated in eyc1 and P1363/Df(2R) Px2 flies relative to wild type at 30% p.d.. Eyc protein levels are likewise elevated relative to wild type at this stage. P3 was then used to screen a Drosophila adult head cDNA library and it identified a single class of cDNA clone, representing a transcript of 1.1 kb. This 1112 bp cDNA was sequenced on both strands to obtain the gene sequence. Genomic sequence of a P1 clone (DS02336) indicates eyc is intronless (Sang, 2002).
That eyc1 and eycP are recessive appears at odds with a hypothesis that suggests transcript elevation causes the eye phenotype: should they not be dominant? Regulation of transcription by interallelic interactions, known in Drosophila and elsewhere, could account for the current observations. The possibility is considered that a 3' suppressor sensitive to epigenetic influences contributes to eyc regulation. Analogous to the trans activity of the yellow enhancer, pairing a wild-type homolog with an eyc mutant may allow recruitment of a suppressor that downregulates transcription of both copies, over-riding decreased binding of a repressive factor by 3' changes in eyc1 and eycP. Further genetic and molecular analysis of eyc will be required to explore this possibility (Sang, 2002).
Since p97 is sensitive to alkylation by N-ethyl-maleimide (NEM), and since in vitro reassembly of Xenopus nuclear membrane is NEM sensitive (Macaulay, 1996; Marshall, 1997), it was speculated that p47/Eyc might be essential for nuclear envelope fusion. Indeed, interference with Eyc function in syncytial blastoderm embryos disrupts nuclear morphology and cell cycle progression. Embryos of eycl39/CyO inter se crosses were stained with an antibody to Lamin, a nuclear envelope protein that has been shown to play an essential role in nuclear envelope assembly. During the first half hour after egg deposition, no differences were observed in the nuclear envelopes of embryos from wild-type or eycl39/CyO crosses, suggesting early nuclear division in eyc nulls proceeds normally using maternally supplied Eyc. At 1.5 to 2 hours, wild-type embryos stained with anti-Lamin show two staining patterns: Lamin is either concentrated around the separated M-phase chromosomes, consistent with an association between Lamin and chromatin, or the interphase nuclear envelope is highlighted. In eycl39/CyO-derived progeny, approximately one quarter of the embryos show no nuclear envelopes at the same stage; occasional embryos showed Lamin aggregates, which may be similar to the annulate lamellae observed in Drosophila lamin Dm0 (Sang, 2002).
Concomitant with loss of the nuclear envelope, the normally dense nuclear DNA seen in zygotic nuclei becomes irregularly fragmented and dispersed. It seems likely that absence of the nuclear envelope results in abnormal chromosome segregation. Probably as a result of destructive mitosis and chromosome dispersal, eyc null nuclei do not migrate to the surface of the embryo and organize the normal hexagonal actomyosin staining pattern at the embryo membrane; cellularization fails in mutant embryos (Sang, 2002).
To further examine Eyc loss-of-function phenotypes, anti-Eyc antiserum was microinjected into the posterior of stage 2-3 embryos and its effects on nuclear division were observed using confocal microscopy 1 hour after injection (~ stage 4-5). A gradient of cortical nuclear organization was found across injected embryos: nuclei were dense and regularly arrayed at anterior ends; nuclei were sparse and poorly organized at the posterior. Occasional clustered nuclei could be found in the affected region (Sang, 2002).
Side views reveal disruption of cellularization in the posterior of anti-Eyc injected embryos. In normal stage 4-5 embryos syncytial nuclei are closely packed and cylindrical. By contrast, the posterior region of anti-Eyc injected embryos shows fewer nuclei; those that are there appear to have a spherical shape, presumably a more relaxed shape in the less crowded environment. It is speculated that the nuclear divisions are slowed in the Eyc-inhibited cytoplasm, perhaps by delays of postmitotic nuclear envelope reassembly (Sang, 2002).
These results indicate Eyc is fundamental to cell cycle progression during embryogenesis. To determine whether Eyc is generally used at different developmental stages and tissues, the EGUF/hid method was applied to generate homozygous eyc null eyes. Eyes are absent in flies in which eycl39 is homozygosed early in the development of the eye primordium, supporting a cell-essential role for Eyc (Sang, 2002).
A cell-essential role for p47/p97 is also suggested by failure to obtain ter94 (a Drosophila p97 homolog) null eye clones, as well as the failure to obtain ter94 loss-of-function germline clones (Sang, 2002).
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date revised: 20 February 2002
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