Summary

Clone O108 Encodes a Novel Peptide of Unknown Function

Clone O108 contains several ORFs, the longest one which codes for a putative protein product of approximately 15 kD with a predicted pI of 5.37. Comparison of the sequence of this clone to the database of known sequences revealed no obvious similarity to genes of known function. The putative protein encoded by O108 contains a consensus ATP/GTP binding site at the C-terminal end. For additional information, please see the publication by (Nadeau, Zhang, Li and O'Neill, 1996).

Analysis of Gene Expression

In situ hybridization of clone O108 to transverse sections through the orchid ovary when ovules are immature shows no hybridization to tissues of the ovule, placenta or pollen tubes prior to maturation of the ovule (not shown). In situ hybridization results (Panels C and D), however, illustrate that there is a large amount of hybridization to mature ovules, a finding consistent with RNA blot hybridization analysis that shows expression of O108 only at 11 WAP when ovules are mature (See Time Line or Mature Ovule). Moreover, when ovules are observed to express O108 transcripts, signal is present only in the outer layer of the outer integument and in the megagametophyte. Panels C and D also show that all ovules in a given area of the ovary do not express the gene at equivalent levels, and interestingly, many ovules do not express the gene at detectable levels. Ovules which do not express O108 (see ovules indicated by arrowheads) are interspersed randomly with those that do, such that examination of numerous sections did not reveal a discernible spatial pattern which might explain this observation. This pattern of expression within the ovule population as a whole indicates that expression of O108 most likely occurs within a narrow developmental window, such that small differences in stage between neighboring ovules results in the presence or absence of O108 transcripts.

Panels E and F illustrate a higher magnification view of numerous ovules containing O108 transcripts, as well as several ovules that do not express O108 transcripts (see arrowhead). These panels also demonstrate that O108 is not present in other tissues of the ovary such as the hair cells which are derived from the inner ovary wall, or the pollen tubes. Panels B and C illustrate a section through the same ovules as shown in panels A and B, but two of the ovules have been sectioned through the megagametophyte and thus show the centrally located O108 hybridization signal. In no instance was signal observed in the embryo sac but not in the integuments. From these findings it appears that O108 expression in the embryo sac must coincide closely with expression in the outer integument.

Panels E, F illustrate longitudinal sections through ovules. In panel E and F, O108 transcripts are localized to the outer cell layer of the outer integument and embryo sac only. Furthermore, the funiculus does not express O108 mRNA. Expression of O108 is turned off or down-regulated following fertilization.