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Fig. 4. Delineation of mRNA cis-elements that regulate ttv
expression. (A) Western blots of staged embryonic extracts from
flies transgenic for the indicated constructs were probed to visualize the
expression of Ttv or GFP. LC, loading control. (i) Truncation of the 5'
UTR to eliminate upstream AUG sequences and removal of the 3' UTR
permits the translation of maternally loaded Ttv-myc transcript in 0-3 hour
embryos (upper band); the endogenous protein (lower band) is not expressed at
this time. (ii-v) matTub>Gal4 was used drive the expression of UAS
constructs in which the GFP coding sequence was flanked by the indicated
regions of ttv. (ii) GFP expression was detected in 0-3 hour extracts
in the absence of ttv regulatory sequences, as well as when only
5' (iv) or 3' (v) UTRs were present. (iii) By contrast, GFP
expression was blocked in early embryos and initiated at 
3 hours, similar
to endogenous Ttv, when the ORF was flanked by both 5' and 3' UTR
sequences. (B-F) Translational regulation visualized in live animals.
(B) GFP fluorescence is absent from a female transgenic for a matTub>Gal4
driver and a construct in which GFP is flanked by ttv 5' and
3' UTRs. (C) By contrast, ovarian expression results in bright
fluorescence in a female transgenic for matTub>Gal4 and a GFP construct
that lacks ttv UTRs. (D,E) Merged images showing DAPI-stained nuclei
and GFP fluorescence in egg chambers. (D) Egg chambers from an animal carrying
matTub>Gal4 and the reporter construct with ttv 5' and
3' UTRs, showing the absence of GFP expression and only yolk
autofluorescence. (E) By contrast, a reporter that lacks ttv UTRs
generates high levels of GFP expression in nurse cells (Nc) and oocytes (Ooc).
Note that expression cannot be detected in follicle cells (Fc). (F) Time-lapse
series of a 5'ttv-UAS-GFP-3'ttv embryo shows
that significant GFP fluorescence develops only after three hours post-egg
laying.
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