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Fig. 3. GAG enzyme synthesis is post-transcriptionally regulated. (A)
Transcripts for enzymes involved in HSPG GAG chain synthesis are maternally
provided. Template RNA isolated from unfertilized eggs supports the generation
of RT-PCR products for all enzymes involved in HSPG GAG chain synthesis. The
UDP glucose dehydrogenase Sgl and the Glucuronic acid transporter Fringe
connection (Frc) act in the synthesis of GAG chain building blocks.
Peptide-o-xylosyltransferase (Oxt), Galactosyltransferase I and II (GalT1,
GalT2), and Glucuronyltransferases AT-1, BS1 and BS2 synthesize the
tetrasaccharide linker; the polymerases Botv, Ttv and Sotv are required for
chain elongation. The glypicans Dlp and Dally encode the core protein
substrate for GAG chain addition, and the N-deacetylase-N-sulfotransferase Sfl
modifies sugar residues on the polymerised GAG chains. Dally mRNA is
undetectable in eggs although an RT-PCR product can be generated using RNA
from 0-24 hour embryos. To rule out amplification of a DNA template, primer
pairs for all genes except GalT2 (which lacks an intron) span one or more
introns. (B) Western blots of staged embryonic extracts were probed to
visualize expression of the indicated proteins. Loading controls are shown
below each panel. (i) Endogenous Sfl is absent from 0-3 hour embryos and is
first detected in 2-4 hour extracts. (ii) Antisera against Ttv reveal that
expression of the endogenous protein follows a similar temporal profile. (iii)
In unfertilized eggs, Sfl is barely detectable at 0-3 hours but is robustly
expressed in 2-4 and 4-7 hour samples. (iv) Ttv expression in unfertilized
eggs mirrors its expression in embryos. (C,D) The ttv
5' UTR shows IRES activity in (C) reticulocyte lysates and (D)
Drosophila S2 cells. Luciferase assays were carried out on
reticulocyte in vitro translation extracts or lysates from cells transfected
with an unmodified bicistronic vector (RSTV), or a vector with the CV 5.1 IRES
(CV) or the ttv 5' UTR (TTV). The ratio of Renilla to Firefly
luciferase was calculated for each sample and the data represented as fold
amplification relative to the values for the empty pRSTF vector, set at 1. In
in vitro translation extracts, the ttv 5' UTR confers a
7.5-fold increase in firefly luciferase expression, comparable to the 8-fold
increase generated by the CV IRES (average of two assays). In cultured cells,
the ttv UTR is more than three times more effective than the viral
IRES, and results in 
108-fold stimulation compared with empty vector. The
graph represents the average values and standard error from five independent
transfection assays.
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