Article Figures & Data
Figures
- Figure 1.
Sequence Comparison Suggests a Role for TTL in Allantoin Biosynthesis.
(A) Alignment of TTL with Urad and Urah protein from zebrafish. Conserved positions are shaded according with the Espript (Gouet et al., 1999) similarity criteria. The sequence motif corresponding to the PTS2 consensus is boxed. Secondary structure elements derived from the three-dimensional coordinates of the Urad domain from Arabidopsis (Kim et al., 2007) and the Urah domain from zebrafish (Zanotti et al., 2006) are drawn above the alignment.
(B) Scheme of the enzymatic pathway for the formation of S-allantoin from uric acid.
[See online article for color version of this figure.]
- Figure 2.
Biochemical Activity of TTL as S-Allantoin Synthase.
(A) Approximate CD spectra of the chiral molecules involved in ureide biosynthesis (θ, molar ellipticity).
(B) Time-resolved CD spectra of the conversion of uric acid (0.1 mM in 100 mM potassium phosphate, pH 7.6) in the presence of urate oxidase and Arabidopsis TTL (10 nM).
(C) Conversion of HIU (left panel; 312 nm) and OCHU (right panel; 257 nm) substrates in the presence of different TTL concentrations: (1) no enzyme, (2) 10 nM, (3) 100 nM, and (4) 200 nM. Substrates were generated immediately before the reaction using uric acid (0.1 mM in 100 mM potassium phosphate, pH 7.6) and urate oxidase (for HIU) or urate oxidase plus zebrafish HIU hydrolase (for OHCU).
- Figure 3.
Alternative Splicing in TTL.
(A) Schematic overview of the transcript variants of the Arabidopsis TTL gene. The intron boundaries are numbered.
(B) Sequence detail of the alternative splicing as deduced by Genewise comparison of the transcript variants with the TTL gene; transcript variants are written as amino acid sequences, codons comprised in exons are written in uppercase letters below the corresponding amino acid, and intronic sequences are written in lowercase letters. The sequence corresponding to the PTS2 consensus is boxed.
[See online article for color version of this figure.]
- Figure 4.
In Vivo Localization of TTL1− and TTL2− Splice Variants.
Fluorescence in the range of 490 to 510 nm (GFP), 542 to 596 nm (YFP), and 649 to 767 nm (chlorophylls) was monitored in transformed Arabidopsis protoplasts using confocal microscopy. Detection channels and three-channel merged images are indicated by column labels. Transformants are indicated by row labels.
- Figure 5.
Conservation of TTL Alternative Splicing in Plants.
(A) RT-PCR analysis of TTL splice variants in O. sativa (line 1) and Arabidopsis (line 2). Total RNA was extracted from whole plants, and PCR products were fractionated on an ethidium bromide–stained agarose gel.
(B) Alignment of the linker region between Urad and Urah domains in various plants. Sequences were retrieved by homology searches in protein and EST databases (accession numbers are reported in Supplemental Table 1 online) and aligned with ClustalW. Conserved positions are shaded according with Espript (Gouet et al., 1999). The sequence motif corresponding to the PTS2 consensus is indicated.
[See online article for color version of this figure.]
- Figure 6.
RT-PCR Analysis of TTL Splice Variants in Different Arabidopsis Tissues.
PCR products were fractionated on an ethidium bromide–stained agarose gel. Total RNA was extracted from plant stem (lane 1), floral buds (lane 2), flower (lane 3), rosette leaves (lane 4), and 3-d-old seedlings (lane 5). The control PCR reaction was conducted in the absence of reverse transcriptase (lane 6). Elongation Factor 1-α (EFα) was used as a reference gene.
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