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First published online 13 September 2006
doi: 10.1242/dev.02582

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Translational control of regA, a key gene controlling cell differentiation in Volvox carteri

¹ Naturwissenschaftliche Fakultät III-Biologie und Vorklinische Medizin, University of Regensburg, D-93040 Regensburg, Germany.
² Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstrasse 25, D-33615 Bielefeld, Germany.

View larger version (50K): [in a new window]   Fig. 1. Histogram showing the ratio and distribution of phenotypes among V. carteri 153-68 transformed with WT (A1), mutant ATG (ATG1-ATG7) and mutant ORF2 (ATG2Stop) regA DNA, respectively, and the observed phenotypes. (A) A histogram indicating the abundance - relative to the WT control, A1 (100%) - of co-transformants exhibiting three different phenotypes: wild-type (WT), somatic regenerator (Reg) and modified somatic regenerator (M-Reg). (B) An adult WT Volvox carteri spheroid containing terminally differentiated somatic cells on the surface and 16 reproductive cells (gonidia) inside the spheroid [picture from Schmitt (Schmitt, 2003)]. (C) The Reg mutant phenotype is characterized by somatic cells that dedifferentiate, grow and redifferentiate to become fully functional gonidia (Kirk et al., 1999). (D) An adult M-Reg spheroid with juveniles ready for hatching. Just a few somatic cells have begun to grow and to redifferentiate, suggesting suboptimal expression of the regA gene.

View larger version (6K): [in a new window]   Fig. 2. Map of the genomic 5' UTR of regA. Grey boxes symbolize exons, and the lines between them represent introns. The locations of the upstream ATGs (ATG1-ATG8) and of the functional start codon (ATG9) are indicated. The transcription start (+1) is marked by a bent arrow. The expanded portions below show the ATG1, ATG2 and ATG7 open reading frames (light grey boxes); the black bars indicate the potential bipartite take-off (E1.1 and E1.2) and landing (E3) sites, the location of the triple-hairpin structure (HPS, lower bar), the region deleted in the S18 construct, and the point where the artificial hairpin (hp) shown in Fig. 6 was inserted.

View larger version (11K): [in a new window]   Fig. 3. Secondary structure predictions for homologous portions of the 5' UTRs of WT regA mRNA and two mutant mRNAs (ATG2 and ATG2Stop). (A-C) Numbering starts at transcription start (+1; Fig. 2). Grey arrows indicate the location of ATG2; black arrows indicate the locations at which stop codons were introduced. The encircled portion indicates the triple hairpin region between positions 215 and 277 that is discussed in the text. Note the secondary structure in this region changed by mutations. The structures and free enthalpies of the whole 5' UTRs (dG in kcal/mole) were calculated by the program mfold [version 3.1, Zuker (Zuker, 2003)].

View larger version (22K): [in a new window]   Fig. 4. Bipartite sequence complementarities of the potential ribosome binding sites. (A) Complementarities of the 3' hairpin of human 18S rRNA and the human adenovirus tripartite leader (3LDR, Ad, C1-C3). Nucleotide positions relative to the +1 start site of transcription are indicated (Yueh and Schneider, 2000). (B) Complementarities of the 3' hairpin of V. carteri 18S rRNA (Rausch et al., 1989) and three regions of the 5' UTR of regA from V. carteri f. nagariensis and V. carteri f. kawasakiensis (Duncan et al., 2006). E1.1 und E1.2 are located in the first exon; E2 is in the second exon (Intron 2 will not be spliced in comparison to nagariensis); and E3 in the third exon. Nucleotide positions relative to the +1 start site of transcription are indicated (Fig. 2).

View larger version (21K): [in a new window]   Fig. 5. The normalized abundance of different phenotypes. Histogram showing the normalized abundance of different phenotypes among organisms co-transformed with the WT regA construct (A1=100%), the mutant construct (S18) lacking the potential landing site, that is complementary to S18 rRNA, and the mutant construct containing a stable hairpin structure (hp; Fig. 6) introduced upstream of the landing site (Fig. 2).

View larger version (11K): [in a new window]   Fig. 6. Secondary RNA structure. Sequence-derived RNA secondary structure (G of -87 kcal/mole) transcribed from the synthetic hairpin introduced into the 5' UTR of regA (Fig. 2).