Functional Divergence of Former Alleles in an Ancient Asexual Invertebrate
Natalia N. Pouchkina-Stantcheva1*,
Brian M. McGee1,
Chiara Boschetti1,
Dimitri Tolleter2,
Sohini Chakrabortee1,
Antoaneta V. Popova3
,
Filip Meersman4
,
David Macherel2,
Dirk K. Hincha3 and
Alan Tunnacliffe1
1 Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK.
2 UMR 1191 Physiologie Moléculaire des Semences, Université d'Angers/INH/INRA, 49045 Angers, France.
3 Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam, Germany.
4 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
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Fig. 1. Genomic organization of A. ricciae lea genes. (A) Schematic representation of Ar-lea-1A and Ar-lea-1B genes within 5-kb Dra I fragments. Introns are depicted as numbered boxes (red) below lines (scaled dark gray) indicating the sequenced region containing each gene. Genomic organization outside these regions (light gray) is deduced from Southern hybridization analysis. Ar-lea-1B fragments indicated by labeled bars (blue) correspond to probes used in Southern hybridizations. (B) Southern hybridization of A. ricciae genomic DNA with lea gene probes. Each panel contains genomic restriction digests with Dra I, Dra I/Eco RI, and Dra I/Nde I, respectively. Size marker positions are indicated. (C) A. ricciae karyotype and FISH with lea gene probe. (Left) The 12 chromosomes of A. ricciae in a single mitotic nucleus from an embryo stained with 4', 6'-diamidino-2-phenylindole (DAPI). (Right) Interphase nucleus hybridized at high stringency to Ar-lea-1A probe labeled with Alexa 488. Red (superimposed, false color): fluorescent signals; blue: DAPI-labeled DNA. Scale bar: 2 µm.
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Fig. 2. Primary and secondary structure of A. ricciae LEA proteins. (A) Alignment of ArLEA1A and ArLEA1B protein sequences showing repeated 11-oligomer motifs. ArLEA1A is 420 residues long with a (predicted) molecular mass of 44.5 kD, whereas ArLEA1B extends for 376 residues with a (predicted) molecular mass of 39.8 kD. Near canonical motifs are orange, green, and yellow; degenerate motifs are gray; highlighted residues differ between the two proteins. The 44-residue indel, shown by dashes, is identical to a more N-terminal sequence whose 11-oligomer motifs are also highlighted orange-yellow-gray-orange. A putative signal peptide is overlined at the N terminus. (B and C) Far-UVCD spectroscopy of ArLEA1A and ArLEA1B in solution and dry state.
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Fig. 3. Bdelloid LEA protein antiaggregation assay. Citrate synthase (CS), with or without LEA proteins or BSA, and the latter proteins alone where indicated, were subjected to two cycles of vacuum drying and rehydration. Light scattering by protein particulates was measured by apparent absorption at 340 nm in the spectrophotometer. Error bars show standard deviation (n = 3); ns, not significantly different (P > 0.05); significant values *P < 0.05 or **P < 0.001.
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Fig. 4. Bdelloid LEA protein membrane association. Infrared spectra of the asymmetric phosphate stretching region of POPC liposomes dried alone or in the presence of ArLEA1A, ArLEA1B, or AavLEA1. Spectra were recorded at 78°C (liquid-crystalline phase). The solid curve comprises both the measured (dots) and fitted absorbance curves. Normalized peaks were fitted into two bands with maxima at 1262 and 1242 cm–1 corresponding respectively to  P=Oasfree (short dashes) and P=Oasbound (long dashes).
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