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Molecular cloning and characterisation of a novel membrane receptor gene from the lobster Jasus edwardsii

Jenny Gaik Imm Khoo^* and Frank Y. T. Sin

Department of Zoology, University of Canterbury, Christchurch 1, Private Bag 4800, New Zealand
^* Present address: Biotechnology Center, University of Connecticut, Storrs, Connecticut, USA

View larger version (92K): [in a new window]   Fig. 1. Fluorogram of L-[35S]methionine-labelled proteins from a wheat germ cell-free translation assay of Xenopus ß-globin mRNA (G), lobster eyestalk poly(A)+ RNA (L) and water control (C). 5 µl of translation product was loaded in lanes 1, 2 and 4, and 10 µl in lanes 3, 5 and 6, on a 12 % polyacrylamide-SDS gel. Protein molecular mass standards (kDa) are indicated on the left. The dried gel was exposed for fluorography for 3 days at -80 °C, with intensifying screens.

View larger version (26K): [in a new window]   Fig. 2. Sequence comparison of the nucleotide sequences and deduced aa sequences of cDNA clones peJK2 and peJK3 (568 bp long). The two clones were isolated from a cDNA library of the lobster eyestalk, and share 96 % sequence identity. The deduced aa sequence is shown above the nucleotide sequence; both the initiation and stop codons are underlined, and the polyadenylation signal AATAAA is indicated in bold-face type The sequence data has been deposited in the GenBank database (accession no. AF112985 and AF112986).

View larger version (21K): [in a new window]   Fig. 3. Hydrophobicity plots of the deduced aa sequences of peJK2 (A) and peJK3 (B) (Kyte and Doolittle, 1982).

View larger version (15K): [in a new window]   Fig. 4. Deduced features of peptides coded by peJK2 (A) and peJK3 (B). Transmembrane helices were predicted and analysed using TopPred 2 (von Heijne, 1992) (light grey boxes), SOSUI (Hirokawa et al., 1998), DAS (Cserzo et al., 1997), and ALOM (Klein et al., 1985; Nakai and Kanehisa, 1992) through the PSORT server. The GPCR motif II was detected in peJK2 only using eMOTIF (Attwood et al., 1997). The positions of the predicted signal peptide cleavage sites (VVA-TM), protein kinase C phosphorylation sites (TNK) and nuclear localization signal, pattern 7 sites (PARRAC) (Hicks and Raikhel, 1995) are also indicated.

View larger version (71K): [in a new window]   Fig. 5. Northern blot analysis of poly(A)+ RNA from epithelial tissue (p), eyestalk (e), gill (g), heart (h), hepatopancreas (hp) and muscle (m). The blot was hybridised to cDNA probe peJK2, and a final high stringency wash of 0.1x SSC/0.1 % SDS at 65 °C was included. The blot was exposed for 2 h (A), 4 h (B) and 14 h (C). The positions of the 28S and 18S rRNA bands are indicated on the right, and single-stranded HindIII-digested DNA size markers (kb) are indicated on the left. The positions of the bands are indicated by arrowheads; large arrowheads indicate the major eyestalk band (0.7 kb).

View larger version (163K): [in a new window]   Fig. 6. In situ hybridization of peJK2 to the longitudinal section of the lobster eyestalk. peJK2 hybridised to the neurosecretory regions of the eyestalk (A), as shown by the dense accumulation of silver grains in the eyestalk section whole mount. Nuclear localization of peJK2 was shown in the neurosecretory cell groups of the medulla externa and interna (A,C), the medulla terminalis (B), the X-organs of the medulla terminalis (D), and above the lamina ganglionaris, in the retina (E). LG, lamina ganglionaris; ME, medulla externa; MI, medulla interna; MT, medulla terminalis; OM ommatidia. Bars, 500 µm (A); 100 µm (B,C), 50 µm (D,E).

View larger version (132K): [in a new window]   Fig. 7. In situ hybridization of peJK2 to lobster body tissue sections. Nuclear and cytoplasmic hybridization of peJK2 is evident as silver grains accumulated over the nuclei and tissue of the epithelium (A) and heart (B). peJK2 hybridization is confined to the nuclei of the muscle fibres (C) and the nuclei and cytoplasm of the cells within the gill lamellae (D). RNase treatment of the controls reduced probe binding to background levels (E–H). cy, cytoplasm; n, nucleus; m, muscle. Bars, 20 µm (A,B,E,F); 50 µm (C,D,G,H).