QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hagedorn, M. Articles by Ziegler, A. Search for Related Content PubMed PubMed Citation Articles by Hagedorn, M. Articles by Ziegler, A.

Molecular characterisation of the smooth endoplasmic reticulum Ca²⁺-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle

Monica Hagedorn¹, Dirk Weihrauch², David W. Towle³ and Andreas Ziegler^1,*

¹ Z.E. Elektronenmikroskopie, Universität Ulm, 89096 Ulm, Germany
² Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60612, USA
³ Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA

View larger version (62K): [in a new window]   Fig. 1. Polymerase chain reaction amplification of the epithelial SERCA cDNA fragment. (A) Amplification of a 513 bp band from total anterior sternal epithelium RNA using the degenerate primers SERCA-F1 and SERCA-R1; 5'-RACE (B,C) and 3'-RACE (D,E) were performed with the constructed adaptor-ligated cDNA pool to clone the full-length SERCA cDNA. The specific primers GSP1 and DAP-st11 (B) and the nested primers GSP2 and DAP (C) were used to amplify the 5'-end. GSP3 and DAP-TRsa (D) and the nested primers GSP4 and DAP (E) were used to amplify the 3'-end. The indicated bands at approx. 500 bp, 1.2 kbp and 2.6 kbp were extracted, purified, reamplified and sequenced. See Materials and methods for details.

View larger version (9K): [in a new window]   Fig. 2. Main strategy of the 5' and 3'-RACE reactions according to the procedure of Matz (2002). Two sequential rounds of polymerase chain reaction (PCR) were performed with adaptor- and sequence-specific primer. Primer sequences and reaction conditions are described in Materials and methods.

View larger version (17K): [in a new window]   Fig. 7. Dendrogram of full-length arthropod and vertebrate SERCAs. Multiple alignment was completed using ClustalW and the neighbor-joining algorithm was then used to construct the tree with Mega2. Comparable results were obtained using related algorithms. GenBank accession numbers are given in parentheses.

View larger version (55K): [in a new window]   Fig. 3. Nucleotide (top) and deduced amino acid (bottom) sequences of the Porcellio scaber smooth endoplasmic reticulum Ca2+-ATPase (SERCA) cDNA. Nucleotides and amino acids are numbered at the sides of the sequence. Start and stop (asterisk) codons are labelled in red. The KILLL-motif (yellow), phosphorylation site and site for probe hybridisation (green), and residues forming calcium-binding site I (grey) and binding site II (blue), and residue D800 (green) bridging sites I and II are labelled. Putative transmembrane regions (M1–M10) are underlined.

View larger version (37K): [in a new window]   Fig. 4. Hydrophobicity plot of Porcellio scaber anterior sternal epithelium SERCA. Hydrophobicity analysis was done by the method of Kyte and Doolittle (1982) with a window of 12 residues using BioEdit. Asterisks indicate putative transmembrane regions.

View larger version (82K): [in a new window]   Fig. 5. In situ hybridisation of cryosections (0.7 µm thick) of the anterior sternal epithelium (ASE) of Porcellio scaber in three different moulting stages (A–D, early premoult; E,F, late premoult; G,H, intramoult) with specific biotinylated probes for SERCA mRNA. (A,B,E,G) Phase contrast micrographs; (C,D,F,H) corresponding fluorescence micrographs. cl, cuticle, n, nucleus. Scale bars, 10 µm. Broken lines in A–D outline the ASE.

View larger version (94K): [in a new window]   Fig. 6. In situ hybridisation of cryosections (0.7 µm thick) of ganglial nervous tissue from Porcellio scaber in three different moulting stages (A,B, early premoult; C,D, late premoult; E,F, intramoult) with specific biotinylated probes for SERCA mRNA. (A,C,E) Phase contrast micrographs; (B,D,F) corresponding fluorescence micrographs. np, neuropil; g, glia. Scale bars, 10 µm.