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First published online June 11, 2007
Journal of Experimental Biology 210, 2070-2081 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004309

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Gill-specific transcriptional regulation of Na⁺/K⁺-ATPase -subunit in the euryhaline shore crab Pachygrapsus marmoratus: sequence variants and promoter structure

Nishad Jayasundara^1,*, David W. Towle^1,, Dirk Weihrauch² and Céline Spanings-Pierrot³

¹ Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA
² Division of Animal Physiology, University of Osnabrück, D-49076 Osnabrück, Germany
³ Université Montpellier II, Adaptation Ecophysiologique et Ontogenese, UMR 5119 CP092, Pl. E. Bataillon, 34095 Montpellier cédex 5, France

View larger version (130K): [in this window] [in a new window]   Fig. 1. Multiple alignment and phenogram of initial 707-nucleotide amplification product of Na+/K+-ATPase -subunit from Pachygrapsus marmoratus (accession no. AF375957) with corresponding partial cDNA sequences from four other arthropods: blue crab Callinectes sapidus (acc. no. AF327439), American lobster Homarus americanus (acc. no. AY140650), mosquito Anopheles gambiae (acc. no. XM_563283), and honey bee Apis mellifera (acc. no. XM_392363). The alignment (A) and phenogram (B) were both generated using the Hein algorithm (Hein, 1990) in the MegAlign component of DNASTAR software and configured with GeneDoc (Nicholas and Nicholas, 1997). The blue background indicates 100% agreement between all five sequences, green 80%, and yellow 60%. Bootstrap values at each node of the phenogram=100.

View larger version (113K): [in this window] [in a new window]   Fig. 2. Complete nucleotide and predicted amino acid sequence of the D form (accession no. DQ173924) of Na+/K+-ATPase -subunit cDNA amplified from gills of Pachygrapsus marmoratus. The C form (acc. no. DQ173925) lacks the 81-nucleotide segment and 27-amino-acid segment indicated in blue. A consensus 14-3-3 binding site RTDSYR (Aitken, 2002), indicated by a rectangle, is found near the N terminus of the D form but not the C form. In-frame stop codons are indicated in red. Ten transmembrane domains predicted by transmembrane hidden Markov modeling (TMHMM) (Sonnhammer et al., 1998) are underlined. Prosite (release 20.3) analysis identified the amino acid sequence DKTGTLT at position 390-396 (bold typeface) as a characteristic P-type ATPase motif including the aspartate that becomes phosphorylated during the transport cycle (Kaplan, 2002). A cAMP- and cGMP-dependent protein kinase phosphorylation site (RRNS) is predicted at position 954-957 (bold typeface). Brackets enclose the 707-nucleotide section initially amplified and aligned in Fig. 1 with corresponding segments from other species.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Multiple alignment of N-terminal amino acids of eight crustacean Na+/K+-ATPase -subunits illustrating two groups, one composed of sequences containing a 14-3-3 protein binding site (RTDSY) close to the N terminus and the other group truncated and lacking this binding site. Accession numbers are given in parentheses: Artemia franciscana Alpha1 (X56650) (Macías et al., 1991), Artemia franciscana Alpha2 (Y07513) (Baxter-Lowe et al., 1989), Pachygrapsus marmoratus C form (DQ173925) (present study), Litopenaeus vannamei EST (CK572083) (O'Leary et al., 2006), P. marmoratus D form (DQ173924) (present study), Fenneropenaeus chinensis EST (BM303114) (Wang et al., 2006), Callinectes sapidus (AF327439) (Towle et al., 2001), Homarus americanus (AY140650) (Parrie and D.W.T., unpublished). The alignment was produced with Multalin version 5.4.1 (Corpet, 1988) and processed with GeneDoc (Nicholas and Nicholas, 1997). Blue background indicates 100% amino acid agreement between the eight sequences, green indicates 75–99%, yellow 50–74%.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Quantitative PCR analysis of Na+/K+-ATPase -subunit mRNA abundance in individual gill preparations from Pachygrapsus marmoratus following transfer for various time periods (2–48 h) from 36 seawater to 10 (A,B) or to 45 (C,D). Values are means ± s.d. (N=3 or 4 measurements at each time point using gills pooled from three or four animals) of data obtained with primers NAKPMF1 and NAKPMR1 designed to detect both isoforms simultaneously (Table 1). Two independent experiments from 2002 (A,C) and 2003 (B,D) are presented. Relative expression levels are indicated in relation to a standard reference (one sample of gill 6 at 48 h after transfer to 10). Significant differences from zero time values (crabs in 36 seawater) are indicated: *P<0.001; P<0.01.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Quantitative PCR analysis of arginine kinase mRNA abundance in individual gills of Pachygrapsus marmoratus transferred from 36 to 10 (A) or to 45 (B) seawater for time periods indicated above the figure. Mean ± s.d. (N=3 or 4 measurements at each time point using gills pooled from three or four animals). Relative expression levels are indicated in relation to a standard reference (one sample of gill 6 at 48 h after transfer to 10). Significant differences from zero time values (36 seawater) are indicated: *P<0.001; P<0.01; P<0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Quantitative PCR analysis of mRNA encoding C and D forms of Na+/K+-ATPase -subunit in individual gills of Pachygrapsus marmoratus transferred from 36 seawater to 10 (A,B) or to 45 (C,D) seawater. Values are mean ± s.d. (N=3 measurements at each time point using gills pooled from three or four animals). Relative expression levels are indicated in relation to a standard reference (one sample of gill 6 at 48 h after transfer to 10). Significant differences from zero time values (animals in 36 seawater) are indicated: *P<0.001; P<0.01; P<0.05.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Genomic DNA sequence of Pachygrapsus marmoratus Na+/K+-ATPase -subunit promoter region including the transcription start site (arrow) and 462 additional upstream nucleotides determined by genome walking. CAAT and TATA box regions are shaded, and predicted binding sites for transcription factors are indicated by labeled rectangles.