Table 1.
Primers used in this study.
Figure 1.
Nucleotide and deduced amino acid sequences of CfCSP.
The nucleotides and amino acids are numbered along the left margin. The putative CSD is boxed. The two consensus RNA binding domains are shaded in gray and four DNA binding sites are marked in italic and bold. The RGG repeat motifs are underlined. The classical polyadenylation signals and the A+U destabilizing elements in the 3′ UTR are marked in bold.
Figure 2.
Multiple alignment of the amino acid sequences for CSD containing proteins.
Cold shock domains are boxed, consensus RNA binding domains (RNP1 and RNP2) and RGG repeats are shaded in gray and consensus DNA binding sites are shaded in dark. Perfectly matched residues, conserved residues, and less conserved residues are indicated by an asterisk (*), a colon (:), and a period (.), respectively. Accession numbers of the CSD proteins are: CfCSP (JN869460), LsYB (AAT97092), ApY1 (NP_001191560), E. coli CSPA (P15277), human YB-1 (P67808) and XlYB3 (CAA42778).
Figure 3.
Phylogenetic tree of CSD containing protein sequences from diverse organisms.
The tree is constructed by the neighbor-joining (NJ) algorithm and the scale bar corresponds to 0.2 estimated amino-acid substitution spersite. Accession numbers of the CSD containing proteins are: E. coli CSPA (P15277), E. coli CSPB (P36995), Lactococcus lactis CSPB (CAA76695), Bacillus subtilis CSPB (P32081), C. elegans LIN-28 (AAC47476), Human YB-1 (I39382), CfCSP (JN869460), Xenopus laevis YB3 (CAA42778), Lymnaea stagnalis YB (AAT97092), Aplysia californica Y1 (NP_001191560), WCSP1 (BAB78536), AtCSP3 (NM_127341), AtCSP2 (NP_195580.1) and NsGRP2 (CAA42622).
Table 2.
The percentage identities and similarities of CSD between CfCSP and other CSD containing proteins.
Figure 4.
CfCSP mRNA expression in different tissues of C. farreri detected by RT-PCR.
CfCSP transcripts level in adductor, gonad, kidney, mantle, hepatopancreas and gill were normalized to that of hemocytes. Vertical bars represented the mean ± S.E. (N = 5), and bars with different letters were significantly different (P<0.05).
Figure 5.
Temporal expression of the CfCSP mRNA after acute cold shock treatment.
The mRNA level of CfCSP relative to β-actin in adductor (A) and hemocytes (B) were measured by RT-PCR. Vertical bars represent the mean ± S.E. (N = 5). (*: P<0.05).
Figure 6.
Lane M: protein molecular standard (kDa); lane 1: negative control for rCfCSP (without induction); lane 2: induced rCfCSP; lane 3: purified rCfCSP.
Figure 7.
Analysis of nuclear acids binding activity of CfCSP by gel shift assay.
The purified rCfCSP proteins were incubated with ssDNA (A), M13mp8 dsDNA RFI DNA (dsDNA) (B) and the in vitro transcribed luciferase mRNA (C) to analyze the effect of CfCSP on the formation of nucleotide-protein complexes. A range of CfCSP fusion proteins from 0 to 300 pmol were used for analysis. Arrow shows nucleotide-protein complex.
Figure 8.
Complementation of cold sensitive growth of BX04 (ΔcspAΔcspBΔcspEΔcspG) with CfCSP and E. coli CSPA.
Overnight cultures of JM109/pINIII, BX04/pINIII, BX04/CfCSP and BX04/CSPA were streaked on LB-ampiciline plates and incubated at 37°C for 12 h (A) or 17°C for 120 h (B).