Characterization of a cDNA encoding a homolog of actin-related protein 4 from a marine red alga Porphyra yezoensis

Abstract

A cDNA (PyARP4) containing an open reading frame for a protein of 573 amino acids was identified in the marine red alga Porphyra yezoensis. The conceptual PyARP4 protein exhibits significant similarity to actin-related protein (ARP) 4 in the terrestrial plant Arabidopsis. Comparison of the deduced amino acid sequence showed moderate sequence identity (30%) to a conventional actin in P. yezoensis, as seen in comparisons between ARP and conventional actins of other organisms. A putative bipartite nuclear localization signal and an actin motif were found within the PyARP4 amino acid sequence. In a phylogenetic analysis, the PyARP4 was found to cluster with the ARP4 of other organisms. The expression level of PyARP4 did not change significantly among four developmental stages of life cycle and was lower than that of a conventional actin. This cDNA therefore may serve as a useful internal standard in gene expression analyses of differentially expressed genes in P. yezoensis.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta)

“…Filamentous sporophytes and leafy gametophytes of P. yezoensis (strain TU-1) were cultured in enriched sealife (ESL) medium as described previously (Kitade et al, 2006). The vegetative sporophytes were induced to form conchosporangia by transferring from a continuous light at 25°C to a photoperiod of light:dark 14:10 h at 15°C under irradiation of 25 μmol photons/m 2 /s.…”

“…Nucleotide sequences of cDNA clones were determined, and conceptual translation performed as has been described (Kitade et al, 2006). Identities of the deduced amino acid sequences were calculated using the BLAST 2 SEQUENCES (http://www.ncbi.nlm.nih.…”

“…The expression level of the actin genes was assessed by semiquantitative RT-PCR analysis as described previously (Kitade et al, 2006). Briefly, the fluorescence from the three amplified fragments was measured using a fluorescent image analyzer (FLA-3000G, Fujifilm, Tokyo, Japan), and the expression value of PyACT2, 3 and 4 was normalized with that of the elongation factor-1α gene as well as the size of the PCR products.…”

“…Total RNA was extracted from vegetative gametophytes as described previously (Kitade et al, 2006). Poly(A) + RNA was prepared using Oligotex ™ -dT30 super (Roche, Tokyo, Japan).…”

“…To elucidate molecular mechanisms underlying the regulation of life cycle, expressed sequence tag (EST) analysis has been applied to both generations to identify differentially expressed genes (Nikaido et al, 2000;Asamizu et al, 2003). In addition, genes have been characterized with normal and subtracted cDNA libraries (e.g., Kitade et al, 2006;Kakinuma et al, 2006) and cDNA macroarray analysis .…”