Identification and characterization of three differentially expressed genes, encoding S-adenosylhomocysteine hydrolase, methionine aminopeptidase, and a histone-like protein, in the toxic dinoflagellate Alexandrium fundyense

Abstract

Genes showing differential expression related to the early G(1) phase of the cell cycle during synchronized circadian growth of the toxic dinoflagellate Alexandrium fundyense were identified and characterized by differential display (DD). The determination in our previous work that toxin production in Alexandrium is relegated to a narrow time frame in early G(1) led to the hypothesis that transcriptionally up- or downregulated genes during this subphase of the cell cycle might be related to toxin biosynthesis. Three genes, encoding S-adenosylhomocysteine hydrolase (Sahh), methionine aminopeptidase (Map), and a histone-like protein (HAf), were isolated. Sahh was downregulated, while Map and HAf were upregulated, during the early G(1) phase of the cell cycle. Sahh and Map encoded amino acid sequences with about 90 and 70% similarity to those encoded by several eukaryotic and prokaryotic Sahh and Map genes, respectively. The partial Map sequence also contained three cobalt binding motifs characteristic of all Map genes. HAf encoded an amino acid sequence with 60% similarity to those of two histone-like proteins from the dinoflagellate Crypthecodinium cohnii Biecheler. This study documents the potential of applying DD to the identification of genes that are related to physiological processes or cell cycle events in phytoplankton under conditions where small sample volumes represent an experimental constraint. The identification of an additional 21 genes with various cell cycle-related DD patterns also provides evidence for the importance of pretranslational or transcriptional regulation in dinoflagellates, contrary to previous reports suggesting the possibility that translational mechanisms are the primary means of circadian regulation in this group of organisms.

FIG. 1

STX biosynthesis and the cell cycle in synchronously growing A. fundyense. Lines show relative distributions of cells in the different cell cycle stages (G₁ [●], S [⧫], and G₂ plus M [▴]). Positive toxin production rates (bars; μ_TOX) were limited to the first 8 to 10 h of the G₁ cell cycle phase. No toxin production was detected for the remainder of the cell cycle. Arrows denote sampling points for the DD analysis protocol; two were during G₁, the first (I) when toxin was accumulated and the second (II) after it stopped, one was during the S phase (III), and the last one was during G₂ (IV). Shaded areas, dark periods of the cycle of 14 h of light and 10 h of darkness under which A. fundyense was synchronized.

FIG. 2

Comparison of inter- and intraspecific DD analysis of dinoflagellates. (A) Three toxic strains of A. tamarense, three nontoxic strains of A. tamarense, and two toxic strains of A. fundyense were compared. The inter- and intrageneric variabilities were high. (The image is a composite of lanes from one gel rearranged for clarity.) (B) Three different cell cycle stages in synchronized cultures of A. fundyense (GtCA28) were analyzed. In contrast to what was seen in panel A, the patterns were identical and hence allowed the identification of differentially expressed bands (Fig. 4).

FIG. 3

DD comparing cells of A. fundyense in three different cell cycle stages (G₁, S, and G₂) and while STX production was turned on or off (I through IV refer to the four sampling points in Fig. 1; double lanes represent duplicate reverse transcription-PCRs). Band A is downregulated, while bands B and C are upregulated, relative to toxin production. Band D is constitutively expressed, while band E is G₁ specific (expressed in early and late G₁).

FIG. 4

RNA dot blot analysis confirming the induction of DD bands A, B, and C (shown from top to bottom) in A. fundyense. Total RNA (10 μg per dot) corresponding to four equivalent sampling points (I through IV; Fig. 1) on the 24-hour cycle immediately following the cycle analyzed by DD was hybridized with cDNA probes generated by PCR of cloned cDNA fragments obtained from the DD gels (see Results).

FIG. 5

Partial alignments of the A. fundyense amino acid sequences determined in this study. (A) S-Adenosylhomocysteine hydrolase aligned with tobacco (N. sylvestris), yeast (S. pombe), and a cyanobacterium (Synechocystis sp.). (B) Methionine aminopeptidase aligned with yeast (S. cerevisiae), a cyanobacterium (Synechocystis sp.), and E. coli. The shaded areas correspond to three of the five highly conserved copper binding regions that have been described for this enzyme (see text). (C) Histone-like protein aligned with two different histone-like protein sequences from C. cohnii. Boxes denote higher than 50% identity (boldface) or similarity (lightface) among organisms.