Relationship between Rubisco activase and Rubisco contents in transgenic rice plants with overproduced or decreased Rubisco content

<p>Overproduction of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; E.C. 4.1.1.39) in rice did not necessarily improve photosynthesis. The reason for this was that a partial deactivation of Rubisco occurred in <i>RBCS</i>-overexpressed rice plants. Since a negative correlation between the amounts of Rubisco activase (RCA) and Rubisco has been reported for plants with overproduced or decreased RCA, the possibility that RCA content declines in <i>RBCS</i>-overexpressed rice plants was considered. The relationship between RCA and Rubisco contents was examined in <i>RBCS</i>-overexpressed and <i>RBCS</i>-antisense rice plants. Whereas the ratio of RCA to Rubisco contents in <i>RBCS</i>-antisense plants increased three- to fourfold as compared with that of the wild-type levels, this ratio decreased 60–70% of the wild-type levels in <i>RBCS</i>-overexpressed rice plants. Thus, an apparent trade-off between the amounts of RCA and Rubisco was observed in <i>RBCS</i>-transgenic rice plants. However, the amounts of several Calvin–Benson cycle enzymes changed in a similar manner to that of RCA in both types of <i>RBCS-</i>transgenic rice plants. When the relationships between the amounts of these enzymes, including RCA, and those of total leaf-N minus Rubisco-N and trichloroacetic acid (TCA)-soluble N were examined, there were no differences between them irrespective of genotypes. These results indicate that the negative correlation between the amounts of RCA and Rubisco in <i>RBCS</i>-transgenic rice plants is the result of a change in N allocation to Rubisco in transgenic rice plants rather than a trade-off. Such a negative correlation was also found for other Calvin–Benson cycle enzymes. In addition, since the amounts of these Calvin–Benson cycle enzymes and RCA were highly correlated with their mRNA levels irrespective of genotype, it is suggested that changes in the amounts of these proteins are regulated at their transcript levels by a change in N allocation to Rubisco.</p

Diversifying Selection on the Thrombospondin-Related Adhesive Protein (TRAP) Gene of <i>Plasmodium falciparum</i> in Thailand

<div><p>Sporozoites of <i>Plasmodium falciparum</i> are transmitted to human hosts by Anopheles mosquitoes. Thrombospondin-related adhesive protein (TRAP) is expressed in sporozoites and plays a crucial role in sporozoite gliding and invasion of human hepatocytes. A previous study showed that the TRAP gene has been subjected to balancing selection in the Gambian <i>P. falciparum</i> population. To further study the molecular evolution of the TRAP gene in <i>Plasmodium falciparum</i>, we investigated TRAP polymorphisms in <i>P. falciparum</i> isolates from Suan Phueng District in Ratchaburi Province, Thailand. The analysis of the entire TRAP coding sequences in 32 isolates identified a total of 39 single nucleotide polymorphisms (SNPs), which comprised 37 nonsynonymous and two synonymous SNPs. McDonald–Kreitman test showed that the ratio of the number of nonsynonymous to synonymous polymorphic sites within <i>P. falciparum</i> was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between <i>P. falciparum</i> and <i>P. reichenowi</i>. Furthermore, the rate of nonsynonymous substitution was significantly higher than that of synonymous substitution within Thai <i>P. falciparum</i>. These results indicate that the TRAP gene has been subject to diversifying selection in the Thai <i>P. falciparum</i> population as well as the Gambian <i>P. falciparum</i> population. Comparison of our <i>P. falciparum</i> isolates with those from another region of Thailand (Tak province, Thailand) revealed that TRAP was highly differentiated between geographically close regions. This rapid diversification seems to reflect strong recent positive selection on TRAP. Our results suggest that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of <i>P. falciparum</i>.</p></div

Catalytic activities of DGKη3 and η4.

<p>(A) Expression of AcGFP alone, AcGFP-DGKη1, η3 and η4 in COS-7 cells. COS-7 cells were transfected with pAcGFP vector alone, pAcGFP-DGKη1, η3 or η4. The cell lysates (12 μg of protein) were analyzed by Western blotting using anti-GFP antibody. (B) The relative activities of DGKη3 and η4 compared to DGKη1. The cell lysates (5 μg of protein/sample) were assayed for DGK activity (triplicate determinations). The background activities were subtracted and then the values were normalized for DGK expression levels visualized by Western blotting. The results are presented as the percentage of the value of DGKη1 and the mean ± S.D. of the values obtained in three separate experiments.</p

Phylogenetic tree (A) and population structure (B) of the TRAP gene of 110 <i>P. falciparum</i> isolates from three geographic regions.

<p>Samples included 32 isolates from Ratchaburi Province, Thailand (investigated in this study), 29 from Tak Province, Thailand, and 49 from Gambia. A, The phylogenetic tree of the TRAP sequences was constructed using the neighbor-joining method. B, Membership coefficients from STRUCTURE analysis (<i>K</i> = 3) were plotted for 110 isolates. Each isolate is represented by a vertical bar displaying the proportion of membership in each of the three hypothetical clusters.</p

DGKη3 is derived from DGKη1.

<p>(A) Schematic representation of the primers used for RT-PCR. (B) RT-PCR analysis was carried out on mRNA prepared from mouse testis using primers A and B. Representatives of three independent experiments are shown. (C) The protein samples (50 μg) from the testes of 10-week-old wild-type (WT) and DGKη-knockout (KO) male mice were analyzed by Western blotting using anti-DGKη and β-actin antibodies. *: non-specific band.</p

Polymorphic amino acid residues of the TRAP gene in 32 <i>P. falciparum</i> isolates.

<p>A, Density of polymorphic amino acid residues in the TRAP gene. A sliding window analysis of amino acid polymorphisms (substitutions) was conducted using a sliding window size of 20 amino acids and a step size of one amino acid. B, Schematic representation of the TRAP domain composition, signal sequence (SS), von Willebrand factor A domain (A domain), thrombospondin type 1 domain (TSP-1), and transmembrane domain (TM). The proline-rich repeat region lies between TSP-1 and TM. Positions of polymorphic amino acid residues are indicated by the thin red line above the box.</p

A new alternative splicing variant, DGKη4.

<p>(A) Schematic representation of the primers used for RT-PCR. (B) The nucleotide sequences and deduced amino acid sequences of DGKη1, DGKη2, DGKη3 and DGKη4 are shown. (C) RT-PCR analysis was carried out on mRNA prepared from mouse testis using primers C and D. Representatives of three independent experiments are shown.</p

Fst value at each SNP.

<p>A total of 94 SNPs were detected in three <i>P. falciparum</i> populations. Fst values were calculated for three population pairs (Ratchaburi and Tak, Tak and Gambia, and Gambia and Ratchaburi). The locations of four TRAP domains, signal sequence (SS), von Willebrand factor A domain (A domain), thrombospondin type 1 domain (TSP-1), and transmembrane domain (TM) were indicated by thick horizontal lines.</p

Subcellular localization of DGKη3 and η4 in NEC8 cells stimulated with osmotic stress.

<p>NEC8 cells were transfected with pAcGFP vector alone, pAcGFP-DGKη1, η3 or η4. After 24 h, the cells were serum starved for 3 h and incubated (A) 0 mM or (B) 500 mM sorbitol in RPMI-1640 for 30 min. The cells were fixed with 3.7% formaldehyde and then mounted onto coverslips. The fixed cells were stained using Alexa 594-conjugated phalloidin and 4’,6-diamino-2-phenyl indole (DAPI). Fluorescence images were obtained using an inverted confocal laser microscope. (C) The percentages of cells exhibiting translocation of AcGFP alone, AcGFP-DGKη1, η3 or η4 to the plasma membrane were scored. More than 20 cells expressing AcGFP alone, AcGFP-DGKη1, η3 or η4 were counted in each experiment. The results are the means ± S.D. of three separate experiments. *<i>P</i><0.05, **<i>P</i><0.01.</p

Distribution of the <i>Z</i>-test statistic for pairwise comparison of the rate of nonsynonymous substitution (<i>d</i>_N) with that of synonymous substitution (<i>d</i>_S) among 32 <i>P. falciparum</i> isolates.

<p>Distribution of the <i>Z</i>-test statistic for pairwise comparison of the rate of nonsynonymous substitution (<i>d</i>_N) with that of synonymous substitution (<i>d</i>_S) among 32 <i>P. falciparum</i> isolates.</p