CiYB1 is a major component of storage mRNPs in ascidian oocytes: implications in translational regulation of localized mRNAs

In ascidian eggs, the existence of several localized maternal cytoplasmic determinants has been proposed and the importance of localized mRNAs for tissue differentiation has been demonstrated. We previously identified the ascidian Y-box proteins (CiYB1, 2 and 3), homologues of which are known to be involved in the storage of maternal mRNA in oocytes of other organisms. In this study, we found that CiYB1 protein is abundant in the gonad, egg, and embryo. Purification of messenger ribonucleoprotein (mRNP) particles from the gonad revealed that CiYB1 was one of their major components. A significant change in the distribution of CiYB1 protein from stored mRNP particles in the gonad to the ribosome fraction in eggs and embryos was observed. This change correlates most likely with the shift of stored maternal mRNAs to polyribosomes. Moreover, we found that CiYB1 colocalized with Cipem and Ci-macho1 mRNAs, which are localized at the posterior end of the embryo at the cleavage stage. Cipem and Ci-macho1 mRNAs were co-immunoprecipitated with CiYB1 in the oocyte and embryo lysates. The formation of a complex between Cipem mRNA and CiYB1 protein resulted in translational repression in the in vitro translation system. Our results indicate that associating with CiYB1 protein contributes to the translational control of the localized mRNA in eggs and embryos.     

Relationships between brood size and offspring body size in an ovoviviparous fish: maternal effects and genetic trade-off

Maternal effects can form an important source of variation in offspring fitness and have important evolutionary and ecological consequences. To explore genetic control for body size of newborn offspring, the present study examined maternal brood size and offspring body size in 14 strains and performed cross experiments in an ovoviviparous fish, guppy. Correlation analyses among the strains indicated that the strains with larger brood size have smaller offspring body size. Diallel and reciprocal crosses among four strains revealed a large maternal, but no paternal, contribution to offspring body size. To examine whether offspring body size is determined by maternal genotypes and whether offspring body size correlates with albinism, backcrosses were performed between a wild-type strain with large offspring body size and an albino strain. Offspring body size differed from both the parental strains in one generation of backcrossing but was not significantly different from the paternal strains after three generations. In the backcross generations, the offspring yielded by albino individuals showed significantly smaller body size than those yielded by normal individuals. These results indicated that offspring body size is determined by maternal genotypes, suggesting that offspring body size is influenced by the maternal albinism gene or the maternal locus linked with the albinism locus. The significant negative correlation between maternal brood size and offspring body size detected through our experiments showed that the maternal genetic character of brood size strongly correlated with offspring body size, indicating genetic trade-off between maternal brood size and offspring body size in the guppy.     

The GURKE gene encoding an acetyl-CoA carboxylase is required for partitioning the embryo apex into three subregions in Arabidopsis

In Arabidopsis, three major regions, which ultimately develop into the two cotyledons, the cotyledon boundaries and the shoot apical meristem (SAM), are formed at the apex of the globular stage embryo. To reveal the molecular mechanism underlying this pattern formation, we isolated a cotyledon-defective mutant from EMS mutagenized lines. This mutant completely lacks cotyledons in the most severe cases, and is allelic to gurke (gk), which was previously reported as a mutant defective in apical patterning of the embryo. To evaluate the morphological effects of the mutation in the GK gene, we investigated the expression patterns in gk embryos of SHOOT MERISTEMLESS (STM), AINTEGUMENTA (ANT) and CUP-SHAPED COTYLEDON1 (CUC1), which are markers of the SAM, cotyledons and cotyledon boundaries, respectively. Expression of all these genes largely overlapped in gk, suggesting a failure to partition the apex of the embryo into the three subregions. Enlargement of the CUC1 expression domain was also observed and may explain the inhibition of cotyledon development in gk. Moreover, we cloned the GK gene, and confirmed that it encodes ACC1, an acetyl-CoA carboxylase which catalyzes malonyl-CoA synthesis. Our results suggest that metabolites derived from malonyl-CoA are required for partitioning of the apical part of the embryo.     

False-positive selection identified by ML-based methods: examples from the Sig1 gene of the diatom Thalassiosira weissflogii and the tax gene of a human T-cell lymphotropic virus

Sexually induced gene 1 (Sig1) in the centric diatom Thalassiosira weissflogii is considered to encode a gamete recognition protein. Sorhannus (2003) analyzed nucleotide sequences of Sig1 using parsimony analysis and the maximum-likelihood (ML)-based Bayesian method for inferring positive selection at single amino acid sites and reported that positively selected sites were detected by the latter method but not by the former. He then concluded that for this type of study, the ML-based method is more reliable than parsimony analysis. Here we show that his results apparently represent false-positive cases of the ML-based method and that there is no solid evidence that this gene contains positively selected sites. We further demonstrate that in the tax gene of human T-cell lymphotropic virus type I (HTLV-I), all codon sites, including invariable sites, can be inferred as positively selected sites by the ML-based method. These observations indicate that the ML-based method may produce many false-positive sites. One of the main reasons for the occurrence of false positives is that in the ML-based method, codon sites are grouped into several categories, with different nonsynonymous/synonymous rate ratios (omegas), on a purely statistical basis, and positive selection is inferred indirectly by examining whether the average omega for each category is greater than 1. In parsimony analysis, however, the evolutionary change of nucleotides at each codon site is examined. For this reason, parsimony-based methods rarely produce false positives and are safer than ML-based methods for detecting positive selection at individual codon sites, although a large number of sequences are necessary.     

Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products

We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.     

Origins and divergence times of mammalian class II MHC gene clusters

The class I and II major histocompatibility complex (MHC) genes are apparently subject to evolution by a birth-and-death process. The rate of gene turnover is much slower in the latter genes than in the former. In placental mammals, the class II region can be subdivided into different orthologous subregions or gene clusters (DR, DQ, DO, and DN), but the origins and evolutionary relationships of these gene clusters are not well established. Here we report the results of our study of the times of origin and evolutionary relationships of these gene clusters in mammals. Our analysis suggests that both class II alpha-chain and beta-chain gene clusters are shared by placental mammals and marsupials, but the gene clusters from nonmammalian species are paralogous to mammalian gene clusters. We estimated the times of divergence between gene clusters in placental mammals using the linearized tree and distance regression methods. Our results indicate that most gene clusters originated 170-200 million years (MY) ago, but that DO beta-chain genes diverged from the other beta-chain gene clusters approximately 210-260 MY ago. The phylogenetic trees for the alpha- and beta-chain genes were not congruent, suggesting that the evolutionary history of the class II gene clusters is more complex than previously thought.     

Efficiencies of fast algorithms of phylogenetic inference under the criteria of maximum parsimony, minimum evolution, and maximum likelihood when a large number of sequences are used

In phylogenetic inference by maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods, it is customary to conduct extensive heuristic searches of MP, ME, and ML trees, examining a large number of different topologies. However, these extensive searches tend to give incorrect tree topologies. Here we show by extensive computer simulation that when the number of nucleotide sequences (m) is large and the number of nucleotides used (n) is relatively small, the simple MP or ML tree search algorithms such as the stepwise addition (SA) plus nearest neighbor interchange (NNI) search and the SA plus subtree pruning regrafting (SPR) search are as efficient as the extensive search algorithms such as the SA plus tree bisection-reconnection (TBR) search in inferring the true tree. In the case of ME methods, the simple neighbor-joining (NJ) algorithm is as efficient as or more efficient than the extensive NJ+TBR search. We show that when ME methods are used, the simple p distance generally gives better results in phylogenetic inference than more complicated distance measures such as the Hasegawa-Kishino-Yano (HKY) distance, even when nucleotide substitution follows the HKY model. When ML methods are used, the simple Jukes-Cantor (JC) model of phylogenetic inference generally shows a better performance than the HKY model even if the likelihood value for the HKY model is much higher than that for the JC model. This indicates that at least in the present case, selecting of a substitution model by using the likelihood ratio test or the AIC index is not appropriate. When n is small relative to m and the extent of sequence divergence is high, the NJ method with p distance often shows a better performance than ML methods with the JC model. However, when the level of sequence divergence is low, this is not the case.     

Calumin, a Ca²⁺-binding protein on the endoplasmic reticulum, alters the ion permeability of Ca²⁺ release-activated Ca²⁺ (CRAC) channels

Store-operated channels (SOC) are Ca(2+)-permeable channels that are activated by IP(3)-receptor-mediated Ca(2+) depletion of the endoplasmic reticulum (ER). Recent studies identify a membrane pore subunits, Orai1 and a Ca(2+) sensor on ER, STIM1 as components of Ca(2+) release-activated Ca(2+) (CRAC) channels, which are well-characterized SOCs. On the other hand, proteins that act as modulators of SOC activity remain to be identified. Calumin is a Ca(2+)-binding protein that resides on the ER and functional experiments using calumin-null mice demonstrate that it is involved in SOC function, although its role is unknown. This study used electrophysiological analysis to explore whether calumin modulates CRAC channel activity. CRAC channel currents were absent in HEK293 cells co-expressing calumin with the CRAC channel components, Orai1 or STIM1. Meanwhile, HEK cells that co-expressed calumin with CRAC channels exhibited larger currents with slower inactivation than cells expressing CRAC channels alone. The current-voltage relationship showed an inwardly rectifying current, but a negative shift in the reversal potential of greater than 60mV was observed in HEK cells co-expressing calumin with CRAC channels. In addition, the permeability coefficient ratio of Ca(2+) over monovalent cations was much lower than that of cells expressing CRAC channels alone. Replacement of Na(+) with N-methyl-d-glucamine(+) in the external solution noticeably diminished the CRAC current in HEK cells co-expressing calumin and CRAC channels. In a Cs(+)-based external solution, CRAC current was not observed in either cell-type. In addition, Ca(2+) imaging analysis revealed that co-transfection of calumin reduced extracellular Ca(2+) influx via CRAC channels. Further, calumin was shown to be directly associated with CRAC channels. These results reveal a novel mechanism for the regulation of CRAC channels by calumin.     

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.