Abstract  The response to cell surface stress in yeast is mediated by a set of five plasma membrane sensors. We here address the relation of intracellular localization of the sensors Wsc1, Wsc2, and Mid2 to their turnover and signaling function. Growth competition experiments indicate that Wsc2 plays an important role in addition to Wsc1 and Mid2. The two Wsc sensors appear at the bud neck during cytokinesis and employ different routes of endocytosis, which govern their turnover. Whereas Wsc1 uses a clathrin-dependent NPFDD signal, Wsc2 relies on a specific lysine residue (K495). In end3 and doa4 endocytosis mutants, both sensors accumulate at the plasma membrane, and a hypersensitivity to cell wall-specific drugs and to treatment with zymolyase is observed. A haploid strain in which endocytosis of the two sensors is specifically blocked displays a reduced fitness in growth competition experiments. If the Mid2 sensor is mobilized by the addition of an endocytosis signal, it mimics the dynamic distribution of the Wsc sensors, but is unable to complement the specific growth defects of a wsc1 deletion. These data suggest that sensor distribution is not the major determinant for its specificity.

Abstract  The mitochondrial genomes of flowering plants are highly recombinogenic and this can lead to altered transcriptional units, even between closely related species. We are interested in the effects that DNA rearrangements have on the generation of mature mRNAs, and to this end we have determined the termini of mitochondrial S7 ribosomal protein (rps7) mRNAs from selected grasses, using circularized-RT-PCR. Although the rps7 mRNAs show a similar size of about 750 nt by northern hybridization analysis and have virtually identical 3? UTRs, their 5? terminal extremities differ among plant species, and this is attributable to genome rearrangements in some but not all cases. In wheat, rice, and barley, the 5? ends are homogeneous for each plant but map to non-homologous sites among the three species. In contrast, the rye, brome and Lolium 5? ends are quite heterogeneous in length even though they are located within conserved genomic regions. Comparative sequence analysis suggests that certain grass lineages have retained an ancestral organization upstream of rps7 that includes a 170-bp block homologous to sequences preceding several other mitochondrial genes, whereas others have undergone independent rearrangements at a recombination-prone site. Our analysis of mature rps7 transcripts revealed two non-silent RNA edits within the coding sequences, and also editing at several sites within the conserved 5? and 3? UTR regions in these plants, raising the possibility of their role in rps7 expression at the post-transcriptional level. Taken together, our observations illustrate the dynamic nature of upstream regulatory cis-elements for mitochondrial rps7 mRNA production in contrast to conservative 3? end-formation signals, during evolution in grasses.

Abstract  Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a new member of the tumor necrosis factor-?-induced protein 8 (TNFAIP8) family that functions as an important factor in the maintenance of immune homeostasis. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine TNFAIP8L2. RH mapping using the IMpRH panel showed that this gene was closely linked to microsatellite marker SW512 in pig chromosome 4. Subcellular localization analysis showed GFP?TNFAIP8L2 fusion protein distributed in nucleus as well as cytoplasm including mitochondria and endoplasmic reticulum. Real-time PCR analysis revealed that porcine TNFAIP8L2 was more highly expressed in spleen than other tissues. To understand its characterization of transcriptional regulation, we cloned approximately 2 kb of 5?-regulatory region upstream to the porcine TNFAIP8L2 translational start site and generated sequential deletion constructs evaluated in dual-luciferase reporter assay. The results demonstrated that its core promoter is 435 base pairs (bp) upstream to the transcription initiation site. Then, site-directed mutation experiment combined with electrophoretic mobility shift assay (EMSA) indicated that M-CAT binding factor (MCBF) and activator protein 1 (AP-1) were important transcription factors for porcine TNFAIP8L2. These findings provide an important basis for further understanding of porcine TNFAIP8L2 regulation and function in swine.

Abstract  In response to environmental phosphate limitation, the transcriptional activator PhoB of Escherichia coli (E. coli) activates transcription of the phosphate regulon (pho regulon) genes that are involved in phosphate utilization. At least 31 of pho regulon genes have been identified and well characterized in E. coli by numerous studies using non-pathogenic K-12 derivative strains. In this study, we searched for PhoB-regulated promoters from a lacZ-fused genomic library of the E. coli O157:H7 Sakai in an attempt to find novel pho regulon genes in the strain. A promoter region located upstream of a gene cluster (ecs0540?ecs0544) that mapped within one of the strain-specific chromosomal regions of the E. coli O157:H7 was identified. By further in vivo analysis with various subclones of the 5?-flanking region, it was suggested that the ecs0540 transcription was regulated by at least two promoters, an upstream PhoB-regulated promoter and a downstream constitutive promoter. S1 mapping and footprinting experiments revealed two transcription start sites and a sequence similar to the consensus sequence of PhoB binding, respectively. Bioinformatic analysis of the ecs0540?ecs0544 genes showed that these genes were highly homologous to the Escherichia fergusonii (E. fergusonii) siiCA-DA operon encoding a 718 kDa giant protein (SiiEA) and its cognate type I secretion system. In addition, a highly repetitive region and motifs that are shared among RTX (repeats in toxin) toxin family were found in the amino acid sequence of these giant proteins. Our finding is the first example of a member of the pho regulon identified in the O157:H7 strain-specific chromosomal region.

Abstract  The transcription factor OsNAC5 in rice is a member of the plant-specific NAC family that regulates stress responses. Expression of OsNAC5 is induced by abiotic stresses such as drought, cold, high salinity, abscisic acid and methyl jasmonic acid. Transactivation assays using rice protoplasts demonstrated that OsNAC5 is a transcriptional activator, and subcellular localization studies using OsNAC5-GFP fusion proteins showed that it is localized to the nucleus. Pull-down assays revealed that OsNAC5 interacts with OsNAC5, OsNAC6 and SNAC1. To analyze the function of OsNAC5 in rice plants, we generated transgenic plants that overexpressed OsNAC5. The growth of these plants was similar to that of control plants, whereas the growth of OsNAC6-overexpressing transgenic plants was retarded. OsNAC5-overexpressing transgenic plants also had improved tolerance to high salinity compared to control plants. By microarray analysis, many stress-inducible genes, including the ?late embryogenesis abundant? gene OsLEA3, were upregulated in rice plants that overexpressed OsNAC5. By gel mobility shift assay, OsNAC5 and OsNAC6 were shown to bind to the OsLEA3 promoter. Collectively, our results indicate that the stress-responsive proteins OsNAC5 and OsNAC6 are transcriptional activators that enhance stress tolerance by upregulating the expression of stress-inducible rice genes such as OsLEA3, although the effects of these proteins on growth are different. Furthermore, because OsNAC5 overexpression did not retard growth, OsNAC5 may be a useful gene that can improve the stress tolerance of rice without affecting its growth.

Abstract  Valproic acid (VPA) causes various therapeutic and biological effects, but the exact mechanisms underlying these effects, however, remain elusive. To gain insights into the molecular mechanisms of VPA action, we performed in fission yeast a genetic screen for mutants that show VPA hypersensitivity and have identified several membrane-trafficking mutants including vas1-1/vps45 and vas2-1/aps1. Here, we describe the isolation and characterization of vas3-1/ric1-v3, a mutant allele of the ric1 + gene encoding a fission yeast homolog of the budding yeast Ric1p, a component of Ypt/Rab-specific guanyl-nucleotide exchange factor (GEF). The Rab GTPase Ryh1 knockout (?ryh1) cells and ?ric1 cells exhibited similar phenotypes. The double knockout ?ric1?ryh1 cells did not display synthetic growth defects. These results are consistent with the notion that Ric1 may be a component of the GEF complex for Ryh1. Overexpression of wild-type Ryh1 and the constitutively active Ryh1Q70L only partially suppressed the phenotypes of ric1-v3 and ?ric1 cells, and they failed to localize to the Golgi/endosomes in ric1-v3 and ?ric1 cells. Furthermore, we isolated vps15 + gene, encoding a serine/threonine protein kinase, as a dosage-dependent suppressor of the temperature-sensitive phenotype of ric1-v3 mutant, but not that of ?ric1 cells. Our results showed that the ric1-v3 mutant allele has some residual functional activity and suggest that Vps15 plays a role in the regulation of Ric1 function. In conclusion, Ric1 is a putative component of GEF for Ryh1 and might be regulated by Vps15. Further studies are needed to reveal the mechanism underlying the regulation.

Abstract  Invertase inhibitors (InvInh) interacted with invertases (Inv) and inhibited their activities involved in reducing sugars (RS) accumulation in cold-stored potato tubers. Understanding their potential contribution to RS accumulation is of both theoretical and practical importance because RS accumulation is a costly postharvest problem for both potato producers and processors. In this study, four genes with significant sequence homology to NtInvInhs were identified from potato and their possible contributions to cold-induced sweetening (CIS) of tubers were investigated together with StInv1, an acid invertase gene previously clarified corresponsive to CIS. Transcripts analysis of these StInvInhs and StInv1 among six potato genotypes with distinct CIS sensitivity indicated that StInvInh2 had a negative power regression to RS increase of the cold-stored tubers while a positive linear regression was obtained with StInv1. The relative expression ratio calculated by StInv1/StInvInh2 performed a very significant correlation to RS accumulation, suggesting a possible interaction between StInv1 and StInvInh2 in response to CIS. The bimolecular fluorescence complementation visualized the interaction between StInv1 and StInvInh2A and with StInvInh2B in both onion epidermal cells and tobacco BY-2 cells and demonstrated that these two inhibitors may be the isoforms of StInvInh2 as the counterparts of StInv1. The recombinant StInvInh2B protein inhibited the activities of soluble acid invertase indicating evidently its inhibitory properties. Our results strongly suggest that the interaction between StInv1 and StInvInh2 may play critical roles in controlling the CIS through posttranslational regulation of StInv1 by StInvInh2 in potato tubers and will provide novel tools and resources for improving CIS tolerance of potatoes.

Abstract  Flowering time is a major determinant for the local adaptation of crops. Hd1 is a key flowering-time gene in rice and is orthologous to the Arabidopsis CONSTANS gene. To elucidate the role of Hd1 in selection, we examined the Hd1 alleles of 60 landraces of Asian cultivated rice (Oryza sativa L.) originating from all regions of Asia, which comprised three cultivar groups, indica, japonica, and aus. The identified alleles were classified into four allele groups. The functional Hd1 alleles in allele groups I and II corresponded to indica and japonica, respectively. Non-functional alleles in these groups were not clearly associated with cultivar groups or locations. Allele groups III and IV corresponded to the aus cultivar group. The ancestry of each cultivar group was identified by the coalescent approach for Hd1 molecular evolution using the haplotype patterns of 14 regions over the 1.1 Mb chromosomal region surrounding Hd1 and the pSINE patterns of two loci, 1.4 and 4.4 Mb apart from Hd1. The haplotype patterns clearly revealed that Hd1 allele migration was caused by multiple and complex introgression events between cultivar groups. The Hd1 haplotypes among dozens of accessions of the wild species O. rufipogon were strongly divergent and only two of the haplotype clusters in O. rufipogon were closely related to those in cultivated rice. This strongly suggested that multiple introgression events have played an important role in the shaping and diversification of adaptation in addition to primary selection steps at the beginning of domestication.

Abstract  Identification of genes for quantitative traits is difficult using any single approach due to complex inheritance of the traits and limited resolving power of the individual techniques. Here a combination of genetic mapping and bulked transcriptome profiling was used to narrow down the number of differentially expressed salt-responsive genes in rice in order to identify functional polymorphism of genes underlying the quantitative trait loci (QTL). A population of recombinant inbred lines (RILs) derived from cross between salt-tolerant variety CSR 27 and salt-sensitive variety MI 48 was used to map QTL for salt ion concentrations in different tissues and salt stress susceptibility index (SSI) for spikelet fertility, grain weight, and grain yield. Eight significant QTL intervals were mapped on chromosomes 1, 8, and 12 for the salt ion concentrations and a QTL controlling SSI for spikelet fertility was co-located in one of these intervals on chromosome 8. However, there were total 2,681 genes in these QTL intervals, making it difficult to pinpoint the genes responsible for the functional differences for the traits. Similarly, transcriptome profiling of the seedlings of tolerant and sensitive parents grown under control and salt-stress conditions showed 798 and 2,407 differentially expressed gene probes, respectively. By analyzing pools of RNA extracted from ten each of extremely tolerant and extremely sensitive RILs to normalize the background noise, the number of differentially expressed genes under salt stress was drastically reduced to 30 only. Two of these genes, an integral transmembrane protein DUF6 and a cation chloride cotransporter, were not only co-located in the QTL intervals but also showed the expected distortion of allele frequencies in the extreme tolerant and sensitive RILs, and therefore are suitable for future validation studies and development of functional markers for salt tolerance in rice to facilitate marker-assisted breeding.

Abstract  MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that collectively regulate the expression of a large number of mRNAs by either promoting destabilization or repressing translation, or both. Therefore, they play a major role in shaping the transcriptomes and proteomes of eukaryotic organisms. Typically, animal miRNAs are produced from long primary transcripts with one or more of hairpin structures by two sequential processing reactions: one by Drosha in the nucleus and the other by Dicer in the cytoplasm. However, deviations from this paradigm have been observed: subclasses of miRNAs, which only partially meet the classical definition of a miRNA, are derived by alternative biogenesis pathways, thereby providing an additional level of complexity to miRNA-dependent regulation of gene expression.

Abstract  The centromere is a genetic locus, required for faithful chromosome segregation, where spindle fibers attach to the chromosome through kinetochore. Loss of centromere or formation of multiple centromeres on a single chromosome leads to chromosome missegregation or chromosome breakage, respectively, which are detrimental for fitness and survival of a cell. Therefore, understanding the mechanism of centromere locus determination on the chromosome and perpetuation of such a locus in subsequent generation (known as centromere identity) is very fundamental to combat conditions like aneuploidy, spontaneous abortion, developmental defects, cell lethality and cancer. Recent studies have come up with different models to explain centromere identity. However, the exact mechanism still remains elusive. It has been observed that most eukaryotic centromeres are determined epigenetically rather than by a DNA sequence. The epigenetic marks that are instrumental in determining centromere identity are the histone H3 variant, CENP-A and the specialized posttranslational modification of the core histones. Here we will review the recent studies on the factors responsible for generating unique centromeric chromatin and how it perpetuates during cell division giving the present-day models. We will further focus on the probable mechanism of de novo centromere formation with an example of neocentromere. As a matter of similitude, this review will include marking extrachromosomal chromatin to be served as a partitioning locus by deposition of CENP-A homolog in budding yeast.

Abstract  Despite the significance of actin in plant growth and development, little is known of the structure, expression and evolution of the actin gene family in woody plants. In this study, we systematically examined the diversification of the actin gene family in Populus by integrating genomic organization, expression, and phylogeny data. Genome-wide analysis of the Populus genome indicated that actin is a multigene family consisting of eight members, all predicted to encode 377-amino acid polypeptides that share high sequence homology ranging from 94.2 to 100% identity. Microarray and real-time PCR expression analysis showed that the PtrACT family members are differentially expressed in different tissues, exhibiting overlapping and unique expression patterns. Of particular interest, all PtrACT genes have been found to be preferentially expressed in the stem phloem and xylem, suggesting that poplar PtrACTs are involved in the wood formation. Gene structural and phylogenetic analyses revealed that the PtrACT family is composed of two main subgroups that share an ancient common ancestor. Extremely high intraspecies synonymous nucleotide diversity of ?syn = 0.01205 was detected, and the ?non-syn/?syn ratio was significantly less than 1; therefore, the PtACT1 appears to be evolving in Populus, primarily under purifying selection. We demonstrated that the actin gene family in Populus is divided into two distinct subgroups, suggesting functional divergence. The results reported here will be useful in conducting future functional genomics studies to understand the detailed function of actin genes in tree growth and development.

Abstract  Modern sugarcane cultivars (Saccharum spp., 2n = 100?120) are complex polyploids derived from interspecific hybridization performed a century ago between the sugar-producing species S. officinarum L. and the wild species S. spontaneum L. Using genomic in situ hybridization, we revealed that between 15 and 27.5% of the genome of modern cultivars is derived from S. spontaneum, including 10?23% of entire chromosomes from this wild species and 8?13% chromosomes derived from interspecific recombination. We confirmed the occurrence of 2n + n transmission in crosses and first backcrosses between these two species and demonstrated that this also can occur in crosses between S. officinarum and modern cultivars. We analysed five S. officinarum clones with more than 80 chromosomes and demonstrated that they were derived from interspecific hybridization supporting the classical view that this species is characterized by 2n = 80. We also illustrated the complementarities between molecular cytogenetics and genetic mapping approaches for analysing complex genomes.

Abstract  Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.