Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome.

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. Here, we found that cryptic promoter activation occurs by at least two different mechanisms using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) without an apparent promoter sequence was expressed from intergenic regions: one mechanism is 'cryptic promoter capturing', in which the LUC ORF captured pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is 'promoter de novo origination', in which the promoter chromatin was newly formed near the 5' end of the inserted LUC ORF. The latter finding raises a question as to how the inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter-LUC constructs, we found that the functional core promoter region, where transcription start sites (TSSs) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the coding sequence alters the local distribution of TSSs in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

Fluorescence visualization of branchial collagen columns embraced by pillar cells.

A collagen column is a structure of the extracellular matrix that helps to maintain the flatness and width of gill lamella. Collagen columns are unique in that they are enfolded by plasma membrane of pillar cells that form two-dimensional vascular networks between parallel sheets of respiratory epithelia. Despite their unique structure and fundamental importance in the physiology of aquatic animals, little is known about their properties and molecular components, owing to the lack of detection methods. In this study we demonstrated that collagen columns can be visualized by staining with fluorescence-labeled concanavalin A (ConA), a lectin that specifically recognizes the trimannoside core of N-glycosylated proteins and histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a fluorescent substrate for transglutaminase. We constructed a three-dimensional image of a pillar cell and visualized the spatial relationship between collagen columns and contractile apparatuses within the pillar cell body. This manuscript contains online supplemental material at (www.jhc.org). Please visit this article online to view these materials.

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.

Null mutation of the MdACS3 gene, coding for a ripening-specific 1-aminocyclopropane-1-carboxylate synthase, leads to long shelf life in apple fruit.

Expression of MdACS1, coding for 1-aminocyclopropane-1-carboxylate synthase (ACS), parallels the level of ethylene production in ripening apple (Malus domestica) fruit. Here we show that expression of another ripening-specific ACS gene (MdACS3) precedes the initiation of MdACS1 expression by approximately 3 weeks; MdACS3 expression then gradually decreases as MdACS1 expression increases. Because MdACS3 expression continues in ripening fruit treated with 1-methylcyclopropene, its transcription appears to be regulated by a negative feedback mechanism. Three genes in the MdACS3 family (a, b, and c) were isolated from a genomic library, but two of them (MdACS3b and MdACS3c) possess a 333-bp transposon-like insertion in their 5' flanking region that may prevent transcription of these genes during ripening. A single nucleotide polymorphism in the coding region of MdACS3a results in an amino acid substitution (glycine-289 --> valine) in the active site that inactivates the enzyme. Furthermore, another null allele of MdACS3a, Mdacs3a, showing no ability to be transcribed, was found by DNA sequencing. Apple cultivars homozygous or heterozygous for both null allelotypes showed no or very low expression of ripening-related genes and maintained fruit firmness. These results suggest that MdACS3a plays a crucial role in regulation of fruit ripening in apple, and is a possible determinant of ethylene production and shelf life in apple fruit.

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater.

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.