Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Synthesis of lipid A type pyran carboxylic acids with ether chains and their biological activities.

Synthesis of lipid A type pyran carboxylic acids having ether chains at both the C-3' and C-4 positions and their bioactivities toward human U937 cells are described.     

Laboratory-induced development of the ice-ice disease of the farmed red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae,Gigartinales, Rhodophyta)

The most common perception of unfavorable environmental factors causing the ice-ice disease in the farmed seaweeds, Kappaphycus and Eucheuma, was demonstrated in this study for the first time using stressful conditions of abiotic factors in a continuous culture system. Light intensity of less than 50 mol photon m^–2 s^–1 and salinity of 20% or less induced ice-ice whitening characterized by short segments at midbranches which were similar to those observed in the Philippine seaweed farms, while temperatures of up to 33–35 °C resulted in wide-scale whitening leading to complete damage of the branches. These effects were preceded by slow growth rates from an optimum of 3.7% d^–1 to almost –2.0% d^–1. Mechanical stress by wound injury did not result to ice-ice whitening similar to the above. Environmental factors observed to trigger ice-ice in the laboratory, although may not necessarily parallel those in the field, may act synergestically to produce similar effects.     

A Modified Micro-Drop Bioassay Using Dwarf Rice for Detection of Femtomol Quantities of Gibberellins

The sensitivity of the micro-drop assay with dwarf rice (Oryzasativa L., cv. Tan-ginbozu and cv. Waito-Q to gibberellins (GAs)was increased conspicuously by the use of assay plants thathas been treated with uniconazole (S-3307), an inhibitor ofthe biosynthesis of GAs. The Tan-ginbozu plants treated withS-3307 responded to 10 fmol/plant of GA₃ (ca. 3.5 pg/plant)and to 30 fmol/plant of gibberellins A₁, A₄, A₇, A₁₉ and A₂₀.Waito-C plants treated with S-3307 responded to 10 fmol of GA₃and to 30 fmol/plant of gibberellins A₁, A₄ and A₇. GibberellinsA₉, A₁₉ and A₂₀ had much less of an effect on the treated Waito-Cplants than did gibberellins A₁, A₃, A₄ and A₇. Furthermore,treatment with S-3307 counteracted the inhibition of growthof both cultivars by abscisic acid. Thus, the modified micro-dropassay should prove very useful for the detection of minute amountsof GAs in plant extracts. (Received October 3, 1988; Accepted March 29, 1989)     

Nondwarf rice seedling bioassay for gibberellins

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.     

Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells.

Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.     

Enhancement of O2- generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A

The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O2- generation in mouse peritoneal macrophages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3-O-beta-hydroxymyristoyl groups are essential for the elevated O2- generation and induction of tumoricidal activity due to lipid X.