Isolation of an Arabidopsis thaliana Mutant,mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation)

We isolated Arabidopsis thaliana mutants that are resistant to ethionine, a toxic analog of methionine (Met). One of the mutants was analyzed further, and it accumulated 10- to 40-fold more soluble Met than the wild type in the aerial parts during the vegetative growth period. When the mutant plants started to flower, however, the soluble Met content in the rosette region decreased to the wild-type level, whereas that in the inflorescence apex region and in immature fruits was 5- to 8-fold higher than the wild type. These results indicate that the concentration of soluble Met is temporally and spatially regulated and suggest that soluble Met is translocated to sink organs after the onset of reproductive growth. The causal mutation, designated mto1, was a single, nuclear, semidominant mutation and mapped to chromosome 3. Accumulation profiles of soluble amino acids suggested that the mutation affects a later step(s) in the Met biosynthesis pathway. Ethylene production of the mutants was only 40% higher than the wild-type plants, indicating that ethylene production is tightly regulated at a step after Met synthesis. This mutant will be useful in studying the translocation of amino acids, as well as regulation of Met biosynthesis and other metabolic pathways related to Met.     

Expression in Yeast of a Fusion Gene Composed of the Promoter of a Heat-Shock Gene from Arabidopsis and a Bacterial Gene for {beta}-Glucuronidase

Production of a functional ß-glucuronidase (GUS) proteinwas induced by exposure of exponentially growing yeast cellsto heat shock after transformation of the GUS gene under thecontrol of the promoter of the heat-shock gene, HSP18.2, fromArabidopsis. Yeast cyr and bcy mutations appeared to have essentiallyno effect. ¹Present Address: Laboratory of Plant Molecular Biology, TheRockefeller University, 1230 York Avenue, New York, NY 10021-6399,U.S.A.     

Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

The Promoter of a Pine Photosynthetic Gene Allows Expression of a {beta}-Glucuronidase Reporter Gene in Transgenic Rice Plants in a Light-Independent but Tissue-Specific Manner

In angiosperms, the expression of the cab gene that encodesthe chlorophyll a/b-binding protein of PSII is light-regulated.However, the pine cab gene is expressed in a light-independentbut cell-type-specific manner. In the present study, the cab-6promoter (1.7 kbp) from pine was fused to a -glucuronidase (GUS)reporter gene and the chimeric gene was introduced into riceprotoplasts by electroporation. The GUS expression was studiedin the resultant transgenic rice plants. Expression of GUS ata substantial level was confirmed in primary leaves of dark-germinatedrice seedlings, and no obvious effect of light on the GUS activitywas observed. The expression of GUS was restricted to photosynthetictissues. The pine cab-6 promoter is, thus, sufficient for inductionof light-independent but cell-type-specific expression in cellsof a monocot, as is the case in the original pine cells. (Received December 17, 1993; Accepted April 22, 1994)     

The Sequence and Expression of the {gamma}-VPE Gene, One Member of a Family of Three Genes for Vacuolar Processing Enzymes in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturationof seed proteins. Southern blot analysis showed that a familyof genes for VPEs in Arabidopsis thaliana was composed of threegenes, for      

Stable Delivery of a Canavalin Promoter-{beta}-Glucuronidase Gene Fusion into French Bean by Particle Bombardment

Shoot apices of French bean (Phaseolus vulgaris L. cv. Goldstar)seeds were bombarded with gold particles coated with plasmidpSAG-734 carrying a chimeric gene that encodes ß-glucuronidaseunder the control of the canavalin gene promoter. The organ-and maturation stage-specific expression of the construct wasobserved histochemically in seeds of transgenic plants (Received July 10, 1996; Accepted October 5, 1996)     

Characterization of a novel mucin sulphotransferase activity synthesizing sulphated O-glycan core 1, 3-sulphate-Gal{beta}1-3GalNAc{alpha}-R

A novel sulphotransferase (sulpho-T) activity from rat colonicmucosa was characterized using O-glycan core 1 substrate, Galß1-3GalNAc     

Effects of overexpression of {beta}1, 4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells

ß1,4-Galactosyltransferase (GalTase) plays a centralrole in the biosynthesis of N-acetyllactosamine-containing oligo-saccharides.However, despite this seemingly important function, little isknown about how changes in the levels of GalTase affect oligosaccharidebiosynthesis. We have examined the effects of overexpressingGalTase on the glycosylation of endogenous glycoproteins inF9 mouse embryonal carcinoma cells. Cells transfected with eitherthe short form of the GalTase cDNA (encoding a protein of 386amino acids) or the long form of the GalTase cDNA (encodinga protein of 399 amino acids) had a 3-fold increase in totalGalTase activity, relative to control F9 cells. Analysis ofpronase-digested glycopeptides obtained from control and transfectedcells after metabolic labelling with [6-³H]galactose revealedno significant qualitative or quantitative differences, as assessedby Bio-Gel P-6 gel filtration chromatography and Tomato lectinaffinity chroma-tography. Furthermore, SDS-PAGE analysis ofimmuno-precipitated [³H]galactose-labelled lysosomal-associatedmembrane protein-1 (LAMP-1) glycoprotein showed no differencein amounts or mobility. Pronase digestion and subsequent analysisof the gel-fractionated LAMP-1 glycoproteins also indicatedno differences between the various cell lines. The inabilityof elevated GalTase activity to affect glycosylation was notdue to limiting levels of GalTase substrates, since an excessof substrates was detectable in lysed cells using either endogenousor exogenous GalTase and UDP-[³H]galactose. Finally, the subcellulardistribution of GalTase, as assessed by sucrose gradient fractionation,was similar between all cell types, thus suggesting that GalTasewas appropriately compartmentalized in the transfected cells.More importantly, GalTase specific activities in the Golgi membranesof the transfected cells were 3–4 times greater than incontrol cells. These results show that selectively increasingGalTase activity does not alter glycoprotein biosynthesis inF9 cells. F9 cells galactosyltransferase glycoprotein biosynthesis     

Expression of a Soybean (Glycine max [L.] Merr.) Seed Storage Protein Gene in Transgenic Arabidopsis thaliana and Its Response to Nutritional Stress and to Abscisic Acid Mutations

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1-1 or abi3-1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.     

磷和葡萄糖及细胞分裂素对拟南芥SEN1基因表达的影响

拟南芥SEN1基因受衰老诱导.将该基因启动子融合报告基因萄聚糖酶(glucuronidase,GUS)基因转入拟南芥,通过染色并测定GUS活性发现,缺氮、缺磷、缺钾诱导叶中SEN1表达,而只有缺磷能导根中SEN1表达.缺磷对根叶中SEN1的诱导被3%葡萄糖和细胞分裂素抑制.3%葡萄糖胺在根和叶中均诱导SEN1表达,外源细胞分裂素不能抑制这种效应.结果表明:SEN1基因可受缺磷信号特异调控,并受糖信号和细胞分裂素负调控;葡萄糖胺能大大促进根和叶中SEN1表达,且不受细胞分裂素的负调控.     

RMA1 an Arabidopsis thaliana Gene Whose cDNA Suppresses the Yeast secl5 Mutation, Encodes a Novel Protein with a RING Finger Motif and a Membrane Anchor

To identify molecules that function in the plant secretory pathway,we screened for Arabidopsis thaliana cDNA clones that complementthe temperature-sensitive (ts), secretion-deficient sec15 mutationof yeast Saccha-romyces cerevisiae. RMA1, one of the genes obtainedin this screening, suppressed not only the ts growth of sec15but also its secretory defect. RMA1 is not a structural homologueof SEC15 but encodes a novel 28 kDa protein with a RING fingermotif and a C-terminal membrane-anchoring domain. Mutationalanalysis indicates that the RING finger motif of RMA1 is importantfor its suppression activity. In Arabidopsis plant, RMA1 isubiquitously expressed. A search for homologous proteins inthe database revealed that Arabidopsis, nematode, mouse andhuman possess close homologues of RMA1. (Received January 5, 1998; Accepted March 9, 1998)     

Mutation of a Gene Essential for Ribosome Biogenesis, EMG1, Causes Bowen-Conradi Syndrome

Bowen-Conradi syndrome (BCS) is an autosomal-recessive disorder characterized by severely impaired prenatal and postnatal growth, profound psychomotor retardation, and death in early childhood. Nearly all reported BCS cases have been among Hutterites, with an estimated birth prevalence of 1/355. We previously localized the BCS gene to a 1.9 Mbp interval on human chromosome 12p13.3. The 59 genes in this interval were ranked as candidates for BCS, and 35 of these, including all of the best candidates, were sequenced. We identified variant {"type":"entrez-nucleotide","attrs":{"text":"NM_006331.6","term_id":"194328698","term_text":"NM_006331.6"}}NM_006331.6:c.400A→G, p.D86G in the 18S ribosome assembly protein EMG1 as the probable cause of BCS. This mutation segregated with disease, was not found in 414 non-Hutterite alleles, and altered a highly conserved aspartic acid (D) residue. A structural model of human EMG1 suggested that the D86 residue formed a salt bridge with arginine 84 that would be disrupted by the glycine (G) substitution. EMG1 mRNA was detected in all human adult and fetal tissues tested. In BCS patient fibroblasts, EMG1 mRNA levels did not differ from those of normal cells, but EMG1 protein was dramatically reduced in comparison to that of normal controls. In mammalian cells, overexpression of EMG1 harboring the D86G mutation decreased the level of soluble EMG1 protein, and in yeast two-hybrid analysis, the D86G substitution increased interaction between EMG1 subunits. These findings suggested that the D-to-G mutation caused aggregation of EMG1, thereby reducing the level of the protein and causing BCS.