Expression of the First N-Glycosylation Gene in the Dolichol Pathway, ALG7, Is Regulated at Two Major Control Points in the G1 Phase of the Saccharomyces cerevisiae Cell Cycle

The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene. To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle. We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of α-factor. ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START. In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by α-factor treatment later in G1. Importantly, deregulated expression of ALG7 resulted in an aberrant α-factor response. Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START.     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae

The Saccharomyces cerevisiae alg3-1 mutant is descilbed as defectivein the biosynthesis of dolichol-linked oligosaccharides (Huffakerand Robbins, Proc. Natl. Acad. Sci. USA, 80, 7466–7470,1983). Man₅GlcNAc₂-PP-Dol accumulates in alg3 cells and EndoH resistant carbohydrates are transferred to protein by theoligosaccharyltransferase complex. In this study, we describethe cloning of the ALG3 locus by complementation of the temperaturesensitive growth defect of the alg3 stt3 double mutant. Theisolated ALG3 gene complements both the defect in the biosynthesisof lipidlinked oligosaccharides of the alg3-mutant and the underglycosylationof secretory proteins. The inactivation of the nonessentialALG3 gene results in the accumulation of lipid-linked Man₅GlcNAc₂and protein-bound carbohydrates which are completely Endo Hresistant. The ALG3 locus encodes a potential ER-transmembraneprotein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrievalsequence. lipid-linked oligosaccharide N-glycosylation synthetic lethality     

The Effects of Cell Cycle Parameters on Cell Wall Regeneration and Cell Division of Cotton Protoplasts (Gossypium hirsutum L.)

Protoplasts of cotton cotyledons were isolated and culturedto undergo cell wall regeneration and cell division. DNA contentand cell cycle parameters of nuclei from cotyledons and/or protoplastswere determined by flow cytometry. The DNA content of cotton,Gossypium hirsutum L., was estimated to be 4·34±0·12pg DNA per nucleus. There was a strong positive correlation between G₂ or Sand G₂,and cell wall regeneration and cell division and a strong negativecorrelation between G₁, and cell wall regeneration and celldivision of cotton cotyledon protoplasts. The cell cycle statusof cotyledons changes during their development; as the cotyledonsenlarge, the proportion of cells in G₀ and G₁ phases of thecell cycle increases. The implication of these results in relationto protoplast growth and development is discussed. Key words: Cell cycle parameters, cell wall regeneration, cell division, flow cytometry, Gossypium     

Cell Cycle Changes in the Shoot Apex of Silene coeli-rosa During the Second and Third Days of Floral Induction

The cell cycle in Silene coeli-rosa shoot apices was measuredto test whether or not early components of the floral stimulus,produced during the 2nd and 3rd long days (LD) of an inductiveLD treatment, resulted in an increase in the duration of G₂phase in constant 20–24 h cell cycles. Plants were grownat 20°C in short days (SD) of 8 h light and 16 h darknessfor 28 d (day 0). Starting on day 0, plants were given SD or3 LD each comprising an identical 8 h day and 16 h photo-extension,or 3 dark-interrupted (d.i.) non-inductive LD, interrupted at1700 h of each day with 1 h of darkness. The cell cycle (percentagelabelled mitoses method) and changes in cell number were determinedin the shoot apical meristem. During days 1–2 of the SDtreatment, the cell cycle and mean cell generation time (MCGT)was 18 and 32 h, respectively, giving a growth fraction of 56%.During days 2–3, the cell cycle and MCGT shortened to15 and 23 h, respectively (growth fraction = 65%). During days1–2 of the LD and d.i. LD treatments, cell cycles andMCGTs were 9–10 and 27–29 h, respectively, resultingin smaller growth fractions (about 33%). Thus, shortened cellcycles and altered growth fractions occurred regardless of whetheror not the treatment was inductive. The LD treatment resultedin a marked shortening of G₁ and, to a lesser extent, S-phase,whilst G₂ remained constant. These changes were consistent withincreases in the proportion of cells in G₂ during the photoextensionof each LD which were suppressed during the comparable periodsof the d.i. LD treatment. The latter treatment resulted in eachphase occupying virtually identical proportions of the cellcycle as in the SD treatment. Thus, the unique cell cycle responsesto the initial part of the inductive LD treatment were increasesin the proportion of cells in G₂ coupled with G₁ and G₂ beingof similar duration. Cell cycle, mean cell generation time, shoot apex, Silene coeli-rosa     

Allocation of Organ Biomass in Perfect and Imperfect Flowers

Perianth M_P, gynoecium M_G, and androecium M_A dry-weight biomass(in g) of 39 species of perfect flowers was measured. Thesedata were pooled with published data from an additional 51 speciesand used to determine size-dependent variations in (M_G and M_A)in terms of the hypothesis that the quotient of M_G and M_A exceeds1·0 for out-breeding (xenogamous) species and less than1·0 for in-breeding (autogamous) species. Ordinary leastsquare regression of the pooled data (n = 90) showed M_G = 0·118M^0·916_P (r² = 0·884) and M_A = 0·186 M^0·975_P(r² = 0·865), indicating that the biomass of the gynoeciumproportionally decrease as floral size increases. The exponentsof these regressions indicate that the ratio of gynoecial toandroecial biomass decreased with increasing floral size suchthat comparatively small flowers (M_P < 0·0021 g) hadM_G/M_A > 1·0 (predicted for 'out-breeders') while comparativelylarger flowers (M_P > 0·0021 g) had M_G /M_A < 1·0(predicted for 'in-breeders'). Thus, on average, the type ofbreeding system was a size-dependent phenomenon. To test whether the biomass of a floral organ-type is a legitimateindicator of gender reproductive effort, the biomass (in g)of stamen filaments M_m and anther sacs M_AS of 39 species wasdetermined. Least square regression of these data showed M_AS= 0·188 M^0·854_fil (r² = 0·967), indicatingthat species with larger stamen filaments, on the average, boreproportionally smaller anther sacs and thereby cautioning againstthe uncritical use of the allocation of biomass to floral organ-typeas a strict gauge of gender-function investment. To determine whether the loss of one gender-function resultsin proportional reallocation of biomass to the remaining gender-function,the size-dependency of androecial and gynoecial biomass wasdetermined for a total of 33 perfect and imperfect flowers ofCucumis melo. Regression of the data obtained from perfect flowersyielded M_A = 0·402 M^1·47_P (r² = 0·898)and M_G = 4·63 M^1·36_P (r² = 0·842). SinceM_G/M_A M^0·11_P , the biomass allocation to the gynoeciumrelative to the androecium decreased with increasing floralsize. This result was consistent with the broad interpecificcomparison based on 90 species with perfect flowers . Regressionof the data for imperfect flowers yielded M_A = 0·151M^1·02_P (r² = 0·675) and M_G = 4·68 M^1·47_P(r² = 0·996), indicating a near allometric relation forthe androecium and a strong positive anisometry for the gynoecium.Thus, for flowers of comparable size, a loss of female genderobtains a modest to significant again in androecial biomasswhereas the loss of male gender yields only a slight increasein gynoecial biomass. Collectively, the results of these studies indicate that biomassallocation patterns are size-dependent phenomena whose complexitieshave been largely ignored in the literature.Copyright 1993,1999 Academic Press Allometry, floral biomass, reproduction     

Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

The human Na⁺-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na⁺-sulfate cotransporter (hSUT-1) and the Na⁺-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn⁵⁹¹ is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced V_max, with no change in K_m. These results suggest that Asn⁵⁹¹ and/or N-glycosylation is critical for transport activity in NaSi-1. antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence     

Effect of Radiation and Temperature on Efficiency of Cereal Leaves during Grain Growth

Grain: leaf ratio, G (the ratio of grain yield to leaf areaduration between ear emergence and maturity), in 15 experimentson wheat and barley in different seasons (Group A experiments)was highly correlated with mean daily radiation, R, mean dailytemperature, T_µ, and mean daily maximum temperature, T_max,during the grain growth period. The regression of G on R accountedfor 81 per cent of the variance of G, and introducing T_µto the regression significantly increased this to 88 per cent.The regression of G on T_max alone accounted for 87 per cent,perhaps because T_max effectively integrates radiation and temperature. When R was varied artificially by shades in two experimentson wheat in different years (Group B experiments) the relationshipbetween G and R was approximately linear in both, but the slopeof the line was less in one year, when R and temperature wereless, than in the other. For this second year, when R and temperatureswere about the middle of the ranges found in Group A experiments,the calculated relationship agrees with the Group A resultsafter correcting values of G for differences of Tu from itsvalue in the shading experiment. A formula relating G and Rderived from the results of both Group B experiments and theobserved correlation of R and temperature in the field, assumingthat the regression of G on R depends on temperature, agreeswith the relationship between G and R in the Group A experiments. It is concluded that differences in radiation and temperatureare about equally responsible for the differences in G foundbetween seasons. The positive effect of temperature on G suggeststhat factors other than leaf photosynthesis, e.g. translocationrate or capacity of the grain to accumulate carbohydrate, areimportant in determining G.     

Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate Amphidinium carteri

Cell cycle phase durations of cultures of Amphidinium carteriin light- or nitrogen-limited balanced growth were determinedusing flow cytometry. For both types of growth rate limitation,the increases in generation time caused by increasing degreesof limitation were due solely to expansion of the G₁ phase ofthe cell cycle. The durations of the S and G₂ + M phases wereindependent of growth rate. Furthermore, when cells were deprivedcompletely of light and nitrogen, they arrested in the G₁ phaseof the cell cycle. The results indicate that light- and nitrogen-dependentprocesses are heavily concentrated in the early part of thecell cycle, while DNA replication and cell division, once initiated,are independent of light or nitrogen supply.     

The Mechanisms of Apple Bud Morphogenesis: Analysis and a Model

Information in the literature suggests that the process of applebud morphogenesis is controlled by the level of growth substances,notably gibberellins in the bud, and by the availability ofgrowth substrate (S). The spur buds of apples must contain acertain minimum number of nodes (N_m) before flower primordiacan be formed, and the process of bud morphogenesis can be describedby a rate of node production with time, t by the equation It is postulated that the parameter k_G is a function of thebalance between the enzymes systems in the bud, the rate ofsynthesis of the enzymes associated with the development offloral bud parts (floral enzymes) being inversely related tothe level of gibberellins (G) in the bud. The parameter k_G isa function of the nutritional state of the bud; a is a rateparameter, and i is a conversion factor. Node production rateis asymptotic with increase in S, but switches from high tolow values as G varies about a critical concentration. The implicationsof the model, which appears to describe most observations, arediscussed, and some suggestions for testing it are put forward.     

Water Import Rate in Tomato Fruit: A Resistance Model

A model of the water import rate in tomato fruit is proposed.It compares the fruit to a hollow sphere (P) with external radiusR and internal radius R_G, corresponding to pericarp, and containingan internal spherical part (G). The pathway limiting water inputrate at any point I at a distance r from the fruit centre wasassumed to be proportional: (a) in P, to the length of the arcwhich has a radius r and which goes from I to the pedicel extension;(b) in G, to r. The water input rate at I was modelled basedon a law similar to Darcy's law which takes into account thedifference between the water potential at entry of fruit andthe water potential at point I. This latter potential was thesum of fruit osmotic potential and pressure potential due toresistance of tissue to deformation. This potential was proportionalto R-r or R_G -r. The model was expressed at fruit level by alaw such that water mass imported per unit time per unit surfacearea of fruit (f_rw) was a linear function of R. The model wascompared to linear regressions of this rate in terms of R whichhad been found during fruit swelling from published results,and which were obtained at different values of nutrient solutionsalinity. The results suggested that water input in tomato fruitis conditioned by passive forces depending on fruit size.Copyright1994, 1999 Academic Press Fruit, growth, model, resistance, salinity, size, tomato, transfer, water     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

Genotypic, Developmental and Drought-Induced Differences in Root Hydraulic Conductance of Contrasting Sugarcane Cultivars

Hydraulic properties of entire root systems and isolated rootsof three contrasting sugarcane clones were evaluated using transpiration-induceddifferences in hydrostatic pressure across intact root systems,root pressure-generated xylem sap exudation, and pressure-fluxrelationships. Regardless of the measurement technique employed,the clones were ranked in the same order on the basis of theirleaf area–specific total root system hydraulic conductance(C_root). All methods employed detected large developmental changesin G_rootroot with maximum values occurring in plants with approximately02 m₂ total leaf area. Genotypic ranking according to G_root,was reflected as a similar ranking according to root length-specifichydraulic conductance (L) of individual excised roots. Genotypicdifferences in G_root and L were consistent with anatomical characteristicsobserved in individual roots. Patterns of G_root, during soildrying and following re-irrigation suggested that the declinein G_root, observed during soil drying occurred within the rootsrather than at the soil–root interface and may have beencaused in part by xylem cavitation in the roots. Key words: Root hydraulic conductance, Saccharum spp, transpiration, root pressure, pressure-flux     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

Flow Cytometric Determination of Nuclear Replication Stage in Seed Tissues

Flow cytometric determination of DNA levels in embryos of fullymatured seeds of various plant species revealed large amountsof 2C DNA signals, indicating that most cells had arrested thecell cycle at the presynthetic G₁ phase of nuclear division.The accumulation of cells at G₁ was found both in orthodox andin recalcitrant (i.e. Castanea sativa) seed species. As recalcitrantseeds are characterized by the absence of maturation drying,the arrest of the cell cycle in the presynthetic phase may notbe linked to the seed water status. Apart from the 2C signal, 4C values were found in the embryoof some seed species (e.g. Raphanus sativus) indicating thatcells were arrested in G₂ Cells arrested in G₂ were primarilylocated in the root-tip region of the embryo. In addition, combinationsof higher C values (i.e. 8C, 12C, 16C and 64C) were observedin the endosperm of Solanum melongena and Lycopersicon esculentum,and in the root-tip cells of Phaseolus vulgaris and Spinaciaoleracea. These mixtures of polyploid nuclei (also called 'polysomaty')may arise from a developmentally controlled cellular endoreduplicationand indicates that in each cell type of the seed the amountof DNA is regulated both spatially and temporally.Copyright1993, 1999 Academic Press Endive, Cichorium endiva, lettuce, Lactuca sativa, egg-plant, Solanum melongena, pepper, Capsicum annuum, tomato, Lycopersicon esculentum, radish, Raphanus sativus, bean Phaseolus vulgaris, spinach, Spinacia oleracea, chestnut, Castanea sativa, beech, Fagus sylvatica, pine, Pinus nigra, DNA content, flow cytometry, seed, nuclear replication stage, C levels, storage     

Potassium Transport Across the Membranes of Chara: III. EFFECTS OF pH, INHIBITORS AND ILLUMINATION

Smith, J. R., Walker, N. A. and Smith, F. A. 1987. Potassiumtransport across the membranes of Chara. III. Effects of pH,inhibitors and illumination.—J. exp. Bot. 38: 778–787. The effects of several treatments, normally used to inhibitelectrogenic proton transport, upon the potassium permeability(P_k) of the membranes of Chara were examined by means of simultaneousmeasurements of the ⁴²K influx (ⁱⁿ_K) and the membrane electricalconductance (G_m). ⁱⁿ_K, P_K and G_mwere found to be substantiallyunaffected when the external pH (pH?) was varied over the range5?0 to 85. However, when pH? was increased to 11 it was foundthat, although G_m increased considerably, both P_k and ⁱⁿ_K decreasedtypically by an order of magnitude. When cells were placed intotal darkness, P_K decreased substantially only after one dayhad elapsed. For the particular experimental conditions used,the inhibitors DES, NaN₃, and La₃₊ were found to alter P_K, whereasDCCD left P_K substantially unaffected. These results suggestthat care must be taken with some common procedures used toexamine the electrical properties of the electrogenic protonpump. Key words: Potassium, pH, illumination, inhibitors