Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.     

Purification and characterization of β-Mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity

Marine bacterium Reinekea sp. KIT-YO10 was isolated from the seashore of Kanazawa Port in Japan as a seaweed-degrading bacterium. Homology between KIT-YO10 16S rDNA and the 16S rDNA of Reinekea blandensis and Reinekea marinisedimentorum was 96.4 and 95.4%, respectively. Endo-1,4-β-D-mannanase (β-mannanase, EC 3.2.1.78) from Reinekea sp. KIT-YO10 was purified 29.4-fold to a 21% yield using anion exchange chromatography. The purified enzyme had a molecular mass of 44.3?kDa, as estimated by SDS-PAGE. Furthermore, the purified enzyme displayed high specificity for konjac glucomannan, with no secondary agarase and arginase activity detected. Hydrolysis of konjac glucomannan and locust bean gum yielded oligosaccharides, compatible with an endo mode of substrate depolymerization. The purified enzyme possessed transglycosylation activity when mannooligosaccharides (mannotriose or mannotetraose) were used as substrates. Optimal pH and temperature were determined to be 8.0 and 70?°C, respectively. It showed thermostability at temperatures from 20 to 50?°C and alkaline stability up to pH 10.0. The current enzyme was thermostable and thermophile compared to the β-mannanase of other marine bacteria.     

Spectral Changes of the γ-Irradiated Glucose Solution

β-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp. XY-214 by ammonium sulfate precipitation and successive chromatographies. The enzyme had a pl of 3.6 and a molecular mass of 52 kDa. The enzyme had the highest level of activity at pH 7.0 and 37°C. The enzyme activity was completely inhibited by Cu²⁺, Hg²⁺, and N-bromosuccinimide. The enzyme hydrolyzed β-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-β-D-xyloside, β-1,4-xylan, β-1,3-glucan, or carboxymethyl cellulose.     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Penicillium sp. 23 alpha-galactosidase: purification and substrate specificity

α-Galactosidase, a glycoprotein with carbohydrate and protein in ratio 1:6, has been isolated from liquid culture of micromycete Penicillium sp. 23 and purified to homogeneous state by ammonium sulphate precipitation followed by ion exchange and gel-filtration chromatography on TSK-gels. The Penicillium sp. 23 α-galactosidase specificity against a series of natural and synthetic substrates has been studied. The enzyme was found to exhibit strict specificity towards the glycon and hydrolyze exclusively α- -galactosides such as p-nitrophenyl-α- -galactopyranoside (p-NPhGal), melibiose, raffinose and stachyose. The configuration at C1 and C4 atoms of substrate as well as substitution at C2 and C6 of substrate made an important contribution to the interaction with the enzyme. The tested α-galactosidase exerted the highest affinity (K_m) with respect to the synthetic substrate p-NPhGal and maximal rate of hydrolysis (V_max), about 10 times higher, comparing with natural substrates (melibiose, raffinose and stachiose). The Penicillium sp. 23 α-galactosidase possesses wide specificity towards α-galactosidase hydrolysis link type, splitting off at varying rates the terminal galactose from disaccharides, attached by α-1,2-, α-1,3- and α-1,6-links. The enzyme is ineffective towards disaccharides with α-1,4-link. The enzyme showed potential to splitting off α-1,3-bound terminal galactose residues from antigens of the human blood group B(III) erythrocytes.     

d-Xylose Isomerase from a Marine Bacterium, Vibrio sp. Strain XY-214, and d-Xylulose Production from β-1,3-Xylan

The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding d-xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K _m values for d-xylose and d-glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out d-xylulose production from β-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of β-1,3-xylanase (TxyA) and β-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of β-1,3-xylan to d-xylose. d-Xylose was then converted to d-xylulose by using XylA from the strain XY-214. The conversion rate of d-xylose to d-xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for d-xylulose production from β-1,3-xylan. Because d-xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from β-1,3-xylan.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins

Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.

Τhe β-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes β-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V₁₀₉VTS₁₁₂ as playing a key role in the enzyme reaction.     

Cloning and characterization of a modular GH5 β-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13

The gene (1272-bp) encoding a β-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant β-1,4-mannanase (rManH) was approximately 44.0 kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. β-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50 °C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498 IU mg⁻¹ towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.     

Cloning, Sequencing, and Expression in Escherichia coli of the New Gene Encoding β-1,3-Xylanase from a Marine Bacterium, Vibrio sp. Strain XY-214

The Vibrio sp. strain XY-214 β-1,3-xylanase gene cloned in Escherichia coli DH5α consisted of an open reading frame of 1,383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed β-1,3-xylan to produce several xylooligosaccharides.     

Examination of the Conditions for Protoplast Isolation from Tobacco Cells Cultured in vitro

Protoplasts were isolated from cultured tobacco cells by removing the cell wall enzymatically. We examined the time courses of treatment with some different concentrations of enzymes (cellulase and macerozyme) and with some different kinds and concentrations of sugars (sucrose, sorbitol and mannitol) which produce good conditions for protoplast isolation. The best conditions for protoplast preparation from tobacco cells cultured in vitro were: use of actively growing cells and isolation of protoplasts with 5% cellulase and 0.1% macerozyme in 0.5% sorbitol.     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Optimization of protoplast yields from the red algae <Emphasis Type="Italic">Gracilaria dura</Emphasis> (C. Agardh) J. Agardh and G. <Emphasis Type="Italic">verrucosa</Emphasis> (Huds.) Papenfuss

This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7 ± 0.7 × 10⁶ cells g⁻¹ fresh wt for G. dura and 1.2 ± 0.78 × 10⁶ cells g⁻¹ fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region, were also observed in G. verrucosa.     

A Simple Determination Method for Herbicides,NIP and CNP in Soil Using Single Ion Monitoring Mass Spectrometry

A purified extracellular endo β-1,3-xylanase (EC 3.2.1.32) from an isolated strain, Aspergillus terreus A-07, was found to hydrolyze 1,3-xylosyl linkages only. When rhodymenan (β-1,4 and β-1.3-linked xylan) was hydrolyzed by β-1,3-xylanase (EF-6), four β-1,4-linked xylooligosaccharide fractions were produced. The main product was β-1,4-xylotriose, with trace amounts of other β-1,4-linked xylooligosaccharides. Successive degradation by β-l,4-xylosidase of the β,4-xylooligosaccharides that were produced from hydrolysis of β-1,3-xylanase on rhodymenan yielded only xylose as the final product.

We compared the action pattern of this enzyme with that of an extracellular endo β-l,4-xylanase (EC 3.2.1.8) of Streptomyces. From a mixture of products of β-1,4-xylanase hydrolysis on rhodymenan, an isomeric xylotriose was isolated by charcoal chromatography after treating with β-1.4-xylosidase. The structure of this isomeric xylotriose was elucidated by methylation analysis and its susceptibility to β-1,4-xylanase, β-1,3-xylanase, and β-1,4-xylosidase. The obtained isomeric xylotriose was identified as 3-O-β-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose (X1→3X1→4X). It has a melting point of 224~225°C and ${\left[\alpha \right]}_{\text{D}}^{20}$(c = 1, H₂O)= —46°.     

三倍体‘银中杨’叶肉原生质体制备的优化

以三倍体杨树品种‘银中杨’（Populus alba×P.berolinensis Yinzhong）无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明：酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BAS,酶解时间为8 h,原生质体产量和活力分别为2.13×10⁷个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高（1.06×10⁷个/g）,可满足进一步的原生质体培养等技术的要求。     

STORAGE AND STRUCTURAL PRODUCTS FORMED DURING PHOTOSYNTHESIS IN THE SIPHONOUS ALGA CAULERPA SIMPLICIUSCULA(CHLOROPHYCEAE)1

The distribution of radioactivity into storage and intermediary metabolites during photosynthesis over periods of up to three hours was followed in the siphonous green alga Caulerpa simpliciuscula C. Ag. After the first hour, almost all the carbon assimilated was recovered as insoluble 1,4-α-glucan. There was rapid movement of carbon into insoluble 1,3-β-glucans during the first 20 min of photosynthesis, but little additional carbon moved into these compounds after that time. This contrasted with the movement of carbon into the soluble 1,3-β-glucan fraction, which continued for 2 h. Sucrose accumulated very little ¹⁴C during the entire period of photosynthesis. There was a very slow transfer of ¹⁴C into lipid throughout the period but the rate was linear. Protein labelling showed a lag of 2 h before it reached the same rate of ¹⁴C accumulation as shown by the lipid initially. It is suggested that the distribution of radioactivity can be explained in part by proposing that the tissue is in the latter stages of recovery from the wounding which look place at the time of dividing the thallus into sections.