Characterization of recombinant endo-1,4-β-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-β-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant K_m of 1.32 mg ml^?1 birchwoodxylan (soluble form) and maximum reaction velocity (V_max) 73.53 mmol min^?1 mg^?1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

     

Genetic analysis of the φC31 -specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo-immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl^? phenotype of J1501, a pgl mutant derivative of the Pgl^tsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl^? phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl⁺ recombinants. Crosses between strains of the same class gave no Pgl⁺ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.     

Restoration of enzyme activity by recessive missense suppressors in the fungus Coprinus.

Summary The acu-1 locus in Coprinus is the structural gene for acetyl-CoA synthetase. Five suppressor gene mutations, which suppress the acu-1,34 missense allele, were induced by mutagen treatment. All five suppressors were shown to have properties expected for tRNA structural gene mutations: they are recessive, they show a gene dosage effect in any doubly heterozygous combination of two sup ⁺ mutations and they are allele specific in action.Crosses between suppressed mutants established that at least four suppressor loci were represented. Doubly suppressed mutants derived from these crosses were used to show that the gene dosage effect is maintained when two sup ⁺ mutations are in cis as well as trans combinations in the two nuclei of the basidiomycete dikaryon.Extracts of the unsuppressed acu-1.34 mutant contained less than 2% of wild type acetyl-CoA synthetase activity whereas extracts of four of the five suppressor strains showed activities ranging from 28 to 37% of wild type. Only a slight increase in activity was detected in the fifth suppressor strain but this was associated with a temperature sensitive sup ⁺ phenotype. All five sup ⁺ mutations restored the ability of the acu-1.34 mutant to induce isocitrate lyase, an enzyme which, under the conditions of growth used, can only be induced when acetyl-CoA synthetase activity is present. Thus all five suppressors act to restore normal acu-1 protein function.     

Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum

Summary Cycloheximide-resistant mutants of Physarum polycephalum were induced in the haploid myxamoebae by the combined action of UV¹ and caffeine (Haugli and Dove, 1972) or by treatment with NMG². Eight independent mutants segregated in a Mendelian fashion (Table 1). Crosses between 6 of the mutants revealed 2 loci, actA and actB, for cycloheximide resistance (Table 2).All mutants are expressed in the plasmodium and are recessive in heterozygotes (Fig. 1 and 2). One mutation, conferring resistance to high levels of cycloheximide, was studied in heterokaryons and found to be incompletely recessive.An in vitro peptide synthesizing system was constructed from ribosomes from Physarum and supernatant factors from Saccharomyces cerevisiae. Cycloheximide strongly inhibited the activity of ribosomes derived from either wild type or mutants at the actB locus. In contrast, ribosomes from mutants at the actA locus were resistant to cycloheximide. Thus, the actA locus operates through the ribosomes.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

Instability at the y-1 locus of Chlamydomonas reinhardtii

Summary Twenty-three spontaneous yellow mutants were isolated from two stable green strains of the unicellular green alga Chlamydomonas reinhardtii. Genetic characterization indicated that 22 of 23 mutants had a mutation at the y-1 locus, and all 22 y-1 alleles were unstable. Crosses designed to follow the inheritance of instability at the y-1 locus showed that instability is caused by a single genetic factor located at the y-1 locus or very close to it.     

Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.     

Membrane lipid composition and restoration of photosynthesis during low temperature acclimation in Synechococcus sp. strain PCC 7942

We compared temperature acclimation of the cyanobacterium Synechococcus sp. strain PCC 7942 and two psbA inactivation mutants, R2K1 and R2S2C3, following shifts from 37 to 25°C. Wild-type cultures incubated in the dark at 25°C showed no chill-induction of lipid desaturation, probably because the lipid acclimation is dependent on photosynthesis. Incubation in the light at 25°C, however, induced considerable increases in membrane lipid desaturation, and within 24 h the monoenoic fatty acids increased from about 46 to about 57%. In parallel with this desaturation the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol (MGDG/DGDG) increased. Both of these lipid changes increase the repulsive forces of the hydrophobic chains of the membrane lipids and thereby alter the physical properties of the membrane. As expected, under irradiation this temperature shift also induced a reversible replacement of the constitutive photosystem II protein, D1:1, with an alternative stress form, D1:2. Photosynthesis decreased to 42% of the control level within the initial 2 h of cold incubation, but later recovered. The D1:2 protein accumulated to high levels between 2 and 4 h after the temperature shift, when desaturation of membrane lipids and MGDG/DGDG ratio had not yet increased significantly. Much of this accumulated D1:2 protein was in a higher molecular mass form, termed D1:2^*, which is probably an unprocessed precursor form of the protein. In contrast to the wild-type cells the psbA inactivation mutants, R2K1 and R2S2C3 did not accumulate any precursor form of D1 protein either at the optimal or low growth temperature. The R2S2C3 mutant strain expresses only the constitutive D1:1 protein and suffered severe photoinhibition following the temperature shift. Nevertheless, R2S2C3 eventually recovered some photosynthetic activity, induced lipid desaturation and slowly resumed growth at 25°C, thus demonstrating acclimation to the lower growth temperature. The R2K1 mutant synthesizes only the D1:2 stress form of D1 protein and maintained oxygen evolution at a high level (ca 70% of a control rate) after the low temperature shift. Chill-induced lipid desaturation and increase in MGDG/DGDG ratio did proceed but, for unknown reasons the strain did not resume growth at the lower temperature. The physical properties of the membrane lipids were not the limiting factor for growth resumption. Our results demonstrate that in the wild-type the chill-induced desaturation of membrane lipids follows after the exchange of the two forms of the D1 proteins, but the D1 exchange results in accumulation of unprocessed D1:2^* polypeptides until the lipid composition later acclimates. We also show that the lipid desaturation process in Synechococcus sp. strain PCC 7942 is dependent upon photosynthetic activity.     

Rice <Emphasis Type="Italic">phot1a</Emphasis> mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 μmol m⁻² s⁻¹), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO₂ uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H₂O₂ in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H₂O₂ accumulation, leading to a reduction in plant growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

The Modular Organisation and Stability of a Thermostable Family 10 Xylanase

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca²⁺ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.     

Thermophilic mutants of Pseudomonas fluorescens

Summary A series of heat tolerant mutants of Pseudomonas fluorescens were obtained which can grow at temperatures up to 54°C, in contrast to a maximum growth temperature of 37°C for the wild type. The minimum temperatures allowing growth of the mutant strains increased to the same extent as their maximum temperatures. Antibiotic sensitivity patterns suggested the mutants had altered ribosomes, but the purified mutant ribosomes showed no significant increase in thermostability. The virulence of the wild and mutant strains for mice correlated with their relative abilities to grow at the mouse body temperature of approximately 37°C.     

dr and spr/sr mutations of Chlamydomonas reinhardtii affecting D1 protein function and synthesis define two independent steps leading to chronic photoinhibition and confer differential fitness

The effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m^?2 s^?1). The herbicide-resistant psbA mutation Ser²⁶⁴→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m^?2 s^?1). The maximum rate of light-and CO₂-saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild-type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4-fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of F_o and a decline in F_v/F_m, quantum yield and maximum rate of CO₂-saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.     

Induction of fruiting in two aggregateless mutants of Dictyostelium discoideum

Five general groups of morphogenetically aberrant mutants of Dictyostelium discoideum were isolated. Each group of mutants was characterized either by the absence of any fruiting structures or by the formation of abnormal fructifications. Among these developmental mutants were two aggregateless isolates, Agg⁻-1 and Agg⁻-2, that could be induced to form normal sorocarps under certain conditions. Sorocarps of the normal D. discoideum type were formed when growing myxamoebae from either of these mutants were allowed to come in contact with myxamoebae of the other mutants, wild-type D. discoideum, D. purpureum, or D. mucoroides. No sorocarps were formed when myxamoebae of Agg⁻-1 and Agg⁻-2 were paired. These two aggregateless mutants, while incapable of aggregating or fruiting when cultivated singly with Escherichia coli B/r on a glucose-salts medium, formed normal fruiting structures after being freed of what appeared to be a product of bacterial growth. The spores produced by Agg⁻-1 and Agg⁻-2 myxamoebae again gave rise to aggregateless clones of the original parental types.     

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis

Objectives

This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results

Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (k_cat/K_m: 44.14 s^?1 mM^?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL.

Conclusions

Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

     

Phosphorylation influences water and ion channel function of AtPIP2;1

The phosphorylation state of two serine residues within the C-terminal domain of AtPIP2;1 (S280, S283) regulates its plasma membrane localization in response to salt and osmotic stress. Here, we investigated whether the phosphorylation state of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phosphomimic and phosphonull AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phosphomimic mutants AtPIP2;1 S280D, S283D, and S280D/S283D had significantly greater ion conductance for Na⁺ and K⁺, whereas the S280A single phosphonull mutant had greater water permeability. We observed a phosphorylation-dependent inverse relationship between AtPIP2;1 water and ion transport with a 10-fold change in both. The results revealed that phosphorylation of S280 and S283 influences the preferential facilitation of ion or water transport by AtPIP2;1. The results also hint that other regulatory sites play roles that are yet to be elucidated. Expression of the AtPIP2;1 phosphorylation mutants in Saccharomyces cerevisiae confirmed that phosphorylation influences plasma membrane localization, and revealed higher Na⁺ accumulation for S280A and S283D mutants. Collectively, the results show that phosphorylation in the C-terminal domain of AtPIP2;1 influences its subcellular localization and cation transport capacity.     

Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺ and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺ condition. The cell wall‐derived pectin homogalacturonan (GalA)₃ partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺ stress.     

Evidence for the existence of 2n gametes in Lotus tenuis Wald. et Kit. (2n=2x=12): their relevance in evolution and breeding of Lotus corniculatus L. (2n=4x=24)

Summary Crosses between male sterile L. corniculatus (2n=4x=24) and L. tenuis (2n=2x=12) plants were performed in order to verify the presence of 2n gametes in L. tenuis. All but one of the plants from these crosses had 2n=4x=24 and the L. corniculatus phenotype; this plant had 2n=2x=12 and the L. tenuis phenotype. The plants also showed good quantity of pollen at tripping, good pollen fertility and good percentage of seed setting in the backcross to L. corniculatus. On the whole, both cytological and morphological observations, showing that all but one of the plants from L. corniculatus x L. tenuis were normal tetraploids, suggest the existence of diploandrous gametes in L. tenuis. On the other hand, haploid parthenogenesis probably gave origin to the dihaploid plant 2n=2x=12.     

Isolation and characterization of five genotypic mutants of chlorate-resistant cyanobacteria unable to utilize nitrate

Spontaneous mutants of the cyanobacteriumSynechococcus PCC 7002 resistant to chlorate were isolated. Either 40mM or 400mM Na₂ClO₃ was used as the selective agent. Putative Chl^r colonies were picked onto medium containing ammonia as the sole N source, then replicaplated to media containing either NH₄ ⁺, NO₂ ^– as N sources. Of 252 putative mutants, 106 were able to use either NH₄Cl or NaNO₂ but not NaNO₃ as their sole source of nitrogen. All of the mutant isolates had generation times similar to wild-type 7002 when grown on either ammonium (3.8–4.1 h/generation) or nitrite (4.5–4.7 h/generation). None had detectable methyl viologensupported nitrate reductase activity and are thus phenotypically NRase^–. The Chl^r mutants had photomediated O₂ production and dark O₂ uptake rates similar to the wild type and responded similarly to selected metabolic inhibitors. They expressed increased levels of phycocyanin (PC) synthesis under normal, nitrogen-replete growth conditions, but rapidly lapsed into a chlorotic state upon a shift to either medium containing nitrate or to N-free medium. Genetic analysis of the Chl⁴ mutants indicated that each could be rescued by direct transformation with chromosomally derived DNA from the wild-type strain. Frequencies of transformation for the mutants were characteristic for single genetic lesions in this cyanobacterium. On the basis of marker rescue by a cosmid library of wild-type DNA, the NRase^– mutants could be grouped into five distinctive genotypic families.     

Chilling-sensitive mutants of arabidopsis

Twenty-one mutants ofArabidopsis thaliana were isolated that developed chlorosis or necrosis upon incubation at low temperature (10°C to 15°C). Crosses among mutants in different phenotypic classes showed that mutants in three of four classes were found in a small number of loci. This article is reproduced fromWeeds World, vol. 1. For electronic access toWeeds World, see PMBR 12(4):302–303.