Chromosomal polymorphisms involving telomere fusion,centromeric inactivation and centromere shift in the ant Myrmecia (pilosula) n=1

Detailed karyological surveys of the ant Myrmecia pilosula species group, which is characterized by the lowest chromosome number in higher organisms (2n=2), were attempted. We revealed that this species has developed highly complicated chromosomal polymorphisms. Their chromosome numbers are in the range 2n=2, 3, and 4, and six polymorphic chromosomes are involved, i.e., two for chromosome 1 (denoted as SM₁ and ST₁), three for chromosome 2 (A₂, A₂, and M₂), and M₍₁₊₂₎ for the 2n=2 karyotype. We suggested that these chromosomes were induced from a pseudo-acrocentric (A ₁ ^M ) and A₂ as follows: (1) A ₁ ^M SM₁ or ST₁ by two independent pericentric inversions; (2) A₂A₂M₂ by chromosomal gap insertion and centromere shift; and (3) ST₁+A₂M₍₁₊₂₎ by telomere fusion, where (3) means that the 2n=2 karyotype was derived secondarily from a 2n=4 karyotype. It is a noteworthy finding that active nucleolus organizer (NOR) sites, in terms of silver staining, are tightly linked with the centromere in this species, and that both the centromere and NOR of A₂ were inactivated after the telomere fusion.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Determination of an ethylene biosynthesis pathway in the unicellular green alga,Haematococcus pluvialis. Relationship between growth and ethylene production

In the freshwater ChlorophyceaeHaematococcus pluvialis, precursors of ethylene biosynthesis cycle are the same as those of higher plants: L-methionine S-adenosylmethionine 1-aminocyclopropane-1-carboxylic acid ethylene. However, the enzymatic complex of the last step of ethylene synthesis-ACCoxidase-differs from that of higher plants. It is stimulated by Co²⁺ (at least 10^-5 M), Mn²⁺ (at least 10^-6 M) and Ag²⁺ (at least 10^-4 M), inhibited by Cu²⁺ (at least 10^-5 M) and not affected by Zn²⁺, Fe²⁺ or Mg²⁺. ACCoxidase is also inhibited by salicylhydroxamic acid and by dark. Ethylene production is more important in young, mobile, green cells in active growth phase than in old, encysted and red cells in stationary growth phase. No peaks in ethylene production or respiration were observed during batch culture, as opposed to the situation with climacteric fruits.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

Pyrimidine dimers as pre-mutational lesions in Escherichia coli WP2 Hcr

Summary Mutation to prototrophy in E. coli WP2 Hcr^- induced by far-UV radiation (F-UV) in an intermediate dose range follows dose-squared kinetics. In a comparable dose-range with near-UV radiation in the presence of acetophenone (N-UV+Acph) mutation induction follows kinetics which are linearly related to dose. The difference in response to the two types of irradiation is a more general one in that it is the same for true revertants, for suppressor mutants, and for several markers.Double-irradiation experiments together with treatment by photoreactivating light (PR) after the first irradiation (i.e. F-UVPRF-UV; N-UV+AcphPRN-UV+Acph; N-UV+AcphF-UV; N-UV+AcphPRF-UV) seem to indicate the following: a) the dose-squared kinetics for F-UV are due to the necessary co-operation of at least two types of pre-mutational lesions, only one of which is photoreversible; b) N-UV+Acph also produces these photoreversible lesions in addition to such photoreversible ones which do not require the co-operation of other types; the production of the former is not indicated by the appearance of visible mutants because the non-photoreversible type, whose co-operation is required to give rise to such mutants, is not produced.     

Specific Status of Propithecus spp.

Controversial taxonomic relationships within Propithecus have consistently made conservation and management decisions difficult. We present a multidisciplinary phylogenetic analysis of Propithecus supporting the elevation of 4 subspecies to specific status: P. diadema perrieri P. perrieri, P. diadema candidus P. candidus, P. diadema edwardsi P. edwardsi, and P. verreauxi coquereliP. coquereli; leaving P. diadema diadema as P. diadema and P. verreauxi verreauxi as P. verreauxi.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline     

Effect of microbial growth on pore entrance size distribution in sandstone cores

Summary The in situ growth of microorganisms in Berea sandstone cores preferentially plugged the larger pore entrances. The largest pore entrance sizes after microbial plugging ranged from 20 to 38 m, compared with 59 to 69 m before plugging. The pore entrance size distribution of plugged cores was shifted to smaller sizes. A mathematical model based on Poiseuille's equation was found to adequately predict permeability reductions (greater than 90%) caused by microbial growth in the large pore entries.Nomenclature Q volumetric flow rate (L ₃/t) - C orifice constant (dimensionless) - A cross-sectional area (L ₂) - g gravity (L/t ²) - h pieziometric head (L) - _s transmittivity (L ²) - R _e Reynolds number (dimensionless) - a constant (dimensionless) - density (M/L ₃) - viscosity (M/Lt) - d diameter (L) - L length (L) - P pressure change (M/L ₂)     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Foam behavior of biological media

Summary The surface tension and foaminess of (a) unlimited, (b) substrate limited, and (c) oxygen transfer limited growth media of Hansenula polymorpha were measured using methanol, ethanol or glucose as a substrate.The time dependence of can be described by the Avrami-Überreiter relationship: log (2.3 log V)=n log t+log b, where V = (_O – _eq/(_t – _eq, and _O, _t and _eq are at t_M=0, t_M=t and t_M (equilibrium value).The constants n and b are functions of the fermentation time t_F as long as the growth is unlimited but they are constant in the state of limited growth. With glucose substrate, the foaminess can be presented as a definite function of the time, t_DG, which is necessary to attain _eq. With alcohol as a substrate no definite (t_DG) function was found.Symbols b constant in Eq. (1) - n constant in Eq. (1) - S substrate concentration - T temperature - t_M time h (measured from the beginning of the determination of the surface tension ) - t_F cultivation time h (measured from the time of inoculation) - t_DG time (min) necessary to attain the equilibrium surface tension ) - X dry biomass concentration (gl^–1) - V (_O – _eq)/(_t – _eq) - V_S equilibrium volume of the foam (cm³) - V_G volumetric gas flow rate during the estimation of (cm³ s^–1) - vvm volumetric gas flow rate with regard to the volume of the medium (min^–1) - w_SG superficial gas velocity (cm s^–1) - _m maximum specific growth rate (h^–1) - V_S/V_G foaminess (s) - surface tension, mMm^–1 (milli Newton m^–1) - _O at t_M=0 - _eq equilibrium surface tension ( at t_M) - _t at t_M=t - HP probes from Hansenula polymorpha cultivation - NLG non limited growth - OTLG oxygen transfer limited growth - SLG substrate limited growth     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.     

Anaerobic degradation of cresols by denitrifying bacteria

The initial reactions in anaerobic metablism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o-or p-cresol as sole organic substrate with a generation time of 11 h; o-or p-cresol was completely oxidized to CO₂ with nitrate being reduced to N₂. A denitrifying Pseudomonas-like strain oxidized m-or p-cresol as the sole organic growth substrate completely to CO₂ with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps:(1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol 4-OH-benzaldehyde 4-OH-benzoate 4-OH-benzoly-CoA benzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating).(2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH₃-benzoyl-CoA as central intermediate as follows: o-cresol 4-OH-3-CH₃-benzoate 4-OH-3-CH₃-benzoyl-CoA 3-CH₃-benzoyl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between ¹⁴CO₂ and the carboxyl of 4-OH-3-CH₃-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH₃-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH₃-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH₃-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH₃-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH₃-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH₃-benzoate and further to 3-CH₃-benzoyl-CoA.(3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4-and 3,4-dimethylphenol were oxidized to 4-OH-3-CH₃-and 4-OH-2-CH₃-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.The hydroxyl and methyl groups are abbreviated as OH-and CH₃-, respectively     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Triplet state EPR of reaction centers from the HisL173→LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His^L173Leu^L173 substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu^L173 mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu^L173 mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His^M200Leu^M200 mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin - EPR electron paramagnetic resonance - LDAO lauryl-dimethylamine N-oxide     

A pyrophosphate bridge links the pyruvate-containing secondary cell wall polymer of Paenibacillus alvei CCM 2051 to muramic acid

The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed:[(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(13)]_ñ11-(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(1O)-PO₂-O-PO₂-(O6)-MurNAc-Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. ³¹P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex.In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.