Structural analysis of water-soluble fractions obtained fromAspergillus fumigatus mycelium

Fractions were prepared from the water-soluble components ofAspergillus fumigatus mycelium either by lectin-affinity chromatography or salt precipitation. While they varied considerably in their amino-acid composition, each contained a preponderance of aspartic and glutamic acids.¹³C-NMR spectroscopy of these fractions, compared with that of polysaccharide obtained by alkaline extraction, indicated the presence of glycoproteins, the polysaccharide components of which contained -d-Galf units that are part of structures chemically different from those obtained by alkali treatment. In two of the three fractions examined, gas-liquid chromatography-mass spectrometry showed marked differences in the contents of non-reducing end-units of -d-Manp and -d-Galf. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the preparations revealed an array of components, which stained to differing extents with silver stain and with Coomassie Blue and many of which were bound by lectins with specificity for different sugars.     

Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica

Summary Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated YlARS1 and YlARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728–1003 by for YlARS1 and 1377–1629 by for YlARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both YlARS elements contained at, or close to, their boundaries a 13 bp sequence, 5-TATATTCAAGCAA-3, which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 by ClaI fragment of YlARS2 contained four stretches of a 17 bp direct repeat sequence, 5-GAAAAACAAAAACAGGC-3, and exhibited the electrophoretic behavior typical of bent DNA.     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

Statistical optimization of a thermostable and neutral glucoamylase production by a thermophilic mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in solid-state fermentation

Glucoamylase production by a thermophilic mold Thermomucor indicae-seudaticae was optimized in solid-state fermentation (SSF) by conventional one variable at a time approach and further statistically using response surface methodology (RSM). Glucoamylase secretion was strongly affected by three variables (moisture ratio, inoculum level and incubation time), and therefore, these three factors were further optimized using response surface methodology. The glucoamylase production in flasks containing wheat bran, under the conditions optimized by RSM, was 455 ± 23 U/g of dry moldy bran (DMB), while the predicted value by a polynomial model was 433.30 U/g DMB. The enzyme titre (455 ± 23 U/g DMB) attained in the validation experiment of this investigation is higher than those reported in the literature. When the large-scale production was attempted in enamel trays, a marginally lower enzyme titres were attained. An overall 1.8-fold increase in glucoamylase production was achieved in SSF due to statistical optimization in comparison with that of one variable at a time approach (250 ± 13 U/g DMB). A 10-fold enhancement in glucoamylase production was recorded in SSF as compared to that in submerged fermentation.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa

Four recently described species, Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200–890 g/g), and F. napiforme (16–388 g/g), and by F. nygamai not obtained from millet in Africa (15–874 g/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300–18200g/g) and the 15 isolates from rice with Bakanae disease (2300–19300 g/g). The taxonomic position of these two uncertain taxa should be re-evaluated.     

Seed flavonoids of thePodalyrieae andLiparieae (Fabaceae)

A study of the phenolic compounds of the closely related papilionoid tribes,Podalyrieae andLiparieae, proved that the flavonoid patterns of hydrolysed seed extracts are remarkably conservative. Butin (7, 3, 4-trihydroxyflavanone), 3-hydroxydaidzein (7, 3, 4-trihydroxyisoflavone), vicenin-2 (6, 8-di--D-glucopyranosyl-5, 7, 4-trihydroxyflavone) and orobol (5, 7, 3, 4-tetrahydroxyisoflavone) were isolated and identified as the major flavonoids. The seeds ofAmphithalea, Coelidium, Liparia, Xiphotheca, Calpurnia, Stirtonanthus andPodalyria accumulated three isoflavone O-glycosides that yielded 3-hydroxydaidzein on hydrolysis. In contrast,Virgilia contained a unique combination of vicenin-2 and orobol. Vicenin-2 was also present inCalpurnia as a major compound, butStirtonanthus insignis was the only other species studied that contained orobol (in trace amounts only). Butein, a chalcone, was reported byHarborne from the seed ofCyclopia subternata. This compound's flavanone analog, butin, was the principal component inCyclopia. A cladistic analysis, using flavonoid, alkaloid and morphological data, showed that the seed flavonoids of thePodalyrieae andLiparieae behave rather poorly as cladistic characters. They are, however, of considerable taxonomic value at the tribal level favouring the opinion that the two tribes should be combined. The apparent absence of flavonoids in the seed ofHypocalyptus supports the suggestion that it should be excluded from theLiparieae. Flavonoids also show that theArgyrolobium-group is very different from the tribeCrotalarieae and support the recent transfer of this group to the tribeGenisteae.     

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-¹⁴C] and [2-¹⁴C] acetate to ¹⁴CO₂ at relatively high rates. All strains tested were N,N,N,N-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.Non-standard abbreviations BSS basal salts solution - BH Beggiatoa heterotrophic medium - BSO Beggiatoa sulfide oxidation medium - TMPD N,N,N,N-tetramethylphenylenediamine - Q8 ubiquinone-8     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Summary We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus' of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3 to 5 exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.Abbreviations HPUra p-hydroxyphenyl azouracil - nt nucleotide - PCR polymerase chain reaction     

Relationship between cyclic AMP level and accumulation of carotenoid pigments in Neurospora crassa

Dark grown mycelial cells of Neurospora crassa bearing mutant genes crisp-I or frost and having a decreased level of cyclic adenosine 3,5-monophosphate contained more carotenoid pigments than the cells with wild alleles of these genes. A transient decrease of the cyclic AMP occurred following photoinduction of carotenoid synthesis during its lag-period. Its intensity correlated with the increase of carotenoid pigment level due to photoinduction. No correlation in the content of cyclic guanosine 5-phosphate with both constitutive level of carotenoids and its photoinduced increase was observed.     

Phenotypic Features of Ferroplasma acidiphilum Strains YT and Y-2

Earlier, we described a new family of mesophilic, strictly autotrophic Fe²⁺-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and the species F. acidiphilum (strain Y^T) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain Y and a new strain, F. acidiphilum Y-2, isolated from dense pulps during oxidation of gold-containing arsenopyrite/pyrite concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk krai) ore deposits, respectively. The G+C content of DNA from strains Y^T and Y-2 comprised 35.1 and 35.2 mol %, respectively; the level of DNA–DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous iron and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. The strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains Y^T and Y-2 were 34–36 and 40–42°, respectively. Comparative analysis of the total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of the total lipids: lipid I, -D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2 : 1. Thus, it was established that the intraspecies phylogenetic divergence within F. acidiphilum is manifested in the two strains by different temperature optima against a background of similarity in other phenotypic properties.     

Activation studies by phospholipids on the purified cytochromec 4:o oxidase ofAzotobacter vinelandii

A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochromec ₄:o oxidase fromAzotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochromec ₄ (3.6 nmol/mg protein) and cytochromeo (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at moderate rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2–4-fold stimulation in TMPD oxidase activity occurred. Other authentic micellar phospholipids also readily activited TMPD oxidase activity in Fraction V. Themaximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.Dedicated to the memory of David E. Green, a fine gentleman, an excellent scientist, and a true scholar. He will be missed by many of his former colleagues.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Observations on the cell-wall compositions of the bracket fungi Laetiporus sulphureus and Piptoporus betulinus

Homogenized tissues and their alkali-soluble and alkali-insoluble fractions of fruiting bodies of the basidiomycetes Laetiporus sulphureus and Piptoporus betulinus were investigated using X-ray diffraction, infrared spectrometry and chemical methods. The presence of (13)--d-glucan, (13)--d-glucan and chitin was established. The relative amounts of these polysaccharides were different in the two species and differences were also found between context and trama. The proportion of (13)--d-glucan was exceptionally high in the context of L. sulphureus (about 78%). In addition, the trama of both species contained a substance resembling a cyclic wax by its X-ray pattern and solubility properties. The substances identified are considered to belong to the hyphal wall