An investigation into the 19-hydroxylation of androstenedione,cortexolone and progesterone by selected fungi

Summary An investigation was performed to recognise a fungus capable of 19-hydroxylating the steroids androstenedione, cortexolone and progesterone, in high yield with little or no side reactions. The fungi selected for study belong to a group which has been previously reported to possess 19-hydroxylation ability. Pellicularia filamentasa f. sp. microsclerotia IFO 6298 and P. filamentosa f.sp. sasakii IFO 5254 both 19-hydroxylated cortexolone, but 19-hydroxylated products were not detected using either androstenedione or progesterone as substrate. When 19-hydroxylating cortexolone, both strains of P. filamentosa produced 11-hydroxycortexolone as a by-product, but P. filamentosa f.sp. microsclerotia IFO 6298 was superior in terms of 19-hydroxylation and the relative amounts of the two products.     

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.     

Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis,in cell cultures and plants of Digitalis lanata EHRH

Summary The in vitro transformation of pregnenolone into progesterone in Digitalis lanata tissues was shown to be catalyzed by a 3-hydroxysteroid dehydrogenase/ketosteroid isomerase (3-HSD). Product formation was monitored by HPLC. The enzyme could be partially characterized and 3-HSD activities were measured in various Digitalis lanata tissues and in cell cultures of other plant species. Since no correlation was observed between biosynthetic competence of the tissue and 3-HSD activity, it was concluded that this enzyme does not play a major role in regulating cardenolide biosynthesis.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - DMF N,N-dimethylformamide - IAA indole-3-acetic acid     

Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.     

Conjugal transfer of Streptococcal antibiotic resistance plasmids intoClostridium acetobutylicum

Summary Streptococcal plasmids pAM1, pVA797, pVA797Tn917 and pAM610 were transferred or mobilized toClostridium acetobútylicum fromStreptococcus sanguis,S. faecalis andS. lactis donors.Clostridum transconjugants were able to retransfer pAM1 and pVA797 to a plasmid-freeS. lactis recipient.     

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes

Hairy root cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.     

Fates and expression ofBacillus subtilis endo-β-1, 4-glucanase gene andAlcaligenes faecalis β-glucosidase gene introduced inEscherichia coli andBacillus cells

Summary Two kinds of cellulase genes coding for endo--1, 4-glucanase and -glucosidase, isolated fromBacillus subtilis andAlcaligenes faecalis respectively, were separately or combinedly put on a newly constructedEscherichia coli-Bacillus shuttle vector plasmid. When the recombinant plasmids having cellulase gene(s) were introduced intoE. coli orBacillus cells, drastic differences in fates and expression of the two genes were observed.     

Synthesis of novel sugars,oligoglucosyl-inositols,and their growth stimulating effect forBifidobacterium

Summary Novel sugars, oligoglucosyl-inositols, which were synthesized using CGTase fromBacillus ohbensis, stimulated the growth ofBifidobacterium. The enzyme catalyzed transglucosylation from -1,4-maltodextrin (donor) tomyo-inositol (acceptor). Of donors examined, -cyclodextrin gave superior oligoglucosyl-inositol yield of 56.6% (w/w) based on the conversion ratio of incubated inositol. Maltosyl-inositol stimulated growth ofB. adolescentis by 194% when compared with glucose.     

Conversion ofN-acetyl-o-toluidine into 4′-hydroxy-N-acetyl-o-toluidine byFusarium verticillioides

Summary Approximately 850 fungus and yeast strains were tested for their ability to hydroxylateN-acetyl-o-toluidine in the 4-position. The strain Y-1 selected as the best producer, was identified asFusarium verticilliides, and accumulated 1.5 mg of 4-hydroxy-N-acetyl-o-toluidine per ml of culture broth.     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Towards a classification ofE. coli ribosomal proteins: A hypothetical ‘small ribosome’ as a primitive protein-synthesizing apparatus

Homologies were searched among the published primary sequences of 51E. coli ribosomal proteins, partly by eye and partly by computer-assisted methods. By employing Moore and Goodman's alignment statistics for evaluating homology levels, 33 out of these 51 ribosomal proteins has been classified into 9 homology groups, some of which being yet tentative and remaining to be further analyzed. Taking it into consideration that most ribosomal protein genes are clustered atstr-stc region,rif region and several other regions, these results strongly suggest that most or all of the contemporary ribosomal proteins must have evolved by repeated gene duplications ofvery few (oronly one) primitive ancestral ribosomal protein gene(s). Thus it is most reasonable to propose that a small ribosome consisting of very few (or only one) ribosomal protein(s) must have existed as a primitive protein-synthesizing apparatus.     

Production and localization of cellulases and β-glucosidase from the thermophilic fungusThielavia terrestris

Summary The enzyme production and localization ofThielavia terrestris strains C464 and NRRL 8126 were compared to determine their optimum temperature and pH for cellulase activity. High levels of intracellular -glucosidase activity were detected in the former strain. The intracellular -glucosidase of both strains were more thermostable than the extracellular enzyme; the half life ofT.terrestris (C464) endoglucanase activity at 60°C was greater than 96 hrs.     

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata Crandon, NC-5339 and P. nigra Betulifolia × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula Erecta) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - NC-XXXX North Central Forest Experiment Station accession number assigned to hybrid poplar clones.     

Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Production of active human interferon-β inE. coli II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid

Summary The maximum level of human interferon- activity was expressed under the control of theE. coli tryptophan promoter whenE. coli cells were induced at late logarithmic growth phase by 3,-indoleacrylic acid (IAA). The level is one order of magnitude higher than that obtained when the cells were induced at early logarithmic or stationary phase. When IAA was subsequently further added, the decrease in the activity observed at a latter period of fermentation was suppressed.     

Glucosylation of methyl β-D-arabinofuranoside with 6′-chloro-6′-deoxysucrose and immobilized Protaminobacter rubrum

Summary Transglucosylation byProtaminobacter rubrum using 6-chloro-6-deoxysucrose (1) and methyl -D-arabinofuranoside (2) as donor and acceptor, respectively, were examined. inhibition caused by 6-chloro-6-deoxy-D-fructose (4) was observed and could be greatly lightened in a borate buffer, where the yield of the disaccharide (3) increased by 1.35-fold.     

Studies of dotted,a regulatory element in maize

Summary Dotted (Dt) is the regulatory element of a two-unit controlling system in maize. Dt causes the inherited change from the recessive a (colorless) to its dominant allele, A (anthocyanin production), during the development of the stalk, leaves, and endosperm. The mutation events are observed as sectors of color in an anthocyaninless background.Since its discovery over 40 years ago, Dt has always been found in the terminal knob of the short arm of chromosome 9. This is puzzling because controlling and regulatory elements in general are not located permanently, but change positions (transpose) within the chromosomal complement. To resolve this seeming discrepancy, transpositions were looked for in a homozygous a Dt stock. Because the frequency of aleurone mutations is exponentially related to Dt dosage, a Dt transposition would result in a greatly increased number of dots if the egg or sperm nucleus contained both the transposed Dt and the Dt remaining on chromosome 9. A total of 6 transposed Dt's (Dt-T) were recovered in this manner. Dt-TA was found linked to the gene Y (yellow endosperm) of chromosome 6. Dt-TB no longer showed linkage with yg2 of chromosome 9, but remains unlocated (the original Dt in this stock is separated from yg2 by 6 or 7 cross-over units.). The remaining transpositions (C-F) assorted independently of Dt on chromosome 9.The transposed Dt's had the same effect as Dt on the frequency and timing of aleurone mutations. An increase in transposition frequency and losses of Dt-T's was characteristic of several of the transposed Dt's. Dt-T's B-F transposed so frequently that testcross ratios of 71 (three Dt' s) and 15 1 (four Dt' s) were observed. No secondary transpositions or losses of Dt-TA were detected. Thus, Dt-TA resembles the original Dt with regard to its transposition frequency and stability.Journal Paper No. J-8333 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1880.     

Isolation and immobilization ofSclerotium rolfsii protoplasts

Summary Protoplasts fromSclerotium rolfsii were prepared usingTrichoderma harzianum lytic enzymes and immobilized in Ca alginate gels. The immobilized protoplasts when incubated with 1% carboxymethylcellulose in osmotically stabilized induction medium, could secrete endoglucanase and -glucosidase. On repeated use the immobilized preparation retained 36% endoglucanase and 26% -glucosidase activity after 5 cycles.NCL Communication No. 3798     

Agrobacterium-mediated transformation and regeneration of kiwi fruit

Summary Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25g/ml kanamycin and 500g/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.     

Regulation of mycotoxin production by Fusarium graminearum: Complementation effects between two mutant types

Summary Starting from anileu auxotroph ofFusarium graminearum producing high levels of the mycotoxin zearalenone, selection after UV irradiation gave low-producing mutants of essentially normal morphology,zea,ileu. Heterokaryons betweenzea,ileu strains and an auxotrophic strainlz,inos derived from the lazy morphological mutant ofFusarium graminearum, which has abnormal morphology and also produces little or n zearalenone, produced significant levels (over 50% of the wild-type level) of mycotoxin. The observation confirms views as to the regulatory nature of thelazy mutation.