Comparative shoot regeneration responses of diploid brassicas and their synthetic amphidiploid products

Comparative shoot regeneration responses of three diploid Brassica species, B. campestris (AA), B. nigra (BB) and B. oleracea (CC) and their synthetic amphidiploid combinations have been investigated. The study indicates that A genome has an inhibitory effect on regeneration as evident from significantly low responses of B. juncea (AABB) and B. napus (AACC) combinations, in comparison with regeneration response of B. nigra and B. oleracea. This inhibition may arise from genomic interactions or from the B. campestris cytoplasm interacting negatively with the alien genome. Significant cytoplasmic influence on regenerability has been observed in B. carinata (BBCC) synthesised from reciprocal crosses of B. nigra and B. oleracea.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - NAA -napthaleneacetic acid - BA 6-Benzylaminopurine     

Sucrose syrup from carob pod

Summary The ripe carob pod (pericarp) is rich in water-soluble sugars, mainly sucrose (63% on total sugars). Sucrose crystallization from aqueous carob extract is prevented by its reducing sugar content. The selective consumption of these sugars by mixed culture ofRhizopus oligosporus andSaccharomyces rouxii gives a sucrose syrup suitable for several uses e.g. sucrose extraction.     

Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L) by using elicitors and abiotic stress

Summary A cell line of carrot (Daucus carota L) which produces anthocyanin was subjected to various elicitors and abiotic stresses: The elicitors tested were culture filtrates (CF) and cell extracts (CE) of certain bacteria and yeasts. The abiotic stresses were salts of certain metal ions. The production increase obtained with cell extracts of Bacillus cereus. Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were 49, 72, 45 and 41% respectively over the control. Maximum elicitation was obtained with elicitor derived from cell extract of the yeast Rhodotorula rubra where it enhanced anthocyanin production by two fold. The abiotic stress agents Ca, Mn, Zn, Co, Fe & V enhanced anthocyanin production. Of all the metal ions tested Ca was the most effective. The elicitation process was governed by the type and level of elicitor.     

<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

Background  

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).     

Species specific shoot regeneration response of cotyledonary explants of Brassicas

A study of shoot regeneration from cotyledons of three basic diploid species of Brassica, B. campestris (AA), B. nigra (BB), B. oleracea (CC) and their amphidiploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC) showed species-specific responses for in vitro shoot regeneration. Analysis of the species mean shoot regeneration response over a range of growth regulator combinations revealed that i) B. campestris is the lowest regenerating species, ii) B. nigra and B. oleracea regenerate with high frequencies, iii) In amphidiploids, the presence of B. campestris component brings down shoot regeneration frequency below the value of B. oleracea in B. napus combination and is additive of the combining genomes in B. juncea combination. In B. carinata regeneration frequencies are less than the parental diploid species, iv) Significant intraspecific genotypic differences were observed for B. nigra and B. oleracea among diploids and B. juncea and B. carinata among amphidiploids, when cotyledons of eighteen genotypes were tested in one growth regulator combination.Abbreviations MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - IAA Indole 3-acetic acid - BA 6-Benzyl aminopurine     

Plant regeneration from protoplasts of Dimorphotheca and Rudbeckia

Protoplasts were isolated from leaves, shoots, cotyledons, ray florets and callus cultures of Dimorphotheca aurantiaca (syn. D. sinuata) (Cape Marigold, Star of the Veldt) and Rudbeckia hirta, R. laciniata and R. purpurea; species of ornamental value. For Dimorphotheca, plants were regenerated from protoplasts of all sources apart from the ray floret, whilst for the Rudbeckia species, although protoplast division was induced in most cases, only leaf mesophyll protoplasts of R. hirta c.v. Marmalade gave plants. The establishment of plant regeneration for these ornamental species, from protoplasts, now provides a basis for their somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - K kinetin - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - f.wt. fresh weight     

Building Better Barrels – β-barrel Biogenesis and Insertion in Bacteria and Mitochondria

β-barrel proteins are folded and inserted into outer membranes by multi-subunit protein complexes that are conserved across different types of outer membranes. In Gram-negative bacteria this complex is the barrel-assembly machinery (BAM), in mitochondria it is the sorting and assembly machinery (SAM) complex, and in chloroplasts it is the outer envelope protein Oep80. Mitochondrial β-barrel precursor proteins are translocated from the cytoplasm to the intermembrane space by the translocase of the outer membrane (TOM) complex, and stabilized by molecular chaperones before interaction with the assembly machinery. Outer membrane bacterial BamA interacts with four periplasmic accessory proteins, whereas mitochondrial Sam50 interacts with two cytoplasmic accessory proteins. Despite these major architectural differences between BAM and SAM complexes, their core proteins, BamA and Sam50, seem to function the same way. Based on the new SAM complex structures, we propose that the mitochondrial β-barrel folding mechanism follows the budding model with barrel-switching aiding in the release of new barrels. We also built a new molecular model for Tom22 interacting with Sam37 to identify regions that could mediate TOM-SAM supercomplex formation.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Summary Three strains ofPichia stipitis and three ofCandida shehatae were compared withPachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Biotransformations by microbial Baeyer-Villiger monooxygenases stereoselective lactone formationin vitro by coupled enzyme systems

Summary The regio- and stereoselective biotransformation of bicyclo (3. 2. 0) hept-2-en-6-one by the NADH-dependent Baeyer-Villiger monooxygenase from camphorgrownPseudomonas putida NCIMB 10007 has been shown to yield a chiral lactone not accessible by curently-used biocatalysts. The biotransformation can be conductedin vitro using two alternative coupled enzyme systems (alcohol dehydrogenase and monooxygenase: formate dehydrogenase and monooxygenase) within situ recycling of NAD⁺/NADH.     

On the steric course of the microbial generation of (Z6)-gamma-dodecenolactone from (10R,S) 10-hydroxy-octadeca-(E8, Z12)-dienoic acid

Summary (Z6)-gamma-dodecenolactone samples produced endogenously inFusarium poae and biogenerated from (10R, S) 10-hydroxy-octadeca-(E8, Z12)-dienoic acid inCladosporium suaveolens show (R) and (S) configurations, respectively.     

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.     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

Extraction of bovine serum albumin with reverse micelles from glucosylammonium and lactosylammonium surfactants

Reverse micelle extraction is still in the stage of laboratory. Major limitation associated with use of synthetic surfactants in reverse micelle extraction process is the unfolding or denaturation of proteins. Sugar surfactants are thought non-toxic and environmentally benign, and can exhibit interesting interfacial properties, but the application of sugar-based surfactants in protein extraction is still limited. In the present study, we extracted bovine serum albumin (BSA) by using reverse micelles from glucosylammonium (GA) and lactosylammonium (LA) surfactants (with dicarboxylate as counter ion). It was found that under optimum condition, (1) the maximum forward extraction efficiency was ca. 86% with GA, while only around 50% with LA, and (2) almost all BSA solubilized in reverse micelles prepared from GA could be recovered into aqueous phase, while the recovery of BSA from the reverse micelles of LA was lower. In addition, the optimum extraction parameters were closely related to surfactant structure. Therefore, the electrostatic interaction, H-bonding and sugar head size should be important for BSA transfer.     

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.     

Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides

Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.Abbreviations MS Murashige and Skoog medium (15) - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Removal of NH3-N from domestic waste water by fungi

Summary Nine species of fungi viz.,Aspergillus niger,A. flavus,A. terreus,Fusarium solani,Mucor sp.,Neurospora crassa,Penicillium janthinellum,Trichoderma harzianum andTrichothecium roseum were evaluated for their potential to remove NH₃–N from domestic waste water. Of the fungi tested,A. flavus was found to be the most effective in the removal of NH₃–N. Maximum reduction (92%) of NH₃–N by this organism was observed at pH 8.0 at 20°C.     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Transformation of the coryneform bacterium cellulomonas flavigena with plasmid DNA via electroporation

Summary Using electroporation we have transformed Cellulomonas flavigena with a shuttle vector (pJA85) derived from the E. coli plasmid pUC8 and the Brevibacterium lactofermentum plasmid pULRS8. Upon transformation this plasmid was found to be stable, not to undergo detectable deletion, and to express antibiotic resistance markers originating in Brevibacterium.     

Improvement of restriction endonuclease cleavage at the termini of PCR products. Application to the Human Papillomavirus type 16 E7 gene

Summary A protocol is described for the evaluation of the cleavage efficiency of the Human Papillomavirus (HPV) type 16 E7 oncogene synthetic ends by restriction endonucleasesBamHI andNdeI. which constitutes a quick and easy mean of optimizing subsequent ligation reactions. The syntheticBamHI site is located at two base pairs from one end, whereas theNdeI site is located four base pairs from the second E7 synthetic gene end.