A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, -naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2–1 M) of -naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 ⁺ protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of -naphthalene acetamide (between 30 and 300 M) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 ⁺ and gene 2 ^– cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.     

Expression of pAMα1 tetracycline resistance gene inCorynebacterium glutamicum: Segregation of antibiotic resistance due to intramolecular recombination

Summary AnEscherichia coli/Corynebacterium glutamicum chimeric plasmid has been constructed containing the tetracycline resistance (Tc^R) determinant from pAM1 and the kanamycin resistant (Km^R) determinant from pBD10. The paAM1 tetracycline resistance determinant is expressed inC. glutamicum although the transformed population segregates readily into tetracycline and kanamycin resistant populations. The segregation event is the result of an intramolecular recombination between the pAM1 and pUB110 regions of the chimera.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.     

Studies on the Dehydration of New 2- (Pentitol-1-YL) Pyridines Having D-Gulo,D-IDO,and L-Manno Configurations

Abstract

Fusion of 2-trimethylsilylpyridine and tetra-O-acetyl-aldehydo-D-xylose or 2,3:4,5-di-O-isopropylidene-aldehydo-L-arabinose led, after removing of the protecting groups, to 2-(pentitol-1-yl)pyridines of D-gulo and D-ido or L-manno configurations. Dehydration of the sugar-chain with D-gulo and D-ido configurations gave the corresponding 2′,5′-anhydro derivatives, whereas 2-(5-O-isopropyl-L-manno-pentitol-1-yl)-pyridine was the only compound formed by dehydration of the sugar-chain with L-manno configuration. Structural proofs are based on ¹H and ¹³C NMR spectra.     

A Simple,Preparative Procedure for N 3-Anisoyluridine and O 6-Diphenylcarbamoylguanosine 2′-O-(Tetrahydropyran-2-YL) Derivatives via The Corresponding 3′,5′-Dibenzoates

Abstract

3′,5′-Di-O-benzoyl-2′-O-(tetrahydropyran-2-yl)uridine and 3′,5′ -di-O-benzoyl-N ²-isobutyryl-2′-O-(tetrahydropyran-2-yl)guanosine are converted into-N ³-anisoyl-2′-O-(tetrahydropyran-2-yl)uridine (less and more polar diastereoisomers in 37% and 42% yields, respectively) and O ⁶-diphenyl carbamoylN ²-isobutyryl-2′-O-(tetrahydropyran-2-yl)- guanosine (less and more polar diastereoisomers in 15% and 59% yields, respectively), respectively, by N ³-anisoylation and O ⁶-diphenylcarbamoylation, followed by 3′,5′-di-O-debenzoylation.     

P1-(Thymidine 5′-)P1-amino-triphosphate: Synthesis,Hydrolysis and Reaction with Cytidine in Alkali1

Abstract

The title compound 1 is prepared from thymidine 5′-phos-phorodiamidate (2) and inorganic pyrophosphate (3) in anhydrous DMF, at 30–32°C. The products of alkaline hydrolysis of 1, at room temperature, are: thymidine 5′-phosphoramidate (4), thymidine 3′-phosphoramidate (8) and thymidine (9) as well as 3 and inorganic trimetaphosphate (10). In 1 N NH₄OH, 1 reacts with cytidine (15) to form cytidylyl-/2^T(3′)-5′/-thymidine (16) and a mixture of cytidine 2′,3′-cyclic phosphate (17) and 9.     

Synthesis of the Selenium Analog of the Cytokinin 6-(3-Methyl-2-Butenylamino)-2-Methylthio-9-(β-D-Ribofuranosyl)Purine

Abstract

6-(3-methyl-2-butenylamino)-2-methylthio-9-(β-D-ribofuranosyl)purine (1) is a naturally occurring nucleoside with potent cytokinin activity. It has been isolated and identified in the t-RNA of E. coli,^1,2 the t-RNA from wheat germ^3,4 and from Staphylococcus epidermidis.⁵ In addition, compound 1 has been found in t-RNA species corresponding to each of six amino acids whose codons start with uridine, i.e., t-RNA^Cys     

Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha citrata were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using ³²P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.Abbreviations aux 1 Tryptophan monoxygenase - aux 2 Indoleacetamide hydrolase - ipt Isopentenyl transferase - tzs Trans-Zeatin secretion locus - 2,4-D 2,4-Dichloro phenoxyacetic acid - oct Octopine - nop Nopaline - suc L,L-succinamopine - T-DNA transferred DNA     

Growth rate and methane affinity of a turbidostatic and oxystatic continuous culture ofMethylococcus capsulatus (Bath)

Summary A turbidostatic and oxystatic fermentation system was used to study the growth kinetic ofMethylococcus capsulatus (Bath). Dissolved oxygen and methane concentrations were measured continuously with membrane inlet mass spectrometry. The specific growth rate was found to increase from 0.25 h^–1 to 0.37 h^–1 and the saturation constant for methane was found to decrease from 71 M to 1.3 M as the copper content of the medium was varied from a very low to a high value.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Triaryl(Oxy)Phosphine Dibromide- A Convenient Reagent for the Preparation of S-Arylthioinosines and N 6, 5′-Disubstituted Adenosine Derivatives from Inosine

Abstract

Inosine isopropylidene 1 reacts with triphenylphosphine/phosphite dibromide and thiophenol to give 5′-bromo-S-phenylthioinosine 5 which is a versatile precursor for 5′,N ⁶-disubstituted adenosine derivatives.     

Insecticide specific emulsifier production by hexachlorocyclohexane utilizingPseudomonas tralucida Ptm+ strain

The culture supernatant ofPseudomonas tralucida (Ptm⁺ strain) produces an agent which can emulsify the insecticide hexachlorocyclohexane (HCH), which was secreted by resting cells. Emulsifying agent is most active with -HCH, and has some activity towards other chlorinated and phosphatic insecticides. The emulsifier is a heat stable macromolecule and associated with growth of the organism on -HCH.     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

Growth and differentiation of callus cultures of Pinus

Segments of hypocotyl and cotyledons of aseptically-grown seedlings of Pinus strobus L. (white pine) and P. echinata Mill (shortleaf pine) were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied on a modified Murashige and Skoog's medium containing nutrients and plant growth regulators. Meristems below the surface of callus tissue of P. strobus could be induced on media supplemented with -naphthaleneacetic acid alone or in combination with certain other plant growth regulators. Occasionally, differentiation of shoot buds also occurred on callus cultures. These shoot buds could be grown in vitro but roots did not develop.Abbreviations ABA abscisic acid - BA 6-Benzyl-aminopurine - 2-ip N⁶-(²-isopentanyl)-adenine - GD Gresshoff and Doy's medium - GE Gamborg and Eveleigh's medium - MS Modified Murashige and Skoog's medium - NAA -naphthaleneacetic acid - SC Sommer and Caldas' medium - TIBA 2,3,5-Triiodobenzoic acid     

Growth,nitrogen fixation,and mass culture of isolatedAnabaena azollae

Summary An independent strain ofAnabaena azollae was evaluated for its potential as a biofertilizer in wetland rice fields. Sustained rapid growth (doubling time=10.5 h) and nitrogenase activity (32 nmol C₂H₄ h^–1 g^–1 chl) was recorded. Mass cultivation (up to 300 litres) for the first time with this species was also achieved.     

31P-15N One-Bond Couplings of Diastereoisomeric Adenosine Cyclic 3′,5′-Phosphoramidates

Abstract

¹J(³¹P¹⁵N) coupling constants of R _p and S _p adeno-sine cyclic 3′,5′-phosphoramidates (1), -N-methylphosphor-amidates (2) and -N,N-dimethylphosphoramidates (3) increase in the order of 1<2<3 and obey the Stec rule (J(R _p)< J(S _p)). A possible interpretation of coupling constant differences based on differences in substituent electronegativities and variation in hybridization at nitrogen atom, is suggested.     

Diacetyl production by co-immobilized citrate-positiveLactococcus lactis subsp.lactis 3022 and homogenized bovin liver in alginate fibers with double gel layers

Summary Diacetyl production by (Citr⁺)Lactococcus lactis subsp.lactis 3022 cells immobilized in Ca-alginate fine fibers with single layer in the presence of catalase was three times higher than that in the absence of catalase. A co-immobilized culture system of the lactic acid bacterial cells (outer) and the homogenized bovine liver (inner layer) in Ca-alginate fibers with double gel layers was developed. The culture system gave high diacetyl productivity (30 mg/l) for ten repeated batch cultures.     

The D-xylose fermenting capacities of immobilizedPichia stipitis andPachysolen tannophilus

Summary The yeastsP. stipitis NRRL Y-7124 andP. tannophilus NRRL Y-2460 were entrapped in -carrageenan beads and used for repeated batch fermentation of D-xylose, in a series of four reactors. The operating conditions finally chosen gave an oxygen coefficient (K_La) of 0.83 min^–1, as measured by the sulphite method. Ethanol yields were 0.40 g/g forP. stipitis and 0.36 g/g forP. tannophilus (respectively 78.4% and 70.5% of the theoretical yields). In spite of its lower retention by the gel,P. stipitis exhibited greater fermenting capacities thanP. tannophilus.     

Pyrazolo[3,4-d)Pyrimidine 2′-Deoxyribo and 2′,3′-Dideoxyribo-Furanosides: Synthesis and Application to Oligonucleotide Chemistry

Abstract

The synthesis of pyrazolo[3,4-d]pyrimidine 2′-deoxyribo-nucleosides with various substituents at C-4 and C-6 (1 4) is described employing either liquid-liquid or solid-liquid phase-transfer glycosylation. From 1a (Z⁸C⁷A_d) and 2b (Z⁸C⁷G_d) the phosphoramidites 12a, b and 15a, b were synthesized. They were used in automated solid-phase synthesis resulting in the oligonucleotides 16 - 25. Deoxygenation (3′-OH) of 1a and 2b yielded pyrazolo[3,4-d]-pyrimidine 2′,3′-dideoxynucleosides isosteric to ddA, ddG, and ddI.