Fluorescent Vital Stains for Complementary Labelling of Protoplasts from Trichoderma Spp

In this study several fluorescent vital stains were evaluated for their ability to provide complementary vital staining of protoplasts of Trichoderma spp. for selection of heterokaryons following protoplast fusion. Tetramethyl rhodamine isothiocyanate and fluorescein isothiocyanate were rejected because they stained only a small proportion of protoplasts. Fluorescein diacetate stained all protoplasts, but the chromophore leaked rapidly from stained cells. A mixture of FluoroBora T and acriflavine stained all cells, but intensity was low and fading upon illumination was rapid. Nile red stained lipid bodies in all cells, but the stain was lost upon protoplast fusion in polyethylene glycol. Rhodamine 6G, on the other hand, stained all cells, fluoresced green, and was stable through fusion and upon illumination. Hydroethidine also stained all protoplasts, and staining was relatively stable through fusion and upon illumination. Hydroethidine fluoresced red and stained nuclei more prominently than the cytoplasm. Rhodamine 6G and hydroethidine were tested on a number of strains to determine whether they were toxic to protoplasts. No toxicity to any strain was noted with rhodamine 6G. Hydroethidine, however, was toxic at the higher concentrations tested, especially when stained protoplasts were exposed to light. When protoplasts were stained with the minimum concentration giving ready visualization and were incubated in darkness, hydroethidine also was nontoxic. Hydroethidine and rhodamine 6G are useful complementary vital stains of Trichoderma protoplasts for visualization of frequency and type (dicell, multicell) of fusion.     

Rapid Chemical Synthesis of Oligodeoxyribonucleotides by the Improved Phosphotriester Method

Abstract

An efficient phosphotriester methodology based on the use of condensing agents in the presence of several new nucleophilic catalysts has been developed.     

The Incorporation of 2,6-Diaminopurine Into Oligodeoxyribonucleotides by the Phosphoramidite Method

Abstract

A method has been developed to allow the synthesis of oligodeoxyribonucleotides containing 2,6-diaminopurine and avoiding depurination.     

定量PCR的荧光技术

荧光定量PCR是在普通PCR基础上,利用荧光技术对核酸进行绝对定量的一项新兴技术,其灵敏度高、特异性高、操作简便和定量准确,已被广泛应用于临床和科研中。为更好地发挥荧光定量PCR的优点,荧光技术领域的研发工作十分活跃。     

Synthesis of Oligodeoxyribonucleotides Containing 2,6-Diaminopurine

Abstract

The preparation of a new protected derivative of 2,6-diaminopurine 2′-deoxyriboside carrying two phenoxyacetyl groups is described. The new derivative is useful to prepare oligonucleotides containing 2,6-diaminopurine and it is deprotected at the same time as the standard protecting groups of the natural bases.     

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.     

Automated Synthesis of Cyclic Oligodeoxyribonucleotides via Phosphoramidite Method

Abstract

The functionalized polyethylene glycol/polystyrene copolymer support 4 was shown to be suitable for a completely automated synthesis of small- to medium-sized cyclic oligodeoxy-ribonucleotides. Syntheses of the linear precursors were achieved by the phosphoramidite method, whereas the cyclization reactions were based on the phosphotriester chemistry.     

Reaction Fields in the Environment of Fluorescent Probes: Polarity Profiles in Membranes

Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains.     

Quantitative Parametrs of Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template

Abstract

The cooperative interactions of oligonucleotides on the complementary template were studied using the quantitative analysis of the template alkylation with the oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl group at 5′-end. The influence of the mismatched nucleotides and the stabilizing N-(2-hydroxyethyl)phenazinium group at the 5′- and 3′-ends of the oligonucleotides on the parameters of cooperativity was evaluated.     

Diastereomer Separation of Azobenzene-Tethered Oligodeoxyribonucleotides and Determination of Their Absolute Configurations by Enzymatic Digestion

Two diastereomers were produced by the introduction of azobenzene-tethering prochiral linker (2,2-bis(hydroxymethyl)propionic acid) in the modified ODN, which had been used for the photoregulation of DNA functions. We found that this modified ODN with sequence 5′-…pNpXpN…-3′ (p = phosphate; N = nucleoside; X = azobenzene residue) could be digested to pX (the phosphate at the 5′ side of X was left) by an over excess of Phosphodiesterase I. By comparing the retention time of pX from the separated diastereomer with that of authentic R- or S-pX on chiral HPLC, absolute configuration could be easily determined.     

A Review: Synthesis of Aryl C-Glycosides Via the Heck Coupling Reaction

In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.     

Coupling of Pressure-Induced Structural Shifts to Spectral Changes in a Yellow Fluorescent Protein

X-ray diffraction analysis of pressure-induced structural changes in the Aequorea yellow fluorescent protein Citrine reveals the structural basis for the continuous fluorescence peak shift from yellow to green that is observed on pressurization. This fluorescence peak shift is caused by a reorientation of the two elements of the Citrine chromophore. This study describes the structural linkages in Citrine that are responsible for the local reorientation of the chromophore. The deformation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the β-barrel scaffold of the molecule, resulting in a slight bending of the β-barrel. The high-pressure structures also show a perturbation of the hydrogen bonding network that stabilizes the excited state of the Citrine chromophore. The perturbation of this network is implicated in the reduction of fluorescence intensity of Citrine. The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the β-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Specific Labelling of Virus Proteins by Quantitative Reaction of Cysteine Residues with <Superscript>35</Superscript>S-Sulphite

POLYPEPTIDES from different sources can be compared conveniently by digesting them with proteolytic enzymes and fingerprinting the resulting smaller peptides. If peptides with identical electrophoretic and chromatographic properties are obtained, the implication is very strong that the sequences of the original polypeptides were, at least in part, the same. The need for such comparisons arises in studies of in vitro polypeptides synthesized in coupled systems directed by viral DNAs. The material synthesized in vitro must be compared with authentic virus-coded material to verify that the system is transcribing DNA to RNA and translating RNA to protein with fidelity. For viruses such as SV40 and polyoma, which can be grown in tissue culture, the virus particles grown in the presence of ³⁵S-methionine are a convenient source of virus-coded proteins. Proteolytic digests of these particles can be compared with digests of ³⁵S-methionine labelled material synthesized in vitro. Preliminary results have shown that, in the case of polyoma virus, matching peptides are obtained from virus particles and polypeptides synthesized in vitro¹.     

Synthesis and Characterization of Oligodeoxyribonucleotides Containing Tandem Base Damage

Abstract

Both N(2-deoxy-β-D-erythro-pentofuranosyl)-formylamine (dβF) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) were introduced in synthetic oligonucleotides at a vicinal position via the solid phase phosphoramidite method in order to investigate the biological and structural significance of such a tandem lesion. Further experiments aimed at determining the enzymatic repair by both E. coli endonuclease III (Endo III) and Fapy-glycosylase (Fpg) were carried out with these synthetic substrates.