DNA sequencing

Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.     

An augmented ribbon model of protein structure

In this paper, work on a new model,an augmented ribbon model, for describing a number of features of primary, secondary, and super-secondary protein structure in a qualitative but mathematically rigorous way is discussed. The structural features that can be treated by the model include connectivity, directionality, chirality, orientation, and proximity which, in many cases, are difficult to deal with using more traditional structural representations (e.g. wireframe, ball-and-stick, or space-filling CPK). In practice, the information encoded in the augmented ribbon model is represented by a labelled, directed graph (digraph) which provides an efficient means for storing and analyzing the information on computers. This opens the way to computer-based analyses of proteins and for the application of similarity methods that have been shown to be quite useful in treating small molecules.     

X-ray, Raman and FTIRS studies of the microstructural evolution of zirconia particles caused by the thermal treatment

Genesis of the structure of zirconia particles prepared by precipitation of amorphous hydrated zirconia by ammonia from the ZrO(NO₃)₂ solution followed by a mild hydrothermal treatment (HTT) of precipitate, washing and calcination under air up to 1000 °C has been studied by X-ray diffraction (XRD), Raman and FTIRS. As revealed by FTIRS of lattice modes, the local structure of amorphous zirconia subjected to HTT is close to that in m-ZrO₂. This helps to obtain nearly single-phase monoclinic nanozirconia (particle size 5-15 nm) already after a mild calcination at 500 °C. Stability of this phase with nanoparticles sizes below the critical value determined by thermodynamic constraints is due to its excessive hydroxylation demonstrated by FTIRS. Dehydroxilation and sintering of these nanoparticles at higher (600-650 °C) temperatures of calcination leads to reappearance of the (111) “cubic” reflection in XRD patterns. Modeling of XRD patterns revealed that this phenomenon could be explained by polysynthetic (001) twinning earlier observed by HRTEM.     

Structure and Stereochemistry of New Sesquiterpene Esters from Echinacea purpurea (L.) MOENCH. (Preliminary communication)

The structure elucidation of four new constituents from the roots of Echinacea purpurea is described. They are shown to be cinnamoyl esters of sesquiterpene alcohols with a germacrane or a guaiane skeleton. First pharmacological results indicate immunological activities.     

A new paradigm for cationic cyclization of iron tricarbonyl diene complexes with pendant alkenes and arenes

A new example of stereospecific cationic cyclization of iron tricarbonyl diene complexes with pendant alkenes and arenes is provided. Protonation of a double bond vicinal to the iron tricarbonyl diene moiety is employed to trigger the cyclization, rather than the previously reported Lewis/protic acid dehydroxylation of diastereomeric alcohols, eliminating one step of separation and avoiding some reactivity problems previously encountered for one of the alcohol diastereoisomers. [reaction: see text]     

Poly[(2,2'-bipyridine)tetrakis(imidazolato)diiron(II)]: structural and spin-state phase transitions and low-temperature magnetic ordering in a unique 2-dimensional material

Poly[(2,2'-bipyridine)tetrakis(imidazolato)diiron(II)] was synthesized by the reaction of ferrocene with imidazole in an excess of 2,2'- bipyridine in a Carius tube at 130 degrees C. Dc magnetic susceptibility studies at an applied field of 1000 G reveal that on cooling from room temperature, the material undergoes two structural phase transitions designated alpha --> beta and beta --> gamma at 151 and 133 K, respectively. On warming, the gamma --> beta and beta --> alpha transitions are observed at 137 and 151 K, respectively; a 4 K thermal hysteresis clearly detectable in the lower temperature beta <--> gamma transition. These structural phase transitions have also been studied by detailed, variable-temperature, ac susceptibility and M?ssbauer spectroscopy techniques. Single-crystal X-ray diffraction studies done at 294, 143, and 113 K reveal 2, 12, and 6 unique iron centers in the alpha-, beta-, and gamma-forms, respectively. All three forms have the same basic structure involving 2D extended double layer sheets (bilayers) of alternating tetrahedral and octahedral irons singly bridged by imidazolate ligands, with the octahedral centers additionally coordinated by 2,2'-bipyridine ligands that occupy positions between the sheets. Magnetic susceptibility and bond length data reveal that in the gamma-phase one in three six-coordinate sites corresponds to spin singlet ground-state iron(II); i.e., the overall alpha --> gamma process involves a spin-crossover transition such that at least 1/6 of the iron sites in the gamma-phase correspond to S = 0. This is supported by the low-temperature M?ssbauer spectra of the gamma-phase, which reveal the simultaneous presence of both S = 2 and S = 0 iron(II) centers. The compound magnetically orders, with a resultant small remnant magnetization, at low temperatures. The magnetic phase transition, studied by a combination of dc and ac susceptibility and M?ssbauer techniques, occurs at T(c) approximately 6.5 K.     

Hyperpolarized 129Xe NMR investigation of multifunctional organic/inorganic hybrid mesoporous silica materials

An extensive study has been made on a series of multifunctional mesoporous silica materials, prepared by introducing two different organoalkoxysilanes, namely 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS) and 3-cyanopropyltriethoxysilane (CPTES) during the base-catalyzed condensation of tetraethoxysilane (TEOS), using the variable-temperature (VT) hyperpolarized (HP) 129Xe NMR technique. VT HP-129Xe NMR chemical shift measurements of adsorbed xenon revealed that surface properties as well as functionality of these AEP/CP-functionalized microparticles (MP) could be controlled by varying the AEPTMS/CPTES ratio in the starting solution during synthesis. Additional chemical shift contribution due to Xe-moiety interactions was observed for monofunctional AEP-MP and CP-MP as well as for bifunctional AEP/CP-MP samples. In particular, unlike CP-MP that has a shorter organic backbone on the silica surface, the amino groups in the AEP chain tends to interact with the silanol groups on the silica surface causing backbone bending and hence formation of secondary pores in AEP-MP, as indicated by additional shoulder peak at lower field in the room-temperature 129Xe NMR spectrum. The exchange processes of xenon in different adsorption regions were also verified by 2D EXSY HP-129Xe NMR spectroscopy. It is also found that subsequent removal of functional moieties by calcination treatment tends to result in a more severe surface roughness on the pore walls in bifunctional samples compared to monofunctional ones. The effect of hydrophobicity/hydrophilicity of the organoalkoxysilanes on the formation, pore structure and surface property of these functionalized mesoporous silica materials are also discussed.     

Diels-Alder reaction of optically active (E)-γ-keto-α,β-unsaturated p-tolylsulfoxides with cyclopentadiene

The Diels-Alder reaction of enantiomerically pure (E)-γ-keto-α,β-unsaturated p-tolylsulphoxides 3 with cyclopentadiene give four easily separable diastereomers. The effect of several Lewis acids on the reaction was studied, finding a high endo selectivity with respect to the carbonyl group and moderate π-diastereoselectivity using BF₃·Et₂O as catalyst. The reactivity of compounds 3 as well as their endo selectivity are both higher than those observed for the corresponding (E)-3-sulfinylacrylates.     

Generation of new landomycins by combinatorial biosynthetic manipulation of the LndGT4 gene of the landomycin E cluster in S. globisporus

A 3 kb DNA fragment from the Streptomyces globisporus 1912 landomycin E (LaE) biosynthetic gene cluster (lnd) was completely sequenced. Three open reading frames were identified, lndGT4, lndZ4, and lndZ5, whose probable translation products resemble a glycosyltransferase, a reductase, and a hydroxylase, respectively. Studies of generated mutants from disruption and complementation experiments involving the lndGT4 gene allowed us to determine that LndGT4 controls the terminal L-rhodinose sugar attachment during LaE biosynthesis and that LndZ4/LndZ5 are responsible for the unique C11-hydroxylation of the landomycins. Generation of the novel landomycins F, G, and H in the course of these studies provided evidence for the flexibility of lnd glycosyltransferases toward their acceptor substrates and a basis for initial structure-activity relationships within the landomycin family of antibiotics.