A new cell-to-cell transport model for Potexviruses

In the last five years, we have gained significant insight into the role of the Potexvirus proteins in virus movement and RNA silencing. Potexviruses require three movement proteins, named triple gene block (TGB)p1, TGBp2, and TGBp3, and the viral coat protein (CP) to facilitate viral cell-to-cell and vascular transport. TGBp1 is a multifunctional protein that has RNA helicase activity, promotes translation of viral RNAs, increases plasmodesmal size exclusion limits, and suppresses RNA silencing. TGBp2 and TGBp3 are membrane-binding proteins. CP is required for genome encapsidation and forms ribonucleoprotein complexes along with TGBp1 and viral RNA. This review considers the functions of the TGB proteins, how they interact with each other and CP, and how silencing suppression might be linked to viral transport. A new model of the mechanism for Potexvirus transport is proposed.     

Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.     

Sonication: A new method for gene transfer to plants

Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed.     

A new method for straightening DNA molecules for optical restriction mapping.

We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.     

A new computational method for the detection of horizontal gene transfer events

In recent years, the increase in the amounts of available genomic data has made it easier to appreciate the extent by which organisms increase their genetic diversity through horizontally transferred genetic material. Such transfers have the potential to give rise to extremely dynamic genomes where a significant proportion of their coding DNA has been contributed by external sources. Because of the impact of these horizontal transfers on the ecological and pathogenic character of the recipient organisms, methods are continuously sought that are able to computationally determine which of the genes of a given genome are products of transfer events. In this paper, we introduce and discuss a novel computational method for identifying horizontal transfers that relies on a gene's nucleotide composition and obviates the need for knowledge of codon boundaries. In addition to being applicable to individual genes, the method can be easily extended to the case of clusters of horizontally transferred genes. With the help of an extensive and carefully designed set of experiments on 123 archaeal and bacterial genomes, we demonstrate that the new method exhibits significant improvement in sensitivity when compared to previously published approaches. In fact, it achieves an average relative improvement across genomes of between 11 and 41% compared to the Codon Adaptation Index method in distinguishing native from foreign genes. Our method's horizontal gene transfer predictions for 123 microbial genomes are available online at http://cbcsrv.watson.ibm.com/HGT/.     

A new method for the automated age-at-death evaluation by tooth-cementum annulation (TCA)

A valid age at death estimation is required in historical and also forensic anthropology. Tooth cementum annulation (TCA) is a method for age at death estimation of adult individuals. The method is based on light microscope images taken from tooth-root cross sections. The age is then estimated by manually counting the cementum incremental lines and adding this to the chronological age at the assumed point of tooth eruption. Manual line counting, however, is time consuming, potentially subjective and the number of individual counts is insufficient for statistical evaluations. Software developed for the automated evaluation of TCA images, that uses Fourier analysis and algorithms for image analysis and pattern recognition is presented here. It involves "line-by-line" scanning and the counting of gray scale peaks within a selected region-of-interest (ROI). Each scanning process of a particular ROI yields up to 400 counts that are subsequently statistically evaluated. This simple and time saving program seeks to substitute manual counting and supply consistent and reproducible results as well as reduce the demand of human error by eliminating unavoidable factors such as subjectivity and fatigue.     

A new type of semi-empirical molecular orbital method for large molecules.

A new type of molecular orbital method is proposed. It is applicable to large molecules containing large conjugated substructures. Only π-electrons in the conjugated part, but all-valence electrons in the non-conjugated part of a molecule, are taken into account explicitly. The Fock matrix elements are evaluated from the semi-empirical values employed in the existing all-valence-electron methods. The examples presented here suggest that the new type of MO method predicts electronic structures which are quite similar to those obtained by complete semi-empirical MO calculations. This new method may make it possible to reasonably well describe the electronic structure of, and interaction between, large molecules using considerably less computation time and core storage than the complete calculation analogs.     

A new method for karyological studies in teleost fishes

A simple method employing CoCl₂, is described which has application in the rapid preparation of fish chromosomes for research and teaching. The benefits of this method include low initial skill level, relatively inexpensive equipment and supplies, and the production of a high number of metaphase spreads.     

A general method for the direct isolation of the 3'-terminal fragments derived from transfer RNA molecules

A general procedure for the isolation of 3′-linked fragments derived from tRNA molecules is described. Purified N-2-naphthoxyacetylglycyl derivatives of the $\text{tRNA}{}_1{}^{\mathrm{Gly}}$ and $\text{tRNA}{}_2{}^{\mathrm{Gly}}$ of yeast were exhaustively digested with RNase T₁ and the 3′-linked fragments (bearing the derivative) were separated from other degradation products (lacking the derivative) by stepwise chromatography on BD-cellulose. Subsequent chromatographic resolution and base-composition analysis allowed tentative identification of the 3′-terminals of $\text{tRNA}{}_1{}^{\mathrm{Gly}}$ and $\text{tRNA}{}_2{}^{\mathrm{Gly}}$ as Gp(Cp,Ap)CpCpA and Gp(Cp,Cp,Up,Ap)CpCpA, respectively. The potential utility of this procedure for development of a novel approach to nucleic acid sequence analysis is discussed.     

A new automated method of image analysis: comparison of ciliary and flagellar beats

A new automated method of image analysis of sperm flagellar (human) and cilia (Dunaliella) bends is developed. This method permits an automatic determination of the line characterizing the flagellum. Two dynamic parameters are measured: the wave propagation velocity and the wave curvature radius. The data reveal similar patterns in the propagation of the principal and reverse waves between flagelated and ciliated cells. Conversely, differences are seen in principal wave curvature due perhaps to the presence of periaxonemal structures in the flagellum, absent in cilium. The identical patterns of reverse wave curvaturei in both systems may be linked to axonemal limitations.     

Kinetic multichannel spectroscopy of biological molecules: decomposition of the spectral matrix

The time-resolved difference spectra after flash excitation of various biological molecules are measured on a gated optical multichannel analyzer. Electron transfer between the photoactive covalent label thiouredopyrene-trisulfonate and the heme of cytochrome c and the photocycle of the E204Q mutant bacteriorhodopsin are studied. The spectral matrices containing consecutive difference spectra are analyzed to reveal the reaction kinetics and individual component spectra. Singular value decomposition combined with stoichiometric analysis and self-modeling is demonstrated as a tool for successful dissection of the matrix, despite the difficulties arising from broad, featureless, overlapping spectra and the overlapping kinetics of the components.     

Fluorescence resonance energy transfer studies of U-shaped DNA molecules

Fluorescence resonance energy transfer studies allow to determine global shape properties of nucleic acids and nucleoprotein complexes. In many DNA-protein complexes, the DNA is more or less bent and the degree of bending can be obtained by FRET. For example, the DNA in complex with the integration host factor (IHF) is kinked by approximately 160 degrees building a U-shaped structure. The two DNA helix ends come close to one another in space in a distance range easily measurable by FRET. The global DNA structure of this complex can be mimicked by introducing two regions with unpaired bases ('bulges') into the DNA each producing a sharp kink of approximately 80 degrees. These U-shaped DNA constructs were used to measure the electrostatic interaction of the two nearly parallel negatively charged DNA helix arms. The electrostatic repulsion between the helix arms, and as a consequence their distance, decreases with growing salt concentration of mono- or divalent cations. This experimental approach also allows the sensitive study of the local structure of DNA sequences positioned between the two bulges.     

A new method for detecting sites of 2'-O-methylation in RNA molecules.

2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.