Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization

We analyzed repeat sequences composition in the genome of cucumber inbred line 9930 using whole-genome shotgun reads. The analysis showed that satellite DNA sequences are the most dominant components in the cucumber genome. The distribution pattern of several tandem repeat sequences (Type I/II, Type III and Type IV) on cucumber chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of the Type III and 45S rDNA provide useful cytogenetic markers, whose position and fluorescence intensity allow for easy identification of all somatic metaphase chromosomes. A karyotype showing the position and fluorescence intensity of several tandem repeat sequences is constructed. The establishment of this FISH-based karyotype has created the basis for the integration of molecular, genetic and cytogenetic maps in Cucumis sativus and for the ultimate genome sequencing project as well.     

Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.     

In vitro assembly of multiple DNA fragments using successive hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.     

Single-stranded DNA binding proteins isolated from mouse brain recognize specific trinucleotide repeat sequences in vitro.

Expansion of trinucleotide repeats (CAG)n and (CGG)n is found in genes responsible for certain human hereditary neurodegenerative diseases. By gel-mobility shift assay, we detected a single-stranded (AGC)n repeat-binding activity primarily in mouse brain extracts and very low or undetectable activity in other tissue extracts. Two (AGC)n-repeat binding proteins, with apparent molecular weights of 44 and 40 kDa, have been purified from mouse adult brain by a DNA affinity column and fast protein liquid chromatography. UV-cross linking of radiolabeled (AGC)n repeats with crude brain extracts and with purified two proteins of 44 and 40 kDa produced identical doublet bands, indicating that these proteins are in fact responsible for the (AGC)n-binding activity in brain extracts. We designated these two proteins TRIP-1 for the 44 kDa protein and TRIP-2 for the 40 kDa protein, where TRIP represents trinucleotide repeat-binding protein. TRIP-1 and TRIP-2 bind to a specific subset of trinucleotide repeat sequences including (AGC)n, (AGT)n, (GGC)n, and (GGT)n repeats but not to various other trinucleotide repeats. A minimum of eight (AGC) trinucleotide repeating units is required for TRIP-1 and -2 recognition and binding. The (AGC)n repeat-binding activity increases in the brain after birth and reaches a plateau within 3 weeks. In the brain, TRIP-1 and TRIP-2 may alter the function of the genes containing the expanded-trinucleotide repeats.     

Spot blot: a hybridization assay for specific DNA sequences in multiple samples

Small aliquots of DNA-containing samples from gradient or column fractions are spotted onto agarose plates containing ethidium bromide, and the DNA is visualized qualitatively. After it is overlaid with a thin agarose cover, the DNA is denatured and transferred to nitrocellulose filters for hybridization. This method, called the "spot-blot assay," is particularly convenient when dealing with large numbers of samples.     

Detection of specific DNA sequences in yeast by colony hybridization

Summary A procedure is described for the detection of specific DNA sequences in Saccharomyces cerevisiae. This method allows a rapid screening of a large number of yeast colonies. The yeast cells of each colony, grown on nitrocellulose filters, are converted, in situ, to protoplasts by snail enzyme, and are then lysed and their DNAs are denatured and fixed on the filter. The presence of the specific DNA sequence is detected directly on the filter by hybridization with a radioactive cRNA.We have used successfully this technique to detect the presence or the absence of specific mt DNA sequences, in ⁺, ^- and ⁰ strains, and to detect the presence or the absence of the 2 m DNA sequences in different strains.     

Organisation of inverted repeat sequences in hamster cell nuclear DNA.

Hamster cell nuclear DNA is shown to contain inverted repeat (foldback) sequences, in some respects similar to the foldback fraction in DNA from other animal cell types. Using electron microscopy the majority of foldback duplexes are shown to be located in simple hairpin-like DNA structures, formed from individual pairs of complementary inverted repeated sequences 50--1000 nucleotides in length, in some cases arranged in tandem, and in other cases separated by intervening sequences, up to 16000 nucleotide residues long. In addition, a novel class of foldback structure, referred to as 'bubbled hairpins' is reported, which appear to be formed from clusters of inverted repeat sequences that are separated from adjacent clusters of complementary inverted repeats by large intervening sequences which vary in length from 5000 to over 20000 nucleotide residues. Due to the special pattern of distribution of these latter inverted repeat sequences, 'bubbled hairpins' are observed only in long foldback DNA. Evidence is presented that the distribution of foldback sequences in hamster cell DNA is highly ordered. The lengths of the intervening single chains in foldback structures appear to vary non-randomly. This gives rise to a localised periodic pattern of organisation that is believed to be a consequence of regular alternating arrangements of foldback and non-foldback sequences in the segments of DNA from which foldback structures are derived.     

Ribosomal DNA sequences detected in malaria parasites by cytochemical hybridization

A procedure in which fluorochrome-labelled RNA is hybridized in situ to homologous DNA sequences was used to investigate the possible application of this method to the human malaria parasite Plasmodium falciparum. Rhodamine-labelled ribosomal RNA stained the nuclei of the parasites after cytochemical hybridization. This result demonstrates that ribosomal RNA genes can be visualised. It was estimated that the hybridization efficiency was greater than 40%.     

Ribosomal DNA sequences detected in malaria parasites by cytochemical hybridization

A procedure in which fluorochrome-labelled RNA is hybridized in situ to homologous DNA sequences was used to investigate the possible application of this method to the human malaria parasite Plasmodium falciparum. Rhodamine-labelled ribosomal RNA stained the nuclei of the parasites after cytochemical hybridization. This result demonstrates that ribosomal RNA genes can be visualised. It was estimated that the hybridization efficiency was greater than 40%.     

Detection of CAG repeat DNA sequences by pyrene-functionalized pyrrole-imidazole polyamides

Five N-methylpyrrole-N-methylimidazole (Py-Im) polyamides possessing a fluorescent pyrene were synthesized by Fmoc solid-phase synthesis using Py/Im monomers and pyrenylbutyl-pyrrole monomer compound 9. The steady state fluorescence of conjugates 1-5 was examined in the presence and absence of (CAG)(12)-containing oligodeoxynucleotides (ODNs) 1 and 2. Of the conjugates, conjugate 1 showed no background emission around 470 nm in the absence of ODNs, and a clear increase of emission at 475 nm was observed upon addition of ODNs 1 and 2. The emission of conjugate 1 at 475 nm increased linearly with the concentration of ODN and the number of CAG repeats. The results indicate that conjugate 1 efficiently forms a pyrene excimer upon binding in the minor groove of DNA.     

Quaternion representation of RNA sequences and tertiary structures

A quaternion representation of nucleotides is proposed, with representation of RNA sequences by vectors whose elements are quaternions. Structure and transition matrices in quaternion representation are defined. Correspondence between diagrammatic technique in complex-number and quaternion representation of nucleotides is delineated.     

Sensitive measurement of unmethylated repeat DNA sequences by end-specific PCR

We describe a new method that is well-suited for the determination of the methylation level of repetitive sequences such as human Alu elements. We have applied the method to the analysis of cell and tissue DNAs and expect it to have wide utility in studies of DNA methylation in cancer and other disease states, in monitoring response to epigenetic cancer therapies and in epidemiological studies. Only 1 ng DNA is needed for a duplex, one-tube real-time PCR in which methylation level and the amount of input DNA are concurrently measured. The relative cutting by the methylation-sensitive enzyme BstUI is compared with that of the methylation-insensitive enzyme DraI to give a measure of DNA methylation. The method depends upon the use of 5'-tailed, 3'-blocked oligonucleotides called facilitator oligonucleotides (Foligos). Only cut DNAs with specific matching sequences at their 3' ends can copy the tails of the Foligos and thus become tagged and available for subsequent PCR. Both the tagging and PCR are carried out by the same enzyme, Taq DNA polymerase. Because amplification only occurs if suitable ends have been generated in the target DNA, we have called this method end-specific PCR (ESPCR). ESPCR avoids the bisulfite treatment step that is usually required to measure methylation.     

Spectroscopic comparison of different DNA structures formed by oligonucleotides

Six different nucleic acid structures including duplex, triplex and quadruplex are formed by oligonucleotides. Their structural properties are studied in detail by four spectroscopic techniques, i.e. CD, UV, NMR and fluorescence. Results are: CD Spectra: The common characteristics is a negative band at 240 nm, and the spectra are different from each other in the range 260-300 nm. Many factors such as chain direction, sugar puckering, orientation of the glycosyl bond, base stacking and sequence can effect their conformation and then show diversity and complexity in the spectra. UV Spectra: The UV spectra of all forms are quite similar, all of them exhibit a sharp positive peak around 210 nm and a broad positive band in the region of 240-280 nm. Although the bands are different in absorbance, the spectra are not characteristic enough to distinguish these forms. In addition, their thermal denaturation is also observed by UV spectrum, different melting curves and points are shown and some thermodynamic information is provided. NMR Spectra: Since the G residues in the six samples all participate in hydrogen bond, the imino proton can not exchange with the solvent freely so as to allow an observable resonance to arise. The resonance number and chemical shift will vary with the change in base-pairing number and mode as well as the whole geometry of its molecule. Fluorescence Spectra: The interaction mechanisms between EB and these structures are different. B type duplex and triplex adopt an intercalative mode in which the efficiency of energy transfer is relatively high and the fluorescence of EB can not be quenched easily. While for the parallel duplex, outside binding is predominant in which energy transfer can hardly happen and most of its fluorescence can be quenched. As for the quadruplex, groove binding is possible, so the efficiency of energy transfer is higher than that in outside binding, but lower than that in intercalative binding, and fluorescence is quenched partly.     

Distribution of inverted repeat sequences in nuclear DNA from Physarum polycephalum.

Inverted repeat sequences, capable of forming stable intra-chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7,000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as 'bubbled' hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non-foldback sequences of variable sizes up to 15,000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single-chain segments of DNA, are not random. Instead, they appear to form a regular, arithmetic series of lengths. These observations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and unform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases extend over distances up to 50,000 nucleotides in length.