Identification of messenger RNA for human type II collagen

The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured. The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis). It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values. A new model is proposed.     

Internal motion of DNA in bacteriophages

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.     

Viral DNA packaging studied by fluorescence correlation spectroscopy

The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Il genere Allium L. in Italia. IV. Studio citofotometrico sul granulo pollinico di Allium Chamaemoly L.

Abstract

The genus Allium L. in Italy. IV. A DNA cytophotometric study on the pollen grain of Allium chamaemoly L. — A cytophotometric analysis of DNA contents in pollen generative and vegetative nuclei of Allium chamaemoly L. was carried out. DNA synthesis in both nuclei was confirmed and a lightly higher DNA amount than 2C in the vegetative nucleus was pointed out. An analysis of the Fast-green stainable histones in the generative and vegetative nuclei was also accomplished. While the generative nucleus had a very high content of Fast-green stainable histone, the vegetative one have nearly no stainable histone. The occurrence of DNA synthesis and the very low histone content suggest the vegetative nucleus is functional and biochemically activ. The higher than 2C DNA content supports the possibility of a DNA amplification process including probably the amplification of ribosomal cistrons in the pollen vegetative nucleus.     

Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.     

Encapsidation and transfer of phage DNA into host cells: from in vivo to single particles studies

A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.     

Structure of virions of the M13 phage containing chimeric B-protein molecules]

We have studied the virion structure of M13 strains (M13B1, M13BOM1, M13BOM2, M13BOL1) with chimeric variants of B-protein. Data concerning the spatial structure of chimeric B-protein molecules and their interaction with intraphage DNA were obtained. The phage contour lengths were measured under electron microscope and the DNA/protein ratios were obtained by spectrophotometry. These data testified that the insertion of foreign peptide affected neither DNA packaging nor the compactness of molecular arrangement of proteins in the virion. By linear dichroism and fluorescence spectra of phages it was determined, that the insert can influence the polarity of amino acid environment and the orientation of amino acids in the B-protein central part. It was shown by quenching of phage fluorescence by KI that the inward or outward amino acids location in the capsid is invariable. The carboxyl residues have been titrated in the phage strains by Auramine O. It was shown that there is no correlation between the number of the titrated carboxyl groups and the number of the carboxyl groups as a whole.     

Flow cytometric cell cycle analysis using the quenching of 33258 Hoechst fluorescence by bromodeoxyuridine incorporation.

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, The cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G1, S and G2 + M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Screening and Identification of a Specific Binding Peptide to Ovarian Cancer Cells from a Phage-Displayed Peptide Library

To select specific binding peptides for imaging and detection of human ovarian cancer. The phage 12-mer peptide library was used to select specific phage clones to ovarian cancer cells. After four rounds of biopanning, the binding specificity of randomly selected phage clones to ovarian cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). DNA sequencing and homology analysis were performed on specifically bound phages. The binding ability of the selected peptides to SKOV3 cells was confirmed by fluorescence microscopy and flow cytometry. After four rounds of optimized biological panning, phage recovery was 34-fold higher than that of the first round, and the specific phage clones bound to SKOV3 cells were significantly enriched. A total of 32 positive phage clones were preliminarily identified by ELISA from 54 randomly selected clones, and the positive rate was 59.3%. S36 was identified as the clone with best affinity to SKOV3 cells via fluorescence microscopy and flow cytometry. A representative clone of OSP2, S36 is expected to be an effective probe for diagnosis and treatment of ovarian cancer.

     

RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA

Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO.     

Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.     

A single-step competitive binding assay for mapping of single DNA molecules

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.     

Fluorescence changes of a membrane-bound dye during bacteriophage T5 infection of Escherichia coli.

The fluorescence intensity of membrane-bound N-phenyl-1-naphthylamine increases dramatically when T5 bacteriophage infect colicin Ib plasmid-containing hosts. This dramatic increase is not seen during normal infections or in infections wherein either the plasmid or the phage contain mutations which allow productive infection to occur. Two smaller increases in fluorescence intensity are seen, however, in all T5 infections in which the characteristic two-step injection of DNA can proceed.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Incorporation, into the DNA, chain of a fluorescent derivative of 2-deoxyuridylic acid during catalysis of synthesis by DNA-polymerase A

A 2'-deoxyuridine 5'-triphosphate analogue with a dansyl (5-dimethylaminonaphtalene 1-sulphonyl) residue in the 5-position of uracyl has been synthesised. This compound substitutes dTTP in the DNA synthesis catalyzed by Klenow's fragment of E. coli DNA polymerase I on the M13mp10 phage DNA as template with synthetic 14-member primer. When the synthesis is terminated by four termination substrates, structure of the synthesised DNA chain can be read. It demonstrates in principle possibility of determination of DNA sequence by means of fluorescence.