On Bacterial and ØX-174 Messenger-RNA

General methods for the chromatography of nucleic acids on benzoylated (naphthoylated) DEAE cellulose are described. This procedure results in well-resolved peaks with good recovery and seems to separate nucleic acids on the basis of their secondary structure almost independently of their molecular weight. These methods have proved to be useful in the analysis of the replicative intermediates of MS-2 RNA, ØX RFDNA, and ØX single stranded DNA, for example. Specific methods for the purification of E. coli. pulse-labeled RNA based on the chromatography on benzoylated DEAE cellulose at pH 7.5 and pH 3.5 were developed. Greater than 60% of the pulse label with less than 4% of the mass label is recovered, and the RNA size distribution in a denaturing solvent (99% dimethyl sulfoxide) after chroma­tography shows that the mass label closely parallels the pulse label as a function of size; there appears to be no selection or degradation. Pulse-chase experiments indicate that a large fraction of both the pulse and mass labels are chased out, suggesting reasonable purity of the mRNA. These data imply that there is a difference, structural or chemical, between mRNA and other known RNAs that allows all E. Coli. mRNA to act as a group regardless of size during the purification. The in vivo ØX mRNA has been studied using the above procedure for purification and analysis. The results show that the ØX mRNA size distribution at early and late times after infection is very broad ranging from a distinct maximum size of 1.7 megadaltons (one genome length of poly- cistronic mRNA), to a peak in the distribution at 0.2-0.3 megadaltons, and with significant mRNA as small as 104 daltons. The only difference observed between the early and late times after infection appears to be in the amount of RNA present: approximately 100 molecules of mRNA per cell are present at 4 min. after infection compared to 1000 molecules per cell at 25 min. after infection. Little if any difference could be found in the size distribution of mRNA made by the strongly polar OP6 ØX mutant or upon infection of the ØX replication-restrict. A variety of experimental approaches showed an absence of significant methylation and 5'triphosphate termini in the mRNA. Attempts were made to ask which RF, parental or progeny, was the template for the transcription of the ØX mRNA. One type of experiment indicated that there was a small amount of pulse labeled RNase-, phenol-, and detergent- resistant RNA specifically attached to the density labeled parental RF. However, other types of direct experiments (such as analysis of non-RNase treated RF in CsCl density equilibrium gradients) demonstrated that neither type of RF was shifted to higher densities due to attached nascent mRNA; nor could significant pulse labeled RNA be detected in the RF region of the CsCl density gradients. Thus, no unambiguous answer can be given to the template question.</p

Telomeres Cluster De Novo before the Initiation of Synapsis: A Three-dimensional Spatial Analysis of Telomere Positions before and during Meiotic Prophase

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stagedependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over

CryoSIM: super-resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultrastructural imaging

Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.Funding: Wellcome Trust (091911/Z/11/Z, 096144/Z/11/Z, 105605/Z/14/Z, 107457/Z/15/Z, 203141/Z/16/Z, 209412/Z/17/Z); H2020Marie Skłodowska-Curie Actions (700184)

Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples

Evidence of Activity-Specific, Radial Organization of Mitotic Chromosomes in Drosophila

A fluorescently labeled protein specifically binding to genes was reproducibly found at the periphery of condensed mitotic fruit fly chromosomes, illustrating preservation of a radial structure between consecutive divisions

Centrality evolution of the charged-particle pseudorapidity density over a broad pseudorapidity range in Pb-Pb collisions at root s(NN)=2.76TeV

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