Electronmicroscopy of Genetic Activity

Hamkalo, Barbara A.; Miller, O. L.

doi:10.1146/annurev.bi.42.070173.002115

Cited by 125 publications

(31 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the lack of a good correlation between the number of rDNA copies on individual chromosomes and their transcriptional output (de Capoa et al, 1988) is consistent with individual competent NORs hosting both active and silent rDNA copies. Furthermore, there is some evidence that active human rRNA genes are clustered within competent NORs (Hamkalo and Miller, 1973). Taken together with the lack of preferred initiation sites (Fig.…”

Section: Discussionmentioning

confidence: 74%

“…By a similar logic, it is possible that replicating the active rRNA genes at the periphery, or outside, nucleoli reduces the risk of collisions between the transcription and replication machineries that can cause DSBs and promote rDNA hyper-recombination. The ribosomal genes are the most highly transcribed genomic loci, with 100-150 PolI molecules loaded onto each active gene (Hamkalo and Miller, 1973), and this represents a serious obstacle for their replication. The existence of a robust mechanism, independent of the RFBs, which coordinates the activity of transcription and replication complexes in the competent NORs is implied by the bidirectionality and low efficiency of RFBs in human rDNA (Lebofsky and Bensimon, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…Only 20-50% of all rRNA genes are transcriptionally active in most human cells (Miller and Bakken, 1972;Gagnon-Kugler et al, 2009). They are believed to exist in a nucleosome-free state, whereas the silent copies are packed as regular chromatin (Hamkalo and Miller, 1973;Sogo and Thoma, 2004). Although the precise organization of active and repressed ribosomal genes within human NORs is unknown, it is clear that some NORs are composed entirely of inactive rRNA genes (silent NORs), whereas other NORs (competent NORs) host active only, or a mixture of active and inactive, rRNA genes (de Capoa et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Dimitrova

2011

Journal of Cell Science

View full text Add to dashboard Cite

SummaryTypically, only a fraction of the ≥600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNA genes coalesce in specialized nuclear compartments -the nucleoli -and are believed to replicate during the first half of S phase. Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, earlyreplicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates to the nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNA units. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. By contrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are fired preferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain this specific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensure streamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosome breaks and rDNA hyper-recombination.

show abstract

Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

Dimitrova

2011

Journal of Cell Science

View full text Add to dashboard Cite

show abstract

“…The distribution of displacements on the long axis of the cell is bell-shaped, with a half-width of Ϸ50-200 nm (measurements in five other cells yielded similar results). The most natural explanation for these observations is that we are looking at an RNA molecule tethered to DNA during transcription and possibly afterward (39). It is interesting to compare the observed behavior with a simple biophysical model of the system, that of a particle tethered to a spot by a spring, with an effective spring constant k sp (40).…”

Section: Results and Discussion Ms2-rna Complexes In Vitromentioning

confidence: 99%

RNA dynamics in live Escherichia coli cells

Golding

Cox

2004

Proc. Natl. Acad. Sci. U.S.A.

273

290

View full text Add to dashboard Cite

We describe a method for tracking RNA molecules in Escherichia coli that is sensitive to single copies of mRNA, and, using the method, we find that individual molecules can be followed for many hours in living cells. We observe distinct characteristic dynamics of RNA molecules, all consistent with the known life history of RNA in prokaryotes: localized motion consistent with the Brownian motion of an RNA polymer tethered to its template DNA, free diffusion, and a few examples of polymer chain dynamics that appear to be a combination of chain fluctuation and chain elongation attributable to RNA transcription. We also quantify some of the dynamics, such as width of the displacement distribution, diffusion coefficient, chain elongation rate, and distribution of molecule numbers, and compare them with known biophysical parameters of the E. coli system.T he bacterium Escherichia coli may well be the most thoroughly characterized model biological system. Much of its metabolism serves as the basic paradigm for DNA replication, RNA transcription, protein synthesis, and gene regulation (1, 2).Recently, techniques have become available that allow us to study central problems in genome organization and expression in individual living cells (3, 4), rather than rely on the averaged properties of large populations. These studies have added to our knowledge in two main areas: the heterogeneity among cells in a supposedly homogeneous population (5-7) and the spatiotemporal organization of macromolecules in the bacterial cell (8). The subcellular location of a variety of different proteins involved in cell division, such as the MinCDE family (9), are now known to be localized in special regions, as is the location of replicating chromosomes (10) and several different plasmids (11,12). Complexes of signaling proteins in Bacillus subtilis and Caulobacter crescentus have likewise been localized to the poles of dividing and differentiating cells (8, 13).To our knowledge, there are no published studies on the localization and timing of mRNA synthesis in living bacterial cells. Our knowledge of RNA transcription and dynamics comes from population studies, or in vitro studies with purified components (14-17). A rare glimpse into cell-to-cell variability in message number was provided by the studies of with Salmonella using whole fixed mounts. However, there is no information at the single-cell level in living cells on message localization: whether mRNA is free to move throughout the cytoplasm, whether movement is active or passive, and, for RNA molecules present in low copy numbers, how the actual number of molecules is a function of cell state. This last information is of particular interest because it bears directly on the question of the regulation of proteins known to be present in low copy numbers (for example, many of the proteins that act to switch genes on and off) and how the cell successfully regulates copy number in the presence of considerable extrinsic and intrinsic noise (see ref. 7 for a review).Here we describe a metho...

show abstract

“…Electron micrographs of transcriptionally active, non-ribosomal chromatin from amphibian lampbrush chromosomes [49] and from the embryo of the milkweed bug [SO] show specific sites for initiation and termination of transcription and suggest rather long primary RNA transcripts for at least some structural genes. These pictures of active transcription units are a strong argument for the idea that eucaryotic mRNAs are transcribed in the nucleus as RNA molecules of larger than their cytoplasmic size and that larger nuclear RNA molecules containing messenger sequences are precursors to mature polysomal mRNAs.…”

Section: Discussionmentioning

confidence: 99%

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Sippel

Groner

Hynes

et al. 1977

European Journal of Biochemistry

View full text Add to dashboard Cite

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98 % formamide. Complementary DNA (cDNA) was prepared from the 8 -14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that :1. All abundant 8 -14 -S poly(A)-containing mRNAs have larger nuclear precursor molecules ; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length.2. 70 % of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S.3 . The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8 -14-S mRNA.To understand the regulation of gene expression in eucaryotic cells it is crucial to know more about nuclear processes involved in the biogenesis of mRNA. Several features of the synthesis and structure of hnRNA indicate its role in mRNA metabolism (for review see [l-51). Indirect evidence first led to the postulate that hnRNA contained precursor molecules to mRNA [6,7]. Hybridization-competition experiments later demonstrated base-sequence homology between polyribosomal mRNA and hnRNA from HeLa cells [8]. Furthermore, it was found that viral-specific RNAs in and polyoma-infected [lo] cells are transcribed as giant RNA molecules. However, owing to the enormous sequence complexity, the large size and the high turnover of hnRNA, a detailed describtion of the nuclear pathway of messenger sequences has not yet Abbreviations. Hepes. 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; AMV, avian myeloblastosis virus; cDNA, DNA copy complementary to mRNA ; hnRNA, polydisperse high-molecularweight nuclear RNA; rot value, concentration of RNA (as mol phosphate/l) times time of hybridization (in s); rotllz value, rot value at which half the hybridizable cDNA is hybridized; vgt value, RNA derived from the amount of liver tissue (g), hybridized in IO-pI assay volume, times the hybridization time (h).Enzyme. S 1 nuclease, single-strand-specific endonuclease from Aspergillus oryzae (EC 3.1.4.21).been possible. The use of molecular hybridization with complementary DNA copies of specific mRNA molecules to identify messenger sequences in steadystate nuclear RNA has resulted in an increased amount of new information, but no general picture has emerged from these studies. For example, larger nuclear precursor RNA molecules for globin mRNA have been found, although with conflicting results as to their largest size [ l l -171. For the case of ovalbumin mRNA, no larger nuclear RNA containing the messenger se...

show abstract

Electronmicroscopy of Genetic Activity

Cited by 125 publications

References 17 publications

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

DNA replication initiation patterns and spatial dynamics of the human ribosomal RNA gene loci

RNA dynamics in live Escherichia coli cells

Size Distribution of Rat Liver Nuclear RNA Containing mRNA Sequences

Contact Info

Product

Resources

About