scholarly journals Analysis of DNA attached to the chromosome scaffold.

1982 ◽  
Vol 93 (2) ◽  
pp. 278-284 ◽  
Author(s):  
M T Kuo
Keyword(s):  
Dna Sequences ◽  
Southern Blotting ◽  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Nonhistone Protein ◽  
Sequence Class ◽  
Restriction Endonuclease Ecori ◽  
Cold Spring ◽  
Endonuclease Ecori ◽  
Chromosome Scaffold

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.

scholarly journals MPE-seq, a new method for the genome-wide analysis of chromatin structure

2015 ◽  
Vol 112 (27) ◽  
pp. E3457-E3465 ◽  
Author(s):  
Haruhiko Ishii ◽  
James T. Kadonaga ◽  
Bing Ren
Keyword(s):  
Dna Sequences ◽  
Chromatin Structure ◽  
Nuclear Dna ◽  
Massively Parallel Sequencing ◽  
Nucleosome Occupancy ◽  
Regulation Of Gene Expression ◽  
Micrococcal Nuclease ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Sequence Bias

The analysis of chromatin structure is essential for the understanding of transcriptional regulation in eukaryotes. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method for the genome-wide characterization of chromatin that involves the digestion of nuclei withMPE-Fe(II) followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. However, there are differences in the cleavage of nuclear chromatin by MPE-Fe(II) relative to MNase. Most notably, immediately upstream of the transcription start site of active promoters, we frequently observed nucleosome-sized (141–190 bp) and subnucleosome-sized (such as 101–140 bp) peaks of digested chromatin fragments with MPE-seq but not with MNase-seq. These peaks also correlate with the presence of core histones and could thus be due, at least in part, to noncanonical chromatin structures such as labile nucleosome-like particles that have been observed in other contexts. The subnucleosome-sized MPE-seq peaks exhibit a particularly distinct association with active promoters. In addition, unlike MNase, MPE-Fe(II) cleaves nuclear DNA with little sequence bias. In this regard, we found that DNA sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). This phenomenon may have affected the analysis of nucleosome occupancy over exons. These findings collectively indicate that MPE-seq provides a unique and straightforward means for the genome-wide analysis of chromatin structure with minimal DNA sequence bias. In particular, the combined use of MPE-seq and MNase-seq enables the identification of noncanonical chromatin structures that are likely to be important for the regulation of gene expression.

DNA is replicated at the nuclear cage

1980 ◽  
Vol 46 (1) ◽  
pp. 365-386 ◽  
Author(s):  
S.J. McCready ◽  
J. Godwin ◽  
D.W. Mason ◽  
I.A. Brazell ◽  
P.R. Cook
Keyword(s):  
Electron Microscopy ◽  
Dna Synthesis ◽  
Hela Cells ◽  
Membrane Structure ◽  
Structural Unit ◽  
Nuclear Dna ◽  
Short Pulses ◽  
Restriction Endonuclease Ecori ◽  
Endonuclease Ecori ◽  
Average Size

Structures resembling nuclei are released when HeLa cells are lysed in a detergent and 2 M salt. These nucleoids, which lack any organized membrane structure, contain all the nuclear DNA packaged within a cage of RNA and protein. Their DNA is supercoiled so that the linear DNA must remain unbroken and looped during lysis. Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA. Pulse-labelled (i.e. newly synthesized) DNA remains preferentially associated with the cages. This association has been confirmed by autoradiography. When nucleoids are prepared for electron microscopy by the Kleinschmidt procedure the DNA spills out to form a skirt around the flattened cage. Labelling, which is restricted to the region of the cage after short pulses, extends out into the skirt as the labelling time increases. A model, based on the premise that replication takes place at the nuclear cage, is presented in the Appendix. The results of the biochemical experiments and electron microscopy both indicate that the average size of the unit of replication is approximately 2 micrometer. This is about one-quarter the size of the average structural unit - the loop. Therefore sequences in the loop must become attached to the nuclear cage prior to the initiation of DNA synthesis.

scholarly journals Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences

2009 ◽  
Vol 104 (3) ◽  
pp. 403-416 ◽  
Author(s):  
Gerardo A. Salazar ◽  
Lidia I. Cabrera ◽  
Santiago Madriñán ◽  
Mark W. Chase
Keyword(s):  
Dna Sequences ◽  
Phylogenetic Relationships ◽  
Nuclear Dna

Phylogenetics and evolution of the aphid genus Uroleucon based on mitochondrial and nuclear DNA sequences

1999 ◽  
Vol 24 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Nancy A. Moran ◽  
Matthew E. Kaplan ◽  
Michael J. Gelsey ◽  
Troy G. Murphy ◽  
Edwin A. Scholes
Keyword(s):  
Dna Sequences ◽  
Nuclear Dna

scholarly journals Evolutionary Relationship between Two Firefly Species,Curtos costipennisandC. okinawanus(Coleoptera, Lampyridae), in the Ryukyu Islands of Japan Revealed by the Mitochondrial and Nuclear DNA Sequences

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Masahiko Muraji ◽  
Norio Arakaki ◽  
Shigeo Tanizaki
Keyword(s):  
Dna Sequences ◽  
Evolutionary History ◽  
Nuclear Dna ◽  
Evolutionary Relationship ◽  
Early Pleistocene ◽  
Ryukyu Islands ◽  
Mtdna Sequences ◽  
Northern Half ◽  
The Ryukyu Islands ◽  
History Of

The phylogenetic relationship, biogeography, and evolutionary history of closely related two firefly species,Curtos costipennisandC. okinawanus, distributed in the Ryukyu Islands of Japan were examined based on nucleotide sequences of mitochondrial (2.2 kb long) and nuclear (1.1-1.2 kb long) DNAs. In these analyses, individuals were divided among three genetically distinct local groups,C. costipennisin the Amami region,C. okinawanusin the Okinawa region, andC. costipennisin the Sakishima region. Their mtDNA sequences suggested that ancestralC. costipennispopulation was first separated between the Central and Southern Ryukyu areas, and the northern half was then subdivided betweenC. costipennisin the Amami andC. okinawanusin the Okinawa. The application of the molecular evolutionary clocks of coleopteran insects indicated that their vicariance occurred 1.0–1.4 million years ago, suggesting the influence of submergence and subdivision of a paleopeninsula extending between the Ryukyu Islands and continental China through Taiwan in the early Pleistocene.

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