scholarly journals Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

1985 ◽  
Vol 5 (6) ◽  
pp. 1287-1294 ◽  
Author(s):  
R I Bolla ◽  
D C Braaten ◽  
Y Shiomi ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Rna Sequences ◽  
S1 Nuclease ◽  
The Core ◽  
Rdna Sequences ◽  
L Cell ◽  
Cytoplasmic Rna ◽  
Rrna Species ◽  
Tight Association

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix

1985 ◽  
Vol 5 (6) ◽  
pp. 1287-1294 ◽  
Author(s):  
R I Bolla ◽  
D C Braaten ◽  
Y Shiomi ◽  
M B Hebert ◽  
D Schlessinger
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Rna Sequences ◽  
S1 Nuclease ◽  
The Core ◽  
Rdna Sequences ◽  
L Cell ◽  
Cytoplasmic Rna ◽  
Rrna Species ◽  
Tight Association

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.

Reconstruction of complexes of histone and superhelical nuclear DNA

1981 ◽  
Vol 50 (1) ◽  
pp. 209-224
Author(s):  
J.M. Levin ◽  
P.R. Cook
Keyword(s):  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
The Core ◽  
Ionic Detergent ◽  
Nuclear Rna ◽  
Core Histones ◽  
Non Destructive ◽  
Rna And Dna ◽  
General Method

When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin. Their DNA is supercoiled and so intact. Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the ‘core’ histones without breaking the DNA. We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling. The superhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones. Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures. These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.

Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1

1979 ◽  
Vol 57 (6) ◽  
pp. 666-672 ◽  
Author(s):  
P. K. Chan ◽  
C. C. Liew
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Base Pairs ◽  
The Core ◽  
Liver Chromatin ◽  
Chromatin Proteins

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate – polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.

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 PTB/hnRNP I Is Required for RNP Remodeling during RNA Localization in Xenopus Oocytes

2007 ◽  
Vol 28 (2) ◽  
pp. 678-686 ◽  
Author(s):  
Raymond A. Lewis ◽  
James A. Gagnon ◽  
Kimberly L. Mowry
Keyword(s):  
Protein Interactions ◽  
Xenopus Oocytes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Rna Transport ◽  
Rna Sequences ◽  
Cytoplasmic Rna ◽  
Embryonic Polarity ◽  
Specific Mrnas

ABSTRACT Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.

scholarly journals Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals.

1988 ◽  
Vol 8 (11) ◽  
pp. 4829-4839 ◽  
Author(s):  
R M Denome ◽  
C N Cole
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Coding Region ◽  
S1 Nuclease ◽  
Cytoplasmic Rna ◽  
Sv40 Origin ◽  
Sv40 Infection ◽  
Simplex Virus ◽  
Polyadenylation Signals

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.

Conformational changes induced by salt in complexes of histones and superhelical nuclear DNA

1981 ◽  
Vol 50 (1) ◽  
pp. 199-208
Author(s):  
J.M. Levin ◽  
P.R. Cook
Keyword(s):  
Free Energy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Salt Concentration ◽  
Nuclear Dna ◽  
The Core ◽  
Ionic Detergent ◽  
Core Histones ◽  
Intercalating Dye ◽  
Negative Supercoiling

When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear DNA, RNA and the ‘core’ histones, but few other proteins characteristic of chromatin. Their DNA is intact. The core histones dissociate on raising the salt concentration. We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli. Both have a higher affinity for superhelical DNA than they do for relaxed DNA. The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively. As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl. This transition, which is reversible in the presence of the arginine-rich histones, occurs without dissociation of these histones from the DNA and so must reflect a conformational change in the complex. In contrast to the results with ethidium, we find that RNA polymerase can detect the presence of some negative free energy of supercoiling in nucleoids containing the core histones. The transformations of the free energy that can assist the binding of ethidium and RNA polymerase are discussed.

Adenovirus chromatin structure at different stages of infection

1981 ◽  
Vol 1 (12) ◽  
pp. 1094-1105
Author(s):  
E Daniell ◽  
D E Groff ◽  
M J Fedor
Keyword(s):  
Dna Synthesis ◽  
Nuclear Dna ◽  
Protein Complexes ◽  
Fragment Size ◽  
Human Adenovirus ◽  
Micrococcal Nuclease ◽  
Base Pairs ◽  
Viral Dna ◽  
Infected Cells ◽  
Cellular Dna

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

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