Higher order metaphase chromosome structure: Evidence for metalloprotein interactions

Lewis, Catherine D.; Laemmli, Ulrich Karl

doi:10.1016/0092-8674(82)90101-5

Cited by 394 publications

(298 citation statements)

References 23 publications

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“…Alternatively, fixation was performed with methanol following the procedure for staining tissue culture cells. Since copper ions in a concentration as low as ID-8 M have been reported to stabilize the chromosomal scaffold [32], we added 1 JlM CuCl 2 into the spreading buffer. In no case topo 11 was detectable on lampbrush chromosomes.…”

Section: Discussionmentioning

confidence: 99%

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

Fischer

Hock

Scheer

1993

Experimental Cell Research

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In somatic cells DNA topoisomerase 11 (topo 11) is thought to be involved in the domain organization of the genome by anchoring the basis of chromatin loops to a chromosomal scaffold. Lampbrush chromosomes of amphibian oocytes directly display this radial loop organization in cytological preparations. In order to find out whether topo 11 may play a role in the organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo 11. Our results indicate that topo 11 is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomalloops. Topo 11 was, however, enriched in the amplified nucleoli of Xenopus oocytes. © 1993 Academic Press, Inc. INTRODUCTIONThe chromatin fiber of eukaryotic cells is folded into a series of loops or domains which are stabilized by a structural framework called nuclear matrix or chromosomal scaffold. The loops are thought to be anchored at their base to the matrix or scaffold via specific attachment sites, the so-called matrix or scaffold attachment regions (MARs or SARs). Loop length is variable, ranging from 5 to 200 kbp of DNA [for reviews see 1, 2]. Unfortunately, the chromatin loop structures cannot be directly visualized in interphase nuclei with cytological methods because the chromatin strands are highly entangled.The situation is different for amphibian lampbrush chromosomes which can be readily examined under almost native conditions by light microscopy [3]. The structural organization of these meiotic chromosomes exhibits a striking similarity to the loop model described above. Each lampbrush chromosome consists of a chromosomal axis from which numerous loops radiate laterally. The axis represents a linear array of chromo-1 To whom correspondence and reprint requests should be addressed at Zoologie I, Biozentrum Am Hubland, D-97074 Wiirzburg, Germany. Fax: 0049 931 888 4252. 255 meres containing highly condensed chromatin, while the lateral loops consist of decondensed, transcriptionally active chromatin covered with numerous nascent transcripts. Because lampbrush chromosome architecture resembles so closely the proposed organization of somatic interphase chromatin and mitotic chromosomes, it is tempting to speculate that the same molecular components might be involved in the maintenance of chromatin loops.One of the best characterized components of the chromosomal scaffold is DNA topoisomerase 11 (topo 11) [4][5][6][7]. Besides its enzymatic activity that creates transient double-strand breaks in duplex DNA and passes DNA strands through one another, the protein has been shown to interact with the MAR and SAR elements [8]. Thus, topo 11 is thought to have catalytic as well as structural functions. Topo 11 is not only associated with the scaffold of mitotic chromosomes but also the cores and centromeric regions of spermatocyte-derived meiotic chromosomes [9]. Recently Luke and Bogenhagen identified by biochemical means topo 11 in the germinal vesicles of Xenopus o...

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Section: Discussionmentioning

confidence: 99%

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

Fischer

Hock

Scheer

1993

Experimental Cell Research

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“…Analysis of non-histone proteins in mitotic chromosomes found Fscaffold protein I_ (Lewis & Laemmli 1982), later identified as topo II , Gasser et al 1986. Electron-microscopic studies have indicated that topo II can bind a crossover of two DNAs (Zechiedrich & Osheroff 1990), and topo II has been observed to be able to recondense protease-decondensed chromosomes (Bojanowski et al 1998).…”

Section: Topoisomerase IImentioning

confidence: 99%

“…Condensins were first characterized in yeast (Strunnikov et al 1993(Strunnikov et al , 1995 and Xenopus (Hirano & Mitchison 1994). It was soon realized that one of the SMCs had been identified as non-histone Fscaffold protein II_ from mitotic chromosomes (Lewis & Laemmli 1982). Electron microscopy (EM) indicates that the two SMCs bind together to form a hinged structure nearly 100 nm in length if extended, suggesting a function as a chromatinYchromatin linker.…”

Section: Topoisomerase IImentioning

confidence: 99%

Micromechanical studies of mitotic chromosomes

2008

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Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin Fnetwork_ or Fgel_ organization, with stretches of chromatin strung between Fcrosslinking_ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.

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“…Mitotic chromosomes were isolated in polyamine-EDTA buffers as described previously (Lewis and Laemmli, 1982), except that the detergents used after cell lysis were 0.1% Ammonyx Lo or 0.1% n-dodecyl-d-maltoside instead of digitonin. Chromosomes used for immunofluorescence ( Figure 6G) were purified up to the glycerol gradient step.…”

Section: Preparation Of Mitotic Chromosome and Scaffoldsmentioning

confidence: 99%

“…CuSO 4 (0.5 mM) was then added under nitrogen gas and incubated for 10 min on ice. For histone depletion, the chromosomes were incubated in 2 M NaCl for 20 min (Lewis and Laemmli, 1982). The insoluble scaffold fraction was pelleted at 6800 ϫ g and solubilized in SDS sample buffer.…”

Section: Preparation Of Mitotic Chromosome and Scaffoldsmentioning

confidence: 99%

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Hudson

Ohta

Freisinger

et al. 2008

MBoC

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We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.

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Higher order metaphase chromosome structure: Evidence for metalloprotein interactions

Cited by 394 publications

References 23 publications

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

Micromechanical studies of mitotic chromosomes

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

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