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èC×EÌÔúÉ¨¢ÄÍ¼añ\~NÌu×EjvÉû[³êÄ¢éB»µÄ×Eªªô·éÛCí¸©\ªÌ¤¿É·³~NÌ46{ÌuªôúõFÌvÉ©ËçêéB¶¨wÌ³ÈÅÍCDNA ªqXg^pN¿Éª©êÄukNI\[vÆÈèC±ÌkNI\[üÛªK¥³µ­©ËçêÄuN}`üÛvÆCXÈéKw\¢ðÂ­élqª}¦³êÄ¢éBµ©µÈªçCÅßÌMÒçÌ SPring­8 ÌúËõðp¢½\¢ðÍÌÊC]ÌKw\¢fÆÍÙÈèCªôúõFÌªkNI\[üÛÌsK¥ÈÜèôÝÅ\¬³êÄ¢é±Æª¾ç©ÉÈÁ½BÜ½C±Ìæ¤ÈsK¥Èû[Í×EÌÔúÉ¨¢Äà¶ÝµÄ¢é±Æð¦µ½B{eÅÍQm DNA Ìû[´ÉÂ¢ÄCÅßÌMÒçÌm©ðÐîµ½¢BÌ¶ÝªmçêÄ«½BÅÍÇÌæ¤ÉµÄõFÌÍ`¬Í¶ßÉ³êéÌ¾ë¤©BDNA Í¼a 2 nm Ì}CiXd×ð

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Fig. 1(Color online) (a) In the textbook model, a long DNA molecule is wrapped around a basic core histone octamer
that consists of H2A, H2B, H3 and H4 histone proteins, and forms a nucleosome. The nucleosome had long been
assumed to be folded into 30­nm chromatin bers before the higher­order organization of mitotic chromosomes
occurs. The images are reproduced with minor modications from Ref. 8. (b) According to a hierarchical helicalfolding model, the 30­nm chromatin ber is progressively folded into larger bers having diameters of 100_150
and 200_250 nm to form nal mitotic chromosomes.

206  úËõ July 2013 Vol.26 No.4
(C) 2013 The Japanese Society for Synchrotron Radiation Researchðà ¡ úËõÉæéqgõFÌÌ\¢ðÍJ[qXgðºÁÄCç¹ñóÉK¥³µ­ÜèôÜê
ÄC¼añ30 nm ÌN}`üÛª`¬³êé±Æðñ¥µ½1)iFig. 1ajB»ÝL­ó¯üêçêÄ¢éõFÌ\¢ÌèàÅÍC±ÌN}`üÛªç¹ñóÉª©êÄñ100 nm ÌüÛðÂ­èCÂ¬É200_250 nmC³çÉÍ
500_750 nm Ìæ¤ÉCK¥³µ¢ç¹ñóÌKw\¢ð`¬·éÆ³êÄ«½2)iFig. 1bjBÀÛCªq¶¨wÌÅàL¼È³ÈÅéu×EÌªq¶¨wvÅÍCß25NÈãÉí½ÁÄ±ÌèàªfÚ³êÄ¨èCÜ½
wZÌ¶¨ II Ì³ÈÉàLÚ³êÄ¢éBqgõFÌÌ X ü¬pUðÍiSAXSj
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ntÌ¨ÌàðÍ·é±ÆªÅ«é½ßC¶¨¿ÉKµ½è@ÅéB±Ì½ßC

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Fig. 2 (Color online) (a) When non­crystal materials are irradiat­
ed with X­rays, small­angle scattering generally reects thesize and spacing of internal structures. (b) Experimental
setting: a chromosome pellet in a quartz capillary tube was
exposed to a synchrotron radiation beam and the scattering
patterns were recorded with a CCD camera or imaging plate.
(c) Typical SAXS patterns of mitotic HeLa chromosomes.
Three peaks at `6, `11 and `30 nm were detected(arrows). (d) Two types of nucleosome positioning: face­to­face, with `6­nm spacing, and edge­to­edge, with `11­nm
spacing. The nucleosome model image (yellow, DNA; red,core histones). Panels (a) and (b) were reproduced from
Ref. 11 with permission. Panels (c) and (d) werereproduced from Ref. 5 with permission.

ÊíCqg|{×EÌ SAXS ªèð¨±È¤ÆC×EàÉ½ÊÉ¶Ý·é¬EÈÇÌ\¢ª30_40 nm Ì­¢

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±ÌÊÍCSAXS É¨¯é30 nm Ìs[Nª{\[qgªôú×EÌNCIdq°÷¾Ï@¯¶

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208  úËõ July 2013 Vol.26 No.4

ðà ¡ úËõÉæéqgõFÌÌ\¢ðÍ

Fig. 3(Color online) (a) Cryo­EM image of the chromosome clusters (cross­sections). Many black dots were observedon the chromosome surface. Note that knife marks (thick arrow) and compression marks (thin arrow) were ob­served. Bars indicate 1 mm. An enlarged image of the region outlined with a box is depicted on the right. A black
dot region is surrounded by a broken line (Ri). A chromosome part is marked as `Xs'. Long and short bars indi­cate 0.5 mm and 30 nm, respectively. (b) Immunostaining with anti­P antibody against a ribosomal component
suggested that the peripheral black dots were ribosome aggregates. DNA (DAPI), a ribosome and merged imagesof two chromosome clusters are shown. Bar indicates 10 mm. (c) Power spectrum (Fourier transform) analysesof the aggregated regions, the chromosome regions and whole cryo­EM images. A 30­nm peak (arrow) was seenin the aggregated region (green dots) and whole images (blue dots). (d) Removal of ribosome aggregates. Isolat­
ed chromosomes were washed in buer A (polyamine{EDTA) and then returned to IB containing 5 mM Mg2{.
Cryo­EM showed that this treatment removed most of the aggregates. Note that knife marks (arrow) were ob­served. Bar indicates 1 mm. The inset shows an enlarged image. Long and short bars indicate 0.5 mm and 30 nm,
respectively. Panels (a)_(d) were reproduced from Ref. 5 with permission.

ÌIðIÈðÂ\É·éÁÙIÈ`¿âCqXg

4fjBÁ¦ÄC×EàÌN}`ÉÍkNI\[ÌCüðîµ½ÁÊÈJjYª¶ÝµCK¥³µ¢

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30 nm N}`üÛð`¬µÄ¢éÌ¾ë¤B»µÄC

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É30 nm N}`üÛª\¬Å«È­Èé±Æªñ³êûCSAXS ÉÚµ¢ÇÒÍCSAXS ÅÍTCYÌo

êÄ¢é12) BÈãÌ±Æ©çCMÒçÍªôúõFÌÉé¨ÌÌêCs[NÆµÄoÅ«È

uÉ¢v30 nm N}`üÛª¶Ý·éÂ\«ÍrÅ¢ÌÅÍÈ¢©CÆ¢¤^âðø©êé©àµêÈ¢B±«È¢àÌÌC­È­ÆàªôúõFÌÌåªÅÍ¶ÝêÉÎµÄCJASRI énÉæévZ@V~µÈ¢¾ë¤Æl¦Ä¢éBG[Vª[VÉæÁÄCÉ¢30 nm üÛÆµÄ¼aûüi15÷jyÑË¶êûüi15÷jÉÎçÂ«ðµÄàiFig. 4ejU Ê Í 30 nm Ì s ¢ s [ N ð ¦ · ± Æ ª Å « ½5) i Fig.

úËõ July 2013 Vol.26 No.4  209

Fig. 4

(Color online) (a) In SAXS analysis, only the 30­nm peak disappeared after removal of ribosome aggregates(left), whereas the other peaks remained. A ribosomal fraction from Xenopus egg extracts (kindly provided by
Dr T Hirano, RIKEN) produced a 30­nm peak (arrow) on SAXS (right). (b) Model explaining the results: the30­nm peak in SAXS comes from regularly spaced aggregates of ribosomes, and not from the chromosomes them­selves. (c) The chicken erythrocyte nuclei produced a sharp 30­nm peak (arrow). In addition, two peaks of `11
and `6 nm were prominent (brackets). (d) Dot­like 30­nm structures in the chicken erythrocyte nucleus wereobserved by cryo­EM. Long and short bars indicate 200 and 30 nm, respectively. (e) Compact bundles of 30­nm
bers, with variations in their diameter (}15÷) and torsion (}15÷), were computationally modeled. The up­
per image is the top view, and the bottom is the side view. (f) The modeled 30­nm ber bundles produced a peakat `30 nm. Panels (a)_(f) were reproduced from Ref. 5 with permission.

úËõÉ¬pU USAXS ÉæéªôúõFÌÌ\¢ðÍ·é±ÆÉµ½13) B±ÌuÍ{¬pUÌ½ßÌà
ÌÅÍÈ¢ªCnSÌª^óºÉèCmCYàá­C

¢¬pªð\ª¾çêéBÀÛC¼ìÆMÒçÍC±Ì¡ÜÅÌÀ±Éæè30 nm ÌN}`\¢Ío³êuðp¢ÄCPêÌqgõFÌÌ CXDM ÉæéC[WÈ¢±Æªí©Á½ªCªôúõFÌÉ³çÉå«ÈK¥

OÉ¬÷µÄ¢½13) B±Ìuðp¢ÄCÉ¬pUIÈ\¢ªªèiUSAXSjð¨±È¤½ßÉÍCTvð^óºéÌ©Æ¢¤^âÍcÁÄ¢½B»Ì½ßC

SAXS ÉæÁÄõFÌÌæSÌð²×é±ÆªÅ«éæÉÝu·éKvª¤ÈªèªKvÅ

ß½ÎpKXÌLs[ðÚÁ½B±ÌÚIÌ½ßCMÒçÍC

éB±Ì½ßCMÒÍqgõFÌðl
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SPring­8 BL29XUL ÅC¼ìi»kåjçÉæÁÄJ­ [àÌn tÌö­ð h®±ÆÉµ ½B±Ì uÌ³ê½Rq[g X üñÜ°÷@ CXDM Ìuðp

USAXS ÅÌ«\ðm©ßé½ßC`1000 nm ÌebN

210  úËõ July 2013 Vol.26 No.4

ðà ¡ úËõÉæéqgõFÌÌ\¢ðÍ

Fig. 5(Color online) (a) USAXS study of latex particles with di­ameters of `1000 nm showed clear fringes over a wide range(`50_1000 nm). The prole is plotted as log (I) versus log(S). (b) A computer simulation of latex particles with di­ameters of `1000 nm produced a pattern highly similar tothat measured. (c) USAXS of human mitotic chromosomes,
no notable structures around 100_150 or 200_250 nm weredetected. (d) USAXS of human interphase chromatin, no
notable structures `100_
150 or 200_
250 nm were detected in
HeLa interphase nuclei. Beyond the `275 nm range (redarrow), the slope in interphase chromatin decreased in mag­nitude. Panels (a)_(d) were reproduced from Ref. 5 withpermission.

Fig. 6

X±qðRg[ÆµÄªèµ½Æ±ëC`50_1000
nm ÌÍÍÅtWÌp^[ª\êé±Æªí©Á(Color online) (a) Chromosomes consist essentially of ir­
regularly folded nucleosome (beads on a string) bers. Con­densins (blue) hold the nucleosome bers (red) globallyaround the chromosome centre. Locally, the nucleosomeber is folded in an irregular or disordered manner, forming
loop structures that are collapsed towards the chromosome
centre (blue). (b) In the interphase nucleus, there are
numerous compact chromatin domains like ``chromatin liq­
uid drops'' (yellow balls). Red, transcribed nucleosome;Green, RNA polymerase and RNA. (c) In the cells, nucleo­
some bers (Red balls and lines) are irregularly folded. The
nucleosomes are uctuating. This nucleosome dynamics
facilitates chromatin accessibility. The nucleosome uctua­tion is the basis for scanning genome information. Panels(a) was reproduced from Ref. 5 with permission.

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E­mail: kmaeshimnig.ac.jpêåF×E¶¨wC¶¨¨wmªðn
1999NCåãåwåw@ãw¤Èm
ÛöC¹C2004NÜÅXCXÉW l[uåwÉ¤õB»w¤¤õÉê
C¤õðoÄ09N©ç»EB»w¤qõ¤õB

Structural study of human chromosomes bysynchrotron radiation beamKazuhiro MAESHIMA
Abstract

Structural Biology Center, National Institute of Genetics,
Mishima, Shizuoka 411_8540, Japan

How is 2­m­long genomic DNA organized into a mitotic chromosome or nucleus? The nucleosomeber has long been assumed to be folded into a 30­nm chromatin ber, and further helically folded
larger ber. To investigate the bulk structure of mitotic chromosomes, we performed small­angle
X­ray scattering (SAXS) at SPring­8. No structural feature larger than 11 nm was detected, evenon a chromosome­diameter scale (`1 mm). We also found a similar scattering pattern in inter­
phase nuclei of HeLa cells in the range up to `275 nm. Our ndings suggest a common structuralfeature in interphase and mitotic chromatins: compact and irregular folding of nucleosome bers
occurs without a 30­nm chromatin structure.

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