John H. Frenster ^{1, @} and Jeannette A. Hovsepian ^{2, @}

Departments of ¹ Medicine and of ² Radiology, Stanford University School of Medicine, Stanford, California  94305, USA,

^@ Present Addresses: RNA Research, Physicians’ Educational Series, Atherton, CA  94027-5446 USA.
Phone:  +1 650 367 6483;   Fax:  +1 650 364 1773;   e-mail:   frenster@euchromatin.net

* Supported in part by a USPHS Research Career Development Award (CA-17857) from the National Cancer Institute to J.H.F.

Recent fluorescence microscopy studies (Parada LA, McQueen PG, and Misteli T, Genome Biology, vol. 5, no. 7, r44 (June 21, 2004), involving a systematic analysis of the spatial positioning of a subset of mouse chromosomes in several tissues, have suggested the possibility of an associative clustering of particular chromosomes in particular tissues during nuclear interphase. We have noted a similar phenomenon in isolated lymphocyte nuclei of calf thymus (Frenster JH, Nature 205: 1341 (1965), and have further analyzed our electron micrographs in order to define the ultrastructure of the phenomena. In the course of the isolation of repressed and active chromatin from interphase calf thymus lymphocytes, a stage is reached in which the nuclei swell to twice their normal size, offering a favorable material for the examination of the structural relations between the active euchromatin microfibrils and the repressed heterochromatin masses. Such swollen nuclei were gently prepared in cation-free isotonic sucrose, and examined for the location, number, shape, caliber, and length of continuous, closed loops of the 10 nm caliber active euchromatin microfibrils. The closed loops appear to be tethered at intervals of 50-100 nm to single masses of repressed heterochromatin chromocenters. The loops extend out into the interior of the nucleus for up to 1 um, forming elongated lariats that contact similar loops from adjacent  chromocenters. At the point of close apposition between opposing loops from distinct chromocenters, dense microcylinders of 20-30 nm caliber are formed, appearing to represent equal contributions from each chromocenter. The microcylinders range up to 100 nm in length before separating into their original calibers of 10 nm each. The fields of such chromosomal apposition range up to 3 um in diameter, and are often elliptical in outline.

PHA activation of normal human lymphocytes.

Without PHA-Activation.                             With PHA-Activation.

Fig. 1a: (left, above). Electron micrograph, X 16,000,  of a normal human blood lymphocyte incubated in vitro in the absence of phytohemagglutinin for 48 hours (Hovsepian JA, and Frenster JH, 2003). The cytoplasm is scanty and composed largely of monosomal ribosomes. The nucleus is compact with a major part of the DNA contained within condensed heterochromatin masses arranged directly underneath the nuclear membrane. Only a small minority of nuclear chromatin is arrayed as euchromatin 10 nm microfibrils. The nucleolus is small and surrounded by additional condensed heterochromatin. (X 16,000).

Fig. 1b: (right, above). Electron micrograph, X 9,000,  of normal human blood lymphocyte incubated in vitro in the presence of phytohemagglutinin for 48 hr (Hovsepian JA, and Frenster JH, 2003). The lymphocyte has undergone lymphoblastic transformation with an increased cytoplasm composed largely of polysomal ribosomes. The nucleus has enlarged with a major part of the DNA contained within extended euchromatin 10 nm microfibrils dispersed throughout the nucleus. The nucleolus has enlarged and is free of any surrounding heterochromatin. The plasma membrane has increased its number of microvillus projections and phagocytic activity. If the activated lymphocyte is now re-suspended in PHA-free incubation medium, the cell will return to its normal basal state within 72 hours of new culture. If PHA is instead added again, the cell will remain activated. (X 9,000).

Isolated nuclei and swollen nuclei from normal calf thymus lymphocytes.

Fig. 2a: Electron micrographs of: Isolated nuclei, X 10,000, and Fig. 2b: swollen nucleus, X 5,000, from normal calf thymus lymphocytes, during isolation of repressed and of active chromatin, (Frenster JH, Allfrey VG, and Mirsky AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci., U.S.A. vol. 50, no. 6, pp. 1026-1032 (December, 1963).)
 

Higher magnification of swollen nucleus from normal calf thymus lymphocyte.

Fig. 3: Electron micrograph, X 15,000,  of the isolated calf thymus lymphocyte nucleus in Fig. 2, swollen after the neutral extraction of nuclear ribonucleoprotein ribosomes and saline-soluble HMG proteins under hypotonic conditions (Frenster JH, et al, 1960), and its re-suspension in cation-free 0.25 M sucrose (Frenster JH, et al, 1963). The condensed heterochromatin masses remain dispersed radially (note euchromatin microfibrils "spilling out" of the areas between the heterochromatin masses at the nucleus margin), and the extended 10 nm euchromatin microfibrils are seen to be continuous with the condensed heterochromatin masses, with a sharp zone of transition between the extended 10 nm microfibrils and the densely-packed fibers of heterochromatin (Frenster JH, 1965).

On  the surface of the central condensed  heterochromatin chromocenter mass, 3 different types of euchromatin-heterochromatin junctions can be seen. Along the lower half-border arc, numerous elongated closed loops of euchromatin can be seen emerging from the heterochromatin mass. Along the upper right border arc, fewer euchromatin microfibrils are seen emerging, and these may be coursing out of the plane of the section. Along the upper left border arc, still less euchromatin microfibrils are seen emerging, and instead, low-density globular structures are observed that may suggest lipid structures.

The closed loops are tethered at intervals of 50-100 nm to single masses of condensed heterochromatin, both in the center of the nuclei and at the nuclear peripheral margins, with little observed contact between adjacent loops. The loops extend out into the interior of the nucleus for up to 1 mm, forming elongated lariats attached to heterochromatin. The loops are of a continuous caliber of 10 nm, are slightly coiled, and may have a total length of up to 2 mm.

Between 20- 50 closed loops of euchromatin can be observed within a single nucleus in a single 80 nm thick electron microscopic section, extrapolating up to  5,000 such loops per complete nucleus. X 15,000.

One or two areas of each isolated nucleus (1-3 o'clock, at right) display areas of an increased number of  extended 10 nm microfibrils in contact with other microfibrils, suggesting a clustered association of 10 nm microfibrils from two or more chromosomes ( Parada, LA, et al, 2004).

At the point of close apposition between opposing loops from distinct chromocenters, dense microcylinders of 20-30 nm caliber are formed, appearing to represent equal contributions from each chromocenter. The microcylinders range up to 100 nm in length before separating into their original calibers of 10 nm each. The fields of such chromosomal apposition range up to 3 um in diameter, and are often elliptical in outline.

Kissing Chromosomes' Contact Points favor Paired Sense-Antisense Transcription by a DNA-DNA Tetraplex.

Fig. 4:  Kissing Chromosomes' Contact Points favor Paired  Sense-Antisense Transcription by a DNA-DNA Tetraplex.

1. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

2. Coudert AE, Pibouin L, Vi-Fane B, Thomas BL, Macdougall M, Choudhury A, Robert B, Sharpe PT, Berda A, and Lezot F, "Expression and regulation of the Msx1 natural antisense transcript during development".

3. Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, Murphy PR, and Casson AG, "Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene".

Such close kissing interactions between non-homologous chromosomes in a DNA-DNA tetraplex could be mediated by an enhancer activity of the new MER121 class of conserved noncoding DNA sequences recently found by:

Kamal M, Xie X, and Lander ES, "A large family of ancient repeat elements in the human genome is under strong selection",

or by the extensive conserved noncoding RNA sequences acting as enhancers as isolated recently by:

Shin JT, Priest JR, Ovcharenko I, Ronco A, Moore RK, C. Burns CG, and MacRae CA, "Human-zebrafish non-coding conserved elements act in vivo to regulate transcription".

Close kissing interactions have also been found between homologous chromosomes by:

Xu N, Tsai C-L, and Lee JT, "Transient Homologous Pairing Marks the Onset of X Inactivation", Science vol. 311, no. 5764, pp. 1149-1152 (February 24, 2006),

and have been proposed for homologous sequences between normal host DNA and oncogenic viral DNA:

Frenster JH, "Model of Single-Stranded Integration of Oncogenic Viral Genomes".
.

References:

1a. Parada LA, McQueen PG,  and Misteli T, "Tissue-specific spatial organization of genomes", Genome Biology, vol. 5, no. 7, r44 (June 21, 2004).

1b. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei".

1c. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

2. Frenster JH, "Ultrastructural Continuity Between Active and Repressed Chromatin", Nature vol. 205: no. 4978, pp. 1341-1342 (March 27, 1965).

3. Frenster JH, Allfrey VG, and Mirsky AE, "Metabolism and Morphology of Ribonucleoprotein Particles from the Cell Nucleus of Lymphocytes", Proc. Natl. Acad. Sci. U.S.A. vol. 46: no. 4, pp. 432-444 (April, 1960).

4. Frenster JH, Allfrey VG, and Mirsky AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci., U.S.A. vol. 50, no. 6, pp. 1026-1032 (December, 1963).

5. Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, and Fraser P, "Active genes dynamically colocalize to shared sites of ongoing transcription".

6. Hovsepian JA, and Frenster JH, "Euchromatin as an Extensile Force within Mammalian Cell Nuclei", Molec. Biol. Cell, vol. 14, supp. p. 93a (November, 2003).

7a. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells", in: "Advances in Cell and Molecular Biology", vol. 3, pp. 1-19 (1974), ed. DuPraw EJ, New York: Academic Press.

7b. Frenster JH, "Ultrastructure and Function of Heterochromatin and Euchromatin", in: "The Cell Nucleus", vol. 1, pp. 565-580, (1974), (Busch H, ed.), New York, Academic Press.

8. Frenster JH, Papalian MM, Masek MA and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", Journal of the National Cancer Institute, Vol. 63, pp. 331-335, Aug. 1979.

9. Volpi EV, Chevret E, Jones T, Vatcheva R, Williamson J, Beck S, Campbell RD, Goldsworthy M, Powis SH, Ragoussis J, Trowsdale J, and Sheer D, "Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei", J. Cell Sci., vol. 113, no. 9, pp. 1565-1576 (2000).

10. Fransz P, de Jong JH, Lysak M, Castiglione MR, and Schubert I, " Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate", Proc. Natl. Acad. Sci., U.S.A., vol. 99, no. 22, pp. 14584-14589 (October 29, 2002).

11. O'Sullivan JM, Tan-Wong SM, Morillon A, Lee B, Coles J, Mellor J, and Proudfoot NJ, "Gene loops juxtapose promoters and terminators in yeast", Nature Genetics, vol. 36, no. 9, pp. 1014-1018 (September, 2004).

12. Muller WG, Rieder D, Kreth G, Cremer C, Trajanoski Z, and McNally JG, "Generic Features of Tertiary Chromatin Structure as Detected in Natural Chromosomes", Molec. Cell. Biol., vol. 24, no. 21, pp. 9359-9370 (November, 2004).

13.  Bystricky K, Laroche T, van Houwe G, Blaszczyk M, and Gasser SM, "Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization", J. Cell Biol., January 31, 2005; 168(3): 375 - 387.

14. Frenster JH, "Selective Control of DNA Helix Openings during Gene Regulation", Cancer Research, vol. 36, pp. 3394-3398 (September, 1976).

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

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E-mail: frenster@euchromatin.net