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 (Chambeyron S,  and Bickmore WA, Genes & Develop., 18: 1119 (2004), have suggested the possibility of a choreographed looping out of decondensed chromatin from chromosome territories during the active transcription of the linearly clustered genes of the Hoxb1-Hoxb9 locus in embryonic stem cells of the mouse. We have noted a similar phenomena 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 favourable material for the examination of the structural relations between the active chromatin microfibrils and the repressed chromatin masses. Such swollen nuclei were gently prepared in cation-free isotonic sucrose, and examined for location, number, shape, caliber, and length of continuous, closed loops within the 10 nm active euchromatin microfibrils. The closed loops appear to be 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 very 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. More than 50 of such closed loops of euchromatin can be observed within a single nucleus in a single 80 nm thick electron microscopic section. The effect of phytohemagglutinin (PHA) activation and withdrawal on euchromatin looping is being studied.

PHA activation of normal human lymphocytes.

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 an isolated calf thymus lymphocyte nucleus 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.

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 transcription association of 10 nm microfibrils from two or more chromosomes ( Parada, LA, et al, 2004).

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.

Fig. 4: Ultrastructural DNase I-sensitive probe analysis of active DNA sites within lymphocytes of a positive lymph node taken from an untreated patient with Hodgkin lymphoma, nodular sclerosis type. Electron-dense reaction products are localized exclusively within the euchromatin portion of the cell nucleus.

(A, left): Monoribosomal lymphocyte. The cytoplasm contains a centriole, and the cytoplasmic ribosomes are largely arrayed in a monosomal manner, with scanty endoplasmic reticulum. X 13,000.

(B, right): Polyribosomal lymphocyte. The cytoplasmic ribosomes are largely arrayed in a polysomal manner, with slightly increased endoplasmic reticulum. X 9,750.

Fig. 5a (Left): Ultrastructural DNase I-sensitive probe analysis of active DNA sites within a positive lymph node biopsied from a patient with Hodgkin lymphoma, nodular sclerosis type. A neoplastic polynuclear Reed-Sternberg cell, shows almost no ultrastructural probes, while 3 surrounding lymphocytes are heavily labeled by ultrastructural probes in their euchromatin microfibrils. Each of the lobes of the Reed-Sternberg cell nucleus are themselves invaginated and at least three large nucleoli are evident. The cytoplasm of the Reed-Sternberg cell is polyribosomal with an occasional thin strand of rough endoplasmic reticulum. X 3,750.

Fig. 5b (Right): Ultrastructural DNase I-sensitive probe analysis of active DNA sites within normal human bone marrow. Undifferentiated stem cell. The nucleus is large, with numerous ultrastructural probes within the euchromatin microfibrils. The cytoplasm is extremely scanty and contains only an occasional mitochondrion. X7,500.

References:

1. Chambeyron S,  and Bickmore WA, "Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription",Genes & Development, vol. 18: no. 10, pp. 1119-1130 (May 15, 2004).

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

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. 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".

Links to RNA and Biological Causality:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

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

For Further Information and Feedback:
Phone:  +1 650 367 6483
E-mail: frenster@euchromatin.net