Presented at the 46th Annual Meeting of the American Society for Cell Biology, San Diego, CA December 9-13, 2006, and published in: Molec. Biol. Cell, vol. 17, suppl., p. 1853a (December, 2006).


"DNase-I Ultrastructural Probe Sites and Kissing Chromosomes". 1

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,

@ Present Addresses: Euchromatin Research, Physicians’ Educational Series, Atherton, CA  94027-5446
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.


NetworkEditor's Perspective: "Giant sites of DNase I-sensitivity may map regions of chromosomal kissing".
Abstract:
Fig. 1: Ultrastructural Probes of DNase I-Sensitive Sites within a human leukemic bone marrow spicule.
Tables of Quantitative Data from Ultrastructural Probes of DNase-I Sensitivity.
   Table 1. Probe sites  within human normal intact marrow differentiating granulocytes.
   Table 2. Probe sites within human normal intact marrow differentiating erythrocytes.
   Table 3. Probe sites  within human Hodgkin lymphoma intact lymph node activating lymphocytes.
Fig. 2: Ultrastructural Probes of DNase I-Sensitive Sites within a human Hodgkin Lymphoma lymph node.
References:
Additional Electron Microscopy Studies of Hodgkin Lymphoma and of Acute Myelocytic Leukemia:
Further Topics:
Other Links:
Further Information:


Abstract:

DNA sites active in  gene transcription can be visualized within mammalian intact interphase cells by high-resolution electron microscopy,  utilizing a probe specific for DNase I-sensitive DNA within euchromatin microfibrils (Cancer Res. 31: 1128 (1971). Such active DNA sites are found exclusively within euchromatin, and range in size from 25 nm (small) to 100 nm (large) in diameter within human bone marrow cells. Through the course of erythrocytic or granulocytic bone marrow maturation, the number of such active DNA sites per cell diminishes, with a shift in size of active DNA sites from large to small diameters as marrow cells mature. When lymph nodes of patients with newly-diagnosed Hodgkin Lymphoma were examined (J. Natl. Cancer Inst., 63: 331 (1979), T-lymphocytes adjacent to neoplastic Reed-Sternberg cells are found to be activated, with a  polysomal cytoplasm and increased numbers and size of active DNA sites compared to non-activated T-lymphocytes at more distance from the neoplastic cells. This T-lymphocyte activation appears to be the reverse of bone marrow maturation, with the diameter of observed active DNA sites within activated T-lymphocytes sometimes exceeding 350 nm (giant). Such giant active DNA sites within activated T-lymphocytes correlate with the clustering of interphse chromosomes first observed within activated T-lymphocytes (Spilianakis CG, et al., Nature 435: 637 (2005), during which chromosomal kissing ( Kioussis D, "Gene Regulation: Kissing Chromosomes", Nature vol. 435: 579 (2005) of related active gene loci on separate chromosomes was detected . We have previously found 20 nm caliber microcylinders formed from 10 nm euchromatin microfibrils from each of two adjacent chromosomes within isolated calf thymus T-lymphocyte interphase nuclei prepared in cation-free isotonic sucrose (ASCB 2005, #1280). The giant (>350 nm) active DNA sites and the 20 nm microcylinders found within T-lymphocyte nuclei may be ultrastuctural manifestations of the earlier described kissing chromosomes within T-lymphocytes.



Fig. 1: Ultrastructural Probes of DNase I-Sensitive Sites within a human leukemic bone marrow spicule.

Electron Micrograph of DNase I-Sensitive Probes within a human leukemic bone marow myelocyte.

Acridine orange and DNase I-sensitive probe method as described previously (Cancer Res. 31: 1128 (1971).
The electron-dense probe reaction products are found within the active euchromatin portion of the leukemic cell nucleus,  from where they can be counted, measured, and arrayed in tables. Magnification (x 12,000).

Tables 1-3.

DNase I-sensitive ultrastructural probe sites within intact human cells.

Small probe sites:    25-100 nm diameter
Large probe sites:  >100 nm diameter
Giant probe sites:   >350 nm diameter

Table 1. Probe sites  within human normal intact marrow differentiating granulocytes.
(n = 123)

.  Percent of cells
 containing sites:		 .  Mean site count
 per positve cell:		 .
    Cell type  Large sites
 (>100 nm diameter) 		  Small sites
 (25-100 nm diameter) 		  Large sites
 (>100 nm diameter)		  Small sites
 (25-100 nm diameter
 Promyelocyte  91.5  100.  9.55  35.5
 Myelocyte  85.9   96.4  8.75  35.1
 Metamyelocyte  47.6   85.7  3.9  36.3
 Band
 granulocyte		  0   20.0  0  8.0
 Segmented
 granulocyte		  0   12.2  0  10.1

Table 2. Probe sites within human normal intact marrow differentiating erythrocytes.
(n = 189)
   .  Percent of cells
 containing sites:		 .  Mean site count
 per positve cell:		 .
   Cell type  Large sites
 (>100 nm diameter)		  Small sites
 (25-100 nm diameter)		  Large sites
 (>100 nm diameter)		  Small sites
 (25-100 nm diameter)
 Proerythroblast  100  100  16.0   8.4
 Early
 erythroblast		   84.7   91.6    8.5  16.3
 Late
 erythroblast		   14.8   17.2    3.4   11.8
 Nucleated
 erythrocyte		    0    8.3     0   12.0

Table 3. Probe sites  within human Hodgkin lymphoma intact lymph node activating lymphocytes.
                                                                            (n = 194).
.  Percent of cells
 containing sites: 		 .  Mean site count
 per positve cell:		 .
   Lymphocytes  Giant sites
 (>350 nm diameter)		  Large sites
 (>100 nm diameter)		  Giant sites
 (>350 nm diameter)		  Large sites
 (>100 nm diameter)
 Monoribosomal  53.2  94.8  2.7  18.1
 Transitional  67.8  100  3.2  18.7
 Polyribosomal  79.5  100  3.5  17.1


Fig. 2: Ultrastructural Probes of DNase I-Sensitive Sites within a human Hodgkin Lymphoma lymph node.

Electron Micrograph of DNase I-Sensitive Probes within 3 lymphocytes surrounding a neoplastic Reed-Sternberg cell within a Hodgkin Lymphoma lymph node biopsied at original diagnosis and staging.

Acridine orange and DNase I-sensitive probe method as described previously (Cancer Res. 31: 1128 (1971), and J Natl Cancer Inst 63, 331-335 (1979). The electron-dense probe reaction products are found within the active euchromatin portions of the 3 activated lymphocyte nuclei, but not in the non-dividing multi-nucleated Reed-Sternberg cell. Magnification (x 8,000).



References:

1. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bome Marrow Cells", Cancer Res 31, 1128-1133 (1971).

2. Frenster JH, Nakatsu SL, and Masek MA, "Ultrastructural Probes of DNA Templates within Human Bone Marrow and Lymph Node Cells", Advances Cell Molec Biol 3, 1-20 (1974).

3. Nakatsu SL, Masek MA, Landrum S, and Frenster JH, "Activity of DNA Templates during Cell Division and Cell Differentiation", Nature 248, 334-335 (1974).

4. Frenster JH, Papalian MM, Masek MA, and Frenster JA, "Electron Microscopic Analysis of Lymph Node Cellular Activity in Hodgkin's Disease", J Natl Cancer Inst 63, 331-335 (1979).

5. Frenster JH, and Hovsepian JA, "Ultrastructure  of Closed Loops within Euchromatin of Isolated Lymphocyte Nuclei", Molec. Biol. Cell, vol. 15, suppl., p. 450a (November, 2004).

6. Spilianakis CG, Lalioti MD, Town T, Lee GR, and Flavell RA, "Interchromosomal associations between alternatively expressed loci", Nature vol. 435, no. 7042, pp. 637-645 (June 2, 2005).

7. Kioussis D, "Gene Regulation: Kissing Chromosomes", Nature vol. 435, pp. 579-580 (June 2,  2005).

8. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes", Molec. Biol. Cell, vol. 16, suppl., p. 1280a (December, 2005).

9. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis".
Cold Spring Harbor Symp. Quant. Biol. vol. 71: page 62, May 31-June 5, 2006.

10. Hovsepian JA, and Frenster JH,  "Chromosome-Chromosome Contact Points and Paired Sense-Antisense RNA Synthesis", RNA 2006, p. 237, June 20-25, 2006, The RNA Society, Bethesda, MD.

11. Lanctôt C, Cheutin T, Cremer M, Cavalli G, and Cremer T, "Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions", Nature Reviews Genetics vol. 8, no. 2, pp. 104-115 (February 2007).

12. Frenster JH, and Hovsepian JA, "DNA-DNA Tetraplex Model of Paired Sense-Antisense RNA Synthesis", RNA2007, p. 186, May 29-June 3, 2007, The RNA Society, Bethesda, MD.



NetworkEditor's Perspective: "Giant sites of DNase I-sensitivity may map regions of chromosome kissing".

These ultrastructural probe studies of human bone marrow cells and lymph node cells reveal decreases in occurence, numbers and and sizes of probes during marrow granulocytic and erythrocytic cell differention, and increases in occurrence, numbers and sizes of probes during lymph node T-lymphocyte activation, correlating with chromosomal kissing observed previously during T-lymphocyte activation in cell culture.



Additional Electron Microscopy Studies of Hodgkin Lymphoma and of Acute Myelocytic Leukemia:

1. Archibald RB, and Frenster JH, "Quantitative Ultrastructural Analysis of In-Vivo Lymphocyte - Reed-Sternberg Cell Interactions in Hodgkin's Disease", Natl. Cancer Inst. Monogr. 36: 239-245 (1973).

2. Masek MA, Rhoades DJ, and Frenster JH, "In-Vivo Macrophage Interactions with Lymphocytes in Hodgkin's Disease", Proc. Am. Assoc. Cancer Res. 14: 8 (1973).

3. Frenster JH, "Ultrastuctural Probes of Gene De-Repression within Human Leukemia and Lymphoma Cells", Proc. 11th International Cancer Congress, Florence, Italy (October, 1974).

4. Rowan RA, Masek MA, Thompson JM, and Frenster JH, "Electron Microscopic Localization of Acid Phosphatase Activity within Hodgkin's Disease Lymph Nodes", Proc. Am. Assoc. Cancer Res. 16: 10 (1975).

5. Frenster JH, Landrum SR, Masek MA, and Wilson LS, "Nuclear Maturation Within Neoplastic Cells In-Vivo", J. Cell Biol. 67: 123a (1975).

6. Frenster JH, Landrum SR, Masek MA, et al, "Comparison of DNA Helix Openings during In-Vivo Mitosis of Normal and Neoplastic Human Cells", Proc. Am. Assoc. Cancer Res. 19, 1-2 (1978).

7. Frenster JA, Papalian MM, Masek MA, et al, "Persistent Euchromatin after DNA Template Inactivation", J. Cell Biol. 79, 110a-111a (1978).

9. Frenster JH, Papalian MM, Masek MA, and Frenster JA, "Asymmetry of Intra-Nuclear Function during Immune Lymphocyte Activation", Biophys. J. vol. 25, no. 2, part 2, p. 228a, Feb. 1979.

9. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites During Neoplastic Cell Differentiation within Hodgkin's Disease Lymph Nodes", Leukemia Reviews International, Rich MA, Editor, Volume 1, pp. 22-23, (Marcel Dekker, Inc. New York/1983).

10. Frenster JH, "Single-Cell Analysis of DNase I-Sensitive Sites during Neoplastic and Normal Cell Differentiation within Human Bone Marrow", Ann. N.Y. Acad. Sci. 567: 334-336 (Aug. 4, 1989).

11. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", in: AACR-NCI-EORTC International Conference: "Molecular Targets and Cancer Therapeutics: Discovery, Development, and Clinical Validation", Washington, DC, November 16-19, 1999, and Published in: Clinical Cancer Research, vol. 5, suppl. l, p. 3855s, (624), (November, 1999).

12. Frenster JH, "Uni-Polar Clustering of Lymphocyte DNA Templates Toward Neoplastic Target Cells within Hodgkin's Disease Lymph Nodes", Proc. Am. Assoc. Cancer Res. vol. 43, p. 1134 (March, 2002).
 



Links to Hodgkin Lymphoma Immuno-Pathology:

Links to RNA and Biological Causality:


Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

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:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).



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euchromatin: "the most active portion of the genome within the cell nucleus".