Shape normalization of 3D cell nuclei using elastic spherical mapping.

Topological analysis of cells and subcellular structures on the basis of image data, is one of the major trends in modern quantitative biology. However, due to the dynamic nature of cell biology, the optical appearance of different cells or even time-series of the same cell is undergoing substantial variations in shape and texture, which makes a comparison of shapes and distances across different cells a nontrivial task. In the absence of canonical invariances, a natural approach to the normalization of cells consists of spherical mapping, enabling the analysis of targeted regions in terms of canonical spherical coordinates, that is, radial distances and angles. In this work, we present a physically-based approach to spherical mapping, which has been applied for topological analysis of multichannel confocal laser scanning microscopy images of human fibroblast nuclei. Our experimental results demonstrate that spherical mapping of entire nuclear domains can automatically be obtained by inverting affine and elastic transformations, performed on a spherical finite element template mesh.

Four-dimensional imaging of chromatin dynamics during the assembly of the interphase nucleus.

Large-scale chromatin organization is likely to play an important role in epigenetic control of gene expression. This implies that after mitosis the correct chromatin organization must be re-established in the nuclei of the two daughter cells. Here we analyze the dynamic behavior of chromatin during the transition from late anaphase to G1 in dividing HeLa cells, which express green fluorescent protein-tagged histone H2B. Time-lapse confocal microscopy was used to image the movement and the decondensation of chromatin as cell division progresses. Typically, time series of over 100 three-dimensional images (4D images) were collected, spanning a time period of up to three hours. Special care was taken to avoid photodamage, since cell cycle progression is exquisitely sensitive to photochemical damage. Quantitative analysis of the 4D images revealed that during the anaphase to G1 transition the movement of chromatin domains relative to other chromatin is remarkably limited. Chromatin dynamics can best be described as a radial expansion of the cluster of chromosomes that is present in late anaphase. We find that decondensation occurs in two phases. First a rapid decondensation by about a factor of two, followed by a slower phase in which part of the chromatin does not decondense any further, whereas the remaining chromatin decondenses further about two fold.

Local UV-induced DNA damage in cell nuclei results in local transcription inhibition.

UV-induced DNA damage causes cells to repress RNA synthesis and to initiate nucleotide excision repair (NER). NER and transcription are intimately linked processes. Evidence has been presented that, in addition to damaged genes, undamaged loci are transcriptionally inhibited. We investigated whether RNA synthesis from undamaged genes is affected by the presence of UV damage elsewhere in the same nucleus, using a novel technique to UV irradiate only part of a nucleus. We show that the basal transcription/repair factor TFIIH is recruited to the damaged nuclear area, partially depleting the undamaged nuclear area. Remarkably, this sequestration has no effect on RNA synthesis. This result was obtained for cells that are able to carry out NER and for cells deficient in NER. We conclude that cross talk between NER and transcription occurs only over short distances in nuclei of living cells.

Sequential assembly of the nucleotide excision repair factors in vivo.

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Light and electron microscopy localization of the 11beta-hydroxysteroid dehydrogenase type I enzyme in the rat.

The 11beta-hydroxysteroid dehydrogenase type I enzyme (11betaHSD1) converts cortisone to cortisol in humans, and 11-dehydrocorticosterone to corticosterone in rodents. In the present study we used a new immunopurified polyclonal antibody, RAH113, to localize 11betaHSD1 at the light and electron microscopy levels in a wide range of rat tissues. 11betaHSD1 staining in the liver was of highest intensity around the central vein and decreased radially. In the lung, 11betaHSD1 was found at highest levels in the interstitial fibroblast, with levels in the type II pneumocyte an order of magnitude lower. RAH113 stained proximal tubules of the renal cortex and interstitial cells of the medulla and papilla. Adrenal 11betaHSD1 was confined to the glomerulosa and medulla, whereas the glucocorticoid-inactivating hydroxysteroid dehydrogenase isoform 11betaHSD2 was present in fascilulata/reticularis. 11betaHSD1 was found in parietal cells of the fundic region of the stomach, but not in the antrum. In the heart, 11betaHSD1 was detected in cells resembling interstitial fibroblasts of the endocardium and in the adventitial fibroblasts of blood vessels. Western blot analysis confirmed the presence of an antigen of the correct size (34 kDa) and intensity consistent with levels of enzyme activity previously reported in these tissues. Brain and testis also displayed the 34-kDa protein, confirming the expression of authentic 11betaHSD1 in these tissues. Electron microscopy of lung and kidney interstitial cells showed that 11betaHSD1 was localized both to the endoplasmic reticulum and the nuclear membrane. These results show that 11betaHSD1 is present in discrete cell populations where it may facilitate intracrine and paracrine glucocorticoid action in addition to its classical role of maintaining circulating glucocorticoids via activity in the liver.

SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA.

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

Nuclear distribution of prothymosin alpha and parathymosin: evidence that prothymosin alpha is associated with RNA synthesis processing and parathymosin with early DNA replication.

Prothymosin alpha and parathymosin are two ubiquitous small acidic nuclear proteins that are thought to be involved in cell cycle progression, proliferation, and cell differentiation. In an effort to investigate the molecular function of the two proteins, we studied their spatial distribution by indirect immunofluorescence labeling and confocal scanning laser microscopy in relation to nuclear components involved in transcription, translation, and splicing. Results indicate that both proteins exhibit a punctuated nuclear distribution and are excluded by nucleoli. The distribution of prothymosin alpha in the nucleus is related to that of transcription sites, whereas the distribution of parathymosin correlates with early replication sites. This implies that prothymosin alpha and parathymosin are involved in transcription and replication, respectively. In addition to the punctate distribution, prothymosin alpha also is found concentrated in 1-6 nuclear domains per cell. These domains are found in more than 80% of randomly growing T24 human bladder carcinoma cells. They have a diameter of 0.2-2.5 microm, their size being inversely related to the number of domains per cell. The domains disappear during mitosis and the protein is excluded from the metaphase chromosomes. Double-labeling experiments associate these prothymosin alpha domains with PML and CstF64 containing nuclear bodies, but not with hnRNP-I containing domains or coiled bodies.

Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L).

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators.

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

Cell cycle regulation of PML modification and ND10 composition.

The nuclear sub-structures known as ND10, PODs or PML nuclear bodies can be rapidly modified by diverse stimuli, and the resultant structural changes correlate with events such as cellular transformation and successful virus infection. We show that the ND10 components PML and Sp100 undergo profound biochemical changes during the cell cycle. Both proteins are conjugated to the ubiquitin-like protein SUMO-1 during interphase, but they become de-conjugated during mitosis and an isoform of PML of distinct electrophoretic mobility appears. This mitosis-specific form of PML is highly labile in vitro, but is partially stabilised by phosphatase inhibitors. Treatment of interphase cells with phosphatase inhibitors induces the production of a PML isoform of similar gel mobility to the mitosis-specific species, and taken together these results suggest that phosphorylation is an important factor in the differential modification of PML during the cell cycle. PML and Sp100 normally tightly co-localise in ND10 in interphase cells, but they become separated during mitosis. Interphase cells treated with phosphatase inhibitors or subjected to heat shock also show structural changes in ND10, accompanied by alterations to the normal pattern of PML modification. Taken with previous findings on the effects of infection by herpes simplex virus and adenovirus on ND10 structure and PML modification, these results suggest that the many factors which have been shown to modify ND10 structure may do so by interaction with the biochemical mechanisms that act on ND10 components during the cell cycle.

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner.

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.

Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins.

The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin Adelta10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for a vertical orientation. Time-lapse studies of 3-D reconstructed interphase cells showed a remarkable stability in both number and location of these structures over time, while the lamina showed considerable dynamic movements, consisting of folding and indentation of large parts of the lamina. Fluorescence recovery after bleaching studies revealed a low protein turnover of both tubular and lamina-associated lamins. Repetitive bleaching of intranuclear areas revealed the presence of an insoluble intranuclear fraction of A-type lamins. Time-lapse studies of mitotic cells showed that reformation of the lamina and the tubular structures consisting of A-type lamins did not occur until after cytokinesis was completed.

Spatial relationship between transcription sites and chromosome territories.

We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

Coiled bodies are predisposed to a spatial association with genes that contain snoRNA sequences in their introns.

Small nucleolar RNAs (snoRNAs) constitute a group of more than 100 different stable RNA molecules that are found concentrated in the nucleolus where they are involved in the maturation of ribosomal RNA. Most snoRNAs are not produced from their own genes but are encoded in the introns of other genes, referred to as snoRNA host genes. Little is known about the mechanisms by which the snoRNAs are produced from introns and how the snoRNAs mature to functional snoRNP complexes in the nucleolus. One class of intron-encoded snoRNAs binds with high specificity to the protein fibrillarin which is found concentrated in the nucleolus, but also in small nuclear domains known as coiled bodies. It has become clear that genes that code for small stable RNAs, e.g., U1, U2 snRNA, and the U3 snoRNA, are often found adjacent to coiled bodies. High concentrations of transcription factors and RNA processing factors in and around coiled bodies indicate that they may be involved in the expression of the adjacent genes. In order to investigate whether coiled bodies could play a role in the synthesis of intron-encoded snoRNAs the distribution of coiled bodies was studied relative to three different snoRNA host genes, i.e., hsc70, RPS3, UHG. All three were found adjacent to coiled bodies at significantly high frequencies (11-19%), compared to control sequences (0-2%), to conclude a preferential association between the snoRNA host genes and coiled bodies. This association could point to a possible role for coiled bodies in the synthesis and/or maturation of snoRNAs. An involvement in snoRNA production could explain the presence of transcription factors, splicing factors, and fibrillarin in coiled bodies.

Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division.

Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.

A bipartite sequence element associated with matrix/scaffold attachment regions.

We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.

Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Randomness of spatial distributions of two proteins in the cell nucleus involved in mRNA synthesis and their relationship.

Randomness and relation between point positions play an important role in archeology, cosmology, geography, and biology. An often-discarded effect is the edge effect, the effect that points are bound to a certain region. Without an appropriate correction, the outcome will be wrong. We studied the problem of randomness by comparing the distribution of interpoint distances with what can be expected for randomly distributed points (pair correlation function in statistics), and applied this to two sets of nuclear proteins inside the cell nucleus. The technique comprised labelling the proteins with a fluorescent dye, recording the fluorescent distribution with a 3D confocal microscope, and detecting the positions of the individual fluorescent spots. Results showed that, apart from studying randomness, the method is well equipped to quantitatively analyze a spot detection procedure, as the resolving power and the subvoxel accuracy were clearly visible. Given the results of assessing the randomness of the general transcription factor BRG1 and the RNA synthesizing protein RNA polymerase II (polII) in the cell nucleus, we concluded that the high intensity spots of the BRG1 protein are regularly spaced. The low intensity spots of the BRG1 protein and the low- and high-intensity spots of the polII protein showed more random behavior. The BRG1 and polII proteins showed correlation; unexpectedly, the relation was also found for the low-intensity spots, which were expected to have a more random behavior.