Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Somatic pairing of chromosome 1 centromeres in interphase nuclei of human cerebellum

Summary Interphase nuclei isolated from paraffin-embedded tissue of four normal brains were hybridized with biotinated repetitive DNA probes specific for the (peri) centromeric regions of chromosomes 1 and 7. Hybridization results were visualized with a peroxidase-DAB system after which the number of specific signals per nucleus was counted using bright field microscopy. Using the probe specific for chromosome 7 (p7t1), both the cerebral and the cerebellar samples showed 2 spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe (pUC1. 77) showed two spots in 69% of the cerebral nuclei. In cerebellar samples, hybridization with pUC1.77 resulted in only one large spot per nucleus in 82% of the cells. The average spot size in nuclei with one signal was about 1.6 times as large as that in nuclei with two signals. These observations suggest that the single large spot in the cerebellar cells is not the result of monosomy of chromosome 1 but that it reflects somatic pairing of the two chromosome 1 centromeres. Based on the size and the fraction of nuclei with one large spot, the small granular neuron is the most likely candidate. The difference between cerebral and cerebellar samples indicates that this somatic pairing of chromosome 1 is a cell-type-dependent phenomenon.     

Size-dependent positioning of human chromosomes in interphase nuclei

By using a fluorescence in situ hybridization technique we revealed that for nine different q-arm telomere markers the positioning of chromosomes in human G(1) interphase nuclei was chromosome size-dependent. The q-arm telomeres of large chromosomes are more peripherally located than telomeres on small chromosomes. This highly organized arrangement of chromatin within the human nucleus was discovered by determining the x and y coordinates of the hybridization sites and calculating the root-mean-square radial distance to the nuclear centers in human fibroblasts. We demonstrate here that global organization within the G(1) interphase nucleus is affected by one of the most fundamental physical quantities-chromosome size or mass-and propose two biophysical models, a volume exclusion model and a mitotic preset model, to explain our finding.     

Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei

Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.     

Distinct patterns of histone methylation and acetylation in human interphase nuclei

To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.     

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.     

Cytogenetics of solid human tumors

The paper presents cytogenetic data available in literature concerning results of the study of malignant somatic cells at chromosomal and genetic levels in the pretumour period and in advanced tumours.     

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (alpha MSH/ACTH[4-11]), was synthesized and labelled in the 3'-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hybridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidin-alkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridization (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Detection of proopiomelanocortin mRNA by in situ hybridization,using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

Summary A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (MSH/ACTH[4-11]), ws synthesized and labelled in the 3-end by use of terminal transferase. Probes tailed with either [³H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

Localization of a mouse centromeric DNA repeat in interphase nuclei

The position of a mouse DNA repeat located near the centromere of mouse chromosomes X, 11, 13, and 17 was examined in interphase nuclei of bone marrow and fibroblast cells by in situ hybridization of 3H- or biotin-labeled DNA probe 70-38. In most laboratory mouse strains this probe recognizes a single repeat cluster (DXWas70) close to the centromere of the mouse X chromosome. In a few mouse strains, a second locus (D11Was70, D13Was70, or D17Was70, depending on the mouse strain) is located near the centromere of an autosome. In interphase nuclei from mouse strains with the X-linked locus only, two distinct sites of hybridization were found in female mice and one in male mice. These two sites remained separated during the different phases of the cell cycle (G1, early S, late S, and G2) as demonstrated by in situ hybridization of the probe to flow-sorted nuclei. In interphase nuclei from mouse strains with both the X-linked locus and an autosomal locus, four distinct sites of hybridization were found in female mice and three in male mice. Further analysis of loci DXWas70 and D17Was70 showed that these loci were often located in the outer region of nuclei from bone marrow and fibroblast cells.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Structural organization of chromosomes in interphase nuclei

The in situ molecular hybridization method has been applied to the detection, at the electron microscope level, of SV40 viral DNA in permissively infected monkey kidney cell cultures. The observations suggest an important role of the host cell nucleolus during the lytic infection with SV40.     

Identification of chromosome markers in interphase nuclei

The chromosome complement of the mosquito Cuilseta longiareolata (2n=6) reveals distinguishable centromeric regions and one telomere of the Y chromosome by using light-induced differentiation and autoradiographic techniques in mitotic and premeiotic interphase nuclei. The localization of these cytological markers and their spatial relationships appear to be very similar in the two types of nuclei and suggest an interphase arrangement where centromeric regions are clustered together in a chromocenter like structure, close to the nuclear membrane, with the telomeres lying on the opposite pole of the nucleus.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.