In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

High-resolution mapping of satellite DNA using biotin-labeled DNA probes

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity- purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.     

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.     

Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization.

We characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric alpha-repeat DNA as chromosome-specific probe, we found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centomeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or beta-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. We hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric alpha-repeat DNA and proximal to beta-satellite DNA.     

Use of non-radioactive DNA probes for the characterization of adult T-cell leukemia cells

DNA fragments were labeled with dinitrophenyl (DNP) residues by the reaction with 2,4-dinitrobenzaldehyde in alkaline condition and the labeled DNA was used as a probe for non-radioactive in situ hybridization. DNP-labeled DNA probes for T cell receptor beta chain, c-myc and HTLV-1 were hybridized in situ to mRNA on cell specimens fixed with Carnoy's fixative. DNA-mRNA hybrids were detected immunohistochemically using anti-DNP antibodies. Cytoplasms of adult T cell leukemia cells were stained with varied intensity when these probes were used. More than 70% of cells were positively stained with T cell receptor probe. However, less than 30% of cells were stained with c-myc and HTLV-1 probes. The present study indicates that non-radioactive in situ hybridization can be used for the characterization and classification of leukemia.     

Denaturation of mouse satellite and ribosomal DNA during hydroxyapatite thermal chromatography of chromatin

Mouse DNA and chromatin were melted on hydroxyapatite and the denaturation profiles of ribosomal and satellite DNAs were followed by hybridization with their complementary RNAs. Neither ribosomal nor bulk DNA had significantly different melting profiles in chromatin as compared to DNA. However, most of satellite DNA eluted at higher temperature from chromatin than from purified DNA. One explanation for the higher melting temperature of mouse satellite DNA in chromatin suggests that the complex between this particular DNA component and at least some proteins in chromatin is more stable than the average DNA-protein interaction.     

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.     

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

Summary Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.     

Radiation-induced DNA breaks in different human satellite DNA sequence areas,analyzed by DNA breakage detection-fluorescence in situ hybridization

Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding-renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.     

Evolution conservatively of SCAR DNA localization in genome isochores of warm-blooded vertebrates

In meiotic prophase I, chromatin fibrils attached to the lateral elements of the synaptonemal complexes form loops. Synaptonemal complex associated regions of DNA (SCARs DNA) are a family of genomic DNA sequences tightly associated with the synaptonemal complex; they are located at the chromatin loop basements. Isochore compositional fractions of the human and chicken genomes were used as 32P labeled probes for hybridization with SCAR DNA isolated previously from the spermatocyte nuclei of the golden hamster Mesocricetus auratus. Nucleotide sequences similar to the golden hamster’s SCAR DNA were found in human and chicken genome isochores. The localization of SCAR DNA in isochore compartments of the examined genomes was established to be evolutionary conservative.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the-A-B-A-B-A-form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant-B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashtion along the DNA strand.     

Activity banding of human chromosomes as shown by histone acetylation

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromsomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromsomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a “snap-shot” of the distribution of gene activity on chromosomes at the time of cell harvest. Edited by: P.B. Moens     

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Saturation of human chromosome 3 with unique sequence hybridization probes

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.