A new application of in situ hybridization: detection of numerical and structural chromosome aberrations with a combination centromeric-telomeric DNA probe

Fluorescent in situ hybridization provides a fast method for detection of specific nucleic acid sequences. We have used high-resolution, single-color fluorescent in situ hybridization with a combination centromeric-telomeric DNA probe, specific for chromosome 1, to investigate the feasibility of simultaneous assessment of numerical and structural chromosome aberrations. The K562 leukemia cell line served as a model.     

Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy

Summary Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A₃, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R-and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.     

Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.     

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

Summary The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.     

High-resolution organization of mouse telomeric and pericentromeric DNA

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.     

Localization of the pig luteinizing hormone/choriogonadotropin receptor gene (LHCGR) by radioactive and nonradioactive in situ hybridization.

The porcine gene for luteinizing hormone/choriogonadotropin receptor (LHCGR) was localized to chromosome 3q2.2----q2.3 using radioactive and nonradioactive in situ hybridization. A computer-assisted image-analysis system was developed which facilitated detection of the position of silver grains and fluorescent spots on the chromosomes after in situ hybridization. Compared with autoradiographic visualization, the nonisotopic procedure proved to be more rapid, precise, and highly specific; however, nonradiographic in situ hybridization was much less efficient than the autoradiographic technique for the detection of unique DNA sequences with small probes. From these results and published gene-mapping data, it was concluded that the synteny between LHCGR and MDH1 observed in man is conserved in the pig genome.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

A new FISH protocol with increased sensitivity for physical mapping with short probes in plants

Fluorescence in situ hybridization (FISH) is a well-established technique used for the detection of specific DNA regions, that has been applied to interphase nuclei, pachytene and metaphase chromosomes as well as to extended DNA fibres. This technique allows the physical mapping of specific DNA sequences both on individual chromosomes and extended fibres. A new FISH protocol is described here that enhances the sensitivity of the method. Probes for small unique DNA sequences of less than 2 kb give high signal-to-noise ratio with this method, and can be visualized easily by means of conventional fluorescence microscopy.     

A cloned repeated DNA sequence in human chromosome heteromorphisms

A sequence derived by ECoRI restriction of human satellite DNA III has been cloned in lambda gt WES. The cloned DNA was used as a template for in vitro synthesis of cRNA, which was hybridized in situ to preparations of human metaphase chromosomes with a range of heterochromatic polymorphisms. Most of the hybridization was found on chromosome 1, and the amount of hybridization was related to the size of the C-band on this chromosome. Hybridization to other chromosomes was not related to the C-band size, although hybridization of total satellite DNA is proportional to C-band size. Total satellite DNAs contain a mixture of sequences, some of which are predominantly located on only one pair of chromosomes. Hybridization in situ is able to discriminate between such chromosome-specific sequences and the bulk of satellite DNA. Further analysis of satellite DNAs may identify sequences specific for every chromosome pair.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Isolation of human chromosome 21 sequences and their application to in situ hybridization

Summary We report the isolation of 50 independent unique sequences from a human chromosome 21 library (identification code LA21 NSO1). These sequences were individually assigned to chromosome 21 using a mouse-human somatic hybrid cell line, WAVR 4d-F94a. Use of these unique clones as a mixture of probes for in situ hybridization of human metaphase chromosomes demonstrated strong signals on chromosome 21. These unique DNA sequences should provide useful tools for structural and functional analysis of human chromosome 21. The use of these sequences for the detection of Down syndrome is discussed.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

A simple method for simultaneous R- or G-banding and fluorescence in situ hybridization of small single-copy genes.

A significant improvement in fluorescence in situ hybridization, enabling the detection of single-copy genes as small as 500 bp directly on banded chromosomes, is presented. The induction of chromosome banding, which does not require additional handling or any system of amplification, is obtained simply by using an alkaline (pH 11) p-phenylenediamine anti-fade solution. As the banding produced is related to the timing of 5-bromodeoxyuridine incorporation, either R- or G-banding, constitutive heterochromatin staining, or chromosome asymmetry can be observed simultaneously with the fluorescent hybridized spots. Results of hybridization of small cDNA probes for the human genes for motilin, thymidylate synthetase, and lymphocyte activation-3 are provided as examples of the high-resolution mapping obtainable with this technique.     

Genes occupy a fixed and symmetrical position on sister chromatids

A high-resolution fluorescence methodology for nonisotopic in situ hybridization was applied to determine the positions occupied by several single-copy genes, DNA sequences, and integrated viral genomes on sister chromatids. The lateral and longitudinal mapping of the probes was performed on prometaphase and metaphase chromosomes. A fixed lateral position, exterior or median in relation to the longitudinal axis of the chromatids, was observed for a given probe, with a symmetrical position of the double fluorescent spots. This position appears to be independent of chromosome condensation stage from prometaphase to metaphase. These observations suggest an opposite helical-handedness conformation of DNA on both chromatids with a mirror symmetry. They support the model of chromosome packaging recently proposed by Boy de la Tour and Laemmli. Moreover, our results indicate that the last stages of chromosome condensation occur by packing down the coils without further coiling.     

Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X chromosome, by dosage analysis, and by in situ hybridization to mouse chromosomes. In mouse strain C57BL/10BK, one fragment appeared to be located only on the X chromosome, while the other fragment had homologous sequences on chromosome 11 in addition to the X chromosome. The latter fragment showed DNA variants between mouse strains, which are potentially useful for mapping. Both fragments cross-hybridized to another mouse species: Mus caroli. In this species, each fragment appeared to be located on the X chromosome, indicating that some X-chromosome repetitive sequences are partially conserved. In addition, one fragment cross-hybridized to human DNA.     

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.     

A PCR product derived from female DNA with regional localization on the Y chromosome.

A 154-bp PCR product amplified from human female DNA mapped onto the Y chromosome under high-stringency in situ hybridization conditions. The female DNA sequence revealed an 89% homology with the HSDYZ1 sequence. When the same primers were used to amplify male DNA, a 154-bp DNA fragment was also obtained, showing a 98% homology with HSDYZ1. However, although the HSDYZ1 sequence is widely distributed along the long arm of the Y chromosome, both of these particular PCR products are di-regionally localized within this distal block of constitutive heterochromatin. In situ hybridization under lower stringency showed that these 154-bp sequences map both onto the autosomes and the Y chromosome. Overall, this paper shows (i) a new class of DNA sequences shared by the autosomes and the Y chromosome; and (ii) a substructured organization of some DNA repeats within the DYZ1 family that forms a large part of the constitutive heterochromatin of the Y chromosome.     

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.     

Sex reversal in the mouse (Mus musculus) is caused by a recurrent nonreciprocal crossover involving the X and an aberrant Y chromosome

Satellite DNA (Bkm) from the W sex-determining chromosome of snakes, which is related to sequences on the mouse Y chromosome, has been used to analyze the DNA and chromosomes of sex-reversed (Sxr) XXSxr male mice. Such mice exhibit a male-specific Southern blot Bkm hybridization pattern, consistent with the presence of Y-chromosome DNA. In situ hybridization of Bkm to chromosomes of XXSxr mice shows an aberrant concentration of related sequences on the distal terminus of a large mouse chromosome. The XYSxr carrier male, however, shows a pair of small chromosomes, which are presumed to be aberrant Y derivatives. Meiosis in the XYSxr mouse involves transfer of chromatin rich in Bkm-related DNA from the Y-Y1 complex to the X distal terminus. We suggest that this event is responsible for the transmission of the Sxr trait.