Rapid and simultaneous detection of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization

This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75°C for 8 min, hybridized for 5 min at 37°C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa. © 1996 Wiley-Liss, Inc.     

In situ hybridization in Actinidia using repeat DNA and genomic probes

 In situ hybridization has been used to probe chromosome spreads of hexaploid Actinidia deliciosa (kiwifruit; 2n=6x=174) and tetraploid A. chinensis (2n=4x=116). When a species-specific repeat sequence, pKIWI516, was used, six hybridization sites were observed in some accessions of tetraploid A. chinensis and all of A. deliciosa. Southern analysis with the pKIWI516 probe revealed that there are two types of tetraploid A. chinensis. Genomic probes from diploid A. chinensis (2n=2x=58) did not differentiate the genomes of hexaploid A. deliciosa and tetraploid A. chinensis, irrespective of the presence or absence of blocking DNA. The results indicate that the genomes of polyploid Actinidia species are similar but not identical. The origin of A. deliciosa is discussed. Received: 29 June 1996 / Accepted: 5 July 1996     

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes

Summary Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (XqterXp22.2::Yq11Y qter) contained in the Chinese hamster x human hybrid cell line 445 x 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.     

Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human x Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.     

Detection of individual human chromosomes by chromosome in situ suppression hybridization using PCR-amplified bacteriophage library probes

We used the polymerase chain reaction (PCR) to prepare chromosome-specific probes from the bacteriophage λ library LA01NS01, prepared at the Los Alamos National Laboratory from flow sorted human chromosome 1. By using oligonucleotide primers flanking the EcoRI insertion site of the Charon 21A vector, we were able to amplify the human sequences preferentially in the library up to 9.1 kb (maximum insert size). The product of the PCR reaction was nick translated with incorporation of biotinylated residues and used with fluorescence in situ hybridization to observe metaphase chromosomes by fluorescence microscopy. This technique allows for a relatively easy method for preparation of chromosome-specific library probes for “chromosome painting.” The quality of the results obtained by this method compares favorably to those obtained by using bulk-purified library inserts. This method offers potential advantages in terms of cost and east of use.     

Reverse in situ hybridization of DNA probes of anomalous chromosomes in diagnosing chromosome pathologies]

The results of analysis of congenital chromosomal pathologies and chromosomal rearrangements upon the occurrence of haematological diseases, which was involved constructing DNA libraries of abnormal chromosomes and subsequent reverse CISS hybridization have been considered. High effectiveness of this approach for analysis of chromosomal translocations, deletions of chromosomal regions, minor extra chromosomes, and large marker chromosomes with complex organization was shown. The possibility of implementation of this approach and its large-scale application in medical and genetic studies of congenital developmental pathologies and chromosomal diagnostics of haematological diseases has been discussed.     

A human chromosome 9-specific alphoid DNA repeat spatially resolvable from satellite 3 DNA by fluorescent in situ hybridization.

We have isolated a DNA clone (pMR9A) that identifies an alphoid DNA subset specific for chromosome 9. This alphoid subset is characterized by a dimeric organization as revealed by Southern blot analysis after digestion with HaeIII, HinfI, or StuI. Nonradioactive in situ hybridization demonstrated that pMR9A hybridizes only to the centromeric region of chromosome 9 and reveals chromosome 9 aneuploidies in interphase nuclei. In addition, the probe detects quantitative differences in alpha satellite DNA on chromosome 9, but these quantitative differences are not correlated with the size of the heterochromatic region. Double-labeling experiments, using a chromosome 9-specific satellite 3 clone and pMR9A, enabled us spatially to distinguish the alphoid and satellite 3 domains on metaphase chromosomes after treatment of the cultures with 5-azacytidine.     

Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes

In situ DNA hybridization using biotin-labelled leptospiral DNA was performed on clinical specimens to investigate its usefulness as a technique for the identification of Leptospira interrogans. The applicability of this test in blood, urine and liver smears was demonstrated. In situ DNA hybridization can be completed in only 4 h and it combines the advantage of visualization of the leptospiral morphology with the specificity of the hybridization reaction. No cross-hybridization was observed with other bacteria. This study shows that hybridization in situ can be simple to perform and may contribute to a rapid diagnosis.     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes

Summary Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were radily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.     

Assignment of 35 single-copy and 17 repetitive sequence DNA probes to human chromosome 3: high-resolution physical mapping of 7 DNA probes by in situ hybridization

Thirty-five single-copy and 17 repetitive sequence DNA probes specific for human chromosome 3 were isolated from human chromosome 3-derived genomic libraries. Seven DNA clones, including three that are polymorphic for BglII or MspI, were mapped by in situ hybridization. Four probes were mapped to 3p subregions and 3 were mapped to 3q subregions. Three of the DNA sequences map to regions overlapping a segment of chromosome 3 (3p14-23) frequently deleted in small cell lung cancer cells. By Southern blot analysis on a deletion hybrid panel, we previously mapped 6 of these probes to three distinct chromosome 3 subregions. Our in situ data support these assignments and more precisely determine the localization of each clone to the following regions: D3S34 (3p14-21), D3S35 (3p21), D3S39 (3p21), D3S40 (3p12-13), D3S37 (3q21-23), and D3S36 (3q21). Clone pL84c, a low repeat sequence clone (approximately 30 copies), was mapped to the 3q21-29 subregion. These DNA clones mapped by in situ hybridization can provide useful landmarks for the ordering and localization of other clones.     

Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.     

High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: Rapid chromosome identification by directly fluorescently labeled alphoid DNA probes

We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5–10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

18p− Syndrome: an unusual case and diagnosis by in situ hybridization with chromosome 18-specific alphoid DNA sequence

Summary A patient with an atypical clinical picture of 18p^– syndrome is described. By the in situ hybridization technique we localized the chromosome 18-specific cloned repetitive sequence to metaphase chromosomes of the patient. The predominant hybridization of the probe was found in pericentromeric regions of homologous chromosomes 18. The amount of pericentromeric DNA measured by in situ hybridization differed between homologous chromosomes; and the number of radioactive grains was statistically greater in the normal chromosome 18 than in the aberrant chromosome 18p^–. The results indicate that this probe may be useful in clinical cytogenetics for identification of aberrant chromosomes, localization of breakpoints, and studies of C-band DNA polymorphism of chromosome 18.     

Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization using 10 chromosome-specific probes

OBJECTIVE: Although information on the cytogenetic characteristics of meningioma tumors has accumulated progressively over the past few decades, information on the genetic heterogeneity of meningiomas is still scanty. The aim of the present study was to analyze by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y in a group of 70 consecutive meningioma tumors. Another goal was to establish the potential associations among the altered chromosomes, as a way to assess both intertumoral and intratumoral heterogeneity. METHODS: For the purpose of the study, 70 patients diagnosed with meningioma were analyzed. Interphase FISH for the detection of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y was applied to fresh tumor samples from each of the patients studied. RESULTS: The overall incidence of numerical abnormalities was 76%. Chromosome Y in males and chromosome 22 in the whole series were the most common abnormalities (46% and 61%, respectively). Despite the finding that monosomy of chromosome 22/22q(-) deletions are the most frequent individual abnormality (53%), we have observed that chromosome gains are significantly more common than chromosome losses (60% versus 40%). Chromosome gains corresponded to abnormalities of chromosomes 1 (27%), 9 (25%), 10 (23%), 11 (22%), 14 (33%), 15 (22%), 17 (23%), and X in females (35%) and males (23%) whereas chromosome losses apart from chromosome 22 frequently involved chromosomes 14 (19%), X in males (23%), and Y in males (32%). Although an association was found among most gained chromosomes on one side and chromosome losses on the other side, different association patterns were observed. Furthermore, in the latter group, monosomy 22/22q(-) was associated with monosomy X in females and monosomy 14/14q(-) was associated with nulisomy Y in males. In addition, chromosome losses usually involved a large proportion of the tumor cells whereas chromosome gains were restricted to small tumor cell clones, including tetraploid cells. CONCLUSIONS: Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, as assessed by interphase FISH.     

Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat-rye chromosome pairing

 Fluorescence in situ hybridization (FISH) with multiple probes has been applied to meiotic chromosome spreads derived from ph1b common wheat x rye hybrid plants. The probes used included pSc74 and pSc 119.2 from rye (the latter also hybridizes on wheat, mainly B genome chromosomes), the Ae. squarrosa pAs1 probe, which hybridizes almost exclusively on D genome chromosomes, and wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH with a two-by-two combination of these probes allowed unequivocal identification of all of the rye (R) and most of the wheat (W) chromosomes, either unpaired or involved in pairing. Thus not only could wheat-wheat and wheat-rye associations be easily discriminated, which was already feasible by the sole use of the rye-specific pSc74 probe, but the individual pairing partners could also be identified. Of the wheat-rye pairing observed, which averaged from about 7% to 11% of the total pairing detected in six hybrid plants of the same cross combination, most involved B genome chromosomes (about 70%), and to a much lesser degree, those of the D (almost 17%) and A (14%) genomes. Rye arms 1RL and 5RL showed the highest pairing frequency (over 30%), followed by 2RL (11%) and 4RL (about 8%), with much lower values for all the other arms. 2RS and 5RS were never observed to pair in the sample analysed. Chromosome arms 1RL, 1RS, 2RL, 3RS, 4RS and 6RS were observed to be exclusively bound to wheat chromosomes of the same homoeologous group. The opposite was true for 4RL (paired with 6BS and 7BS) and 6RL (paired with 7BL). 5RL, on the other hand, paired with 4WL arms or segments of them in more than 80% of the cases and with 5WL in the remaining ones. Additional cases of pairing involving wheat chromosomes belonging to more than one homoeologous group occurred with 3RL, 7RS and 7RL. These results, while adding support to previous evidence about the existence of several translocations in the rye genome relative to that of wheat, show that FISH with multiple probes is an efficient method by which to study fundamental aspects of chromosome behaviour at meiosis, such as interspecific pairing. The type of knowledge attainable from this approach is expected to have a significant impact on both theoretical and applied research concerning wheat and related Triticeae. Received: 21 February 1996 / Accepted: 12 July 1996     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization

The major histocompatibility complex (SLA) of the domestic pig (Sus scrofa) was regionally mapped to 7p12----q12 by in situ hybridization with an SLA class I-specific recombinant DNA probe. This localization contradicts linkage data suggesting a possible assignment of the SLA locus to porcine chromosome 15.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.