Fluorescence in situ hybridization and Y ring chromosome

Summary Investigations by fluorescence in situ hybridization and a Y-specific probe (Y190) of a male patient with a Y ring chromosome, 46,X,r(Y) showed four bright fluorescent spots within the ring. Thus, using this technique, it is possible to suggest that the ring originates from the duplication of the short arms of the Y chromosome.     

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.     

Nonisotopic in situ hybridization as a method for nondisjunction studies in human spermatozoa

Human spermatozoa were studied with a nonradioactive in situ hybridization method. Using a chemically modified DNA probe and immunocytochemical reactions for visualization, it was possible to obtain hybridization signals in 31 of 32 semen samples. Positive hybridization reactions, depending on cell accessibility, varied from 40% to over 90% for the different samples. Using a chromosome 1-specific DNA probe, disomy for this chromosome was found in 0.67% of all accessible sperm cells.     

Absence of Y-specific DNA sequences in human 46,XX true hermaphrodites and in 45,X mixed gonadal dysgenesis

Summary A search for Y-specific DNA sequences has been performed in a sample of seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case and compared with a sample of 11 XX males. Using six Y-specific DNA probes no hybridization signal was obtained in the hermaphrodite group; in contrast, all XX males gave a positive signal with at least one probe. This difference is statistically highly significant. We conclude that the aetiology of true hermaphroditism is different from that of the XX male syndrome. As all cases of the hermaphrodite group are positive for the serological sex-specific antigen (Sxs) it is concluded that this antigen can be present even in the absence of Y-specific DNA.     

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.     

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions

Summary In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.     

Demonstration of spontaneous XX/XY chimerism by DNA fingerprinting

Summary The M13 bacteriophage probe, which makes possible the establishment of DNA fingerprints, was used to study a phenotypically normal woman with a 46XY karyotype and her twin brother. Identical fingerprints and positive hybridzation with a series of Y-specific probes were obtained on blood samples from the siblings. DNA from a skin biopsy of the woman yielded a clearly different pattern and displayed no Y-specific hybridization, indicating that she is a spontaneous chimera. This study illustrates the use of DNA fingerprinting as a powerful and simple aid to the diagnosis of natural chimerism.     

Detection of single-copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization

DNA sequences homologous to single-copy genes were labelled with biotinylated dUTP or digoxygenin-labelled dUTP and hybridized to chromosome spreads. The hybridization signals were visualized with fluorescent avidin- or antibody-conjugates. This method allowed the detection of DNA targets on metaphase chromosomes as small as 1.4 kb. The hybridization signals were identified as fluorescent spots on both sister chromatids. Using an 18S rDNA probe as marker to identify chromosomes II and III it was possible to assign single-copy genes to these chromosomes. In the line V30 the endogenous chalcone synthase gene (chsA) was mapped at the distal end of the short arm of chromosome 5. The cDNA probe for this single-copy gene was 1.4 kb. In contrast, in the lines Mitchell and V26 chsA was localized at the distal end of the long arm of chromosome 3, suggesting that a chromosomal rearrangement had taken place. In a transformed Petunia uidA, transgenes were detected using a 2.7 kb probe. One transgene was mapped on one of the homologues of chromosome II proximal to the ribosomal genes. This homologue could be distinguished from the other by having the ribosomal genes at the distal end of the long arm. Using multicolour fluorescence in situ hybridization it was shown that it is possible to detect the endogenous chsA genes and both transgenes simultaneously.     

Use of repetitive DNA for diagnosis of chromosomal rearrangements

Summary We have used two repeated DNA fragments (3.4 and 2.1 kb) released from Y chromosome DNA by digestion with the restriction endonuclease Hae III to analyze potential Y chromosome/autosome translocations. Two female patients were studied who each had an abnormal chromosome 22 with extra quinacrine fluorescent material on the short arm. The origin of the 22p+ chromosomes was uncertain after standard cytologic examinations. Analysis of one patient's DNA with the Y-specific repeated DNA probes revealed the presence of both the 3.4 and 2.1 kb Y-specific fragments. Thus, in this patient, the additional material was from the Y chromosome. Analysis of the second patient's DNA for Y-specific repeated DNA was negative, indicating that the extra chromosomal segment was not from the long arm of the Y chromosome. These two cases demonstrate that repeated DNA can distinguish between similar appearing aberrant chromosomes and may be useful in karyotypic and prenatal diagnosis.     

Characterization of human heterochromatin by in situ hybridization with satellite DNA clones

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.     

Improved detection of DNA aneuploidy in primary breast cancer using quantitative DNA image analysis in combination with fluorescent in situ hybridization technique

Summary To obtain more information about the relationship between numerical aberrations of chromosome 1 and the overall DNA content of breast cancer cells, fluorescent in situ hybridization with a pericentromeric probe for this chromosome and image analysis based densitometry were carried out on imprints of benign (15 cases, mainly fibroadenomas) and malignant breast disease (31 invasive ductal carcinomas out of 45 cases). The most pronounced aneuploidy was observed in invasive ductal and lobular carcinoma cases both by in situ hybridization and DNA content (76.7 and 75.0% were aneuploid). The frequency of cells with two spots for chromosome 1 was 48.3 and 51.5%, respectively, as compared to 80.3% in control lymphocytes. There was a weak overall correlation (r² = 0.83) between DNA content and copy number of chromosome 1 in the malignant samples, although some of the DNA diploid/near diploid carcinomas showed a marked aneusomy for this chromosome. Also, some aberrations were present in the benign breast disease samples. Classification of cases by a linear discriminant analysis was most accurate when both techniques were combined (77% of cases correctly classified, according to anatomo-pathological diagnosis). The variables which received the highest weight in the linear discriminant function are the percentage DNA-diploid cells and the fraction of cells with two spots for chromosome 1. The sensitivity and sources of error of both techniques is considered.     

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.     

Detection of Y-specific repeat sequences in normal and variant human chromosomes using in situ hybridization with biotinylated probes

In situ hybridization with a cloned human Y-specific repeat, pY3.4, derived from the 3.4-kb HaeIII repetitive sequences, is useful in identifying Yq-autosome translocations. In this study nonradioactive procedures were also employed to detect the sites of hybridization. Using a biotinylated probe and either immunofluorescence or horseradish peroxidase reaction, the chromosomes of three probands and members of their families with probable Y-autosome translocations were examined. It was found that not all such translocations can be correctly diagnosed based on conventional banding analysis. The present data indicate the importance of chromosome-specific probes in studying chromosome rearrangements in man.     

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.     

Characterization of a (Y;4) translocation by DNA hybridization

Summary A phenotypically normal male with azoospermia was found to have a translocation between the short arm of the Y chromosome and the distal long arm of a chromosome 4. By cytogenetic analysis it could not be determined whether the translocation was reciprocal, nor whether it was balanced. In situ DNA hybridization with two pseudoautosomal and one Y-specific probe demonstrated that the breakpoint was on distal Yp and that there was Y chromosome material on 4q. Thus the translocation was reciprocal and could be characterized as t(Y;4)(pll;q32). There was no evidence for loss of Y-DNA sequences as judged by Southern blotting with Y-DNA probes. Thus the translocation may be balanced. We conclude that DNA hybridization can be used to refine considerably the cytogenetic analysis of such translocations.     

Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter’s karyotype: a FISH analysis

Meiotic segregation of the sex chromosomes was analysed in sperm nuclei from a man with Klinefelter’s karyotype by three-colour FISH. The X- and Y-specific DNA probes were co-hybridized with a probe specific for chromosome 1, thus allowing diploid and hyperhaploid spermatozoa to be distinguished. A total of 2206 sperm nuclei was examined; 958 cells contained an X chromosome, 1077 a Y chromosome. The ratio of X : Y bearing sperm differed significantly from the expected 1 : 1 ratio (χ² = 6.96; 0.001 < P < 0.01). Sex-chromosomal hyperhaploidy was detected in 2.67% of the cells (1.22% XX, 1.36% XY, 0.09% YY) and a diploid constitution in 0.23%. Although the frequency of 24,YY sperm was similar to that detected in fertile males, the frequencies of 24,XX, 24,XY and diploid cells were significantly increased. A sex-chromosomal signal was missing in 4.26% of the spermatozoa. This percentage appeared to be too high to be attributed merely to nullisomy for the sex chromosomes and was considered, at least partially, to be the result of superposition of sex-chromosomal hybridization signals by autosomal signals in a number of sperm nuclei. The results contribute additional evidence that 47,XXY cells are able to complete meiosis and produce mature sperm nuclei. Received: 6 November 1996     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Rapid methods to visualize Y-specific repeated DNA sequences in cytological preparations

We described rapid methods to detect Y-specific repeated DNA sequences in cytological preparations using in situ hybridization. A human Y chromosome specific DNA probe with an insert equivalent to that in pHY2.1 was labelled with [alpha-32P]dCTP or photobiotin, and hybridized to chromosome preparations. Signals were visualized specifically on Y chromosomes after 1 day's autoradiography or a couple of hours treatment with streptavidin alkaline phosphatase/BCIP/NBT. These methods are useful for molecular confirmation of Y-autosomal translocations.