Satellite DNA Probe Research Articles

Trisomy 12 is a rare cytogenetic finding in typical chronic lymphocytic leukemia

We have studied 61 cases of B-chronic lymphocytic leukemia (CLL), combining cytological features, conventional cytogenetics and in situ hybridization (ISH). The comparison of these results constitutes the main subject of this study. The patients were cytologically classified according to the FAB criteria as: chronic lymphocytic leukemia (CLL) typical type (48 cases) and CLL atypical types (13 cases). Chromosome analysis was carried out on lymphoid cells from peripheral blood. The following mitogens were used: phytohemagglutinin (PHA) 5%, pokeweed (PWM) and lipopolysaccharide from E. coli. The ISH was performed with a biotinlabeled, chromosome 12-specific alpha satellite DNA probe, pSP12-1. Trisomy 12 was not found in any of the 48 patients with the typical type of CLL and in contradistinction it was present in some patients with atypical types. This study emphasizes the great importance of a closer link between hematological morphology and the cytogenetic approach.

Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male.

Meiotic segregation of gonosomes from a 46,XY/47,XXY male was analysed by a three-colour fluorescence in situ hybridisation (FISH) procedure. This method allows the identification of hyperhaploid spermatozoa (with 24 chromosomes), diploid spermatozoa (with 46 chromosomes) and their meiotic origin (meiosis I or II). Alpha satellite DNA probes specific for chromosomes X, Y and 1 were observed on 27,097 sperm nuclei. The proportions of X- and Y-bearing sperm were estimated to 52.78% and 43.88%, respectively. Disomy (24,XX, 24,YY, 24,X or Y,+1) and diploidy (46,XX, 46,YY, 46,XY) frequencies were close to those obtained from control sperm, whereas the frequency of hyperhaploid 24,XY spermatozoa (2.09%) was significantly increased compared with controls (0.36%). These results support the hypothesis that a few 47,XXY germ cells would be able to complete meiosis and to produce mature spermatozoa.

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide.

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.

Analysis of radiation-induced micronuclei in two-cell human-hamster embryos using telomeric and centromeric FISH probes.

Simultaneous, fluorescent in situ hybridization using a centromeric human alpha satellite DNA probe and a telomeric DNA probe was used to analyze the chromosome content of micronuclei induced in two-cell human-hamster embryos by in vitro gamma-ray irradiation of human spermatozoa. In unirradiated samples, about 26% of micronuclei were centromere positive, indicating that both structural chromosome aberrations and numerical changes are involved in the spontaneous production of micronuclei. After exposure of spermatozoa to radiation, a significant increase in the number of micronuclei was found. About 77% of induced micronuclei contained only telomeric signals suggesting that they originated from acentric fragments. However, both centromere-positive and centromere-negative micronuclei increased with radiation dose. These results are consistent with the well known clastogenic effect of ionizing radiation and with its weak aneugenic effect.

Detection of centromeres in vinblastine- and radiation-induced micronuclei of human lymphocytes using FISH with an α satellite pancentromeric DNA probe

Fluorescence in situ hybridisation (FISH) with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in micronuclei of human lymphocytes induced by gamma irradiation and by Vinblastine sulfate. In a cytokinesis-block micro-nucleus assay a dose-dependent increase of micronuclei was detected for both agents. 72-89% of vinblastine-induced micronuclei, but only 7-48% of radiation-induced micronuclei showed centromere-positive fluorescence signals. Vinblastine treatment frequencies of centromere-negative micronuclei did not increase compared to control values, nor did frequencies of centromere-positive micronuclei in irradiated lymphocytes. Since FISH with an alpha satellite DNA probe allows the direct detection of centromeric DNA sequences the spindle damaging or clastogenic effectiveness of a compound can be easily and reliably examined in a cytokinesis-block micronucleus assay in human lymphocytes.

Classification of X-ray-induced Robertsonian fusion-like configurations in mouse splenocytes.

A characteristic karyotypic feature of mouse chromosomes is the presence of large blocks of heterochromatin in the vicinity of the centromeres. Breakage inside this centromeric heterochromatin might result in the formation of abnormal chromosomes, very similar to the metacentric chromosomes derived from Robertsonian fusion. X-rays are very efficient in inducing these Robertsonian fusion-like configurations (RLC) in cultured mouse splenocytes. Observed frequencies of these RLC increase in a linear-quadratic manner with dose. Two types of RLC were found. The first type (approximately 70% of induced RLC) has heterochromatin only in the middle of the chromosome and appears as a metacentric chromosome, whereas the other type (approximately 30%) has a heterochromatic block inside the chromosome arm (and has the appearance of a dicentric chromosome). Induced RLC are difficult to classify as either a stable or unstable aberration, based only on traditional cytogenetic techniques such as C-banding. Here we describe a cytogenetic approach to achieving better insight into the molecular organization of RLC. We utilized two-colour fluorescence in situ hybridization (FISH) using a combination of mouse minor satellite DNA probe and telomeric probe. Over 90% of RLC did not have detectable minor satellite arrays inside the interstitial heterochromatin. Consequently, most of the RLC of the first type should be classified as acentric fragments and those of the second type as translocations. The chance of inducing true Robertsonian fusions in mouse splenocytes by X-rays is < 2.5% based on the total RLC observed.

Effects of 5-azacytidine on the centromeric region of human fibroblasts studied by CREST staining and in situ hybridization on cytokinesis-blocked cells.

Several interphase methodologies have been applied in this study to investigate whether chemically induced undercondensation of the pericentromeric region of human fibroblast chromosomes promotes structural or numerical aberrations at the cell cycles following treatment. To achieve this aim, the effects of the hypomethylating agent 5-azacytidine (5- azaC) were studied on cytokinesis-blocked binucleated fibroblasts. This approach allowed the distribution of whole chromosomes or chromosome fragments in the daughter nuclei and micronuclei of treated cells to be followed, since the daughter nuclei of a single mitosis are maintained in one cytoplasm by treatment with the actin inhibitor cytochalasin B. Antikinetochore staining and in situ hybridization with an alpha-satellite probe capable of detecting all human centromeres on 5-azaC-induced micronuclei in binucleated fibroblasts indicated that the predominant effect of the chemical is to induce micronuclei lacking centromeres, suggestive of induced chromosome aberrations. Double in situ hybridization with alphoid and classical satellite DNA probes specific for chromosome 1 on binucleated fibroblasts was used to discriminate induced aneuploidy from breakage effects in the target area of hybridization. The results showed that undercondensation of the pericentromeric heterochromatin of human fibroblast chromosomes by treatment with 5-azaC produces structural chromosome aberrations involving the classical satellite DNA, whereas there was no evidence that the chemical induced chromosomal aneuploidy.

Loss of telomeric sites in the chromosomes of Mus musculus domesticus (Rodentia: Muridae) during Robertsonian rearrangements.

Mouse chromosomes possessing multiple Robertsonian rearrangements (Rb chromosomes) have been examined using fluorescence in situ hybridization with the telomeric consensus sequence (TTAGGG)n. No hybridization signals were detected at the primary constriction of Rb chromosomes. This observation leads us to conclude that the formation of Rb chromosomes in the mouse is invariably associated with the loss of telomeric regions. More significantly, a further alteration in regions flanking the primary constrictions was observed after hybridizing with a minor satellite DNA probe to Rb chromosomes. It seems likely that the breakpoints required for a Robertsonian process do not include telomeric sites exclusively but extend to the adjacent pericentromeric regions of the original acrocentric chromosomes. In contrast to previous reports, these observations demonstrate the elimination of substantial amounts of chromosomal DNA during the formation of mouse Rb chromosomes.

Monosomy of chromosome 18 detected by fluorescence in situ hybridization in colorectal tumors.

At least two different evolutional pathways of colorectal cancer, namely the adenoma-carcinoma sequence and de novo carcinogenesis, have been indicated. However, whether there is a difference between them in affected chromosomes and genes has not yet been elucidated. Chromosomal examination is expected to provide a clue to an answer to this question. In this study, the relation of aberrations in chromosome 18 to type of colorectal cancer was examined. Numeric aberrations in chromosome 18 were investigated in 71 colorectal tumors by means of fluorescence in situ hybridization, using an alphoid satellite DNA probe specific for the pericentromeric region on chromosome 18. The loss of one chromosome 18 was found in 33% (6 of 18) of patients with early cancer, excluding those with cancer in an adenoma. The loss was frequent in early colorectal carcinomas without foci of adenoma (four of six patients, 67%), whereas monosomy 18 was not significant in those with foci of adenoma except for patients with a hereditary background. Specimens exhibiting monosomy 18 were macroscopically classified as flat lesions, but the converse was not true. No adenoma showed monosomy 18. It was encountered in 44% of nine cancers with invasion to the muscularis propria. However, no significant subpopulation of monosomy 18 was present in cancers penetrating through the serosa or adventitia in which polysomic populations were often identified alternatively. Furthermore, tetrasomy for this chromosome was exclusive in these advanced cancers. The loss of chromosome 18 is closely related to colorectal cancers without foci of adenoma.

Cross‐hybridization of the chromosome 13/21 alpha satellite DNA probe to chromosome 22 in the prenatal screening of common chromosomal aneuploidies by fish

In a routine application of commercially available centromeric DNA probes for the prenatal screening of common trisomies involving the autosomes 13, 18, and 21, and sex chromosomes, four cases of discrepancy between fluorescence in situ hybridization (FISH) results and follow-up cytogenetic analysis were observed from a total of 516 cases of amniocentesis. In three of these cases, the results were false negative, and in one false positive. In this case, amniocentesis was performed because of a positive triple test in a 34-year-old woman with previous infertility treatment. The alpha satellite DNA probe for chromosomes 13/21 revealed five signals in 50 per cent of uncultured amniocytes, while standard cytogenetic analysis showed a normal karyotype. FISH analysis on metaphase chromosomes demonstrated the location of the additional signal in the centromeric region of chromosome 22. This additional signal was also present in the centromeric region of chromosome 22 of the mother, providing evidence for a possible inherited polymorphism in chromosome 22 responsible for unspecific hybridization with the alpha satellite probe for chromosomes 13/21 in this case. The observed polymorphism in centromeric regions may contribute to unreliability of the use of the 13/21 alpha satellite probe for prenatal screening by FISH.

Pulsed-field gel analysis of 5S and satellite DNA in barley

Pulsed-field gel electrophoresis was used to study the variability of clustered tandemly repeated sequences in barley. Twelve spring barley cultivars were investigated with a heterologous 5S DNA probe and the 118 base pair barley satellite DNA probe HVT01. On a per fragment basis, the 5S probe was 5 times and the barley satellite probe 6.7 times more variable than single- or low-copy RFLP markers, demonstrating their usefulness for cultivar distinction.

Analysis by fluorescence in situ hybridization with a mouse gamma satellite DNA probe of isolated micronuclei induced in mice by two clastogens and two spindle poisons.

The presence of centromeric DNA was studied in micronuclei isolated from the blood of male ddY mice after five weekly intraperitoneal injections of mitomycin C (MMC), 1-beta-D-arabinofuranosylcytosine (Ara-C), colchicine (COL) or vinblastine sulfate (VBL). In agreement with our earlier findings, about half of the micronuclei isolated from vehicle control mice showed centromere signals as analyzed by fluorescence in situ hybridization (FISH) with a mouse major (gamma) satellite DNA probe. In an earlier experiment with mice acutely exposed to the same chemicals, the clastogens MMC and Ara-C did not reduce the proportion of micronuclei with centromere signals. In the present study, however, MMC and Ara-C decreased the proportion of micronuclei with centromeres. In contrast, the spindle poisons COL and VBL increased the proportion of micronuclei that contained centromeres.

Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes. Fluorescence in situ hybridisation with a specific classical satellite DNA probe, showed that this differential labelling was dependent on a greater sensitivity of chromosome 9 constitutive heterochromatin to exonuclease III digestion in amniocytes than in lymphocytes, thus indicating qualitative differences in this region between both human cellular materials.

Potential use of buccal smears for rapid diagnosis of autosomal trisomy or chromosomal sex in newborn infants using DNA probes

Buccal smears from 3 women and 1 man were probed with alpha satellite DNA probes for chromosomes 8, 18, X, and Y. Buccal smears were also collected from an adolescent phenotypic female with uterine agenesis, as well as from newborn infants with suspected trisomy 18 and trisomy 21. The clinical cases were confirmed with conventional cytogenetic studies of peripheral lymphocytes. Overall probe efficiency at detecting expected chromosome number in interphase cells was found to be 71% +/- 6.8%. Higher than expected n-1 signal numbers may be due to karyopyknotic intermediate epithelial cells present in all collected samples. Overall probe efficiency was found to be consistent using alpha satellite and cosmid probes, both of which accurately reflected the modal copy number of the target chromosomes. False trisomy was less than 1%. This study suggests DNA probes can be used in buccal smears for rapid diagnosis of trisomies and chromosomal sex in newborns, but because of high rates of false hypoploid signals, probed buccal smear specimens may not be accurate at diagnosing mosaicism.

Detection of i(17q) chromosome by fluorescent in situ hybridization (FISH) with interphase nuclei in medulloblastoma

Medulloblastomas are the most frequent primitive neurectodermal tumors in children. An isochromosome for the long arm of 17, i(17q), is found in 30% of medulloblastomas. For some authors, this abnormality is observed in cases with a shorter survival time. In our cytogenetic studies of 30 medulloblastomas, we observed i(17q) in only three cases, a monosomy 17 in two cases, a monosomy 22 in four cases, nonspecific numerical or structural abnormalities in five cases, and normal karyotypes in 12 cases. We compared the results of karyotypic analysis after culture and FISH with a chromosome 17 alpha satellite DNA probe on interphase nuclei in five cases of medulloblastoma. In one case, i(17q) was only observed in four cells in karyotypic analysis, in three cases a normal karyotype was found, and in one case karyotypic analysis was impossible. In all of these cases, i(17q) was observed in a great number of nuclei by FISH on interphase nuclei. Our study shows that the FISH on interphase nuclei permitted us to observe i(17q) in the cases where it was not or could not be completely detected by karyotypic analysis. The association of these two techniques is required to detect i(17q), an abnormality whose prognosis value in medulloblastomas is now recognized.

Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.

Flow-cytometric quantification in human gliomas of alpha satellite DNA sequences specific for chromosome 7 using fluorescence in situ hybridization.

Cell suspensions prepared from freshly frozen tissue specimens were used to examine aberrations in the number of chromosome 7 signals in 10 human gliomas. Nuclear DNA was hybridized in vitro with an alpha satellite DNA probe specific for the centromeric regions of chromosome 7, using fluorescence in situ hybridization (FISH). The intensity of the fluorescence signal from the hybridized probe was measured, together with the nuclear DNA content, by flow cytometry. The mean probe fluorescence of all nuclei was compared to the mean copy number per nucleus found with microscopic scoring. Moreover, the mean probe fluorescence ratio of DNA aneuploid nuclei relative to DNA diploid nuclei (FISHa/FISHd) was calculated to determine how the numerical aberration of chromosome 7 signals contributes to the DNA ploidy of the sample. The results from the flow-cytometric analysis and from microscopic evaluation were compatible. One of four tumors with DNA diploidy had a higher average intensity of FISH signal and a broader coefficient of variation in FISH signal than normal brain tissue; this was shown to be due to the gain of chromosome 7 signals. Although FISHa/FISHd correlated with DNA indices (P < 0.01), there were some disparities, probably due to other complex genotypic associations involving several gains or losses of chromosomes. Thus gain of chromosome 7 in gliomas is related to both DNA ploidy change and chromosome specific gain. It is concluded that flow-cytometric quantification of FISH is useful in investigating numerical aberrations of chromosomes and nuclear DNA content simultaneously.

Interphase Cytogenetics of Lung Tumors Using In Situ Hybridization: Numerical Aberrations

ObjectivesSince conventional cytogenetic analysis for bronchogenic carcinogenesis is limited by the difficulty to get enough number of high quality metaphase spreads, the development of new method to overcome above problems is strongly needed. Therefore, the introduction of non-radioactive in situ hybridization (ISH) with pericentromeric chromosome probes gave us the way to investigate the genetic events during carcinogenic process. We applied this method on lung cancer tissue to validate the possibility of this method for general usage and to analyze numerical chromosome aberration status and their clinical correlations.MethodsA set of satellite DNA probes specific for chromosome 3, 7, 9, 11, and 17 was hybridized directly to paraffin-embedded tissue section of 30 non-small cell lung cancers. Mean chromosome index of each chromosome and frequency of polysomy for each chromosome were calculated.ResultsMean chromosome indices for chromosome 3, 7, 9, 11, and 17 were 1.10, 1.13, 1.17, 1.12, and 1.17. respectively. Polysomy for a set of chromosomes was detected in all 30 cases except 4 cases which showed hypoploidy only for chromosome 3 or 7 in 2 cases and diploidy only for chromosome 3 or 11 in 2 cases. Among the set of chromosomes, mean chromosome index and polysomy frequency for chromosome 9 & 17 were significantly higher than that for others. Mean chromosome index or polysomy pattern for each chromosome was not much different among cell types or clinical stages.ConclusionsOur results show that chromosome ISH can be used to screen for numerical chromosome aberrations on paraffin tissue sections and further studies for ISH analysis with different probes on same tumor area or double-target ISH in large scale are needed to confirm above results and to elucidate the specific meanings.

Gender Preselection in Humans? Flow Cytometric Separation of X and Y Spermatozoa for the Prevention of X-Linked Diseases

Human X- and Y-chromosome-bearing spermatozoa were separated based on their DNA content, using modified flow cytometric cell sorting technology. The resulting separation purity of the X-bearing from Y-bearing spermatozoa was evaluated using in-situ hybridization with alpha satellite DNA probes for the X- and Y-chromosomes. In the putative X-enriched-sorted populations, an average of 82% of the spermatozoa showed a hybridization signal with the X probe. Similarly, in the Y-sorted population 75% gave a signal with the Y probe. Sorted X- and Y-bearing spermatozoa were found to maintain their viability for several hours after sorting. These results demonstrate that the human sperm sex ratio can be significantly shifted to favour the selection of female-producing (X) spermatozoa or male-producing (Y) spermatozoa when spermatozoa are flow cytometrically sorted on the basis of DNA content. We propose that flow cytometrically sorted human spermatozoa, used in conjunction with in-vitro fertilization or intra-oviductal insemination, could be used by families who are at risk for X-linked diseases to preferentially produce female offspring. Sorted spermatozoa could also be used to pre-select for male offspring if that were medically indicated.

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells.

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10(-3) M MTX-res H2 and the 5 x 10(-7) M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band of m as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a 3H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.