DNA Probes For Chromosomes Research Articles

Comparison of γ-ray induced dicentric yields in human lymphocytes measured by conventional analysis and FISH

In an earlier work stable aberrations and dicentrics were determined by fluorescence in situ hybridization (FISH) after various doses of 137Cs γ-rays. No corresponding calibration curve for dicentrics is available for determinations in terms of the conventional analysis as performed in our laboratory, In view of the potential for the application of chromosome painting to human biological dosimetry, it is desirable to determine such a calibration curve and this and the comparison of the resulting data to those obtained in terms of the FISH method is the objective of the present communication. In the study it is found that the linear-quadratic dose response curves for dicentrics, that are determined by the two methods, are significantly different, although the different target sizes are accounted for. A similar problem was found earlier for X-rays. It does not appear that the difference is due to technical difficulties in the FISH method, that has been improved by employing in addition to the whole chromosome DNA probes, a pan-centrometric DNA probe

A fluorescence in situ hybridization (FISH) analysis with centromere‐specific DNA probes of chromosomes 3 and 17 in pleomorphic adenomas and adenoid cystic carcinomas

Aberrations of chromosomes 3 and 17 were studied by FISH using centromere-specific DNA probes in 11 salivary adenoid cystic carcinomas (ACC) and 8 salivary pleomorphic adenomas (PA), with 3 lymph nodes as controls. Two hybridized signals were detected in 92.8 +/- 2.7% of controls, 73.2 +/- 7.0% of PA and 66.8 +/- 7.9% of ACC cells for chromosome 3, and in 90.4 +/- 2.3% of controls, 59.5 +/- 25.0% of PA and 44.8 +/- 20.2% of ACC for chromosome 17. More than 3 hybridized signals, which indicate polysomy, were observed in 3.1% of controls, 15.5% of PA and 22.9% of ACC cells for chromosome 3, and in 1.2% of controls, 10.3% of PA and 23.1% of ACC cells for chromosome 17. A single hybridized signal was much more frequent for chromosome 17 than for chromosome 3. These findings suggest that polysomy of both chromosomes occurs during the development of salivary gland tumors, and its frequency is increased in adenoid cystic carcinoma as compared to pleomorphic adenoma. In addition, monosomy of chromosome 17 could possibly be significant in salivary gland tumors.

Comparison of periodontal disease in HIV seropositive subjects and controls (ll).

The aims of this study were to compare the prevalence of suspected periodontal pathogens in subgingival plaque from 29 HIV seropositive and 27 control subjects and to determine the association of these bacteria with periodontal destruction. Subgingival plaque was collected from the mesiobuccal sites of all teeth, except 3rd molars. Bacteria were identified and enumerated using non-isotopic whole chromosomal DNA probes and a colony lift method. At baseline, HIV seropositive subjects had significantly higher mean % of Porphyromonas gingivalis than control subjects. This difference could be attributed to a subgroup of HIV seropositive subjects with widespread attachment loss. No correlations were observed between the mean %s of DNA probe species and mean attachment loss, CD4 and CD8 T lymphocyte counts or CD4: CD8 ratio. No significant microbiological differences were detected between active and control sites in HIV seropositive subjects on a longitudinal basis. There appeared to be an inverse relationship between the mean %s of P. gingivalis and V. parvula, with respect to progression of HIV infection. The ability of microbiological parameters to predict site-specific breakdown in HIV seropositive subjects requires further investigation.

Marker chromosome 21 identified by microdissection and FISH

A child without Down syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with alpha-satellite DNA probes for acrocentric chromosomes, and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21.

Chromosome painting in highly irradiated Chernobyl victims: a follow-up study to evaluate the stability of symmetrical translocations and the influence of clonal aberrations for retrospective dose estimation.

Follow-up fluorescence in situ hybridization (FISH) measurements of symmetrical translocations were performed in peripheral blood lymphocytes from 12 highly irradiated victims of the Chernobyl nuclear power plant accident biannually, between September 1991 and July 1994, to investigate the persistence of these aberration type with time post-exposure. Translocations were determined using biotin-labelled painting DNA probes for human chromosomes 1, 4 and 12 and a digoxigenin-labelled alpha-satellite pancentromeric DNA probe. In 11 of 12 cases the translocation frequencies remained fairly constant during the observation period, which allows to generate comparable dose estimates on the various sampling times. In one case (no. 9) the existence of a cell clone containing the consistent chromosome rearrangement t(1;13) (q25;q14) was identified using FISH in rehybridized slides with a digoxigenin-labelled painting DNA probe for chromosome 13 and a separate G-banding analysis. To obtain reliable dose estimates, total translocation frequency has to be corrected for the high contribution (16.5-23.5%) of this clonal translocation.

Extended abstract: Chromosome rearrangements involved in transformation of human T‐Cell leukemia virus type I infected human lymphocytes

AbstractFollowing human T‐cell leukemia virus type I (HTLV‐I) infection, human lymphocytes (Coculture‐5) acquired capacity to grow continuously in the presence of cytokine. Coculture‐5 cells transformed (UV‐1) following ultraviolet (UV) irradiation. Coculture‐5 and UV‐1 cells had abnormalities of chromosomes 1 and 7. Coculture‐5 cells also transformed (Coculture‐15) following cocultivation with UV‐1 cells. In Coculture‐15, 18 structural abnormalities were observed. Of these, 6 were of chromosome 1 and 5 were of chromosome 7. The marker chromosome of UV‐1 cells, i(7q), was seen in up to 80% of Coculture‐15 cells. All UV‐1 cells examined had i(7q) with single centromeric G‐band, whereas 60% of Coculture‐15 cells had i(7q) with double centromeric G‐bands. By fluorescence in situ hybridization (FISH), an alpha satellite centromeric DNA probe for chromosome 7 gave a single band in i(7q) in UV‐1 and single or double bands in i(7q) in Coculture‐15 cells. These findings suggest that integration of HTLV‐I proviral DNA into chromosomal DNA causes fragility of certain chromosomes of infected cells and leads them to transformation. © 1995 Wiley‐Liss, Inc.

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.

DNA aneuploidy in prostatic adenocarcinoma: A frequent event as shown by fluorescence in situ DNA hybridization

Fluorescence in situ hybridization (FISH) using specific DNA probes for chromosomes 1, 7, 10, and Y was performed on 53 prostatic tissue samples obtained from 33 radical prostatectomy specimens and two benign control specimens. The 53 samples from carcinomatous prostates included 33 cancerous and 20 noncancerous samples. Additionally, four metastatic lymph node specimens were examined. Clonal chromosome abnormalities were observed in 78% of the tumors studied. They were detected in a higher proportion in stage pT2 and pT3 tumors (86% and 88%, respectively) compared with stage pT1 tumors (25%). No stage pT4 tumor was analyzed. There was evidence of remarkable focal intratumoral heterogeneity documented by the study of two samples from the same tumor in three of six cases. Comparing FISH determined ploidy patterns with DNA flow cytometry (FCM) in 22 samples, FISH showed aneuploidy whereas FCM showed none.

Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells.

We describe the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase-Fast Red immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.

Numerical abnormalities of chromosome 7 in human prostate cancer detected by fluorescence in situ hybridization (FISH) on paraffin‐embedded tissue sections with centromere‐specific dna probes

Fluorescence in situ hybridization (FISH) using chromosome-specific alpha-satellite DNA probes for chromosomes 7, 8, and 12 was performed on paraffin-embedded tissue sections and touch imprint preparations of 53 cases of human prostate cancer. Subsequent haematoxylin and eosin (H & E) staining of the hybridized tissue sections allowed unambiguous assignment of hybridization signals either to tumour or to non-tumorous parenchyma. Fifty-three cases of human prostate cancer were evaluated for numerical aberrations of chromosome 7. Scoring 200 cells of tumour and non-tumorous parenchyma in each case revealed abnormalities exclusively in tumour parenchyma in 41 cases (77 per cent). Ten of 41 cases (24 per cent) showed trisomy 7, and 15 cases (37 per cent) monosomy 7 or trisomy 7 in combination with monosomy 7, respectively. Sixteen cases (39 per cent) exhibited polysomy 7 in cells of the tumour parenchyma. In the tumour tissue in one case, different polyploid clones (triploid, tetraploid) and polysomy 7 could be identified by double hybridization with chromosome-specific DNA probes for chromosome 7, plus 8 or 12. The indicated numerical aberrations of chromosome 7 were correlated with 78 per cent of advanced pathological stages or poorly differentiated tumours (pT3/4 or G3) of prostate carcinomas. A statistical analysis of the data revealed significant relationships of particular numerical abnormalities of chromosome 7 to different pathological categories (pT, G, pN) of tumour classification. For the T-classification, the frequency of cells carrying polysomy 7 and polysomy 7/+7 increases significantly from pT1 to pT3/4 (P = 0.022).(ABSTRACT TRUNCATED AT 250 WORDS)

Monospermic polyploidy and atypical embryo morphology

Monospermic cleavage-arrested human embryos were analysed by fluorescence in-situ hybridization using specific DNA probes for chromosomes X, Y and 18 simultaneously. Two groups of monospermic polyploid embryos could be distinguished: (i) embryos (n = 13) with only one large cell surrounded by smaller blastomere-sized extracellular fragments. These embryos were polyploid and frequently polyploid mosaic. (ii) Embryos developing from larger than normal oocytes were triploid or triploid mosaics (n = 4). Atypical morphology was not seen in eight other polyploid monospermic embryos. The atypical morphologies described here are quite rare, but are genetically uniform. Polyspermic embryos are the only other known example of a dual genetic-morphological abnormality.

Chromosome painting analysis of X-ray-induced aberrations in human lymphocytes in vitro.

Chromosomal rearrangements in human lymphocytes induced by X-rays (0, 0.5, 1.0 and 2.0 Gray) were analyzed using chromosome painting. DNA probes for human chromosomes 1, 3 or 4 alone, and a combination of 1 and 4, were used for analysis. The frequency of cells with rearrangements, i.e. reciprocal translocations, dicentrics, insertions, tricentrics and fragments, involving chromosome 4 increased with dose in both 48 and 72 h cultures. The number of translocations per cell also increased with dose at 48 and 72 h. Dicentrics increased with dose in 48 h but not in 72 h cultures. The estimated genomic frequency of aberrations per cell was comparable with results in banded cells. No difference was shown on the detection efficiency of chromosome rearrangements among the various DNA probes used. Since this technique does not necessarily require well-spread metaphases for analysis, it is possible to increase the number of analyzable metaphases compared with the banding technique. Chromosome painting is a simpler, more objective and more practical method for detecting chromosome rearrangements than conventional banding analyses.

Phylogenetics of Salmonella enteritidis

The phylogenetics of Salmonella enteritidis is reviewed. Data from RFLP typing with cloned chromosomal DNA, rRNA genes and insertion sequence probes are described. Human isolates of this serovar exhibit a high degree of genotypic homogeneity. Multilocus enzyme electrophoresis defines S. enteritidis as a polyphyletic serovar closely related to S. dublin, S. gallinarum and S. pullorum. Two main classes of virulence plasmids are found in S. enteridis. We report a comparative study of genotype for three principal clones of S. enteritidis, six other serovars carrying the g flagellar antigen, and S. pullorum. It is suggested that S. enteritidis occupies an ancestral and pivotal position among these Salmonellae.

Simultaneous hybridization and subsequent colour detection of subgingival bacterial DNA on colony lifts

This paper reports the development of a protocol allowing hybridization and detection of DNA fixed to nylon colony lifts from up to three species of bacteria simultaneously. Half ml samples of serial dilutions of pure cooked-meat broth (CMB) cultures of Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia were grown on trypticase soy blood agar (TSBA) plates for 7 days in an anaerobic chamber. From the same CMBs a further set of dilutions was completed that contained all three species. Samples from these dilutions produced mixed-growth TSBA plates following anaerobic incubation for 7 days. After incubation, colony counts on pure-growth TSBA plates were enumerated by colony counter. Colony counts of C. ochracea, A. actinomycetemcomitans and P. intermedia on mixed-growth TSBA plates were enumerated by nylon colony lift, simultaneous hybridization with non-isotopic whole chromosomal DNA probes and alkaline phosphatase substrates generating three colours. The results indicate that the protocol correctly identified and differentiated between the three species on mixed-growth TSBA plates. The proportions of each species and mean total colony count expected by counting pure plates were in agreement with the proportions of each species and total colony counts enumerated by DNA probes on mixed growth plates. The development of simultaneous hybridization and multicolour detection will result in improved data recovery from dental plaque samples, in addition to reducing the cost and labour required in current colony-lift protocols, without affecting the specificity or sensitivity of the probes used.

Heterogeneity in bladder cancer as detected by conventional chromosome analysis and interphase cytogenetics

Thirty transitional cell carcinomas (TCCs) of the bladder were examined by classical chromosome counting to establish range, modal number, and percentage of metaphases with 2n, 3n, 4n, and ≥ 5n chromosomes. In addition, fluorescence in situ hybridization (FISH) was applied to interphase nuclei to detect the percentage of tumor cells showing polyploidization and chromosome imbalance. In FISH, centromere-specific DNA probes for chromosomes 1, 7, 9, and 11 were used. The tumors were analyzed flow cytometrically to determine the DNA index (DI). Fourteen of 21 cases (67%) having a DI = 1 showed, after classical chromosome counting, in addition to a diploid model number, some cells with a 3n and 4n chromosome count. With FISH, eight cases (38%) showed a low percentage of cells with multiple signals for each of the probes, thus indicating polyploidization. In 13 (62%) cases, an imbalance between different chromosomes was detected. In nine tumors having a DI of 1.6 to 1.9, classical chromosome counting showed low percentages of ≥ 5n cells in four cases, in addition to a triploid modal number. With FISH in six cases, a low percentage of cells showed five or more signals for each of the chromosomes, indicating polyploidization. In all cases, a chromosome imbalance was detected. With classical chromosome counting not all tumors can be analyzed. With FISH, small percentages of polyploid cells are not recognized. Both methods complement each other in that chromosome counting allows readier detection of heterogeneity in DNA-diploid tumors after polyploidization, whereas FISH allows efficient recognition of the chromosomes involved in the process of imbalance.

A Fluorescence in Situ Hybridization Map of Human Chromosome 21 Consisting of 30 Genetic and Physical Markers on the Chromosome: Localization of 137 Additional YAC and Cosmid Clones with Respect to This Map

A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome. Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map. For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter. The location of the markers was established with a standard error of +/- 1.9 Mb using from 9 to 63 FL measurements for each probe. The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome. Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome. The cosmids, chosen at random from a flow-sorter chromosome 21 cosmid library, show some biases in chromosome distribution.

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella choleraesuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Radiation-induced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe.

Fluorescence in situ hybridization with composite whole chromosome-specific DNA probes for human chromosomes 1, 4 and 12 and a degenerate alpha-satellite pancentromeric DNA probe labelled with digoxigenin was used to measure symmetrical translocations and dicentrics induced in vitro by 137Cs gamma-rays (0-6.0 Gy) in peripheral lymphocytes. Despite subtracting our mean background translocation frequency of 0.0016 per cell (11,411 cells scored from 11 individuals) from induced frequencies, about 1.3-1.8-fold more translocations were found than dicentrics at a given dose. Translocation frequencies determined only in stable cells agree well with total translocation frequencies determined also in cells containing additional unstable chromosomal changes. The linear quadratic calibration curve generated for total stable translocations is based on approx. 17,000 cells. The suitability of this curve for biological dosimetry of human radiation exposure can now be evaluated in comparison with dose estimates based on a conventional dicentric dose-response curve.

Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats

A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific — differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled wh probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

Isolation and characterization of Y chromosome DNA probes

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46, XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests althouth in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.