Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)     

Evidence of chromosomal inversion using fluorescence in situ hybridization to stretched DNA

The resolution of fluorescence in situ hybridization techniques (FISH) can be improved using techniques of DNA stretching. The so-called DIRVISH technique has been used to demonstrate the existence of an inversion involving a small chromosomal segment of the long arm of chromosome 14. This inversion was suspected, but not proven, in patients with familial Alzheimer disease. Two-colour FISH using YAC and cosmid probes allowed us to limit the rearranged region around YAC 964e2, which encompasses the Presenilin 1 (PR1) gene. The existence of small-sized inversions within the genome becomes, thus, open to microscope analysis.     

A complex chromosomal rearrangement detected prenatally and studied by fluorescence in situ hybridization

We report of case of a complex chromosomal rearrangement detected prenatally and studied with traditional banding methods and fluorescence in situ hybridization. The combination of these techniques showed that four chromosomes were involved in the translocation. Nine breakpoints were proposed to explain these results. Some of the findings could only be detected with fluorescence in situ hybridization, demonstrating the usefulness of this technique in characterizing chromosomal abnormalities that would otherwise be difficult to interpret correctly with classical cytogenetics alone.     

Localization of the beta-globin gene by chromosomal in situ hybridization

A 3.7-kilobase (kb) genomic clone of the human beta-globin gene, including 1.5-kb upstream and approximately 0.5-kb downstream, was utilized in chromosomal in situ hybridization for precise mapping of the beta-globin locus on peripheral blood lymphocyte-derived metaphases from a normal male, and for further evaluation of a clonal t(7;11) (q22;p15) translocation on bone marrow-derived metaphases from a 46-year-old male with erythroleukemia. Analyses of 205 midmetaphases from a normal male hybridized with the tritium-labeled beta-globin probe and stained with quinacrine mustard dihydrochloride revealed approximately 12% of spreads to have silver-grain deposition over the p15 band of chromosome 11. Of the 365 silver grains observed to be located on or beside chromosomes, 25 (approximately 7%) grains were localized in band p15. Karyotype analysis of a bone marrow specimen from the patient with erythroleukemia revealed hypodiploidy with various unidentified marker chromosomes as well as a presumably balanced translocation between 7q and 11p . Chromosomal in situ hybridization showed localization of silver grains at the junction between chromosomes 7 and 11 as well as to the normal chromosome 11, indicating that the beta-globin locus had not been translocated in the chromosomal rearrangement. This case demonstrates the value of chromosomal in situ hybridization in the definition of chromosome rearrangements and provides further evidence for the localization of the beta-globin gene to 11p15 .     

Using fluorescence in situ hybridization (FISH) in genome mapping.

Fluorescence in situ hybridization (FISH) provides one of the most effective and rapid approaches for assigning and ordering DNA fragments within single eukaryotic chromosome bands. These techniques have wide applications not only for the mapping of the human genome and the genomes of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.     

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.     

A and C genome distinction and chromosome identification in brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization

The two genomes (A and C) of the allopolyploid Brassica napus have been clearly distinguished using genomic in situ hybridization (GISH) despite the fact that the two extant diploids, B. rapa (A, n = 10) and B. oleracea (C, n = 9), representing the progenitor genomes, are closely related. Using DNA from B. oleracea as the probe, with B. rapa DNA and the intergenic spacer of the B. oleracea 45S rDNA as the block, hybridization occurred on 9 of the 19 chromosome pairs along the majority of their length. The pattern of hybridization confirms that the two genomes have remained distinct in B. napus line DH12075, with no significant genome homogenization and no large-scale translocations between the genomes. Fluorescence in situ hybridization (FISH)-with 45S rDNA and a BAC that hybridizes to the pericentromeric heterochromatin of several chromosomes-followed by GISH allowed identification of six chromosomes and also three chromosome groups. Our procedure was used on the B. napus cultivar Westar, which has an interstitial reciprocal translocation. Two translocated segments were detected in pollen mother cells at the pachytene stage of meiosis. Using B. oleracea chromosome-specific BACs as FISH probes followed by GISH, the chromosomes involved were confirmed to be A7 and C6.     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Experimental genome evolution: large-scale genome rearrangements associated with resistance to replacement of a chromosomal restriction-modification gene complex

Type II restriction enzymes are paired with modification enzymes that protect type II restriction sites from cleavage by methylating them. A plasmid carrying a type II restriction-modification gene complex is not easily replaced by an incompatible plasmid because loss of the former leads to cell death through chromosome cleavage. In the present work, we looked to see whether a chromosomally located restriction-modification gene complex could be replaced by a homologous stretch of DNA. We tried to replace the PaeR7I gene complex on the Escherichia coli chromosome by transducing a homologous stretch of PaeR7I-modified DNA. The replacement efficiency of the restriction-modification complex was lower than expected. Some of the resulting recombinant clones retained the recipient restriction-modification gene complex as well as the homologous DNA (donor allele), and slowly lost the donor allele in the absence of selection. Analysis of their genome-wide rearrangements by Southern hybridization, inverse polymerase chain reaction (iPCR) and sequence determination demonstrated the occurrence of unequal homologous recombination between copies of the transposon IS3. It was strongly suggested that multiple rounds of unequal IS3-IS3 recombination caused large-scale duplication and inversion of the chromosome, and that only one of the duplicated copies of the recipient PaeR7I was replaced.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

A fluorescence in situ hybridization technique for retrospective cytogenetic analysis

Fluorescence in situ hybridization (FISH) is being used increasingly in clinical practice; however, current FISH techniques require fresh material, and there is considerable variation in hybridization efficiency between laboratories. We have modified a FISH technique described by Pinkel et al. (1986) that works not only on freshly G-banded material but also on cytogenetic preparations ranging in age from 2 wk to 12 yr. We have tested this technique on several centromeric alphoid satellite probes (D1Z5, D7Z1, D17Z1, DXZ1, and DYZ3) and one noncentromeric minisatellite probe (D1Z2). Our average hybridization efficiency on freshly banded preparations for these probes is consistently greater than 90%. The combination of higher efficiency and the ability to perform hybridization on previously G-banded material makes this a valuable technique for retrospective analyses.     

Detection of chromosomal aneuploidy in endometriosis by multi-color fluorescence in situ hybridization (FISH)

Endometriosis affects 10–15% of women of reproductive age and is a common cause of infertility and pelvic pain. Although endometriosis is characterized by abnormal growth or turn-over of cells, the genetic changes involved remain unclear. We employed a multi-color fluorescence in situ hybridization (FISH) strategy to determine the incidence of somatic chromosomal numeric alterations in severe/late stage endometriosis. Using alpha-satellite sequence-specific DNA probes for chromosomes 7, 8, 11, 12, 16, 17, and 18, simultaneous two- and three-color FISH were performed to evaluate the frequency of monosomic, disomic, and trisomic cells in normal control and endometriotic tissue specimens. In one of four endometriosis samples studied, a significantly higher frequency of monosomy for chromosome 17 (14.8%, χ² ₄ = 53.3, P < 0.0001) and 16 (8.8%, χ² ₄ = 11.4, P < 0.05) was observed. An increased number of cells with chromosome 11 trisomy (14.8%, χ² ₄ = 96.2, P < 0.0001) were detected in a second case. In a third case, a distinct colony of nuclei with chromosome 16 monosomy (14.1%, χ² ₄ = 21.39, P < 0.005) was detected. Acquired chromosome-specific aneuploidy may be involved in endometriosis, reflecting clonal expansion of chromosomally abnormal cells. That candidate tumor suppressor genes and oncogenes have been mapped to chromosomes 11, 16, and 17 suggests that chromosomal loss or gain plays a role in the development and/or progression of endometriosis. Received: 27 December 1997 / Accepted: 14 April 1997     

Thirteen loci physically assigned to sheep Chromosome 2 by cell hybrid analysis and in situ hybridization

Sheep x hamster cell hybrids containing sheep metacentric Chromosome (Chr) 2 were produced by fusing blood leukocytes from normal sheep with hamster auxotrophic Ade F-minus mutants. Cell clones that were isocitrate dehydrogenase 1 (IDH1) positive were cytogenetically characterized, confirming that they contained sheep Chr 2. The following loci were newly assigned by Southern hybridization to sheep Chr 2: lipoprotein lipase (LPL), glycoprotein-4-beta galactosyltransferase 2 (GGTB2), neurofilament light polypeptide (68 kDa; NEFL), surfactant-associated protein 2 (SFTP2), lymphocyte-specific protein tyrosine kinase (LCK), and nebulin (NEB). These new assignments and the in situ localization of gelsolin (GSN) to sheep Chr 2pter-p24 are consistent with the predicted homology of cattle Chr 8 (U18) with sheep Chr 2p, and of cattle Chr 2 (U17) with sheep 2q. In addition, the assignment by cell hybrid analysis of loci previously mapped to Chr 2 in sheep, viz., cholinergic receptor, nicotinic, delta polypeptide (CHRND), collagen type III alpha 1 (COL3A1), fibronectin 1 (FN1), isocitrate dehydrogenase (IDH1), and villin 1 (VIL1), confirmed the localization of sheep syntenic group U11 to this chromosome. By nutritional selection and complementation of the hamster auxotrophic Ade F mutation, the multifunctional enzyme locus phosphoribosylaminoimidazolecarboxamide formyltransferase (AICAR transformylase)/IMP cyclohydrolase (inosinicase) (provisionally given the symbol PRACFT) has also been newly assigned to sheep Chr 2. This report significantly extends the number of loci physically mapped to sheep Chr 2 and confirms its close homology with cattle Chrs 2 and 8.     

The use of DNA hybridization in situ for identifying chromosomal rearrangements in the karyotyping of cell lines]

A complex karyological analysis of three human cell lines (PLC-PRF-5, MT-4 and U937) was carried out that involved traditional methods of G-, C-banding and silver staining, in addition to the in situ hybridization technique using 4 alpha-satellite DNA probes: DNA specific for centromeric regions of chromosome 11, 6, 13, and 21, and 14 and 22. The application of this additional method allowed to identify, prove or detalize the structure of 13 markers in PLC-PRF-5 cells, 1 marker in MT-4 cells, and 3 markers in U937 cells. The results show that the in situ hybridization method would be successful in cell line karyotyping for a more objective identification of some markers difficult for analysis.     

High-resolution fine mapping and fluorescence in situ hybridization analysis of sun, a locus controlling tomato fruit shape, reveals a region of the tomato genome prone to DNA rearrangements

The locus sun on the short arm of tomato chromosome 7 controls morphology of the fruit. Alleles from wild relatives impart a round shape, while alleles from certain cultivated varieties impart an oval shape typical of roma-type tomatoes. We fine mapped the locus in two populations and investigated the genome organization of the region spanning and flanking sun. The first high-resolution genetic map of the sun locus was constructed using a nearly isogenic F(2) population derived from a cross between Lycopersicon pennellii introgression line IL7-4 and L. esculentum cv Sun1642. The mapping combined with results from pachytene FISH experiments demonstrated that the top of chromosome 7 is inverted in L. pennellii accession LA716. sun was located close to the chromosomal breakpoint and within the inversion, thereby precluding map-based cloning of the gene using this population. The fruit-shape locus was subsequently fine mapped in a population derived from a cross between L. esculentum Sun1642 and L. pimpinellifolium LA1589. Chromosome walking using clones identified from several large genomic insert libraries resulted in two noncontiguous contigs flanking sun. Fiber-FISH analysis showed that distance between the two contigs measured 68 kb in L. esculentum Sun1642 and 38 kb in L. pimpinellifolium LA1589, respectively. The sun locus mapped between the two contigs, suggesting that allelic variation at this locus may be due to an insertion/deletion event. The results demonstrate that sun is located in a highly dynamic region of the tomato genome.     

Cross-species bacterial artificial chromosome-fluorescence in situ hybridization painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species.     

Genetic analysis of chromosomal rearrangements in the cyclops region of the zebrafish genome.

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.     

Strand-specific fluorescence in situ hybridization: the CO-FISH family

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5'-->3' towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5'-->3' orientation of DNA sequences with respect to the pter-->qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.