Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes.

Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.     

Fluorescence in situ hybridization (FISH)--application in research and diagnostics

The methods of molecular cytogenetics, in particular fluorescence in situ hybridization (FISH), are widely applied in cytogenetics for identification of numerical and structural chromosomal abnormalities, which are difficult to detect by routine cytogenetic techniques. Due to many advantages, FISH is used in research (gene mapping, gene expression studies, interspecies chromosome homology), and clinical diagnostics (chromosomal aberrations analysis in pre- and postnatal diagnostics, oncology). The techniques of in situ hybridization (ISH) are often employed in addition to classical banding techniques, in case where banding pattern is not reliable. This paper focuses on particular clinical examples, where FISH was successfully used to identify structural and numerical chromosomal aberrations.     

Cytogenetic analysis of human solid tumors by in situ hybridization with a set of 12 chromosome-specific DNA probes

We have applied fluorescent in situ hybridization (FISH) to assess the presence of numerical chromosome aberrations in fresh specimens of human solid tumors of varying histology. For this purpose, a set of 12 biotinylated chromosome-specific, repetitive alpha-satellite DNA probes (for chromosomes 1, 6, 7, 9, 10, 11, 15, 16, 17, 18, X and Y) were hybridized directly to isolated interphase nuclei. Utilizing this approach, we found numerical chromosome changes in all tumors. FISH ploidy profiles were in accordance with flow cytometric DNA histograms of these tumor cells.     

Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).     

Spectral karyotyping and fluorescence in situ hybridization analysis of de novo partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter)

An 8-year-old boy presenting with hypotonia, moderate mental retardation, developmental delay, and psychomotor retardation is reported. Magnetic resonance imaging of the brain at age 3 years revealed a Dandy-Walker variant. Cytogenetic analysis of the peripheral blood revealed a derivative chromosome 12 with unknown additional material attached to the distal region of the long arm of chromosome 12. The parental karyotypes were normal. Spectral karyotyping (SKY) using the 24-color SKY probes and fluorescence in situ hybridization (FISH) using the specific 7p, 7q, 12p, and 12q telomeric probes confirmed a duplication of distal 7p and a deletion of terminal 12q. The karyotype of the proband was designated as 46,XY.ish der(12)t(7;12) (p21.2;q24. 33)(SKY+, 7pTEL+, 12qTEL-). The present case provides evidence for the association of partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter) with a cerebellar malformation and the usefulness of SKY and FISH in the identification of a de novo aberrant chromosome resulting from an unbalanced translocation.     

Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice

The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions). No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.     

Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates

Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)₈ (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra. Lu Ma and Sheng-Mei Wu contributed equally to this work.     

Developmental patterns of ceramide glucosyltransferase (GlcT-1) expression in the mouse: in situ hybridization using DIG-labeled RNA probes.

Glycosphingolipids (GSLs) play significant roles in a variety of cell membrane events, including cellular interactions, signaling, and trafficking. Ceramide glucosyltransferase (glucosylceramide synthase, GlcT-1, EC 2.4.1.80) catalyzes the initial step in GSL synthesis, the transfer of glucose from UDP-glucose to ceramide. The reaction product of glucosylceramide serves as a core structure for over 400 species of GSLs. The enzyme is a key regulatory factor controlling intracellular levels of ceramide and GSLs. Appearance and differential distribution of GlcT-1 mRNA during mice postimplantation embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes, coupled with alkaline phosphatase detection. On E9, tissues of the mesencephalon, myelecephalon, diencephalons, and telencephalon expressed GlcT-1. On E11, it was expressed to a detectable extent in various tissues including mesencephalon, myelecephalon, diencephalon, telencephalon, nose, lung, liver, vertebra, tail, spinal cord, and tongue. The expression patterns of E13 were similar to those of E11, except that the heart and stomach became positive. On E15, a specific signal for GlcT-1 was detected in all organs of the embryo. These results provide the first evidence that GlcT-1 is differentially expressed during postimplantation embryogenesis.     

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.     

Genomic in situ hybridization analysis between Rubus coreanus and its relatives in Rubus (Sect. Idaeobatus)

To further investigate the phylogenetic relationship between Rubus coreanus and its relatives in the section Idaeobatus, we used genomic in situ hybridization (GISH) to ascertain the degree of their genomic homology. Genomic DNA from R. parvifolius and R. inopertus hybridized throughout the centromeric and sub-terminal regions on 14 and 12 chromosomes of R. coreanus, respectively. The probes from R. niveus and R. ellipticus var. obcordatus gave robust signals at the same region of eight chromosomes. R. ellipticus and R. pinfaensis generated strong signals at the centromeric and sub-terminal parts of six chromosomes. The hybridization signals from the R. tsangii and R. corchorifolius probes existed only at the telomeric parts of four chromosomes. The two signals at the sub-terminal region on chromosome 6 of R. coreanus might be 45S rDNA repeats. These results indicated that R. coreanus and R. parvifolius shared many repeat sequences. It could be deduced that the genome of R. parvifolius was most closely related to that of R. coreanus among the species tested, R. inopertus came next, while R. tsangii and R. corchorifolius showed the farthest relationship. The phylogenetic relationships between R. parvifolius and R. coreanus, as well as among the five subsections were mainly discussed.     

In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with RRNA-targeted peptide nucleic acid probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.     

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera)

American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death.     

Translocation t(1;17)(q12;q25) with a clinical picture like of a proximal deletion of 1q: identification by in situ hybridization with chromosome 1-specific satellite DNA probes

Summary The authors report a case of a balanced 1;17 translocation with breakpoints located in the secondary constriction of chromosome 1. This translocation is associated with pathological symptoms similar to those observed following a proximal deletion of 1q. We request contact with colleques who have observed similar, or related, cases of translocation with breakpoints in heterochromatic regions of human chromosomes.     

Fluorescent in situ hybridization (FISH) analysis of the relationship between chromosome location and nuclear morphology in human neutrophils

Human neutrophil nuclei typically consist of three of four large heterochromatic lobes joined by thin, DNA-containing filaments. In addition, some lobes exhibit appendages of various sizes and shapes. Classical genetic and cytological studies suggest that some appendages contain specific chromsomes. The studies reported here provide the first detailed analysis of the spatial relationship between individual chromosomes and recognizable structures in neutrophil nuclei using fluorescent in situ hybridization. Analysis of DNA sequences in chromosomes 2, 18, X, and Y demonstrate that specific lobes in a population of neutrophil nuclei do not have a fied chromosome content. This result implies that chromosomes partition randomly among lobes during neutrophil differentiation. However, neutrophil nuclear topography is not entirely fortuitous. For instance, none of the sequences probed in this study mapped to a filament and most centromeres lie in clusters near the nuclear periphery. In addition, one of the X chromosome centromeres in females and the Y chromosome centromere in males consistently associate with specific nuclear appendages found in a subset of neutrophil nuclei. Chromosomes 2 and 18 occupy discrete nd separate territories within individual lobes and neither territory ever extends into a filament. Surprisingly, the sizes of these territories are not proportional to chromosome length, suggesting that individual neutrophil chromosomes vary in their degree of compaction. These results are discussed in the light of models that attempt to explain nuclear morphology in terms of chromosome spatial organization. Received: 10 April 1997 / Accepted: 14 April 1997     

Chromosomal differences between European and North American Atlantic salmon discovered by linkage mapping and supported by fluorescence in situ hybridization analysis

ABSTRACT: BACKGROUND: Geographical isolation has generated a distinct difference between Atlantic salmon of European and North American Atlantic origin. The European Atlantic salmon generally has 29 pairs of chromosomes and 74 chromosome arms whereas it has been reported that the North American Atlantic salmon has 27 chromosome pairs and an NF of 72. In order to predict the major chromosomal rearrangements causing these differences, we constructed a dense linkage map for Atlantic salmon of North American origin and compared it with the well-developed map for European Atlantic salmon. RESULTS: The presented male and female genetic maps for the North American subspecies of Atlantic salmon, contains 3,662 SNPs located on 27 linkage groups. The total lengths of the female and male linkage maps were 2,153 cM and 968 cM respectively, with males characteristically showing recombination only at the telomeres. We compared these maps with recently published SNP maps from European Atlantic salmon, and predicted three chromosomal reorganization events that we then tested using fluorescence in situ hybridization (FISH) analysis. The proposed rearrangements, which define the differences in the karyotypes of the North American Atlantic salmon relative to the European Atlantic salmon, include the translocation of the p arm of ssa01 to ssa23 and polymorphic fusions: ssa26 with ssa28, and ssa08 with ssa29. CONCLUSIONS: This study identified major chromosomal differences between European and North American Atlantic salmon. However, while gross structural differences were significant, the order of genetic markers at the fine-resolution scale was remarkably conserved. This is a good indication that information from the International Cooperation to Sequence the Atlantic salmon Genome, which is sequencing a European Atlantic salmon, can be transferred to Atlantic salmon from North America.     

Mapping of apolipoprotein A-1 and ceruloplasmin genes on human, rat and mouse chromosomes by hybridization in situ with specific human DNA probes

In situ hybridization was carried out on metaphase-prometaphase chromosomes of PGA-stimulated lymphocytes and bone marrow cells obtained from laboratory rats and mice. Plasmid cloned sequences of human apolipoprotein A-1 (Apo A-1) and ceruloplasmin (CP) cDNA fragments have been used as specific probes labelled in nick-translation reaction with 3HdTTP and 3Hd ATP. The data of our study suggest that Apo A-1 is localized in 11q14-22, 9 A2-4 and 5q36 areas in men, mice and rats, respectively. The DNA sequences of human CP cDNA most probably occupy 3q23-25, 13q24-26 and 15q13-20 areas. Heterologous in situ hybridization of other species with DNA probes does not always give reliable results in gene mapping. Thus, the data of heterologous hybridization should be considered with caution.     

Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes.

Fluorescence in situ hybridization using simultaneously a combination of DNA probes for the telomeric hexamer repeat (TTAGGG) and the centromerically repeated murine gamma-satellite DNA was applied to analyze the nature of radiation-induced micronuclei in mouse NIH 3T3 fibroblasts. After subtraction of spontaneously occurring micronuclei independent from the dose and time after irradiation, approximately 22% of the radiation-induced micronuclei did not reveal any hybridization signal. Approximately 17% showed one centromeric hybridization signal and about four telomeric signals, suggesting their origin from whole chromosomes. Almost 60% of radiation-induced micronuclei had telomeric signals only, suggesting their origin from acentric fragments. A fraction of micronuclei were found to contain two or more acentric fragments. Micronuclei derived from whole chromosomes or from multiple acentric fragments might, together with DNA synthesis in micronuclei, explain the occurrence of radiation-induced micronuclei with DNA contents greater than the largest chromosome arm.     

RFLP analysis and genomic in situ hybridization (GISH) in somatic hybrids and their progeny between Lycopersicon esculentum and Solanum lycopersicoides

 RFLP (restriction fragment length polymorphism) and GISH (genomic in situ hybridization) analyses were employed to identify the chloroplast and nuclear genomes of the somatic hybrids and progeny between tomato ‘Ohgata zuiko’ and Solanum lycopersicoides (‘LA 2386’). A random distribution of the chloroplast genotype was determined using a cloned 19.6-kb BamHI fragment (Ba1) of tobacco chloroplast DNA. Eight selected hybrids were analyzed for their chromosomal compositions; 4 were tetraploids (2n=48) with an equal number of chromosomes derived from each parent as accurately determined by GISH, and the other 4 were hexaploids, containing an average of two sets of tomato chromosomes and one set from the wild parent. RFLP analysis with six tomato nuclear probes of known chromosomal locations revealed no major variation among the 44 hybrid plants surveyed. However, it also showed the presence of both parent-specific alleles and the loss of some and the presence of a few non-parental alleles, indicating rearrangement and/or recombination of the nuclear DNA. The relevance of the molecular and cytological methods and the potential use of somatic hybrids for plant breeding are demonstrated. Received: 20 July 1997 / Accepted: 6 October 1997     

Subchromosomal localization of two human cytokeratin genes (KRT4 and KRT15) by in situ hybridization.

The genes for human cytokeratins 4 and 15 (KRT4 and 15) are assigned to the p11.2----q12 region of chromosome 12 (cytokeratin 4) and to the q21----q23 region of chromosome 17 (cytokeratin 15), respectively.