Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.     

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

RAPID DETECTION OF GERMINATING BACILLUS CEREUS CELLS USING FLUORESCENT IN SITU HYBRIDIZATION

Methods for the specific detection of Bacillus spores are needed in many situations such as the recognition of food poisoning. This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples.
B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus -specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra heat-treated milk samples. FISH-labeled cells were detected by using flow cytometry, and the results were confirmed by fluorescence microscopy. The optimal condition allows the detection of B. cereus spores in less than 2 h. Overall, a ninefold reduction in total time for detection was achieved when comparing with previous works. Therefore, the permeabilization and hybridization optimizations mentioned in this study are major improvements for the detection time of B. cereus spores.

PRACTICAL APPLICATIONS

By using the optimized conditions of germination/outgrowth, permeabilization and hybridization, the detection of 10³ cfu/mL of Bacillus cereus spores using fluorescent in situ hybridization is possible within 2 h in milk sample.     

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.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ～ 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ～ 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8–8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley-Liss, Inc.     

Molecular quantification of symbiotic dinoflagellate algae of the genus Symbiodinium

The dinoflagellate microalga Symbiodinium is the dominant algal symbiont in corals and related marine animals. To explore the incidence of mixed infections, methods employing real-time quantitative polymerase chain reaction (QPCR) and fluorescence in situ hybridization (FISH) were developed. In experiments focusing on Symbiodinium clades A and B, QPCR and FISH results were well correlated and generally more precise and sensitive than those from the endpoint PCR-restriction fragment length polymorphism analysis (PCR-RFLP) traditionally used for this application, thus increasing the detected incidence of mixed infections. For example, the prevalence of mixed infections in the sea anemone Condylactis gigantea was 40% by PCR-RFLP and 80%-90% by QPCR and FISH. However, the use of QPCR and FISH was limited by inter-host variation in the rRNA gene copy number per Symbiodinium cell, precluding any single conversion factor between QPCR signal and Symbiodinium cell number; and one FISH probe that gave excellent hybridization efficiency with cultured Symbiodinium yielded variable results with Symbiodinium from symbioses. After controlling for these caveats, QPCR studies revealed that field-collected hosts previously described as universally unialgal bore up to 1.6% of the alternative clade. Further research is required to establish the contribution that algal cells at low density in symbiosis and external to the symbiosis make to the minor clade.     

Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH).

Herein we report the results of the first major prospective study directly comparing aneuploidy detection by fluorescence in situ hybridization of interphase nuclei with the results obtained by cytogenetic analysis. We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copy-like signal when used in conjunction with suppression hybridization. A total of 526 independent amniotic fluid samples were analyzed in a blind fashion. All five probes were analyzed on 117 samples, while subsets of these five probes were used on the remaining samples (because of insufficient sample size), for a total of over 900 autosomal hybridization reactions and over 400 sex chromosome hybridization reactions. In this blind series, 21 of 21 abnormal samples were correctly identified. The remaining samples were correctly classified as disomic for these five chromosomes. The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization/detection allowed accurate chromosome enumeration in uncultured human amniotic fluid cells, consistent with the results obtained by traditional cytogenetic analysis.     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy

A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT + BCIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetylaminofluorene (AAF] were used. The advantages and disadvantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.     

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Functional significance of genetically different symbiotic algae Symbiodinium in a coral reef symbiosis

The giant sea anemone Condylactis gigantea associates with members of two clades of the dinoflagellate alga Symbiodinium, either singly or in mixed infection, as revealed by clade-specific quantitative polymerase chain reaction of large subunit ribosomal DNA. To explore the functional significance of this molecular variation, the fate of photosynthetically fixed carbon was investigated by (14)C radiotracer experiments. Symbioses with algae of clades A and B released ca. 30-40% of fixed carbon to the animal tissues. Incorporation into the lipid fraction and the low molecular weight fraction dominated by amino acids was significantly higher in symbioses with algae of clade A than of clade B, suggesting that the genetically different algae in C. gigantea are not functionally equivalent. Symbioses with mixed infections yielded intermediate values, such that this functional trait of the symbiosis can be predicted from the traits of the contributing algae. Coral and sea anemone symbioses with Symbiodinium break down at elevated temperature, a process known as 'coral bleaching'. The functional response of the C. gigantea symbiosis to heat stress varied between the algae of clades A and B, with particularly depressed incorporation of photosynthetic carbon into lipid of the clade B algae, which are more susceptible to high temperature than the algae of clade A. This study provides a first exploration of how the core symbiotic function of photosynthate transfer to the host varies with the genotype of Symbiodinium, an algal symbiont which underpins corals and, hence, coral reef ecosystems.