Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition.     

Gene mapping by fluorescent in situ hybridization

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

Assignment of linkage groups to pea chromosomes after karyotyping and gene mapping by fluorescent in situ hybridization

Chromosomes of the pea (Pisum sativum L.) were submitted to fluorescent in situ hybridization (FISH) with probes specific for the oligonucleotides (AG)₁₂, (AC)₁₂, (GAA)₁₀, and (GATA)₇ and for the genes encoding 25S rRNA, 5S rRNA and the storage proteins legumin A, K and vicilin. A fourth 5S rRNA gene locus, apparently specific for an accession of the cultivar Grüne Victoria, was newly detected. This allowed all seven chromosome pairs to be distinguished by FISH signals of rRNA genes. The same was possible using a combination of oligonucleotide probes or of oligonucleotides and rRNA gene-specific probes in multicolour FISH. Rehybridization with the 5S rRNA gene-specific probe allowed us to assign vicilin genes to the short arm of chromosome 5, the single legumin A locus to the long arm of chromosome 3 and the legumin B-type genes (exemplified by legumin K) to one locus on the short arm of chromosome 6. Correlation of these data with an updated version of the pea genetic map allowed the assignment of most linkage groups to defined chromosomes. It only remains to be established which of linkage groups IV and VII corresponds to the satellited chromosomes 4 or 7, respectively. Received: 13 February 1998; in revised form: 3 April 1998 / Accepted: 7 April 1998     

PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)     

Differentiation of two very similar glaucomid ciliate morphospecies (Ciliophora, Tetrahymenida) by fluorescence in situ hybridization with 18S rRNA targeted oligonucleotide probes

Conventional, morphological identification of ciliates and other protozoa needs considerable experience and often is difficult as various staining methods must be applied. New molecular techniques, such as fluorescence in situ hybridization (FISH) with gene probes, are powerful means to overcome this problem. As a test case, the morphology of two very similar, and thus difficult to differentiate ciliate morphospecies, Glaucoma scintillans and Glaucomides bromelicola, were compared. They were then distinguished by applying the Ciliate-FISH technique with a set of eight 18S rRNA targeted oligonucleotide probes, four of which have been developed for specific detection of G. scintillans. The remaining four probes were designed to detect G. bromelicola in order to prove probe specificities by binding to the homologous target region of the probes mentioned before. The tests resulted in a clear and easy differentiation of the two species by fluorescence signals of three of the four tested probe pairs. Thus, FISH techniques are very useful for the identification and detection of protozoa and might be of great help studying geographical distributions of known taxa.     

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.     

Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum , a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans ), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3–2.1% of the total rRNA in the digesters, 2.6–6.6% in soil, 1.5–3.3% in human faeces and 2.5–6.2% in pig colon samples.     

Chromosomal repatterning in crocodiles: C,G and N-banding and the in situ hybridization of 18S and 26S rRNA cistrons

The chromosomes of Crocodylus porosus, C. johnstoni and Caiman crocodilus have been analysed using C, G and N-banding techniques and the in situ hybridization of 18S+26S rRNA cistrons derived from Xenopus. It is clear that in addition to the gross structural changes (robertsonian rearrangements and pericentric inversions) which are known to distinguish these crocodiles, numerous other modes of repatterning have occurred. These involved both the heterochromatic and euchromatic portions of the genome. They appear to be associated with the gross structural changes which have been established, and involve two distinct forms of chromatin transformation.In addition, the in situ hybridization of 18S+26S rRNA cistrons onto these crocodilian chromosomes has localized the site of nucleolus organizer activity to the C-band positive G-band negative secondary constrictions present in all three species. The significance of these results is discussed.     

Use of specific DNA probes for mapping the ceruloplasmin gene on rat chromosomes by direct in situ hybridization

Specific DNA-probes representing the fragments of chromosomal ceruplasmin gene (lambda RCp-1, lambda RCp-2, lambda RCp-3) and its cDNA copy of the corresponding mRNA were heavily labelled with 125J dCTP (the specific activity of the probes varying from 1.5 X 10(7) to 3.4 X 10(8) dpm). These probes were used for in situ hybridization on metaphase chromosomes. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 653 metaphase plates from bone marrow cells of laboratory rats. The results of in situ hybridization were very similar for all four specific DNA-probes tested and allow to assign ceruplasmin gene to the q13 region of chromosome 7. The local increase of silver grain count over chromosome 15 was also registered and discussed.     

In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes

One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973-4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083(T) was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA of E. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.     

Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization

AIMS: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. METHODS AND RESULTS: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. CONCLUSION: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.     

Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from gamma-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from alpha-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4', 6'-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for alpha-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. alpha-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha2 (GFRalpha2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFRalpha2 mRNA and virtually all GFRalpha2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFRalpha2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.     

Cyto-evolution of Boronia genomes revealed by fluorescent in situ hybridization with rDNA probes.

The physical location of the 25S-26S rDNA sequences was examined in 11 taxa of nine species of Boronia. In diploid species, two rDNA sites were detected in Boronia clavata (2n = 14), Boronia pinnata 'White' (2n = 22), and Boronia chartacea (2n = 32); four in Boronia megastigma (2n = 14) and Boronia denticulata (2n = 18); six in Boronia pinnata 'Pink' (2n = 22); and eight in Boronia molloyae (2n = 16). Eleven sites were found in Boronia heterophylla 'Red' and 'Near White' (2n = 15), but only two active nucleolar organizer regions (NORs) were observed. In polyploid species, Boronia pilosa (2n = 44) had four rDNA sites, while Boronia coerulescens (2n = 72) had six. Most of the rDNA sequences were terminal, but a few were interstitial. There were also differences in signal intensity indicating that the gene copies between and within rDNA sites might be different. The result suggests that considerable chromosome rearrangements have occurred during Boronia cyto-evolution, leading to variation among Boronia taxa in rDNA copy number, site number, and location. These changes together with dysploid reduction during cyto-evolution have made the Boronia genome considerably diverse in chromosome number, genome organization, and chromosome structure.     

Fluorescence in situ hybridization: applications in cytogenetics and gene mapping

Unique sequences, chromosomal subregions, or entire genomes can be specifically highlighted in metaphase or interphase cells by fluorescence in situ hybridization (FISH). This technique can be used to identify chromosomes, detect chromosomal abnormalities or determine the chromosomal location of specific sequences. FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.     

Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes

Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.     

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.