The identification of microorganisms by fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes facilitates the rapid and specific identification of individual microbial cells in their natural environments. Over the past year there have been a number of methodological developments in this area and new applications of FISH in microbial ecology and biotechnology have been reported.     

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.     

Influence of temperature on the detectibility and chromosomal distribution of specific DNA sequences by in situ hybridisation

Hybridising certain AT-rich satellite complementary RNAs (cRNAs) to their homologous chromosomal DNA sequences at different temperatures of incubation results in a different dispersion of autoradiographic label throughout the karyotypes. The temperature at which most label, or cRNA-DNA hybrid formation, exists corresponds to the optimal rate temperature for the hybridisation of these same satellite cRNA-DNA hybrids as determined by RNA excess filter hybridisation. It is likely that the in situ hybridisation results can therefore be explained by the fact that there is a similar temperature-dependence on the rate of hybrid formation for both in situ and RNA excess hybridisation. This should have important implications for the designing of in situ hybridisation experiments in general.     

Identification of the rice D-genome chromosomes by genomic in situ hybridisation

 The 24 rice D-genome chromosomes were identified among the 48 chromosomes of O. latifolia, which comprise the C- and D-genomes, using genomic in situ hybridisation (GISH). The B-genome chromosomes were also discriminated from the C-genome chromosomes in O. minuta (BBCC) by GISH. A comparison of the differences in the fluorescence intensity between the C and D genomes within O. latifolia (CCDD), and between the B and C genomes within O. minuta, indicated that the overall nucleotide-sequence homology between the B and C genomes is less than that between the C and D genomes. The origin of the D genome and the phylogenetic relationship of the D genome among the rice genomes are discussed, based on the results obtained. Received: 5 June 1997 / Accepted: 19 June 1997     

Oligo-riboprobes. Tools for in situ hybridisation

In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. Moreover, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes ("oligo-riboprobes"). These probes can be labelled to very high (10(9) cpm/micrograms) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise beta (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from beta preprotachykinin cDNA.     

Fluorescent in situ hybridisation to amphioxus chromosomes

We describe an efficient protocol for mapping genes and other DNA sequences to amphioxus chromosomes using fluorescent in situ hybridisation. We apply this method to identify the number and location of ribosomal DNA gene clusters and telomere sequences in metaphase spreads of Branchiostoma floridae. We also describe how the locations of two single copy genes can be mapped relative to each other, and demonstrate this by mapping an amphioxus Pax gene relative to a homologue of the Notch gene. These methods have great potential for performing comparative genomics between amphioxus and vertebrates.     

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.     

Genome reorganization in Nicotiana asymmetric somatic hybrids analysed by in situ hybridization

In situ hybridization was used to examine genome reorganization in asymmetric somatic hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by fusion of gamma-irradiated protoplasts from one of the parents (donor) with non-irradiated protoplasts from the other (recipient). Probing with biotinylated total genomic DNA from either the donor or the recipient species unequivocally identified genetic material from both parents in 31 regenerant plants, each originating from a different nuclear hybrid colony. This method, termed genomic in situ hybridization (GISH), allowed intergenomic translocations containing chromosome segments from both species to be recognized in four regenerants. A probe homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat (5'-TTTAGGG-3')n, identified telomeres on all chromosomes, including 'mini-chromosomes' originating from the irradiated donor genome. Genomic in situ hybridization to plant chromosomes provides a rapid and reliable means of screening for recombinant genotypes in asymmetric somatic hybrids. Used in combination with other DNA probes, it also contributes to a greater understanding of the events responsible for genomic recovery and restabilization following genetic manipulation in vitro.     

The identification of human chromosomes by quinacrine fluorescence after hybridisation in situ

Summary A method is described for producing fluorescent bands on human chromosomes by staining with quinacrine after hybridisation in situ. The advantages of the method include the elimination of artefacts arising from staining before hybridisation, the fact that there is no reduction in sample number between staining and autoradiography, the ease with which autoradiographic grains can be identified and counted, and the reduction in exposure time.Offprint requests to: S.S. Lawrie     

Regulation of glial development by cystatin C

Cystatin C (CysC) is an endogenous cysteine proteases inhibitor produced by mature astrocytes in the adult brain. Previously we isolated CysC as a factor activating the glial fibrillary acidic protein (GFAP) promoter, and showed that CysC is expressed in astrocyte progenitors during development. Here we show that protease inhibitor activity increased daily in conditioned medium, and that this activity was mainly a result of CysC released from primary cultured cells. Human CysC added to the culture medium of primary brain cells increased the number of GFAP-positive and nestin-positive cells. Human CysC also increased the number of neurospheres formed from embryonic brain, and thus it increases the number of neural stem/precursor cells in a manner similar to glycosylated rat CysC. The addition of a neutralizing antibody, on the other hand, greatly decreased the number of GFAP and glutamate aspartate transporter (GLAST)-positive astrocytes. This decrease was reversed by the addition of CysC but not by another cysteine protease inhibitor. Thus, the promotion of astrocyte development by CysC appears to be independent of its protease inhibitor activity. The antibody increased the number of oligodendrocytes and their precursors. Therefore, CysC modifies glial development in addition to its activity against neural stem/precursor cells.     

Histone gene expression during the cell cycle studied by in situ hybridisation

Cloned sea urchin histone gene DNA sequences have been in situ hybridized to histone RNA sequences in the cytoplasm of unsynchronized populations of Friend erythroleukemic cells, HeLa S3 and Chinese Hamster Ovary cells. S phase cells were detected by [³H]thymidine labelling of cell cultures prior to preparation for in situ hybridization. Autoradiography of the hybridized preparations has shown that in unsynchronized cells histone sequences are present in abundance in the cytoplasm of S phase cells only.     

Multi-colour brightfield in situ hybridisation on tissue sections

 We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase) reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining, the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer. Accepted: 27 June 1997     

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C beta(1)

Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type beta(1) is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCbeta(1) under different stimuli implicate PLCbeta(1) as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCbeta(1) in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35-36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCbeta(1) cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCbeta(1). By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCbeta(1) on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35-36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCbeta(1) could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCbeta(1), which has been shown to behave as a key signalling intermediate in the control of the cell cycle.     

Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization

 Three lines of the tetraploid wheat Aegilops ventricosa Tausch (2n=4x=28), which contains good resistance to eyespot, were analysed using fluorescent in situ hybridization. Probes used included rDNA, cloned repeated sequences from wheat and rye, simple-sequence repeats (SSRs) and total genomic DNA. The banding patterns produced could be used to distinguish most chromosome arms and will aid in the identification of Ae. ventricosa chromosomes or chromosome segments in breeding programmes. All lines had a single major 18S-25S rDNA site, the nucleolar organizing region (NOR) in chromosome 5N and several minor sites of 18S-25S rDNA and 5S rDNA. A 1NL.3DL, 1NS.3DS translocation was identified, and other minor differences were found between the lines. Received: 11 August 1998 / Accepted: 28 November 1998     

Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).     

Selective binding of nucleotide probes by eosinophilic cationic protein during in situ hybridisation

During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IB, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe–eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.     

The nucleolar organisers of tetraploid and hexaploid wheats revealed by in situ hybridisation

Summary The technique of in situ hybridisation of cloned ribosomal DNA has been used to establish the numbers of nucleolar organising sites in a range of tetraploid and hexaploid wheats.