In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

In situ immunocytochemical detection of potyviral proteins in plant cells

A method for in situ protein immunodetection using a peroxidase labeling system is described for detecting functional and structural proteins encoded by potato virus Y (Tunisian isolate) in plant tissues. Such Potyviruses are characterized by the accumulation of inclusion bodies containing viral encoded proteins other than coat protein. These proteins are functional at early stages of infection, making them easy to detect. Data are compared to those obtained by immunofluorescence techniques. Our technique can be used as a preliminary method for rapid detection of virus infection using antibodies directed against functional proteins.     

In situ nick translation of meiotic chromosomes to demonstrate homologous heterochromatin heterogeneity.

The in situ nick translation procedure performed on fixed meiotic chromosomes partially cleaved with restriction endonucleases shows a different staining of homologous heterochromatic regions, which could be explained through a differential restriction endonuclease cleavage. Mutations occurring before massive tandem duplication and involving those DNA motifs that produce these heterochromatic blocks, together with the absence of DNA recombination that characterizes these particular regions, could explain the observed results. This method for chromosome labelling is most useful to demonstrate a certain level of heterochromatin heterogeneity that is present in the genome of living species but remained cryptic to other techniques that are also able to induce longitudinal differentiation of the chromosomes.     

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White^® resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White^® embedded tissues to be sectioned at 10 μm providing excellent signal contrast.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

In situ nick translation of human metaphase chromosomes with the restriction enzymes MspI and HpaII reveals an R-band pattern.

Several restriction enzymes (HindIII, HaeIII, MspI, HpaII, EcoRI, KpnI, and NotI) were evaluated for their ability to induce bands in human metaphase chromosomes during in situ nick translation. MspI and HpaII were able to induce a completely developed R-band pattern. Preferential cleavage of R-band chromatin is due to the presence of unmethylated CpG-residues present in CpG-rich islands, which are apparently unevenly distributed and mainly concentrated in R-bands.     

In situ characterization of mercury-resistant growth-promoting fluorescent pseudomonads

Pseudomonas fluorescens strains PRS9 and GRS1 (wild type) were made mercury resistant PRS9Hg(r) (147 microM HgCl2) and GRS1Hg(r) (55 microM HgCl2), respectively, in King's medium by enrichment selection and their in situ root colonization studies were carried out. Mercury resistant mutant of PRS9 was stable and resulted in significant increase in root and shoot fresh weight (P < 0.05). Both the mutants are positive for indoleacetic acid (IAA), 'P' solubilization and siderophore production. PRS9, potent 'P' solubilizer, exhibited higher 'P' solubilization as compared to GRS1. After 2 weeks of inoculation, the population level of wild type PRS9 and its mercury resistant mutants has increased (50 fold). Mercury resistance has no adverse effect on the growth promoting properties of mutants besides being comparable in its morphological and physiological properties with their wild type counterpart. Furthermore, mercury resistant character facilitates rhizospheric competition and thus helpful for establishment of growth promoting strains where metal ions are either limiting and/or present at toxic level.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.     

An improved method for in situ nick translation of human chromosomes with biotin 11-labelled dUTP detected by biotinylated alkaline phosphatase

Nick translation of the DNA of conventionally prepared human metaphase chromosomes using DNase I and biotin dUTP combined with streptavidin-phosphatase-detection assay produced a banding-like appearance. This pattern seems to be due to differences in DNase I sensitivity along the chromosomes. The Y chromosome could be clearly distinguished from the other chromosomes because of its intensely dark labelled heterochromatic region. In addition to DNase I concentration, hypotonic treatment seems to be an important methodological factor influencing band resolution. Together with recently published similar methods these results indicate that in situ nick translation using biotinylated nucleotides may develop into a useful technique to overcome several problems of human cytogenetics.     

In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry

Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers, they can react in situ with a non-fluorescent 1,8-naphthalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of model and cell membranes.     

In situ immunoassays for gene translation products in phage plaques and bacterial colonies

A series of simple, in situ immunoassays have been developed which can be used in screening for translation products of genes cloned in vitro recombination experiments with either phage or plasmid vectors. Antigen-antibody complex formation occurring within a vector-phage plaque can be used to detect the production of a specific protein from an amplified gene. Immunoassays of colonies lysed in situ either by λ prophage induction or by biochemical means afford a much higher level of sensitivity than the plaque assay probably adequate to detect the production of a few molecules of protein per cell.     

A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

In vitro antitrypanosomal activity of plant terpenes against Trypanosoma brucei

During the course of screening to discover antitrypanosomal compounds, 24 known plant terpenes (6 sesquiterpenes, 14 sesquiterpene lactones and 4 diterpenes) were evaluated for in vitro antitrypanosomal activity against Trypanosoma brucei brucei. Among them, 22 terpenes exhibited antitrypanosomal activity. In particular, α-eudesmol, hinesol, nardosinone and 4-peroxy-1,2,4,5-tetrahydro-α-santonin all exhibited selective and potent antitrypanosomal activities in vitro. Detailed here in an in vitro antitrypanosomal properties and cytotoxicities of the 24 terpenes compared with two therapeutic antitrypanosomal drugs (eflornithine and suramin). This finding represents the first report of promising trypanocidal activity of these terpenes. Present results also provide some valuable insight with regard to structure–activity relationships and the possible mode of action of the compounds.     

In situ monitoring of heart rates in shore crabs Carcinus maenas in two tidal estuaries: effects of physico-chemical parameters on tidal and diel rhythms

Heart rates were monitored in situ in the shore crab, Carcinus maenas, in relation to variations in depth, salinity, oxygen tension, temperature, light intensity and pH. Experiments were performed in the Looe Estuary, Cornwall, England and in Batson Creek in the Salcombe-Kingsbridge Estuary, Devon, England. Experiments in the Looe Estuary were conducted in the vicinity of a storm water storage discharge whereas the experiments in Batson Creek were performed on a clean site. Tidal rhythms in heart rates were commonly detected but diel rhythms in heart rate were also observed frequently. Both types of rhythm were more evident in animals from Batson Creek than from Looe. In Batson Creek, 12 out of 15 crabs expressed tidal rhythms in heart rate, whereas 6 out of 15 crabs expressed diel rhythms. In the two studies in the Looe Estuary, 6 out of 15 crabs and 3 out of 15 crabs expressed tidal and diel rhythm in heart rate, respectively. At both experimental sites, heart rates were positively correlated with increasing changes in depth and salinity, whereas heart rates were negatively correlated with light intensity. In addition, heart rates appeared to be positively correlated with increasing oxygen tension in the experiments performed in the Looe Estuary. The study suggests that depth and oxygen availability are more important to in situ heart rates in shore crabs within tidal estuaries than are salinity, light intensity and pH. Also, sewage discharge appears to cause an acute increase in heart rate, which may affect expression of biological rhythms in shore crabs.     

In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.