Syrian hamster 5S rRNA genes are assigned to 6q2 with PNA-FISH by an R-banded karyotype

A karyotype for the Syrian hamster is proposed based on an R-banding pattern. R-bands were obtained by BrdU incorporation into the cells followed by a combined DAPI and propidium iodide staining of the fixed metaphase spreads. In situ hybridisation was performed with two biotinylated 18-mer PNA (peptide nucleic acid) probes complementary to sequences within the 5S rRNA gene. The 5S rRNA gene repeats map to chromosome 6q2. The present PNA-FISH procedure is an abbreviated and simpler version of that previously published.     

Identifying the chromosomes of the A- and C-genome diploid Brassica species B. rapa (syn. campestris) and B. oleracea in their amphidiploid B. napus

Oilseed rape (Brassica napus L.) is an amphidiploid species that originated from a spontaneous hybridisation of Brassica rapa L. (syn. campestris) and Brassica oleracea L., and contains the complete diploid chromosome sets of both parental genomes. The metaphase chromosomes of the highly homoeologous A genome of B. rapa and the C genome of B. oleracea cannot be reliably distinguished in B. napus because of their morphological similarity. Fluorescence in situ hybridisation (FISH) with 5S and 25S ribosomal DNA probes to prometaphase chromosomes, in combination with DAPI staining, allows more dependable identification of Brassica chromosomes. By comparing rDNA hybridisation and DAPI staining patterns from B. rapa and B. oleracea prometaphase chromosomes with those from B. napus, we were able to identify the putative homologues of B. napus chromosomes in the diploid chromosome sets of B. rapa and B. oleracea, respectively. In some cases, differences were observed between the rDNA hybridisation patterns of chromosomes in the diploid species and their putative homologue in B. napus, indicating locus losses or alterations in rDNA copy number. The ability to reliably identify A and C genome chromosomes in B. napus is discussed with respect to evolutionary and breeding aspects. Received: 13 July 2001 / Accepted: 23 August 2001     

Expression of a clone specific DNA sequence from Staphylococcus aureus in Escherichia coli.

Staphylococcus aureus produces a large number of factors thought to contribute to virulence, although the precise role of some of these individual factors is not clearly defined. To investigate whether specific virulence factors might be responsible for the selection and dominance of certain genotypes of methicillin- and multiply resistant S. aureus (MRSA), the method of subtractive hybridisation was used to identify conserved DNA sequences associated with the clinical, clonal populations of S. aureus. The findings described in this report indicate that the method of subtractive hybridisation is a valuable tool to identify clone specific virulence factors, which might be of potential as diagnostic markers and as alternative vaccine targets.     

Location of repetitious DNA in the chromosomes of the desert locust (Schistocerca gregaria)

A modified Giemsa staining technique and the in situ hybridisation technique, have been used to investigate the localisation of highly repeated sequences in the karyotype of the locust Schistocerca gregaria. The centromeric regions are stained densely with Giemsa and further Giemsa-stained bands occur at the telomeric region of the short (S) chromosomes. RNA complementary to repetitious DNA hybridised to loci scattered along the whole length of the chromosomes, with concentrations of grains at the centromeric regions of all the chromosomes and also at the telomeric regions of the S chromosomes.     

Identification of Enterococcus spp. based on specific hybridisation with 16S rDNA probes

The conventional methods for routine enterococci species identification are usually based on phenotypic characteristics. However, in recent years, some studies have defined specific probes based on both 16S and 23S rRNA genes for the identification of some Enterococcus spp. A set of probes based on the 16S rRNA gene has been developed in order to evaluate the usefulness of a six-step biochemical key for species level identification of enterococci. Probe specificity has been evaluated with type collection and environmental strains by dot blot hybridisation. A high correlation was obtained between biochemical key and hybridisation identifications. This set of probes provides a confirmative method for phenotypic species identification.     

The Unique Karyotype of Henochilus wheatlandii, a Critically Endangered Fish Living in a Fast-Developing Region in Minas Gerais State, Brazil

Henochilus wheatlandii, the only species of this genus, is critically endangered and was considered extinct for over a century. The rediscovery of this fish in 1996 made it possible to study its phylogenetic relationships with other species in the subfamily Bryconinae. The aim of this study was to characterise the karyotype of H. wheatlandii. Standard staining, C-positive heterochromatin and nucleolar organiser region (NOR) banding, chromomycin A(3) staining, and fluorescent in situ hybridisation (FISH) using 5S rDNA and 18S rDNA probes were conducted on nineteen specimens collected in the Santo Antonio River, a sub-basin of the Doce River in Ferros municipality, Minas Gerais State, Brazil. Henochilus wheatlandii shared the same diploid number and chromosome morphology as other species of Bryconinae. However, its heterochromatin distribution patterns, NOR localisation, and FISH patterns revealed a cytogenetic profile unique among Neotropical Bryconinae, emphasizing the evolutionary uniqueness of this threatened species.     

Lack of correspondence between CMA3-, Ag-positive signals and 28S rDNA loci in two Iberian minnows (Teleostei, Cyprinidae) evidenced by sequential banding

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations.     

Physical mapping of rDNA genes establishes the karyotype of almond

The localisation of ribosomal RNA genes on chromosomes of almond (Prunus amygdalus, 2n = 16) was studied by fluorescence in situ hybridisation. Simultaneous double-colour hybridisation with both 18S–5.8S–25S and 5S rDNA probes demonstrated that all chromosomes can be identified. In spite of the small size, differences in length between chromosomes that hybridised with the same rDNA probe as well as between chromosomes without hybridisation signal are apparent. Chromosomes were ordered in the karyotype according to their length. The 18S-5.8S-25S rDNA genes were detected in subdistal positions of chromosomes 2, 3, and 8. Sites located on chromosomes 2 and 3 carry a higher number of repeats than the site of chromosome 8. The 5S rDNA genes were found proximally located on chromosomes 5 and 7, the signal on chromosome 5 showing higher intensity than the signal on chromosome 7. Chromosomes 1, 4, and 6 show no hybridisation signal.     

Schistosomiasis at Loum, Cameroun; Schistosoma haematobium, S. intercalatum and their natural hybrid.

A survey of 500 schoolchildren in Loum in 1968 revealed an overall infection rate of 54.2% with Schistosoma intercalatum and this was the only species of schistosome encountered. In 1972 a number of children were found to be passing schistosome eggs in their urine and these eggs ranged in shape and size from the forms characteristic for S. haematobium to those of S. intercalatum. Preliminary laboratory studies demonstrated that hybridisation between the two species was occurring. Subsequent field surveys showed that the snail hosts for the two parasites (B. rohlfsi for S. haematobium and B. forskali for S. intercalatum) were both present in the river Mbette and its tributaries in Loum and the distribution of the two snail species coincided closely with the distribution of the schistosomes in the human population. Detailed study of a small group of children passing hybrid eggs in their urine revealed that few of them were passing eggs in their faeces and that those eggs which were found in faeces were not viable. Analysis of schistosome egg-shape by plotting cumulative size-frequency data on probability paper demonstrated that the graph obtained from a natural hybrid series was different from that given by a known mixture of the two separate species. The hybrid series included a number of exceptionally large eggs resembling those of S. bovis but isolation of these eggs and subsequent laboratory passage of the parasites showed that they were part of the series and were not evidence of the presence of a third species. Hybridisation experiments in the laboratory showed that the cross S. haematobium male X S. intercalatum femal is fully viable but that the reverse mating is not successful, thus accounting for the failure of the faecal eggs recovered from children with hybrid infections. Histological results from laboratory passaged hybrids suggest that the Ziehl-positive staining reaction of the egg-shells of S. intercalatum may be a recessive character. The observations reported here indicate that S. haematobium has only recently become established in Loum and that it is, through introgressive hybridisation, replacing the indigenous S. intercalatum. A suggested explanation for the change in the parasite fauna is offered and this depends upon ecological changes resulting from forest clearance and agricultural development providing improved conditions for the spread of B. rohlfsi, the snail host for S. haematobium. It is suggested that, in contrast to recent reports on the spread of S. intercalatum, this species is in fact retreating and being replaced by S. haematobium in areas where forest clearance is taking place. In conclusion it is suggested that introgressive hybridisation of this kind may have been responsible for the evolution of certain characteristic local strains of African schistosomes.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome

In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.     

Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli.

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.     

Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.     

The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.     

Curtis' Substitute for Van Gieson Stain

A triple staining method is described in which nuclear staining is by Weigert's hematoxylin. The cytoplasmic and collagen staining is effected by the Curtis substitute for Van Gieson, in which ponceau S is substituted for acid fuchsin. Nuclear staining is sharper than with Delafield's hematoxylin. The red of the collagen fibers is probably not subject to fading. Unlike Van Gieson, this method gives staining of reticular as well as collagen fibers. The advantages of the method are its simplicity and reliability. The use of this method is made possible by a new source of reliable samples of the ponceau S called for in this method.