Direct‐geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)‐targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct‐geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct‐geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome‐labelled polynucleotide gene probes and rRNA‐targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct‐geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy‐control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct‐geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.     

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.     

Isolation of denitrifying bacteria from hydrocarbon-contaminated Antarctic soil

In this study, we report the isolation of denitrifiers from hydrocarbon-contaminated Antarctic soils. Seventy-two isolates were obtained from soils that had received a fertilizer treatment to stimulate hydrocarbon degradation. All isolates, except one, belonged to the genus Pseudomonas. The one exception was a member of the Microbacteriaceae, which was also, coincidentally, the only isolate negative for the nirS gene. The diversity of the 16S rRNA and nosZ genes was assessed by denaturing gradient gel electrophoresis and sequencing. There was a slight correlation between the 16S rRNA and nosZ operational taxonomic units. Surprisingly, many isolates contained nosZ on plasmids and, to the best of our knowledge, this is the first report of nosZ being extra-chromosomally present in Pseudomonas spp.     

Non‐target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non‐target rRNA sequences. We show here that probe target sites containing single‐base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non‐target sites resulting from single‐base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.     

Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

Nitrous oxide (N₂O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N₂O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10¹ and 10² target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10⁵ to 10⁷ target copies g⁻¹ of dry soil, whereas genes for 16S rRNA were found at 10⁸ to 10⁹ target copies g⁻¹ of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.     

Presence of Two Different Active nirS Nitrite Reductase Genes in a Denitrifying Thauera sp. from a High-Nitrate-Removal-Rate Reactor

The nirS nitrite reductase genes were studied in two strains (strains 27 and 28) isolated from two denitrifying reactors and characterized as Thauera according to their 16S rRNA gene sequences. Strain 28 contains a single nirS sequence, which is related to the nirS of Thauera mechernichensis, and strain 27 contains two nirS sequences; one is similar to the nirS sequence from Thauera mechernichensis (gene 2), but the second one (gene 8) is from a separate clade with nirS from Pseudomonas stutzeri, Azoarcus species, Alcaligenes faecalis, and other Thauera species. Both genes were expressed, but gene 8 was constitutively expressed while gene 2 was positively regulated by nitrate.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Vertical profiles of water and sediment denitrifiers in two plateau freshwater lakes

The present study investigated the abundance, richness, diversity, and community composition of denitrifiers (based on nirS and nosZ genes) in the stratified water columns and sediments in eutrophic Dianchi Lake and mesotrophic Erhai Lake using quantitative PCR assay and high-throughput sequencing analysis. Both nirS- and nosZ denitrifiers were detected in waters of these two lakes. Surface water showed higher nosZ gene density than bottom water, and Dianchi Lake waters had larger nirS gene abundance than Erhai Lake waters. The abundance of sediment nirS- and nosZ denitrifiers in Dianchi Lake was larger than that in Erhai Lake. nirS richness and diversity and nosZ richness tended to increase with increasing sediment layer depth in both lakes. The distinct structure difference of sediment nirS- and nosZ denitrifier communities was found between in Dianchi Lake and Erhai Lake. These two lakes also differed greatly in water denitrifier community structure. Moreover, phylogenetic analysis indicated the presence of several different groups of nirS- or nosZ denitrifiers in both lakes. The novel nirS denitrifiers were abundant in both Dianchi Lake and Erhai Lake, while most of the obtained nosZ sequences could be affiliated with known genera.

     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene phylochip and mapping of signal intensities

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.     

Molecular, Biochemical, and Physiological Approaches forUnderstanding the Ecology of Denitrification

One of the major challenges in microbial ecology for the future is to establish links between structural and functional biodiversity. This is particularly difficult when one is interested in a phylogenetically diversified function such as denitrification. The data banks are very rich in functional gene sequences (nirS in this study), but most of them were obtained from not yet cultivated bacteria, and thus must be supplemented by sequences of organisms from the environment for which we could associate a taxonomic position and physiological characteristics. Combined analysis including molecular (16S-rRNA or nirS genes), physiological, and biochemical approaches was carried out on a bacterial set of 89 strains isolated from marine sediment. The denaturing gradient gel electrophoresis (DGGE) technique was successfully applied on unclamped polymerase chain reaction (PCR) products of nirS genes to compare the picture of the biodiversity obtained with 16S rRNA and nirS genes. The diversity of nirS genes and denitrifier characteristics were found within several of the 16S rDNA phylotypes. In contrast, the nirS phylotypes were no diverse both with respect to 16S rDNA and to physiology and biochemistry of denitrification. Sequences of the nirS PCR products were very close to marine environmental clones and were analyzed within the same phylogenetic tree.     

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     

GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms

Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH – a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA‐targeted catalysed reporter deposition – fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T_m of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia.     

Isolation and functional analysis of denitrifiers in an aquifer with high potential for denitrification

Aquifers are among the main freshwater sources. The Raigón aquifer is susceptible to contamination, mainly by nitrate and pesticides, such as atrazine, due to increasing agricultural activities in the area. The capacity of indigenous bacteria to attenuate nitrate contamination in different wells of this aquifer was assessed by measuring denitrification rates with either acetate plus succinate or nitrate amendments. Denitrification activity in nitrate-amended assays was significantly higher than in unamended assays, particularly in groundwater from wells where nitrate concentration was 33.5 mg L⁻¹ or lower. Furthermore, groundwater denitrifiers capable of using acetate or succinate as electron donors were isolated, identified by 16S rRNA gene sequencing and evaluated for functional denitrification genes (nirS, nirK and nosZ). Phylogenetic affiliation of 54 isolates showed that all members belonged to nine different genera within the Proteobacteria (Bosea, Ochrobactrum, Azospira, Zoogloea, Acidovorax, Achromobacter, Vogesella, Stenotrophomonas and Pseudomonas). In addition, isolate AR28 that clustered separately from validly described species could potentially belong to a new genus. The majority of the isolates were related to species belonging to previously reported denitrifying genera. However, the phylogeny of the nirS and nosZ genes revealed new sequences of these functional genes. To our knowledge, this is the first isolation and sequencing of the nirS gene from the genus Vogesella, as well as the nosZ gene from the genera Acidovorax and Zoogloea. The results indicated that indigenous bacteria in the Raigón aquifer had the capacity to overcome high nitrate contamination and exhibited functional gene diversity.     

Recognition of individual genes in diverse microorganisms by cycling primed in situ amplification

Cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) was developed to recognize individual genes in a single bacterial cell. In CPRINS, the amplicon was long single-stranded DNA and thus retained within the permeabilized microbial cells. FISH with a multiply labeled fluorescent probe set enabled significant reduction in nonspecific background while maintaining high fluorescence signals of target bacteria. The ampicillin resistance gene in Escherichia coli, chloramphenicol acetyltransferase gene in different gram-negative strains, and RNA polymerase sigma factor (rpoD) gene in Aeromonas spp. could be detected under identical permeabilization conditions. After concentration of environmental freshwater samples onto polycarbonate filters and subsequent coating of filters in gelatin, no decrease in bacterial cell numbers was observed with extensive permeabilization. The detection rates of bacterioplankton in river and pond water samples by CPRINS-FISH with a universal 16S rRNA gene primer and probe set ranged from 65 to 76% of total cell counts (mean, 71%). The concentrations of cells detected by CPRINS-FISH targeting of the rpoD genes of Aeromonas sobria and A. hydrophila in the water samples varied between 2.1 x 10(3) and 9.0 x 10(3) cells ml(-1) and between undetectable and 5.1 x 10(2) cells ml(-1), respectively. These results demonstrate that CPRINS-FISH provides a high sensitivity for microscopic detection of bacteria carrying a specific gene in natural aquatic samples.     

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)–FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD–FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD–FISH in detecting low copy targets.     

Chromosome‐specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

Jute (Corchorus spp.), as a natural fibre‐producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome‐specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600 bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C. olitorius. Subsequently, chromosome‐specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome‐specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome‐wide physical maps for any recalcitrant plant species like jute.     

Detection of single-nucleotide polymorphisms in the <Emphasis Type="Italic">p53</Emphasis> gene by LDR/RCA in hydrogel microarrays

To find single-nucleotide polymorphisms (SNPs) in the human genome, three modern technologies of molecular genetic analysis were combined: the ligase detection reaction (LDR), rolling circle amplification (RCA), and immobilized microarray of gel elements (IMAGE). SNPs were detected in target DNA by selective ligation of allele-specific nucleotides in microarrays. The ligation product was assayed in microarray gel pads by RCA. Two variants of microarray analysis were compared. One included selective ligation of short oligonu-cleotides immobilized in a microarray with subsequent amplification with a preformed circular probe (a common circle). The probe was especially designed for human genome research. The other variant employed immobilized allele-specific padlock probes, which could be circularized as a result of selective ligation. Codon 72 SNP of the human p53 gene was used as a model. RCA in microarrays proved to be a quantitative assay and, in combination with LDR, allowed efficient discrimination of alleles. The principles and prospects of LDR/RCA in microarrays are discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 30–39.Original Russian Text Copyright © 2005 by Kashkin, Strizhkov, Gryadunov, Surzhikov, Grechishnikova, Kreindlin, Chupeeva, Evseev, Turygin, Mirzabekov.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999