Chromosomal aberrations in Crepis capillaris cells detected by FISH

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.     

Karyotyping of Brassica amphidiploids using 5S and 25S rDNA as chromosome markers

Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.     

Molecular and cytological characterization of ribosomal RNA genes in Chenopodium quinoa and Chenopodium berlandieri.

The nucleolus organizer region (NOR) and 5S ribosomal RNA (rRNA) genes are valuable as chromosome landmarks and in evolutionary studies. The NOR intergenic spacers (IGS) and 5S rRNA nontranscribed spacers (NTS) were PCR-amplified and sequenced from 5 cultivars of the Andean grain crop quinoa (Chenopodium quinoa Willd., 2n = 4x = 36) and a related wild ancestor (C. berlandieri Moq. subsp. zschackei (Murr) A. Zobel, 2n = 4x = 36). Length heterogeneity observed in the IGS resulted from copy number difference in subrepeat elements, small re arrangements, and species-specific indels, though the general sequence composition of the 2 species was highly similar. Fifteen of the 41 sequence polymorphisms identified among the C. quinoa lines were synapomorphic and clearly differentiated the highland and lowland ecotypes. Analysis of the NTS sequences revealed 2 basic NTS sequence classes that likely originated from the 2 allopolyploid subgenomes of C. quinoa. Fluorescence in situ hybridization (FISH) analysis showed that C. quinoa possesses an interstitial and a terminal pair of 5S rRNA loci and only 1 pair of NOR, suggesting a reduction in the number of rRNA loci during the evolution of this species. C. berlandieri exhibited variation in both NOR and 5S rRNA loci without changes in ploidy.     

Molecular Cytogenetics of the Genus Arabidopsis: In situ Localization of rDNA Sites, Chromosome Numbers and Diversity in Centromeric Heterochromatin

We have used in situ hybridization to determine the number ofsites of rDNA in species in the genus Arabidopsis. A. wallichii(2n = 16) has one major pair of sites and one minor pair ofsites, while A. pumila and A. griffithiana (both 2n = 32) havesix major and two minor rDNA sites. A. thaliana (2n = 10) isknown to have two pairs of rDNA sites. a highly repeated para-centromericsequence from A. thaliana, pAL1, is absent in the other threespecies. Hence the A.thaliana genome is not present (or thecentromeric DNA has evolved substantially) in the polyploidspecies A. pumila and A. griffithiana. Analysis of Arabidopsisspecies is a valuable complement to the large programmes forgenetic analysis of A. thaliana.Copyright 1993, 1999 AcademicPress Arabidopsis, centromeric DNA, maps (genetic), nuclear architecture, repetitive DNA, ribosomal DNA, rDNA, evolution, Brassicaceae, Crucifereae, in situ hybridization     

Distribution of 5S and 35S rRNA gene sites in 34 Chenopodium species (Amaranthaceae)

Studies on Chenopodium chromosomes are scarce and restricted mainly to chromosome number estimation. To extend our knowledge on karyotype structure of the genus, the organization of 5S and 35S rRNA genes in Chenopodium chromosomes was studied. The rDNA sites were predominantly located at chromosomal termini, except in a few species where 5S rDNA sites were interstitial. The majority of the diploid species possessed one pair each of 35S and 5S rDNA sites located on separate chromosomes. Slightly higher diversity in rDNA site number was observed in polyploid accessions. One or two pairs of 35S rDNA sites were observed in tetraploids and hexaploids. Tetraploid species had two, four or six sites and hexaploid species had six or eight sites of 5S rDNA, respectively. These data indicate that, in the evolution of some polyploid species, there has been a tendency to reduce the number of rDNA sites. Additionally, polymorphism in rDNA site number was observed. Possible mechanisms of rDNA locus evolution are discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ??, ??–??.     

An Improved Nonfluorescent Detection System for in Situ Hybridization in Plants

Molecular cytogenetics, particularly the localization of DNA sequences by in situ hybridization, has increased our understanding about the genomic structure of plants and animals. We demonstrate here the application of an improved nonfluorescent in situ hybridization system detection (DAKO GenPoint system) to plant chromosomes. Using this system, highly repetitive 18S-25S rRNA genes were mapped on Vicia faba chromosomes (2n = 12). The modified method of this horseradish peroxidase based enzymatic detection system gave satisfactory results that are comparable to fluorescent signal detection.     

The ability to bind albumin is correlated with nitric oxide sensitivity in Moraxella catarrhalis

Moraxella catarrhalis is sensitive to NO generators e.g. S -nitroso- N -acetylpenicillamine (SNAP) and sodium nitroprusside (SNP), but can spontaneously develop higher SNP tolerance. Using SDS-PAGE of outer membrane proteins and immunoblotting for serum albumin, we found that the wild strain bound more blood-medium-derived albumin than the SNP-resistant variant did. There was a negative correlation between NO tolerance and the presence of serum albumin in the medium. We suggest that M. catarrhalis can change its surface properties to avoid binding albumin and thereby increase its resistance to NO. Growth of Moraxella is affected by iron, and that may have influenced our results. Using chrome azurol S plates as an indicator, we noted that both albumin and SNP have a strong affinity for iron(III).     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Physical mapping of rDNA loci in Brassica species.

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.