DAPI-banding Patterns Research Articles

Karyotype analysis of Nicotiana kawakamii Y. Ohashi using DAPI banding and rDNA FISH

Prometaphase cells were used to analyze the karyotype of Nicotiana kawakamii Y. Ohashi by means of sequential Giemsa/CMA/DAPI staining and multicolor fluorescence in situ hybridization with 5S and 18S rDNA. Observation of the DAPI-stained prometaphase spreads indicated that N. kawakamii had six pairs of large chromosomes, one pair of medium-sized chromosomes and five pairs of small chromosomes. The six pairs of large chromosomes possessed remarkable DAPI bands, and each could be identified from both the DAPI banding pattern and the length of the short arm. The DAPI banding pattern was approximately identical to the CMA and Giemsa banding patterns. Hybridization signals of the 18S rDNA probe were detected on two pairs of large chromosomes. In addition, two pairs of small chromosomes were identified based on the position of the 5S rDNA signals. An idiogram of N. kawakamii chromosomes was produced based on DAPI bands and rDNA loci.

DNA content, rDNA loci, and DAPI bands reflect the phylogenetic distance between Lathyrus species.

The nuclear DNA content, the proportion of A+T base pairs, the chromosomal positions of 5S and 25S rRNA genes, as well as of DAPI (4,6-diamidino-2-phenylindole) bands are described for seven species belonging to three different sections of the genus Lathyrus. These data and chromosome measurements allowed to establish precise idiograms and to discriminate most of the chromosome pairs of the seven species. The karyotypic features correlate well with the phylogenetic distances between these species.

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.

Position effect of translocations involving the inactive X chromosome: physical linkage to XIC/XIST does not lead to long-range de novo inactivation in human differentiated cells

Given the reported long-range cis-inactivating effect of the XIST gene in early embryonic development and the lack of requirement of X-chromosome-specific elements for propagating the inactive state, there exists the possibility of cis inactivation of autosomal material after de novo translocation to an inactive X chromosome (Xi) in differentiated cells. We have analyzed de novo radiation-induced translocations between the Xi and autosomes to study the maintenance and spreading of X-chromosome inactivation (X inactivation) in relation to the position of the X-inactivation center (XIC)/XIST in differentiated cells. Autosome/Xi translocations were detected by fluorescence in situ hybridization (FISH). The activation status of the chromosomes involved in the translocation was determined by simultaneous immunocytogenetic studies using antibodies against either BrdU incorporated at late S phase or acetylated histone H4. The position of XIC/XIST in the reciprocal products of the translocation was determined by XIST-specific FISH and computer enhancement. In other experiments, the Xq13 region carrying XIC/XIST was localized by computer enhancement of the DAPI banding pattern. Our study in differentiated cells provides a visual demonstration that physical separation from XIC/XIST does not result in reactivation of inactive X-chromosome material and that X inactivation is not spread to the translocated autosomes irrespective of the position of XIC/XIST. This observation suggests that physical linkage to XIC/XIST does not lead to de novo inactivation of autosomal material.

FISH mapping of the IGF2 gene in horse and donkey-detection of homoeology with HSA11.

Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of this human chromosome with ECA12 and ECA7. The novel ECA12 comparative painting results are thus in accordance with the localization of the equine IGF2 gene. Comparison of the hitherto known physical locations of IGF2 in different species, viz. human, cattle, sheep, horse, and donkey, shows that this gene tends to maintain a terminal location on the chromosome arm.

The use of combined FISH/GISH in conjuction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosome integration site and subgenomic allocation of a transgene in-sert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4x = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosome location of transgenes influences expression levels.

The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco

We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4',6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosomal integration site and subgenomic allocation of a transgene insert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4 chi = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosomal location of transgenes influences expression levels.