R-banding produced by DNase I digestion of chromomycin-stained chromosomes

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.     

Supernumerary heterochromatic segments associated with the nucleolar chromosomes of Pyrgomorpha conica (Orthoptera) contain methylated rDNA sequences

The two nucleolus organizing chromosome pairs of the grasshopper Pyrgomorpha conica can carry a proximal supernumerary heterochromatic segment. We employed different cytological techniques to characterize and analyze the possible origin of this segment. The supernumerary segment and the nucleolus organizing regions (NORs) show similar responses after C-banding plus either Giemsa or acridine orange, and chromomycin A₃/distamycin A staining to detect GC-rich chromosome regions. Fluorescence in situ hybridization with a biotinylated rDNA probe demonstrated that the segment originated by amplification of the rDNA genes. However, as the silver staining indicates, the ribosomal genes present in the segment are not active since no nucleolus is formed. The use of in situ digestion with the isoschizomeric MspI and HpaII restriction endonucleases and subsequent Giemsa, ethidium bromide or chromomycin A₃/distamycin A staining, suggests that the segment has been inactivated by DNA methylation.     

Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.     

Chromosome banding in salmonid fishes: nucleolar organizer regions in Oncorhynchus

Chromosome banding patterns obtained by silver staining and chromomycin a3 (CMA3) staining were analyzed in six species of Oncorhynchus: O. tshawytscha, O. kisutch, O. keta, O. nerka, and O. gorbuscha from North America and O. masou from Japan. Four different chromosomal locations of the nucleolar organizer regions (NORs) were found in different species. In O. tshawytscha, O. kisutch, and O. masou the NORs comprised the entire short arms of one medium-sized acrocentric chromosome pair. In O. nerka the NORs were found in an interstitial band on the short arms of one submetacentric chromosome pair and in O. gorbuscha proximal to the centromere on one metacentric chromosome pair. In O. keta the NORs were found on the telomeres of one small submetacentric chromosome pair. As in the related genera Salmo and Salvelinus chromomycin A3 positive bands were found at the same sites as the AgNORs in all species. Salmonid fish are assumed to be ancestral tetraploids and the considerable differences in chromosome number between different species are thought to be the result of chromosomal fusions after tetraploidization. In all members of the genus Oncorhynchus the rearrangements have resulted in the consolidation of the NORs on a single chromosome pair. The possible significance of intra- and inter-species NOR polymorphisms is discussed.     

Karyotypic characterization and constitutive heterochromatin in the grasshopper Stiphra robusta (Orthoptera proscopiidae)

Conventional analysis, C-banding, silver nitrate and base specific fluorochrome staining with chromomycin A3 (CMA3) were used to analyse the meiotic chromosomes of the grasshopper Stiphra robusta. Diploid numbers of 2n = 19 in the males and 2n = 20 in the females were observed. The chromosome complement comprised a graded series of uniarmed chromosomes, and the X chromosome was medium sized. The nucleolar organizer regions, restricted to the bivalent chromosomes 6, 7 and 8, were CMA3 positive.     

Characterization of the canine karyotype by counterstain-enhanced chromosome banding

The application of the counterstain-contrasted fluorescent banding technique to canine chromosomes provided an improved capability to highlight specific heterochromatic regions and to produce well defined banding patterns both on mitotic and meiotic chromosomes. Triple staining with chromomycin A3 - distamycin A - DAPI revealed the occurrence of DA - DAPI positive heterochromatin in chromosomes 33, 36, 37, and 38. Pachytene nuclei present more favourable material for the detection of very small amounts of DA - DAPI material than mitotic division stages. Counterstain-enhanced chromomycin R-banding greatly facilitated chromosome identification. A standard R-band karyotype of Canis familiaris is proposed and described in some detail. DAPI - actinomycin D staining produced a QFH-type banding pattern and enhanced differentiation of some polymorphic regions.     

Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

Ribosomal RNA expression in a mammalian hybrid,the hinny

The expression of nucleolus organizer region (NOR) activity in diploid cells was investigated in a model mammalian hybrid system, the hinny (female ass x male horse), by sequential Ag-NOR and chromomycin A3/distamycin A/DAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes. In the majority of cases we found non-expression of the horse-derived NOR chromosomes in the hinny. However, in one case there was strong NOR expression on horse-derived chromosome no. 1.     

Modeling heterochromatin regions in transgenic mice

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.     

Nucleotide composition of constitutive heterochromatin of Pleurodeles waltlii

Complementary to previous works implying other staining or labeling techniques, this study used chromomycin A3, a specific fluorochrome for G and C bases, to show that in Pleurodeles waltlii Michah. (Amphibian, Urodele) the mitotic chromosome specific heterochromatin consisted of a DNA particularly rich in either of the two types of bases and the near constant presence of argyrophilic proteins. The more the DNA was rich in A and T bases, the more the argyrophilic proteins were abundant.     

Genetic activity of the G- and R-band DNA of human mitotic chromosomes

The concept of genetic inactivity of G-band DNA had been reinvestigated using the modified approach of Korenberg et al (1978). Coefficients of correlation and partial correlation between the relative gene density (g'), the relative G-band material richness (kH/C) and the relative chromosome size (s') were calculated. The kH/C was calculated as the ratio of brightness of fluorescence of chromosomes stained by Hoechst 33258 (Hi) and by chromomycin A3(Ci). The kH/C is the characteristics of G-band chromosome richness, because G-bands become bright after Hoechst 33258 staining and R-bands are bright after chromomycin A3 staining, while no significant C-bands in chromosomes which may be stained by these fluorochromes are discovered. For the kH/C determination the flow cytometry data of Langlois et al (1982) were used. The relative size of chromosomes was determined, based on the flow cytometry data of Young et al (1979). According to Korenberg, the "gene density" (g') in a chromosome was calculated as a ratio of the number of genes located in the chromosome before 1984 (Human Gene Mapping 7) to the relative size of this chromosome. Correlation between the "gene density" and the G-band richness was rs = -0.65. Out of 107 genes located in either G- or R-bands (Human Gene Mapping 7), 90 were mapped in the R-band and only 17 were ascribed to the G-band in metaphase chromosomes. The data on gene replication time show that all genes of the general cell activity and a portion of tissue-specific genes replicate during the early S-phase, together with R-band materials. These three independent lines of evidence are consistent with the notion that the R-band DNA is more genetically active than G-band DNA. The nature of "junk" DNA of G-bands is discussed.     

Band karyotypes and specific types of heterochromatins in several species of European percid fishes (Percidea,Pisces)

The chromosomes of the European Percidae (Lucioperca lucioperca L., Gymnocephalus cernuus L., Gymnocephalus schraetser L. and Perca fluviatilis L.) were analyzed by means of silver staining chromomycin A₃/distamycin A/DAPI and DAPI/actinomycin D fluorescence banding techniques. The nucleolus organizer regions (NORs) were localized at the satellite stalks of chromosome no. 16 in Lucioperca lucioperca and Perca fluviatilis, and of chromosome no. 18 in both Gymnocephalus species. Bright chromomycin A₃ fluorescence clusters were associated with them.Bright distamycin A-DAPI and DAPI/actinomycin D heterochromatic blocks were detected in Lucioperca lucioperca and the Gymnocephalus species.     

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling.

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short fragment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A3 (CMA3) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.     

Cytogenetic analysis of an asymmetric potato hybrid

An asymmetric potato hybrid and its parental lines were cytogenetically examined. DAPI (4'-6-diamidino-2-phenylindole) staining was used to count chromosomes in all analysed lines and revealed the presence of minichromosomes in the hybrid genome. Fluorescent in situ hybridization (FISH) with rDNA sequence as a probe helped to determine the ploidy level of analysed lines and revealed that none of the minichromosomes contains rDNA repeats. CMA (chromomycin A3) band occurred to be a new chromosome marker that identifies potato chromosome No.1. It was possible to detect a deletion in one of four chromosomes No. 1 of the asymmetric potato hybrid. On the basis of these analyses a karyotype of the asymmetric hybrid was constructed.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

Characterization of heterochromatin by sequential counterstain-enhanced fluorescence in three domestic bird species: Meleagris gallopavo, Columba livia domestica, and Anser anser L

The karyotypes of three avian species--Meleagris gallopavo, Anser anser L., and Columba livia domestica--were investigated by means of counterstain-enhanced fluorescence techniques (chromomycin A3/distamycin A/DAPI followed by DAPI/actinomycin D staining). A heterochromatin characterization of macro- and microchromosomes was performed. CMA3-positive (GC-rich) regions in the turkey included the telomeres of chromosomes 1, 3, 4, and Z. In the goose, the chromosome 2 was also CMA3-bright at the telomeres. The W chromosome possessed large amounts of CMA3-bright material on the short arm in both the turkey and the goose. Two types of centromeric heterochromatin were distinguished on acro- to telocentric chromosomes 6 to 14 in the pigeon. The microchromosomal heterochromation of the turkey and goose was GC-rich but had a high degree of variation. In the pigeon, several microchromosomes possessed predominantly AT-rich heterochromatin.     

Counterstaining human chromosomes for flow karyology

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.     

Cytogenetics of bisexual/unisexual species of Poecilia. III. The karyotype of Poecilia formosa, a gynogenetic species of hybrid origin.

Chromosomes of the Amazon molly, Poecilia formosa, a unisexual species of hybrid origin, were investigated by C-banding, silver staining, and fluorescent staining with DAPI, quinacrine dihydrochloride, and chromomycin A3. Analysis of heterochromatin distribution indicates that chromosomes similar to the W chromosome of P. latipinna are not present in the unisexual species. Therefore, morphologically differentiated sex chromosomes do not form the basis of the unisexuality in P. formosa. The number and location of nucleolar organizer regions vary in P. formosa and do not correlate well with those of the parental species.     

Karyotype analysis, banding, and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea : Scarabaeidae)

Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic chromosomes of Gymnopleurus sturmi (Scarabaeoidea : Scarabaeidae) are described. The karyotype is distinguished by a pericentric inversion polymorphism in chromosome 3, which is either acrocentric or subtelocentric. Silver staining (Ag-NOR) and chromomycin A3 (CMA3), failed to study the detection of nucleolar organizer regions (NORs), due to the extensive silver and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped the ribosomal RNA genes (rDNA). FISH with the all-human telomeric sequences (TTAGGG)n revealed a lack of homology between the telomeric probe and the telomeres of G. sturmi. This suggests that the telomeric hesanucleotide (TTAGGG)n is not so conserved within eukaryotes as it has been hypothesized.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.