High-resolution idiogram of Giemsa R-banded human prophase chromosomes

The schematic representation of RHG-banded chromosomes (R-banding was produced by heat denaturation followed by Giemsa staining (RHG) in the 850-band range per haploid set, was prepared showing the relative position, the specific size, and the characteristic staining intensity for each band. To this idiogram was adapted the new International Standard Cytogenetic Nomenclature. Our aim was to produce a realistic idiogram which could help in the preparation of R-banded prophase karyotypes and in the localization of chromosomal rearrangements. A comparative analysis of bands at prophase and metaphase revealed certain aspects of the dynamics involved in chromosome condensation and in R-band organization. The effect of chromosome elongation on the appearance of R-bands within heterochromatic regions has also been discussed.     

A simple method to sequentially reveal Q- and C-bands on the same metaphase chromosomes

The Q-C staining procedure, i.e., to treat QM stained preparations with a modified ASG method, conveniently provides Q? and C? (instead of the expected G?) bands on the same metaphase chromosomes. This procedure is especially useful for karyological study of heteroploid cells and interspecific cell hybrids in which extensive chromosomal rearrangements and complex karyotypic compositions are present. A few examples of karyological interest are reported. C-bands, which are either Q+ or Q?, can be divided into two to three subsegments in human chromosomes 1, 9, and 16. These subsegments are connected by a Q?/C?element. Interstitial C-bands could have originated mostly from a C-band without the kinetochore or with the“non-functional” kinetochore.“Double Minutes” are of two kinds, Q+/C+ and Q?/C+. Mechanism for the production of C-bands by the Q-C procedure is briefly discussed.     

Localization by FISH of the 31 Texas nomenclature type I markers to both Q- and R-banded bovine chromosomes

A series of 31 marker genes (one per chromosome) were localized precisely to both Q- and R-banded bovine chromosomes by fluorescence in situ hybridization (FISH), as a contribution to the revised chromosome nomenclature of the three major domestic bovidae (cattle, sheep and goat). All marker genes except one (LDHA) are taken from the Texas Nomenclature of the cattle karyotype published in 1996. Homologous probes for each marker gene were obtained by screening a bovine BAC library by PCR with specific primer pairs. After labeling with biotin, each probe preparation was divided into two fractions and hybridized to bovine chromosomes identified either by Q or R banding. Clear signals and good quality band patterns were observed in all cases. Results of the two series of hybridizations are totally concordant both for Q and R band chromosome numbering and precise band localization. This work permits an unambiguous correlation between the Q/G- and R-banded 31 bovine chromosomes, including chromosomes 25, 27 and 29 which remained unresolved in the Texas Nomenclature (1996). Hybridization of the chromosome 29 marker gene to metaphase spreads from a 1;29 Robertsonian translocation bull carrier showed a positive signal on the short arm of this rearranged chromosome, confirming that the numbering of this long-known translocation in cattle is correct when referring to the Texas Nomenclature (1996). Taking into account that cattle, goat and sheep have very similar banded karyotypes, the data presented here will help to establish a definite and complete reference chromosome nomenclature for these species.     

CT banding of human chromosomes

Summary This paper decribes a study of the role of certain cations in the alkaline pretreatment step used in the CT technique for chromosome band formation. Treatment of human chromosomes with ammonium bases or with the hydroxides of the monovalent alkali metals Na, K, or Li resulted in their rapid disintegration, unless very short treatment periods or diluted solutions were used. In the latter cases a subsequent staining produced a weak G-banding pattern. The chromosomes appeared to be much less sensitive to treatment with the hydroxides of the divalent alkaline earch metals Ba, Sr, Ca, and Mg. Staining after exposure to these hydroxides yielded R-banding patterns. The reduced alkali sensitivity of the chromosomes and the reverse banding pattern formation observed are probably the result of a chromatin stabilization by the divalent cations of the alkaline earth metals. It is proposed that not only in the R-band formation with the hydroxides of the alkaline earth metals but also in that obtained by other techniques, chromosome stabilization plays an important role.     

CT banding of human chromosomes

A technique is described for staining centromeric areas and reverse, mainly telomeric bands in human chromosomes. With this "CT" technique karyotyping of C-banded metaphases is possible without previous or subsequent use of other banding methods. The method consists of an alkaline pretreatment at 60 degrees C with Ba(OH)2, followed by salt incubation in 2 X SSC at 60 degrees C and staining with the cationic dye "Stains-all". In a series of experiments the influence of the variables in the procedure was studied, with the following main results: 1) Ba(OH)2 treatment alone and subsequent staining produces a distinct reverse banding pattern in which the secondary constriction of chromosome 9 is positive. 2) The 2 X SSC incubation in the CT procedure causes the Ba(OH)2 induced reverse bands to become weaker; the centromeric regions, however, become very prominent. 3) If the temperature of the 2 X SSC treatment is raised to 85 degrees C, the CT technique results in a specific staining of the short arm regions of some probably variant acrocentric chromosomes. The interphase nuclei of individuals possessing such acrocentrics usually show very distinct chromocentres after this treatment; in the polymorphs these chromocentres are often situated along the nuclear membrane. The mechanisms which may form the basis of the staining results obtained, and the possible significance in human cytogenetics of the techniques described, are discussed briefly.     

The ultrastructure of R-banded chromosomes

Electron microscopy has been used to study the fine structural organization of R-banded chromosomes prepared by treatment of the chromosomes with a hot NaH₂PO₄ solution. The results indicate that there is a structural basis for R-banding with this technique. In comparison to untreated control chromosomes, the R-banded chromosomes had a greatly reduced electron density, suggesting that the heat treatment has a general adverse effect on chromosome structure. Chromatin fibers formed a coarse, irregular network throughout the chromosome and were often enlarged, probably as a result of the fusion of two or more native fibers. The chromatin fibers were more aggregated and had an increased electron density in the R-band regions of the chromosome than in the interbands. This indicates that the treatment has a differential effect on the structure of bands and interbands. A comparison of the ultrastructure of R- and G-banded chromosomes demonstrated that the distribution of aggregated chromatin was reversed by these two types of banding techniques; however, the treatments producing R-banding appeared to induce less extreme differences in the degree of chromatin condensation in band and interband regions than those giving rise to G-banding. It is suggested that alterations of DNA-protein interactions may arise from the differential denaturation of proteins and/or DNA in R-band and interband regions during the heat pretreatment. Such differential alterations in DNA-protein interactions may induce localized changes in the organization of chromatin and may account for the subtle morphological differences observed between the band and interband regions.     

Localization of actin-related sequences by in situ hybridization to R-banded human chromosomes

Using a cytoplasmic actin cDNA probe we have localized a number of actin sequences in the human genome using a novel in situ hybridization technique. Metaphase chromosomes treated to produce R-bands were directly annealed with ¹²⁵I-labeled actin probe. Under these conditions many regions of the genome were apparently denatured enough to be capable of hybridizing with the probe. Most of the actin sites detected in prior experiments using chromosome preparations, which had been completely denatured, were recognized in this experiment. The major advantage of this method over standard in situ hybridization techniques is the marked increase in the resolution of subregional localization.     

Prometaphase banding of human chromosomes with basic fuchsin

Summary A technique is described for the production of detailed and richly contrasting G-band patterns in human prometaphase chromosomes with the aid of the triphenylmethane dye basic fuchsin. The usefulness of this method is illustrated by its application for the precise analysis of two chromosome 11 rearrangements. It is also demonstrated that high-resolution banding with basic fuchsin can reveal bands not present in the international standard idiogram of human prophase chromosomes (ISCN 1981). The technique described can also be used for easy recognition of the late replicating X chromosome, which stains darker than its early replicating homologue. A preliminary analysis of the late replicating X chromosomes in a 49,XXXXY individual suggests that the three supernumerary X chromosomes do not necessarily replicate synchronously.     

Influence of Q- and G-banding on the Feflgen-stainability of human metaphase chromosomes

Synopsis In this paper, model experiments on chicken red blood cell nuclei are described concerning the influence of methanol-acetic acid fixation and irradiation at different wavelengths, with and without prior Atebrin staining on subsequent Feulgen-stainability. In addition, data are reported on the influence on Feulgenstainability of Giemsa-banding procedures, illumination of unstained chromosomes at 220 and 515 nm and exposure of unstained and Atebrin-stained chromosomes to illumination at 440 nm.The ASG and especially the trypsin-Giemsa technique appeared to reduce markedly Feulgen-stainability. The same holds true for Atebrin fluorescence of chromosomes. The data are discussed in relation to their implications for the assumed cause of the Q- and G-banding phenomena. Techniques are described that allow reliable Feulgen DNA measurements of individual chromosomes after application of either G- or Q-banding.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Heterochromatic banding patterns in the chromosomes ofBrimeura (Liliaceae)

The two species of the genusBrimeura (Liliaceae),B. amethystina andB. fastigiata, show a strikingly disjunct geographical distribution. Both species have a bimodal chromosome complement, 2n = 28, which includes 10 large (L) and 18 small (S) chromosomes. Most of the L-chromosomes possess heterochromatic segments which show enhanced quinacrine fluorescence. The resulting banding patterns are species-specific. Two triploid plants have been found inB. fastigiata.     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes

Direct detection of fluorescent in situ hybridization signals on R-banded chromosomes stained with propidium iodide is a rapid and efficient method for constructing cytogenetic maps for species with R-banded standard karyotypes. In this paper, our aim is to establish an R-banded rabbit karyotype nomenclature that is in total agreement with the 1981 G-banded standard nomenclature. For this purpose, we have produced new GTG- and RBG-banded mid-metaphase karyotypes and an updated version of ideograms of R-banded rabbit chromosomes. In addition, to confirm correlations between G- and R-banded chromosomes, we have defined a set of 23 rabbit BAC clones, each containing a specific gene, one marker gene per rabbit chromosome, and we have localized precisely each BAC clone by FISH on both G- and R-banded chromosomes.     

A simple banding technique for identification of human metaphase chromosomes

Summary A simple technique of Giemsa staining is described giving characteristic banding patterns of human metaphase chromosomes; these patterns can be used to identify all 24 chromosome types.

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Zusammenfassung Wir beschreiben eine einfache Methode, die mit geringem Zeitaufwand charakteristische Bandenmuster in menschlichen Metaphase-Chromosomen ergibt. Die Muster ermöglichen die Identifizierung aller 24 Chromosomentypen.

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Director: Prof. Dr. W. Fuhrmann

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Supported by the Deutsche Forschungsgemeinschaft and the Sonderforschungsbereich 35.     

Activity banding of human chromosomes as shown by histone acetylation

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromsomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromsomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a “snap-shot” of the distribution of gene activity on chromosomes at the time of cell harvest. Edited by: P.B. Moens