Banding pattern analysis of human chromosomes by use of a urea treatment technique

A new technique to reveal the banding pattern of human chromosomes is described. Slides prepared by the routine air drying technique were treated with urea-Sörensen buffer solution for ten minutes at pH 6.8 at 37° C. Individual pairs of all human chromosomes exhibited a characteristic banding pattern by this technique, and by its use the karyotypes were analysed. With the exception of some minor differences the banding patterns obtained by the present technique appeared to be identical with those obtained by the Giemsa staining and quinacrine fluorescence methods carried out by previous workers.Contribution No. 877 from the National Institute of Genetics, Japan. Supported by grant-in-aid No. 92332 from the Ministry of Education of Japan.     

Banding pattern observed in human chromosomes by the modified BSG technique

Summary A successful modification of the BSG technique to reveal C and R bands simultaneously in human chromosomes is described. Conventional air dried preparations were treated first with 0.1 N HCl for 30 min at room temperature, then denatured in freshly prepared 3% aqueous solution of Ba(OH)₂8H₂O for 10 min at 50°C. After rinsing, the slides were incubated for 1 h at 60°C in 2xSSC, and stained with Giemsa. The striking intense staining pattern could be observed in chromosome No. 19.The factors involved in the present technique were analyzed changing the concentrations of the reagents and the treatment time. It was evident that R, T and C bands correspond to a progressive destruction of the chromosome sructure mainly by the Ba(OH)₂8H₂O solution.     

A new differential technique for staining the heteropycnotic X-chromosome in female mice

The technique described here is the result of a successful attempt to identify the heteropycnotic X-chromosome at metaphase in female mice. A new modification of the commonly used air-drying technique for spreading chromosomes was employed using bone marrow cells. After treatment with hypotonic solution of 0.5% KCl at 50 °C for 30 min, all the chromosomes except one of the two X-chromosomes were only palely stained with Giemsa solution (pH 6.9) or quinacrine mustard. This X-chromosome with high stainability was not observed in males.     

ASTRIN - a new trichrome staining technique

The new trichrome stain ASTRIN, composed of alcian blue, safranine, thionine and resorcin, differentiates myocytes from fibrocytes, mature from immature cartilagineous elements, heterochromatic from euchromatic state of nuclear chromatin, and detects neurosecretions. The great differential staining potential of ASTRIN is in all probability due to the dissimilar diffusion rates of its components, for no new dye molecule arises from the combination, as shown by spectrophotometric and thin-layer chromatographic analysis.     

Heterogeneous staining of rat hepatocyte nuclei by HBFP technique

Synopsis The non-enzymatic histochemical technique Haematoxylin-Basic Fuchsinpicric acid (HBFP) was studied in fresh-frozen and Carnoy-fixed, paraffin-embedded rat liver sections. The hepatocyte nuclei fell into two populations and showed either a crimson red or purple staining in frozen as well as paraffin sections. The heterogeneous staining of the rat heptocyte nuclei was also present when the tissue sections were stained by Methyl Green-Pyronin stain. The differing nuclear staining was present in the isolated nuclei also. The HBFP technique, therefore, appears potentially useful when applied to liver and other tissues as well.     

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

Banding patterns of the chromosomes of two new karyotypes of the owl monkey, Aotus, captured in Panama.

Two new chromosome complements of Aotus trivirgatus griseimembra are described making a total of five different karyotypes observed in this subspecies inhabiting Panama and the northwestern part of Colombia, South America. Detailed comparisons of the G-banded chromosomes of these five karyotypes suggest that the polymorphism of chromosome numbers 56 and 55 in Panamanian Aotus and 54, 53, and 52 in Colombian Aotus stems primarily from a Robertsonian translocation mechanism involving pairs B13 and B14 (or A1). A second Robertsonian translocation mechanism involving pairs B28 and B29 (or A2) constitutes the karyotypic differences between the two chromosomal races.     

Combination of differential sister chromatid staining,G-banding pattern,and X-inactivation pattern

Summary A method is presented for obtaining a combination of differential sister chromatid staining, G-banding and X-chromosome inactivation pattern. The result of this method enables a precise localization of the sister chromatid exchange points to particular bands of individual chromosomes.     

Banding pattern analysis of initial structural chromosome alterations induced by n-methyl-n'-nitro-n-nitrosoguanidine in Syrian hamster cells

Cultured secondary Syrian hamster embryo cells exposed to 0.5 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) microgram/ml medium exhibited chromatid type of aberrations consisting of gaps, breaks and exchanges. Although no specific chromosome or chromosome segment was preferentially affected, chromosomes belonging to the larger groups tended to more often involved. G-band analysis demonstrated that 80% of the lesions occurred in negative bands, 9% involved the centromere, 3% were on non-banded heterochromatin, and approximately 8% of the lesions could not be definitely categorized by G-band analysis. Whether the lesions occur at positive bands or at the interface between negative and positive bands is difficult to discern by the G-band resolution. The Y chromosome compared to autosomes of similar size rarely had lesions. X chromosome damage was found in both the euchromatic and heterochromatic arms. However, both sex chromosomes, as well as an autosome (E20) which is heterochromatic on its long arm, were not found joined to the chromatids of other chromosomes, further emphasizing that chromosomes with large heterochromatic areas are isolated in terms of chromatid exchange events. The analysis of MNNG induced chromosome damage indicates that the negative bands are the primary site of damage and points of exchange.     

105种人类染色体新核型的细胞遗传学报道

为了探讨异常染色体的遗传效应, 采用细胞培养、G显带及C显带的方法, 根据人类遗传学国际命名体制(ISCN 2009)对染色体核型命名, 对2009年1月至2012年7月就诊广西壮族自治区妇幼保健院检出的新核型进行细胞遗传学及临床分析。在受检者中检出105种人类染色体新核型, 经检索国内外文献未见报道。其中易位86例, 倒位10例, 衍生染色体6例, 重复染色体1例, 等臂染色体1例, 部分重复和缺失1例。结果显示, 染色体异常是导致流产、不孕不育、先天畸形、智力低下、闭经等疾病的重要原因。     

A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4,6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.     

Uranyl-EDTA-hematoxylin: a new selective staining technique for nucleolar material.

This paper deals with the visualization of nuclear structures in glutaraldehyde fixed, acetic acid flattened preparations from Chironomus salivary glands, by means of an uranyl mordanting followed by hematoxylin staining. Under these conditions all the nuclear structures (bands, Balbiani rings, and nucleoli) were deeply stained. Treatment with 0.1 M EDTA for at least 30 sec after uranyl mordanting completely prevents the following hematoxylin staining in all the structures but the nucleolus. With increased EDTA extraction times (60-90 sec) the central region (composed of pars fibrosa) in spontaneously or experimentally segregated nucleoli showed the highest capacity for retaining uranyl ions. This selective staining of the nucleolar (possibly proteinic) material proved also efficient in cells from Drosophila testes and Allium roots.     

Study of constitutive heterochromatin with a new and simplified fluorescence staining technique

A new fluorochrome that preferentially binds itself to the centromeric or the constitutive heterochromatin is described. This stain allows an easy assay, through fluorescence, of the repetitive DNA or bands, supposedly composed of constitutive heterochromatin, in insectivores, rodents and man, without following the in situ hybridization of Pardue & Gall (1970) or the DNA denaturation-renaturation processes of Arrighi & Hsu (1971). The staining patterns with this derivative of a Benzimidazole compound (Hoechst 33258) are induced in the chromosomes without incubation or pretreatment with SSC and are identical to those produced by other techniques. This stain may eventually contribute to elucidating the hitherto unknown molecular mechanisms involved in the relationship between the repetitious DNA sequences and the banding patterns, and to interpreting the mechanisms responsible for the chromosomal rearrangements and aberrations involving the peri-and non-pericentric regions of the chromosomes.Paper read by P.K.S. at the NATO Advanced Study Institute on Comparative Biology of Primates Turin 7–19 June 1972. Supported by the Alexander von Humboldt-Stiftung, Bonn-Bad Godesberg.     

Banding isolated metaphase chromosomes by a sequential fluorescent G/Q technique

Summary G/Q-banding is a new, rapid, fluorescent technique for banding isolated chromosomes that incorporates characteristics of both G- and Q-banding. G-bands, while easily characterized, are often inconsistent when using isolated chromosomes, and Q-bands, while reliable, fade rapidly under UV exposure, making prolonged observation and photography difficult. G/Q-banding combines these techniques to sequentially utilize quinacrine staining over Giemsa banding to produce slow-fading fluorescent G/Q-bands. The background fluorescence in G/Q preparations fades quickly under continued UV exposure, while the chromosomes remain brightly banded and can be observed and photographed for at least five minutes. G/Q-banding was extended to whole cell chromosome spreads and produced results identical to those obtained with isolated chromosomes. Whole cell karyotypes indicate that G/Q-bands generally correspond to Q-bands. Advantages of G/Q-banding as a unique and universal technique over current double-staining procedures are discussed.     

A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

Karyotype analysis of clone L929 murine fibroblasts by using differential chromosome staining

Karyological analysis of mouse fibroblasts L929 has been carried out using the differential staining of chromosomes (44-58% of the total chromosome number), and their derivatives, i.e. markers of the particular clone. Normal, non-rearranged chromosomes are mainly present in 1-3 copies, while the markers are available as a single copy only. The frequency of occurrence of diverse chromosomes differs from cell to cell, the total number of chromosomes in the cells being not constant. The modal class consists of 62-64 chromosomes. Two new chromosome markers were found after a repeated karyological analysis one year after the cultivation of cells under the standard conditions. A possible role of some chromosome aberrations in the process of transformation of mouse fibroblasts is discussed. The particular attention is given to alteration of chromosome 15.