The role of chromosomal proteins in the C-banding of Allium cepa chromosomes.

When chromosomes of Allium cepa are subjected to a C-banding procedure (incubation in saturated barium hydroxide followed by phosphate buffer at 60 degrees C for 1 h) and then treated with Giemsa stain, bands appear at the telomeres of all chromosomes. Microspectrophotometric measurements of Feulgen-DNA content, demonstrated that the C-banding procedure extracted DNA from the nuclei. Staining of banded chromosomes with several DNA-specific stains showed that this loss was differential, with the band DNA exhibiting more resistance to extraction than that of the rest of the chromosome. The C-banding procedure did not extract chromosomal proteins, however, and no difference in mass per unit length could be detected by Nomarski optics between band and interband regions. Several experiments demonstrated that chromosomal proteins play a significant role in C-banding. First, treatment of chromosomes with pronase before C-banding resulted in the elimination of differential staining with Giemsa. Furthermore, in preparations where the DNA was completely hydrolysed with hot TCA, the remaining chromosomal proteins were found to exhibit a differential affinity for Giemsa stain. Amido black staining demonstrated that total chromosomal protein was uniformly distributed after the hot TCA digestion, but the proteins localized in the telomeres had a greater affinity for the Giemsa stain than the bulk of the chromosomal proteins. When the TCA-digested chromosomes were subjected to the C-banding procedure before staining, the differential affinity of the telomeres for the Giemsa stain was lost. Thus, C-banding appears to be the result of a complex interaction between protein and DNA in which the greater resistance to extraction of the band DNA is necessary to stabilize and preserve chromatin protein which exhibits a differential affinity for Giemsa stain.     

A Modified Giemsa C-Banding Technique For Hordeum Species

A Giemsa C-banding technique with a hot 1 N HCI hydrolysis step has been developed for barley chromosomes. This step makes it easy to obtain well separated C-banded chromosomes. To compare this technique with other C-banding techniques, chromosomes of H. vulgare cv. York were stained by both this technique and a modification of the technique of Kimber et al (1976). With respect to centromeric and intercalary bands, both techniques produce a similar banding pattern, but telomeric bands observed by the modified technique of Kimber et al (1976) were not detected by our procedure. This indicates that telomeric heterochromatin may be different chemically and/or structurally from the centromeric and intercalary heterochromatin and its appearance dependent upon the C-banding technique. The procedure described provides a relatively rapid technique for C-banding of barley chromosomes.     

A technique for C-banding plant chromosomes with Wright stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.     

A Technique for C-Banding Plant Chromosomes with Wright Stain

A technique is presented for C-banding plant chromosomes with a modified Wright stain. This procedure consistently produces brightly stained, well defined telomeric and interstitial heterochromatic bands, identifiable centromeric constrictions, and lightly stained euchromatic areas on chromosomes of rye.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

C-band morphology of T(8;9)/8;9 in Blattella germanica

Summary Centromere position and each arm of the T(8;9)/8;9 quadrivalent at late pachytene can be recognized by C-banding. The chromosome 9 breakpoint lies immediately adjacent to the centromeric C-band; that of 8, in the general region of the centromere but the relationship to centromeric bands was not determined since the latter stain very faintly. Chromosome 9 differs from no. 8 in the presence of a complex series of intercalary bands, heavy centromeric bands and, overall, a larger amount of C-band material. Possible implications of these differences with respect to chromosome breakability and the nature and distribution of mutant loci are noted. Earlier identification of adjacent-1 and adjacent-2 metaphase I ring orientations, made on the basis of orientation pattern, was confirmed.     

Visible light observations on the kinetochore of the Indian muntjac, Muntiacus muntjac, Z

We report here a silver stain technique (Kt stain) for locating the kinetochore (centromere body) without concomitant staining of C-band material. We compare our observations with those obtained from C-banding, Cd (centromeric dot) banding, and electron micrographs, and we report preliminary observations on Indian muntjac centromeres.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

中国两种掌突蟾(锄足蟾科Pelobatidae,无尾目Anura)的细胞遗传学研究

本文对分布于云南境内的两种Leptolalax——L.ventripunctatus和L.alpinus的常规Giemsa核型、C-带和Ag-NORs作了研究,结果表明L.ventripunctatus的2n=22,20M 2T,NF=42,1对Ag-NORs位于5(?),并呈现异形现象,该区域亦显C-带正染;L.alpinus 2n=24,14M 4SM 6T,NF=42,1对Ag-NORs位于No.8短臂端部,并有随体联合现象。两种的着丝点区域均呈现C-带正染。     

Chromosome homology and heterochromatin in goat,sheep and ox studied by banding techniques

Peripheral blood lymphocyte metaphase chromosomes of three Bovoidean species have been studied using Quinacrine fluorescence and Giemsa banding techniques to give Q-, G-, and C-banding patterns. Q- and G-banding characteristics, coupled with chromosome length, enabled all of the chromosomes in each of the chromosome complements to be clearly distinguished, although some difficulties were encountered with the very smallest chromosomes. A comparison of G-banding patterns between the species revealed a remarkable degree of homology of banding patterns. Each of the 23 different acrocentric autosomes of the domestic sheep (2n=54) was represented by an identical chromosome in the goat (2n=60) and the arms of the 3 pairs of sheep metacentric autosomes were identical matches with the remaining 6 goat acrocentrics. A similar interspecies homology was evident for all but two of the autosomes in the ox (2n=60). This homology between sheep metacentric and goat acrocentric elements confirms a previously suggested Robertsonian variation. The close homology in G-banding patterns between these related species indicates that the banding patterns are evolutionarily conservative and may be a useful guide in assessing interspecific relationships. —The centromeric heterochromatin in the autosomes of the three species was found to show little or no Q-or G-staining, in contrast to the sex chromosomes. This lack of centromeric staining with the G-technique (ASG) contrasts markedly with results obtained with other mammalian species. However, with the C-banding technique these regions show a normal intense Giemsa stain and the C-bands in the sex chromosomes are inconspicuous. The amount of centromeric heterochromatin in the sheep metacentric chromosomes is considerable less than in the acrocentric autosomes or in a newly derived metacentric element discovered in a goat. It is suggested that the pale G-staining of the centromeric heterochromatin in these species might be related to the presence of G-Crich satellite DNA.     

Heterochromatin diversity in Cymbidium, and its relationship to differential DNA replication

1. 1. DNA was extracted from aseptical cultures of protocorms of the orchid Cymbidium and analysed by thermal denaturation. The denaturation profiles revealed an AT-rich fraction of about 18% of total DNA.

2. 2. Mitotic chromosomes and diploid and endopolyploid nuclei of in vitro cultured protocorms and root tips were differentially stained with quinacrine (Q), 33258 Hoechst (H) and a novel compound 4′-6-diamidino-2-phenylindole (DAPI) [10], as well as by the Giemsa C-banding technique. The centromeric regions display very bright fluorescence with all three fluorochromes and stain intensely following the Giemsa procedure. It is proposed that the AT-rich fraction of the Cymbidium DNA is located within the centromeric heterochromatin.

3. 3. In interphase nuclei differential Q, H, and DAPI fluorescence both within and between the chromocenters occurs. In nuclei with enlarged chromocenters, i.e. with amplified DNA in heterochromatin [2], the increased size of chromocenters is mainly caused by enlargement of the less brightly fluorescing fractions of the heterochromatin. The proportion of the very brightly fluorescing heterochromatin is similar in all nuclei (about 7%).

4. 4. A comparison of nucleolar size and differential Giemsa staining of the nucleolus organizers showed that there is no disproportional increase of nucleoli and nucleolus organizing heterochromatin during endopolyploidization. That means, there is no indication for amplification of ribosomal DNA.

5. 5. Electron micrographs particularly of heterochromatin-rich nuclei revealed areas of different chromatin density within and between the chromocenters. However, the differences in fiber packaging density are much smaller than the observed differences in fluorescence brightness.

6. 6. The data obtained are interpreted as evidence for differential replication of AT-rich and non-AT-rich heterochromatin. It is suggested that DNA amplification [2, 4] is restricted to a non-AT-rich component which apparently is located neither in the brightly fluorescent centromeric nor in the nucleolus-associated heterochromatin.

     

Differences in DNA composition along mammalian metaphase chromosomes

Denaturation of chromosomal DNA in situ can be achieved without disruption of chromosomal morphology by heating slides at 25–90° C in 10–95% formamide in SSC. The extent of denaturation is proportional to formamide concentration and temperature. Reassociation of denatured DNA is prevented with formaldehyde. — The DNA in the paracentromeric constrictions in human chromosomes 1, 9 and 16 denatures earlier than in any other regions, as shown by the red colour with acridine orange. When the temperature or formamide concentration is raised a red and green banding pattern emerges in which regions known to stain brightly with quinacrine mustard are red whereas other regions are green. The last regions to turn red are the short arms of some acrocentric chromosomes. Since A+T-rich DNA denatures before G+C-rich DNA, it is inferred that QM-bright areas are rich in A+T. Similar results are obtained with mouse and Microtus agrestis cells. — Reassociation of chromosomal DNA denatured by heat and formamide occurs if no formaldehyde is used. In human cells, kinetic studies on reassociation indicate that the highest degree of repetition is in the DNA of the distal half of the Y chromosome. Next in degree of repetition are the paracentromeric constrictions, the short arm regions of some of the acrocentric chromosomes, and all the centromeric regions. Highly repetitious DNA is found in all mouse centromeric regions except that of the Y chromosome. Constitutively heterochromatic segments of X and Y and the autosomal centromeric regions of Microtus agrestis also contain repetitious DNA. — It is proposed that differential base content and susceptibility to denaturation of DNA contribute to or at least accompany Q-, G- and R-banding. The degree of C-banding is related to repetitious DNA. The human Y chromosomal DNA is probably A+T-rich and exceptionally repetitious, exhibiting spontaneous reassociation under many experimental conditions.     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

小熊猫染色体异染色质的显示

以培养的小熊猫外周淋巴细胞为实验材料,结合Ｃ－显带技术及ＣＭＡ３／ＤＡ／ＤＡＰＩ三竽荧光杂色的方法,对小熊猫的染色体组型、Ｃ－带带型及ＣＭＡ３／ＤＡ／ＤＡＰＩ荧光带带型进行了研究,发现：（１）经Ｃ－显带技术处理,可在小熊猫染色体上呈现出一种极为独特的Ｃ－带带型。在多数染色体上可见到丰富的插入Ｃ－带及端粒Ｃ－带。而着丝区仅显示弱阳性Ｃ－带;（２）除着丝粒区外,ＣＭＡ３诱导的大多数强荧光带纹与Ｃ－阳性     

New C-band protocol by heat denaturation in the presence of formamide

C-banding techniques detect the presence of constitutive heterochromatin, which is usually located in centromeric regions of chromosomes in the majority of analysed species. The common method for C-banding used over the last 30 years involves treatment with a mild alkali barium hydroxide 5% Ba(OH)2 at 50 degrees C for 5-15 min and subsequent incubation in salt solution (2 x SSC at 60 degrees C for 1 h). We here present a new, easy and reliable technique for C-banding, which basically involves heat denaturation of chromosomal DNA in the presence of formamide and incubation in 2 x SSC at room temperature.     

Cytogenetic study of Anopheles albitarsis (Diptera: Culicidae) by C-banding and in situ hybridization

The C-banding pattern and the size and location of the nucleolar organizer regions (NORs) are described for the first time in Brazilian populations of Anopheles (Nyssorhynchus) albitarsis sensu lato. C-banding revealed variation in the size of the centromeric heterochromatic blocks in autosomal chromosomes and in the acrocentric (X) and puntiform (Y) sex chromosomes. Fluorescence in situ hybridization showed that the NORs were located in the pericentromeric region of the sex (XX/XY) chromosomes and that this coincided with the number and location of centromeric constitutive heterochromatin blocks previously revealed by C-banding. The NORs varied in size among the homologues of the three populations. These findings of the populations studied support the hypothesis that the stability of NORs in the A. albitarsis complex is characterized by the presence of clustered and conserved sites in a unique pair of chromosomes.     

簇毛麦染色体的改良C—分带

运用改良的C-分带技术,使簇毛麦(Haynaldia villosa Schur)V染色体组的7对染色体全部显示出特异性的丰富带纹,包括与N-带相似的近着丝粒带以及N-带所不具有的末端带和亚端带。这些带纹使V组7对染色体之间以及簇毛麦染色体与小麦染色体之间能明确地相区分。     

Differential silver staining of chromatin in metaphase chromosomes

The silver techniques used to demonstrate nucleolar organizer regions and cores in chromosomes can also differentially stain chromatin within chromosomes. Direct silver staining of mouse and human chromosomes resulted in preferential staining of centromeric regions and non-nucleolar secondary constrictions, both of which are composed of constitutive heterochromatin. After C-banding, these regions were no longer silver-stainable, suggesting that the biochemical constituents (presumably non-histone proteins) which contain the reaction sites for silver are extracted during the banding treatment. Light and electron microscopy of chromosomes G-banded with trypsin and then silver-stained revealed heavier deposits of silver over the condensed aggregates of chromatin within the band regions than over the more dispersed interband chromatin. At the ultrastructural level, chromatin fibres were covered with silver grains, indicating that there are many reaction sites for this metal along the fibres. These results suggest that the degree of silver staining in any region of the chromosome may be contingent upon the concentration of chromatin in that region. This finding may have important implications concerning the nature of the silver-stained core-like structure in chromosomes. If a preferential dispersion of chromatin fibres occurs at the periphery of the chromosome during slide preparation, leaving the central region of each chromatid relatively undispersed, this difference in the concentration of chromatin may account for the differential silver staining of these regions and the consequent appearance of a core-like structure.     

Karyotyp und Heterochromatinmuster des rumänischen Hamsters (Mesocricetus newtoni)

In the Romanian hamster (2n=38) a number of whole chromosome arms is heterochromatic. This offers the opportunity to test the effect of some recently developed differential staining techniques upon heterochromatin. It is shown that the late replicating segments are stained by the C-banding technique. A method for exclusively demonstrating centromeric heterochromatin is described. With this, only 8 autosome pairs and the X-chromosome show centric heterochromatin. There is a good agreement between the multiple banding pattern produced by fluorescent and Giemsa stain.

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Stipendiat der Alexander-von-Humboldt-Stiftung.     

Effects of DAPI on human leukocytes in vitro.

DAPI (4'-6-diamidino-2-phenylindole), a fluorochrome specific for AT-rich DNA, was supplied for 24 h at various concentrations to human leukocytes in culture. This treatment caused the appearance on the chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1,9, and 16, a short region on the long arm of chromosomes 1 and 2, and the distal heterochromatic part of the long arm of the Y chromosome were despiralized. The despiralization pattern of DAPI is compared with those previously obtained with Hoechst 33258 and Distamycin A.