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.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Isolation of specific human metaphase chromosomes

The human karyotype can be subdivided into seven fractions containing specific chromosomes to provide material for recombinant DNA research. The isolated metaphase chromosomes are sorted according to size by velocity zonal centrifugation, and specific chromosome groups are further purified by electrostatic deflection in a flow microfluorometer. Rapid improvements in technology should soon provide preparations of single chromosomes.     

Differential spiralization along mammalian mitotic chromosomes

The morphology of human metaphase chromosomes of peripheral blood lymphocytes taken from normal persons of both sexes and cultured at the final stages of the S-period in the presence of 5-bromodeoxyuridine (BUdR), or 5-bromodeoxycytidine (BCdR) was studied. It was observed that the chromosomes of the complement were capable of responsing to the treatment with analogs by the appearance of extended segments along their length. The pattern of segmentation was constant and specific for a given chromosome, serving as a basis for its identification, and appeared to be similar for both analogs. — Autoradiography of such chromosomes performed with ³H-thymidine (³H-TdR), ³H-deoxycytidine (³H-CdR), and ³H-BUdR showed that the extended chromosomal segments are late replicating. In accordance with this correlation, the most regular and distinctive segmentation was observed in chromosomes having large late replicating regions, such as Nos. 4, 6, 9, 13, 16, X, and Y. — A comparative analysis of the BUdR-induced differential spiralization pattern and banding pattern obtained with the G-staining technique was carried out. A good correspondence between the extended segments and Giemsa-positive bands was found. The data are discussed in relation to the mechanism of differential staining of metaphase chromosomes.     

Differential spiralization along mammalian mitotic chromosomes

Morphology of chromosomes replicating in the presence of 5-bromodeoxyuridine was studied using long-term cultures of Chinese hamster cells (line Blld-ii-FAF28). The cytological effect of the analog administered in various concentrations, at different stages of the S period, and during one and two successive mitotic cycles was studied. — The main cytological manifestation of the BUdR action consisted in spiralization delay of certain chromosome regions. The degree of the delay was dependent on the time interval between the introduction of the agent and mitosis, as well as on the agent's concentration. With prolongation of the interval, the spiralization delay diminished and disappeared being therefore always observable only in late replicating chromosome regions. Increased concentration of BUdR (in the range of 25 to 400 g/ml) produced enhancement of the delay of chromosome spiralization. — After two successive reproduction cycles in the presence of BUdR, a great number of metaphases contained chromosomes the sister chromatids of which showed unequal spiralization delay. Autoradiography of ³H-BUdR distribution showed that the sister chromatid with a more pronounced underspiralization corresponds to the chromatid incorporating BUdR into both strands of the DNA molecule. — Mechanisms of the effect observed, as well as chemical influence on chromosome spiralization as a usefull tool of displaying linear chromosome differentiation, are discussed.     

Semiconductor nanocrystal probes for human metaphase chromosomes

To improve signal stability and quantitation, an optically stable, novel class of fluorophore for hybridization analysis of human metaphase chromosomes is demonstrated. Detection of hybridization sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide [(CdSe)ZnS]. Fluorescence of nanocrystal fluorophores was significantly brighter and more photostable than organic fluorophores Texas Red and fluorescein. Thus, semiconductor nanocrystal fluorophores offer a more stable and quantitative mode of fluorescence in situ hybridization (FISH) for research and clinical applications.     

DNA catenation maintains structure of human metaphase chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.     

Cytochemistry of RNA in metaphase chromosomes of human PHA-blasts

RNA which is resistant in situ to ribonuclease in solutions of high ionic strength was demonstrated in interphase nuclei and metaphase chromosomes of human PHA-blasts. Since the synthesis of this RNase-resistant RNA is not affected by actinomycin D it is likely that it is of a non-ribosomal type. The resistance to ribonuclease is confirmed in part by the formation of RNA-protein complexes. Two features are characteristic of the localization of this type of RNA within the chromosome: (1) its localization in the telomeric regions; (2) its symmetrical distribution in sister chromatids.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 1182 mitoses of 21 patients with trisomy 13 and in a control group. In addition, 173 trisomic mitoses were compared with the same number of diploid mitoses in a case of mosaicism.The number of mitoses with associations was no higher in the trisomic cells than in cells with normal karyotypes. Some differences were observed in the frequency of associations per cell and of the types of associations in the patient group and in the trisomic cells of the mosaic case. The number of associations in which more than two acrocentric chromosomes were involved was unexpectedly low in the cells with a supernumerary chromosome 13.The result are interpreted as suggesting the existence of a compensatory mechanism activated by the additional acrocentric chromosome.Parts of this work are included in the doctoral (MD) thesis of DM     

High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes

Summary In photomicrographic negatives of cytochemically stained human metaphase preparations, images of individual chromosomes were scanned interactively with a Zeiss SMP interfaced to a PDP-12 computer.By means of the CHROSCAN computer program spot-scanning of selected chromosomes was performed in a direction perpendicular to the length axis, each measured value being corrected for background absorbance taken on both sides of the chromosome image. Plotting of the integrated absorbance values of each transversal scanline results in a graphic representation of the absorbance distribution over the chromsome length. Following this procedure, longitudinal curves were obtained which showed the characteristic patterns obtained after Q or G banding, and, in the case of Feulgen-staining, represented quantitative variations of DNA mass along the individual chromosomes. For Feulgen-stained chromosomes, the total integrated absorbance value and the ratio of integrated absorbance in the long arm over the total integrated absorbance, correspond with the DNA-absorbance and-arm ratio values per chromosome respectively.The results of investigations concerning the reproducibility and accuracy of cytochemical Feulgen staining and of the photographic procedures are presented, together with total integrated Feulgen-DNA absorbance and arm ratio values for a number of human chromosomes.For several chromosomes, Feulgen absorbance arm ratio measurements were found to result in values more constant over different metaphases when the long arm was considered to start at the lowest dip in the longitudinal absorbance curve, than when the microscopically observable primary constriction was taken to represent the centromere. The results indicate that the present method allows accurate photometry of naturally absorbing, or of stained or fluorescent objects, with measuring intervals of 0.16 . In addition it is shown that the arm ratio values and total DNA content can serve as very constant parameters for karyotype analysis.Supported by grant nr. 28-16915 of the Praeventiefonds, 's Gravenhage.     

Localization of chromosomal RNA in human G-banded metaphase chromosomes

Chromosomal RNA with an 11% dihydropyrimidine content was extracted from human placental chromatin. Under appropriate conditions, this RNA showed wide-spread in situ hybridization to metaphase chromosomes. This included preferential hybridization to the telomeric regions and heterochromatic short arms of acrocentric chromosomes as well as significant hybridization to Q- and G-positive bands.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 2715 mitoses of 90 meningiomas with different numbers of acrocentric chromosomes. In cells with monosomy 22, a significant increase of mitoses with associations was observed in comparison to cells with a normal karyotype. The number of associating acrocentric chromosomes was highly significantly increased. This surplus was not only caused by a highly significant increase of associating G chromosomes but also of D chromosomes. The loss of further acrocentric chromosomes had no significant influence on the number of mitoses with associations or the number of associating chromosomes. Based on the well-known correlations between the nucleolus organization and the association pattern, the results seem to indicate a compensation mechanism among the nucleoles organizing regions (NOR's) which keeps the supply of nucleolar material constant and simultaneously causes a higher association tendency between the remaining acrocentric chromosomes. The increase of associations in the 22 monosomic cells was interpreted as a overcompensation after the loss of only one NOR.