Uneven extraction of protein in Chinese hamster chromosomes during G-staining procedures

To elucidate the role of chromosomal protein in G-band production, changes of protein distribution in chromosomes were studied in situ at each step of G-staining procedures. As a highly specific stain for protein, dansyl Cl was used, which conjugated with amino groups in polypeptide to emit bright fluorescence under UV irradiation, so that the pattern of fluorescence of dansyl-stained chromosomes was expected to reflect the distribution of protein. Uniform fluorescence pattern observed in untreated, dansyl-stained chromosomes indicated even distribution of protein in the ordinary air-dried chromosomes. The pattern of fluorescence representing the distribution of chromosomal protein after pretreatments of G-staining showed brighter outlines of chromatids, reduced fluorescence of chromosome body, and a slight difference in intensity along chromosome arms which corresponded to G-bands. This correspondence was confirmed when Giemsa stain was removed from G-banded chromosomes and the chromosomes were stained with dansyl Cl. The resulting dim fluorescence pattern conformed to G-bands previously observed in the same chromosomes. Similar events were observed in HCl-extracted chromosome slides, although the fluorescence was considerably reduced in this case. Our results inferred that chromosomal protein was partially lost during pretreatments of G-staining, that acid-soluble protein assumed less significant role in G-staining mechanism, and that uneven deprivation of acid-insoluble protein may occur during G-staining procedures.     

植物染色体G-带的初步研究

本文首次报道了川百台(Lilium davidii)、华山松(Pinus armardii)和七叶一枝花(Paris polyphylla)等植物染色体G-带研究结果。本试验的G-带与以往的C-带不同,C-带每条染色体上一般只有1-4条带,多分布在着丝点附近,而G-带则多达几十条,分布在整条染色体上,带纹清晰,前期染色体带呈颗粒状,中期染色体呈明显的带状,与哺乳动物染色体G-带很相似。G-带的数目取决于染色体浓缩的程度。前期染色体带纹数目是中期的三倍,接近人类高分辨带水平。对G-带带纹采用了自动光谱分析,波峰数值与带纹相符。作者同时介绍了胰酶法在植物染色体G-带中的应用。认为此方法既适合动物亦适用于植物。但植物G-带显示的关键可能不在胰酶法本身,而在合适的分裂时期及染色体处理技术。     

Occurrence of G-banding in metaphase chromosomes of Encarsia berlesei (Hymenoptera: Aphelinidae).

Well-defined G-bands were obtained on somatic metaphase chromosomes of Encarsia berlesei using trypsin and warm 2x SCC in sequence. The G-banded pattern allowed rapid identification of all five metacentric chromosomes, which appeared uniformly lighted when stained with DAPI fluorochrome dye. It is stressed that ageing affects G-banding in this insect species; in fact, good banded chromosomes were obtained on 1-month air-stored chromosomes. Evidence for asynchronous condensation on the chromosomes of this species is also provided.     

Why plant chromosomes do not show G-bands

Summary Giemsa techniques have refused to reveal G-banding patterns in plant chromosomes. Whatever has been differentially stained so far in plant chromosomes by various techniques represents constitutive heterochromatin (redefined in this paper). Patterns of this type must not be confused with the G-banding patterns of higher vertebrates which reveal an additional chromosome segmentation beyond that due to constitutive heterochromatin. The absence of G-bands in plants is explained as follows: 1) Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of comparable length. At such a high degree of contraction vertebrate chromosomes too would not show G-bands, simply for optical reasons. 2) The striking correspondence of pachytene chromomeres and mitotic G-bands in higher vertebrates suggests that pachytene chromomeres are G-band equivalents, and that this may also be the case in plants. G-banded vertebrate chromosomes are on the average only 2.3 times shorter in mitosis than in pachytene; the chromomeric pattern therefore still can be shown. In contrast, plant chromosomes are approximately 10 times shorter at mitotic metaphase; their pachytene-like arrangement of chromomeres is therefore no longer demonstrable.     

Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

Studies on G-bands and Macrocoils in Plant Chromosomes

Four different methods including trypsin urea, SDS and NaOH are presented for the in situ induction of G-bands and macrocoils on the chromosomes of Secale cereale, Hordeum vulgare and Vicia faba. The bands obtained were numerous and along the whole chromosome, the number of the G-bands was much interrelated with the condensation of chromosomes. The bands of homologous chromosomes in some cells were matchable. The G-banded chromosomes in late prophase have nearly reached high resolution level. When incubation periods were beyond critical time for G-banding, macrocoils were often revealed. Gyre number changed with chromosome condensation and the direction of coils has showed different patterns. Transformation of G-bands into macrocoils was first reported in plant chromosomes. Some chromosomes showing G-bands under light microscope appeared spiral patterns under scanning electron microscope. In this paper the relationship between G-bands and macrocoils in plant chromosomes is also discussed.     

浮游病毒的电镜观察

直接用戊二醛固定水样中的浮游病毒,通过超速离心使已固定的浮游病毒沉淀到覆有Formvar膜和碳支持膜的铜网上,经醋酸双氧铀染色后,利用透射电镜对湖水中的浮游病毒进行观察．结果可观察到球形、杆状和蝌蚪状等形态各异的浮游病毒颗粒及球形病毒的囊膜子粒、杆状病毒的核衣壳、具有不同尾部的蝌蚪状病毒等的精细超微结构．从而建立了一种简便、快捷和高效研究浮游病毒的电镜方法．     

Morphology of microtubules in rabbit platelets

Summary The morphology of the microtubular wall in rabbit platelets fixed in glutaraldehyde and osmic acid solution and stained with uranyl acetate, or lead hydroxide, or doubly stained, is variable. In cross section, the wall may appear as a uniformly dense annulus, an annulus containing nodular densities about 35 Å in diameter or as a series of contiguous subunits with a circular cross sectional profile about 70 Å wide. The authors relate the varying morphology to the intensity of staining and equate the nodular and circular subunits. Rotational analysis suggests that there are 12 ± 2 subunits in the microtubular wall.We thank Professor A. C. Ritchie for his criticism of the paper and Mrs. M. Lorber and Mrs. M. Mezari for technical assistance. — This work was supported by grants from the Medical Research Council of Canada and the Ontario Heart Foundation.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.     

Dependence of heterochromatin differential staining on the time of its reduplication and the degree of condensation]

A comparison of the chromosomes banding pattern after G-and C-staining with the time of DNA reduplication and the degree of chromosome condensation, was carried out using Chinese hamster metaphase chromosomes. Chromosome condensation was studied under 5-bromodeoxyuridine and 5-bromodeoxycytidine treatment. All the chromosomal segments stained with C-technique are also stainable with G-technique, while only some G-positive segments are capable to be C-bands. C-bands are heterochromatic segments characterized by extremely late replication and great delay in condensation under the analog action, while G-bands are segments with earlier labelling and irregular decondensation. The data obtained suggest a close correlation between the capability of chromosomal region of G- and C- staining and the degree of its heterochromatinization.     

High resolution bands in maize chromosomes by G-banding methods

Summary It was demonstrated that G-bands are unequivocally present in plant chromosomes, in contrast to what had been formerly believed by plant cytologists. Maize chromosomes prepared by an enzymatic maceration method and treated with trypsin or SDS showed clear G-bands spreading along the chromosomes. The most critical point during the G-banding procedures was the post-fixation with glutaraldehyde solution. Banding patterns were processed by using the chromosome image analyzing system and a clearer image was obtained. Gbanding technique and the image manipulation method described here can be applied to many plant species, and would contribute new information in the field of plant cytology and genetics.     

Zur Ultrastruktur der Chromosomen des Menschen

Zusammenfassung Dünnschnitte von hypoton vorbehandelten Metaphase-Chromosomen des Menschen zeigen im Elektronenmikroskop unregelmäßig und vielfach gefaltete Fibrillen von ca. 200–250 Å Durchmesser. Nach Uranylacetatkontrastierung ist vorwiegend das Zentrum der Fibrillen dargestellt, während nach Phosphorwolframsäurebehandlung die Peripherie kontrastiert ist. Die Fibrillen erscheinen durch die hypotone Vorbehandlung wahrscheinlich verdickt.Als wahrscheinlichste Deutung der Bilder wird angenommen, daß eine Fibrille, die aus einem DNS-Doppelschraubenmolekül mit einem Histongerüst besteht, in viele kleine Falten und Schlingen gelegt, einen dickeren Strang, der einem chromatid entspricht, aufbaut. In der Metaphase ist dieser Strang noch zusätzlich in große Windungen gelegt.

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Ultrastructure of chromosomes

Summary Electron microscopy of sections of hypoton pretreated human metaphase chromosomes reveal irregular and multiple folded fibrils ca. 200–250 Å thick. After staining with uranyl acetate the central core of the fibrils is contrasted, whereas after treatment with phosphor tungsten acid the periphery is stained. The fibril appears to be thickened due to the hypotonic pretreatment.The most propable interpretation seems to be, that one fibril, made up of a DNA double helix and a histon-frame, is laid into many irregular minor foldings building up a strand corresponding to a chromatid. In metaphase this strand is laid into major coils.

     

Fluorescent staining of L cell centromeres and chromocenters with 1-dimethylaminonaphthalene-5-sulfonyl chloride and G-bandings

Fluorescent staining patterns of L cell chromosomes with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride) were studied. Ordinary air-dried L cell metaphase chromosomes exhibited relatively uniform and bright yellowish green fluorescence by dansyl-staining under the fluorescence microscope. However, after the chromosome preparations were treated with 10 mM NaCl for 24 h at 4 °C, which produced distinctive G-bands with Giemsa-staining, the centromeric regions and several interstitial regions of some particular chromosomes were clearly fluorescent but other regions showed only dull fluorescence. After the treatment of chromosome slides with cupric sulfite reagent, which converts sulfhydryls and disulfides to thiosulfates chromosomes showed clear G-bands which were indistinguishable from those after 10 mM NaCl treatment. By dansyl-staining, however, the cupric sulfite-treated chromosomes exhibited very faint fluorescence on their contour alone, and neither centromeric regions nor some interstitial regions of marker chromosomes had distinctly bright fluorescence.Although Giemsa-staining disclosed dark chromocenters in approx. 75% of interphase nuclei irrespective of pretreatments, dansyl-staining revealed bright chromocenters in approx. 60% of interphase nuclei in control slides, in about 40% of nuclei in 10 mM NaCl-treated slides, and in only about 30% of nuclei in cupric sulfite-treated preparations.These observations indicated that in the air-dried chromosome preparations, the distribution of protein over the metaphase chromosome is relatively uniform along its length, and that G-bands in the chromosome and Giemsa-staining of chromocenters in interphase nuclei are not significantly affected by apparent loss of protein from the preparations. It was also suggested that particular protein may be associated with the centromeric regions of L cell chromosomes. Some technical details of dansyl fluorochroming and the significance of the observations were discussed.     

G-band-like structures and centromeric asymmetry in the BrdU containing mouse chromosomes

Mouse cells cultured in the presence of BrdU or BrdC for one replication cycle were stained in a 4Na-EDTA Giemsa solution which stains BrdU-containing chromatin preferentially (Takayama and Tachibana, 1980). With this treatment clear bands (B-bands) were revealed along the length of the chromosomes. The B-banding patterns were identical with the G-banding patterns of this species except for the centromeric region in which lateral asymmetry of Giemsa staining was seen. The concomitant occurrence of the lateral asymmetry with the B-banding supports the assumption that the B-bands visualized by the present technique reflect the BrdU-rich chromatin regions differentially localized along the chromosomes. Most of the chromosomes constituting the mouse karyotype showed their own characteristic appearance of the asymmetry, but in some of them the asymmetry was not clear and the Y did not show any specific, centromeric staining. The marked coincidence of the B- and G-banding patterns seems to provide evidence for the involvement of AT-rich chromatin in the induction of positive G-bands. The present technique also seems quite useful to analyze chromosomes of some species in which ordinary G-banding techniques have been known to bring about only unsatisfactory results.     

Scanning electron microscopy of the G-banded human karyotype

The chromosome structure of human metaphases was observed in the scanning electron microscope (SEM) after exposure to G-banding techniques for light microscopy (LM). Individual chromosomes showed an inherent specificity of quaternary coiling. Circumferential grooves along the chromatids demarcated the individual gyres of the coils, which were shown to correspond to the LM G-banding pattern. An increased number of quaternary coils was observed in prometaphase chromosomes, which were shown to be correlated with the high resolution LM bands. We propose that the observation of G-bands relies on LM visualization of quaternary structure by accumulation of Giemsa stain between the coils.     

Collagen formation — observations on its intracellular packaging and transport

Summary Odontoblasts, osteoblasts and fibroblasts of young rats were examined in the electron microscope after staining thin sections either with lead citrate alone or with uranyl acetate prior to lead citrate.With lead citrate alone, collagen fibrils in the extracellular matrix stand out as lucent structures against a moderately electron dense background. Within the cells, lucency is restricted to certain dilated portions of the Golgi saccules as well as to the secretory granules located nearby and in the secretory pole of the cells. The lucency present in these compartments may be attributed to fibrils that are similar to the lucent collagen fibrils in the extracellular matrix. Other cellular compartments, e.g. the rough ER, do not display lucency.When preparations are stained with uranyl acetate prior to lead citrate, lucency is observed neither in the matrix nor in the cells. In the matrix, collagen fibrils are easily identifiable by their cross banded pattern. In the odontoblasts, dilated portions of Golgi saccules between the outer and inner face contain filaments aligned in parallel that are approximately 3 000 Å in length. In saccules on the inner face filament aggregates are present, some of them exhibiting a cross banding pattern. In secretory granules, however, the contents appear rather homogeneous.It is suggested that filament aggregates of collagen can assemble in the Golgi apparatus from filamentous units. These are transported through the cell by way of secretion granules and are discharged to the extracellular matrix by exocytosis.This investigation was supported by grants of the Medical Research Council of Canada. The author wishes to express appreciation to Dr. C. P. Leblond for his guidance in the course of this work.     

植物染色体高分辨G-带技术研究

本文对植物染色体高分辨 G-带技术进行了比较系统的研究,并首次运用改良的尿素法在野生一粒小麦、玉米、蚕豆、吊兰、川百合等多种植物上诱导出 G-带,带纹清晰,数目多,分布在染色体全长上。前期染色体带呈颗粒状,中期染色体呈明显带状,与哺乳动物染色体 G-带很相似。G-带的数目取决于染色体浓缩程度,中期染色体一条深带到晚前期可显示出2.67条亚带。作者同时比较了胰酶法与尿素法的显带效果。认为两种方法显示的带纹基本相同,尿素法比胰酶法作用温和,显带时间长达数分钟,易于掌握,重复性高,具有更高的应用价值。     

水稻染色体标本制备的风油精法

水稻的染色体较小,不同的染色体在形态上较难区分。常规的压片技术由于很难使染色体分散,且也不能完全排除细胞质的干扰,因而很不适用于水稻染色体核型分析及显带。Kurata 等(1978)采用酶解与火焰干燥技术制备水稻染色体标本,获得清晰的染色体图象,从而成功地进行了水稻染色体的核型分析。陈瑞阳等(1982)参照人类染色体