Trypsin G- and C-banding for interchange analysis and sex identification in the chicken

The trypsin banding methods were applied to feather pulp and embryonic material of the chicken. Two contrasting types of chromosomal banding patterns were obtained by varying the duration of trypsin treatment. A short time treatment shows a G-banding pattern which has characteristic and distinctive bands along the chromosome arms. Prolonging the trypsin treatment causes the G-banding pattern to disappear, and only the centromeres and the W chromosome remained heterochromatic which is characteristic of the C-banding pattern. The application of the G-banding pattern analysis was used to identify regions of chromosomes involved in rearrangements. The simplified trypsin technique which produces the C-banding pattern makes it relatively easy to identify the W sex-chromosome and determine sex in avian species.     

C- and G-bands of the opossum chromosomes: terminal sequences of DNA replication.

The sex chromosomes of the opossum, Didelphys virginiana, are the only elements that exhibit C-banding. In contrast, the sex chromosomes as well as the autosomes bear specific G-Bands. However, unlike other mammalian species different types of G-banding are observed if the chromosomes are pretreated with trypsin and SSC solution The SSC-pretreated chromosomes show discrete bands only when stained with Giemsa at certain pH values. An asynchronous pattern of terminal DNA replication is observed among the three C-banding regions of the X-chromosome. The inter- and intrapositive G-banding areas of the chromosomes are not always late in DNA replication in comparison to those negatively stained G-banding areas.     

Proteins in chromosome banding

Nuclei were isolated from Chinese hamster cells, treated with hypotonic KCl, fixed in acetic methanol, and either air-dried in glass tubes (in situ) or left in suspension (in vitro). These preparations were then exposed to a variety of G-banding treatments, including the 2 × SSC, urea, NaCl-urea, and trypsin methods. The proteins extracted into the treatment solution and those remaining in the nuclei were analyzed by SDS polyacrylamide gel electrophoresis. The three former treatments extracted specific subsets of the total nuclear nonhistone proteins into the treatment solution. Some of the extracted nonhistones were common to all treatments while others were unique to a particular treatment. Variable amounts and types of the histones were also extracted by these treatments, but significant quantities of all of these proteins still remained in the nuclei afterwards. The trypsin treatment appeared to degrade some of the nonhistones, while other non-histones, as well as the histones, were relatively resistant to trypsin digestion. Although there were a few differences in the residual proteins found in the nuclei after the various G-band treatments, the overall electrophoretic patterns of these proteins were generally similar. The results indicate that the G-banding techniques induce specific and reproducible changes in the proteins of isolated nuclei. If these banding treatments induce similar changes in the proteins of mitotic chromosomes, such alterations might be involved in mechanisms of chromosome banding.     

Effects of acetic acid-alcohol,trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes

Summary In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irrversibly changed the conformation of HI and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73–212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.     

Changes in elemental composition of human chromosomes during a G-banding (ASG) and a C-banding (BSG) procedure.

Human chromosomes fixed in methanol-acetic acid have been examined by X-ray microanalysis, before, during and after a G-banding and a C-banding procedure. Phosphorus (representing mainly DNA), sulphur and calcium are the most prominent elements in untreated chromosomes. In the G-banding procedure, the calcium is lost during 2 x SSC treatment. In the C-banding procedure, calcium is lost in the preliminary HCl treatment. During the following barium hydroxide treatment a large amount of barium becomes attached to the chromosomes, but is lost again during the subsequent 2 x SSC treatment. In both banding techniques Giemsa staining produces large peaks for sulphur (thiazine dyes) and bromine (eosin), showing that both types of dyes are involved in the staining. Reduction in the phosphorus peak during these procedures may be partly due to extraction of DNA and other chromosomal components, but could also be due to absorption of phosphorus X-rays by heavy elements (barium and bromine).     

Study of the human male meiosis

Summary Results obtained from a meiotic study of 250 pachytene cells from four normal human males are presented.G-banding patterns for pachytene bivalents, obtained using trypsin treatment, are also presented; a comparative study between G-banding patterns of pachytene bivalents and G-banding patterns of mitotic chromosomes, and a study of chromomere counting and distribution, have also been made.     

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.     

The occurrence of G-bands in the mitotic chromosomes of the amphibian Xenopus muelleri

The mitotic chromosomes from cultured cells of Xenopus muelleri were G-banded with trypsin and/or with trypsin/urea. These amphibian chromosomes were not found to be more highly contracted at metaphase than those of mammals or reptiles and trypsin G-banding was more easily induced than in the case of most reptilian chromosomes. The organization of vertebrate chromosomes into distinct early replicating (R-bands) and late replicating (G-bands) replicon clusters may be characteristic of eucaryotes in general.     

Configurational changes in chromatids from helical to banded structures

Induction of configurational changes in the helical chromatids of air dried chromosomes was used to explore the mechanism of G-banding. From the water-Giemsa stained metaphase spreads of Chinese hamster cells, chromosomes having clearly helical chromatids were selected and photographed. Then the chromosomes were decolorized, treated with trypsin, and restained with saline-Giemsa (1 x SSC). Such procedures were repeatedly carried out upon the same chromosomes. Subsequent examination of the chromosomes showed that configurational changes from a helical structure to a banded structure had occurred. Some chromosomes revealed a variety of transitional changes between these two configurations. During the repeated G-banding treatments, the distances between bands along the same chromatids changed each time. The results obtained seem to indicate that the G-banding results from locally induced compaction of chromosomal materials along the chromatids.     

The effect of chromosome banding techniques on the proteins of isolated chromosomes

Experiments were undertaken to determine the effect of various chromosome banding treatments on the histone and nonhistone proteins of isolated, fixed, air-dried metaphase chromosomes. Chromosome preparations were exposed to G-banding (SSC, urea, NaCl-urea, or trypsin), R-banding (Earle's balanced salt solution), and C-banding (NaOH or Ba(OH)₂) treatments, and the extracted and residual proteins were examined by SDS polyacrylamide gel electrophoresis. The results indicate that each of the banding treatments induce characteristic alterations in the chromosomal proteins. The residual proteins left in chromosomes after the diverse G-banding treatments were generally similar to one another, indicating that treatments inducing the same type of banding have similar effects on the chromosomal proteins. This was also true for the two different C-banding treatments. On the other hand, the residual protein patterns seen after the G-banding treatments were strikingly different from those seen after R-banding, which in turn differed from those seen after C-banding. The treatments inducing different types of banding therefore produce markedly different effects on the chromosomal proteins. These protein alterations may have an important influence on the induction of chromosome bands.     

The mechanism of C- and G-banding of chromosomes

A series of biochemical, staining and electron microscopy techniques were utilized to investigate the mechanisms of C- and G-banding. These led to the following conclusions.

1. 1. The treatment of fixed chromosomes with 0.07 N NaOH for 30 to 180 sec removes from 16 to 81% of the DNA from the chromosomes.

2. 2. On average, the complete C-band technique removes 60% of the DNA.

3. 3. This DNA is preferentially extracted from the non-C-band regions.

4. 4. In marked contrast to this, all G-band techniques (except 1) removed less than 9% of the chromosomal DNA.

5. 5. Most of the G-band techniques, including those using trypsin, remove very little protein from the chromosomes.

6. 6. Feulgen staining indicated that neither C- nor G-banding can be explained on the basis of different amounts of DNA along the length of the chromatid.

7. 7. Treatment of chromosomes with alkali or prolonged treatment with trypsin tends to destroy G-bands, while C-bands remain.

8. 8. The combined use of acridine orange and Giemsa staining indicate that, (a) repetitious DNA in situ renatures in seconds while non-repetitious DNA renatures in minutes; (b) Neither C- nor G-banding depends upon the differential renaturation of DNA for its effect.

9. 9. G-banding is more delicate and relatively mild conditions allow staining of both C- and G-bands. To obtain only C-bands the chromosome must be treated more harshly to disrupt or destroy the G-bands.

10. 10. DNA-non histone protein interactions probably play an important role in the production of both C- and G-banding.

     

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.     

Structure of human chromosomes visualized at the electron microscopic level

A newly developed technique allows cytological (light microscope level) chromosome preparations to be examined at the electron microscopic level. Ultrathin (50 nm) sections of highly condensed Hela cell metaphase chromosomes show the characteristic mitotic chromosome morphology. In addition a fibrous network (presumably chromosome fibers) can be seen within them. Fibers appear to be gathered at foci along each chromatid. Treatment of chromosomes with trypsin in a trypsin/G-banding procedure reduces the amount of staining material at the electron microscopic level and results in more prominent foci. Thicker (100 nm) sections of less condensed chromosomes prepared from human lymphocytes display a banding pattern similar to G-banding, even without pretreatment with proteases.     

The human complement after trypsin pretreatment as compared to the Paris standard.

A composite G-banding diagram after trypsin pretreatment of metaphase chromosomes from 5 different individuals (2 males and 3 females) is presented and compared with the Paris diagram. The patterns obtained by the present technique were very similar to those previously reported. It was found that the darkly staining bands were much more consistent in appearance than the lightly staining bands and that there was little individual variation.     

An approach to the G-Banding Technique in Rye Chromosome

The G-banding technique has not yet been broken through in studying plant chromosomes. in this paper, we have described a new banding method in Secale cereale. The rye root tips were treated with actinomycin D (40-100 μg/ml) for two hours and with colchicine (0.01%) for 0.5 hour and then fixed with methanol-acetic acid (3:1). After cell wall degradation by cellulase and pectinase, the chromosome sample were made by a hypotonic and flame-drying method (hypotonic treatment→preparation of cell suspension→dropping suspension on slide flame-drying). Following an air-drying period of about a week, the slides were incubated in trypsin-EDTA solution (0.01–0.05%) at 30℃ for 10–15 sec. and subsequently stained with Giemsa. Lots of deep stained bands along the arms of many prophase and late prophase chromosomes were seen. The position of them was obviously different from that of the C-band and the number of them was approximately in proportion to the longitude of chromosomes. Such bands were not seen in metaphase chromosomes. We thought it preferable to use prophase chromosomes to probe G-banding technique in plant and this paper has proposed a possible way for studying G-banding technique in plant chromosome. We also discuss why metaphase chromosomes of plant do not show G-bands.     

恙螨染色体分带的初步研究

本文报道应用胰酶法对微红纤恙螨Leptotrombidium rubellum,苍白纤恙螨L. pallidum和小板纤恙满L. scutellarc进行G带带型分析.三种恙螨染色体分别显示17、21、19条深带带纹,用CS-190机对每条显带的染色体进行薄层扫描,结果每一条深带显示一个峰,对微红纤螨和巨螯齿恙螨Odontaearus majestivus进行BSG法的C显带实验,均未见到带纹,从C带结果及对敬红纤慧螨染色体扫描电镜的初步观察结果,提示恙螨染色体可能为泛着丝粒类型,本文根据恙螨染色体的分带情况,探讨了几种恙螨间的亲缘关系以及恙螨染色体的研究在分类上的意义.     

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.     

A rapid, simple and reliable combined method for G-banding mammalian and human chromosomes

A simple and reliable method for G-banding chromosomes from human and mammalian cells is described. This rapid method combines hot saline and trypsin treatments and yields high quality G-bands in both bone marrow and cultured cells.     

Microfluorometric studies on chromosomes

The fluorescent stains, dansyl chloride and fluorescamine, were used to indicate the amount of protein in G-banded and unbanded chromosomes 1 of the Chinese hamster relative to the average amount of protein in human erythrocyte. Fluorescence was found to be proportional to protein mass up to the equivalent of three erythrocytes. In G-banding, trypsin digestion resulted in an average protein loss of 35.4% compared with unbanded chromosomes.     

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。