Chromosome organisation in the Australian plague locust,Chortoicetes terminifera

In Chortoicetes terminifera, G-banding, produced by the trypsin treatment of air-dried slides followed by Giemsa staining, leads to light staining gaps at the secondary constrictions on autosomal pair 6 and regions proximal to the centromere on the long arms of pair 4. The variable short arms of two of the three smallest pairs were usually flared and lightly stained after treatment. In contrast to the relatively minor response of the normal chromosome set to G-banding, the large supernumerary chromosomes of C. terminifera show a spectacular series of dark bands alternating with lightly stained gaps. Two G-band variants of the B-chromosome were found in a laboratory stock. These patterns of G-banding are discernable both at mitosis in adults and embryos of both sexes and at all stages of male meiosis. Some regions which are gaps after G-banding appear as dark bands after C-banding. Consequently the supernumerary chromosome is mainly darkly stained with C-banding. In addition the centromeres and some telomeres are C-banded along with narrow interstitial bands and polymorphic heterochromatic blocks. — C-banding was not always successful, the technique often yields a mixture of G- and C-banding. The disparity of banding between the normal complement and the B-chromosome implies that whatever the source of origin of the B it has undergone spectacular changes in organisation since its origin.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

Zinc Chloride Methylene Blue, Ii. Preparation of Giesma and Wright Type Low Azure B Blood Stains from Dichromate Oxidized Commercial Dye

TO determine the amount of K₂Cr₂O₇ required to produce optimal Giemsa type staining, six 1 g amounts (corrected for dye content) of zinc methylene blue were oxidized with graded quantities of K₂Cr₂O₇ to produce 4, 8, 12, 16, 20 and 24% conversion of methylene blue to azure B. These were heated with a blank control 15 minutes at 100 C in 60-65 ml 0.4 N HCI. cooled, and adjusted to 50 ml to give 20 mg original dye/ml. Aliquots were then diluted to 1% and stains were made by the “Wet Giemsa” technic (Lillie and Donaldson 1979) using 6 ml 1% polychrome methylene blue, 4 ml 1% cosin (corrected for dye content), 2 ml 0.1 M pH 6.3 phosphate buffer, 5 ml acetone, and 23 ml distilled water. The main is added last and methanol fixed blood films are stained immediately for 20-40 min.

For methylene blue supplied by MCB 12-H-29, optimal stains were obtained with preparations containing 20 and 24% conversion of methylene blue to azure B. With methylene blue supplied by Aldrich (080787), 16% conversion of methylene blue to azure B was optimal. Eosinates prepared from a low azure B/methylene blue preparation selected in this way give good stains when used as a Wright stain in 0.3% methanol solution. However, when the 600 mg eosinate solution in glycerol methanol is supplemented with 160 mg of the same azure B/methylene blue chloride the mixture fails to perform well. The HCI precipitation of the chloride apparently produces the zinc methylene blue chloride salt which is poorly soluble in alcohol. It appears necessary to have a zinc-free azure B/methylene blue chloride to supplement the probably zinc-free eosinate used in the Giemsa mixture.     

Characterization of yellow grouper Epinephelus awoara (Serranidae) karyotype by chromosome bandings and fluorescence in situ hybridization

The cytogenetics of yellow grouper Epinephelus awoara was studied using multiple cytogenetic markers [Giemsa staining, C-banding, Ag-NORs and fluorescence in situ hybridization (FISH)]. Giemsa staining results showed that the karyotypic formula of E. awoara was 2n = 48a, FN (fundamental number) = 48. Faint C-bandings were only detected at the centromeric regions of chromosome pair number 24, being almost indiscernible on the other chromosome pairs. After Ag-NOR staining, one pair of nucleolar organizer regions (NOR) was observed in the subcentromeric region of pair number 24. FISH results showed that 5S rDNA was located at a pair of medium-sized chromosomes, while 18S rDNA appeared at the same location in the subcentromeric region of pair number 24 where Ag-NORs were detected. The telomeric sequence (TTAGGG)(n) detected by FISH was located at both ends of each chromosome. The results suggested that E. awoara has retained general karyotypic structure stability during the evolutionary diversification process.     

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.     

Influence of the C-banding procedure on DNA and proteins of mouse chromosomes: I. A microdensitometric study

Cytochemical quantitative methods were used to investigate DNA protein contents of mouse metaphase plates during an alkaline C-banding procedure ( Sumner et al., 1971). Cytochemical stains and reactions for DNA and for total protein content were used to quantitatively assess the sequential involvement (losses) of DNA and protein during the appearance of the classic C-banding pattern which was monitored with Giemsa staining. The data point the preferential loss of DNA from euchromatic regions of chromosomes as the main cause of the C-banding pattern appearance. The effect of chromosomal protein is more likely indirect and perhaps tied to some specific interaction with centromeric DNA that contributes to DNA retention in C-bands. Following the C-banding procedure it was possible to differentially stain the centromeric area with Feulgen and GCA and even with non-fully specific stain for DNA such as methylene blue.     

Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization

An intensive karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) was carried out with three different methods. These included Feulgen staining, Giemsa C-banding, and fluorescence in situ hybridization (FISH). The mitotic chromosomes of the cucumber (2n = 2x = 14) were characterized, based on the length and arm ratio values. A C-banding analysis showed dark stains on the centromeric, telomeric, and intercalary regions of the chromosomes, except that chromosome 2 had a heavy staining in the long arm. Bicolor FISH, using 45S and 5S rDNA probes, provided additional information to identify cucumber chromosomes. The signals for 45S rDNA were detected on the pericentromeric regions of chromosomes 1, 2, and 4. The signals for 5S rDNA were on the short arm of chromosome 5. Similar band patterns (as the C-banding) were observed when the chromosomes were counter-stained with 4',6-diamidino-2-phenyoindole (DAPI). The data implied that the karyotype of the Korean cucumber cultivar is peculiar and different from previous reports.     

Observations on specific giemsa staining of the Y and on selective oil destaining of the chromosomes.

The human Y chromosome can be differentially stained with Giemsa using simple procedures. This phenomenon is strikingly to that observed with quinacrine fluorescence. The specific Giemsa-Y stain may be selectively removed by the action of an oil. The same oil, under certain conditions, selectively removes Giemsa stain from all chromosomes, resulting in R- and T-banding patterns. These bands, which are obtained through subtraction of dye from Giemsa-stained chromosomes, allow slides to be further processed.     

Reverse differential staining of sister chromatids after substitution with BUdR and incubation in sodium phosphate solution

Chinese hamster strain cells were cultured in the presence of BUdR and air-dried on slides. The chromosome preparations were incubated in 1 M NaH₂PO₄ at 88 °C for 4–6 min and stained with Giemsa. The reverse type of sister chromatid differential staining occurred, in which unifilarly BUdR-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Feulgen reaction performed on the same chromosomes after removing Giemsa stain showed the same type of differential staining.     

X-linked heterochromatin distribution in the holocentric chromosomes of the green apple aphid <Emphasis Type="Italic">Aphis pomi</Emphasis>

Chromatin organization in the holocentric chromosomes of the green apple aphid Aphis pomi has been investigated at a cytological level after C-banding, NOR, Giemsa, fluorochrome staining and fluorescent in situ hybridization (FISH). C-banding technique showed that heterochromatic bands are exclusively located on X chromosomes. This data represents a peculiar feature that clearly contradicts the equilocal distribution of heterochromatin typical of monocentric chromosomes. Moreover, silver staining and FISH carried out with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; CMA₃ staining reveals that these silver positive telomeres are the only GC-rich regions among A. pomi heterochromatin, whereas all other C-positive bands are DAPI positive thus containing AT-rich DNA.     

黄颡鱼染色体的C─带、Ag—NORs、荧光带和复制带显带的研究

采用Giemas染色、C─带、Ag—NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明,黄颡鱼只有部分的染色体呈现阳性C─带,可分为三类,其中NORs区是染色最深、染色面积最大的区域,为深染居间C─带。其Ag－NORs位于m5q末端。CMA3染色显示NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C─带、Ag—NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致,C─带阳性区和中复制带浅染区具有对应性。     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

Centromeric banding (C) of sequentially Q- and R-banded human chromosomes

Summary A modified C-banding technique is described that produces C bands on human chromosomes after sequential Q and R banding and retains good chromosome morphology. Despite the considerable exposure to UV light during sequential Q and R bandings, clear C bands could still be achieved. Employing the present technique, Q, R, and C polymorphisms can be recorded on a single metaphase.     

宜昌百合根尖染色体C-带分析

利用Giemsa C-带方法对宜昌百合（Lilium leucanthum(Baker) Baker）进行研究。结果表明：宜昌百合（L. leucanthum）的染色体数目为2n＝2x=24,单套染色体的条带总数目为21条。其带型公式为：2n＝24＝6C+2CI+2I+2CI⁺+2CI⁺+4I⁺+2I⁺+2T⁺+2I+S。宜昌百合（L. leucanthum）每条染色体上都显示出显著的特征带,且带纹的深浅差异明显。宜昌百合（L. leucanthum）的强带主要集中在着丝点及附近区域。通过Giemsa C-带方法可以将宜昌百合（L. leucanthum）的每条染色体区分开。     

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.     

Human cells in suspension 2

Summary When PHA stimulated human lymphoid cells are allowed to proliferate in vitro and chromosomal slides are prepared from the culture after 48,72, 96,120, and 144h of growth, gradual changes in chromosome morphology can be observed after traditional Giemsa staining of the slides. Keeping culture conditions, colcemid exposure, and fixation procedure constant for all samples, it is found that average chromosome length decreases with increasing culture time. A shift from high frequencies of subbanded chromosomes (sample 48 h and sample 72 h) to high frequencies of unbanded and G banded chromosomes (sample 120 h and sample 144 h) takes place simultaneously with the general compaction of the chromosomes. Examination of trypsin-induced G bands as well as examination of untreated G banded chromosomes from all samples clearly indicate that the basic G band pattern is not altered during proliferation and differentiation, although the progressive compaction of the chromosome observed with increasing culture time results in a phenomenon similar to that observed during mitosis, where the compact late metaphase chromosome after trypsin treatment exhibits fewer but more prominent bands than the prophase/prometaphase chromosomes. Thus the progressive compaction of metaphase chromosomes observed during in vitro aging seems to resemble the condensation processes during the G₂ phase and mitosis.It has been suggested that the chromomeres serve as centers for chromosome condensation during mitosis, probably mediated by a sulphydryl-disulphide transition in chromosomal proteins. The data presented here further suggest that the chromomeres may also serve as centers for chromosomal differentiation, presumable by a mechanism similar to that acting during chromosome condensation in mitosis.     

茅舍血厉螨核型及染色体的C带、G带的研究

本文首次报道了一种革螨——茅舍血厉螨核型及染色体C带、G带的研究。用剖腹取卵法、玻璃纸压片、Giemsa染色,经分析茅舍血厉螨的核型,单倍体n=7,二倍体2n=14。 用氢氧化钡—吉姆萨技术显示茅舍血厉螨染色体C带。在第1、2、4、5染色体上出现恒定的C带部分,第3、6、7染色体上出现不恒定的C带部分。根据C带带型,茅舍血厉螨着丝点的位置可分为近中区域(sm),近端区域(St),末端区域(t)和末端点(T)四类。 G带分析用胰蛋白酶—吉姆萨技术显示。 本文对茅舍血厉螨的核型、革螨染色体研究中螨卵的收集方法和染液的改进、C带带型与着丝粒位置的确定和G带显带问题进行了讨论。     

The effect of restriction enzyme digestion of human metaphase chromosomes on C-band variants of chromosomes 1 and 9

Human metaphase chromosomes, fixed on slides, have beent treated with 8 different restriction endonucleases and 29 combinations of 2 restriction enzymes prior to staining with Giemsa. The endonucleases AluI and DdeI and the combinations AluI + DdeI, AluI + HaeIII, AluI + HinfI, and AluI + MboI have then been used to digest metaphase chromosomes of nine individuals with C-band variants of chromosomes 1 or 9, obtained by the CBG technique. The restriction enzyme resistant chromatin of the paracentromeric regions of chromosomes 1 and 9 has been measured and compared with the corresponding CBG-bands. The size of the enzyme resistant chromatin regions depend upon the type of enzyme(s) used. Treatment with AluI + MboI was the only digestion that acted differently on different chromosome pairs. However, within one pair of homologous chromosomes, all digestions revealed the same variations as conventional C-banding.