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.     

Banded chromosomes of the owl monkey, Aotus trivirgatus.

Chromosomes of the owl monkey, Aotus trivirgatus, with 2n=54, 53, or 52, have been stained to show quinacrine (Q-) and Giemsa (G-) bands, and a karyotypic arrangement has been proposed based on lengths, centrometric index, and banding pattern. C-bands were present at the centromeric region of every chromosome and over the entire short arm of certain acrocentric chromosomes; 5-methylcytosine was concentrated in the same regions. Bright Q-bands at the telomeric ends of the short arms of some chromosomes probably represent a second type of repetitive DNA. Ag-staining showed that only the chromosomes bearing a secondary constriction are nucleolus organizer chromosomes.     

Feulgen hydrolysis and chromosome banding in Chilocorus (Coleoptera: Coccinellidae).

Prolonged Feulgen hydrolysis of chromosomes of Chilocorus orbus Csy. and C. stigma Say produces banding patterns that are the reverse of those revealed with quinacrine; brightly fluorescing regions are unstained, but nonfluorescent regions remain relatively darkly stained. This differential reactivity at hydrolysis times that otherwise yield intense Feulgen staining confirms the need for caution in the determination of DNA values with the Feulgen reaction in material with well-defined quinacrine bands. The coincidence of DNA-specific Feulgen bands with Q-, G-, and C-bands supports the view that, in Chilocorus at least, bands reflect differences in DNA composition along the chromosome.     

Bandign isolated metaphase chromosomes by a sequential flurescent G/Q technique.

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.     

The structural organization of mouse metaphase chromosomes

The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5-GAAAAATGA-3 (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA      

Counterstain-enhanced chromosome banding

Summary Chromosome staining, in which at least one member of a pair or triplet of DNA binding dyes is fluoescent whereas the others act as counterstain, is reviewed. Appropriately chosen combinations of fluorescent dyes and counterstains can be employed to enhance general chromosome banding patterns, or to induce specific regional banding patterns. Some pairs of dyes which exhibit complementary DNA binding specificity, A-T/G-C or G-C/A-T, provide enhanced definition of positive or reverse banding patterns. Dye combinations of the type A-T/A-T, that include two DNA stains with similar specificity but non-identical binding modes, produce a specific pattern of brightly fluorescnet heterochromatic regions (DA-DAPI bands). In man, the method highlights the C bands of chromosomes 1, 9, 15, 16, and the Y. Certain dye triplets of the type G-C/A-T/A-T, which include two spectroscopically separated fluorescent stains with reciprocal DNA base pair binding specificites and a non-fluorescent A-T binding counterstain, can be used to highlight selectively, in the appropriate wavelength ranges, either R bands or DA-DAPI bands.Applications of these techniques in human cytogenetics are described. The potential of the new methodology for detecting and analysing specific chromosome bands is demonstrated. The mechanisms responsible for contrast enhancement and pattern induction are reviewed and their implications for chromosome structure are discussed as they relate to the banding phenomenon and to the DNA composition of chromosomes.     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Studies of Chromosome Banding with Giemsa in Vicia faba and Allium cepa

Giemsa dye is a complex mixture containing methylene blue, its oxidation products-azure Ⅰ, Ⅱ, Ⅲ, and their eosinate. The results of our experiments have demonstrated that staining with methylene blue alone can give a faint trace of banding as well as azure Ⅰ, Ⅱ. No bands are obtained with eosin. Nevertheless, good chromosome bandings can be often produced by staining with methylene blue-eosinate or azure Ⅱ-eosinate. These data indicate that eosinate has an important effect for the formation of C-banding on plant chromosomes. In our experiments, the treatments of chromosomes with trypsin or papain have also resulted in good C-banding pattern when slides are stained with Giemsa. We found that the slides untreated with proteinase showed homogeneous intense chromosome staining and, on the contrary, the slides treated with proteinase led to palestaining chromosomes and presenting bandings. It has shown that proteinase, especially trypsin, not only can remove a large amount of chromosomal protein but also can remove DNA and results in C-bandings. Treated properly with trypsin and followed by the Feulgen staining, chromosomes can also produce the C-bandings, but chromosomes treated overtime with trypsin are stained more palely in Feulgen reaction or lead to colourlessness. The above results have further proved that trypsin technique removes large amounts of chromosome DNA and removes less from the C-band regions than from the non-band regions. In this paper we mainly discussed the effects of protein on mechanism of plant chromosome banding. We consider that the production of plant C-banding is probably due to the differential accessibility of nucleoprotein between euehromatin and heteroehromatin regions. It brings about selective removal of nucleoprotein from the chromosome arms. We have compared the effect of trypsin with papain and pepsin on producing bands. Good bands are produced by Giemsa staining chromosomes with trypsin, but no bands are obtained by staining chromosomes treated with pepsin. So the results have expressed that histones are possibly playing more important role in C-bandings.     

Chromosome regions containing DNAs of known base composition,specifically evidenced by 2,7-di-t-butyl proflavine

After staining by a new proflavine derivative (2,7-di-t-butyl proflavine, DBP), which specifically binds to the A-T base pairs of DNA by an external process, the constrictions of the human chromosomes 1, 16 and to a lesser extent 9 and the centromeric regions of the chromosomes (except the Y) of Mus musculus are brightly fluorescent. These chromosome regions are known to contain repetitive DNAs rich in A-T. On the contrary, the centromeric regions of the autosomes of Bos taurus, which contain a G-C rich DNA, are faintly fluorescent. The arms of the chromosomes of the three species display a banding similar to, but fainter than, the Q-banding. These results are discussed in correlation with physico-chemical studies on the binding and fluorescence processes of the dye bound to DNA and to nucleohistone. The staining properties of DBP are compared to those of quinacrine, quinacrine mustard and proflavine, three intercalative dyes which are also supposed to reveal the A-T base pairs along the chromosomes, but are faintly fluorescent on the human and murine A-T rich regions. This comparison leads us to discuss the mechanisms responsible for the chromosomal banding in relation to DNA base composition and repetitiveness, protein distribution and packing of the chromatin fibers, along the chromosomes.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

Ultraviolet irradiation of methanol: acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immuno-fluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA-RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species.     

R-banding produced by DNase I digestion of chromomycin-stained chromosomes

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.     

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。     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding.

By using a DNA fragment immunoassay, the binding of simian virus 40 (SV40) and polyomavirus (Py) large tumor (T) antigens to regulatory regions at both viral origins of replication was examined. Although both Py T antigen and SV40 T antigen bind to multiple discrete regions on their proper origins and the reciprocal origin, several striking differences were observed. Py T antigen bound efficiently to three regions on Py DNA centered around an MboII site at nucleotide 45 (region A), a BglI site at nucleotide 92 (region B), and another MboII site at nucleotide 132 (region C). Region A is adjacent to the viral replication origin, and region C coincides with the major early mRNA cap site. Weak binding by Py T antigen to the origin palindrome centered at nucleotide 3 also was observed. SV40 T antigen binds strongly to Py regions A and B but only weakly to region C. This weak binding on region C was surprising because this region contains four tandem repeats of GPuGGC, the canonical pentanucleotide sequence thought to be involved in specific binding by T antigens. On SV40 DNA, SV40 T antigen displayed its characteristic hierarchy of affinities, binding most efficiently to site 1 and less efficiently to site 2. Binding to site 3 was undetectable under these conditions. In contrast, Py T antigen, despite an overall relative reduction of affinity for SV40 DNA, binds equally to fragments containing each of the three SV40 binding sites. Py T antigen, but not SV40 T antigen, also bound specifically to a region of human Alu DNA which bears a remarkable homology to SV40 site 1. However, both tumor antigens fail to precipitate DNA from the same region which has two direct repeats of GAGGC. These results indicate that despite similarities in protein structure and DNA sequence, requirements of the two T antigens for pentanucleotide configuration and neighboring sequence environment are different.     

Cellular factors required for multiple stages of SV40 DNA replication in vitro.

Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells. Upon fractionation of this extract, two essential replication factors have been identified. One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB). The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin. These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.