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.     

Staining of human metaphase chromosomes with fluorescent conjugates of polylysine

The interaction of polylysine and partially substituted dansyl, fluorescein, and quinacrine conjugates of polylysine with cytological preparations of human metaphase chromosomes has been studied by fluorescence microscopy. The fluorescence intensity along chromosomes stained with the dansyl and fluorescein conjugates exhibits little variation, suggesting that regions capable of binding these polycations are nearly evenly distributed. In contrast, the quinacrine derivatives of polylysine stain the chromosomes in a banded fluorescence pattern resembling that observed following quinacrine or quinacrine mustard treatment.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

High resolution chromosome analysis: one and two parameter flow cytometry

Isolated mammalian chromosomes have been quantitatively classified by high resolution flow cytometry. Chinese hamster chromosomes stained with 33258 Hoechst and excited in the UV showed a fluorescence distribution in which the 14 types of Chinese hamster chromosomes were resolved into 16 groups seen as distinct peaks in the distributions. Chinese hamster chromosomes were also stained with both 33258 Hoechst (HO) and chromomycin A3 (CA3); the two dye contents were measured by selective excitation in the UV and at 458 nm in a dual beam flow cytometer. The resulting two parameter distribution (HO versus CA3) showed 10 chromosome groups¹. Human strain LLL 761 chromosomes stained with HO and excited in the UV showed a fluorescence distribution in which the 23 types of human chromosomes were resolved into 12 groups. Human chromosomes stained with both HO and CA3 and measured in the dual beam flow cytometer produced two parameter fluorescence distributions which showed 20 groups. The chromosomes associated with each group were determined by quinacrine banding analysis of sorted chromosomes and by DNA cytophotometry of preidentified metaphase chromosomes. The relative HO and CA3 stain content and frequency of occurrence of chromosomes in each group were determined from the fluorescence distributions and compared to the results from DNA cytophotometry. The chromosome to chromosome variations in HO and CA3 staining are attributed to variations in chromosomal base composition.     

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.     

Dansyl chloride-stained nucleolar organizers and core-like structures in Chinese hamster metaphase chromosomes

When chromosomes containing both BrdU-substituted and unsubstituted regions were treated with hot NaH₂PO₄ at high or low pH and then stained with dansyl chloride, brightly fluorescent nucleolar organizer regions (NORs) and core-like structures were apparent in the chromosomes. These structures closely parallel the appearance of the same structures in silver-stained chromosomes. Since dansyl chloride is a protein-specific fluorochrome, the distribution of fluorescence suggests that the NORs and central zone of each chromatid contain higher concentrations of protein relative to other chromosome regions. The fluorescent core structures are interpreted to be artefacts of the NaH₂PO₄ pretreatment induced by changes in the concentration of chromatin (including protein) between the chromatin-dense center and more dispersed peripheral region of each chromatid.     

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.     

Electron microscopy of G-banded human mitotic chromosomes

Trypsin-treated human metaphase chromosomes stained with Giemsa and uranyl acetate showed clear, reproducible band structures under the transmission electron microscope (TEM). The banding pattern observed with TEM corresponded very closely to the G-band pattern visualized by light microscopy. The TEM images were used for karyotype analyses. Trypsin-treated chromosomes stained with uranyl acetate alone also showed clear G-bands under TEM. Shadow casting in addition to uranyl acetate staining revealed more structural detail of the chromosomes. Chromosome fibers, 200 Å–300 Å in diameter, were observed in the interband regions. Most chromosomes showed the major G-bands under the higher TEM magnification wit0out any trypsin treatment.     

Some factors affecting the action of restriction endonucleases on human metaphase chromosomes

We have investigated whether restriction endonucleases produce bands on human chromosomes by extracting DNA, using staining methods which are stoichiometric for DNA. Restriction enzymes that produce C-band patterns appear to remove DNA extensively from chromosome arms. In general, however, those restriction enzymes that produce G-bands do not extract DNA from chromosomes, and their effects are believed to be due to conformational change in the chromosomal DNA; in these cases, the chromosomal regions affected appear to be determined by the chromosome structure and not by the specificity of the enzyme. DNA loss from chromosomes due to digestion by restriction enzymes may in some cases be uniform, although a G-banding pattern is visible after Giemsa staining.     

Heterochromatin recognition and analysis of chromosome variation in Scilla sibirica

The heteroohromatin of Scilla sibirica, consists of two distinct types: 1) showing enhanced Quinacrine fluorescence and located near the centromere of all the chromosomes of the complement, and 2) with reduced Quinacrine fluorescence and located in various positions along the chromosomes. After a denaturation-reannealing treatment both heterochromatin types are stained by Giemsa, and by acetic-orcein. Acetic-orcein, however, tends to stain preferentially the reduced fluorescence segments. An analysis of chromosome variation in a population of twenty plants, reveals that all the plants are unique in their heterochromatic segment endowment. All the chromosomes are polymorphic but there is a certain constancy for band patterns in individual chromosome types, and for the number of bands per chromosome complement.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

Mapping of DNAase I sensitive regions on mitotic chromosomes

We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of chromosomes.     

Chromosome homology and heterochromatin in goat,sheep and ox studied by banding techniques

Peripheral blood lymphocyte metaphase chromosomes of three Bovoidean species have been studied using Quinacrine fluorescence and Giemsa banding techniques to give Q-, G-, and C-banding patterns. Q- and G-banding characteristics, coupled with chromosome length, enabled all of the chromosomes in each of the chromosome complements to be clearly distinguished, although some difficulties were encountered with the very smallest chromosomes. A comparison of G-banding patterns between the species revealed a remarkable degree of homology of banding patterns. Each of the 23 different acrocentric autosomes of the domestic sheep (2n=54) was represented by an identical chromosome in the goat (2n=60) and the arms of the 3 pairs of sheep metacentric autosomes were identical matches with the remaining 6 goat acrocentrics. A similar interspecies homology was evident for all but two of the autosomes in the ox (2n=60). This homology between sheep metacentric and goat acrocentric elements confirms a previously suggested Robertsonian variation. The close homology in G-banding patterns between these related species indicates that the banding patterns are evolutionarily conservative and may be a useful guide in assessing interspecific relationships. —The centromeric heterochromatin in the autosomes of the three species was found to show little or no Q-or G-staining, in contrast to the sex chromosomes. This lack of centromeric staining with the G-technique (ASG) contrasts markedly with results obtained with other mammalian species. However, with the C-banding technique these regions show a normal intense Giemsa stain and the C-bands in the sex chromosomes are inconspicuous. The amount of centromeric heterochromatin in the sheep metacentric chromosomes is considerable less than in the acrocentric autosomes or in a newly derived metacentric element discovered in a goat. It is suggested that the pale G-staining of the centromeric heterochromatin in these species might be related to the presence of G-Crich satellite DNA.     

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.     

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.     

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.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Genetic activity of the G- and R-band DNA of human mitotic chromosomes

The concept of genetic inactivity of G-band DNA had been reinvestigated using the modified approach of Korenberg et al (1978). Coefficients of correlation and partial correlation between the relative gene density (g'), the relative G-band material richness (kH/C) and the relative chromosome size (s') were calculated. The kH/C was calculated as the ratio of brightness of fluorescence of chromosomes stained by Hoechst 33258 (Hi) and by chromomycin A3(Ci). The kH/C is the characteristics of G-band chromosome richness, because G-bands become bright after Hoechst 33258 staining and R-bands are bright after chromomycin A3 staining, while no significant C-bands in chromosomes which may be stained by these fluorochromes are discovered. For the kH/C determination the flow cytometry data of Langlois et al (1982) were used. The relative size of chromosomes was determined, based on the flow cytometry data of Young et al (1979). According to Korenberg, the "gene density" (g') in a chromosome was calculated as a ratio of the number of genes located in the chromosome before 1984 (Human Gene Mapping 7) to the relative size of this chromosome. Correlation between the "gene density" and the G-band richness was rs = -0.65. Out of 107 genes located in either G- or R-bands (Human Gene Mapping 7), 90 were mapped in the R-band and only 17 were ascribed to the G-band in metaphase chromosomes. The data on gene replication time show that all genes of the general cell activity and a portion of tissue-specific genes replicate during the early S-phase, together with R-band materials. These three independent lines of evidence are consistent with the notion that the R-band DNA is more genetically active than G-band DNA. The nature of "junk" DNA of G-bands is discussed.     

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.