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.     

Factors influencing Giemsa band formation of human chromosomes

Summary Factors influencing a Giemsa banding method in which slides are treated with NaOH and then incubated in phosphate buffer were investigated. The study indicated that the removal of chromosomal proteins during fixation in acetic methanol is important for band formation. When fixatives containing formalin were used no banding occurred. Histones do not appear to be involved in band formation as neither of the two histone staining methods tested gave banding patterns. The age of the slide preparations was important, the best banding occurring on slides a week old. Romanovsky stains were the only stains to give banding, other stains resulted in distorted chromosomes. The composition of the incubation buffer had little effect on the quality of the banding. However liquid scintillation analysis of the phosphate buffer in which ³H-thymidine labelled preparations had been treated, revealed that thymidine is removed during incubation in buffer, and suggests that the degradation of thymidine is an important factor in band formation.This work was supported by the W. H. Travis Trust and the Canterbury and Westland Division of the Cancer Society of New Zealand.     

Histone gene transposition in the phylogeny of the Drosophila obscura group

The chromosomal location of the histone genes was determined in seven species of the Drosophila obscura group by in situ hybridization. Histone genes occur on more than one site per genome and on non-homologous chromosome elements. In addition, the metaphase karyotypes and the banding pattern of the polytene chromosomes were compared. Based on chromosomal characters, the cladogenesis of the D. obscura group was established. From the distribution of histone sites in different species, analysed in this paper and in previous studies, the phylogenetic history of histone gene transposition was derived. The molecular mechanisms responsible for the generation of new histone sites are discussed.     

The role of proteins in the production of different types of chromosome bands

Experiments have been carried out to try and answer two questions on the role of proteins in chromosome banding: firstly, what degree of protein extraction is required before banding can be produced; and secondly, to what extent are redistribution and reorganization of chromosomal components required for the production of banding. Partial extraction of all histones, and of a group of non-histones with molecular weights mainly between 50,000 and 70,000 appears to be necessary before G-, C- or R-banding can be produced. More extensive 'dehistonization' to produce chromosome scaffolds inhibits the production of all types of bands. Protein-protein and protein-DNA cross-linking inhibits all types of banding tested, the degree of inhibition being roughly related to the degree of cross-linking, but not apparently to the type of cross-linking. The results of both sets of experiments indicate that chromosome banding of all types is dependent on the prior loss from chromosomes of a specific set of proteins, and on some alteration of the arrangement of remaining chromosomal components during the banding procedure.     

A comparative study on the effect of various detergents in human chromosome G-banding prior to tryptic digestion

The effect of treatment of chromosomes with various detergents prior to tryptic banding was investigated. The pre-treatment improved the resolution of banding for most of the chromosomes. The effectiveness of the detergents varied greatly. The number of chromosome pairs with optimal banding found after the use of different detergents was as follows: 19 of the possible 23 for emulphogene, 15 for Nonidet, 9 for Triton X-100, 10 for Tween 40, 4 for Tween 80, 4 for DOC and 18 for SDS compared to 3 for trypsin without using detergent. Optimal banding was as defined by the Paris Conference (1971) map. The improvement of banding was shown to be due to detergent-induced changes in the chromosomal proteins. When the chromosomes were treated first with trypsin followed by the detergent no improvement of chromosomal banding was observed. The detergents showed a degree of specificity towards individual chromosomes; certain chromosomes were found to be better banded with a particular detergent. Pretreatment of chromosomes with a combination of two detergents — simultaneously or consecutively — was found not to be additive. After such treatment the chromosome bands were disrupted. Pretreatment with different detergents sometimes changed the topography of chromosome banding, i.e., the relative location of the bands from the centromere. These findings suggest that the proteins attached to the DNA of the chromosomes were removed or loosened from different sites by the various detergents. — For chromosomes 8, 9, 20 and X, additional bands not reported previously were detected.On sabbatical leave from the Weizman Institute of Science, Rehovot, Israel     

Study of human chromosomes. IV. Labeling of chromosomal proteins with the amino group specific fluorescent reagent fluorescamine

We describe a method for labeling chromosomal proteins with an amino-group-specific fluorescent reagent, fluorescamine. Chromosomes thus labeled appear either as uniformly fluorescent or as haloes in structure depending on the proteins remaining after treatment with acid-alcohol fixation. Using fluorescamine as a probe, we demonstrate that there is a substantial loss of labeled proteins during the chromosomal preparation and also during the trypsin treatment used in the banding of chromosomes.     

Evolution of histone gene loci in chironomid midges.

In the present study we have localized the histone genes in the chromosomes of 16 different Chironomus species as well as in Prodiamesa olivacea, Glyptotendipes barbipes, and Acricotopus lucidus. In the genus of Chironomus we find four, five, or six different "major" chromosomal loci hybridizing with a histone gene cluster probe isolated from the genome of Chironomus thummi. These major histone gene loci probably contain clustered histone gene repeating units ("clustered" loci). They are located on one and the same chromosome arm in all but one of the species investigated. This shows that the histone gene clusters are rather conservative in their location over a long period of evolution. The comparison of the histone loci pattern from the chromosomes of the different chironomid species shows that there is good agreement with previously established chromosome maps and phylogenetic studies based on the chromosomal banding pattern. Stringent in situ hybridization with various histone gene containing clones suggest that the "clustered" histone gene loci are organized in a locus-specific way. In addition to the linked "clustered" histone gene loci, we found an isolated histone gene group ("orphon") present on chromosome IV in most Chironomus species. This gene group might be organized differently from the histone gene repeating unit described previously.     

Selective interaction of 5'-bromodeoxyuridine substituted DNA with different chromosomal proteins.

Chromosomal proteins selectively interact with 5'-bromodeoxyuridine (BrdUrd) substituted DNA relative to unsubstituted DNA. The relative affinities of chromosomal proteins for BrdUrd-DNA and unsubstituted DNA were measured by both thermal chromatography on hydroxylapatite and selective retention on nitrocellulose filters. Certain chromosomal proteins have a high affinity for hydroxylapatite; thus, during thermal chromatography of chromatin, the single-stranded DNA component percolates across a bed of adsorbed proteins as it elutes. We have measured the relative affinities of Brd-Urd-DNA and normal DNA for chromosomal proteins by chromatographing appropriate mixtures on hydroxylapatite. The results show that, under these conditions, the histone components, rather than the nonhistone chromatin proteins, retard the BrdUrd-substituted DNA. In addition, the individual histones vary in the degree of their affinity for BrdUrd-DNA in the order H3 greater than H4 greater than H2A greater than H2B greater than H1. We have used the property that protein-DNA complexes have a preferential affinity for nitrocellulose filters over naked DNA to measure the selective binding of BrdUrd-DNA and unsubstituted DNA's to both histone and nonhistone chromosomal proteins at low temperatures. The histones selectively retained BrdUrd-DNA on filters in the order H4 greater than H2A greater than H3 greater than H2B greater than H1. Using this assay, the nonhistones displayed greater selectivity toward BrdUrd-DNA than the histone fraction. We interpret these results to mean BrdUrd-containing DNA has a specific affinity for certain chromosomal proteins with BrdUrd-DNA may be the basis for selective inhibition of cytodifferentiation by the thymidine analogue, BrdUrd.     

Reassociation of histone H1 with nucleosomes.

The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.     

Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

Background  

Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.     

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.     

Inhibition of histone acetyltransferase by glycosaminoglycans

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (>8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an approximately 50-fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an approximately 50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation.     

Basic chromosomal proteins in lower eukaryotes: Relevance to the evolution and function of histones

Summary The occurrence of basic chromosomal proteins in lower eukaryotes provides a useful approach to the study of histone evolution and function in higher eukaryotes. The histones of higher plants and animals are very similar and some are nearly identical, suggesting a high degree of evolutionary conservation within this group of proteins. However, a literature survey reveals that in the lower eukaryotes the histone situation is quite variable. The ciliates, and the true and cellular slime molds possess basic chromosomal proteins that are very similar to the histones of higher plants and animals. Various other lower eukaryotes possess basic chromosomal proteins that resemble at least some of the major histone fractions, and some microorganisms possess basic chromosomal proteins that bear little or no relationship to higher plant and animal histones. Since histones play a major role in the control of gene expression and the maintenance of chromosome structure in higher organisms, the evolution of these proteins represents a major change in the packaging of DNA and the mode of regulating gene expression in eukaryotes.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

Histone localization in polytene chromosomes by immunofluorescence

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.     

Geographic variability of the polytene chromosome banding sequence of non-biting midge <Emphasis Type="Italic">Chironomus pseudothummi</Emphasis> Str. (Diptera,Chironomidae)

The karyotypes and chromosomal polymorphism of Chironomus pseudothummi were investigated in different parts of its areal. It was established that chromosomal variability in the natural populations of this species was represented mainly by the inversion polymorphism of arm G. Only rare and unique inversions were found to be heterozygous in arms C, D, and E. In total, 14 banding sequences of polytene chromosomes form the banding sequence pool of C. pseudothummi. Geographic differences in the distribution of chromosomal banding sequences throughout the areal were established. The presence of banding sequences pstG1 and pstG2 is characteristic of European populations. The banding sequence pstG1 disappeared completely with a simultaneous increase in the frequency of pstG2 and with the appearance of a new inversion banding sequence pstG3 in Siberian populations. Differences in the set of rare and unique inversions in arms C, D, and E between the West-European and West-Siberian populations have been revealed.     

A single-column chromatographic system for the analysis and preparation of high mobility group proteins 1 and 2 and other chromosomal proteins using nondenaturing solvents

One-step chromatography on a Mono S column allows the purification of high mobility group (HMG) proteins 1 and 2 under nondenaturing conditions. Chromatography of HMG1 and -2 on Mono S can be achieved with three of the most widely employed extraction techniques for chromosomal proteins, 0.35 M sodium chloride, 0.74 M perchloric acid, and 0.4 N sulfuric acid. In each case HMG1 and -2 are purified away from the other chromosomal proteins, histone H1, and core histones, and are resolved into both their reduced and oxidized forms. Additionally histone H1 and the core histones are fractionated on Mono S, thus the entire complement of chromosomal proteins can be analyzed in a single rapid chromatographic step.