Chromosome banding: specification of structural features of dyes giving rise to G-banding

HistochemicaI Journal 14, 911-928 (1982)

Chromosome banding" specification of structural features of dyes giving rise to G-banding

D I A N A C U R T I S 1 a n d R I C H A R D W . H O R O B I N 2

1Sub-Department of Medical Genetics (Academic Division of Medicine), 117 Manchester Road, Sheffield $10 5DN, U.K. 2Department of Human Biology and Anatomy, The University, Sheffield $10 2TN, U.K.

Received 9 November 1981 and in revised form 18 February 1982

Summary Metaphase chromosomes were stained in a routine G-banding procedure with 39 basic dyes of varied structures substituted for the Giemsa stain. Staining outcomes were categorized as: overstained, differentially stained, trivially or unstained. Certain structural features of the dyes were described numerically, namely, largest conjugated fragment (LCF), conjugated bond number (CBN) and cationic weight. The staining outcomes were compared to these numerical structural parameters, and structure-staining correlations sought. Dyes with large conjugated systems (and high LCF values) were seen to be overstained; dyes with low LCF values were often non-staining. At intermediate LCF values, the more hydrophobic dyes (with high Hansch values) stained differentially; the more hydrophilic dyes failed to stain. Expressed numerically, 89% of the dyes with the following characteristics stained differentially: 30/> LCF/> 10; Hansch ,r > -5.0. It was concluded that contributions to dye-chromosome affinity included coulombic forces and van der Waals attractions and that the selectivity of G-banding was largely due to hydrophobic bonding. Induction of bands could be due to the loss of hydrophilic histones, amplifying underlying variations in the hydrophobic-hydrophilic character of the chromosome structure. Relatively hydrophobic si~es include AT-rich DNA and disulphide-rich proteins.

The effects on Romanowsky G-banding of chemically modifying chromosomes were in keeping with this model. Overstaining resulted from formation of either hydrophobic or conjugated derivatives or both, whereas trivial or non-staining arose from the formation of hydrophilic derivatives. Intriguingly, the efficacy of the dyes used for Q-banding also correlated positively with their hydrophobic character.

In t roduc t ion

The deve lopmen t of ch romosome banding techniques const i tuted a major technical advance in bo th diagnostic and exper imental cytogenetics. These staining me thods generate chromosome-specif ic pat terns of bands, facilitating the identification of

0018-2214/82/060911-18504.66/0 �9 1982 Chapman and Hall Ltd.

912 CURTIS and HOROBIN

individual chromosomes. Though the early banding procedures used fluorescent dyes (Q banding), later methods used Romanowsky stains, such as Giemsa's (hence G-bands). Numerous technical variations and developments followed, enabling the location of complex structural rearrangements to be visualized. A review of the many technical variations, the practical and conceptual disagreements, and the goals of this work has been provided by the Nobel Symposium on Chromosome Identification - - Technique and Applications in Biology (Caspersson & Zech, 1973). Since that time, technical variations, conflicting hypotheses and the range of applications have all proliferated. However, the overall picture does not seem to have altered radically.

The applications of G-banding, though substantial, are limited by two significant factors, one technical and one theoretical. Firstly, G-banding is notoriously capricious. This is demonstrated by the extensive and continuing output of technical variations, some minor and some not so, all of which are considered by their originators to be necessary improvements of existing recipes (for a review, see Lubs et al., 1973). In fact, few of the practical factors controlling G-banding are well defined. Unfortunately, papers written with a critical intent (Barnett et al., 1980; Curtis & Horobin, 1975) tend to be regarded by practitioners as scientific luxuries, rather than providing information pertinent to the production of more consistent routine methodologies. The second limiting factor is the gross lack of agreement concerning the mechanism of G-banding; this topic is discussed more fully below.

Nature of the G-banding process A wide variety of proposals have been put forward concerning the mechanism of the G-banding process. An encyclopaedic account is out of place, but some illustration of the range and variety of suggestions, ranging from the molecular to the chromosomal, is required.

Initially, no doubt influenced by the similarity in appearance of Q- and G-bands, attention was directed to the role of the DNA. The possibilities that G-banding depended only on variations in ratios of GC to AT base pairs or only on denaturation-renaturation of DNA were considered, to be soon dismissed (Sumner et al., 1971; Comings, 1973). Another DNA-based proposal, still espoused in modified form, was that Giemsa staining occurred only when DNA phosphate groups lay at some critical distance apart. This enabled, it was suggested, Thiazine cations to bind to adjacent phosphate groups and single Eosin anions would bind to pairs of cations separated by a critical bridging distance (Sumner & Evans, 1973; Sumner 1980). Most other explanations of G-banding have evoked the participation of chromosomal proteins in addition to DNA. It is generally considered that the pre-staining treatments remove much histone but little DNA or non-histone protein, and that G-banding in some way reflects the DNA-non-his tone protein interaction (Comings et al., 1973). Several suggestions as to the nature of the significant processes or protein components have been made. One proposal is that there is selective denaturation of polypeptide chains, with DNA-denatured protein binding blocking Thiazine binding (Comings, 1975).

Structure-staining correlations and G-banding 913

Another suggestion is that staining portions of the G-bands correspond to protein of high disulphide content, with non-staining interbands having high sulphydryl content (Sumner, 1974), although it has recently been suggested that detection of these groupings in chromosomes may be beyond the sensitivity limits of cytochemical methods (Weise, 1980). At the other end of the range, G-banding explanations can focus on the whole chromosomes. Thus it has been suggested that banding can result from the influence of local condensation of chromatin, modulated by the pre-staining treatments (Krej~i et al., 1980). In keeping with this general idea, though not in fact with the detailed argument, certain electron microscopical preparations show more chromatin strands in the regions of bands than in the interbands (for a summary, see Comings, 1978). Other electron microscopical studies are more ambiguous (Gormley & Ross, 1972; Evans et al., 1973), including a recent SEM study alleging G-banded chromosomes to be grooved (condensed chromatin?) at the band positions (Harrison et aI., 1981). Finally, Sumner (1980) has proposed that G-band staining corresponds to the more hydrophobic regions of the chromosome, and suggested disulphide and thiol groups were the hydrophobic and hydrophilic entities giving rise to the variation.

Though such ideas are sometimes regarded as exclusive, many workers now- consider that G-banding is a complex process and so may be influenced by various observable and manipulable factors. Exploration of this situation has consequently been complicated, since experimental manipulations will usually produce multiple effects. In the work reported here, we have attempted to vary only the nature of the staining reagent, whilst keeping other factors unaltered.

The variety and effects of pre-staining treatments There is a wide range of pre-staining treatments for inducing G-bands. Inorganic salts, organic solvents, alkali, detergents, heat, proteases, nucleases and protein denaturants have all, in some laboratory, given rise to banding (for a review, see Lubs et al., 1973). Equally noteworthy is how often other workers, in other laboratories, fail to repeat these methods successfully: this applies to the favoured methods of the present authors also. After such a listing, it may be pointed out that some pre-treatments, for example, using acids (Comings & Avelino, 1974), do not yield G-bands.

A possible explanation for the technical difficulties lies in the observation that several band-inducing pre-staining treatments generate a range of staining results, depending on the period of treatment (Schnedl, 1973). This has been clarified, and beautifully illustrated, by Barnett et al. (1980; see especially their Fig. 2). Adopting the nomenclature of these workers, we can say that with increased time of exposure to the banding reagent there is a transformation of staining pattern through dark, to dull (both completely stained, but without differential banding), and then through spiral, G-banded and hybrid (all with varying degrees of differential staining), and finally to contour (virtually unstained). The crucial stages are thus over-staining, differential staining (in varying degrees), and understaining. From this viewpoint, we can see why certain workers obtain banding, whilst others do not.


The variety of dyes which yield G-bands Although G-banding originated with Giemsa staining, other Romanowsky stains were soon used also. From there it was a small step to testing the components of the Romanowsky mixture, that is, the acid dye Eosin Y, and Methylene Blue and its basic-dye oxidation products. Eosin does not stain, but Methylene Blue (Scheres & Merkx, 1976), Azur B (L6ber et al., 1978) and Methylene Violet Bernsthen (Curtis & Horobin, 1975) have been reported as yielding bands. In addition, several other basic dyes have been used to produce G-band type staining patterns, for example, Brilliant Cresyl Blue and Brilliant Cresyl Violet (Sumner & Evans, 1973), Crystal Violet, New Fuchsin, Pararosaniline and Rosaniline (Scheres & Merkx, 1976), Pseudoisocyanine (Vagner-Capodano et al., 1976), Pyronin (Tabuchi et al., 1979). RO4 (Bottiroli et al., 1979) and Stains-All (Scheres, 1974).

Such studies show that G-banding may be obtained with basic dyes of a variety of structures, but not with the acid dye Eosin. Further conclusions are uncertain, since the conditions of use varied, as did the band-inducing pre-staining treatments. In addition, the identities of the dye are not always certain (cf. Marshall, 1978). Overall, analysis is limited by the lack of any systematic variation in the structure of the dyes used.

Value and use of structure-staining correlation Objective correlation of~chromosome staining with dye structure requires a description of structure in numerical terms. Such numerical descriptions will permit a graphical display of results, and also facilitate selection of a range of dyes with widely differing but defined characteristics. Application of a structure-staining correlation approach to the G-banding problem, analogous to the widely used structure-biological activity relationships in pharmacology, was encouraged by the recent successful use of the method to other aspects of histochemistry (Horobin & Flemming, 1980; Horobin, 1981).

The fundamentals of structure-staining correlation have been recently discussed (Horobin, 1980a), so a brief introduction will suffice. The numerical expression of some structural features of histochemical significance is straightforward; for instance, electric charge and size expressed as the molecular or ionic weight. The hydrophobic character of dyes, significant in the present context as an indicator of the possible occurrence of hydrophobic bonding, may be represented numerically using Hansch ~ values. These values, devised and used by chemical pharmacologists (Hansch et al., 1973), permit paper and pencil estimation of the overall hydrophobic or hydrophilic nature of molecules and ions. Another useful numerical structural parameter is conjugated bond number (CBN), an indicator of the polarity and polarisability of dyes, and hence of possible dye-tissue van der Waals attractions. This parameter is obtained from the structural formula by counting the number of bonds involved in the conjugated system. A varient parameter, the largest conjugated fragment (LCF), involves a count of the number of bonds in the largest planar conjugated unit in the dye. This may well be appropriate when the tissue substrate itself contains planar conjugated structural units, the aromatic rings of the bases in DNA, for example.


These numerical parameters describing the structures of dyes are readily obtained. Some are found directly by inspection of structural formulae (for example, charge, conjugated bond number, largest conjugated fragment). Others follow inspection and a little simple arithmetic, for example, using tabulations of atomic weights to obtain molecular or ionic weights, and using fragmental Hansch ~ values to estimate the hydrophobic character.

Definition of the current project We attempted to study the effects of systematic variation of staining reagent structure on the G-banding process. Consequently, both the pre-staining band-inducing procedure and the staining conditions were usually kept constant. All dyes used were basic, that is, the coloured entity was cationic, though of varying charge magnitude. Within this limit, dyes of a wide range of structures were investigated. Some were small, some large, as indicated by cationic weights. Some were hydrophilic, some hydrophobic, as indicated by Hansch 7r values. Other dyes were of considerably varied polarizability-polarity, as indicated by the conjugated bond number and largest conjugated fragment parameters.

In this way, we hoped to probe the influence of staining rate, hydrophobic bonding, and van der Waals attractions on G-banding. We anticipated that this would offer some insight into possible variation of permeability, hydrophobicity and polarity-polarizability along chromosome arms.

Materials and methods

Preparation of chromosomes Routine short term cultures of phytohaemmagglutinin-stimulated lymphocytes were prepared from various blood samples obtained from normal individuals. Colcemid (0.02/~g/ml) was added to the cultures for 4 h prior to culture termination at 72 h. Cells were swollen in hypotonic Hanks solution and then fixed in 3:1 v/v absolute ethanol:glacial acetic acid. Slides of metaphase chromosomes were prepared by dropping the fixed cell suspension onto ice-cold wet slides followed by air drying.

Dyes The dyes used in this study, together with their sources, are listed in Table 1.

Methods of staining The standard method of staining with Leishman Stain was as follows. Leishman stain was prepared as 0.15% w/v in fresh methanol by heating a stirred solution to boiling for 5 rain and then cooling before use. Slides were stained with a freshly prepared dilution of 1:3 v/v stain: Gurr's pH 6.8 buffer for 2 rain. Stain was washed off with Gurr's buffer and the slides were blotted dry.

All other dyes were prepared routinely in a similar manner but at a concentration of 0.075% w/v in methanol, since Leishman powder is a mixture of Thiazines and Eosin. Staining was as described. Additionally, the effect of increased staining times of 5 rain, 30 rain, 2 h, 24 h, 48 h was recorded for some dyes.

916 CURTIS and H O R O B I N

Induction of banding involved a slight modification of the method of Seabright (1971), as used routinely in the laboratory of one of us (D.C.).

Investigation of the purity of Pyronin Y Ethanolic extraction of a sample of the crude dye was carried out. The insoluble residue was investigated by combustion, a flame test, and by wet testing with silver nitrate and iodine solutions.

Calculation of structural parameters Structural formulae of the dyes were obtained from standard sources, for example, Conn's Biological Stains (Lillie, 1977) and The Colour Index (Society of Dyers and Colourists, 1971). Inspection of the formulae gave CBNs directly. For example, Thionin contains two phenyl rings with bridging conjugated sulphur and nitrogen atoms (6+ 6+2+ 2), and carries two conjugated amino groups (1+1). Hence the CBN is 18. The LCF value of this planar dye is also 18. In a dye such as Crystal Violet, where steric hindrance prevents all portions of the conjugated system lying in one plane, the LCF value is 8, whilst the CBN is 24. Molecular models were used to assess the planarity, or otherwise, of dye structures. Inspection of structural formulae, together with tabulations of the Hansch n values of molecular fragments (Hansch et al., 1973), permitted the calculation of overall Hansch n values. For instance, the hydrophobic groups present in Thionin are two phenyl rings (2 x + 2.0) and one thioether (1 x + 0.1). The hydrophilic groups are one ring nitrogen (1 x -0.5), and two amino groups, one of which is protonated (1 x -1 .2 + 1 x -7.5). This sums to -5 .1 , the overall Hansch ~ value for Thionin.

Results

Staining of chromosomes by the standard method The resul ts are s u m m a r i z e d in Table 1.

Table 1. Some structural and staining characteristics of dyes used in the present study.

Dye name (Colour Index number or name) Source Observed staining

Alcian Blue 2G (C.I. Ingrain Blue 4) Alcian Blue 8G (C.I. 74240) Alcian Yellow G (C.I. Ingrain Yellow 1) Astra Blue 6GLL [C.I. Basic Blue 140(S)] Astrazone Pink FG (C.I. 48015) Astrazone Red 6B (C.I. 48020) Azur B (C.I. 52010) Basic Fuchsin (C.I. 42500)

Manufacturer's gift

Manufacturer's gift

George T. Gurr

Manufacturer's gift

Edward Gurr

Edward Gurr

Gift of Professor D. Wittekind

Searle/Gurr Diagnostic

0

0

0

0

N

N

D

D

Structure-s ta ining correlations and G-banding

Table 1 (continued).

917


Bismark Brown R (C.I. 21010) Bis-N-methylacridine Celestin Blue (C.I. 51010) Chrysoidin Y (C.I. 11270) Ethyl Violet (C.I. 41600) Gallamin Blue (C.I. 51045) Gallocyanfil (C.I. 51030) Iodine Green (C.I. 42556) Janus Blue G (C.I. 12211) Janus Green B (C.I. 11050) Janus Red B (C.I. 26115) Malachite Green (C.I. 42000) Methyl Green (C.I. 42585) Methylene Blue (C.I. 52015) Methylene Green (C.I. 52020) Methylene Violet (Bernsthen) (C.I. 52041) Neocyanine Neutral Red (C.I. 50040) New Methylene Blue (C.I. 52030) Night Blue (C.I. 44085) Nile Blue (C.I. 51180) Pinacyanol (C.I. ed. 1-808) Pyronin Y (C.I. 45005) Safranin O (C.I. 50240)

George T. Gurr

Ralph Emanuel George T. Gurr

Edward Gurr

George T. Gurr

George T. Gurr

George T. Gurr

George T. Gurr

Edward Gurr

British Drug Houses

George T. Gurr

George T. Gurr

National Aniline

George T. Gurr

George T. Gurr

Matheson, Coleman and Bell

Eastman Eastman

British Drug Houses

Edward Gurr

George T. Gurr

Searle/Gurr Diagnostic

National Aniline

George T. Gurr

N

N N

N

D

N

N

N

0

D

D

N

N

D!

D

D!

N D

D

D

D

D

N

D

918

Table 1 (continued).

CURTIS and HOROBIN


Stains-All Eastman D Thionin Edward Gurr O (C.I. 5200) Victoria Blue B Raymond A. Lamb D (C.I. 44045) Victoria Blue 4R Raymond A. Lamb D (C.I. 42563)

N, non- or trivially stained; D, differentially stained; D!, banded; O, overstained.

Investigation of the purity of Pyronin Y Some 75% of the crude dye proved insoluble in ethanol. This residue was not flammable, and gave a positive flame test for sodium. An aqueous solution of the residue gave a precipitate with silver nitrate, but no reaction with iodine solution. The residue was thus probably substantially sodium choride.

Discussion

Searching for structure-staining correlations The outcomes of chromosome staining, listed in Table 1, are grouped into three categories: overstained, differentially stained, and trivially or unstained. The assignment of a staining outcome to one of these three categories was not always straightforward, especially with dyes giving rise to weak and non-differential staining. It was not apparent whether such dyes should be regarded as overstained or as unstained. However, certain of these, namely Alcian Blue 2G, Alcian Blue 8G and Alcian Yellow, were of large particle size, with high cationic weights and LCF values. Since staining rates are usually diffusion-controlled, and so are inversely proportional to the dye particle size, the staining of these dyes at least may well have been rate limited (cf. Goldstein & Horobin, 1974). We attempted to test this possibility with various dyes by prolonging the time of staining. Some large particled dyes (with high cationic weights and LCF values) did then stain more intensely. No small particled dyes (with low cationic weights and LCF values) showed increased staining. Interpretation, however, was made difficult by the tendency of the chromosomes fixed in acetic acid-methanol to disintegrate on prolonged exposure to solvents. The final assessments were as given in Table 1.

One way of seeking structure-staining correlations m the data of Table 1 was to take a single structural parameter at a time, and plot frequency diagrams for the overstaining, differential staining, and non-staining groups of dyes. This was done with the Hansch rr, CBN (conjugated bond number), LCF (largest conjugated fragment), and cationic


weight structural parameters. In the frequency diagram involving CBNs, dyes of all three staining categories showed much the same distributions. In the diagrams involving Hansch n and LCF values, however, there were clear indications of correlations. Thus, the Hansch n values of the dyes giving rise to differential staining tended to be more positive than those of the non-staining dyes. Furthermore, the LCF values of the differentially staining dyes tended to be lower than the LCF values of the overstaining dyes, but higher than the values of non-staining dyes. In the frequency diagram involving cationic weights, the dyes in all three staining categories largely overlapped. There was a small group of overstaining dyes with cationic weights substantially higher than the main population. This high-weight group, however, provided no further information, since these dyes are those already noted as possessing exceptionally high LCF values.

Encouraged by the apparent significance of LCF and Hansch n values, improved correlations were sought by considering these two parameters simultaneously. This was done by plotting the dyes on a Hansch n-LCF diagram (see Fig. 1). This procedure yields a much clarified picture. Thus, high and low LCF values (namely areas I and 4 on Fig. 1) correlate well with overstaining and trivial or non-staining respectively. At these extreme LCF values, Hansch n values appear non-predictive. However, at intermediate LCF values, Hansch n values are seen to be predictors of staining character. Thus, low Hansch n values (area 2 of Fig. 1) correlate with non-staining, whereas high Hansch n

t

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70

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�9 O v e r s t a i n e d

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o Tr iv ia l ly or u n - s t a i n e d

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Fig. 1. The basic dyes of the present study plotted onto a largest conjugated fragment (LCF)-Hansch n diagram. The significance of areas 1-4 is discussed in the text.


values (area 3) correlate with differential staining. To simplify subsequent discussion, area 3 will be termed the 'differential staining area'.

The boundaries of the differential staining area are not in fact rigid, as Fig. 1 may imply. However, it is very convenient to draw such lines in Hansch n-LCF parameter space. The effects of altering such boundaries may be illustrated numerically. Consider defining the differential staining area boundaries precisely as in Fig. 1, that is, 30/> LCF

10; Hansch n >t -5 . In this instance 1.6/18 (that is, 89%) of the dyes falling into the differential staining area give rise to differential staining. Of the dyes falling outside the area, 17/20 (that is, 81%) fail to stain differentially. Now consider the effects of slightly adjusting the boundaries of the differential staining area, namely to 30 ~ LCF /> 8; Hansch n ~ -5 .5 . Now 20/27 (that is, 74%) of the dyes falling within the differential staining area give rise to differential staining. None of the 12 dyes outside the area give differential staining.

Although the CBN structural parameter had failed to correlate with the staining categories, an improved correlation might be found if the CBN and Hansch n parameters were considered simultaneously. When the dyes are plotted on a CBN-Hansch n diagram, then similar structure-staining correlations to those discussed above are found. However, correlations are less clear with CBN than if the LCF parameter is used; the high CBN area contains dyes of overstaining, differentially staining, and non-staining character.

Limitations of the structure--staining correlation approach A conceptually trivial, practically significant, and inevitable source of error is the impure nature of many commerical dyestuffs (Horobin, 1980b). As an example consider the Pyronin Y sample used in the present work, in which the dye was categorized as 'non-staining'. When this dye was extracted with absolute ethanol, it transpired that some three quarters of the sample was inorganic salt. Of course, a diminution in dye concentration will be expected to decrease the staining intensity; but quite independently so will the presence of salt (Goldstein & Horobin, 1974). Consequently, increasing the concentration of crude dye will not necessarily increase the staining intensity.

The question of the precision of the structural parameters is more fundamental. For a variety of reasons, several of the parameters are innately imprecise. Consider for instance the calculation of LCF values. One stereochemical feature influencing the extent of conjugation is the planarity of the dye molecule. However, it is not possible, in any rigorous way, to evaluate planarity by simple pencil and paper methods. In this work, we made use of molecular model building in order to identify, by inspection, the largest planar portion of the molecule; the LCF can then be calculated.

Finally, it must be emphasized that the staining system is complex, and that dye structure is certainly not the only important practical variable. Staining times, dye concentrations, presence of co-solutes, the nature of the solvents, and pre-staining treatments of the chromosomes can all grossly alter the final picture.


The mechanism of the G-banding of chromosomes First note the possible influence of dye charge. All the dyes giving rise to the coloration of chromosomes in the staining system used by us are, in fact, basic dyes whose coloured components are cations. Only one acid dye, whose coloured component is anionic, was studied. This dye, Eosin Y, does not stain even though it falls within the differential staining area of the Hansch ~-LCF diagram.

The structure-staining correlations of the earlier section raise several mechanistic questions. For instance, why should the overall pattern of staining intensity correlate positively with LCF values? This association of the size of a conjugated system with the intensity of staining has a plausible mechanistic explanation, namely van der Waals attractions. These non-specific intermolecular forces will tend to increase with rises in polarizability and polarity of the dye; and such rises will be reflected by increases in LCF values. A related question is why should the LCF values be a sharper measure of staining intensity than are the CBNs? Here an explanation may be that amongst the probable tissue binding sites are the bases of the DNA. For optimum van der Waals attractions the planar conjugated system of the dye must be almost in contact with the planar conjugated system of the base. Hence, it is not the size of the overall conjugated system of a dye which is significant but rather the size of the largest planar portion, and so the LCF value proves important, not the CBN.

We may now consider a further question. Why at intermediate LCF values do Hansch rr values distinguish between those dyes giving differential staining (high Hansch 7r values) and those dyes giving no staining (low Hansch rr values)? A plausible explanation again suggests that the nature of the substrate is important. Thus, if the substrate contains hydrophobic groups, uptake of hydrophobic dyes may be facilitated by hydrophobic bonding. Such a contribution to affinity will not occur with more hydrophilic dyes, and increasing Hansch 7r values indicate increasing hydrophobic character. This is in keeping with the conclusions drawn by Sumner (1980), on the basis of a different experimental approach, concerning the role of hydrophobic effects in G-banding. Finally, note that at high LCF values, staining is less influenced by Hansch rr values and occurs with both hydrophobic and hydrophilic dyes. This probably reflects the fact that with dyes possessing very extensive conjugated systems, contributions to dye-tissue affinity from van der Waals attractions are sufficient to ensure coloration even in the absence of hydrophobic bonding.

We are, therefore, suggesting that the basis of the selectivity underlying the differential staining of chromosomes, and thus of G-banding itself, is hydrophobic bonding. The overall dye-tissue affinity of G-banding also involves van der Waals attractions and coulombic attractions.

Applications to other G-banding procedures It is apparent that staining outcomes depend on factors additional to the structure of the dye. The variant G-banding systems of other workers are good examples of different recipes which produce banding. If our numerical specification for differential staining


70

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n

L L L~ , - I

60

50

40

30

20

10

O i -20

D i f f e r e n t i a l s ta in ing �9 a rea

. . . . . . . . . . . . . . . . . . . . . . . . .

O 0 �9 �9

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-15 -10 -5 O * 5 +10 §

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Fig. 2. Basic dyes used by other workers to obtain G-band type staining, plotted onto a largest conjugated bond (LCF)-Hansch ,r diagram. For comparison the differential staining area derived from Fig. 1 is also shown.

fits dyes used in other systems then any conclusions drawn concerning the implications of the proposed staining mechanism for chromosome structure will be much more convincing.

A direct check of the generality, or otherwise, of the numerical specification of the structure of 'differential staining dye' was carried out as follows. LCF and Hansch rr values were calculated for the 13 dyes noted in the Introduction as giving G-band type staining. The dyes were then plotted on an LCF-Hansch ~ diagram (Fig. 2), which shows that all 13 of the dyes fall within or on the periphery of the differential staining area, with the boundaries, namely, 30 i> LCF/> 10; Hansch rr ~> -5 .5 , which have previously been described.

Taking into account the wide variety of pre-staining treatments and staining procedures involved in the use of these 13 dyes, this overlap of results between different studies is dramatic. It is clear that a relatively hydrophobic dye structure provides a major contribution to the selectivity of G-banding in a variety of systems.

Implications for chromosome structure The implications of the suggestion that the selectivity of G-banding is due to hydrophobic bonding is that there occurs along the banded chromosome arm a marked variation in hydrophobic-hydrophilic character. Two questions then arise. First, is the


band-inducing pre-treatment required to generate this hydrophobic-hydrophilic variation? Secondly, what are the hydrophobic entities in the chromosome?

It is not necessary to assume that the induction of banding (in our case by trypsinization) actually generates hydrophobic groupings. Consider the following. The band-inducing step is usually regarded as selectively removing much of the extremely hydrophilic histone. Non-histone protein, and DNA, are less labile (Comings & Avelino, 1974). The band-induction can, therefore, be regarded not as, in any absolute sense, a generation of hydrophobic material but rather as increasing the relative hydrophobic character by a widespread removal of hydrophilic protein.

What might the hydrophobic groupings of G-bands be? It is fascinating to find that various proposals made by previous workers, noted in the Introduction, offer plausible answers. We may start with the original suggestion that G-bands correspond to the AT-rich DNA. On comparing the Hansch rr value of an AT pair with that of a GC pair, we find the AT pair to be more hydrophobic. Moving to evidence implicating non-histone chromosomal protein in banding, note that the proteins of G-bands are disulphide-rich, whilst non-staining interbands are rich in thiol groups. The marked fall in aqueous solubility on the conversion of thiols, such as cysteine, to disulphides, such as cystine, is well known (for example, Dawson et al., 1969). Hence disulphide-rich chromosomal regions could well be relatively hydrophobic.

To summarize, the coulombic requirement for basic dye reflects the role of the DNA phosphate anions; G-banding does not occur in the absence of DNA. The involvement of van der Waals attractions may well be substantially non-specific. However, the fact that staining is better predicted by LCF values than by CBN values again suggests a role for DNA. The participation of hydrophobic bonding in selective staining probably reflects the participation of both DNA and non-histone proteins. G-banding is, as has previously been emphasised, a complex process.

Effects of histochemical blockade on G-banding The staining mechanism previously proposed permits prediction of the effects on G-banding of prior chemical modification of the chromosomes. As noted by Sumner (1974), an absence of banding may be due to overstaining, that is, the complete chromosome stains, or to understaining, that is, no staining occurs.

Specifically, the LCF-Hansch ~r model predicts that overstaining is induced by chemically modifying chromosomes so as to increase the LCF number, by introducing derivatives with aromatic rings, and to increase the Hansch rr value, by adding either hydrophobic groups or removing hydrophilic groups or both. Understaining, on the other hand, is induced by lowering the LCF number by removing aromatic rings or converting them to non-aromatic derivatives, and lowering the Hansch rr value by either adding hydrophilic groups or removing hydrophobic groups or both.

These predictions can be checked by utilizing published studies of the effects of such chemical modifications on G-banding when using Romanowsky stains (Sumner, 1974, 1980; Utakoji, 1973). We estimated the probable effects of the chemical modifications on


Table 2. The predicted and observed effects of chromosomal chemical modifications on G-banding

Reagents and Expected changes* in Observed chemical Predicted effect $ t modification LCF Hydrophobic effectt on on expected$ number character banding banding

Benzoyl chloride: forms amides from amines +

Bis(acetatomercuri-methyl) dioxane: forms thiomercuri derivatives from thiols 0

Mercury Orange: forms thiomercuri derivatives from thiots +

Oxidation of thiols to disulphides by a mercuric chloride-iodine sequence 0

Acetic anhydride: forms amides from amines 0

N, N'-p- phenylene dimaleimide: forms thioethers from thiols +

Dithioerythritol, mercaptoethanol, and thioglycollate: reduce disulphides to thiols 0

Hypochlorite: oxidative deamination 0

+ OS OS

+ OS OS

+ OS OS

+

+

OS OS

OS PS

? 0

US US

US US

*+, increased; 0, no change; - , decreased. +OS, overstaining; US understaining; PS, partial overstaining; 0, no change. SReagents and observations are from Sumner (1974, 1980) and Utakoji (1973).

the LCF and Hansch 7r values of typical protein and nucleic acid groupings. To do this, we used both tabulated solubility values and also calculations of the type previously used in the present paper. This procedure could not be carried out on a few reagents, due to ambiguities concerning their mode of action. The predictions made and the experimental observations are shown in Table 2.

The similarity of prediction and observation (see Table 2) does provide further support for the LCF-Hansch ~ based model. Nevertheless, the details of cause and effect remain ambiguous. For example, it is at least plausible that, as hinted by published micrographs (Sumner, 1974), the addition of either aromatic or hydrophobic groups enhances chromosomal condensation, with the addition of hydrophilic groups having the reverse effect. Such changes in chromosomal condensation could then influence


staining. In such a case the influences of aromatic and hydrophobic character would be indirect, rather than as suggested above.

Hydrophobic effects and Q-banding? It is generally considered that whilst G-banding reflects differential dye uptake, Q-banding reflects the differential quenching of fluorescence of dye which has previously been taken up more or less uniformly along the chromosome (for example, Sumner, 1977; Comings, 1978). Accepting this explanation, nevertheless both G- and Q-banding could reflect, at least in part, variation in hydrophobic character along chromosome arms.

Thus, the fluorescence of Quinacrine can certainly be enhanced by a hydrophobic environment, whether constituted by an organic solvent (L6ber et al., 1976) or a hydrophobic mounting medium (Sumner, 1977). This latter effect is seen when the hydrophobic character o f the mountant is estimated using Hansch rr values; use of dielectric constants for this purpose by the original author gave a less clear-cut picture. Since both hydrophobic solvents and hydrophobic mountants favour fluorescence, why not hydrophobic substrates too? On this basis, as discussed previously, since both AT-rich DNA and non-histone proteins are relatively hydrophobic, both these substrates could contribute towards the enhancement of fluorescence.

Finally, note a further less explicable correlation between Q-banding and hydrophobicity. Several studies of Q-banding have compared fluorescent dyes of different structures. Such investigations include the original publications of Caspersson and his collaborators (Caspersson et al., 1969a, b), work by Tsou's group (Tsou et at., 1975), and also a collaborative study involving the laboratories of both Tsou and Comings (Comings et aI., 1978). No firm conclusions seem to have emerged from these efforts, except the appreciation that passable, if not good, banding can be obtained with dyes of very varied structure. Thus, banding occurred with molecules containing four, three or two fused aromatic rings, the latter being a binuclear cyanine, and in the presence or absence of bulky side chains.

We calculated the Hansch rr values for these dyes. In each of the three studies we found a strong correlation between effective banding and hydrophobic character. Taking as an example the study of Tsou et al. (1975), the intensity of banding achieved with different dyes was stated to vary from 'the highest' (for which dye the Hansch 7r value was = + 1.1), through 'in between' (Hansch~r = + 0.1), to 'less' (Hanschrc = - 2.7). Finally, a staining system 'without banding' resulted from use of a dye whose Hansch rc value was = - 2.9.

Without further experimentation, the significance of this correlation is unclear. Effective Q-banding could arise from selective quenching of fluorescence or from selective uptake of dye. The former mechanism has been plausibly argued for certain dyes (Sumner, 1977; Comings, 1978). The latter mechanism remains largely unchecked. These two mechanisms are not, of course, exclusive; both could operate simultaneously in some cases.


Conclusions

(1) It is possible to predict which basic dye will give differential coloration of chromosomes in G-banding systems. Such dyes have conjugated systems of moderate size and are rather hydrophobic.

(2) Numerically, 89% of the basic dyes with the following characteristics stained differentially in our study: 30 > LCF, that is, largest conjugated fragment value, > 10; and Hansch ~ > 5.0. Of dyes outside this differential staining area in LCF-Hansch 7r parameter space (that is, dyes with 10 > LCF > 30; and with Hansch rc < -5 .0) , 81% did not stain differentially.

(3) All dyes of known structure reported by other workers to give G-band type staining fall into, or onto the immediate periphery of, the differential staining area as defined above.

(4) This structural specification is consistent with the sources of dye-chromosome affinity including coulombic forces and van der Waals attractions, and with specificity being highly influenced by hydrophobic bonding.

(5) This selectivity-controlling role of hydrophobic bonding is congruent with two previous proposals, that G-bands contain predominantly AT-rich DNA and disulphide-rich protein.

(6) Chemical modifications of chromosomes reported in the literature which increase either aromatic or hydrophobic character or both induce overstaining, whilst modifications increasing hydrophilic character reduce staining.

(7) Intensity of Q-banding reported in the literature correlates well with the hydrophobic character of the dye, as assessed by calculated Hansch 7r values.

Acknowledgements

We wish to thank Professor F. W. D. Rost of the University of New South Wales, Australia, for discussions leading to the concept of LCF; Professor R. Barer and Dr. C. E. Blank of the University of Sheffield for provision of facilities; and also Bayer, Imperial Chemical industries and Professor D. Wittekind of the University of Freiburg, Germany, for gifts of dyestuffs.

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Chromosome banding: specification of structural features of dyes giving rise to G-banding