Bandeo Cromosomico

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Copyright~ 1978by AnnualReviewsInc. All rights reserved
MECHANISMS OF
CHROMOSOME BANDING
AND IMPLICATIONS
FOR CHROMOSOMESTRUCTURE
o3132
1David E. Comings
Department of Medical Genetics, City of Hope National
1500 East Duarte Road, Duarte, California
91010
Medical Center,
CONTENTS
INTRODUCTION
........................................................................................................
HISTONES
AND
CHROMOSOME
BANDING
......................................................
C-BANDING--EXTRACTION
OF NON-C-BAND
DNA......................................
G-BANDS=CHROMOMERES
OF MEIOTICCHROMOSOMES
..........................
MAJOR Q-BANDS ARE COMPOSED OF SEVERAL SMALLER
CHROMOMERES
........................................................................................
GIEMSA STAINING--SIDE STACKING OF THIAZIN DYES ON DNA ........
G-BANDING--ENHANCEMENT OF THE CHROMOMEREPATTERN ........
Q-BANDING--DETECTION
OFAT-RICH
DNA
..................................................
HOECHST33258 BANDING--DETECTION
OF AT-RICH DNA......................
R-BANDING
DETECTION
OFGC-RICH
DNA
....................................................
Q- or R-BANDING BASED ON EARLY REPLICATING DNA IN R-BANDS
ANDLATEREPLICATING
DNAIN Q- or G-BANDS
......................
CHROMOSOMEBANDS DELINEATE THREE TYPES OF CHROMATIN ....
IMPLICATION
OF CHROMOSOME BANDING FOR CHROMOSOME
STRUCTURE
..............................................................................................
25
26
27
29
30
30
31
33
35
35
37
38
39
INTRODUCTION
The different chromosome
banding procedures have allowed impressive
advances
genetics,
to be made in clinical
and vertebrate
cytogenetics,
somatic cell
and chromosome mapping. These fields
can progress
whether or
~Supported by NIH Grant GM15886..

25
0066-4197/78/1215-0025501.00
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COMINGS

not we have an understanding of the mechanismsof chromosomebanding.

The same cannot be said of the field of chromosomestructure. With the
discovery of nu bodies, or nucleosomes, and the greatly increased understanding of the relationship between DNAand histones that they have
provided, the next major level in chromosomestructure to be attacked is
the higher order arrangement of the chromatin fiber. A clear understanding
of what chromosomebands are and what they represent in terms of underlying DNAbase composition, function, and structure will be essential to
understanding how chromosomesare put together.
HISTONES AND CHROMOSOME

BANDING
Histones have been frequently implicated as a major factor in chromosome
banding (1-8). The major reason for believing that histones are not involved
in banding is that methanol-acetic acid-fixed, air-dried chromosomes
treated with 0.2 N HCIfor up to 4 hr showuniform staining with Giemsa.
If these chromsomesare then treated with trypsin or appropriate salts,
typical banding occurs (9). This conclusion assumes that all the histones
have been removed from the chromosomesby a combination of methanolacetic acid fixation and HCItreatment. Whenmouseliver nuclei are treated
in this manner, then scraped off the slides, SDSgel electrophoresis shows
that all the histones have been removed (9). Other investigations have
indicated that methanol-acetic acid fixation alone removes most of the
histones (8, 10-14). By contrast, on the basis of studies with antihistone
antibodies Pothier et al (15) concluded that H4 histones were not completely removed from metaphase chromosomesby methanol-acetic acid and
0.2 N HCI, since they observed staining with anti-H4 antibodies. An alternative explanation is that the large amountsof historic used in the immunization procedure (5 mg) contained nonhistone proteins and that the
bandingwas the result of antibodies to nonhistones. Studies by Bustin et al
(14) using antihistone antibodies produced by much smaller amounts
histone (20 #g), coupled with RNA,indicated that histones H2A,H3, and
H4 are completely removed after only 5 sec of treatment with methanolacetic acid. These histones and H1 histone were removed by standard
methanol-acetic acid fixation. Only H2Bremained after fixation. Fluorescent antibody staining of all histones occurred whenisolated chromosomes
were fixed with gluteraldehyde. All histones were uniformly distributed
along the chromosomeswith no evidence for banding patterns, even when
1:50 dilutions of the antisera were used.
Brown et al (6) observed that exposure of banded metaphase chromosomes to solutions of H1 and H2Ahistone abolished the banding pattern
and chromosomestaining. It was concluded that selective removal of H1
and H2Ahistone from the bands during fixation was necessary for intense
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MECHANISMS OF CHROMOSOMEBANDING
27
staining of the bands and that the interbands were poorly stained because
these histones were still present. This, however,is inconsistent with the fact
that chromosomes
that are initially fixed in formaldehydeor alcohol alone,
and never exposedto methanol-acetic acid, stain an intense uniform purple.
Also, whenadded at sufficient concentrations, all histones are capable of
abolishing Giemsa staining of chromosomes(6a). Somebanding, although
of less than optimal quality, has been produced in chromosomesthat were
first treated with formaldehydeto prevent the removal of histones by the
banding procedures (12).
H1 histone appears to be associated with the inter nu body DNA(16)
and may be involved in chromosomecondensation. With two lysine-rich
regions separated by a hydrophobiccore, H1 histone is ideally suited to a
role of pulling together two separate DNAmolecules. Thus, if any histone
was involved in banding this Wouldbe a likely candidate. Yet this is precisely the histone that is most readily removedby the methanol-acetic acid
fixation.
The above observations makeit unlikely that histones play an important
role in chromosomebanding. A remaining possible role of histones in
banding is discussed under C-banding.
C-BANDING--EXTRACTION
OF
NON-C-BAND
DNA
In 1970 Pardue & Gall (17) noted that the centromeric heterochromatin
mouse chromosomes, rich in satellite DNA,stained more intensely with
Giemsafollowing treatment for in situ hybridization. This procedure consisted of exposing fixed chromosomesto 0.07 N NaOHfor 5 min, then
2 SSC(0.3 MNaCl, 0.03 Mtrisodium citrate) at 66C for overnight. This
treatment was modified by Arrighi & Hsu (18) to produce the C-banding
procedure.
Because of the association of C-bands with satellite-rich constitutive
heterochromatin,it was initially felt that the Giemsa-positiveregions represented areas of reassdciated, highly repetitious DNA(19-23). In fact, the
technique was often referred to as a denaturation-renaturation procedure.
There are several reasons, however,to believe that this is not the primary
mechanism of C-banding.
1. SomeC-band heterochromatin does not contain highly repetitious or
satellite DNA(24-25).
2. Studies with acridine orange staining indicate that there is no correlation
between C-band positive versus C-band negative regions and doubleversus single-stranded DNA(26-27).
3. Double-stranded DNAdoes not bind greater amounts of dye than single-stranded DNA(28).
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In an extensive study of the mechanismsof C-banding, Comingset al (26)

showed that the feature most clearly associated with C-banding was the
extraction of non-C-band DNAand retention of C-band DNAon the
chromosome.The Giemsa stain was simply side stacking on the DNAthat
remained, resulting in intense staining of constitutive heterochromatin.
Radioisotope labeling studies showed that an average of 60%of the DNA
was being extrac*.ed from chromosomesduring the C-band procedure. Feulgen staining, CsCl centrifugation of the extracted and nonextracted DNA,
and electron microscopy showedthat the DNAwas preferentially extracted
from the non-C-bandregions. This has been confirmed in other laboratories
(29-33).
This raises the question, why is the C-band DNAmore resistant to
extraction than non-C-band DNA?There are several possibilities:
(a)
DNA-nonhistoneprotein interactions, (b) DNA-histoneinteractions, (c)
greater compaction of DNAof constitutive heterochromatin, or (d) more
renaturation of C-band DNA.
Since a preliminary step in C-banding consisted of exposure of the
chromosomesto 0.2 N HCI for 30 min, it seemed unlikely that a DNAhistone interaction was involved in C-banding. This conclusion was further
strengthened by the fact that exposure to 0.2 N HCI for up to 4 hr, a
procedure that appeared to removecompletely all histones from fixed interphase nuclei, also did not affect C-banding(9). These findings emphasized
a probable role of DNA-nonhistone
protein interactions. Becauseof this we
have been quite interested in examiningthe nonhistone proteins of constitutive heterochromatin. Whenheterochromatin is isolated by the hypotonic
Frenster procedure (34), the nonhistone proteins of heterochromatin are
similar to those of euchromatin but some heterochromatin unique bands are
seen (35, 36). At least some of these are nuclear matrix proteins (37).
However,when heterochromatin is isolated by an isotonic procedure that
prevents the dispersion of the heterochromatin, there is a remarkable deficiency of nonhistone proteins in the heterochromatin fractions and no
proteins that are unique to the heterochromatin (36).. The apparent absence
of heterochromatin-unique proteins was confirmed by the electrophoresis of
Drosophila virilis, D. americana,and D. ezoana larval nuclei. These species
contain 40%, 35%, and 0%satellite DNArespectively and there were no
nonhistone proteins present in the D. virilis and D. americanathat were
missing in the D. ezoana (36).
Musichet al (38) found a prominent nonhistone protein in Green monkey
satellite chromatin isolated by restrictive endonucleases. These proteins
have the eleetrophoretie mobility of nuclear matrix proteins and this is
consistent with the tendency for heterochromatin DNAto bind preferentially to the undersurface of the nuclear membrane(39). DNasedigestion
studies of isolated heteroehromatin, freed of histones by acid treatment,
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showit to be more resistant than euchromatin(40). This is also consistent

with the binding of nuclear matrix proteins to heterochromatic DNA.
Studies with the fluorescent protein binding compound,dansyl chloride,
also show retention of protein as well as DNAin the hetcrochromatin of
C-banded chromosomes(41). These findings are all consistent with DNAnuclear matrix protein interaction being responsible for the relative resistance of constitutive heterochromatin DNAto extraction during the
C-band procedure. Somerecent studies of Holmquist(42) have resurrected the possibility
DNA-histoneinteractions from what was an apparently safe entombment.
Holmquist found that treatment with 0.2 N HCI wasimportant to obtaining
good C-banding. This suggested that depurinati0n of the DNAmight be
involved. Studies with the replicative form of t~x DNAindicated that 0.2
N HCI caused 0.2 depurinations/min per l06 daltons. If the aldehyde
produced by depurination was reduced with NAB4,no banding occurred.
Bleomycin, which removes bases at neutral pH, resulted in good C-banding
without acid treatment. Theseresults are consistent with an interaction of
the sugar aldehyde, procuced as a result of depurination, with protein
(especially lysine) via a Schiffbase. This DNA-proteincomplexmaypreferentially occur in constitutive heterochromatin because it is more condensed
than other parts of the chromosomes,or because the heterochromatic DNA
may be more intimately involved with nuclear matrix proteins than other
parts of the chromosomes. The staining of C-banded chromosomes with
antihistone antibodies might resolve the question of whether a DNAhistone complex or a DNA-non-histonecomplex is involved.
Exposure of the chromosomesto alkaline conditions such as 2 X SSCat
pH 8 (29, 43) or to Ba (OH)2(44) appears to be important in the subsequent
extraction of non-C-band DNA(26, 32, 45). The pH of the SSC in the
presence of glass slides increased from 7.to 9 during the 65C incubation
(43).
Although rapid renaturation of satellite DNAmight make C-band DNA
more resistant to extraction (46), studies in which chromosomesin the
process of C-banding are fixed in formaldehyde and then examined by
acridine orange staining (26) suggest the C-band DNAremains singlestranded until the end of the 2 SSC, 60C treatment.
G-BANDS = CHROMOMERESOF MEIOTIC

CHROMOSOMES
In the early days of G-banding,one of the questions of greatest interest was
whether the banding procedures were inducing the G-bands or whether
they were simply enhancing bands that were already present. With the
appearance of reports that banding could occasionally be producedby little
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COMINGS
or no treatment of the chromosomes(47), by Feulgen staining (48, 49),

by special methods of examining G2-treated or -untreated chromosomes,
such as phase microscopy (3, 50) UVmicroscopy (50), or electron microscopy (51-53), the suspicion arose that the bands were there all the time and
they simply needed enhancement. The final confirmation of this idea came
from the observation that the G-bandpatterns corresponded exactly to the
chromomere pattern of meiotic chromosomes (54-56). Since meiotic
chromosomesare severalfold more extended than mitotic chromosomes,
and since meiotic pairing enhances the chromomeres,they are mucheasier
to see during pachytene than during mitosis. It has been suggested that the
reason plant chromosomes rarely show G-bands but do have meiotic
chromomeresis that the ratio of meiotic to mitotic chromosomelength in
plants is 8 to 18 compared to 2.3 for humans (57). Thus, some of the
fundamental questions about G-banding are (a) why do chromomeres exist? (b) what do they represent? (c) are they the same as or different
the bands of Dipterian polytene chromosomes?(d) what is their relation
active and inactive DNA?Someof these questions can be answered by
studies of the properties of G-band versus R-band DNA(see below).
MAJOR G-BANDS ARE COMPOSED OF SEVERAL

SMALLER CHROMOMERES
A second observation that needs emphasis is the lesson learned from the
examination of extended prometaphase chromosomes (58-60), or
chromosomesextended by treatment during the G2 period with rifampicin
or amphoteriein B (D. Shafer, personal communication). These preparations showthat instead of approximately 350 bands in the Paris nomenclature, there are in fact 2-3 thousand or more bands or chromomeres.
Examination of metaphase chromosomes taken up in 4Mammoniumacetate, then water-spread and examinedby electron microscopy, suggests a
muchlarger number of chromomeres(61; D. E. Comingsand T. A. Okada,
unpublished observations).
GIEMSA STAINING--SIDE
DYES ON DNA
STACKING OF THIAZIN
Before discussing the mechanisms of G-banding some aspects of Giemsa

staining of chromatin should be emphasized. Giemsais a complex mixture
of dyes of various degrees of oxidation. Formulas of some of the various
commercially available Giemsapreparations can be found in Comings(62).
Basically they consist of methylene blue, Azure B, Azure A, Azure C, and
thionin. These represent the thiazin molecule with 4, 3, 2, 1, and 0 methyl
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groups respectively. Eosin is also present. These thiazins are positively

charged planar molecules that interact ionically with the phosphate groups
of DNAand side stack along the molecule (62). Maximum
binding occurs
whenthere is an equal molar ratio of thiazin dye to DNA
phosphate groups.
The stacking of these dyes results in hypochromismjust as when DNA
bases are stacked. This results in a decreased optical density or molar
extinction coefficient of the dye. Anadditional characteristic is that there
is a shift in the peak absorbance to lower wave lengths. This is termed
metachromasia(63) and results in a change in the color of the dye from
light blue (650 nm) to a dark magenta (560 to 600 nm) (62). While
presence of eosin results in a slight lowering of the wavelength of this
complex (to 550 nm), contrary to an earlier assumption (64) eosin is
necessary for the production of G-bands. G-banding can be produced with
methylene blue and the azures alone without eosin (62, 65-67). Thionin,
which gives the poorest metachromasia, stains chromosomesuniformly
without G-banding(62). Althougheosin is not necessary, generally superior
banding occurs in the presence of eosin. A thiazin-eosin complex does
appear to be important for Giemsa11 banding (68), which is characteristically much pinker than G-bands. With excess dye, plus pure DNA,the
dye/phosphatemolar ratio is 1.0 indicating that whenthe dye is present in
excess all the phosphate groups are bound with dye (28, 69-71). In the
presence of unfixed chromatin, the ratio is 0.5~).6 (28, 69-71) indicating
about half of the phosphatesare free to bind to DNA.This is consistent with
the nu body structure of chromatin. In the presence of methanol-acetic
acid-fixed chromatin, the ratio is about 0.8 (28) indicating the fixation
procedure removes protein and allows more phosphate groups to interact
with dye. Interestingly, whenfixed ehromatin is treated with hot salts, as
for G-banding, the ratio drops back to 0.5 suggesting that some of the
proteins have been denatured and nowmore efficiently cover some sections
of the DNA(28). The possible relevance of this to G-banding mechanism
is discussed below.
Sometheories of chromosomebanding have suggested that the Giemsa
interacts primarily with chromosomalproteins (72). The in vitro studies
indicate that the only significant binding is with DNA(28).
G-BANDING--ENHANCEMENT
CHROMOMERE PATTERN
OF THE
The mechanismof G-bandingis the least clear of the banding techniques.

It is essentially an enhancementof the basic chromomerepattern. It is the
precise mechanism by which this enhancement is brought about that is
unclear.
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COMINGS
A direct.role of Giemsain producing the bands has often been proposed

(50, 73, 74). The best wayto dissect this factor from others is to examine
chromosomesbefore and after banding treatment by a procedure that does
not use Giemsa. This can be done by electron microscopy. Burkholder (75,
76) has shownthat unstained, methanol-acetic acid-fixed chromosomesare
uniformly electron-dense and show no banding pattern. However, if they
are treated with trypsin, as in trypsin banding, electron-dense bands and less
dense interbands can be seen. Similar results are obtained with scanning
electron microscopy(77). Since no Giemsaor uranyl acetate is used in these
experiments, there is either (a) a rearrangement of the chromatinfibers or
(b) a loss of chromatin from the interband regions. Because experiments
with 3H-thymidine-labeled chromatin (26, 31) and Feulgen staining (64)
indicate a very minimal loss of DNAwith trypsin or ASGbanding, the
major factor is presumably a rearrangement of the chromatin with some
pulling away from the interbands and relative accumulation in the bands.
This rearrangement may be aided by extraction of Ca2+ from the chromosome during G-banding (78). Rearrangement alone, however, may not
adequate to explain G-banding completely for the following reasons: (a)
The relative difference in intensity of the bands and interbands as seen by
electron microscopy is much less than that seen with Giemsa banding.
(b) Whentrypsin-banded chromosomesare stained with Feulgen (16,
or with thionin (62) there is poor or no banding. Whenthe same chromosomes are stained with Giemsa there is excellent banding. Clearly the
Giemsahas an important enhancing effect on the ability to see bands. As
mentionedin the section above, the thiazin dyes are able to stack onto the
DNA.This is verified by scanning electron microscopy (79, 80) and Normaskyoptics (33a), which show a DNA+ dye mass at the bands, and little
mass at the interbands. Thus, the reason for the Giemsa-positive bands is
clear: Chromatinis present whoseDNAis free to side stack with the thiazin
dyes. The feature that needs explanation is whyis there little or no staining
of the interband regions. There are two major possibilities: 1. There is less
DNApresent. 2. The DNAis covered and unavailable for interaction with
the dye. Both factors are probably true. There are four reasons to believe
that there is less DNApresent: 1. The basic structure of the metaphase
chromosome is one of chromatin-dense chromomeres plus chromatin
sparse interchromomeres. 2. The electron microscopy studies suggest this
is enhancedby some degree of chromatin rearrangement. 3. The trypsin and
hot salt treatments probably do extract someof the interband DNA.4. The
distribution of protein in G-bandedchromomeres,as determined by dansyl
chloride staining, matches that of the G-bands(81).
The possibility that the DNAof the interbands regions is also blocked
by protein was alluded to above. In vitro studies with methylene blue show
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that treatment of fixed chromatin with hot salts, as in the G-banding

procedures; results in a decrease in the number of phosphate groups on
chromatin available for interaction with dye (28). Presumably the mechanism is a denaturation of the nonhistone proteins that are especially prevalent in the euchromatic R-bands. Under proper conditions these proteins
maygo from an ordered folded tertiary structure with limited sites for
interacting with DNA,to a denatured randomcoil that has manymore sites
for ionic binding to DNAand covers more DNAphosphate groups, after
the denaturing conditions have been removed.In this regard it is interesting
that the one feature that all the G-bandingprocedures (82) have in common
is the ability to denature protein, or break S-S bonds (83-85).
In conclusion, G-banding occurs because there is a basic chromomere
structure in the metaphase chromosomewaiting to be enhanced. This enhancement occurs by inducing some degree of rearrangement of the fibers
away from the R-bands toward the G-bands, possibly some extraction of
R-band DNA,and covering over the R-band DNAwith denatured nonhistone proteins, followed by the markedenhancementof this pattern through
the ability of thiazin dyes to side stack on available DNA.
One aspect of G-banding that is not completely explained by the above
is that even though centromeric heterochromatin also presents as a prominent chromomerein meiotic chromosomes,these areas in mitotic chromosomes are sometimes poorly stained by G-banding but well stained by
C-banding (30). The C-band region on the humanNo. 9 chromosomeis
example. Here brief trypsin treatment produces G-bandingwith poor staining of the heterochromatin on No. 9, while more prolonged treatment
results in C-banding with intense staining of this heterochromatin (30).
Because the DNAwas obviously present in this region during the Gbanding, the most likely reason for poor staining is poor access of the dye
to DNA,either because of unusually intense condensation of this heterochromatin, or because of masking by proteins. A similar phenomenonhas
been described with centromeric heterochromatin in the mouse. Here
Giemsa staining of fixed chromosomesshows poor staining of the heterochromatin. After C-banding these same regions stain intensely (26).
Q-BANDING--DETECTION
OF AT-RICH
DNA
The initial rationale of Caspersson and his group in using quinicrine mustard for staining chromosomeswas that the mustard moiety would preferentially interact with GC-rich DNA(86, 87). It was soon found, however,
that quinacrine alone was just as effective. Then Weisblum&deHaseth (88,
89) showed that in vitro, AT-rich DNAmarkedly enhanced the fluorescence of quinacrine while GC-rich DNAquenched the fluorescence of
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COMINGS

quinacrine. This observation was verified in numerouslaboratories (90-96).

In addition, Ellison & Barr (97) showedthat a region of very AT-rich DNA
in Samoaia leonensis fluoresced intensely with quinacrine. While this
seemed to be a clear explanation of Q-banding, some of the following
nagging questions and apparent inconsistencies persisted:
1. In vitro studies with chromatin suggested that nonhistone proteins could
also be playing a role by limiting the access of quinacrine to GC-rich
DNA(94, 95, 98).
2. The isolation of specific satellites has failed to give the in vitro effect on
quinacrine fluorescence expected on the basis of their ATcontent (95,
96, 99, 100).
3. In many different organisms the heterochromatic centromeric DNA,
containing satellite sequences that were not GC-rich, nonetheless stained
very poorly with quinacrine (99, 101-103).
Twoexplanations were offered for these observations: 1. Someproteins
were limiting the access of quinacrine to the satellite DNA
(95). 2. There
were some strategically
placed GCbase-pairs that were quenching the
quinacrine fluorescence despite general AT-richness of the DNA(89, 104).
Thesealternatives are still not definitively settled and must await the study
of the effect of synthetic DNAsof knownbase sequence on the in vitro
fluorescence of DNA.
In the calf, the centromeric heterochromatin stains poorly with a quinacrine (105), and contains GC-richsatellites (106). These isolated satellites
quench quinacrine flourescence as expected (96).
An additional problem was whether there was a suflicient degree of
variation in the base composition of main band DNAto account for the
relative variation in quinacrine fluorescence seen in Q-banding(95, 99, 100).
This was studied in detail by Comings& Drets (96) who concluded that
change in base composition along the chromosomeof 6% in AT-richness
was adequate to result in a 50%change in relative quinacrine fluorescence.
The quinacrine fluorescence changes with the fourth power of the base
composition of DNA(90, 96). This is adequate to explain the quinacrine
fluorescence of all but the brightest Q-bands. Withthe very bright Q-bands
(constitutive heterochromatin), the effect of protein on the access of quinatrine to DNAmay still be important. Studies with tritiated quinacrine
suggest there is no more quinacrine bound to the bright Y than other
chromosomes(107), while studies with X-ray microanalysis suggest there
maybe increased amountsof quinaerine over some Q-bright areas (78). The
existence of other compoundssuch as 2,2,-di-t-butyl proflavine and 4,-6diamidino-2-phenylindole (DAPI) (108), which are knownto bind preferentially to AT-rich DNAor fluoresce more in the presence of AT-rich DNA
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and to give Q-bandingpatterns (109), is consistent with the importance

base composition in Q-banding.
In vitro studies with daunomycin, an antibiotic that also gives good
Q-banding(110), provided some additional insights into the mechanism
Q-banding. This antibiotic showedthe same degree of quenching of fluorescence in the presence of DNAsvarying in GCcontent from 40 to 100%.
There was only slightly less quenching with DNAof 30%GCcontent. By
contrast there was muchless quenching of fluorescence in the presence of
poly(dA-T) (96, 111). Since the mean base content of mammalianDNA
40%GC, this suggested that the most important sequences in producing
Q-banding were very AT-rich (70 to 100%AT). Since GC-rich, early
replicating DNAprobably has a base composition of between 43 and 48%
GCwhile the late replicating DNA
is 39 to 41 GCcontent (96), this implies
that Q-bandingmay be due to relatively small stretches of quite AT-rich
DNApresent in the G-bands or chromomeres.This is a potentially important aspect of mammaliangenomeorganization that deserves further study.
As discussed below, such AT-rich regions mayplay an important role in
formation of chromomeres through the interaction with nuclear matrix
protein.
HOECHST 33258
OF
AT-RICH
BANDING--DETECTION
DNA
In vitro studies with Hoechst 33258 also show that its fluorescence is
enhanced by AT-rich DNAwhile the enhancement is less with GC-rich
DNA(96, 112-114). This time the correlation with chromosomestaining
is muchmore satisfying since the regions knownto contain AT-rich satellite
DNAreally do stain much more brightly with Hoechst 33258 (115, 116).
In vitro studies suggest that Hoechst 33258 is bound to DNAby hydrophobic binding in the large groove (114, 117) and not by side stacking
intercalation. In this regard it is similar to dibutyl proflavine (118). This
compoundalso has the interesting effect of causing a markedelongation of
centromeric heterochromatin when added to the culture before the chromosomes are harvested (119). As with quinacrine, chromosomeproteins may
play a minor role in Hoeehst 33258 banding (113, 120).
R-BANDING DETECTION OF GC-RICH DNA

R-banding or reverse banding was first described by Dutrillaux &Lejeune
in 1971 021). Chromosomeswere placed in a 20 mMphosphate buffer pH
6.5 at 87C for l0 rain, then stained with Giemsa. Whenexaminedby phase
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COMINGS
contrast microscopy the G-bands were pale and the interbands (R-bands)
were stained. This is a particularly useful procedure for examining exchanges or deletions involving the distal ends of chromosomessince the
telomeres stain well. A modification of the procedure, termed T-banding,
stains only telomeres (122). Subsequentstudies demonstratedthat staining
the treated chromosomeswith acridine orange resulted in excellent Rbanding (26, 123-129). Whenphotographed in color .the R-bands were
green, the remaining portions of the chromosomewere orange or red. When
photographedwith black and white film the R-bands were dark, the rest of
the chromosomelight. It was also found that alkali could be used instead
of heat treatment (26, 125, 130). Dutrillaux & Covic (131) reported
detailed examination of the factors involved in producing R- and T-bands.
The following observations indicate that the relative GC-richness of Rbands is the major factor involved in R-banding.
1. The only treatments that consistently give R-bandingare those capable of selectively denaturing the AT-rich DNAof the G-bands and leaving
the GC-rich DNAof the R-bands in a native configuration, These treatments include incubation of the chromosomesin various buffers at temperatures of 76 to 95C (127, 131) and incubation of the chromosomes
in alkali
plus formaldehyde (26, 125, 130). Bobrow& Maden(124) have reported
one of the few exceptions to this statement. They observed R-banding in
some chromosomestreated only with trypsin and stained with acridine
orange. We(D. E. Comingsand H. E. Wyandt, unpublished results) were
unable to confirm this. Manyof the parameters involved in acridine orange
monitoring of thermal denaturation of DNAin fixed nuclei have been
studied by flow cytofluorometry (132, 133).
2. Acridine orange stains single-stranded DNAred and double-stranded
DNAgreen (134). Wheneentromerie heteroehromatin known to contain
AT-rich satellite DNAis R-bandedit stains red by acridine orange (22, 26,
124, 125, 135). Whencentromeric heterochromatin containing GC-rich
satellite DNAis R-banded it stains green (136). These are the results
expected from the fact that AT-rich DNAdenatures more readily than
t3C-rich DNA.
3. Verification of the assumption that base composition is the major
factor in R-banding comes from the use of fluorescent compoundsthat
preferentially bind to GC-rich DNAor sfiow greater fluorescence in the
presence of GC-rich than AT-rich DNA.Such R-banding has been produced with the chromomycinantibiotics chromomycinA3 (137, 138), olivomycin (139), and mithramycin (140). Schweizer (138) found that
chromomycin A3-stained chromosomes were treated with DNase I, then
stained with Giemsa, the chromomycin-A3fluorescent-positive bands now
stained darkly. This is most likely due to the protection against DNAseI
by chromomycin A3 bound to the GC-rich DNA. Since chromomycin-
Annual Reviews

37
A3 fluorescence fades rapidly, this has the advantage of makinga permanent

Giemsa stain of the R-banded chromosomes.
One aspect of R-bandingthat is unclear is whyR-bands can be seen with
Giemsastaining (enhancedby phase contrast) especially since thiazin dyes,
if anything, showbetter side stacking to denatured than to native DNA
(28,
33, 66). One possible explanation is that at the temperatures used, the
denatured AT-rich DNAof the G-bands is partially extracted while the
native DNAof the R-bands is not. This is consistent with the studies on
C-bandingthat indicate denaturation of DNAis an important first step to
subsequent extraction of non-C-band DNA(26, 32).
Q- OR R-BANDING BASED ON EARLY

REPLICATING DNA IN R-BANDS AND LATE
REPLICATING DNA IN Q- OR G-BANDS
WhenDNAthat has been labeled with H3-thymidine during various times
in the S period is centrifuged to equilibrium in CsC1with C14 thymidine
labeled DNAfrom unsynchronizedcultures, it is apparent that early replicating DNAis GC-rich and late replicating DNAis AT-rich [see (141) for
refs.]. In the prebanding era, light microscopeautoradiography of chromosomeslabeled with H3-thymidinelate in the S period showeda high density
of grains over certain humanchromosomes, such as No. 13 (142). After
chromosomebanding showed that these regions had major G-bands while
chromosomeregions rich in R-bands contained early replicating DNA,it
implied that G-bandswere composedof AT-rich, late replicating DNAand
R-bands were composedof GC-rich, early replicating DNA(143). Combinations of autoradiography and banding studies show that R-bands are
early replicating and G-bandsare late replicating within the limits of resolution obtainable with autoradiography (144-148). The demonstration
Latt (149) that BrdU-containing DNAquenched the fluorescence of Hoechst 33258(or acridine orange), allowed muchhigher resolution for studying the relationship between the time of replication and G- or R-bands.
Virtually all of these studies have shown that R-bands contain earlyreplicating DNAand Q- or G-bands contain late-replicating
DNA(148,
150-157). Only a few have not found this relationship (158, 159). Thus,
under the proper circumstances of BrdUincorporation, Q-, G-, or R-bands
can be produced simply on the basis of the different timing during the S
period of the replication of the DNA
of these bands. Dutrillaux et al (156)
found that the widest humanR-bands were the earliest to replicate, the
brightest Q-bandswere the latest to replicate, and there was probably little
overlap in Q- vs R-bandreplication. This is consistent with the presence of
two distinct portions of the S-phase as indicated by H3-thymidinecounting
of synchronized diploid Cells (160).
Annual Reviews
38
COMINGS

CHROMOSOMEBANDS DELINEATE THREE TYPES

OF CHROMATIN
In a review of chromatin structure
and function in 1972 (143) I suggested
that the information
available
at that time suggested the presence of three
major types of chromatin:
euchromatin,
constitutive
or C-band heterochromatin,
and intercalary
or G-band heterochromatin.
In the succeeding
years the distinction
between these types of chromatin
has become more
precise (161). Table 1 presents their major characteristics.
The part I wish
to emphasize here is the remarkable difference
between G-band and R-band
chromatin.
The development
of the BrdU-Hoechst procedure
for fine mapping of DNA replication
times (see above) has further
emphasized
the
precise correlation
between early replicating,
GC-rich DNA with R-bands,
and late replicating,
AT-rich DNA with G-bands. This raises the question
of why is there such a remarkable degree of nonrandomness in the distribution GC- and AT-rich DNA? Why is the former early
replicating
and the
later
late replicating?
Why is the AT-rich DNA organized
into chromomeres while the GC-rich, active chromatin tends to be interchromomeric?
Also, do these two types of DNArepresent
two sets of basically
similar
DNA with simply a different
mean base composition,
or does the AT-rich
DNA contain
weird stretches
of very AT-rich
DNA?
Table 1 Three major types of chromatin in chromosome bands
Relation to bands
Location
Condition during
interphase
Genetic activity
Timing of DNA
replication
AT content of DNA
Repetitiousness of
DNA
DNAmethylation
Relation to pachytene chromomeres
Centromedc
constitutive
heterochromatin
Intercalary
"heterochromatin"
In C-bands
Usually centromeric
In G-bands
Chromosome arms
Condensed
Inactive
Condensed
Probably inactive
In R-bands
Chromosome arms
Usually
dispersed
Usually active
Late S
GC-rich, neutral or
AT-rich depending
on satellite
DNA
Usually satellite
DNA
Late S
AT-rich
Early S
GC-rich
Moderately repetitious and

unique
+
Moderately repetitious and

unique
Intercalary
chromomeres
lnterchromomeric
Frequently highly
methylated
Centromeric
chromomere
Euchromatin
Annual Reviews
MECHANISMSOF CHROMOSOME
BANDING
39
IMPLICATION OF CHROMOSOMEBANDING
FOR CHROMOSOMESTRUCTURE

Someof the major questions concerning chromosomestructure, such as the

question of uninemyversus multinemy,and the organization of the chromatin fiber, are nowresolved. There is general agreement that chromosomes
are uninemic (143, 162-164) and the discovery of the nu body (165)
nucleosomes (16) has enormously improved our understanding of the rela-
o) DNA
b) Extended
c) Condensed d) Chromomeresand
Nucleosomes
Nucle~somes
Interbond Chromatin
250 A Fiber
0.7- ].2
h) Compact
Unbanded
Chromosome
0.2-0..5~m
g) Spiralized
Chromosome
f) Chromosome
Bands
t~igure 1 Model of mammalian chromosome structure.

sion
e) Clusterinqof
Chromomeres
from Elsevier/North-Holland
Biomedical
Press,
See text.
[From ref.
Amsterdam.]
(167).
By permis-
Annual Reviews

40
COMINGS
tionship of DNAto histones in the chromatin fiber. An important remaining question is how the chromatin fiber is arranged into a metaphase
chromosome. Prebanding models of chromosome structure
suggested a
chromatin fiber folded upon itself,
progressing from one telomere to the
other (143, 166). The presence of chromosome bands requires some modification of this model. A recently proposed modification (167) is shown
Figure 1. Here the 250 /~ fiber composed of condensed nucleosomes is
arranged into a series of loops or chromomeres by the association of portions of the DNAwith the nuclear matrix, nonhistone proteins.
Filter
binding assays suggest there is a preferential association of nuclear matrix
proteins with AT-rich DNA

(D. E. Comingsand A. Wallach, unpublished
results). Thesesmall loops wouldconsist, at least in part, of relatively
AT-rich, late replicating DNA,while the DNA
between the loops would
be composedof relatively GC-rich,early replicating DNA.Smallchromomeres would associate to form larger chromomeresvisible as bands in
metaphasechromosomes.
Further rationale for this modelhas been presentedelsewhere(167). Therecently described"scaffoldingproteins" of the
metaphasechromosomes
(168) could represent that portion of the intranuclear matrix (37, 169) whichremainsboundto the metaphasechromosomes
organizing the chromomeres.
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BANDING
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Annual Reviews

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

Ann.Rev. Genet. 1078.12.25-46

~Supported by NIH Grant GM15886..

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

not we have an understanding of the mechanismsof chromosomebanding.

HISTONES AND CHROMOSOME

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

In an extensive study of the mechanismsof C-banding, Comingset al (26)

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

showit to be more resistant than euchromatin(40). This is also consistent

G-BANDS = CHROMOMERESOF MEIOTIC

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

or no treatment of the chromosomes(47), by Feulgen staining (48, 49),

MAJOR G-BANDS ARE COMPOSED OF SEVERAL

Before discussing the mechanisms of G-banding some aspects of Giemsa

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

groups respectively. Eosin is also present. These thiazins are positively

The mechanismof G-bandingis the least clear of the banding techniques.

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

A direct.role of Giemsain producing the bands has often been proposed

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

that treatment of fixed chromatin with hot salts, as in the G-banding

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

quinacrine. This observation was verified in numerouslaboratories (90-96).

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

and to give Q-bandingpatterns (109), is consistent with the importance

R-BANDING DETECTION OF GC-RICH DNA

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Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

A3 fluorescence fades rapidly, this has the advantage of makinga permanent

Q- OR R-BANDING BASED ON EARLY

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

CHROMOSOMEBANDS DELINEATE THREE TYPES

Moderately repetitious and

Moderately repetitious and

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

Someof the major questions concerning chromosomestructure, such as the

t~igure 1 Model of mammalian chromosome structure.

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

proteins with AT-rich DNA

8. Retief, A. E., Ruchel,R. 1977.Histones

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

cytological preparations of Chinese

the production of C bands. Chromosoma 41:175-82

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

depurination and B-elimination. Z Cell

chromosomes do not show G-bands.

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

toluidine blue. Biochim. Biophys. Acta

metaphase nucleus. Exp. Cell Res.

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

Molecular basis of chromosomebanding. I. Effect of mouseDNAfractions

fluorescent probe specific for ATconcentration

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

mammalian metaphase chromosomes.

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

dine suppression of 33258 Hoechst

Annu. Rev. Genet. 1978.12:25-46. Downloaded from arjournals.annualreviews.org

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