band methods 2

Karyotype Analysis andChromosome BandingWendy A Bickmore, MRC Human Genetics Unit, Edinburgh, Scotland, UK

A series of reproducible bands across metaphase chromosomes can be revealed by some

treatments. These chromosome bands not only allow the identification of normal and

abnormal chromosomes but they also tell us about fundamental aspects of the chromatin

structure and compartmentalization of the genome.

Methods of Chromosome Banding

Nearly all methods of chromosome banding rely onharvesting chromosomes in mitosis. This is usuallyachieved by treating cells with tubulin inhibitors, such ascolchicine or demecolcine (colcemid), that depolymerizethe mitotic spindle and so arrest the cell at this stage.Excessively long incubations with Colcemid result inovercondensed chromosomes that band poorly and more-over some cell types, especially those from the mouse,eventually escape theColcemid block and proceed throughthe cell cycle.Chromosome banding methods are either based on

staining chromosomes with a dye or on assaying for aparticular function. The most common methods of dye-based chromosome banding are G- (Giemsa), R- (reverse),C- (centromere) and Q- (quinacrine) banding. Bands thatshow strong staining are referred to as positive bands;weakly staining bands are negative bands. However thestaining patterns are not black and white, dierent bandsstain to dierent intensities (Francke, 1994). G-positivebands are usually just called G-bands and likewise for R-positive (R-) bands. Positive C-bands contain constitutiveheterochromatin. Q-bands are considered equivalent toG-bands.Themost widely used function-based bandingmethod is

replication banding and is based on the fact that dierentbands replicate theirDNAat dierent times during Sphaseof the cell cycle. Generally, R-band DNA is replicatedearlier thanG-bands (Dutrillaux et al., 1976).G-bands alsocorrespond to the condensed chromomeres of meioticchromosomes and R-bands to the interchromomericregions.

Uses of Chromosome Banding

G- andR-banding are themost commonly used techniquesfor chromosome identication (karyotyping) and foridentifying abnormalities of chromosome number, trans-locations of material from one chromosome to another,and deletions, inversions or amplications of chromosome

segments. This has had an invaluable impact on humangenetics and medicine and the power of this approach hasbeen augmented by combining cytogenetics with uores-cence in situ hybridization (FISH). The detection ofchromosome deletions associated with disorders, veryoften contiguous gene syndromes, provided some of therst disease gene localizations in humans. Similarly,translocations have been important in pinpointing thelocation of disease-associated genes and the characteristictranslocations associated with some leukaemias is impor-tant, not only for understanding the molecular basis ofthese cancers, but also for their diagnosis and prognosis.One of the best examples of this is the translocationbetween human chromosomes 9 and 22 t(9;22)(q34:q11) or the Philadelphia chromosome diagnostic of chronicmyelogenous leukaemia (CML).Comparisons of chromosome banding patterns can

conrm evolutionary relationships between species andalso reveal changes in karyotype that may have beenimportant in speciation. The banding patterns of human,gorilla and chimpanzee chromosomes are almost identical,though human chromosome 2 is the result of a fusionbetween two great ape chromosomes. There are alsoextensive similarities between human chromosome bandsand those of lower primates.

Number and Size of Bands

Idealized diagrams (ideograms) of G-banded chromo-somes are published as standard reference points forchromosome banding. The G-bands are usually portrayedin black and the R-bands in white. Bands are numberedconsecutively away from the centromere on both the short(p) and long (q) arms (Figure 1). The total number of bandsor resolution in the human karyotype depends on howcondensed the chromosomes are, and at what stage ofmitosis they are in. A 350-band resolution corresponds tochromosomes late in metaphase. High-resolution ideo-grams (approximately 12502000 bands) have also beenproduced for human chromosomes in mid-prophase

Article Contents

Secondary article

. Methods of Chromosome Banding

. Uses of Chromosome Banding

. Number and Size of Bands

. Basis for G-/R-banding

. BandsReflect the Domain Organizationof the Genome

. Extreme Chromosome Bands

1ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

(Yunis, 1981). When a low-resolution band is subdivided,the number of each subband is placed behind a decimalpoint following the rst band designation. For example themost distal low-resolution bandon the short armof humanchromosome 11 (11p15) can be subdivided into bands11p15.1, 11p15.2, 11p15.3, 11p15.4 and 11p15.5 at higherresolution (Figure 1). A 2000-band resolution chromosomeband may contain 1.5Mb of DNA, while a 300-bandresolution band will contain 710Mb of DNA. A skilledcytogeneticist may be able to spot a deletion of 510Mb ofDNAdependingon its location, but at amolecular level thehuman genome probably comprises 4 2000 bands.

Basis for G-/R-banding

G-banding involves staining protease-treated chromo-somes with Giemsa dye and is thought to result frominteractions of both DNA and protein with the thiazineand eosin components of the stain. The most common R-bandingmethod involves heat denaturing chromosomes inhot acidic saline followed byGiemsa staining. Thismethodis thought to preferentially denature AT-rich DNA and to

stain the under-denatured GC-rich regions. T-bandingidenties a subset of R-bands the most intensely stainingones by employing either a more severe heat treatmentthanR-banding. It is thought to identify theGC-richest R-bands, of which approximately half occur at telomeres inthe human genome, hence the name.The need to combine chromosome banding with

uorescence in situ hybridization has meant that bandingtechniques using uorescent dyes has become morepopular. Q-banding involves staining with quinacrinewhich reacts specically with certain bases. Quinacrineintercalates into chromosomal DNA irrespective ofsequence, but uoresces brighter in regions of AT-richDNA. There are a number of other molecules whoseuorescence is inuenced by the base composition of theDNA to which they are bound. In addition to quinacrine,other commonly used uorochromes with a specicity forAT-rich DNA include Hoechst 33258, DAPI (4-6-diamidino-2-phenylindole) and daunomycin. The uores-cence of Hoechst and DAPI is not quenched by guanineand so they give less distinct bands than those produced byquinacrine; however, daunomycin uorescence is greatlyquenched by DNA with a GC content of 4 32%. DAPIstaining has the advantage that it is very resistant to fadingand that its excitation and emission spectra are compatiblewith reportermolecules andlters commonly used inFISH(Figure 2).Fluorochromes with a preference for GC-rich DNA

include chromomycin and 7-amino actinomycin D. Thesedyes give an R-band-like pattern.

Bands Reflect the Domain Organizationof the Genome

Techniques used to reveal chromosome bands enhance aninherent pattern of chromosome organization. A chromo-some band is a manifestation of a chromatin domain withfunctional and structural characteristics that are homo-geneous and distinctive over a long enough stretch to beseen down the microscope. G- and R-banding reectdierences in chromatin structure and base compositionbetween dierent regions of the genome. Fluorochromebanding also reects variation in base composition alongchromosome length.A rather dierent sort of banding is that seen after

staining of polytenized (interphase) chromosomes fromsome tissues ofDipteran insects. The basis for this bandingis not well understood but arises through the alignment, inregister, of many thousands of copies of the chromosome.

Patterns in DNA sequence

Banding patterns can arise as a consequence of dierencesin the DNA sequence along chromosomes. R- and

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Figure 1 G-band ideograms of human chromosome 11 at (from left toright) 350, 550 and 850 band resolution.

Karyotype Analysis and Chromosome Banding

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

G-banding patterns are revealed on human chromosomesby FISHwithAlu and LI interspersed repeats, respectively(Korenberg and Rykowski, 1988). A similar distributionhas also been reported for SINEs (short interspersedelements) and LINEs (long interspersed elements) onmouse chromosomes. However, molecular studies of thehuman genome including sequencing show that both SINEand LINE repeats can be found in close proximity to eachother. To reconcile these dierences it is suggested that theSINEs located in R-bands are those that have retroposedmost recently and hence are closest in sequence to theprogenitor copy. These will therefore hybridize better tothe SINE probes that are based on the consensus repeatsequence than to those that are more diverged, and hencewill produce stronger FISH signals than their G-bandcounterparts.Fractions of DNA of diering base composition

(isochores) produce banding patterns on human meta-phase chromosomes with the most GC-rich fractions

highlighting T-bands (Saccone et al., 1993). Molecularanalyses on a ner scale reveal that, although there is ageneral tendency forG-bands tobequiteAT-rich,R-bandscan contain both AT-rich isochores and GC-rich iso-chores.A fraction of DNA (CpG islands) that contains the 5

ends of approximately 50% of mammalian genes alsoproduces a T- and R-banding pattern on chromosomestelling us that genes are not uniformly distributed along thechromosomes length. Most islands appear to reside in T-and R-bands (Craig and Bickmore, 1994) (Figure 3). Asimilar clustering of CpG islands into early-replicating R-band compartments is seen in rodent genomes (Cross et al.,1997). Chromosomes of a nonmammalian vertebrate (thechicken) show a striking concentration of CpG islands indistinctive parts of the karyotype themicrochromosomes rather than on the macrochromosomes.Examination of genome databases suggests that CpG

island clustering can be extrapolated to gene density itself.

Figure 2 DAPI-stained chromosomes from mouse embryonic stem cells (male). The staining approximates to a G-band pattern, with the C- and G-bandsstained more intensely by DAPI than the R-bands. The constitutive heterochromatin around the centromeres of these acrocentric chromosomes stains veryintensely with DAPI.



Hence the highest densities of genes in our genomes arelocated in the T- and R-bands. This explains why humanchromosomes with a high G-band content, e.g. 13, 18 and21, are seen as viable trisomies in the population, whereastrisomies for small but T-/R-band-rich chromosomes (e.g.chromosome 22) are lost early in embryonic development.Whole genome sequencing has conrmed the low genedensity of human chromosome 21 compared to that of 22(Dunham et al., 1999; The Chromosome 21 Mapping andSequencing Consortium, 2000).There has been a conservation of chromosome organi-

zation at the chromosome band level in terms of relativegene density, time of DNA replication and bandingcharacteristics over 100 million years of mammalianchromosome evolution. This suggests either the inuenceof a strong selective pressure to maintain these character-istics together in particular chromosome bands or the

action of common mechanisms linking the properties ofgene density, replication time and band type.

Patterns in DNA replication

Dierent regions of the genome replicate at dierent timesduring S phase. The relationship between timing ofreplication and chromosome banding is usually studiedby incorporating pulses of the thymidine analogue 5-bromo-2-deoxyuridine (BrdU) into cells during denedstages of S phase and then examining chromosomes in thesubsequentmetaphase. Sites of BrdU incorporation can bedetected with antibodies that detect the presence of BrdUin denaturedDNA (Figure 3). T-bands replicate on averageearlier than ordinary R-bands, and DNA in G-bands isreplicated even later (Dutrillaux et al., 1976).

Figure 3 The location of CpG islands in the human karyotype. Left-hand chromosome of each set: CpG islands (red) painted by FISH onto humanmetaphase chromosomes that have been replication banded so that the G (late replicating)-bands are green (sites of BrdU incorporation). Right-handchromosome of each set DAPI-stained (blue) chromosomes with a late-replication banding pattern (green).



Aspects of the primary DNA sequence or chromatinstructure in dierent types of band could inuence theirreplication time. Also dierences in replication time couldinuence some characteristics of chromosome bands, e.g.base composition or chromatin structure. Sites of tran-scription at the G1/S boundary may seed the assembly ofthe rst replication factories in early S phase and hence themost transcriptionally active regions of the genome, andthe regionswith the highest concentrations of genes, wouldtend to be the ones to be replicated rst.

Patterns in chromatin structure

Several banding techniques, especially G-banding, suggestthat there are both qualitative and quantitative variationsin the interaction of DNA and proteins along the length ofmetaphase chromosomes. The chromatin of active genes isgenerally considered to be more accessible to nucleaseattack than is inactive chromatin. Consistent with thisnucleases preferentially digest R-bands and T-bands ofintact mitotic chromosomes, with G-bands and C-bandsrefractive to digestion. The extent of chromatin packagingin the interphase nucleus also diers between chromosomebands. C-band positive heterochromatin remains visiblycondensed through interphase. FISH has shown that overthe 150 kb to 1Mb size range G-band chromatin is moretightly packaged than that of R-bands.The N-terminal tails of core histones H3 and H4 can be

modied by acetylation of lysine residues. A consequenceof this acetylation may be to facilitate access of proteinssuch as transcription factors to the DNA. Histones can bedeacetylated or acetylated throughout the cell cycle bynuclear acetyltransferase and deacetylase enzymes. Im-munouorescence of mammalian metaphase chromo-somes with antibodies raised against each of theacetylated forms of H4 has shown that the dierentlymodied forms are found preferentially in dierent regionsof the chromosome.Mono-acetylated (Lys16) H4 is foundthroughout euchromatin, whereas acetylation at Lys8 andLys12 occurs mainly in R-bands. Acetylation at Lys5 isfound in the most highly modied (tri- and tetra-acetylated) forms of histone H4. Antibodies to this H4isoform produce a good banding pattern on metaphasechromosomes, especially from cells that have been brieyexposed to histone deacetylase inhibitors. Bright immuno-uorescence is seen over T-/R-band regions of thekaryotype and only faint staining is seen in G-bands(Jeppesen and Turner, 1993). Hence the distribution ofhistone acetylation on mammalian metaphase chromo-somes mirrors that of genes.The radial loop/scaold model of chromosome struc-

ture proposes that higher order chromosome packagingarises through the arrangement of the 30-nm chromatinbre into loops tethered to a proteinaceous chromosomescaold that runs centrally down the length of the

chromosome. The two major protein components of themitotic scaold are topoisomerase IIa Sc I (170 kDa) andSc II (135 kDa). Sc II is a member of the structuralmaintenance of chromosomes (SMC) family of proteins.Immunouorescence analysis shows that topoisomerase

IIa is not uniform along the chromosome length. It isparticularly concentrated in regions of centromeric hetero-chromatin and there is stronger staining of G-bands thanR-bands. This might indicate that chromosome loopanchoring sites to the scaold are the least frequent in R-bands. The pattern of immunouorescence with anti-ScIIantibodies on vertebrate mitotic chromosomes is verysimilar to that of topoisomerase II the chromosome axesare lit up and centromeres are particularly brightly stained.Specic sites along the DNA associate with the

chromosome scaold. Such sequences are generallyreferred to as SARs (scaold attachment regions). Theycontain oligo(dA)-oligo(dT) tracts and uoresce brightlywith daunomycin. The path of aligned SARs along thescaold at the core of metaphase chromosomes, as denedby daunomycin staining is G-band like and colocalizeswith topoisomerase II immunouorescence (Saitoh andLaemmli, 1994). This might mean that G-bands havesmaller loops, and hence more frequent scaold attach-ments thanR-bands and chromosome painting with SARssuggests that there are indeed more of these in G-bandsthan in R-bands (Craig et al., 1997). However, thesestaining patterns could also result from dierences in thepath of the SARs between dierent types of band, with thechromosome scaold being more tightly coiled within G-bands and straighter within R-bands.Variations in the density of meiotic chiasmata along

mammalian chromosomes are apparentwhen physical andgeneticmaps are compared. T-bands have the highest ratesof exchange, followed by mundane R-bands, then G-bands. Heterochromatin (C-bands) shows the lowest ratesof recombination. R-bands are both the sites of synapticinitiation and the preferred sites of crossing-over inmammals and other vertebrates.

Evolution of chromosome bands

Whereas Q-, G- and R-banding patterns have only beenobserved in some eukaryotes, replication banding is almostuniversal among living organisms possessing chromo-somes large enough to see bymicroscopy, suggesting that itis a fundamental consequence of, or requirement for, thecompartmentalization of complex genomes.Chromosomes frommost mammals and birds can beG-

and R-banded. In addition, most reptilian chromosomesband with G- and R-banding techniques to some extent.With amphibia, sh and plants, some species bandwhereasothers do not. The lowest vertebrates with reported goodG-banding are the bony sh.



Evolutionary analysis of chromosome banding patternssuggests that the rst cytogenetically detectable compart-mentalization that arose in the genomes of eukaryotes wasthe temporal control of replication and dierences inchromatin packaging and the segregation of some chro-mosomal domains into heterochromatin. Ability to be G-banded (and we will assume here that this is a reection ofdierences in chromatin structure on mitotic chromo-somes) followed later. Fluorochrome banding seems tohave appeared on the scene last of all.

Extreme Chromosome Bands

All groups of eukaryotes that have chromosomes largeenough to be visualized contain a proportion of hetero-chromatin, visualized by C-banding, and even simpleeukaryotes with microscopic chromosomes have hetero-chromatin at a molecular level. It seems unlikely that thereis a completely strict dividing line between heterochroma-tin and euchromatin wemight consider the very gene-richT-bands and gene-free constitutive heterochromatin as theopposite extremes of a continuum of chromatin avours.In humans, the main C-bands are on the long arm of the

Y-chromosome and close to the centromeres of chromo-somes 1, 9 and 16 (pericentric). The size of these C-bandsdiers between dierent individuals. Smaller C-bands arefound at the centromere of each chromosome and on the parms of the ve acrocentric chromosomes (13, 14, 15, 21,22). In the mouse, the main blocks of visible heterochro-matin are found close to the centromere of each chromo-some (Figure 2). The amount of heterochromatin at thesesites also varies between dierent strains ofMus musculus.InDrosophila the main sites of constitutive heterochroma-tin are found at the chromocentre, telomeres and on thefourth/Y-chromosome. At the molecular level all of thesesites are characterized by the presence ofmiddle and highlyrepetitive sequences, often in tandem arrays of satellites.Classical satellites (I, II and III) locate to the pericen-tromeric C-bands of human chromosomes 1, 9, 16 and thelong arm of the Y; a-satellite is located at the centromeres.It is the minor satellite repeat that is found at mousecentromeres, with major satellite being found beyond this,between the centromere and telomere. In Drosophila,satellite DNA at the chromocentre is interspersed withstretches of more complex DNAs (both unique andmoderately repetitive DNA, including transposable ele-ments).Heterochromatin is normally associated with repression

of transcription and recombination, and with late replica-tion. In Drosophila, the constitutive heterochromatin isunderrepresented in polytene chromosomes. No endogen-ous genes or CpG islands have yet been mapped to regionsof C-banded constitutive heterochromatin in the humanormouse genomes, and transgenes that integrate close to

constitutive heterochromatin are frequently silenced. Afew endogeneous genes in Drosophila are found in aheterochromatic location and moreover they cease tofunction when moved to euchromatic locations.C-band-positive constitutive heterochromatin has a

distinctive chromatin structure. It is the site of mostmethylation in human andmouse chromosomes and so the5MeCpG-binding protein MECP2 is highly concentratedover constitutive heterochromatin in vertebrates in mostcell types. Constitutive heterochromatin is also associatedwith very low levels of histone acetylation; with theexception that the chromocentre of Drosophila is enrichedin histones acetylated at Lys12, and that in mammalianembryonic stem cells prior to dierentiation histone H4within the pericentromeric heterochromatin is acetylated.A set of distinctive chromosomal proteins can also befound concentrated in heterochromatin. Many of theseproteins have motifs, such as the chromobox, that arecharacteristic of proteins involved in the formation ofmultiprotein heterochromatin complexes. Antibodiesagainst M31, a chromobox containing protein that is ahomologue of Drosophila heterochromatin protein HP1,highlight the pericentric heterochromatin of mouse andhuman chromosomes.

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Craig JM, Boyle S, Perry P and Bickmore WA (1997) Scaold

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Further Reading

Craig JM and Bickmore WA (1993) Chromosome bands avours to

savour. BioEssays 15: 349354.

Craig JM and Bickmore WA (1997) Chromosome Bands: Patterns in the

Genome. Heidelberg: Springer-Verlag.

Gosden JR (1994) Chromosome analysis protocols. In: Walker JM (ed.)

Methods in Molecular Biology, vol. 29. Totawa: Humana Press.

Mitelman F (ed.) (1995)An International System for Human Cytogenetic

Nomenclature. Basel: S Karger.

Sumner AT (1990) Chromosome Banding. London: Unwin Hyman.



band methods 2

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