Journal of Biological Sciences and Medicine
Journal home page: www.jbscim.com
∗ Corresponding author
Email address: r_24ras@rediffmail.com (Dr. Rashmi Srivastava)
11
Review Article
Open Access
Understanding heterochromatin characterization through chromosome
banding
Rashmi Srivastava* and Nidhi Mishra
Department of Zoology, University of Allahabad, Allahabad- 211002, India
Received 3 December 2015; Accepted 17 December 2015; Available online 31 December 2015
Abstract
Since the late 1920s, after its finding as the condensed region of chromatin, heterochromatin has been the subject of interest and widely explored and analyzed for unrevealing its characteristics and functional importance. The advent of several chromosomal banding techniques by the time made this task heterochromatin characterization very simple. These can be enlisted as C-banding, G-Banding; some fluorescence banding techniques such as Q-banding; R-Banding, AgNOR banding and other advanced methods that renders higher resolution to the heterochromatin on the pretreatment to chromosomes with DNA ligands and restriction endonucleases. The molecular cytogenetic methods including fluorescence in situ hybridization and its variations flourished later enhanced the capacity to understand the heterochromatin constitution more specifically at gene level. In this review, a description of various banding techniques used for chromosomal characterization of heterochromatin has been provided with a special emphasis to elucidate the components of eukaryotic heterochromatin.
Keywords: Heterochromatin; Chromosome banding; Giemsa; FISH
Introduction
The whole genetic content of a cell resides into the nucleus where it remains packaged into the thread like structures containing certain histone proteins and various non-histone proteins. These structures have an affinity with colour and known as chromatin bodies as the chromatin word is derived from the latin word = chroma means colour (Flemming 1880a; Waldeyer, 1888). After staining with giemsa, chromatin shows a species specific light and dark colour pattern representing decondensed and condensed regions of the chromosome respectively. The decondensed region was further termed as euchromatin and condensed region which stayed condensed through-out the cell cycle as heterochromatin by Hietz (1928). He
propounded that euchromatin is genicly active, and heterochromatin is genicly passive i.e., the heterochromatic chromosomes contain no genes or somehow passive genes (Heitz 1929). Later, this idea proved to be one of the foundations of several pioneering works in the field of chromatin research. Heterochromatin was further subdivided into constitutive and facultative heterochromatin.
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that are silenced during the course of cell cycle (Gall et al. 1971; Gatti and Pimpinelli 1992; Elgin and Reuter 2013). In several organisms, heterochromatin is found at the telomeres and in domains enclosing the centromeres (Alberts et al. 2002). In addition, some species specific heterochromatin regions have also been well-known, for instance the silent-mating-type loci in yeasts and the inactive X chromosome in female mammals (Miele et al. 2009; Heard and Disteche 2006).
Several cytological techniques have been utilized time and again for the characterization of heterochromatin. In the present review, an attempt has been made to summarize the selected banding techniques being used for the identification and characterization of heterochromatin both at cytological and at genetic levels.
Constitutive Heterochromatin
The constitutive heterochromatin is repetitive in nature and forms major constituents of centromeres and the telomeres (White 1973; Bedo 1975; López-Fernández et al. 2004). Constitutive heterochromatin contains satellite DNA, which consists of large numbers of short tandemly repeated sequences such as Alpha- satellite DNA, DNA satellite I, II and III which have an important role in the formation of the highly compact structures of the constitutive heterochromatin (John and Miklos 1979). The constitutive heterochromatin is stable in nature and conserves its heterochromatic properties throughout all stages of development during the cell cycle (Higgs et al. 2007); highly polymorphic because of the instability of the satellite DNA, and this polymorphism may affect the size and localization of the heterochromatin with no apparent phenotypic outcome (Miklos and John 1979). Constitutive heterochromatin is intensely
stained and shows a very high affinity to C-banding technique which is the consequence of the very rapid renaturation of the satellite DNA following denaturation (Hsu 1973). In many organisms, the centromere is embedded in a section of special centric heterochromatin that perseveres throughout the interphase, contains a centromere- specific variant H3 histone CENP-A (centromere protein A) and additional proteins that pack the nucleosomes into particularly dense arrangements forming the kinetochore, required for the attachment of the mitotic spindle (Alberts et al. 2002).
Facultative Heterochromatin
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chromatin structures (Lyon 2003). Unlike the constitutive heterochromatin, the facultative heterochromatin is not rich in satellite DNA, non-polymorphic and negatively stained by C-banding (Hsu 1973).
Chromosome Banding for
heterochromatin characterization
The condensation of chromosomes occurs at the metaphase state of the mitotic cycle. The higher order organization of chromosomes constitutes the DNA and proteins which are important genetic end points. Several imaging procedures have been used to read out the chromosomes- for instance light microscopy, fluorescence microscopy and other advanced imaging tools involving fluorescence in situ hybridization (Sumner 1973; Schweizer 1976a, 1976b; Maltempi and Avancini 2001). Different staining techniques facilitate the suitable visualization of chromosomes with the help of these above mentioned imaging techniques that increases the resolution of chromosomes accordingly. The staining procedures have been immensely developed in the past decades and help to study the karyotype of multicellular organisms (Alberts et al. 2002). The applicability of staining methods have improved more with the practice of certain treatments and give rise to a distinct pattern known as banding, producing differential staining along the length of a chromosome hence provide a more specific chromosomal study. Banding techniques provide a better identification of chromosomes because each chromosome is specific and produces distinctive bands from other (Gustashaw 1991).
A plentiful of stains have been used that bind to chromosomes, such as some visible light dyes like Giemsa and Orcein being used in the light microscopy, whereas fluorochromes like the Quinacrine, DAPI, Hoechst, Ethidium bromide etc. used in the
fluorescence microscopy. Most of these stains commonly are the organic compounds having large aromatic groups that bind or intercalate to major or minor grooves of the DNA of chromosomes (Holmquist 1975).
Heterochromatin is an important fragment in the study of genomes of several distinct organisms. Hence, the characterization of heterochromatin of different organisms is one of the intriguing aspects in cytological analysis, owing to which different methods have been developed time and again to characterize them. For example, C-banding, G-Banding, some fluorescence banding techniques such as Q-banding, R-Banding, AgNOR banding, advanced banding methods which provide higher resolution including DNA ligands and restriction endonucleases. However, with the course of time, molecular cytogenetic methods like fluorescence in situ hybridization succeeded as a more specific characterization method of heterochromatin at gene level. Some other advancements include the multicolour FISH, spectral karyotyping etc. Certain banding techniques or procedures used for the characterization of constitutive and facultative heterochromatin of the eukaryotic organisms ranging from dipteran insects, plants and humans are summarized in the Table 1.
It is important to note that all these banding techniques being used for chromosomal analysis depends upon the stages of cell cycle which promises of the resolution of the bands rendering to the chromosome condensation. An overview of these banding procedures used for the characterization of heterochromatin is as follows-
C-Banding
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repetitive DNA at the centromeres of the chromosomes, replicates late in the cell
cycle than other chromatin and shows special characteristics of stability under
Table 1: Different banding procedures utilized for the characterization of heterochromatic
regions of the chromosomes
Banding techniques
Stains/compounds/ enzymes
Chromosomal regions References
CH FH Euchromatin
C-banding Giemsa/ Trypsin + +/- - Kaul et al. (1978, 1989); Tewari et al. (1986); Maltempi et al. (1996); Maltempi and Avancini (2000) G- banding Giemsa + + - Maltempi and
Avancini (2000) Q-banding Quinacrine mustard + +/- - Kaul et al. (1978,
1989) LIB Quinacrine mustard/
methylene blue
+ +/- - Bultmann and Mezzanotte (1987); Srivastava et al. (2013)
R-banding Chromomycin A3 or Olivomycin
- - + Schweizer (1976a); Schmid and Guttenbach (1988) N-banding Giemsa/ Silver nitrate + + *** Kaul et al. (1978,
1989) Replication
banding
DAPI/ Hoechst 33258, Ethidium bromide, AO, Actinomycin D,
Mithramycin
+ + - Kaul et al. (1978, 1989); Rai et al. (1985); Tewari et al. (1988)
Counter- staining
DAPI/Actinomycin D + + - Agrawal et al. (2010)
Restriction endonuclease banding
Giemsa/Hind III, Alu I, Hae III, Mbo I, Hpa II, Dpn I, Msp I, Hinf I
+ +/- ** Marchi and
Mezzanotte (1990); Martinez et al. (1994)
FISH Propidium
iodide/ antibiotin/ RAG-FITC
+ - *** Maltempi and Avancini (2000)
FISH= fluorescence in situ hybridization; CH= Constitutive heterochromatin; FH= Facultative heterochromatin; C = C- banding; Q= Quinacrine; G = Giemsa; DAPI = 4'-6-diamidino 2-phenyl indole; H = Hoechst 33258; N= N- banding; LIB=Light Induced Banding; AO=Acridine Orange; RAG-FITC = Rabbit Anti-Goat IgG and fluorescein isothiocyanate; + = presence of band; - = absence of band; +/- = differential staining according to species; **= digestion of euchromatin; ***= euchromatic nucleolar organizer embedded in large blocks of constitutive heterochromatin.
the conditions of heat and chemical exposure (Sumner 1973). This property of tightly condensed heterochromatin is
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and all other chromatin remains pale (Lee and Collins 1977; Maltempi and Avancini 2001). The C- banding shows dark staining and had an affinity towards centromeres, telomere and constitutive heterochromatin.
Q-Banding
Q-banding technique includes the staining of chromosome with fluorescent stain- Quinacrine mustard and imaging in fluorescent microscopy for the study of AT-rich sequences on the heterochromatin of chromosomal regions (Ellison and Barr 1972).The regions of the genome in which the bases adenine and thymine were relatively abundant (AT- rich) produce intense fluorescence, while regions containing abundant guanine and cytosine residues (GC- rich) fluoresced more weakly (Caspersson et al. 1969; Comings et al. 1975). Some other fluorescent stains yield similar banding patterns as that of Quinacrine, such as Hoescht 33258, DAPI (4’, 6’-diamidino-2-phenylindole) and diimidazolinophe- nylindole (DIPI) (Schnedl et al. 1977). The Q banding technique is useful for detecting heteromorphisms, predominantly the chromosomal polymorphism.
G-Banding
The Giemsa (G-) banding was introduced for cytological studies that employed the common stain Giemsa subsequent to various chemical and enzymatic treatments like trypsin to remove chromosomal proteins of the air- dried chromosome preparations (Comings et al. 1973). After G- banding, all the chromosome pairs stains in a characteristic light and dark banding pattern, where the dark bands are termed as G bands that nearly correlate to GC or AT base-pair composition and repetitive DNA sequences. The intense, giemsa stained regions found in G-banded chromosomes moderately
corresponds to intense fluorescent regions of Q-banded chromosomes (Ennis 1974).
R-Banding
R-banding is also denoted as the reverse (R-) banding and produces an opposite pattern when compared to G- or Q- banding. GC specific dyes for instance, chromomycin A3 or olivomycin, which specifically stain the gene-rich chromatin, produces fluorescent reverse banding patterns, thus enhancing the ability to visualize small structural rearrangements in some portions of the genome (Schweizer 1976a; Schmid and Guttenbach 1988).The dark and light bands are reversed when the chromosomes are treated with heat, special chemicals or DNA ligands before staining. Reverse banding is applied to improve and enhance the contrast of chromosome banding for a better visualization of sequence specific regions on chromosome. Sometimes, this banding proves to be an advantage over G and Q bands, by facilitating the differentiation between heterochromatic and euchromatic regions of chromosomes.
Light Induced Banding (LIB)
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Replication banding
Replication banding is also known as high-resolution banding and involves the applicability of a variety of compounds that intercalate into or bind to the DNA molecule, inhibiting condensation of chromosomes in the dividing cells (Hsu 1973; Cornforth and Bedford 1983; Regen et al. 1994). For example, certain DNA ligands like Hoechst 33258, Ethidium bromide, acridine orange, actinomycin D and Mithramycin that characterize heterochromatic areas of chromosomes (Stokke and Steen 1986; Schweizer 1976b; Tomiyasu and Testa 1988; Leemann and Ruch 1978; Schnedl et al. 1977; Bella et al. 1986; Santos and de Souza 1998; Srivastava and Gaur 2015). Using the replication banding method, both R- and G-banded patterns can be obtained for chromosomal analysis (Schmid and Guttenbach 1988).
Restriction endonuclease banding
Restriction endonucleases/enzymes have been found to digest mitotic chromosomes and produce variable patterns of chromatin staining, with the fact that utmost variability is found at heterochromatic regions of the pericentromeric areas (Bianchi et al. 1985; Marchi and Mezzanotte 1990). Restriction endonuclease banding is beneficial in the study of chromosomal polymorphisms of a population, identification of marker chromosomes and the parental origins of individual homologues (Babu 1988; Martinez-Lage et al. 1994; Moore and Best 2001).
Some additional banding procedures
Some other specified chromosomal banding techniques used in the characterization of heterochromatic regions are- NOR or silver staining (active Nucleolar Organizer Regions), Sister Chromatid exchange (SCE),
G11- banding for the localization of pericentromeric region of chromosome 9, distamycin/ DAPI banding (Funaki et al. 1975; Maltempi et al. 1996; Souza et al. 1998; Das and Khuda-Bukhsh 2007; Baydemir et al. 2015).
Fluorescence in situ hybridization
In this techniques, fluorescent labelled DNA probes for specific sequences are hybridized to fixed chromosomes to locate and enumerate the DNA sequences of interest, and thus called as fluorescence in situ
hybridization (FISH). Apart from fluorescent labelling, other labelling methods, both isotopic and non-isotopic are also used and visualized after fluorescent staining in fluorescence microscope for the specific chromosomal characterization (O’Keefe et al. 1996; Liehr 2009; Geurts and de Jong 2013; Pita et al. 2014). The FISH technique is used to visualize the gene enriched chromosomal regions such as the rDNA or the nucleolar organizer regions of chromosomes (Souza et al. 1998; Maltempi and Avancini 2001).
Conclusion
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Conflicts of Interest
All contributing authors declare no conflicts of interest.
Acknowledgements
We would like to thank our teachers and fellow researchers for providing necessary guidance in the course of this study.
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