Chromosome banding (C banding)

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Types of bandings

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Chromosome Banding techniques (C banding):

Chromosome Banding techniques (C banding) Mahantesh Ist year Phd PALB 4054

CONTENT :

CONTENT

INTRODUCTION :

INTRODUCTION Analysis of human and plant chromosomes For identification of chromosome that differs morphologically Banding techniques are based on identification of chromosomes segment that consists of rich AT and GC region First step in this is denaturation followed by slow renaturation During slow renaturation the heterochromatin region can be identified which consists of repetitive DNA sequences

History :

History In 1968, Casperson first published paper describing the use of quinacrine mustard to stain the chromosome and there by opened the era of chromsome banding Vicia faba The Paris Conference (1971) was the first effort to give nomenclature for chromosome banding for various species and thus adopted for non-human species as well.

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Paris Conference in 1971 defined Chromosome bands as A part of chromosome which is clearly distinguishable from its adjacent segment by virtue of its darker or lighter staining ability Region defined as area of chromosome lying between two adjacent bands

Chromosome banding :

Chromosome banding The artificial production of such bands by treatment with specific dyes is referred to as chromosome banding The pattern of chromosome banding is highly specific in each chromosome of a species Thus it is useful tool for identification of chromosomes

Factors or Causes of occurrence of Bands :

Factors or Causes of occurrence of Bands Occurrence of repetitive DNA Difference in base composition of DNA Differences in the protein component Differences in the degree of packing of DNA complex

Karyotype Analysis through Banding :

Karyotype Analysis through Banding Three key features used in karyotyping Chromosome size or length : This is the easiest way to tell two different chromosomes apart. Banding pattern : The size and location of bands on chromosomes make each chromosome pair unique. Centromeric position : Centromeres are regions in chromosomes that appear as a constriction.

Nomenclature of Chromosome bands :

Nomenclature of Chromosome bands Nomenclature based on Paris Conference, 1971 and International System of Human Cytogenetic Nomenclature require following information Arm symbol Region number Band number within region Chromosome number Genome Designation

ISCN:

ISCN International System for Human Cytogenetic Nomenclature Each area of chromosome given number Lowest number closest (proximal) to centromere Highest number at tips (distal) to centromere

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Nomenclature : p (peptic) arm = short arm; q=long arm Bands numbering proceeds outwards form the centromere e.g. bands p11 or p23

Band and Region number:

Band and Region number Lowest number given nearer to centromere (proximal) Highest number given distal end from centromere Chromosome bands given number (1-9) If number exceeds 10 chromatids divided into two parts and each part numbered serially Eg , P11 and q23

Karyotyping :

Karyotyping Mainly five types of banding techniques were used karyotype analysis Q Banding – Quinacrine Mustard G Banding – Giemsa stain R Banding – Reverse C Banding – Centromeric (Heterochromatic) N Banding – Nuclear organizer Region

SUB-CLASSIFICATION OF BANDING METHODS ISCN (1985):

SUB-CLASSIFICATION OF BANDING METHODS ISCN (1985) Three letters code to describe banding techniques 1 st Letter – Type of banding 2 nd Letter- General technique 3 rd Letter – The stain E.g. QFM – Q banding by Fluorescence using Quinacrine dye

CONSTANT HETEROCHROMATIC BAND :

CONSTANT HETEROCHROMATIC BAND Visible during interphase and throughout the cell division and relatively constant in size These bands are associated with highly repetitive or satellite DNA in heterochromatic region of chromosomes It is equivalent to C, G11, N band General types of Bands

Facultative Band:

Facultative Band Not distinguishable during interphase varying in size At metaphase they occupy whole chromosome and fusion of band as a result of chromosome contraction and bringing so close to each other These are equivalent to G, Q and R band This cant be produced by any particular type of DNA but can be with meiotic chromosomes where its condensation happen during division Kinetechore : These are pairs of dots presents a centromere and it is defined as Cd band

Q-Banding :

Q - Banding Quinacrine Mustard is a fluorescent and alkyalating agent was first chemical used Caspersson , 1968 (Now Quinacrine dihydrochloride ) Quinacrine intercalate with chromosome DNA ( alkalize Guanine) and quenches for more fluorescence at AT rich region Fluorescence of Quinacrine indication of A-T richness of DNA Bright Bands – A-T rich region Light Bands – G-C rich region

G Banding:

G Banding Drets and Shaw, Patil et al ., Seabright and Sumner et al ., independently developed in 1970 for animal species G bands defined as chromosomal staining techniques which gives darker and lighter bands along the euchromatic part of the chromosome Stain darker at A-T rich and lighter at G-C rich region

R Banding :

R Banding Developed by Dutrillaux and Lejeune in 1971 Mild denaturation by heat and subsequent staining with Giemsa revealed a banding pattern reverse to G banding Whereas mild denaturation with flurochrome dye ( acridine orange/ olivomycin ) produce fluroscent R banding reverse to Q banding The R bands fluorscent bright green and the non-R bands show a faint red colour R banding accomplished by heat treatment (85°C) for 5-10 minutes in physiological saline solution A-T richer DNA denatures and reduces affinity with Giemsa

C Banding :

C Banding Pardue and Gall independently developed in 1970 for staining of constitutive heterochromatin in mouse C banding located next to centromere region (Sat DNA) in many species like Drosophila, Guinea Pig rat etc. C bands located next to the centromeres and secondary constrictions of chromsomes 1,9 and 16 in man ( Arrighi and Hsu, 1971)

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Differential staining involves heat/alkali/acid treatment to denature the cellular DNA and based on principle of DNA renaturation kinetics The denatured repetitive DNA of heterchromatin renatures under prescribed conditions faster than unique DNA Such opportunity used for differential staining

Variants of C banding:

Variants of C banding CT Band Cd Band Centromeric -telomeric bands Centromeric dot bands Bands are located near telomeric region For each chromatin two dots Used in animals and plants Used in human beings

C Banding Protocol :

Pre-treatment of roots : 24 hours with cold water and 3 hour with Colchicine (0.05%) Fixation : Ethanol (99%) and Glacial Acetic Acid in 3:1 for three days in room temperature Acetic Acid (45%) treatment : 2-3 minutes and squashed on slides Ethanol (99%) treatment : few minutes and air dried C Banding Protocol

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Incubation: 0.2 M HCl at 60° C for 2 minutes on water bath and rinse with distill water Saturated barium hydroxide solution for 7 minutes at room temperature 2 x SSC buffer (0.3M Nacl+0.03M Trisodium citrate) solution for 60 minutes at room temperature Staining 1-5% Giemsa stain treatment for 30 minutes, air dried treated with xyline and mounted on paramount

Modified C banding :

Modified C banding Pre treatment of roots : cold water ( 24 hours) Fixation : Ethanol (99%) and Glacial Acetic Acid in 3:1 (3 days at room temperature) Acetocarmine staining : 1% for 2-3 hours Squashing with 45% acetic acid Acetic acid (45%) treatment : at 50° C for 5-10 minutes air dried overnight Hot phospate buffer (1M NaH2PO4) : at 94° C for 2 minutes Saturated Barium Hydroxide treatment : 0.3M Nacl+0.03M Trisodium citrate solution for 3-10 minutes at 50° C Giemsa staining : 15-20 minutes, rinse with water and air dried

Applications of C Banding technique:

Applications of C Banding technique C banding is highly valuable for identification of heterochromatic region of chromosome in plants and animals Highly effective for identification of meiotic choromosomes in mammals Identification of aneuploids , translocation and other structural rearrangement

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Useful for precise physical mapping Phylogenetic conclusion of interspecific and intergeneric hybrids Very useful in identification of bivalents and centromere position in diakinesis

Identification of Monosomics in oat by C banding:

Identification of Monosomics in oat by C banding Oat x maize hybridization produces large number of monosomics and structural aberrations ( Jellen et al., 1997) Comparison of C band and modified C band with previously described karyotype able to identify missing chromosome and structural aberrations Cytological screening observed 18 new monosomics and 3C-14 reciprocal translocation

C Banding in Maize:

C Banding in Maize A biometrical analysis of C-banded and Feulgen stained somatic metaphases of maize stocks with different knob Constitutions has shown that large bands alter arm lengths of mitotic chromosomes. A representative diagram of the knobless maize somatic karyotype and of a high knobbed stock are presented Margaride and Vosa , 1990

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Figure 1 C-banded mitotic metaphase in stock 18 of the Zapalote Chico race. The dark stained bands correspond to knobs. Knobs at 6L 2 and 6L 3 and 8L 1 and 8L 2 in the pachytene stage appear as single bands in metaphase chromosomes. Margaride and Vosa , 1990

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Figure 2: C-banded mitotic metaphase in the hybrid 18 x 371 A-4 Margaride and Vosa , 1990

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Diagrammatic representation of the C-banded karyotype of (a) Zapalote Chico (stock 18) and (b) of the basic maize somatic karyotype without bands obtained from Ceremonial maize data. Margaride and Vosa , 1990

Conclusion:

Conclusion The representative stock of Zapalote Chico used in this study, can be considered as illustrative of band positions in maize somatic chromosomes and may be useful in the future standardization of maize somatic cytogenetics Margaride and Vosa , 1990

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Fig. 1: Somatic cell of the barley variety ‘Emir’ with 14 weit -spread, Giemsa C-banded metaphase chromosomes. Scale: 10µ Linde , 1995

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Fig. 2. Giemsa C-banded barley karyogram . The homologous chromosomes (nos. 1 to 7) of ‘Emir’ barley arranged pairwise according to their banding patterns. Scale: 10 µ Linde , 1995

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Fig. 3. ldiogram of the chromosomes of barley showing relative sizes and positions of C-bands (solid regions) in the variety ‘Emir’. The relative lengths and arm ratios of the chromosomes are from TULEEN (1973) (chromosomes 1 to 3) and TJIO and HAGBER(1951) (chromosomes 4 to 7). Linde , 1995

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Chromosome Description 1 Centromeric bands of medium size in both arms, the band in the short arm being the larger. In the short arm one fairly small proximal band. 2 In the short arm two proximal bands, the one close to the centromere being small. In the long arm two proximal bands, the one closest to the centromere being the larger, and a very small distal band. 3 In the short arm a fairly large centromeric band and close to it a rather small band. In the long arm a medium-sized band near the centromere and a fairly small band. 4 The most heavily banded chromosome of the complement. Large centromeric bands, the one in the short arm the largest band of any barley chromosome. 5 The short arm has a medium-sized centromericband . In the long arm one small band at or near the centromere and two medium-sized interstitial bands, one being subterminal . 6 The short arm has a fairly large centromericband and close to it a fairly small band. At the satellite a large band. The long arm has two small bands, one centromeric and one close to it. 7 Two large centromeric bands. The short arm further carries a very small proximal band and a band at the secondary constriction Linde , 1995

Conclusion:

Conclusion Each of the seven barley chromosomes has its distinctive Giemsa C-banding pattern thus allowing identification. Interstitial bands are present in most, and terminal bands in all chromosome arms. The squashing method employed, resulting in well-spread, well-flattened, complete metaphases, is considered essential for obtaining successful C-banding. Linde , 1995

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Fig. 1. Giemsa banding pattern variants of the chromosomes of twenty barley lines. Figure-cum-capital letter refers to a banding pattern variant of a chromosome. Broken lines indicate centromere position Linde , 1995

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Fig. 2. Drawing of the banding pattern variants of the chromosomes of twenty barley lines. Figurecum -capital letter refers to a banding pattern variant of a chromosome. Relative lengths and arm ratios of chromosomes according TULEEN(1973) (chromosomes 1 to 3) and TI10 and HAGBERG (1951) (chromosomes 4 to 7). Banding pattern of chromosome 1 reversed relative to LINDE-LAURSEN (1995). Linde , 1995

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Fig 1: Giemsa C-banding of rye chromosomes. The seven chromosomes are, from left to right, 1R to 7R. Fig 2: Giemsa C-banding of two B-chromosomes in a somatic cell of rye. The arrow denotes the centromeric position. Fig 3: Heteropycnotic regions stained by Giemsa in rye somatic- interphase nucleus. Gill and Kimber , 1990

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Figure 1.Leishman C-banding of Vigna vexillata ( TVNu 73). Adetula et al., 2005

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Figure 2. Idiogram of C-Banded chromosomes of V. vexillata TVNu 73 . Adetula et al., 2005

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Chromosome no. Type of bands 1 Dark centromeric band 2 Interstitial band on short arm And terminal band on long arm 3 Centromeric band Terminal band on short arm 4 Centromeric band 5 Two centromeric band 6 Telomeric band on short arm 7 Telomeric band on short arm 8 Centromeric band 9 Telomeric band on short arm 10 Telomeric band on each of long and short arm 11 Telomeric band on short arm Adetula et al., 2005

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Fig. 1. C-banding karyotypes of (a) Arrhenatherum parlatorei GRA 633/93, (b) A. thorei GRA 579/94, (c) A. album PI 254870 (Iraq) and (d) A. elatius PI 251946 (Austria). Crystal et al., 2005

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