DNA and DNA and

ChromosomeChromosome

Dr. Dr. ThilakavathyThilakavathy KarrupiahKarrupiah

Function of the genetic materialFunction of the genetic material

•• The genotypic function, The genotypic function, replicationreplication –– store genetic store genetic

information and transmit the information from parents to information and transmit the information from parents to

offspring accuratelyoffspring accurately

•• The phenotypic function, The phenotypic function, gene expressiongene expression –– genetic genetic

material dictate the growth and differentiation of the organismmaterial dictate the growth and differentiation of the organism

•• The evolutionary function, The evolutionary function, mutationmutation –– undergo change so undergo change so

that organisms can adapt modifications in the environmentthat organisms can adapt modifications in the environment

ChromosomeChromosome

Nucleic AcidsNucleic Acids ProteinsProteins

DNADNA RNARNA

Nucleic AcidsNucleic Acids

•• Two types of nucleic acids Two types of nucleic acids –– DNADNA and and

RNARNA

•• Composed of repeating subunits Composed of repeating subunits --

nucleotides nucleotides

•• Each nucleotide is composed ofEach nucleotide is composed of

•• A phosphate groupA phosphate group

•• A fiveA five--carbon sugar (pentose)carbon sugar (pentose)

•• A cyclic nitrogenA cyclic nitrogen--containing containing

compound (base)compound (base)

•• Two type of pentose Two type of pentose –– deoxyribonucleic deoxyribonucleic

acidacid and and ribonucleic acidribonucleic acid

•• Five type of bases Five type of bases –– adenineadenine (A), (A),

guanineguanine (G), (G), thyminethymine (T), (T), cytosinecytosine (C) (C)

and and uraciluracil (U)(U)

•• DoubleDouble--ring bases ring bases –– A and G A and G �������� purinespurines

•• SingleSingle--ring bases ring bases –– C, T and U C, T and U ��������

pyrimidinespyrimidines

DNA structureDNA structure

•• Discovered by Watson and Crick (1953)Discovered by Watson and Crick (1953)

•• Right handed Right handed double helixdouble helix

•• Two polynucleotide chains are coiled Two polynucleotide chains are coiled

about one another in a spiralabout one another in a spiral

•• Polynucleotide consists of nucleotides Polynucleotide consists of nucleotides

linked together by linked together by phosphodiesterphosphodiester

bondsbonds

•• Two polynucleotide strands are held Two polynucleotide strands are held

together in helical form by together in helical form by hydrogen hydrogen

bonding (HB)bonding (HB)

•• Specific baseSpecific base--pairingpairing

•• A with T (2 HB) and G with C (3 HB)A with T (2 HB) and G with C (3 HB)

DNA structureDNA structure

•• The The complementaritycomplementarity of two of two

strands of double helix makes strands of double helix makes

DNA uniquely suited to store DNA uniquely suited to store

and transmit genetic and transmit genetic

information from generation to information from generation to

generationgeneration

•• Base pairs in DNA are stacked Base pairs in DNA are stacked

about 0.34nm apartabout 0.34nm apart

•• 10 base per turn (36010 base per turn (360oo) of ) of

double helixdouble helix

•• The sugarThe sugar--phosphate phosphate

backbones of the two backbones of the two

complementary strands are complementary strands are

antiparallelantiparallel

•• One strand go from One strand go from 55’’ �������� 33’’

and the other from and the other from 33’’ �������� 55’’

•• DNA stability count on the DNA stability count on the

large number of hydrogen large number of hydrogen

bonds between base pairsbonds between base pairs

DNA structure: Alternate formDNA structure: Alternate form

•• Three forms of DNA structureThree forms of DNA structure

•• AA--DNADNA

•• BB--DNADNA

•• ZZ--DNADNA

•• AA--DNA DNA –– in high concentration in high concentration

of salts or in a partially of salts or in a partially

dehydrated statedehydrated state

•• BB--DNADNA –– under physiological under physiological

conditions (in the aqueous conditions (in the aqueous

protoplasm of living cells with protoplasm of living cells with

low concentration of salts)low concentration of salts)

•• ZZ--DNADNA –– discovered by xdiscovered by x--ray ray

diffraction analysis of crystals diffraction analysis of crystals

form by DNA form by DNA oligomersoligomers

(existence in living cells is not (existence in living cells is not

proven)proven)

Basic features of DNA replicationBasic features of DNA replication

•• In human In human –– DNA synthesis at 3,000 nucleotides per minuteDNA synthesis at 3,000 nucleotides per minute

•• In bacteria In bacteria –– DNA synthesis at 30,000 nucleotides per minuteDNA synthesis at 30,000 nucleotides per minute

•• Fidelity of DNA replication Fidelity of DNA replication –– 1 mistake per billion nucleotides incorporated1 mistake per billion nucleotides incorporated

•• DNA synthesis involves three stepsDNA synthesis involves three steps

•• Chain Chain initiationinitiation

•• ChainChain extensionextension or or elongationelongation

•• Chain Chain terminationtermination

SemiconservativeSemiconservative Replication Replication

•• In In semiconservativesemiconservative replication, each replication, each

of the parental strands is conserved of the parental strands is conserved

and serves as a template for the and serves as a template for the

synthesis of the new complementary synthesis of the new complementary

strandstrand

•• The base sequence in each progeny The base sequence in each progeny

strand is determined by the hydrogenstrand is determined by the hydrogen--

bonding potentials of the bases in the bonding potentials of the bases in the

parental strandparental strand

SemiconservativeSemiconservative DNA ReplicationDNA Replication

The mechanism of The mechanism of semiconservativesemiconservative DNA replication DNA replication –– based on E. colibased on E. coli

•• DNA DNA helicasehelicase unwind DNA molecules using energy derived from ATPunwind DNA molecules using energy derived from ATP

•• Once DNA strands are unwound by DNA Once DNA strands are unwound by DNA helicasehelicase, they are maintain through the , they are maintain through the

coating of coating of SSB proteinsSSB proteins

•• DNA DNA primaseprimase catalyzes the synthesis of short (10 catalyzes the synthesis of short (10 -- 60nt) RNA strands that are 60nt) RNA strands that are

complementary to the template strandscomplementary to the template strands

•• DNA polymerase III DNA polymerase III then uses the free 3then uses the free 3’’--hydroxyls of the RNA primers to hydroxyls of the RNA primers to

extend the chainsextend the chains

•• In lagging strand In lagging strand –– DNA polymerase III terminates an Okazaki fragment when it DNA polymerase III terminates an Okazaki fragment when it

bump into the RNA primer of the preceding Okazaki fragmentbump into the RNA primer of the preceding Okazaki fragment

•• RNA primers are excised and replaced with DNA chains. This step RNA primers are excised and replaced with DNA chains. This step is is

accomplished by accomplished by DNA polymerase I DNA polymerase I –– possesses 5possesses 5’’ �������� 33’’ exonucleaseexonuclease activityactivity

•• DNA DNA ligaseligase catalyzes the formation of a catalyzes the formation of a phosphodiesterphosphodiester linkage between the linkage between the

adjacent Okazaki fragmentsadjacent Okazaki fragments

Diagram of a replication Diagram of a replication

fork in E. coli showing fork in E. coli showing

the major components of the major components of

the replication apparatusthe replication apparatus

Telomerase: Replication of chromosome terminiTelomerase: Replication of chromosome termini

SemiconservativeSemiconservative DNA ReplicationDNA Replication

Unique aspects of eukaryotic DNA replicationUnique aspects of eukaryotic DNA replication

•• Shorter RNA primer and Okazaki fragmentsShorter RNA primer and Okazaki fragments

•• DNA synthesis takes place within a small portion of the cell cycDNA synthesis takes place within a small portion of the cell cycle. Not le. Not

continuously as in prokaryotescontinuously as in prokaryotes

•• Contain multiple origins of replicationContain multiple origins of replication

•• Two different DNA polymerases function at each replication forkTwo different DNA polymerases function at each replication fork

•• DNA polymerase alpha DNA polymerase alpha –– discontinuous replication of the lagging stranddiscontinuous replication of the lagging strand

•• DNA polymerase delta DNA polymerase delta –– catalyzes the replication of the leading strandcatalyzes the replication of the leading strand

INTRODUCTION

• Greek; chroma = colour, soma = body

• Thread-like structures or “coloured body” under light microscope

• Cytogenetics – the study of chromosomes and cell division

• Discovered in the second half of nineteenth century by W. Waldeyer

• < 1950s – 48 chromosomes in human

• 1956 – 46 chromosomes (using more reliable techniques)

• Made up of i) DNA

ii) histone proteins

iii) non-enzyme proteins (DNA replication enzymes

and transcription factors)

iv) protein specific to certain cell types

• ~1.5 – 2 meters long – DNA of all the chromosomes in a single cell

• The DNA in chromosomes is highly coiled

• Individual chromosomes identified using special stains

• Light staining regions – euchromatin

• Dark staining regions – heterochromatin (highly coiled)

• Heterochromatin at the centre - centromere

• Heterochromatin at the tip – telomere (TTAGG repeats)

• During cell division – each chromosomes consist of 2 identical strands

known as chromatids or sister chromatids

• Sister chromatids join at centromere

• Morphologically chromosomes classified according to centromere

position

i) metacentric – centromere located centrally

ii) acrocentric – centromere located at the terminal

iii) submetacentric – centromere located at intermediated

position

• Satellite – bloblike ends extend from a thinner, stalk-like bridge

-- sometimes found on acrocentric chromosomes

-- genes coding for ribosomal RNA and proteins

• 22 pairs are autosomes; X and Y are sex chromosomes

• Members of a pair of chromosomes are known as homologues

• Somatic cells have diploid (2n) complement of 46 chromosomes

• Gametes (ova & sperm) have a haploid (n) complement of 23

chromosomes

• Cells with 4 of each chromosomes are tetraploid (4n) – human liver

cells. Cells with 8 of each chromosomes are octaploid (8n)

• Basic number of chromosomes varies among species

• Chromosome numbers unrelated to the size and biological complexity

• Muntjac (tiny asian deer) has only 3 chromosomes in its genome

• some species of ferns have many hundreds

• 10 – 40 chromosomes in average in most species

CLASSIFICATION

• Individual chromosomes differ in position of centromere, length and

presence and absence of satellites

• Chromosomes divided into groups according to the above parameters

• 7 groups - A – G

• A = 1 – 3; B = 4 – 5; C = 6 – 12 + X; D = 13 – 15; E = 16 – 18;

F = 19 – 20; G = 21 – 22 +Y

CHROMOSOME NOMENCLATURE

• Idiogram – standard chromosome banding pattern

• Divided into two arms [p (short, top) & q (long, bottom)]

• The arms divided into region

• The region subdivided into bands

• The bands subdivided into subbands

• Numbering always from the centromere outwards

• 15q11.2 = Chromosome 15, long arm, region 1, band 1, subband 2

• Normal male – 46,XY; normal female – 46,XX

• Male Down syndrome – 47,XY,+21

• Female Cri du chat – 46,XX,del(5p) or 46,XX,5p-

• 46,XY,t(2;4)(p23;q25) – translocation between short arm of chromosome 2 at

region 2 band 3 and the long arm of chromosome 4 at region 2 band 5

SEX CHROMOSOMES• X and Y chromosomes

• crucial role in sex determination

• found by McClung, Stevens, Sutton and Wilson in early twentieth century

• some animal species eg. Grasshoppers – females have one extra chromosome

than males, thus called X chromosome. The females have 2 sex chromosomes

(XX), males have 1 (X @ XO)

• Other animals including humans, males and females have same number of

chromosomes

• numerical equality due to presence of a chromosome in male, called Y

chromosome

• Y chromosome pairs with X chromosome during meiosis

• Y chromosome is smaller than the X chromosome, centromere located close

to the end

• Y chromosomes carries genes for testis determining factor (SRY) and

maintaning spermatogenesis

X INACTIVATION• Human females have 2 alleles for every gene on the x chromosome and males

have only one

• This inequality is balanced by a mechanism called X inactivation - operates in

all mammals

• Found by Dr. Mary Lyon in 1961

• the process of X inactivation is also called Lyonisation

• occurs at 15 -16 days gestation when the embryo consists of ~ 5000 cells and

irreversible

• The inactivation occurs randomly

• the same X chromosome is inactivated in all daughter cells.

• can be observed at cellular level. Turned off X chromosome absorbs stain

much faster than the active X.

• nucleus of a female cell in interphase has one dark-staining X chromosome,

called a Barr body

• Read process of X inactivation

VISUALISING CHROMOSOMES

• Extra or missing chromosomes are detected by counting

• Combinations of stains and DNA probes can distinguish 2

chromosomes exchanging parts among the 24 different types of human

chromosomes

• Chromosomes are displayed in a size-order chart called a karyotype

OBTAINING CELLS FOR CHROMOSOME STUDY

• Any cell except a mature red blood cell (lacks a nucleus)

• Most common – peripheral blood (lymphocytes)

• Cells from tumour – to check chromosome abnormalities – determine

which drugs are likely to be more effective

• Blood borne cancers (leukaemia and lymphoma) – bone marrow cells

• 1950’s cells from hair roots used for chromosome studies

• Olympic athletes donate lining cells from insides of their cheeks to have

their sexes confirmed

• First successful foetal karyotype in 1966 by amniocentesis. Chorionic

villus sampling and cordocentesis provide foetal cells for karyotyping

CHROMOSOME PREPARATION

• In 1923, Theophilus Painter published sketches of human

chromosomes – concluded numbered 48

• Accepted for 30 years – but others found the number to be anywhere

from 38 – 48 due to difficulty in visualising.

• In 1951, by accident dilemma to untangle the chromosomes was solved

• A technician mistakenly washed the WBC less concentrated (than the

interiors of the cells) salt solution.

• Water rushed into the cells, making them swell and separates the

chromosomes

• In 1953, Levan & Tijo dropping the cell-rich fluid on a slide using a

pipette makes the cells burst open and freed the mass of chromsomes.

• In 1956, Levan & Tijo settled the matter on the number of

chromosomes in diploid human cell – 46

• The same year Hamerton & Ford identified the expected 23

chromosomes in human germ cells.

• Then the karyotype was born

CHROMOSOME STAINING AND BANDING

• Late 1960s to early 1970s, chromosome spreads were stained with

Feulgen’s reagent – a purple dye reacts with DNA sugar molecules, or

with aceto-carmine, a deep red dye (Figure 7.2)

• Quinacrine - intercalating agent, fluorescent (view under UV)

• Can identify particular chromosomes in a cell and analyse

chromosomal structural abnormalities

• The staining procedure is called Q-banding, the bands it produces are

called Q bands (Figure 7.3)

• The most popular non-fluorescent staining technique uses Giemsa stain

• Mixture of dyes – named after the inventor, Gustav Giemsa

• Chromosomes treated with trypsin to denature the protein content -

banding

• The nature of banding pattern depends on how the chromosomes were

prepared prior to staining

• G banding – gives dark bands corresponding to the bright bands of

quinacrine

• R banding – reverse pattern, dark bands correspond to light G bands.

The chromosomes are heat-denatured before staining with giemsa

• C banding – stains the region around the centromere. The

chromosomes are pretreated with acid followed by alkali prior to G

banding

Fluorescent In Situ Hybridisation (FISH)• Latest diagnostic tool in cytogenetics; gives rapid results

• A portion of single stranded DNA (probe) anneal with its

complementary sequence

• Interphase cells can be used; interphase cytogenetics

• Visualisation under UV

• Centromeric probe – repetitive sequences in or around centromere of a

specific chromosome

- for common aneuploidy syndromes (trisomy 21,

18, 13)

- non-dividing cells in interphase

• Chromosome-specific unique sequence probes

– for a particular single locus

- identifying tiny submicroscopic deletions and duplications

- gene mapping studies

• Telomeric probes – identifying deletion and translocations on the

telomeric region. Simultaneous analysis (24 chromosomes’ probes

developed) of every chromosome using a single microscope slide per

patient.

• Whole chromosome paint probes

– probes from different parts of a particular chromosome.

- entire chromosome fluoresces (painted).

-characterising complex rearrangements eg. Subtle translocations,

origin of additional chromosome materials (small

supernumerary markers or rings)

� Read on reverse painting,CGH

• Multicolour FISH – whole human chromosome paint probes

- multicolour human karyotype

- homologous chromosomes have the same unique

colour

- for detecting subtle chromosome rearrangements,

small supernumerary markers and ring

chromosomes

• Chromosomal microarray

CGH Array