Introduction Genetics

7/30/2019 Introduction Genetics

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Number of chromosomes

Normally, all the individuals of a species havethe same number of chromosomes. Closely related species usually have similar

chromosome numbers.

Presence of a whole sets of chromosomes iscalled euploidy.

It includes haploids, diploids, triploids,tetraploids etc.

Gametes normally contain only one set ofchromosome – this number is called Haploid

Somatic cells usually contain two sets ofchromosome - 2n : Diploid

What is so special about chromosomes?

1.They are huge:One bp = 600 dalton, an average chromosome is 107 bplong = 109- 1010 dalton !

(for comparison a protein of 3x105 is considered very big.

2. They contain a huge amount of non-redundant information (it is not just a big repetitivepolymer but it has a unique sequence) .

What is so special about chromosomes?

1.They are huge:One bp = 600 dalton, an average chromosome is 107 bplong = 109- 1010 dalton !

(for comparison a protein of 3x105 is considered very big.

2. They contain a huge amount of non-redundant information (it is not just a big repetitivepolymer but it has a unique sequence) .

3. There is only one such molecule in each cell. (unlike any other molecule when lost it cannot be re-synthesized from scratch or imported)

Philosophically - the cell is there to serve,protect and propagate the chromosomes.

Practically - the chromosome must be protectedat the ends - telomers

and it must have “something” that will enable it

to be moved to daughter cells - centromers

Genome Complexity

Chromosome structure

The DNA compaction problem

The nucleosome histones (H2A, H2B,H3, H4)

The histone octamere Histone H1 the linker histone

Higher order compactions

Chromatin loops and scaffolds (SAR)

Non histone chromatin proteins

Heterochromatin and euchromatin Chromosome G and R bands

Centromeres

The nucleosome histones (H2A,H2B, H3, H4)

Chromatin loops and scaffolds(SAR)

Non histone chromatin proteins Heterochromatin and euchromatin

Chromosome G and R bands

Centromeres

Take 4 meters of DNA (string) and compact them into

a ball of 10M. Now 10M are 1/100 of a mm and a

bit small to imagine – so now walk from here to the

main entrance of IIC let say 400 meters and try to

compact it all into 1 mm.

This compaction is very complex and the DNAisn’t just crammed into the nucleus but is organized

in a very orderly fashion from the smallest unit -

the nucleosome, via loops, chromosomal domains

and bands to the entire chromosome which has a

fixed space in the nucleus.

The nucleosome histones (H2A,H2B, H3, H4)

Chromatin loops and scaffolds(SAR)

Non histone chromatin proteins Heterochromatin and euchromatin

Chromosome G and R bands

Centromeres

The basic structural unit of chromatin, the nucleosome, wasdescribed by Roger Kornberg in 1974.

Two types of experiments led to Kornberg’s proposal of the

nucleosome model. First, partial digestion of chromatin with micrococcal nuclease (an

enzyme that degrades DNA) was found to yield DNA fragmentsapproximately 200 base pairs long .

In contrast, a similar digestion of naked DNA (not associated withprotein) yielded a continuous smear randomly sized fragments.

These results suggest that the binding of proteins to DNA inchromatin protects the regions of DNA from

nuclease digestion, so that enzyme canattack DNA only at sites separated by

approximately 200 base pairs.

Electron microscopy revealed that chromatin

fibers have a beaded appearance, with the beadsspaced at intervals of approximately 200 basepairs.

Thus, both nuclease digestion and the electronmicroscopic studies suggest that chromatin iscomposed of repeating 200 base pair unit, which

were called nucleosome.

individual nucleosomes = “beads on a

string”

The histone octamere

Histone H1 the linker histone

Telomeres

Centromeres

Heterochromatin and euchromatin

Chromosome G and R bands Centromeres

Euchromatin and Heterochromatin

Chromosomes may be identified by regions that stain in a

particular manner when treated with various chemicals.

Several different chemical techniques are used to identify certain chromosomal regions by staining then so that they

form chromosomal bands. For example, darker bands are generally found near the

centromeres or on the ends (telomeres) of the chromosome, while other regions do not stain as strongly.

The position of the dark-staining are heterochromatic

region or heterochromatin.

Light staining are euchromatic region or euchromatin.

Heterochromatin is classified into two groups:(i) Constitutive and (ii) Facultative.

Constitutive heterochromatin remainspermanently in the heterochromatic stage, i.e., itdoes not revert to the euchromatic stage.

In contrast, facultative heterochromatin consistsof euchromatin that takes on the staining andcompactness characteristics of heterochromatin

during some phase of development.

R-bands are known to replicate early, to contain most

housekeeping genes and are enriched in hyperacetylated

histone H4 and DNase I-sensitive chromatin.

This suggests they have a more open chromatin conformation,

consistent with a central AT-queue with longer loops that reach

the nuclear periphery.

In contrast, Q-bands contain fewer genes and

are proposed to have loops that are shorter and more tightly

folded, resulting in an AT-queue path resembling a

coiled spring.

Staining and Banding chromosome

Staining procedures have been developed in the past twodecades and these techniques help to study the karyotype inplants and animals.

1. Feulgen Staining:

Cells are subjected to a mild hydrolysis in 1N HCl at 600

Cfor 10 minutes.

This treatment produces a free aldehyde group indeoxyribose molecules.

When Schiff’s reagent (basic fuschin bleached withsulfurous acid) to give a deep pink colour.

Ribose of RNA will not form an aldehyde under theseconditions, and the reaction is thus specific for DNA

2. Q banding:

The Q bands are the fluorescent bands observed

after quinacrine mustard staining and observation withUV light.

The distal ends of each chromatid are not stained by thistechnique.

The Y chromosome become brightly fluorescent both inthe interphase and in metaphase.

3. R banding:

The R bands (from reverse ) are those located in thezones that do not fluoresce with the quinacrine mustard,that is they are between the Q bands and can be

visualized as green.

4. G banding: The G bands (from Giemsa) have the same

location as Q bands and do not require fluorescentmicroscopy.

Many techniques are available, each involving somepretreatment of the chromosomes.

In ASG (Acid-Saline-Giemsa ) cells are incubatedin citric acid and NaCl for one hour at 600C and arethen treated with the Giemsa stain.

5. C banding:

The C bands correspond to constitutiveheterochromatin. The heterochromatin regions in a chromosome

distinctly differ in their stainability from euchromaticregion.

Centromeres

Telomeres

3n – triploid

4n – tetraploidThe condition in which the chromosomes sets

are present in a multiples of “n” is PolyploidyWhen a change in the chromosome number does

not involve entire sets of chromosomes, butonly a few of the chromosomes - isAneuploidy.

Monosomics (2n-1) Trisomics (2n+1) Nullisomics (2n-2) Tetrasomics (2n+2)

Organism No. chromosomes

Human 46 Chimpanzee 48 Dog 78 Horse 64

Chicken 78 Goldfish 94 Fruit fly 8 Mosquito 6 Nematode 11(m), 12(f) Horsetail 216 Sequoia 22 Round worm 2

Organism No. chromosomes

Onion 16 Mold 16 Carrot 20

Tomato 24 Tobacco 48 Rice 24 Maize 20 Haploppus gracilis 4 Crepis capillaris 6

C di i i h h b “ i i ” i DNA

Can distinguish chromosomes by “painting ” – using DNAhybridization + fluorescent probes – during mitosis

Karyotype: is the general morphology of thesomatic chromosome. Generally, karyotypesrepresent by arranging in the descending order

of size keeping their centromeres in a straightline.

Idiotype: the karyotype of a species may berepresented diagrammatically, showing all themorphological features of the chromosome;such a diagram is known as Idiotype.

Chromosomes may differ in the position of theCentromere, the place on the chromosome

where spindle fibers are attached during celldivision.

In general, if the centromere is near the middle,

the chromosome is metacentric

If the centromere is toward one end, thechromosome is acrocentric or submetacentric

If the centromere is very near the end, thechromosome is telocentric.

The centromere divides the chromosome intotwo arms, so that, for example, an acrocentric

chromosome has one short and one long arm, While, a metacentric chromosome has arms of

equal length.

All house mouse chromosomes are telocentric, while human chromosomes include bothmetacentric and acrocentric, but no telocentric.

Autosomal pair Sex chromosome

Diploid No. of No. of X Y

(2n) metacentrics acrocentric or telocentric

Cat 38 16 2 M M

Dog 78 0 38 M A

Pig 38 12 6 M M

Goat 60 0 29 A M

Sheep 54 3 23 A M

Cow 60 0 29 M M

Horse 64 13 18 M A

M – Metacentric; A – Acrocentric

Centromeres and Telomeres

Centromeres and telomeres are two essentialfeatures of all eukaryotic chromosomes.

Each provide a unique function i.e., absolutely

necessary for the stability of the chromosome. Centromeres are required for the segregation of

the centromere during meiosis and mitosis.

Teleomeres provide terminal stability to thechromosome and ensure its survival

Centromere The region where two sister chromatids of a chromosome

appear to be joined or “held together” during mitaticmetaphase is called Centromere

When chromosomes are stained they typically show a dark-stained region that is the centromere.

Also termed as Primary constriction

During mitosis, the centromere that is shared by the sisterchromatids must divide so that the chromatids can migrate toopposite poles of the cell.

On the other hand, during the first meiotic division thecentromere of sister chromatids must remain intact

whereas during meiosis II they must act as they do during mitosis.

Therefore the centromere is an important component of chromosome structure and se re ation.

As a result, centromeres are the first parts of chromosomes to be seen moving towards theopposite poles during anaphase.

The remaining regions of chromosomes lag behind and appear as if they were being pulledby the centromere.

Kinetochore

Within the centromere region, most species haveseveral locations where spindle fibers attach, andthese sites consist of DNA as well as protein.

The actual location where the attachment occursis called the kinetochore and is composed of both DNA and protein.

The DNA sequence within these regions is

calledCEN

Typically CEN DNA is about 120 base pairs long and consists of several sub-domains, CDE-

I, CDE-II and CDE-III. Mutations in the first two sub-domains have no

effect upon segregation,

but a point mutation in the CDE-III sub-domain completely eliminates the ability of thecentromere to function during chromosome

segregation. Therefore CDE-III must be actively involved in

the binding of the spindle fibers to thecentromere.

The protein component of the kinetochore isonly now being characterized.

A complex of three proteins called Cbf-III

binds to normal CDE-III regions but can notbind to a CDE-III region with a point mutationthat prevents mitotic segregation.

Telomere

The two ends of a chromosome are known as

telomeres. It required for the replication and stability of the

chromosome.

When telomeres are damaged or removed due tochromosome breakage, the damaged chromosomeends can readily fuse or unite with broken ends of other chromosome.

Thus it is generally accepted that structuralintegrity and individuality of chromosomes ismaintained due to telomeres.

McClintock noticed that if two chromosomes were

broken in a cell, the end of one could attach to the

other and vice versa.

What she never observed was the attachment of thebroken end to the end of an unbroken

chromosome.

Thus the ends of broken chromosomes are sticky,

whereas the normal end is not sticky, suggestingthe ends of chromosomes have unique features.

Telomere Repeat Sequences

until recently, little was known about molecular structure of

telomeres. However, during the last few years, telomeres have

been isolated and characterized from several sp.

Species Repeat Sequence

Arabidopsis TTTAGGG

Human TTAGGG

Oxytricha TTTTGGGG

Slime Mold TAGGG

Tetrahymena TTGGGG Trypanosome TAGGG

The telomeres of this organismend in the sequence 5'-

TTGGGG-3'. The telomerase adds a series

of 5'-TTGGGG-3' repeats tothe ends of the lagging strand.

A hairpin occurs when unusualbase pairs between guanineresidues in the repeat form.

Finally, the hairpin is removedat the 5'-TTGGGG-3' repeat.

Thus the end of thechromosome is faithfully replicated.

Tetrahymena - protozoaorganism.

RNA Primer - Short stretches of

ribonucleotides (RNA substrates) found on th

lagging strand during DNA replication. Helpsinitiate la in strand re lication

Prokaryotic and Eukaryotic

Chromosomes

Not only the genomes of eukaryotes are morecomplex than prokaryotes, but the DNA of

eukaryotic cell is also organized differently from that of prokaryotic cells.

The genomes of prokaryotes are contained insingle chromosomes, which are usually circular DNA molecules.

In contrast, the genomes of eukaryotes are

composed of multiple chromosomes, eachcontaining a linear molecule of DNA.

Although the numbers and sizes of chromosomes vary considerably between different species, their basicstructure is the same in all eukaryotes

Organism Genome ChromosomeSize (Mb)a numbera

Arabidopsis thaliana 70 5Corn 5000 10Onion 15,000 8Lily 50,000 12Fruit fly 165 4Chicken 50,000 39

Mouse 1,200 20Cow 3000 30Human 3000 23

a – both genome size and chromosome numbers are for haploid cells

P k ti h

Prokaryotic chromosome

The prokaryotes usually have

only one chromosome, and itbears little morphologicalresemblance to eukaryoticchromosomes.

Among prokaryotes there is

considerable variation ingenome length bearing genes.

The genome length is smallestin RNA viruses

In this case, the organism is provided with only a few genesin its chromosome.

The number of gene may be ashigh as 150 in some larger

bacteriophage genome.

In E.coli , about 3000 to 4000 genes are organizedinto its one circular chromosome.

The chromosome exists as a highly folded andcoiled structure dispersed throughout the cell.

The folded nature of chromosome is due to theincorporation of RNA with DNA.

There are about 50 loops in the chromosome of E.coli.

These loops are highly twisted or supercoiledstructure with about four million nucleotide pairs.

During replication of DNA, the coiling must berelaxed.

DNA gyrase is necessary for the unwinding thecoils.

Bacterial Chromosome

Single, circular DNA molecule located in thenucleoid region of cell

Supercoiling

Helix twists on

itself in the opposite

direction; twists to

the left

Most common type

of supercoiling

Mechanism of folding of a bacterial

chromosome

There are many supercoiled loops (~100 in E. coli) attached to acentral core. Each loop can be independently relaxed or condensed.

Topoisomerase enzyme – (Type I and II) that introduce or remove

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