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Chromatin and Chromosome Structure Assist. Prof. Dr. Betul Akcesme

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Chromatin and Chromosome Structure

Assist. Prof. Dr. Betul Akcesme

1) Higher order structure of DNA

• Chromatin: material of which chromosomes are made of (DNA + proteins –histones)

• Nucleosomes are basic building units of chromatin

• Each nucleosome is consisted of: 8 histone molecules (histone octamer) and DNA (≈146 bp

long)

• Core histones: H2A, H2B, H3, H4

• “sealing histone” –H1 (lies outside of core histoneoctamer); purpose in connection of

nucleosomes in fibrous structures of higher order

• LINKER DNA–DNA molecule that “links” 2 nucleosomes (approx. 50 bp long, legth varies

between 8-114 bp)

Histones

• Histones: consisted mostly of basic, positively charged

amino acids like lysine and arginine

• Classified into 5 types: H1, H2A, H2B, H3and H4

Nucleosomes look like "beads on a string“ in the electron microscope

X-ray diffraction analyses of crystals

Structure of a nucleosome core

particle

• DNA bends sharply at

several places as it

wraps around the core

histone octamer

• Base sequence dictates

preferred nucleosome

positions along the DNA

Fig. 12.6

Higher order structure of DNA

• Secondary structure: the solenoid

• Solenoid structure has 6-7 nucleosomes per turn

• This is stabilized by interactions the highly variable C-and N-terminal regions

of the H1 histone and possibly the high mobility group (HMGs)

• This level of supercoiling produces a fiber of approximately 30nm in diameter

(solenoid fiber)

Secondary structure: the solenoid

Tertiary structure: loop structure

• When metaphase chromosomes are entirely depleted of histones–the residue

is an axial fibrous network –PROTEIN SCAFFOLD

• Protein scaffold is surrounded by DNA fibers which are organized into

LOOPS that radiate in all directions

• Loops contain between 5 –200 kb of DNA (63 kb)

• The loops are held together at their bases by non-histone proteins (HMGs)

Tertiary structure: loop structure

• What defines a DNA loop?

• SARs (scaffold-associated regions)

and MARs (matrix-associated

regions)

• Define loops of various sizes and are

usually found in non-transcribed regions

• Cleavage site for DNA topoisomerase II

(chromosome condensation –unwinding)

The radial loop-scaffold model for higher levels of

compaction

Several nonhistone proteins (NHPs) bind to chromatin every 60-100 kb and

tether the 300 Å fiber into structural loops

Other NHPs gather several loops together into daisy like rosettes

11

Fig. 12.7b

The radial loop-scaffold model for higher levels of compaction (cont)

Condensins may further condense chromosomes into a compact bundle for

mitosis

12

Fig. 12.7c

rosettes compressed

into a compact bundle)

Quaternary structure: folding into chromosome

• Quaternary structure also involves the scaffold structure

• Scaffold structure contains NO histones, but a mix of 30 non-

histone proteins (HMGs)

• HMGs involved: Sc1 and Sc2

• Sc1: DNA topoisomeraseII (topoII)

• Sc2: no role has been assigned to it so far

• TopoII: involved in the release of stress during transcription and

replication (chromosome condensation?)

• Loopes–hexamerrosettes –coil (30 rosettes) –chromatides

13

Figure 12.8 Experimental support for the radial loop–scaffold model

• The two ends of each DNA loop appear to attach to adjacent locations in the protein scaffold.

14

Figure 12.2 Chromosome scaffold

Chromosome compaction from interphase

to metaphase chromosomes.

initial looping and gathering compresses

the genetic material sufficiently to fit into the nucleus and to allow the placement of each chromosome

15

many structural loops, which are anchored together in rosettes in some areas.

By metaphase, the height of looping, gathering, and bundling achieves a 250-fold compaction of the roughly 40-fold-

compacted 300 Å fiber, giving rise to the highly condensed, rodlike shapes

Models of chromosomal structure and their fibrilary structure:

Nucleosomal fiber(11 nm)

Solenoid fiber (30 nm)

Chromatin fiber (300-700 nm)

Metaphase chromosome (1400 nm)

Levels of eukaryotic chromosomal organization

17

2) Chromosomes: form and function

• Each chromosome contains a single, duplex

of DNA, folded into a fiber that runs

continuously throughout the chromosome

• The replication of chromosome(s) is

semiconservative

• Individual chromosomes can be seen only

during mitosis (and meiosis)

• Each chromosome comprises 2 sister

chromatids

A pair of homologous chromosomes as seen at

metaphase

Allele (alternative form of a gene/marker)

Locus(position of a gene or DNA marker)

Chromosomes are cellular structures that transmit

genetic information

• Breeding experiments and microscopy provided evidence for the chromosome theory

of inheritance

• Proper development relies on accurate transmission of genes and accurate

maintenance of chromosome number

• The abstract idea of a gene was changed to a physical reality by the chromosome

theory

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et

al., 4th ed., Chapter 4

Evidence that genes reside in the nucleus

1667 - Anton Van Leeuwenhoek

• Microscopy revealed that semen contain spermatozoa ("sperm

animals")

• Hypothesized that sperm may enter egg to achieve fertilization

1854 – 1874

• Direct observations of fertilization through union of nuclei of eggs

and sperm (frog and sea urchin)

• Conclusion: something in the nucleus must contain the hereditary

material

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et

al., 4th ed., Chapter 4

22

Evidence that genes reside in chromosomes

• 1880s – improved microscopy and staining techniques

• Long, threadlike bodies (chromosomes) visualized in the nucleus

• Movement of these bodies followed through cell division

• Mitosis - nuclear division that generates two daughter cells

containing the same number and type of chromosomes as parent cell

• Meiosis - Nuclear division that generates gametes (egg and sperm)

containing half the number of chromosomes found in other cells

23

Diploid versus haploid: 2n versus n

• Most body cells are diploid (each

chromosome pair has one

maternal and one paternal copy)

• Meiosis haploid (n) gametes

• In Drosophila, 2n = 8, n = 4

• In humans , 2n = 46 and n = 23

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et

al., 4th ed., Chapter 4

25

Fig. 4.2

Fertilization is the union of haploid gametes to

produce diploid zygotes

• Fertilized eggs carry matching sets of chromosomes, one set from

maternal gamete and one set from paternal gamete

• Gametes are haploid (n) – carry only a single set of chromosomes

• Zygotes are diploid (2n) – carry two matching set of chromosome

• Mitosis ensures that all cells of developing individuals have identical

2n chromosome sets

26

Metaphase chromosomes can be classified by centromere

position

Metacentric chromosome – centromere is in the middle

Acrocentric chromosome – centromere is near one end

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et

al., 4th ed., Chapter 4

27

Fig. 4.3

Homologous chromosomes are matched in size,

shape , and banding patterns

• Homologs contain the same set of genes, but can have different alleles for

some genes

• Nonhomologs carry completely unrelated sets of genes

• Karyotype – micrograph of stained chromosomes arranged in homologous pairs

(see Fig 4.4)

• Sex chromosomes – unpaired X and Y chromosome

• Autosomes – all chromosomes except X and Y

• Cells of each species have a characteristic diploid number of chromosomes

e.g. D. melanogaster, 2n = 8; D. obscura, 2n = 10; D. virilis, 2n = 12; sweet peas, 2n = 14; goldfish, 2n =

94; dogs, 2n = 78

28

Karyotype of a human male

• Photos of metaphase human chromosomes (2n = 46, n = 23)

• Each homologous pair arranged in order of decreasing size

29

Fig. 4.4

Karyotype is a set of fully

compacted homologous chromosomes

• Different levels of packaging

compact the DNA in human

metaphase chromosomes

10,000-fold

• the centromere, region and

telomeres of each

chromosome become

visible.

30

KARYOTYPE

Various staining techniques

reveal a characteristic banding

pattern for each metaphase

chromosome

The karyotype of a human female examined by high-resolution G-banding

• Metaphase chromosomes

stained with Giemsa have

alternating bands of light and

dark staining

• Each band is contains many

DNA loops and ranges from 1

to 10 Mb in length

31

Fig. 12.9

Banding patterns on each

chromosome are highly

reproducible

important ??

Locations of genes in relation

to chromosomal bands

• Short arm = p arm

• Long arm = q arm

• Within each arm, light and dark

bands are numbered

consecutively

32

Fig. 12.10

Banding patterns and identification of genetic disease

• G-banding karyotype of the patient. It is illustrated the interstitial deletion on the long arm of chromosome 11 (Papoulidis et al, 2015)

33

every time a

chromosome

replicates,

whatever underlies

its banding pattern is

faithfully

reproduced.

they are an intrinsic property of each chromosome,

determined by the DNA sequence

itself.

Standardization of human chromosomes –

banding patterns

Banding techniques – enabled correct identification of every

chromosomal pair in karyotype

The most often used technique is – G-banding (G-Giemsa) on

chromosomes

There are interrellated arrangement of light and dark bands along every

chromosome

This arrangment is characteristic for particular pair of chromosomes

G-banding became a standard procedure for identification of

metaphase chromosomes in human karyotype

Standardization of human chromosomes –

banding patterns

Chromosomes in metaphase can be identified using

certain staining techniques – called BANDING

Cells are cultured and stopped in metaphase to

maximize the number of suitable cells

They are then spread on a slide, stained with a suitable dye

and visualized in the microscope

Most conventional cytogenetic analyses depend on the

karyotyping of banded metaphase chromosomes

Human chromosomes classification

• KARYOTYPE : is the representation of entire metaphase

chromosomes in a cell

▫ The number and structure of the chromosomes in the nucleus of a cell

▫ The karyotype is identical in all the diploid cells of an organism

• KARYOGRAM: a graphic representation of a karyotype

• Graphic representation of chromosomal set where chromosomes are

arranged by their size

• IDIOGRAM: a diagram representing the characteristic features of

the chromosome set

Karyotype–karyogram-idiogram

Compaction of DNA into chromatin presents a

problem

How do proteins access bases within the genome to perform

their functions?

• 1) chromatin structure is dynamic and can change to allow access

of specific proteins when they need to act.

• 2) variations exist in the molecules making up the basic

chromatin structure, and these variants recruit proteins that are

necessary for chromosomal functions.

38

3) Euchromatin and heterochromatin

Euchromatin and heterochromatin

• Genetically active • Less condensed

Is assumed that euchromatin is:

Location: chromosome arms

39

Heterochromatin

Made up of repeat sequences that vary in length

• Primary constriction (centromere) • Telomeres • Secondary constriction (nucleolar

organizing region)

Locations:

40

Heterochromatin

Inheritance is strictly mendelian

Contain tandemly repeated DNA

Some repeat sequences have significantly different DNA composition to the rest of the genome (being more GC or AT rich) – SATELLITE DNA

Made up of simple repeats

Doesn’t code for proteins

Little or no use for somatic cells but in some way important to germ cells!

41

Chromosomal packaging and function

▫Heterochromatin – highly condensed, usually inactive

transcriptionally

• Darkly stained regions of chromosomes

•Euchromatin – relaxed, usually active

transcriptionally

• Lightly stained regions of chromosomes

42

Properties of euchromatic and heterochromatic regions (Shiv & Sarah, 2007)

43

44

Figure 12.12 Stained heterochromatin.

human metaphase chromosomes were stained by a special C-banding

technique that darkens the heterochromatin, most of which localizes to

regions surrounding the centromere.

Structural parts of chromosomesTelomeres

Telomeres

Centromeres

Kinetochores

Nucleolar organizing regions (NORs)

Telomeres

The end of chromosomes

Made up of both DNA and proteins

Essential component in the control of chromosome integrity

Fundamental function of telomeres: to stop degradation of the chromosomes

during replication

sequence motif of telomeres: (TTAGGG)n is highly conserved across

phylogenies

46

Telomeres – mode of action

Prevent shortening of chromosomes

Without telomeres a chromosome would be shortened with every round of division

Telomeres are formed by enzyme telomerase

• To maintain cell and chromosome integrity • Involved in programming cellular senescence • Cause of cessation of cell division etc.

The effects of loosing telomeres:

47

Telomeres are "caps" that protect the ends

of eukaryotic chromosomes

• Telomeres consist of specific

repetitive sequences and don't

contain genes

Species-specific sequences

e.g. TTAGGG in humans, TTGGGG in

Tetrahymena

250-1500 repeats with variable number

between different cell types

• Prevent chromosome fusions and

maintain integrity of chromosomal

ends

48

Fig. 12.17

Replication at the ends of

chromosomes

• After removal of the last RNA primer,

DNA polymerase cannot replicate

some of the sequences at the 5' end

• DNA synthesis occurs only in 5'-to-3'

direction

• Without a special mechanism, DNA

would be lost from every new DNA

strand at each cell cycle

49

Fig. 12.18

Telomerase is a ribonucleoprotein that

extends telomeres • Telomerase RNA is

complementary to telomere

repeat sequences

• Serves as template for

addition of new DNA repeat

sequences to telomere

• Additional rounds of telomere

elongation occur after

telomeres translocate to

newly-synthesized end

50

Fig. 12.19

Centromeres and kinetochores

Centromere – the primary constriction of

chromosomes

Characterized by particular repeat sequences of DNA

(satellite DNA) and specific associated proteins (Cenp)

This is the last point of separation of sister chromatids

during cell division!

51

Centromere: primary constriction

Made of satellite DNA of approx. 170 bp long

Size of repeats vary: from 5 bp – 170 bp

Often highly conserved repeats

DNA is associated with centromere proteins

Called: CENPs

Important for chromosome function and integrity

Site of attachment of the kinetochores

52

Kinetochores

• Is the anchor point for spindle fibers

• 2 kinetochores are present at each chromosome, facing each pole

• Characterized by the presence of CENTROMERE-ASSOCIATED PROTEINS:

▫ CENP-A

▫ CENP-B

▫ CENP-C* (vital for normal functioning)

▫ CENP-D

• The kinetochore is specifically the binding site for the microtubules which pull the

sister chromatids apart during anaphase of cell division!

53

Nucleolar organising regions (Nors)

54

• Are the site of ribosome formation!

• rRNA is transcribed and processed in the nucleolus

• In the human karyotype NORs are to be found associated with satellited chromosomes (ch 13, 14, 15, 21 and 22)

• NORs are essential for normal development

Characteristics of centromeres

• Appear as constrictions in chromosomes

• Can occur almost anywhere on a chromosome, except at the very ends

• Can be in middle of chromosome (metacentric) or near one end of the

chromosome (acrocentric)

• Consist of satellite DNAs, which are repetitive, noncoding sequences

• Tandem repeats of 5 – 300 bp long, can extend over megabases of DNA

• Have different chromatin structure and higher-order packaging than other

chromosomal regions

• Predominant human satellite DNA is a-satellite

171 bp repeat, present in > 1 Mb block of tandem repeats in the centromeric

region of each chromosome

55

56

Figure 12.1 A chromosome’s centromere. Color-enhanced scanning electron micrograph of a human metaphase chromosome and interpretive drawing show that the centromere corresponds to the major constriction of mitotic chromosomes.

•Hold sister chromatids together (through action of cohesin)

•Attachment sites for chromosome segregation machinery

(through formation of kinetochore)

Action of cohesin during mitosis

• Cohesin is a protein complex that holds sister chromatids during

metaphase along the arms and in the centromere region.

• At anaphase, cohesin at the centromer is enzymatically cleaved and sister

chromatids are released from each other

57

Fig. 12.20a

One hypothesis proposes that cohesin encircles the two helixes of the sister chromatids to keep them together. Mutations in any of cohesin’s subunits result in chromosome segregation errors.

Action of cohesin during meiosis I

• At anaphase I, cohesin along chromosome arms is enzymatically

cleaved but cohesin at centromeres is not cleaved

• Shugoshin protects centromeric cohesin from degradation

58

Fig. 12.20b

Action of cohesin during meiosis II

• After entry into metaphase II, shugoshin is removed and

centromeric cohesin is degraded

59

Fig. 12.20c

Structure of centromeres in higher organisms

• Centromeres hold sister chromatids together and contain information for

construction of a kinetochore

• Cohesin binds sister chromatids together at the centromere

60

Fig. 12.21

One chromosome pair determines sex in grasshoppers

W. S. Sutton studied meiosis in great lubber grasshoppers

Before meiosis, testes cells had 24 chromosomes

• 22 in matched pairs (autosomes) and 2 unmatched (large = X and smaller = Y)

After meiosis, two types of sperm were formed

• 1/2 of sperm had 11 chromosomes and an X

• 1/2 of sperm had 11 chromosomes and a Y

After meiosis, only one type of egg was produced

• All had 11 chromosomes plus an X

61

4) Sex chromosomes and sex determination

The great lubber grasshopper

• Fertilization of egg with sperm carrying an X XX female

• Fertilization of egg with sperm carrying a Y XY male

• Sutton concluded that the X and Y chromosomes determine sex

62

The X and Y chromosomes determine sex

in humans

• Children receive an X chromosome from their mother, but either an X or Y chromosome from

their father

• Results in 1:1 ratio of females-to-males

63

Fig. 4.6

Females have 23 pairs of

chromosomes, Two chromosome of each

pair identical size and shape

Males have one

unmatched pair of

chromosome

Sex determination in fruit flies and humans

• In Drosophila, ratio of X chromosomes to autosomes determines gender

• In humans, presence or absence of Y chromosome determines gender

• Abnormal numbers of X or Y chromosomes have different effects in humans

and flies

64 Table 4.1

Mechanisms of sex determination differ between

species

• Heterogametic sex – gender with

two different kinds of gametes (e.g.

XY males in humans, ZW females in

birds)

• Homogametic sex – gender with

one type of gamete (e.g. XX females

in humans, ZZ males in birds)

• In some species, gender is

determined by environment (e.g.

temperature) 65

Table 4.2

EXTRA INFORMATION ABOUT

CHROMOSOMES……

Polytene chromosomes

Lampbrush chromosomes

B-chromosomes

Artificial chromosomes

Polytenechromosomes and polyteny

• Special feature of dipteran flies

(e.g. Drosophyla)

• Clearly visible during interphase

• Constructed by

endoreduplication process

• Puffing: the formation of

decondensed DNA associated with

the bands – Balbiani rings

(actively transcribed sections of

DNA)

Polytenechromosomes

B chromosomes

They are typically smaller than the members of the regular complement

transmitted in a non-Mendelian pattern

not necessary for normal development and reproduction

Contain large proportions of heterochromatin

Don’t carry major genes

Don’t pair with A chromosomes during cell division

Lampbrush chromosomes

• Unusual morphology

• Can be found in oocites of

amphibians, in oocites of humans, in

Drosophila (spermatocytes)

• Discovered in 1882

• Hypothesis: each loop represents a

single gene forming a single

polypeptide

• Number of loops reflects the number

of genes that are active at the time

Lampbrush chromosome from the cell nucleus of an ovarial egg from Triton sp., a salamander.

• A model for the structure of a lampbrush chromosome

• Chromomeres: highly condensed and in general not expressed until unfolding

Artificial chromosomes

Artificially constructed

First artificial chromosome is YAC (yeast artificial chromosome)

BAC (bacterial artificial chromosome)

Can take up to 2 Mb of DNA

• REPLICATION AND SEGREGATION OF DNA