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Curso Bsico de Biologa Celular 2011

Structure of the chromosomes

Dr. Juan Pedro Luna Arias juanplunaa@yahoo.com

What is the material within a cell that contains the genetic information?

Discovery of the transforming principle by Frederick Griffith (1928)

Isolation of the transforming principle by Oswald Avery, Colin MacLeod y Maclyn McCarty (1944)

Lytic cycle of bacteriophage T2

Phage T2

Alfred D. Hershey and Martha Chase (1952)

Progenie contained 30% of the initial 32P input

Less than 1% of initial 35S input was in progenie

DNA is the genetic material

What is the structure of DNA?

1953 Watson and Crick determined the structure of DNA DNA and its Building blocks: Guanine (G), Adenine (A), Cytosine (C), Thymine (T). Polarized strand, 5->3

Base inside, sugar outside

DNA structure is a double helix

A severe problem of packaging

1. Largest human chromosome: ~3 x 108 bp. How long is it?

3 x 108 bp x 0.34 nm/bp x 1 m/109 nm = 0.102 m ~ 10 cm 2. A typical cell = 10 m = 10 x 10-6 m


3. Therefore the DNA must be compacted ~104 fold

What is the strategy of the cell to compact DNA?

Genes are carried by Chromosomes. Two plant cells visualized by light microscope. DNA was stained with DAPI.

(Interphase)

Chromosome Structure changes during the cell cycle

Cells in G1, S or G2 are said to be in Interphase, where the chromosomes are in a more dispersed conformation

A cell in M phase or Mitosis has more condensed chromosomes

Chromosomes
Chromosomes are discrete units used by the cells to organize its nuclear DNA. Each chromosome is a single DNA molecule. The DNA is not bare (not naked). It is coated and organized by a wide variety of proteins. Collectively, the DNA and protein assemblage that constitutes the chromosomes are referred as chromatin.

Plant Species
Arabidopsis thaliana (diploid) Rye (diploid)

Chromosomes Number
10 14

Maize (diploid)
Einkorn wheat (diploid) Durum wheat (tetraploid) Bread wheat (hexaploid)[

20
14 28 42

Potato (tetraploid)
Cultivated tobacco (diploid) Adder's Tongue Fern (diploid)

48
48 approx 1,400

Chromosome Number (2n) in some organimsms


Species
Common fruit fly
Dove Earthworm Octodrilus complanatus

Chrom. Num.
8
78 36

Species
Guinea Pig
Garden snail Tibetan fox

Chrom. Num.
64
54 36

Domestic cat
Laboratory mouse Rabbit Hares Gorillas, Chimpanzees Elephants Donkey Dog Goldfish

38
40 44 48 48 56 62 78 100-104

Domestic pig
Laboratory rat Syrian hamster Human Domestic sheep Cow Horse Kingfisher Silkworm

38
42 44 46 54 60 64 132 56

The collection of all the chromosomes is referred to as a Karyotype:

Human Karyotype as shown by traditional chromosome banding

example mFISH

Chromosome Genes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X (sex chromosome) Y (sex chromosome) Total 4,220 1,491 1,550 446 609 2,281 2,135 1,106 1,920 1,793 379 1,430 924 1,347 921 909 1,672 519 1,555 1,008 578 1,092 1,846 454 32,185

Total bases
247,199,719 242,751,149 199,446,827 191,263,063 180,837,866 170,896,993 158,821,424 146,274,826 140,442,298 135,374,737 134,452,384 132,289,534 114,127,980 106,360,585 100,338,915 88,822,254 78,654,742 76,117,153 63,806,651 62,435,965 46,944,323 49,528,953 154,913,754 57,741,652

Sequenced bases[47] 224,999,719 237,712,649 194,704,827 187,297,063 177,702,766 167,273,993 154,952,424 142,612,826 120,312,298 131,624,737 131,130,853 130,303,534 95,559,980 88,290,585 81,341,915 78,884,754 77,800,220 74,656,155 55,785,651 59,505,254 34,171,998 34,893,953 151,058,754 25,121,652

3,079,843,747 2,857,698,560

How is a chromosome organized?

Levels of chromatin structure


Larger DNA structures mediate more compaction.

Euchromatin: transcribed and less condensed Loops of 30-nm fibers seen at interphase Heterochromatin: more condensed, genes silenced, replicated later in S phase.

A lysed interphase nucleus

A mitotic chromosome

Levels of chromatin structure


Larger DNA structures mediate more compaction.

Euchromatin: transcribed and less condensed Loops of 30-nm fibers seen at interphase Heterochromatin: more condensed, genes silenced, replicated later in S phase.

Heterochromatin consist of packed chromosomal regions.

Heterochromatin Two forms


Constitutive heterochromatin
Remains compacted DNA which is permanently silenced Around centromere Distal arm of Y chromosome in male mammals Highly repeated sequences Few genes

Facultative heterochromatin
Specifically inactivated specific times

Example X and Y chromosomes


Males have small Y and large X chromosome Very few genes in common Single copy of each gene Females have two X Only one is transcriptionally active Other condensed Barr body

Chromatin exists in two forms: condensed and extended.

1. Beads-on-a-string (low salt) 2. 30-nm fiber (0.15 M KCl)

DNA and associated proteins is called chromatin.

Associated proteins are primarily histones


Nucleosomes, repeating units of DNA and histones. Nucleosomes are composed of 146 bp of DNA wrapped around a core particle of 8 histone proteins Core is 2 copies each of histones H2A, H2B, H3, H4

Histones H1 and H5 are linker histones


In total the histones interact with 186 bp of DNA

Allison, Fundamental Molecular Biology

Five classes of histones distinguished by their arg/lys ratio


Histone % arg H1 H2A H2B 1 9 6 % lys 29 11 16 Conservation Variable Moderately varied Moderately varied

H3
H4

13
14

10
11

Highly conserved
Highly conserved

Structural Organization of the Core Histones

The Assembly of the Core Histones

Notice the long tails of the octamer

The bending of DNA in a nucleosome 1. Flexibility of DNAs: A-T riched minor groove inside and G-C riched groove outside 2. DNA bound protein can also help

Nucleosomes are histone complexes

H2A, yellow; H2B, red; H3, blue; H4, green

What are the functions of nucleosomes?

1) Organization of chromatin 2) Transcriptional regulation 3) DNA replication 4) DNA repair 5) Recombination 6) Chromosome segregation

Nucleosomes without H1 histone

Nucleosomes with H1 histone

Assembly of nucleosome

The 30nm fibers are gathered into loops or domains These loops begin & end with AT-rich sequences anchored to a fibrillar network called the nuclear matrix Organization beyond this level is seen only during mitosis A fully compacted mitotic chromosome is packed 10 000 fold

SWI/SNF & HATs recruited by TF open DNA for RNAP & GTFs

RNAP & GTFs assembled on a promoter recruit SWI/SNF & HATs for elongation

Insulators (Boundry Elements)

Locus Control Regions (LCR)

LCR, cis-elements required for maximal transcription of -globin genes Multiple sequences with enhancer-like properties Flanked by insulators ensuring a euchromatin state

Insulators organize the chromatin by establishing separate compartments of higher-order structure

Each chromosome has three fundamental elements:

1) Replication origin.
2) Centromere.

3) Telomere. The level of compactation observed in chromosomes in mitosis is 10,000 fold


The level of compactation observed in chromosomes in interphase is 1,000 fold

Functional DNA sites -- Telomere & Centromere

Schematic and electron micrograph of X chromosome. Telomeres protect the ends. Centromere is at the primary constriction. It mediates chromosome cohesion, spindle attachment and chromosome segregation.

Types of chromosomes: Autosomes

Gonosomes (Sex chromosomes)

Human karyotype

X
C

D Y F

Kinetochore mediates attachment to the spindle

Centromere (DNA segment) is at the primary constriction. The kinetochore is a huge, complicated protein complex with several layers. The outer layer provides attachment sites for microtubules.

Sequences required for chromosome replication and segregation in mitosis

Mitosis

Meiosis

Structural maintenance of chromosomes: cohesin condensin

Centromers

Protenas no histonas proveen un andamiaje estrucutural para las hebras grandes de cromatina

FISH con diferentes sondas revela los loops de la cromatina

Lampbrush chromosomes (amphibian oocyte, immature eggs)

A model for the structure of a lampbrush chromosome Chromomeres: highly condensed and in general not expressed until unfolding

A polytene chromosome from Drosophila salivary gland Dark bands and interbands

Electron Microscope image of Drosophila polytene chromosome

Chromosome puffs Folding and refolding at a time course of 22 hours

RNA synthesis in Chromosome puffs Red: newly synthesized BrUTP; Blue: old ones diffused

RNA synthesis in Chromosome puffs

RNA synthesis in Chromosome puffs

Model of RNA synthesis in Chromosome puffs

A model for the structure of an interphase chromosome

Position Effects on Gene Expression Heterochromatin: condensed Euchromatin: loose

Chromatin decondensation appears to require two types of protein complexes each made of several polypeptide subunits: 1. Histone acetylase complexes. These are often referred to as HATS for histone acetylases. 2. Chromatin remodeling factors. These are often refered to as Swi/Snf factors because they were first identified as yeast mutants defective in mating type switching and in the ability to metabolize sucrose (sucrose nonfermenting).

Chromatin remodeling factors use energy from ATP hydrolysis to rearrange the packing of nucleosomes in higher order chromatin structures There are several different chromatin remodeling complexes in cells. Some of these bind to activation domains and de-condense the associated chromatin. Some bind to repression domains and condense the associated chromatin.

Histone Deacetylases Chromatin Remodeling Complexes

Histone Acetylases Chromatin Remodeling Complexes

Modificaciones de los extremos aminos de las histonas

Acetilacin Fosforilacin Metilacin Ubiquitinacin ADP-ribosilacin

Para qu sirven estas modificaciones?

TELOMERES

What are they? Why are they important? Telomere shortening and the end-replication problem Telomerase Telomere hypothesis of aging

Telomeres

Ends of linear chromosomes


Centromere

Telomere

Telomere

Repetitive DNA sequence (TTAGGG in vertebrates) Specialized proteins Form a 'capped' end structure

Telomeres 'cap' chromosome ends

Why are telomeres important?

Telomeres allow cells to distinguish chromosomes ends from broken DNA

Stop cell cycle! Repair or die!!

Homologous recombination
(error free, but need nearby homologue)

Non-homologous end joining


(any time, but error-prone)

Why are telomeres important? Prevent chromosome fusions by NHEJ (non-homologous end joining)

NHEJ

FUSION BRIDGE
Mitosis

BREAKAGE Fusion-bridge-breakage cycles

Genomic instability
Cell death OR neoplastic transformation

Telomeres also provide a means for "counting" cell division

Proliferative capacity

Finite Replicative Life Span "Mortal" Number of cell divisions

Infinite Replicative Life Span "Immortal"

The End Replication Problem:


Telomeres shorten with each S phase 5' 3' 3' 5'

5' 3' 5'

5'

3'

DNA replication is bidirectional Polymerases move 5' to 3' Requires a labile primer

Ori

Each round of DNA replication leaves 50-200 bp DNA unreplicated at the 3' end

Telomere also provide a means for "counting cell division: telomeres shorten with each cycle

20

Telomere Length (humans)

Telomeres shorten from 10-15 kb (germ line) to 3-5 kb after 50-60 doublings (average lengths of TRFs)
Cellular senescence is triggered when cells acquire one or a few critically short telomeres.

10

Normal Somatic Cells

(Telomerase Negative)
Cellular (Replicative) Senescence

Number of Doublings

TELOMERASE: Key to replicative immortality Enzyme (reverse transcriptase) with RNA and protein components Adds telomeric repeat DNA directly to 3' overhang (uses its own RNA as a template) Vertebrate repeat DNA on 3' end: TTAGGG Telomerase RNA template: AAUCCC

TELOMERASE:

Key to replicative immortality

+ TELOMERASE

Overcomes telomere shortening and the endreplication problem

Expressed by germ cells, early embryonic cells


Not expressed by most somatic cells (human) May be expressed by some stem cells, but highly controlled Expressed by 80-90% of cancer cells Remaining still need to overcome the end replication problem; do so by recombinational mechanisms -ALT (alternative lengthening of telomeres) mechanisms

Telomere Length and Cell Division Potential

20

Germ Cells (Telomerase Positive)

Telomere Length (humans)

10

Normal Somatic Cells

+ Telomerase

(Telomerase Negative)

Cellular (Replicative) Senescence

Number of Doublings

Schematic model of silencing at yeast telomeres

* = Hyperacetylated

TELOMERE STRUCTURE
5 3

Telomeric t loop

5'

3'
NUCLEAR MATRIX

Telomeric proteins: TRF1 TRF2 TIN2 RAP1 TANKS 1,2 POT1 etc

Alteration of chromatin by incorporation of histone variants

H2A.z

is generally associated with transcribed regions of DNA

ATP-dependent chromatin remodeling complexes isolated from nuclear extracts

Position/slide/condense nucleosomes Facilitate factor binding

Model for epigenetic control of heterochromatin assembly in yeast

Important role boundary elements (SAR/MAR/LCR/) Heterochromatic structures are inherited during cell division !! Role in differentiation and reprogramming.

Example of a repressor that recruits a histone methylase to repress transcription

Mammals and plants also DNA methylation -> inherited, maintained during replication.

10.7 Repressors and activators can direct histone deactylation at specific genes

Por mucho tiempo se haba pensado que las modificaciones en las histonas poda alterar la estructura de la cromatina por la influencia de los contactos histona-DNA o histona-histona.

Se cree que este dominio de la cola de las histonas es un lenguaje que ahora se refiere como cdigo histona

Acetilacin

Fosforilacin

Metilacin

Variante de H3 especfica de centrmero

Mechanism of chromatine based silencing in other regions yeast and in other eukaryotes is different! Enzymes containing histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity have been found in protein complexes with repressing and activating activity respectively

The histone code Post-translational modifications of the histones N-terminal tails

In yeast, histone acetylation can precede or follow remodeling

As a result general TFs can bind, and the gene is transcribed

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