ubaiii biologia molecular 1º ano 2013/2014. 29/nov/2012mjc-t09 sumário: capítulo x. o núcleo...

UBAIII Biologia Molecular

1º Ano2013/2014

29/Nov/2012MJC-T09

Sumário: Capítulo X. O núcleo eucariota e o controlo da

expressão genética Comossomas e cromatina Epigenética Organização do núcleo

2

Expressão genética O que é? Todas as células do nosso organismo têm o

mesmo genoma? Todas têm a mesma expressão genética?

29/Nov/2012MJC-T093

Cromossomas Molécula única de DNA 2 metros de comprimento no total

5cm/cromossoma Têm de estar

acessíveis a interação com proteínas transcriptoras e replicadoras;

Separados fisicamente uns dos outros sem se “enlearem”

Aparecem e desaparecem dependendo da fase do ciclo celular.

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Cromatina Conjunto de DNA e Histonas Nucleosomas

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Tipos de histonas Octâmeros de histonas Histona linker H1 Outras variantes Ligações com outras moléculas

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Níveis superiores de empacotamento

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Super enrolamento

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Topoisomerase II

Heterocromatina e eucromatina Vizualização Vs. Transcrição

29/Nov/2012MJC-T099

Heterocromatina constitutiva:telómeros e centrómerosefeito de posiçãosequências bloqueadoras ou

Heterocromatina facultativa:Cromossoma X das fêmeas (ambos estão activos durante a oogenese)mosaicismo em humanos

(daltonismo)

Formação de heterocromatina Depende da modificação

específica das histomas O código das histonas

Acetilação (lisina) “liga” Metilação (lisina ou

arginina) “desliga” Fosforilação (serina)

Lisinas acetiladas abundantes na H3 da eucromatina

As mesmas lisinas metiladas em heterocromatina.

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Exemplos de proteínas que se ligam a histonas As alterações de alguns resíduos das histonas

levava: Ligação de proteínas específicas Alteração da interacção entre histonas

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Todas as histonas H4 acetiladas estão a verde. Conclusão?

Cariotipagem

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Telómeros

TTAGGGAATCCC

Repetidas 500 a 5000 vezes

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Problemas na replicação de elementos lineares

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Modificação da extremidade

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Telomerase-Uma transcriptase reversa

Telomerases não estão sempre activas (expressas).Menor expressãoenvelhecimento e apoptoseExpressão aumentada possibilidade da formação de tumores.

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Centrómeros

São heterocromatina constitutiva ou facultativa?171pbs 500kbasesTêm proteínas associadas

(H3 é CENP-A ligação dos MT)

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Epigenética Nem sempre a hereditariedade depende da

sequência de DNA. Há características determinadas epigeneticamente

(por associação a proteínas específicas). Ex: inactivação dos cromossomas X das fêmeas

Mecanismos epigenéticos normalmente podem reverter.

Mecanismos epigenéticos muito associados a histonas São herdadas aleatoriamente As que são sintetizadas de novo têm de ser

“codificadas” com as acetilações, fosforilações e metilações correctas.

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Núcleo eucariota não é um saco!

É um organelo organizado As fibras de

cromatina estão organizadas num domínio específico dentro do núcleo

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Organização do núcleo eucariota

Dirigida pelas proteínas do envelope nuclear.

A transcrição ocorre em zonas específicas.

Genes envolvidos nos mesmos processos mas estão localizados em cromossomas diferentes são muitas vezes transcritos ao mesmo tempo e interagem

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Recursos utilizados

Capítulo 6 Karp 6ª Edição. Secção 6.1 Capítulo 12 Karp 4 e 5ª Edição. Secção 12.1 Capítulos 7 e 8 do Biologia Celular e

Molecular. Azevedo e Sunkel.

29/Nov/201222 MJC-T09

MJC-T12

Sumário:

Controlo ao nível da transcrição O papel dos factores de transcrição como reguladores

da transcrição. Estrutura dos factores de transcrição

12/dez/2013

DNA and histones are organized into repeating subunits called nucleosomes.

Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker.

DNA is wrapped around the core complex.

The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.

Control of Gene Expression in EukaryotesChromosomes and Chromatin

H3H4H2B

H2A

H3H2B

Nucleosomal organization of

chromatin: Schematic diagram

(top) and EM of Drosophila cell nucleus with

nucleosomes along DNA strand

(bottom)

© 2013 John Wiley & Sons, Inc. All rights reserved.

DNA and histones are organized into repeating subunits called nucleosomes.

Each nucleosome includes a core particle of supercoiled DNA and histone H1 serving as a linker.

DNA is wrapped around the core complex.

The histone core complex consists of two molecules each of H2A, H2B, H3, and H4 forming an octamer.


3D structure of a nucleosome from X-ray

crystallography. Core particle at two views (top) and schematic of half of a

core particle (side)


Histone modification is one mechanism to alter the character of nucleosomes.

DNA and histones are held together by noncovalent bonds. Ionic bonds between negatively charged phosphates of the DNA

backbone and positively charged residues of the histones. Histones, regulatory proteins, and enzymes dynamically mediate

DNA transcription, compaction, replication, recombination, and repair.



Higher Levels of Chromatin Structure A 30-nm filament is

another level of chromatin packaging, maintained by histone H1.

Chromatin filaments are organized into large supercoiled loops.

The presence of loops in chromatin can be seen: In mitotic chromosomes

form which histones have been extracted.

In meiotic lampbrush chromosomes from amphibian oocytes.

Control of Gene Expression in EukaryotesHigher Levels of Chromatin Structure

30-nm fiber: EM of a fiber (left) and two packaging models (middle, right).


Higher Levels of Chromatin Structure A 30-nm filament is

another level of chromatin packaging, maintained by histone H1.

Chromatin filaments are organized into large supercoiled loops.

The presence of loops in chromatin can be seen: In mitotic chromosomes

form which histones have been extracted.

In meiotic lampbrush chromosomes from amphibian oocytes.


Chromatin loops: a higher level of chromatin structure. EM: of a

mitotic chromosome (left) and model for cohesin in maintaining

loops (right)© 2013 John Wiley & Sons, Inc. All rights reserved.

Higher Levels of Chromatin Structure A nucleus 10 m in

diameter can pack 200,000 times this length of DNA within its boundaries.

Packing ratio of the DNA in nucleosomes is approximately 7:1.

Assembly of the 30-nm fiber increases the DNA-packing ratio to 40:1.

Mitotic chromosomes represent the ultimate in chromatin compactness with a ratio of 10,000:1.


Levels of organization of chromatin.


Heterochromatin and Euchromatin Euchromatin returns to a dispersed state after mitosis. Heterochromatin is condensed during interphase.

Constitutive heterochromatin remains condensed all the time. Found mostly around centromeres and telomeres. Consists of highly repeated sequences and few genes.

Facultative heterochromatin is inactivated during certain phases of the organism’s life. Is found in one of the X chromosomes as a Barr body through X

inactivation. X inactivation is a random process, making adult females

genetic mosaics.

Control of Gene Expression in EukaryotesHeterochromatin and Euchromatin


Calico cat cloning: Random inactivation of the X chromosome in different cells

during embryonic development creates a mosaic of tissue patches.

Control of Gene Expression in Eukaryotes Heterochromatin and Euchromatin

• Facultative heterochromatin is inactivated during certain phases of the organism’s life.

– Is found in one of the X chromosomes as a Barr body through X inactivation.– X inactivation is a random process, making adult females genetic mosaics.

Inactivated X chromosome or Barr body (arrows).


The Histone Code and Formation of Heterochromatin The histone code hypothesis states that the activity of a chromatin

region depends on the degree of chemical modification of histone tails.

Histone tail modifications influence chromatin in two ways: Serve as docking sites to recruit nonhistone proteins. Alter the way histones of neighboring nucleosomes interact.

Control of Gene Expression in EukaryotesThe histone code

The “histone code.” Histones can be

modified by addition of methyl, acetyl, and

phosphate groups


The majority of modified amino acids reside on the N-termini of H3 and H4. Each of the bound proteins possesses an activity that alters the structure and/or function of the

chromatin. Heterochromatin has many methylated H3 histones, which stabilize the compact nature of the chromatin. Small RNAs and specific enzymes play a role in histone methylation.

Control of Gene Expression in EukaryotesThe histone code

Proteins that bind selectively to modified H3 or H4 residues


Correlation between transcriptional activity and histone acetylation. Chromosomes labeled with fluorescent antibodies to acetylated histone H4 stain all chromosomes except the inactivated X (arrow).

Control of Gene Expression in EukaryotesHistone modification

Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin.

Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans.

Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1)

Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state,


Control of Gene Expression in EukaryotesHistone modification

Removal of the acetyl groups from H3 and H4 histones is among the initial steps in conversion of euchromatin into heterochromatin.

Histone deacetylation is accompanied by methylation of H3K9 histone methyltransferase (SUV39H1 in humans.

Methylated H3K9 binds to proteins with a chromodomain, for example heterochromatic protein 1 (HP1)

Once HP1 is bound to the histone tails, HP1-HP1 interactions facilitate chromatin packaging into a heterochromatin state,

Model of possible events during the

formation of heterochromatin

Histone deacetylaseHistone methyltransferase


The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists in

its most highly condensed state. Staining mitotic chromosomes can

provide useful information. A karyotype is a preparation of

homologous pairs ordered according to size.

The pattern on a karyotype may be used to screen chromosomal abnormalities.

Control of Gene Expression in EukaryotesThe Structure of a Mitotic Chromosome

Procedure to prepare mitotic chromosomes

for microscopic observation

from leukocytes


The Structure of a Mitotic Chromosome Chromatin of a mitotic cell exists in

its most highly condensed state. Staining mitotic chromosomes can

provide useful information. A karyotype is a preparation of

homologous pairs ordered according to size.

The pattern on a karyotype may be used to screen chromosomal abnormalities.

Control of Gene Expression in EukaryotesThe Structure of a Mitotic Chromosome

Human mitotic chromosomes labeled with

different specific fluorescent dyes.

The stained chromosomes of

a human male arranged in a

karyotype


Telomeres The end of each chromosome is called a telomere and is

distinguished by a set of repeated sequences. New repeats are added by a telomerase, a reverse

transcriptase that synthesizes DNA from a DNA template. Telomeres are required for the complete replication of

the chromosome because they protect the ends from being degraded.

Telomerase activity is thought to have major effects on cell life.

Control of Gene Expression in EukaryotesTelomeres

In situ hybridization with a DNA

probe (TTAGGG) to

locate telomeres on

human chromosome

Proteins can bind to

telomeres: RAP1 in

yellow, DNA in blue








The end-replication problem: Generation of single stranded overhangs that shorten DNA







Control of Gene Expression in Eukaryotes Telomeres

The single-stranded overhang is not free but forms a loop. The loop is a binding site for telomere-capping proteins that protect the ends of the chromosomes

and regulate telomere length.







Control of Gene Expression in Eukaryotes Telomeres

The importance of telomerase in maintaining chromosome integrity.

Chromosomes from a telomerase knockout mouse cell shows some

chromosomes lack telomeres entirely (stained yellow) and some have fused

to one another at their ends© 2013 John Wiley & Sons, Inc. All

rights reserved.

Telomeres In somatic cells, telomere lengths are reduced each cell division

to limit cell doublings. A critical point occurs from telomere shortening when cells stop

their growth and division. In contrast, cells that are able to resume telomerase expression

continue to proliferate. These cells continue to divide and do not shown normal signs of

aging. Approximately 90% of human tumors have cells with active

telomerase.


Telomerase dynamics during normal and abnormal growth. Limited

telomerase levels in somatic cells reduces the amount of cell doublings

compared to germ cells, unless telomerase is reactivated.


Centromeres The centromere is located at the site markedly indented on a chromosome. Centromeres contain constitutive heterochromatin. Centromeric DNA is the site of microtubule attachment during mitosis. DNA sequence is not important for centromere structure and function. Histone H3 variant CENP-A is found in the centromeres to potentially function in

kinetochore assembly.

Control of Gene Expression in EukaryotesCentromeres

Scanning electron micrograph of a mitotic chromosome with the

centromere marked by a distinct indentation.


Epigenetics: There’s More to Inheritance than DNA Epigenetic inheritance depends on factors other than DNA sequences. X-chromosome inactivation is an example, since the two X chromosomes can have identical DNA sequences, but one is inactivated and the other is not. An epigenetic state can usually be reversed; X chromosomes, for example, are reactivated prior to formation of gametes. Differences in disease susceptibility and longevity between genetically identical twins may be due, in part, to epigenetic differences that appear

between the twins as they age. Parental histones determine the chemical modifications found in the newly synthesized histones. As heterochromatin is replicated, a histone methyltransferase labels the newly synthesized H3 molecules added into the daughter nucleosomes. Euchromatic regions tend to contain acetylated H3 tails, a modification transmitted from parental chromatin to progeny chromatin.

Control of Gene Expression in EukaryotesEpigenetics


The Nucleus as an Organized Organelle Chromatin fibers of an interphase chromosome are not diffuse and

random, but are concentrated into distinct territories. Genes are physically moved to nuclear sites called transcription factories

where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but

reside on different chromosomes interact within the nucleus.

Control of Gene Expression in EukaryotesNuclear organization

3D map of all of the chromosomes present in a human fibroblast nucleus. Each chromosome,

represented as an identifiable color, is found to occupy a distinct territory within the nucleus.



Localizing specific chromosomes within an interphase nucleus. More

active chromosomes, those that have more protein-coding genes,

are centrally located in the nucleus.




where transcription machinery is located (e.g., hormone induction). DNA sequences that participate in a common biological response but


Control of Gene Expression in Eukaryotes Nuclear organization

Breast cancer cells treated

with estrogen co-activate

genes on Chr2 and Chr21

Model of how different DNA regions could be organized

for gene expression



where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but




Antibody staining against an mRNA processing factor

shows 30-50 distinct sites Time course of viral gene expression in

infected cells showing splicing factors (orange) compared to integration site (white arrow)



where transcription machinery is located (e.g. hormone induction). DNA sequences that participate in a common biological response but



The Human Perspective: Chromosomal Aberrations and Human Disorders

A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNA-damaging agents.

Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Inversions involve the breakage of a chromosome and

resealing of the segment in a reverse order. Translocations are the result of the attachment of all or

one piece of one chromosome to another chromosome.

The effect of inversion. Crossing

over between a normal chromosome

(purple) and one containing an

inversion (green)

Translocation. Exchange between chr12 (bright blue) and chr7 (red) in

human cells


The Human Perspective: Chromosomal Aberrations and Human Disorders

A chromosomal aberration is loss or exchange of a segment between different chromosomes, caused by exposure to DNA-damaging agents.

Chromosomal aberrations have different consequences depending on whether they are in somatic or germ cells. Deletions result when there is loss of a portion of a

chromosome. Duplications occur when a portion of a chromosome

is repeated.

Translocation and evolution. If the only two ape chromosomes that have no counterpart in humans are hypothetically fused, they match

human chromosome number 2, band for band.


Recursos utilizados

Capítulo 6 Karp 7ª Edição. Secção 6.4 ou 12.4 Capítulo 12 Karp 4 e 5ª Edição. Secção 12.4

MJC-T12 12/dez/2013

ubaiii biologia molecular 1º ano 2013/2014. 29/nov/2012mjc-t09 sumário: capítulo x. o núcleo...

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