ubaiii biologia molecular 1º ano 2013/2014. 29/nov/2012mjc-t09 sumário: capítulo x. o núcleo...
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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.
29/Nov/2012MJC-T094
Cromatina Conjunto de DNA e Histonas Nucleosomas
29/Nov/2012MJC-T095
Tipos de histonas Octâmeros de histonas Histona linker H1 Outras variantes Ligações com outras moléculas
29/Nov/2012MJC-T096
Níveis superiores de empacotamento
29/Nov/2012MJC-T097
Super enrolamento
29/Nov/2012MJC-T098
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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29/Nov/201212 MJC-T09
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
29/Nov/201221 MJC-T09
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.
Control of Gene Expression in EukaryotesChromosomes and Chromatin
3D structure of a nucleosome from X-ray
crystallography. Core particle at two views (top) and schematic of half of a
core particle (side)
© 2013 John Wiley & Sons, Inc. All rights reserved.
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.
Control of Gene Expression in EukaryotesChromosomes and Chromatin
© 2013 John Wiley & Sons, Inc. All rights reserved.
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).
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
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.
Control of Gene Expression in EukaryotesHigher Levels of Chromatin Structure
Levels of organization of chromatin.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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).
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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,
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
The end-replication problem: Generation of single stranded overhangs that shorten DNA
© 2013 John Wiley & Sons, Inc. All rights reserved.
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 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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
The mechanism of action of telomerase. Gap in complementary strand
filled by DNA polymerase (carries DNA primer).© 2013 John Wiley & Sons, Inc. All
rights reserved.
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 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.
Control of Gene Expression in EukaryotesTelomeres
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
Control of Gene Expression in EukaryotesNuclear organization
Localizing specific chromosomes within an interphase nucleus. More
active chromosomes, those that have more protein-coding genes,
are centrally located in the nucleus.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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 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
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
Control of Gene Expression in EukaryotesNuclear organization
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)
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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
© 2013 John Wiley & Sons, Inc. All rights reserved.
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.
© 2013 John Wiley & Sons, Inc. All rights reserved.
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