cold spring harbor symposia on quantititive biology vol 83, 1993 from the chromosomal loops and the...
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Cold Spring Harbor Symposia on Quantititive BiologyVol 83, 1993
From the Chromosomal Loops and the Scaffold to the Classic Bands of Metaphase
ChromosomesY. Saitoh AND U.K. Laemmli
Apresentação: D. Safe AND J.L. Carvalho
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Loops Cromossômicos
. Dificuldades de estudo
. Estrutura dos cromossomos nativos:
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Loops Cromossômicos
Conceito sólido
Primeiras bruxarias. Microscopia eletrônica de cromossomos
metafásicos que tiveram suas histonas removidas através de tratamento com tampões
. Secções dos cromossomos
. Técnicas de espalhamento
. Imunofluorescência (anticorpos anti Topo II)
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Loops Cromossômicos
Scaffold
Halo (loops)
Cromossomos desdobrados
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• Scaffold = bases dos loops em cromossomos desdobrados (região heterocromática)
• Características do scaffold
Loops Cromossômicos
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Topoisomerase II e SARs
• Proteína SC1 (localização, ligação às SARs e função)
• SARs = Scaffold-associated regions (função e características)
- A tracts- Alterações na estrutura do cromossomo
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Bandas Cromossômicas
Bandas Q
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• Modelo clássico: discos empilhados, densidade gênica, tempo de replicação, seqüências repetitivas, conformação da cromatina.
• Tipos: C, Q/G e R
Bandas Cromossômicas
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• Hipóteses para o bandeamento
- Acessibilidade do corante
- Riqueza
- Superestrutura (?)
Bandas Cromossômicas
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• Bandas Q/G
- Mais heterocromática
- “Rica” em AT
- 20% dos genes
Bandas Cromossômicas
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• Bandas R
- Replica primeiro
- “Rica” em GC
- Digerida por tripsina
- Genes housekeeping
Bandas Cromossômicas
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• Dois tipos de loops: Q/G e R
• Fila AT
• Estrutura do cromossomo compacto
Modelo Loop/Scaffold de cromossomos nativos
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Modelo Loop/Scaffold de cromossomos nativos
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Empacotamento dos loops cromossômicos: possibilidades e
ferramentas.
• Polaridades bioquímica e morfológica
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Detecção da Base e Corpo dos Loops
• Soro específico• Fluorocromos AT-específicos
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Daunomicina e a detecção das bases dos loops
• DNA muito rico em AT (>65%)
• Fluorometria:
- Excesso de poli (dA.dT), (dA).(dT) ou (dI).(dC): sinal residual de 45-55%
- Excesso de poli (dC.dG), (dC).(dG): sinal residual de 1-2%
- Excesso de SAR (74% AT): sinal residual de 10-15%
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YOYO e a detecção do corpo dos loops
• Cora tanto a base quanto o corpo
• MG interage com fragmentos de DNA ricos em AT
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Bandas Q e R são complementares e formadas por enovelamento
diferencial da fila AT
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• Densidade de DNA e acessibilidade do corante.
• Complementaridade entre as bandas Q e R
• Aparência “puff-like” das bandas R
• Uso de corantes consecutivamente no mesmo cromossomo
Bandas Q e R são complementares e formadas por enovelamento
diferencial da fila AT
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Bandas Q e R são complementares e formadas por enovelamento
diferencial da fila AT
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Complementaridade de Sub-bandas
• Bandas Q com estrutura preservada não são coradas igualmente por daunomicina
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Complementaridade de Sub-bandas
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Complementaridade de Sub-bandas
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Evidências Relacionam a Fila AT ao Scaffold
• Fila AT como sinal óptico gerado pelo complexo scaffold/SARs
• Daunomicina e SARs
• Topoisomerase II, HMG-I(Y) e a fila AT
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Evidências Relacionam a Fila AT ao Scaffold
Topo II
Dauno
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Sub-bandas Giemsa e o Scaffold
• Cromossomos alongados pré-matafásicos
• Relaçao 1x1 entre sub-bandas giemsa e AT-coils