Date post: | 15-Aug-2015 |
Category: |
Technology |
Upload: | andres-ramirez |
View: | 167 times |
Download: | 1 times |
Einat zalckvar, Christina Paulus, Desiree Tillo, Alexandra Absach-Nitzsche, Yaniv Lubling, Carla Winterling, Nicholas Strieder, Katrin Mucke, Felicia Goodrum, Eran Segal, Michael Nevels.
Nucleosome maps of the human
Cytomegalovirus genome reveal
A temporal switch in chromatin Organization linked to a major
IE protein
Sara Puche GómezAndrés Felipe Restrepo RamírezMedicine studentsIII SemesterMolecular BiologyUPB
INTRODUCTION
Associated diseases:• Infectious mononucleosis• Congenital Infection Cytomegalic inclusion disease• Pneumonitis• Gastrointestinal disease
INTRODUCTION
Chromatin
Complex between
eukaryotic DNA and proteins
Constitutes chromosom
e
Have histone and non-histone
proteins
Basic structure is
the nucleosome
INTRODUCTION
146 pairs of DNA coiled 1,65 times
Histones H2A, H2B,
H3 AND H4, and them
with H1 form chromatoso
me
Nucleosome
INTRODUCTION
Its the first set of viral gene products to be expressed
Not require the novo protein synthesis for their expression
IE 2: essential transcriptional transactivator. Genes activated and inhibits downstream IE gene
transcription through their specific binding to DNA
IE 1: promiscuous transactivator. Inactive
histone deacetylase (HDAC) to repress gene expression
can viral
IE Protein
INTROCUTION
Cytomegalovirus
Have IE protein
Which expresses
to help
The virus to enter the cell nucleus
And use proteins
To form nucleosom
es
And then chromatin
So then is incorporat
ed in
DNA
OBJECTIVE
To investigate the role of the human cytomegalovirus IE protein in the temporal
organization of the nucleosome and involvement in the function and structure of chromatin
MATERIALES Y METODOS
PCR
• No es una tecnica analìtica, es una metología• Desnaturalización del DNA para dar dar hebras sencillas• Hibridación específica de la hebra sencilla con un primer• Replicación de la hebra sencilla por una DNA polimerasa a partir del primer
anterior
PCRq
• Basada en el analisis digital de imagen mediante cualquier programa de analisis de imagen, permite realizar una precisa cuantificación de bandas de ácidos nucléicos desarrollados en gel de agarosa
PCR real time
• Mas seguro que la PCR, procesos como la amplificación y la detección se dan simultaneamente y por fluorescencia se puede detectar la producción de DNA en el momento
Materiales y metodos
Southern blot• Identificar
secuencias especificas de DNA
Microarreglos• Analizar la
expresión diferencial de genes, monitorizándose los niveles de miles de ellos de modo simultaneo
Digestión• Rompimiento
especifico y reproducible, de segmentos de DNA con enzimas de restricción, para luego recombinar ese DNA obtenido
RESULTS
FIGURA 1
cambios masivos en la organización del nucleosoma en todo el genoma hCMV durante la infección productiva.
FIGURA 1
FIGURA 2
Cambios temporales IE1-dependientes en la organización del nucleosoma en el genoma del hCMV
FIGURA 2
FIGURA 3
Efectos IE1-dependientes en la ocupación nucleosomal a través del hCMV genoma
FIGURA 3
FIGURA 4
Correlación negativa entre cambios IE1 dependientes en la organización y expresión del nucleosoma en el genoma del hCVM
DISCUSIÓN
REFERENCES WHAT HE OR SHE SAY? AGREE/DISAGREE
Nevels M et al
herpesvirus genomes are
commonly considered to
formregular
nucleosomal arrays resembling
bulk cellular chromatin in
Latently infected cells
¿Why?
This type of chromatin organization has not been formally shown for hCMV or any β-herpesvirus.
1
Herpesvirus histones inside virus particles
Nonnucleosomal structure of viral genomes
nuclei of cells infected with herpes viruses
2
REFERENCES WHAT HE OR SHE SAID?
AGREE / DISAGREE
Greaves RF et al
Disruption of IE1 delays
transcriptionduring
productive hCMV infection
AGREE
REFERENCESWHAT HE OR SHE
SAID?AGREE/DISAGREE
Sekinger et al
Genome-wide analyses in yeast have indicated
thatnucleosome occupancy
correlates poorly with
transcriptionIt was found significant negative correlations betweenchanges in nucleosome occupancy and changes in gene
expression between WT and dlIE1 strains 96 h postinfection.
REFERENCES WHAT HE OR SHE SAY?
AGREE/DISAGREE
Munch K et al
IE1 is notconsidered to bind DNA directly , the viral protein associates with chromatin and nucleosome modifying factorsIE1 on nucleosome occupancy
and organization might be mediated through direct interaction
between the viral protein and chromatin components
AGREE
conclusions
Increasingly more importance is been given to the complex role of proteins in the chromatin organization and gene expression
understand the mechanism during productive hCMV infection would help to develop new treatments for patients worldwide.
In order to properly understand and analyze a research article is necessary to study deeply the experimental models and the basis of the used techniques to understand the housed results, and the choice of appropriate techniques is essential to get accurate results consistent with the investigation purpose.
The Chip on chip technique is needed to confirm the results because it show the interaction between DNA and nuclear proteins as histones
MAPAS
Sara Puche GSara
GRACIAS