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West Chester University’s
Department of Biology Presents…
Brittany Roundtree
Molecular Biology
Graduate Presentation
October 27,2010
This Presentation Is Based On the
Experimental Research Taken From the
BMC Biochemistry Research Article:
“Gene Expression Profile of HIV-I TAT Expressing
Cells: A Close Interplay Between Proliferative
and Differentiation Signals”
Cynthia de la Fuente2, Francisco Santiago2, Longwen Deng2, Carolyne Eadie2,
Irene Zilberman2, Kylene Kehn2, Anil Maddukuri2, Shanese Baylor2, Kaili Wu2,
Chee Gun Lee1, Anne Pumfery2 and Fatah Kashanchi2
Human Immunodefiency Virus
Shows two H.I.V. virus particles
Central Dogma of Molecular Biology
IMPORTANT!!!
H.I.V. does not follow the rules of the
Central Dogma of Molecular Biology
because it is a…..
RETROVIRUS
What are these scientists trying to
find?
• They want to explain the effect that Tat (TransActivator of Transcription) has on HIV-1
infected cells and Tat expressing cells.
• Cellular changes associated with this gene
The Culprit: Tat
•
•
•
HIV gene
Down regulate mannose receptor- spread of virus
Has been known to repress host cellular genes and involve itself in immunosuppression
–
•
•
•
Increases levels of HIV RNA
Past research has focused on Tat’s ability to activate HIV-1 LTR (HIV-1 Long Terminal Repeat)
In-vivo effects of Tat
–
•
Example: Xenopus embryo – delay of gastrulation, suppression of two early genes important for gastrulation
(Xbra,gsc)
In-vitro effects of Tat
–
•
Example: MHC -1 (Major Histocompatibility Complex Type 1) - displays proteins that are present within the cell to
Cytotoxic cells and if there are foreign peptides present, these cells will recognize them and kill them
Example: EDF-1 (Endothelial-related Factor-1) – regulates endothelial cell differentiation. Addition of Tat during
transcription resulted in the inhibition endothelial growth
Contains protein transfer domain
– Allows Tat to enter cells across cell membrane
– Mechanism of Transfer: UNKNOWN
Important Information About Cell
Culture Methods
•
ACH2 cells : HIV-1 infected CD4 lymphocytic cells (plays a role in cellular immunity)
containing wild type DNA
*Cell Lines have a proviral sequence
• CEM T Cell: Parental cell for ACH2 cell
• TAR: Point mutation on Chromosome 37, which causes it to not respond to Tat.
Although it does not respond to Tat, it is capable of making infectious viruses
when certain stimuli are present. (TNF, PHA,PMA,etc)
• H9 Cells: CD4+ lymphocytes ; control integrated vector without Tat open reading
frame
• H9/Tat Cells: CD4+ lymphocytes; integrated Tat expression vector
• U1: monocytic clone; has two copies of viral genome from parent U973 cells
• All cells were cultured at 37˚C up to 105 cells per ml in RPMI-1640 media
*Contained 10% Fetal Bovine Serum treated with mixture of 1%
streptomycin and penicillin antibiotics and 1% L-glutamine
How the Cell Cycle was Analyzed
for Experimental Purposes
•
Blockage of HeLa cells with Hydroxyurea (prevents proliferation of HeLa cells) for 14 h
•
Cells were then released by washing (2x) with phosphate-buffered saline (PBS; helps
maintain a constant Ph) and adding complete medium.
•
Suspended cells were treated with 1% serum for 48 hrs prior to addition of hydroxyurea.
•
Collection of supernatants and analyzed by usage of an IL-8 ELISA
•
Cells were washed with PBS and fixed by adding 50 ml of 70% ethanol
•
FACS analysis : Fluorescence – Activated Cell Sorting
–
–
–
–
Cells Strained with Cocktail of Propidium Iodide Buffer (PL)- helps to determine cell cycle
PBS
NP-40 – can be used to determine cytoplasm content
PI
Purification of RNA
• 1.Cells grown to mid-log phase
• 2.Pelleted
• 3.Washed (2x) with cold D-PBS (maintains cell
culture media)
• 4. Total RNA extraction on ice using Trizol
Reagent
• 5.Purified RNA was analyzed on 1% agarose
gel (Quality and Quantity Purposes)
Glass Slide Microarray Method
• 1.2400 known human genes were arrayed onto a microarray
glass slide into four separate grids (A,B,C and D). Each
contained 600 genes respectively
• All genes were 2200 bp cDNAs
• 3 plant control genes were used to balance Cy-3 and Cy-5
fluorescence signals
Why Use DNA Microarray Analysis?
• 1. Price
• 2. Ability to study many genes simultaneously
• 3. Speed
• Information about the DNA Sequence is not
required
Figure 1
Uninfected HIV-1 Cells
Latently Infected HIV-1 Cells
Shows all genes that
were activated above
1 fold. (139 genes)
Shows all genes
that were
expressed below 1
fold. (449 genes)
Controls
Brief Description of Northern Blot
Procedure
1.
2.
3.
4.
5.
Gather sample that you wish to extract RNA from and isolate it by treating it with formaldehyde
Electrophoresis with agarose gel. At this point, the RNA will separated by its size.
Transfer of RNA to membrane, which is also termed as Northern Blotting
Fix RNA to the membrane by using either Ultraviolet Light or Heat (IMMOBOLIZE IT!!)
Soak the membrane in a hybridizing buffer. The usage of a hybridizing buffer will prevent the fluorescence of unreactive binding groups. Also, add labeled probes (antibodies specific to protein) to the membrane and incubate.
6. Wash off the excess hybridizing buffer
7. Detection of labeled RNA
Note: In this experiment, instead of X-Ray film, they used a Phosphorlmager cassette. Although a X-Ray film can be
used, the detection time of Phosphorlmager is quicker.
Figure 2
1
2
1. Shows 695 genes that were up-regulated above 1 fold
2. Shows 1705 genes that were down-regulated below 1
fold
Summary of Tables 1, 2 and 3
= Genes that Were Up-Regulated
= Genes that Were Down-Regulated
Total
Receptor
Genes
Translation
Genes
Signal
Transduction
Genes
Genes in
Cytoskeleto
n
Cell Cycle
Genes
DNA
Repair/Repl
ication
Genes
Transcriptio
n Genes
Chromatin
Remodeling
Genes
DNA
Binding
Genes
8
46
1
7
8
5
5
4
6
53
0
6
0
5
6
9
3
8
61
46
7
7
13
11
14
7
14
What is the significance of the box highlighted in yellow?
Figure 3
Figure 4
Figure 5
Figure 6
Conclusions
• More than 2/3 of cellular genes were down-regulated by Tat
• Genes belonged to receptor,co-receptor, and co-activator
pathways that are part of serine/threonine receptor tyrosine
kinase, Ras/Raf/MEK/ERK (MAPK)cascade, which play a role in
proliferative and differentiation signals
• HIV-1 accessory spliced doubly spliced messages (TAT), may
control host genome in latently infected cells and determine
both viral transcription and possibly the fate of posttranscriptional events
BBibliography