S. W. A. T.

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S. W. A. T.
Students
Working
Against
TAT
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West Chester Universitys Department of Biology
Presents

• Brittany Roundtree
• Molecular Biology
• Graduate Presentation
• October 27,2010

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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

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Human Immunodefiency Virus
Shows two H.I.V. virus particles
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Central Dogma of Molecular Biology
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IMPORTANT!!!H.I.V. does not follow the rules of
the Central Dogma of Molecular Biology because it
is a..RETROVIRUS
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What are these scientists trying to find?

• They want to explain the effect that Tat
  (Trans-Activator of Transcription) has on HIV-1
  infected cells and Tat expressing cells.
• Cellular changes associated with this gene

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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
• 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
• Increases levels of HIV RNA
• Past research has focused on Tats 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 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

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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 37C 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

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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

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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)

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(No Transcript)
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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

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Why Use DNA Microarray Analysis?

• 1. Price
• 2. Ability to study many genes simultaneously
• 3. Speed
• Information about the DNA Sequence is not required

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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
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Brief Description of Northern Blot Procedure

• 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 un-reactive binding groups. Also,
  add labeled probes (antibodies specific to
  protein) to the membrane and incubate.
• Wash off the excess hybridizing buffer
• 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.

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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

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Summary of Tables 1, 2 and 3 Genes that Were
Up-Regulated
Genes that Were Down-Regulated
Receptor Genes Translation Genes Signal Trans-duction Genes Genes in Cytoskeleton Cell Cycle Genes DNA Repair/Replication Genes Transcription 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
Total 61 46 7 7 13 11 14 7 14
What is the significance of the box highlighted
in yellow?
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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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
  post-transcriptional events

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Have a safe and Happy Halloween!!!
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BBibliography