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Nucleic Acids and Gene Expression Control
Structure of nucleic acids
Nucleic acids contain the information for determining the aa sequence and hence the structure and
function of all the proteins of a cell. The DNA contains all the information required to build the cells.
The information coded in the DNA is arranged in units called genes. In the process of transcription,
the information stored in DNA is copied into ribonucleic acid (RNA) to be later translated to a
protein.
DNA and RNA are very similar. Both are composed by monomers called nucleotides. A DNA
molecule is 109 nucleotides long, while RNA ranges from a few bases up to many thousands. DNA
and RNA consist each from four different nucleotides. All nucleotides consist of an organic base
linked to a five-carbon sugar that has a phosphate group attached to carbon 5. In RNA the sugar is
ribose, in DNA deoxyribose.
A nucleic acid strand has a end-to-end chemical orientation, the 5’ end has a phosphate group and
the 3’ end has a hydroxyl group on the 3’ carbon of the sugar. Phosphodiester bonds link nucleic
acids into long polynucleotides that can twist and fold into three dimensional conformations
stabilized by noncovalent bonds. Phosphodiester bond is a link between the phosphate group and
two other molecules over two ester bonds. CUAL ES EL ESTER??.
DNA structure
DNA consists of two associated polynucleotide strands that wind together to form a double helix.
The two negatively charged phosphate bonds are on the outside of the double helix and the bases
project into the interior, where they are linked by hydrogen bonds. The orientation of the strands
are antiparallel. Watson-Crick base pairs (bp) are formed between A and T, and G and C. This
complementarity is consequence of the size, shape and chemical composition of the bases,
although other pairs are usually found. Thousands of these hydrogen bonds contribute to the
stabilization of DNA molecules. The helix usually makes a complete turn every 3.6 nm, including
10.5 bp per turn. This is referred to as the B form of DNA.
NCBI->
Colors in chromosome represents G-staining (Giemsa staining, which stains phosphate groups
from the chromosome). The more A-T bonds, the darker the stain.
The numbers on the left-most side refer to cytogenetics markers. The light-blue lines represent
assembled contig sequences. Contig sequences are assemblies of several clones. These clones
were direct sequenced. We can inspect the sequences of these clones.
The next gray histogram depicts the amount of ESTs aligned to a region
A UniGene entry is a set of transcript sequences that appear to come from the same transcription
locus (gene or expressed pseudogene), together with information on protein similarities, gene
expression, cDNA clone reagents, and genomic location.
Genes Map includes known and putative genes placed as a result of alignments of mRNAs to the
contigs, and gene predictions. All of them are identified, not as UniGene, which is made from ESTs
(it’s possible to see diference clearly at 1/100th of chromosome). Colors (blue, brown, etc)
represent the quality of the alignment between the gene entry and the genomic sequence.
Orientation shows the DNA strand on which the gene is located.
The "genome" of any given individual (except for identical twins) is unique; mapping "the human
genome" involves sequencing multiple variations of each gene.
Despite all the popular press articles saying that the genome was "complete", as of 2003 it is still
incomplete and it clearly won't be finished for many more years. First, it is important to realize that
the central regions of each chromosome, known as centromeres, are highly repetitive DNA
sequences that are difficult to sequence using current technology. The centromeres are millions
(possibly tens of millions) of base pairs long, and for the most part these are entirely unsequenced.
Second, the ends of the chromosomes, called telomeres, are also highly repetitive, and for most of
the 46 chromosome ends these too are incomplete. we have completed about 92% of the genome
Great Britain, as well as numerous other groups from around the world broke the genome into
larger pieces; approximately 150,000 base pairs in length. These pieces are called "bacterial
artificial chromosomes", or BACs, because they can be inserted into bacteria where they are
copied by the bacterial replication machinery. Each of these pieces was then sequenced
separately as a small "shotgun" project and then assembled. The larger, 150,000 base pair chunks
were then stitched together to create chromosomes. This is known as the "hierarchical shotgun"
approach, because the genome is first broken into relatively large chunks, which are then mapped
to chromosomes before being selected for sequencing.
The Evidence Viewer (ev) displays graphically the GenBank and RefSeq cDNAs that
align to the genome in a particular region, along with a density plot for ESTs. The
positions of any mismatches or insertions/deletions are marked, the multiple pairwise
sequence alignments are provided, and computed translations are shown.
The Sequence Viewer (sv) is the Entrez graphical display option for any nucleotide
sequence, focused on the gene indicated. By default, a 2-kb section of sequence is shown
below the representation of the features, but that limit can be increased at the bottom of
the page. It is also possible to zoom and navigate in the display.
Sequence Download (seq) provides the same function as the Download/View Sequence link
provided at the top of the Maps page. The scope of the sequence passed to the tool
corresponds to what is being viewed on the page. When connected to a gene feature, the
scope corresponds to that gene. The tool allows the user to alter the sequence scope and
to select a report format (e.g., FASTA, GenBank, ASN.1). For the human and mouse
genomes, a link is also provided to the Human Mouse Homology Map (hm).
Model Maker (mm) displays the evidence for exons in a genomic region by diagramming
the exons predicted from the alignment of cDNAs, from ab initio models (the default),
and from alignment of ESTs (after an explicit selection). To facilitate construction of
your own model transcript or transcripts, the splice junctions and the exons they connect
are displayed, and the coding potential of any combination of exons can quickly be
evaluated using ORFfinder. The sequence can also be edited, and the results can be saved
or downloaded.
Molecular definition of a gene
A gene is defined as the entire nucleic acid sequence that is necessary for the synthesis of a
functional gene product (RNA). Therefore a gene includes more than the coding region. A gene
also includes all the DNA sequences required for the synthesis a RNA transcript. Besides the
coding region, an eukaryotic gene consists of: enhancers (50 kb from coding region), sequences
specifying 3’ cleavage and polyadenylation (poly A sites) and splicing sites.
A transcription unit includes the coding region – which extends from the cap site to the poly(A) site
– and associated control regions. The primary transcript is composed from exons, which will be
translated into proteins, and introns, which will not be translated into proteins and contain many
regulatory sequences for controlling different processes.
Transcription of protein coding genes
Transcripts units are ‘copied’ from a DNA which serves as a ‘template’. This template determines
the order in which ribonucleoside triphospates (rNTP) monomers are polymerized to form an RNA
chain which will be complementary to the template. Complementary strands are in opposite
directions, so if RNA is synthesized from 5’ to 3’, the DNA template will be copied from 3’ to 5’.
Bases in the template DNA strand base-pair with complementary incoming rNTPs, which then are
joined in a polymerization reaction catalyzed by RNA polymerase II (for pre-mRNA). Polymerization
involves formation of phosphodiester bonds between the 3’ oxygen in the growing RNA chain on
the alpha-phosphate of the next nucleotide precursor. As a consequence, RNA molecules are
always synthesized in the 5’->3’ direction.
(1) During transcription initiation, RNA polymerase recognizes and binds to a specific site, called
promoter, in dsDNA. Polymerases require various protein factors, called general transcription
factors, to help them locate promoters and initiate transcription. (2) After binding to a promoter,
RNA polymerase melts the DNA strands in order to make the bases in the template strand
available for base pairing with the bases of the rNTPs. Approximately 14 bp are melted around the
transcription initiation site, which is located on the template strand within the promoter region. (3)
Transcription initiation finishes when the first two ribonucleotides are linked by a phosphodiester
bond.
(4) After several nucleotides have been polymerized RNA polymerase dissociates from the
promoter DNA and general TFs. During the stage of elongation, RNA polymerase moves along the
template DNA one base at a time, opening the dsDNA in front of its direction of movement and
hybridizing the strands behind it. One nucleotide at a time is added to the 3’ end of the nascent
RNA chain by the polymerase. The 14 bp melted region is called transcription bubble.
(5) During termination, the completed RNA molecule, or primary transcript is released from the
RNA polymerase and the polymerase dissociates from the template DNA. Once released, an RNA
polymerase is free to transcribe a new gene.
Regulation of transcription
Environmental changes induce changes in gene expression. Regulation of transcription initiation is
the most common form of gene control in eukaryotes.
An extended chromosome is 2 meters long, and it has to be condensed into a cell nuclei, which
size is ~10 um. This packing is very organized. Transcription-active areas of the chromosome are
less condensed than inactive areas. The level of condensation determines whether a region can be
accessed by transcription factors and polymerases.
The most abundant proteins associated with DNA are histones, a family of basic proteins present
in all nuclei. Histones are rich in positively charged basic aa, which interacts with phosphate groups
of DNA. Nucleosomes are composed of DNA wrapped around octameric histones and a H1
histone, and are the primary structural unit of chromatin. Nucleosomes are packed into a solenoid
arrangement, with six nucleosomes per turn. Loops of solenoid chromatin are associated with a
flexible chromosome scaffold protein, which in turn is compacted in a solenoid form.
Inactive genes are assembled into condensed chromatin, which inhibits the binding of RNA
polymerases and general transcription factors required fro transcription initiation. Activator proteins
bind to control elements near the transcription start site as well as kilobases away and promote
chromatin decondensation and binding of RNA polymerase to the promoter. Repressor proteins
bind to alternative control elements, causing condensation of chromatin.
A DNA sequence that specifies where RNA polymerase binds and initiates transcription of a gene
is called a promoter. Transcription from a particular promoter is controlled by DNA-binding proteins,
termed transcription factors. TFs regulating expression can bind at regulatory sites tens of
thousands of base pairs upstream or downstream from the promoter, and they induce the
positioning of RNA polymerase at the start site. As a result, transcription from a single promoter
may be regulated by binding of multiple TFs to alternative control elements, permitting complex
control of gene expression. There are about 2000 different TFs in the human organism.
In many transcribed genes, there is a conserved sequence TATA box, 25-35 bp upstream of the
start site. A mutation in this sequence drastically reduces transcription of these genes. Mutating the
sequence upstream of the start sites and checking the expression level of a gene may give us
information about the sequences necessary for gene activation. These are found both proximal and
distal to the initiation site.
Activators and repressors that bind to specific sites in DNA regulate expression by two general
mechanisms. First, they act in concert with other proteins to modulate chromatin structure, thereby
influencing the ability of general TFs to bind to promoters. DNA is wrapped around histones,
forming nucleosomes. Acetylation (acetyl groups are acid) of histones influence the relative
condensation of chromatin. Acetylation of lysine neutralizes the positive charge normally present,
thus reducing affinity between histone and (negatively charged) DNA which renders DNA more
accessible to transcription factors. Activator and repressors also interact with a large multiprotein
complex called the mediator of transcription complex, or simple mediator. This complex binds to
RNA polymerase and directly regulates assembly of the transcription preinitiation complex, which is
formed by the polymerase and many other proteins, and is necessary for transcription initiation.
The mediator also has acetylase activity.
A transcription factor is composed of separable functional domains, a DNA-binding domain, which
binds to a specific DNA sequence, and an activation domain which interacts with other proteins to
stimulate transcription from a nearby promoter. Many TFs form heterodimers, and interact with
DNA in this form. The resulting combinatorial possibilities increase the number of potential DNA
sequences that a family of TFs can bind. Three factor monomers could combine to form six dimeric
factors. Some inhibitory factors bind to TFs, blocking their binding to DNA. As a result of this kind
of combinations, the 2000 human TFs can differentially regulate the tens of thousands of human
genes.
The transcription-control regions of most genes contain binding sits for multiple TFs factors that
affect transcription in different ways. Transcription of such genes varies depending on the particular
repertoire of TFs expressed and activated in a particular cell at a particular time.
Nature Reviews Neuroscience 3, 921-931 (2002)
REGULATION OF TRANSCRIPTION FACTORS BY NEURONAL ACTIVITY
Anne E. West, Eric C. Griffith & Michael E. Greenberg
Only a subset — in the range of 15 to 300 — of the genes expressed in the nervous system show
activity-dependent upregulation11, 12. The number of genes whose expression is decreased in
response to synaptic activity is an order of magnitude lower, indicating that gene induction is the
favoured process for activity-regulated changes in neuronal function12
CREB: a multifunctional transcription factor
The activity of CREB is induced in many cell types in response to a variety of extracellular signals;
in neurons, these include neurotransmitters, neuromodulators and neurotrophic factors. More than
100 genes have functional CREs in their promoters, consistent with a role for CREB in many
biological processes22. (CRE site, ATGACGTCAT)
CREB-dependent transcription is induced when activated signal-transduction pathways trigger the
phosphorylation of CREB. Membrane depolarization or an elevation of cAMP strongly induces the
phosphorylation of CREB at Ser133. Many kinases can phosphorylate CREB at Ser133. but
specific kinases predominate in response to different stimuli. In neurons, there is strong in vivo
evidence that the nuclear protein kinase CaMKIV is crucial for the rapid activity-dependent
phosphorylation of CREB at Ser133 . However, pharmacological-inhibition studies indicate that
prolonged activity-dependent phosphorylation of CREB at Ser133 is mediated by calciumdependent activation of the Ras–MAPK (mitogen-activated protein kinase) pathway. Eventually,
calcium influx also leads to the dephosphorylation of CREB at Ser133 through calcium-dependent
activation of the protein phosphatases PP1 and PP2A23, 24, although the mechanism by which
neuronal activity regulates these phosphatases is unclear.
Fig: A few minutes after stimulation. In response to neurotransmitters, calcium flows into the cell
through synaptic and extrasynaptic ligand- and voltage-gated channels, including NMDA (Nmethyl-D-aspartate) receptors (NMDARs) and L- and N-type voltage-gated calcium channels
(VGCCs). active propagation of membrane depolarization from the dendrites to the cell body;
calcium-induced release of calcium from internal stores; direct translocation of the calciumsignalling molecule calmodulin from peripheral channels into the nucleus; and local
calcium/calmodulin (CaM)-dependent activation of signalling pathways near the calcium channels,
followed by the translocation of kinases to the nucleus. Once the calcium signal reaches the
nucleus through one of these pathways, calcium/CaM-dependent protein kinase IV (CaMKIV)
rapidly phosphorylates CREB at Ser133. However, if the Ras–MAPK (mitogen-activated protein
kinase) pathway is not activated, CREB phosphorylation is transient and transcription is not
activated. Several minutes later. The Ras–MAPK pathway is activated by both NMDARs and LVGCCs. Activated MAPK phosphorylates and activates pp90 ribosomal protein S6 kinase (Rsk),
then both MAPK and Rsk translocate to the nucleus. In the nucleus, Rsks phosphorylate CREB at
Ser133
One possibility is that calcium elevation in the nucleus is required to activate CREB kinases and
phosphatases. Alternatively, signalling pathways near the plasma-membrane calcium channels
might be activated locally by calcium influx and then translocate in an activated form to the nucleus.
The localized elevation of calcium just beneath sites of calcium entry is sufficient to trigger the
phosphorylation of CREB at Ser133 even when the global cytoplasmic calcium rise is blocked,
consistent with the hypothesis that signalling pathways are activated close to or at calcium
channels.
A sequence known as an IQ DOMAIN in the carboxy-terminal tail of the 1C subunit of the L-VGCCs
was found to be crucial for the L-VGCC-dependent activation of CREB. The calcium-signalling
molecule calmodulin binds the IQ domain in a calcium-dependent manner48, and mutations in the
IQ domain that block calmodulin binding also block the activation of CRE-dependent gene
transcription while retaining normal calcium influx. These mutations selectively inhibit the ability of
calcium entering through the L-VGCC to activate the Ras–MAPK pathway, and the activation of
channels with IQ-domain mutations fails to induce the prolonged Ras–MAPK-dependent CREBSer133 phosphorylation that is associated with gene transcription. As calmodulin binding is
selectively required for activation of the Ras–MAPK pathway, it is possible that components of this
pathway51 are also localized to this channel complex. Further analysis of the signalling molecules
that are selectively associated with different types of channel might reveal the root of the L-VGCC's
privilege in the activation of CREB (??).However, the NMDAR-mediated calcium-dependent
activation of PP1 can oppose this signal, triggering the dephosphorylation of CREB53. Hardingham
et al.54 dissociated the functions of distinct NMDAR populations in the cell, showing that activation
of synaptic NMDARs leads to the phosphorylation of CREB, whereas activation of extrasynaptic
NMDARs dephosphorylates and inactivates CREB. Intriguingly, glutamate-induced activation of the
NMDAR-dependent phosphatase is inhibited by ifenprodil, a selective inhibitor of the NR2B subunit
of the NMDAR54. As the NR2A and NR2B subunits have been reported to be differentially
distributed between synaptic and extrasynaptic sites, the selective association of signalling
molecules with these subunits might specify whether calcium influx through this receptor leads to
the activation of CREB kinases or CREB phosphatases.
Integration of activation signals
Ser133 phosphorylation enhances CREB ability to recruit the transcriptional coactivator
CREB-binding protein (CBP). CREB is also phosphorylated at Ser 129, 142 and 143.
Phosphorylation of Ser 142 and 143 disrupts CREP-CBP interaction. Some of the stimuli
that phosphorylate Ser133 also phosphorylated Ser 142,143. This indicates that CREB
have some other mechanisms, or that they associate by other domains. Ser 142,143 are
phosphorylated by some specific stimuli, such as membrane depolarization and glutamate
stimulation, but not in response to cAMP elevation or GFs. Ser 142,143 phosphorylation
may confer specific complex formation that regulates specific genes.
NFAT and NF-kB
Signal transduction may be initialized in synapstic boutons and expand to the nucleus.
One way of doing that is by TFs relocalization, as is the case for NFAT and NF-kB.
VGCC and NMDAR activates the phosphatase calcineurin, which dephosphorylates
NFAT and translocates it to the nucleus, where it acts in complex with NFATn.
Calcineurin, in the immune system, is activated by other signals, as antigen-receptors.
NF-kB is a family with several members that form homo and heterodimers and stimulate
gene activation. NF-kB is regulated by the interaction with specific inhibitor proteins as
IkBs (inhibitors of kB). Different stimuli induces phosphorylation (IKK (kinase)),
ubiquitination, and degradation of IkB and release of NF-kB.
Regulation of chromatin architecture
CBP and p300 are HATs. This elements are recruited to interact with different TFs such
as CREB following depolarization. Myocite-enhancer factor 2 (MEF2) family members
are controlled by Ca at several levels. Different CamKs dissociate HDACs from MEFs
and translocate the formers to the cytoplasm. CamK phosphorylates two residues
conserved in all HDACs proteins. Phosphorylation of these sites generate a docking-site
for 14-3-3 chaperone protein. Docking of 14-3-3 expose a nuclear export signal (NES) in
HDACs, resulting in HDAC release from MEFs.
The co-repressor calcineurin-binding protein 1 (cabin1) is another Ca regulated repressor
of MEF2 activity. It attaches some HDAC to MEF2. CaMK binding to cabin1, disrupts
its complex with MEF, and apparently also exports the repressors outside the nucleus.
GABAA receptors: building the bridge between subunit mRNAs, their
promoters, and cognate transcription factors Janine L. Steiger and Shelley J. Russek
As the major inhibitory neurotransmitter in the adult brain, γ-aminobutyric acid (GABA) elicits rapid
synaptic responses by stimulating the ionotropic type A GABA (GABAA) receptor. GABAA receptors
belong to a superfamily of ligand-gated ion channels that mediate fast inhibitory neurotransmission
in the mammalian brain. Stimulation of pentameric GABAA receptors by GABA triggers the opening
of an integral Cl− channel. There are at least 19 different receptor subunits, which have been
grouped by amino acid sequence homology into eight subclasses: α1–6, β1–3, γ1–3, δ, , π, θ, and
ρ1–3 (Rabow et al., 1995; Wilke et al., 1997 and Bonnert et al., 1999). The extensive diversity of
GABAA receptor subunits may allow for the assembly of thousands of possible pentameric
combinations;
The enormous heterogeneity of inhibitory synapses suggests that receptor diversity may fulfill
specific functional requirements of synaptic transmission. Given the fundamental role of GABA A
receptors in controlling neuronal excitability, it is critical to understand the molecular mechanisms
by which they are regulated. At the cellular level, GABAergic function is controlled by changes in
neurotransmitter synthesis, vesicular storage, neurotransmitter release, neurotransmitter re-uptake,
and postsynaptic receptor clustering. The significance of genetic control over GABAergic
transmission is underscored by the temporal and region-specific expression of receptors in the
developing and mature brain
Pharmacological analyses of recombinant GABAA receptors suggest that the structural
heterogeneity of GABAA receptor subtypes may determine the functional properties of receptors
expressed at the cell surface
Genomic mapping
Changes in the function of the GABAA receptor may reflect the differential regulation of the genes
coding for the GABAA receptor subunits. Each GABAA receptor subunit is encoded by homologous,
yet distinct genes. Many of the subunit genes are organized in β-α-α-γ and β-α-γ gene clusters on
different chromosomes. The fact that GABAA receptor subunit genes are found in clusters strongly
suggests that such an arrangement facilitates their coordinate, developmental, and/or tissuespecific expression; although, at this time, there is no direct evidence to suggest that this is indeed
the case.
For example: The β1-α4-α2-γ1-subunit genes are localized to chromosome 4p14–q12. the α2, α4,
and β1 genes are highly expressed in the hippocampal formation of the adult rat (mostrar foto
Wisden 1992)
The physical linkage of GABAA receptor subunits in the genome may be required for coordinate
gene regulation. In theory, the intergenic region may contain regulatory elements that directly
influence the transcription of two loci. Although subunit mRNA levels in some brain regions closely
correlate with the expression of neighboring subunit genes in GABAA receptor gene clusters,
coordinated expression of GABAA receptor subunit genes in the human hippocampal dentate gyrus
cannot be explained by their position on the chromosome (Brooks-Kayal et al., 1999). In individual
human dentate granule cells, α4, β2, and δ gene expression is tightly correlated; yet, each subunit
is localized to a different gene cluster. Furthermore, α4-subunit mRNA is abundant in the thalamus,
while the gene products of its neighbors α2, β1, and γ1 are barely detectable (Wisden et al., 1992).
Thus, the coordinate expression of GABAA receptor subunit gene clusters alone cannot account for
the choice of GABAA receptor subtypes in the nervous system.
(Fig: Coordinated expression of GABAR subunit mRNAs within individual DGCs. Correlation
analysis was performed among the hybridization signals (integrated intensity of pixels normalized
to background) of 14 different GABAR subunits in each cell. The hybridization of 4-, -, and 2subunit mRNAs in individual DGCs correlates positively with each other (top row). 2 mRNA
hybridization also correlates with 3-subunit mRNA (bottom row, left), whereas -mRNA
hybridization correlates strongly with that of 1-mRNA (bottom row, right). Subunits demonstrating
correlation coefficients (r) with a Bonferroni adjustment of >0.90 and a p value <0.001 were
considered to be highly correlated. None of the other 86 potential subunit pairings was found to be
highly correlated)
The expression of many of this subunits is correlated with steroids, ethanol, in epilepsy and in other
neurodegenerative diseases.
While the DNA sequence of individual subunit promoters is certainly not similar, short DNA motifs
are conserved in the promoter regions of specific α-, β-, and γ-subunit genes. We have constructed
promoter-specific regulatory element libraries using computationally derived information about
individual transcription factors and their corresponding consensus sites.
For example:
In chronically epileptic dentate granule cells, a selective decrease in α1 and β1 subunit mRNA is
accompanied by an increase in the levels of α4, β3, δ, and ε mRNA. First, different regulatory sites
within the α1 and α4 promoters may mediate differential changes in their gene expression during
epileptogenesis. The idea is to check what TFs affects the upregulated and which the
downregulated. In particular, MAZF, ZBPF, EGRF, and ZF5 consensus sites are specifically
present in the α4, β3, δ, and subunit promoters. Previously, it has been demonstrated that the
expression of EGRF family proteins is markedly induced in an animal model of TLE. Similarly, the
α1 and β1 subunit promoters contain regulatory elements that recognize forkhead domain factors
(FKHD), OCTP, and serum response element binding factors (SRFF) transcription factors. The
FKHD- and OCTP-specific sites are not present in α4, β3, δ, and promoter sequences spanning
200-bp upstream from the transcriptional start site.
Does this happens in epilepsy?
Previously, it has been demonstrated that the expression of EGRF family proteins is markedly
induced in an animal model of TLE. The same is true for SRFF.
DNA Microarrays
PTSD prognosis by gene expression profile in blood cells
Post-traumatic stress disorder (PTSD) is a maladaptive response to life-threatening events,
consisting of a symptom triad of re-experiencing of the traumatic event, avoidance and numbing,
and increased vigilance and arousal. Early treatment might prevent PTSD,11 but known risk
factors12 and early PTSD symptoms13 do not effectively predict chronic PTSD, and therefore have
limited use in guiding early treatment. Biological alterations may underlie the onset severity and
persistence of PTSD symptoms.3, 4, 14 Such alterations are likely to be associated with differential
gene transcription, during or after exposure to the triggering event. Acute stress exposure has
been shown to induce long-term expression differences in the rat brain for cholinergic15 and
neuroendocrine genes.16, 17 While direct sampling of the brain is not possible in humans, peripheral
blood cell gene expression may provide a surrogate indicator of differential response to stress and
subsequent PTSD. Supporting this tenet, acute psychological stress is associated with immune
activation,18 and persistent immune alterations have been linked with chronic PTSD.
A Peripheral Blood Mononuclear Cell (PBMC) is a blood cell having a round nucleus, such as a
lymphocyte or a monocyte. A lymphocyte is a natural killer cells (NK cells). The small lymphocytes
are the T cells and B cells. Monocytes are responsible for phagocytosis (ingestion) of foreign
substances in the body. Monocytes which migrate from the bloodstream to other tissues are called
macrophages.
Total RNA extracted from PBMCs was used for sample preparation and hybridization as
recommended by the manufacturer of the arrays
Using oligonucleotide arrays (Affymetrix HU95A), we measured gene expression profiles from
PBMCs sampled immediately after trauma at the ER and 4 months after the trauma (M4) from
these 24 subjects.
After signal quantitation and normalization, we identified a set of 4512 active transcripts that are
expressed and show some variance among the collected profiles. We identified 4512 'active'
transcripts as those that had at least one value between 50 and 7500, one 'Present' call by
Microarray Analysis Suite 5.0 (Affymetrix, Santa Clara), and changed in at least one sample twofold or more from the geometric mean of all samples.
We first determined whether gene expression patterns could distinguish PTSD from control
subjects. PTSD is a complex disorder showing a spectrum of severity. To get the most informative
view of the core phenotype, we focused, for this comparison, on the difference between subjects
exhibiting the consistent phenotype (subjects with full persistent PTSD (1–8 Table 1) and subjects
showing no PTSD criteria at any time (14–19)
We identified differentially expressed genes using three test statistics: TNoM, t-test and Info27; the
significance of each score was determined as described previously.26 Genes that had a P-value
<0.05 in all three scoring methods were considered differentially expressed.
Unsupervised hierarchical clustering distinguishes the clinical status at 1 and 4 months (1a). When
only M4 samples were analyzed, all subjects were classified into two clusters, one containing
PTSD subjects and the other control subjects (1c). A similar pattern (with one misclassified subject)
is evident in clustering of samples taken at ER, hours after trauma (Figure 1c), suggesting that
gene expression patterns at the immediate aftermath of trauma may be informative of the later
development of the PTSD phenotype.
To better characterize the differences between PTSD and control subjects, we identified genes that
are differentially expressed between the two groups. Of the 4512 active transcripts, we find a
signature of 656 transcripts that are differentially expressed between PTSD and control samples
(see Materials and methods and Supplementary Methods). This number is significantly larger than
expected by chance (P=0.007, Figure 1d).
To further explore the predictive abilities of these gene expression signatures, we used the Naïve
Bayesian classifier. The leave-one-out cross validation (LOOCV) procedure was used to evaluate
the classification accuracy of the classifier in either M4 samples or ER samples (see Materials and
methods). The classifier was able to classify correctly eight out of nine M4 samples (Figure 1g) and
nine out of 11 ER samples (Figure 1h).
In contrast, the groups did not show statistically significant differences in type of trauma, trauma
severity scores, age, gender, ethnic origin and psychiatric comorbidity
To gain better understanding of these informative transcripts, we examined their functional
classifications. We identified several functional groups that are represented in these signatures
(Figure 4a) Notably, we observe downregulation of transcripts encoding for proteins that are
involved in transcriptional activation, and cell cycle and proliferation among affected subjects (eg
whether defined as consistent PTSD or according to PTSD IES symptoms severity). We also
observe distinct expression signatures for transcripts involved in immune activation, signal
transduction and apoptosis. To attempt a quantitative analysis, we considered the annotations
available through the Gene Ontology (GO) database. The percentage of GO annotation was
calculated among the informative subset of genes that separate PTSD from controls, and was
compared to the percentage among all 4512 active genes on the chip. Significantly increased
representations (P<0.0005) of genes involved in RNA metabolism and processing, as well as
nucleotide metabolism, was found in the consistent PTSD signature (Figure 4b).
REGULATION OF TRANSCRIPTION FACTORS BY NEURONAL ACTIVITY
Anne E. West, Eric C. Griffith & Michael E. Greenberg Nature Reviews Neuroscience 3,
921-931 (2002)
Translation – Protein synthesis
The genetic code is encoded in a three-nucleotide sequence, or codon, which is read from a
specified starting point in the mRNA. The four bases give 64 possible codons, 61 of them specify
one of the 20 aa and three are stop codons that signal the end of translation. The initiation codon is
ATG which codifies Met. The sequence of codons that runs from a specific start codon to a stop
codon is called a reading frame, and it specifies the linear sequence of aa in the future polypeptide
chain.
Translation requires tRNAs. tRNAs are a group of 30-40 molecules ~80 nucleotides long, folded
into a stem-loop arrangement. One of the stems contain an anti-codon, complementary to a unique
codon. Despite so, the base-pairing is not perfect, but is degenerated in the 3’ base of the mRNA.
Attachment of the appropriate aa to tRNA is catalyzed by a specific aminoacyl tRNA synthetase. 20
different synthetases recoifnize one amino acid each and all its compatible tRNAs. The enzymesa
link an aa to the free 2’ or 3’ hydroxyl of the adenosine at the 3’ terminus of tRNA molecules. The
aa is linked to the tRNA by a high energy bond which will drive the formation of peptide bonds
linking adjacent aa in the frowing polypeptide chain.
The efficiency of translation depends by the ribosome, which directs elongation of a polypeptide at
a rate of three to ffive aa per second. The ribosome is a very big complex, formed by four rRNA
molecules and 83 proteins, organized into two large subunits. During translation, a ribosome
moves along an mRNA chain, interacting with various protein factors and tRNAs.
RNA structure
The hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA. As a result RNA
is cleaved into mononucleotides by alkaline solution, RNA can participate in helixes formation
composed of DNA-RNA or RNA-RNA. Most RNAs are single stranded and exhibit a variety of
conformations, carrying out different functions in a cell. Two secondary structures in singlestranded (ss) RNA are formed by pairing of complementary bases. “hairpins” formed by pairing
bases within 5-10 nucloetides of each other, and “stem-loops” by pairing of bases separated
by >10 nucleotides. These simple folds cooperate to form more complicated tertiary structures, as
a “pseudoknot”. These tertiary structures have, as proteins, catalytic capacities, participating in
functions such as mRNA splicing.