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RNAi
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Gene Silencing and Inactivation
By: Brad Bullock
April 22nd, 2005 - bama.ua.
pdf
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Primer on
Molecular
Genetics - primer.pdf
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| Genomic Medicine — A Primer
Alan E. Guttmacher, M.D., and Francis S. Collins, M.D., Ph.D.
- NEJM
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GLOSSARY:
Amino acids: peptide building blocks of
proteins.
Bases: a building block of DNA and RNA. There are five different
bases or nucl: Adenine, Thymine, Guanine, Cytosine (ATGC).
DNA stands for Deoxyribonucleic acid. It's a double-stranded chemical instruction manual for
cell structure and everything a cell does: grow, divide, even when and how to die.
RNA stands for Ribonucleic acid. RNA are single- stranded chemical instructions double-stranded, messenger RNA
(mRNA) carries single pages of instructions out of the nucleus to places they're needed throughout the cell. RNAi
stands for RNA interference Polypeptides: chains of amino acids. Proteins are made up of several or many polypeptides.
Proteins: Molecules that make up the unique cell structure
(skin, heart, long, immune cell) and carry out activities throughout the body,
such as pumping blood (heart cells), to migration (immune
cells).
encoded protein: the recipe for protein as contained in
messenger RNA.
Ribosomes are message centers throughout the cell where the information from DNA arrives in the form of messenger RNA.
Here the RNA message gets translated into a form the ribosome can understand and tells it which protein building blocks it needs and in what order to assemble them. SNPs
(polymorphisms) (markers of biologic diversity)
... "the DNA sequences of two unrelated humans
vary at millions of bases. ... we are each thus
heterozygous at about 3 million bases. Many efforts are
currently under way, ... to catalogue these variants,
commonly referred to as "single-nucleotide polymorphisms"
(SNPs), and to correlate these specific genotypic
variations with specific phenotypic variations relevant
to health.
Some SNP–phenotype correlations occur as a direct
result of the influence of the SNP on health. More
commonly, however, the SNP is merely a marker of
biologic diversity that happens to correlate with
health because of its proximity to the genetic factor that
is actually the cause." - NEJM
Transcription
is the process where messenger RNA information is obtained from DNA
information. Translation is the
process where messenger RNA information is interpreted in the
Ribosome and used to assemble the end product - the unique proteins
that determine cellular activity.
Resources:postmodern.com
Methylation
can cause the over- or under- expression of genes
DNA methylation - is a hallmark of
cancer. Specifically, it is the addition of a methyl group to specific
cytosines (the components of DNA) that regulates gene activity.
One analogy for methylation is that of a car
with an ignition that is filled with a glue (methyl). The
ignition (the gene) cannot be turned off so the car runs continuously. Scientists
are looking for agents that can turn off or inhibit the undesirable
methylation (remove the glue that is specific to the tumor
promoting, or tumor silencing, genes).
In summary, malignant behavior (loss of growth control or inability to die) can
be caused by damaged genes (i.e., translocations) and also by
methylation which changes which genes are turned on or off.
Hypermethylation can cause oncogenes to produce
proteins that cause malignant behavior.
Hypomethylation can quiet genes that normally
suppress cancer (tumor suppressor genes).
In this modification, methyl (CH3) groups are
added to the DNA of specific genes within the cell at specific
sites. These groups sit on the DNA and block certain proteins from
binding. In this way, the DNA methylation could be stopping the cell
from operating normally, Teitell said.
"By silencing cellular genes, this type of
modification is damaging a cell's ability to sense its environment
and may be causing it to grow uncontrollably," Teitell said. - unisci.com
Chromatin Remodeling - Protein fibers called
histones that interact with DNA. Drugs that inhibit histone
deacetylases, for example, may enable expression of tumor suppressor
genes.
Illustration of
mRNA transcription
- Click to enlarge
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Here
we provide basic terms
to help you to
understand how genes and gene expression contributes to cancer, and
how recent insights may lead to new drugs that target the underlying
causes of malignancy.
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DNA
stands for Deoxyribonucleic acid. It is the "instruction manual"
for the manufacture of cell proteins that determine cell structure
and activity.
DNA exists in all cells except mature erythrocytes.
The DNA "manual" contains thousands of pages called genes.
DNA is made up of bases (nucleotides) that are arranged in a specific sequence. The specificity of the sequence accounts for the production of a specific protein. |
| Genes contain
the instructions or recipes for the assembly of unique
proteins. Some of these proteins are related to cell growth
and survival.
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RNA:
In order for DNA to produce a protein, an intermediate step is required.
... In this step, DNA is transcribed into
RNA. The sequence of mRNA that encodes a protein is oriented in only one direction, which is known as the "sense" orientation. |
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Oncogenes
are mutated and/or over-expressed versions of normal genes
that can cause a cell to lose growth restraints, fail to
differentiate (mature to a next stage), or prevent the cell from initiating
the programmed cell death process called apoptosis. Some
oncogenes turn on other genes that can promote malignant behavior. |
| Tumor
suppressor genes are genes that protect cells from
becoming a cancer. They may put the brakes on accelerated growth, or initiate
cell death when DNA damage is detected. Therefore, the low expression of tumor suppression genes can lead to
cancer. |
| Epigenetic
refers to the regulation of gene expression - how,
when, where, how much, etc..
Two key areas of epigenetic control
are
Chromatin Remodeling
and
DNA
Methylation (see Sidebar for details) |
| Targeted
therapeutics may be designed to offset the over-expression of oncogenes, or
the silencing of tumor suppressor genes ... that is, treatments
can influence epigenetic regulation
of cells.
* "Over expression of some genes may be responsible for some forms of cancer;
gene silencing and inactivation techniques could be a cure for these diseases."
- bama.ua.edu
pdf
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Antisense RNA experiments lead to the discovery of
RNAi. |
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RNA Interference
[RNAi] - post-transcriptional gene silencing.
"The antisense approach to gene silencing involves injecting an organism with RNA sequence complementary to mRNA transcribed from a target gene. The antisense RNA and sense mRNA hybridize and block translation and the production of an encoded protein. The presence of dsRNA duplex led to what we now recognize as an RNA interference effect."
Source: miami.edu
Details on RNAi: macalester.edu |
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Gene
silencing is any method of interfering with transcription
or translation of a gene and the protein it produces.* |
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Gene
inactivation is any method that modifies the DNA or
proteins around the DNA that prevents transcription * of DNA
into RNA and thus the encoded protein.
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Molecular Biology:
the path from DNA (information) to proteins (biological activity)
| DNA
> |
RNA
> |
>
Protein |
| Original
instruction manual |
Transcribed
page of the manual |
End
product |
| Alphabet
( blocks) of information: |
Alphabet
(blocks) of information:
|
Assembled
sequence of amino acids |
| Double
stranded information |
Single
stranded information |
Functioning
product. Information on how protein is made is not possible at
this point |
| Error
detection and repair |
No
error protection or repair |
No
error protection or repair |
| Location:
within the nucleus |
Location:
transcribed outside nucleus to areas of cells where it
can be translated into assembly of proteins |
Location:
varied cell locations |
| Cancer:
a mutation in the DNA - one, or more, of the four letters of the DNA is replaced by another letter, or due to deletion or insertion of one or more letters, in one or more DNA
regions |
Cancer:
the transcription and translation of mutated areas of DNA |
Cancer:
abnormal amounts or types of protein that causes loss of
growth control or prolonged survival of the cells - malignant
behavior |
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Polymorphisms are
variations in DNA sequence between individuals.
There are about 60,000 polymorphisms in human genome
But why does it matter to patients?
A big reason for the variable toxicity and efficacy of drugs
is that we are
not the same .... on a genetic level. Our cells may
produce different
enzymes and amounts based on these normal genetic variations
(polymorphisms) ... the recipes for cellular proteins. Enzymes,
for example, that degrade, or fail to degrade, a given drug.
Vincristine for example: The variable half life of
vincristine accounts for
the toxicity it can produce when the body does not degrade it
fast enough.
Anyhow, it's possible to identify the polymorphisms that
influence drug
metabolism so that we can give the "right drug, at the
right dose, to the
right patients." So we can avoid
unproductive toxicities; and select drugs with much more
assurance that it will do more good than harm. In
future, but also now .. for some drugs, for some known
polymorphisms, when there are available devices to use ...
More on the subject: "Genetic variations in
the response to drugs can cause measurable differences in
clinical endpoints such as rates of cure, morbidity, side
effects, and death. Data in this category demonstrate that
genetic variability in the context of a drug effect
significantly changes medical outcomes. These data sets are
different from pharmacodynamics data sets, which may show a
difference that is not sufficiently significant to alter
practice or policy." pharmgkb.org
Example in the literature
for leukemia: "Inter-individual variability in
response to methotrexate could be caused by variable
concentrations of thymidylate synthase. We investigated the
possible association between a tandem-repeat polymorphism in
the thymidylate synthase promoter, of which a triple repeat is
associated with increased expression of thymidylate synthase,
and outcome of acute lymphoblastic leukaemia in 205 children
treated with methotrexate.
We obtained DNA samples from
buccal epithelial cells, peripheral blood, or bone marrow in
remission, and analysed them for the polymorphism by PCR
amplification. Individuals who were homozygous for the triple
repeat had a poorer outlook than those with other genotypes
(odds ratio 4.1, 95% CI 1.9-9.0, p=0.001). Genotyping of
thymidylate synthase might make it possible to individualize
treatment for patients with acute lymphoblastic leukaemia."
PMID: 11937185 - ncbi.nlm.nih.gov
11937185t
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Glossary of Terms
Pharmacodynamics the
study of the relationships between the concentration of a drug
at its site(s) of action and the magnitude of the biological
or physiological effect that is achieved.
Pharmacokinetics - the
study of the bodily absorption, distribution, metabolism and
excretion of drugs.
Drug - A chemical or
biological substance used in the diagnosis,
treatment, or prevention of a disease or phenotype, or as a
component of a
medication.
Molecular assay - an
experiment in which the characteristics of a molecule (or
ensemble of molecules) are measured.
Cellular Assay - an
experiment in which the response or characteristics of a cell
(or population of cells) is measured.
Genotype - is the
internally coded, heritable information carried by the
organism. Variation in genotype represents differences in
sequence within a species, such as SNPs, the location or the
number of
repeats, deletions, or critical splice sites.
Phenotype - the
observable properties of an organism produced by the
interaction of the genotype with the environment. For
pharmacogenetics, the "environment" is often defined
as exposure to a drug, although it may include other variables
as well.
Categories of Pharmacogenetics Knowledge -
http://www.pharmgkb.org
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