Molecular genetics is
primarily concerned with the inter-relationship between the information
macromolecules DNA (deoxyribonucleic
acid) and RNA
(ribonucleic acid) and how these molecules are used to synthesize polypeptides,
the basic component of all proteins.
As the vast majority of gene
expression is dedicated to polypeptide synthesis,
proteins are the major functional end-points
of the DNA template and account for the majority of the dry weight of a cell.
The term protein is derived from the Greek proteios, meaning 'of the
first rank' and reflects the important roles of proteins in diverse cellular
functions, as enzymes, receptors, storage proteins, transport proteins,
transcription factors, signaling molecules, hormones, etc.
composed of one or more polypeptide molecules which may be modified by the
addition of various carbohydrate side chains or other chemical groups.
Like DNA and RNA, polypeptide
molecules are polymers consisting of a linear sequence of repeating units, in
this case amino acids.
The latter consist of a positively charged amino
group and a negatively charged carboxylic acid (carboxyl) group connected by a
central carbon atom to which is
attached an identifying side chain.
Charged molecules are highly soluble in water.
and RNA are
negatively charged (polyanions) because of the
charges present in their component nucleotides.
Depending on their
amino acid composition, proteins may carry a net positive charge (basic
proteins) or a net negative charge (acidic proteins).
bonding potential of water molecules means that molecules with polar groups
(including DNA, RNA
and proteins) can form multiple interactions with the water molecules, leading
to their solubilization.
Thus, even electrically neutral proteins are
often readily soluble if they contain an appreciable number of charged or
neutral polar amino acids.
The linear backbone of a
DNA molecule and of an
consists of alternating sugar residues and
within a cell normally exist as single molecules, the structure of DNA is a
double helix in which two
DNA molecules (DNA
strands) are held together by weak hydrogen bonds to form a DNA
DNA can adopt different types of helical
Genetic information is encoded by the linear sequence of
bases in the DNA strands (the primary structure).
hydrogen bonding permits RNA-DNA duplexes and double-stranded RNA formation
which are important requirements for gene expression.
can occur between bases within a single DNA or RNA molecule.
process of DNA synthesis (DNA replication), the two DNA strands of each
chromosome are unwound by a helicase enzyme and each DNA strand directs the
synthesis of a complementary DNA strand to generate two daughter DNA duplexes,
each of which is identical to the parent molecule.
DNA replication is
initiated at specific points, which have been termed origins of replication.
According to their needs, different cells transcribe different segments
of the DNA (transcription units) which are discrete units, spaced irregularly
along the DNA sequence.
Only a portion of the RNA made by transcription
is translated into a polypeptide.
The expression of genetic
information in all cells is mostly a one-way system: DNA specifies the
synthesis of RNA and RNA specifies the synthesis of polypeptides, which
subsequently form proteins.
Because of its universality, the DNA >
RNA > polypeptide (protein) flow of genetic information has been described
as the central dogma of gene expression
shape and structure of proteins is a crucial aspect of molecular gene
expression and links our understanding of gene expression to the biology of the
While primarily concerned with protein molecules that act on DNA
and RNA sequences, such as transcription factors and histones, the study of
gene expression also focuses on where in the cell expression is
In fact, the modulation of gene expression can occur in the
nucleus, the cytoplasm, or even at the cell membrane due to
the impact of proteins on RNA in those cellular
In his Nobel lecture, given shortly
after he joined the Rockefeller Institute for
Medical Research, Edward L. Tatum outlined the concepts fundamental to his
one-gene, one-enzyme (understood today as one-gene, one-polypeptide)
processes in all organisms are under genetic control;
processes are resolvable into a series of individual reactions;
reaction is controlled in a primary fashion by a single gene - 1:1
correspondence of gene and biochemical reaction exists;
mutation of a
single gene results only in an alteration in the ability of the cell to carry
out a single primary chemical reaction.
Geneticist George W. Beadle and
the biochemist Edward L. Tatum were awarded the Nobel Prize largely through the
auspices of the Rockefeller Foubdation, for this hypothesis which turned out to
be entirely false !!!
A cell uses the DNA molecule in the nucleus as a
template for protein production.
The cell sends a 'messenger RNA' = mRNA
into the nucleus to retrieve the encoding.
The mRNA takes the copied
"recipe" out of the nucleus to the ribosome, which is where proteins are
In eukaryotic cells (the kinds of cells found in plants and
animals), however, something very interesting happens before the mRNA leaves
Some parts of the mRNA are cut away, and the remaining
parts are then stitched back together.
The parts of the mRNA that are
cut away never leave the nucleus, so they are called introns (they stay IN the
Introns regulate the amount of the various proteins that are
"For a while, geneticists didn't know the purpose of
introns, so in typical evolutionary fashion, many decided that they had no
purpose, and introns were lumped into the category of "junk DNA." As we have
learned more about genetics, we have learned that the evolution-inspired idea
of "junk DNA" is, itself, junk, although some evolutionists still cling to it."
- Jay L. Wile
The remaining parts that are stitched together are called
exons (they EXit the nucleus).
Each exon represents a "module" of useful
If the cell stitches the exons together in one way, it
makes one protein.
If it stitches the exons together in another way, it
makes a different protein.
As a result, a single gene can actually produce
many different proteins.
molecular biology is based on this
Genetically modified organisms
and the entire petrochemical
pharmaceutical industry is
based on a theory that has
turned out to be entirely false !!!
We should not be surprised at
Rockefeller's hand in this as John D. Rockerfeller's father made his fortune
selling "patent" medicine.
William Avery "Bill" Rockefeller, Sr.
(November 13, 1810 May 11, 1906) was
an American confidence
man who went by the alias of Dr. William Levingston.
He worked as a
traveling salesman who identified himself as a "botanic physician" and sold
His sons, John
Davison Rockefeller (July 8, 1839 May 23, 1937) and
William Avery Rockefeller, Jr.
(May 31, 1841 June 24, 1922), were Standard Oil
Several systems for induction of transgene expression in
plants have been described recently.
Inducible systems were used mainly
in tobacco, rice, Arabidopsis,
tomato, and maize.
Inducible systems offer researchers the possibility to deregulate gene
expression levels at particular stages of plant development and in particular
tissues of interest.
The more precise temporal and spatial control,
obtained by providing the transgenic plant with the appropriate chemical
compound or treatment, permits to analyze also the function of those genes
required for plant viability.
Specific mutation of a gene can be
achieved by a two-step process.
Introduction of loxP sites around a
functionally essential genomic part followed by a cell type-specific Cre
recombinase-mediated excision of the loxP flanked sequence.
strategy can be used for cell type-specific overexpression of a transgene, when
a strong overall expressing promoter is separated from the coding region of a
gene of interest by loxP flanked 'STOP' sequences.
In both scenarios, a
Cre recombinase transgene provides spatial control.
Cre expression has been switched on and recombination has occurred, the
resultant change in gene expression is, in most cases,
In the quest for the
development of pharmacological switches that control gene expression, no system
has been reported that regulates at the translational level but several systems
have been constructed:
-Tetracycline-inducible transgenic systems
[tetracycline transactivator (tTA) or 'Tet-Off' and reverse tetracycline
transactivator (rtTA) or 'Tet-On'] allow for reversible temporal regulation of
transgene expression .
Between these, rtTA is better suited for rapid
induction of gene expression.
-To permit small-molecule control of
transgene translation a farnesyl transferase inhibitor-responsive translation
initiation factor was constructed.
This artificial protein is a
three-component chimaera consisting of the ribosome recruitment core of the
eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the
R17 bacteriophage coat protein and the plasma membrane localization CAAX motif
of farnesylated H-Ras.
This membrane-delocalized translation factor is
inactive unless liberated in the cytosol.
inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus
increases the cytoplasmic levels of the eIF4G fusion protein, which is then
capable of inducing translation of the second cistron of a bicistronic
messenger RNA containing an R17-binding site in its intercistronic space.
- The concept of cisgenesis could become a promising approach in future
However, cisgenesis depends on the availability of
effective tools for the production of marker-free genetically modified (GM)
The development of such plants was recently shown to be
possible using a heat shock inducible Flp/FRT recombinase based transformation
system allowing the excision of the marker gene from the genome of GM apple
- A new laser mediated
method heat shocks groups
of cells allowing precise spatio-temporal control of gene expression without
requiring knowledge of specific enhancer sequences.
Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to
achieve a high level of foreign gene expression in insect cells, as well as for
efficient gene transduction into mammalian cells without any replication.
In addition to permitting efficient gene delivery,
baculovirus has been shown to induce host innate immune responses in various
mammalian cells and in mice.
barrier to the clinical application of adenovirus gene therapy for diseases
that require stable transgene expression is the immunogenicity of recombinant
adenovirus, which ordinarily limits the duration of its effects to a period of
about 2 weeks.
If tolerance to adenovirus could be induced then
transgene expression could be prolonged if T lymphocytes underwent thymic
selection in the presence of adenovirus antigens.
The ability to
achieve unresponsiveness to a recombinant adenovirus after inoculation of the
thymus in neonates extends the paradigm of intrathymic tolerance induction.
T cells are exquisitely poised to respond rapidly to pathogens and have
proved an instructive model for exploring the regulation of inducible genes.
Individual genes respond to antigenic stimulation in different ways,
and it has become clear that the interplay between transcription factors and
the chromatin platform of individual genes governs these responses.
Understanding of the complexity of the chromatin platform and the
epigenetic mechanisms that contribute to transcriptional control has expanded
dramatically in recent years.
These mechanisms include the
presence/absence of histone modification marks, which form an epigenetic
signature to mark active or inactive genes.
These signatures are
dynamically added or removed by epigenetic enzymes, comprising an array of
histone-modifying enzymes, including the more recently recognized
chromatin-associated signalling kinases.
chromatin-remodelling complexes physically alter the chromatin structure to
regulate chromatin accessibility to transcriptional regulatory factors.
The advent of genome-wide technologies has enabled characterization of
the chromatin landscape of T cells in terms of histone occupancy, histone
modification patterns and transcription factor association with specific
genomic regulatory regions, generating a picture of the T-cell
The use of
transgenic animals in the field of molecular genetics is
Molecular genetics experiments use only model organisms
with known regulatory DNA sequences, i.e. enhancers, that drive gene expression
at particular times in development and in particular cells.
By reducing the variables it
becomes easier to predict mutational effects with transgenic animals.
It is extremely difficult
to predict how these genetic switches would function in animals outside of
those specifically bred for the purposes of these
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