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molecular genetics

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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.

Proteins are 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.

Polymers: "Man-Made Miracles" 1954 BF Goodrich

Charged molecules are highly soluble in water.

Both DNA and RNA are negatively charged (polyanions) because of the organophosphate 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).

The hydrogen 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 RNA molecule consists of alternating sugar residues and organophosphate groups.

Whereas the RNA molecules 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 duplex.

DNA can adopt different types of helical structure.

Genetic information is encoded by the linear sequence of bases in the DNA strands (the primary structure).

Intermolecular hydrogen bonding permits RNA-DNA duplexes and double-stranded RNA formation which are important requirements for gene expression.

Hydrogen bonding can occur between bases within a single DNA or RNA molecule.

During the 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.

Gene Expression

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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 molecular biology.

The 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 cell.

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 modulated.

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 subregions.

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) hypothesis:

all biochemical processes in all organisms are under genetic control;

biochemical processes are resolvable into a series of individual reactions;

each 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 made.

In eukaryotic cells (the kinds of cells found in plants and animals), however, something very interesting happens before the mRNA leaves the nucleus.

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 nucleus).

Introns regulate the amount of the various proteins that are being made.

"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 information.

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.

ALL of molecular biology is based on this fatal error.

Genetically modified organisms and the entire petrochemical pharmaceutical industry is based on a theory that has turned out to be entirely false !!!

HW Bush taught how to genetically modify organisms at Monsanto

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 elixirs.

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 co-founders.

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.

The same 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.

Once Cre expression has been switched on and recombination has occurred, the resultant change in gene expression is, in most cases, irreversible.

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.

Farnesyl transferase 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 apple breeding.

However, cisgenesis depends on the availability of effective tools for the production of marker-free genetically modified (GM) plants.

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 plant tissue.

- 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.

-The baculovirus 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.

-The major 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.

In addition, 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 epigenome.

The use of transgenic animals in the field of molecular genetics is standard.

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 experiments.

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