Recombinant DNA Technology

Recombinant DNA is a form of artificial DNA which is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences which would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.


The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

Introduction

Because of the importance in DNA in the replication of new structures and characteristics of living organisms, it has widespread importance in recapitulating via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that have effects upon cellular or organismal processes. Through the use of recombinant DNA, genes that are identified as important can be amplified and isolated for use in other species or applications, where there may be some form of genetic illness or discrepancy, and provides a different approach to complex biological problem solving.

Applications and methods
Cloning and relation to plasmids
The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell which remain identical. In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine which contain recombinant DNA inserted into a structure known as a plasmid.

Plasmids are extrachromosomal self replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), contain genes related to catabolism and metabolic activity, and allow the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria is then used as a viral vector, which is encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects and increase the amount of cells with the recombinant DNA present within them.

The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism. As a general definition of plasmids, the definition is that they contain three common features -- a replicator, selectable marker and a cloning site.The replicator or "ori" refers to the origin of replication with regards to location and bacteria where replication begins. The marker refers to a gene which usually contains resistance to an antibiotic, but may also refer to a gene which is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA. The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs. Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception. In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.

Chimeric plasmids
When recombinant DNA is then further altered or changed to host additional strands of DNA, the molecule formed is referred to as "chimeric" DNA molecule, with reference to the mythological chimera which consisted as a composite of several animals. The presence of chimeric plasmid molecules is somewhat regular in occurrence as throughout the lifetime of an organism the propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells which all contain copies of the original chimeric DNA.

In the production of chimeric plasmids, the processes involved can be somewhat uncertainas the intended outcome of the addition of foreign DNA may not always be achieved and may result in the formation of unusable plasmids. Initially, the plasmid structure is linearised to allow the addition by bonding of complimentary foreign DNA strands to single-stranded "overhangs" or "sticky ends" present at the ends of the DNA molecule from staggered, or "S shaped" cleavages produced by restriction endonucleases.

A common vector used for the donation of plasmids originally was the bacterium Escherichia coli and later, the EcoRI derivative which was used for it's versatility with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol and spectinomycin; later being replaced by the pBR322 plasmid.In the case of EcoRI, the plasmid can anneal with the presence of foreign DNA via the route of sticky-end ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase , which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on blunt ends. Both sticky-end, or overhang ligation and blunt-end ligation can occur between foreign DNA segments, and cleaved ends of the original plasmid depending upon the restriction endonuclease used for cleavage













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Rnai Discovery Animation

RNA interference (also called "RNA-mediated interference", abbreviated RNAi) is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid inhibits the expression of genes with complementary nucleotide sequences. Conserved in most eukaryotic organisms, the RNAi pathway is thought to have evolved as a form of innate immunity against viruses and also plays a major role in regulating development and genome maintenance.

The RNAi pathway is initiated by the enzyme dicer, which cleaves double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and base-pairs with complementary sequences. The most well-studied outcome of this recognition event is a form of post-transcriptional gene silencing. This occurs when the guide strand base pairs with a messenger RNA (mRNA) molecule and induces degradation of the mRNA by argonaute, the catalytic component of the RISC complex. The short RNA fragments are known as small interfering RNA (siRNA) which are perfectly complementary to the gene to which they are suppressing as they are derived from long dsRNA of that same gene or MicroRNA (miRNA) which are derived from the intragenic regions or an intron and are thus only partially complementary. The RNAi pathway has been particularly well-studied in certain model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the flowering plant Arabidopsis thaliana.

The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms; synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.

Historically, RNA interference was known by other names, including post transcriptional gene silencing, transgene silencing, and quelling. Only after these apparently-unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. RNAi has also been confused with antisense suppression of gene expression, which does not act catalytically to degrade mRNA but instead involves single-stranded RNA fragments physically binding to mRNA and blocking translation. In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans, which they published in 1999











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Protein Synthesis Animation

Protein biosynthesis (Synthesis) is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription which are then used for translation. Protein biosynthesis, although very similar, differs between prokaryotes and eukaryotes.





Amino acid synthesis


Amino acids are the monomers which are polymerized to produce proteins. Amino acid synthesis is the set of biochemical processes (metabolic pathways) which build the amino acids from carbon sources like glucose. Not all amino acids may be synthesised by every organism, for example adult humans have to obtain 8 of the 20 amino acids from their diet.


The amino acids are then loaded onto tRNA molecules for use in the process of translation.


Transcription is the process by which an mRNA template, carrying the sequence of the protein, is produced for the translation step from the genome. Transcription makes the template from one strand of the DNA double helix, called the template strand. Transcription takes place in 3 stages.
Transcription starts with the process of initiation. RNA polymerase, the enzyme which produces RNA from a DNA template, binds to a specific region on DNA that designates the starting point of transcription. This binding region is called the promoter. As the RNA polymerase binds on to the promoter, the DNA strands are beginning to unwind.
The second process is elongation. RNA polymerase travels along the template (noncoding) strand, synthesizing a ribonucleotide polymer. RNA polymerase does not use the coding strand as a template because a copy of any strand produces a base sequence that is complementary to the strand which is being copied. Therefore DNA from the noncoding strand is used as a template to copy the coding strand.
The third stage is termination. As the polymerase reaches the termination stage, modifications are required for the newly transcribed mRNA to be able to travel to the other parts of the cell, including cytoplasm and endoplasmic reticulum, for translation. A 5' cap is added to the mRNA to protect it from degradation. In eukaryotes, poly-A-polymerase adds a poly-A tail onto the 3’ end for stabilization, protection from cytoplasmic hydrolytic enzymes, and as a template for further processes. Also in eukaryotes (higher organisms) the vital process of splicing occurs at this stage by the spliceosome enzyme. It removes the introns (non-coding bits of genetic material) and glues together the exons (the segments that code for a specific protein).
The mRNA now exits the nuclear pore to be translated.


Translation
Protein translation involves the transfer of information from the mRNA into a peptide, composed of amino acids. This process is mediated by the ribosome, with the adaptation of the RNA sequence into amino acids mediated by transfer RNA. Numerous initation and elongation factors also play a role.

Translation requires a lot of energy, with the hydrolysis of approximately 4 NTP --> NDP per amino acid added. (This includes the aminoacylation of the tRNA. Thus, gene expression is highly regulated to ensure that only proteins that are required are translated.

Translation involves 3 processes: initiation, elongation, and termination.

Initiation in Prokaryotes

The initiation of protein translation involves the assembly of the ribosome and addition of the first amino acid, methionine.
The 30S ribosomal subunit attaches to the mRNA, mediated by IF-1 and IF-3 (initiation factors). The 30S ribosome brings with it the P and A site, but the A site is blocked by IF-1 to prevent binding of tRNA. It aligns to the Shine-Dalgarno sequence, which positions the first codon (AUG) in the P site.
Next, the specific aminoacyl-tRNA for N-formylmethionine (F-Met) is brought into the P site by IF-2. The anticodon of this tRNA will bind to the AUG codon on the mRNA. Note: this is the only tRNA brought into the P site; all successive aminoacyl-tRNAs will be brought to the A site for peptide elongation.
The 50S ribosomal subunit is then brought in to complete the ribosome, and with it, IF-1, IF-2, and IF-3 come off the complex. The A and P site are completed, and the 50S subunit also brings the E (exit) site.

Initiation in Eukaryotes

The initiation of protein translation in eukaryotes is similar to that of prokaryotes with some modifications.
A complex of proteins will connect the 5'cap and 3'PolyA tail, and this complex will recruit the ribosome subunits.
There is no Shine-Dalgarno sequence in eukaryotes. Instead, the ribosome scans along the mRNA for the first methionine codon. Similarly, there is no N-formylmethionine in eukaryotic cells.

Elongation

Elongation of protein biosynthesis is fairly similar between prokaryotes and eukaryotes. The following is a description of elongation in prokaryotes.
Elongation proceeds after initiation with the binding of an aminoacyl-tRNA to the A site, which is the next codon in the mRNA. The aminoacyl-tRNA is brought to the ribosome through a series of interactions with EF-Tu (an elongation factor). This step involves the hydrolysis of GTP: EF-Tu-GTP --> EF-Tu-GDP (The hydrolyzed GDP is switched for GTP through another series of reactions with EF-Ts.)
The next aminoacyl-tRNA binds to the codon, and the C-terminus of the F-Met undergoes nucleophilic attack by the N-terminus of the second amino acid. The F-Met is now connected to the second amino acid through a peptide bond.
The first tRNA (for F-Met) is now uncharged. The entire ribosome complex moves along the mRNA through the action of another elongation factor (EF-G) and the hydrolysis of GTP --> GDP.
The first tRNA is now in the E site and comes off from the ribosome, while the second tRNA, with the nascent peptide chain, is in the P site. Step 1-4 will repeat as successive amino acids are added.


Termination

Termination of protein biosynthesis occurs when the ribosome comes across a stop codon, for which there is no tRNA. At this point, protein biosynthesis halts and one of three release factors will bind to the stop codon. (Note: In eukaryotes, there is only one release factor that will bind to all three stop codons.) This induces a nucleophilic attack of the C-terminus of the nascent peptide by water - this hydrolysis releases the peptide from the ribosome. The ribosome, release factor, and uncharged tRNA then dissociates and translation is complete.


Events following Protein Translation
The events following biosynthesis include post-translational modification and protein folding. During and after synthesis, polypeptide chains often fold to assume, so called, native secondary and tertiary structures. This is known as protein folding.

Many proteins undergo post-translational modification. This may include the formation of disulfide bridges or attachment of any of a number of biochemical functional groups, such as acetate, phosphate, various lipids and carbohydrates. Enzymes may also remove one or more amino acids from the leading (amino) end of the polypeptide chain, leaving a protein consisting of two polypeptide chains connected by disulfide bonds.













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Rna to Protein Lecture

Part 1



Part 2

MicroRNA Animation

MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. They were first described in 1993 by Lee and colleagues , yet the term microRNA was only introduced in 2001 in a set of three articles in Science (26 October 2001)












Formation and processing




The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps.

Most pre-miRNAs don't have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for such selectivity. One could be that dsRNAs longer than 21 base pairs activate interferon response and anti-viral machinery in the cell. Another plausible explanation could be that thermodynamical profile of pre-miRNA determines which strand will be incorporated into Dicer complex. Indeed, aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5'- or 3'-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5' end. The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate. After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC complex. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA





Cellular functions




The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. miRNAs may also target methylation of genomic sites which correspond to targeted mRNAs. miRNAs function in association with a complement of proteins collectively termed the miRNP.

This effect was first described for the worm C. elegans in 1993 by Victor Ambros and coworkers (Lee et al., 1993). As of 2002, miRNAs have been confirmed in various plants and animals, including C. elegans, human and the plant Arabidopsis thaliana. Genes have been found in bacteria that are similar in the sense that they control mRNA abundance or translation by binding an mRNA by base pairing, however they are not generally considered to be miRNAs because the Dicer enzyme is not involved.

In plants, similar RNA species termed short-interfering RNAs siRNAs are used to prevent the transcription of viral RNA. While this siRNA is double-stranded, the mechanism seems to be closely related to that of miRNA, especially taking the hairpin structures into account. siRNAs are also used to regulate cellular genes, as miRNAs do.

Detecting and manipulating miRNA signalling




The activity of an miRNA can be experimentally blocked using a locked nucleic acid oligo, a Morpholino oligo or a 2'-O-methyl RNA oligo. Steps in the maturation of miRNAs can be blocked by steric-blocking oligos. The target site of an miRNA on an mRNA can be blocked by a steric blocking oligo




miRNA and cancer




miRNA has been found to have links with some types of cancer.

A study of mice altered to produce excess c-myc — a protein implicated in several cancers — shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days.

Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off.

By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer





miRNA and heart disease





miRNA has been shown to be related to heart disease. Mice were created that were deficient in a muscle-specific miRNA and these mice had high rate of the most common congenital heart disease -- ventricular septal defects characterized by the holes between the left and the right ventricles of the heart. Such mice also show hyperplasia (an increase of the number of cardiac muscle cells that leads to heart enlargement) and abnormalities in cardiac conduction.

Control of Gene Expression

This lecture begins by illustrating how cells differ in the proteins theye xpress. The same cell may express different proteins at different times in its life, and in multi-cellular organisms, different types of cells typically express different suites of proteins. Given that all cells in an organism have the same set of genes, how do these differences in protein expression arise? The lecture outlines the history of the original experiments done by Jacques Monod and François Jacob in the late 1950s and early 1960s using bacteria to discover the basic mechanisms of gene regulation.


Differences in cell function often produce different cell shapes, but the critical difference is the different protein mix each type of cell contains.

Though many thousands of proteins are coded for in a typical eukaryotic cell, most cells contain only a fraction of that number at any one time. This is the case even in single-celled bacteria. This situation makes sense because making proteins is expensive and many proteins do not store well. This leads to the question of how cells control protein production.
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Part 3







We know that making proteins requires the transcription of DNA into mRNA, then the translation of mRNA into proteins; thus, we could alternatively ask why all genes in a cell are not always transcribed and translated.

The general answer is that cells regulate gene expression by controlling the conditions necessary for transcription and translation.


The complexity of gene expression means that there are many possible points where cells can control it.

A. In addition to transcription and translation, the initial mRNA transcript must be processed before it is translated, and the final polypeptide must often be further modified by enzymes before it is functional.
B. Cells could theoretically control gene expression at any of these points,but the majority of gene regulation happens at the level of transcription—specifically, by controlling mRNA synthesis.C. This is the case primarily for efficiency; it “costs” cells less to prevent the process from starting in the first place.


III. Control of transcription mechanisms involves genetic “lock-and-key”mechanisms, which work differently in prokaryotes and eukaryotes.

A. To a first approximation, the “switch” that turns transcription on and off is a lock-and-key mechanism.
B. The lock is a specific sequence of nucleotide bases on the DNA that is distinct from but often located physically adjacent to the gene it controls. Each gene has one or more of these regulatory regions, which normally occur “upstream” from the gene.
C. The key is usually a protein with the right shape (including not only physical shape but the correct physical and chemical properties of amino acids) to fit the DNA lock. In general, only one protein will bind to a particular regulatory region.
D. In order to start transcription, RNA polymerase must bind to promoter sites on the DNA molecule.The binding of RNA polymerase to a promoter is not specific; thus, regulatory regions and proteins generally act in two ways to control gene expression.

1. In prokaryotic cells, regulatory regions typically lie in between promoter sites and genes. Regulatory proteins bound to regulatory regions physically block RNA polymerase from reaching the gene.This represents negative control and is typical of prokaryotic cells.
2. The other type of control depends on the fact that RNA polymerase cannot always bind efficiently to the promoter by itself. In this case regulatory proteins nteracting with regulatory regions affect the ability of RNA polymerase to bind to a promoter. In the most general case, regulatory proteins (called “transcription factors”) facilitate the binding of RNA polymerase, resulting in positive control, though these proteins can have either a positive or negative effect on binding in specific cases.
3. These types of controls create two contrasts: physical blockage of RNA polymerase versus an effect on its ability to bind to a promoter region on the DNA, and negative control versus positive control.

E. Though the lock-and-key mechanis m for gene regulation can be compared to turning on a car, there are some important differences.
1. The “driver” (RNA polymerase) is not selective as to which “car” it chooses. RNA polymerase simply tries to transcribe every gene.
2. Some genes will be exp ressed only if the key is not in the regulatory lock.
3. Some genes require a combination of many different keys to be expressed, and some keys must be in their locks, while others must be out.

IV. In the late 1950s and early 1960s, Jacques Monod and François Jacob investigated how gene regulation works in prokaryotes.
A. The bacteria Escherichia coli (E. coli for short) requires several enzymes to metabolize lactose. Biochemists discovered in the early 20th
century that E. coli produces these enzymes only when lactose is available, suggesting that lactose induces enzyme production.
B. This observation led Jacques Monod and François Jacob to the question of how lactose could induce gene expression; they found mutant forms
of E. coli that differed in how they expressed lactose-digesting enzymes.
1. Two kinds of mutants were unable to digest lactose; in one case, an enzyme involved in lactose breakdown itself was defective, while
in another case, an enzyme that brings lactose into the cell was defective. These mutations fit into the expected pattern: A
mutation in a gene causes a coding error that produces a dysfunctional protein, which cannot do its job.
2. A third kind of mutant always produced lactose-digesting enzymes, regardless of whether lactose was present or not. This type of mutation produced a gain of constant function rather than a loss of function. Monod and Jacob inferred that this third type of mutant must have a mutation in some protein involved in
controlling gene expression, not in the genes coding for the enzymes themselves.
C. Monod and Jacob’s idea was revolutionary at the time, because it pointed to a protein whose sole function was to regulate the expression
of other genes.
D. In E. coli, the regulatory protein seemed to inhibit enzyme production.After some debate, physicist Leo Szilard prevailed with the idea that the regulatory protein acted as a repressor that normally preventedproduction of the lactose-digesting enzymes. Szilard’s model suggested that lactose acts as an inducer by disabling the repressor protein; that is,expression of the lactose-digesting enzymes is under negative control.