Chapter 9

RNAi Mechanism

1. An abnormal gene, cancer gene, or virus gene is transcribed in a host cell. 2. siRNA binds mRNA. 3. RISC breaks down the RNA complex. 4. No protein expression occurs.

Restriction Enzyme & Recombinant DNA steps 4-5

4. The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur. 5. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of recombinant DNA.

Interrupted gene

A gene in which the coding sequence is not continuous due to the presence of introns.

capitalist Robert Swanson cold-called Boyer's lab

Genentech staff successfully created their first product: chemically synthesized genes for human insulin.

Recombinant DNA Technology

Insertion or modification of genes in organisms to produce desired proteins.

Recombinant DNA: Key Terms

So, a competent bacterium can be transformed, making it a transgenic organism expressing recombinant DNA.

Competence

The ability of a cell to take up extracellular ("naked") DNA from its environment.

Biotechnology

The use of microorganisms, cells, or cell components to make a product (foods, antibiotics, vitamins, enzymes)

Cloning is the production of identical copies of DNA

-Gene cloning is production of many identical copies of the same gene. -If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product. -Cloned genes have many research purposes -Humans can be treated with gene therapy

Bacterial Transformation with Recombinant DNA steps5-7

5. Cells with gene of interest are cloned/ Goal may be to make protein product of gene 6A. Copies of gene are harvested. (Gene for pest resistance is inserted into plants or gene alters bacteria for cleaning up toxic waste) 6B. Cells make a protein product 7. Copies of protein are harvested. (Amyase, cellulase, and other enzymes prepare fabrics for clothing manufacture or human growth hormone treats stunted growth)

So, how do you ensure that you select only those bacteria that have a plasmid with your gene of interest?

Blue-White screening

Recombinant DNA

DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.

Protein creation

Everything in our bodies is made from proteins manufactured according to the genetics recipes found in DNA. Once DNA's instructions have been exported out of the nucleus in mRNA, cellular structures called ribosomes process them and translate them into the protein building blocks of bone cells, brain cells-indeed, all cells.

The Relationship between Genes and Proteins

Genes store information for producing all cellular proteins

The cell nucleus

Inside the nucleus of every cell in our bodies is DNA, the famous double helix archive of coded information. Even though it carries genetic information that gets transmitted throughout the cell, the DNA master code never actually leaves the nucleus. The nucleus membrane surrounding the nucleus has openings called nuclear pore complexes, but you won't see DNA traveling through them.

Alternative splicing

Many primary transcripts can be processed by two or more pathways so that a sequence that acts as an intron in one pathway becomes an exon in an alternate pathway. As a result of this process, called alternative splicing, the same gene can code for more than one polypeptide.

Viral RNA

Sometimes a virus invades a cell and deposits its own RNA. A virus's sole purpose is to make genetic copies of itself as quickly as possible, and it soon takes over the cell, forcing it to make viral proteins. When the viruses, its RNA has a long, double-stranded shape. The double-stranded shape of viral RNA provides a powerful warning signs that an invader has penetrated the cell.

Transformation

The genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous DNA from its surroundings.

RNA splicing

The process of excising introns from RNA and connecting the exons into a continuous mRNA.

Gene of interest

is inserted into bacteria, only after bacteria is made "competent" = in a state that allows it to take up outside DNA.

Two enzymes are required to introduce foreign DNA into vector DNA

- A restriction enzyme - cleaves DNA, and - A DNA ligase enzyme - seals DNA into an opening created by the restriction enzyme

Polymerase Chain Reaction (PCR)

- Amplifies a targeted sequence of DNA - Create millions of copies of a single gene or a specific piece of DNA in a test tube.

PCR: Used to

-Clone DNA for recombination -Amplify DNA to detectable levels (i.e., forensics) -Sequence DNA -Diagnose genetic disease -Detect pathogens

Restriction Enzymes

-Enzymes that cut DNA at specific points -Cleaves vector (plasmid) and foreign (human) DNA. -Cleaving DNA makes DNA fragments ending in short single-stranded segments with "sticky ends." -The "sticky ends" allow insertion of foreign DNA into vector DNA.

The sequences of DNA comprising an interrupted protein-encoding gene are divided into the two categories

-Exons are the sequences represented in the mature RNA. A mature transcript starts and ends with exons that correspond to the 5' and 3' ends of the RNA. -Introns are the sequences that are removed when the primary RNA transcript (pre-mRNA) is processed to give the mature RNA. Human genes contain on average 9 introns.

An Overview of the Flow of Information through the Cell

-Messenger RNA (mRNA) is an intermediate between a gene and a protein -Transcription is the process by which RNA is formed from a DNA template -Translation is the process by which proteins are synthesized in the cytoplasm from an mRNA template

A vector

-Self-replicating DNA used to carry the desired gene to a new cell -Plasmids (small accessory rings of DNA from bacteria) are common vectors

Exons and Introns

-The exon sequences are in the same order in the gene and in the RNA, but an interrupted gene is longer than its mature RNA product because of the presence of the introns. -Introns are removed by a process called "RNA splicing."

Exon shuffling

-The movement of genetic "modules" among unrelated genes - Genetic rearrangements require breaks in DNA molecules, which can occur within introns without introducing mutations that might impair the organism -Over time, exons can be shuffled independently in various ways, allowing a nearly infinite number of combinations -As a result of exon shuffling, evolution need not occur only by the slow accumulation of point mutations but might also move ahead by "quantum leaps" with new proteins appearing in a single generation.

Prokaryotic genes

-The simplest form of a gene is a length of DNA that is colinear with a protein. -Bacterial genes are almost always of this type, in which a continuous sequence of 3N bases encodes a protein of N amino acids. -"What you see is what you get."

When is DNA fingerprinting used?

-Usually used to measure number of repeats of short sequences -Used in paternity suits, rape cases, corpse ID, etc.

PCR Requires: DNA polymerase

-Utilizes a heat-stable DNA polymerase (Taq polymerase) from bacteria living in hot springs. -Withstands the temperature necessary to separate double-stranded DNA. -A supply of nucleotides for the new, complementary strand

Eukaryotic genes:

-Where's the missing mRNA? -In the 1960s it was found in eukaryotes that mRNA is first synthesized as long precursor transcript that is subsequently shortened, or "pruned." -Thus, eukaryotic genes are much longer than the functional transcripts they produce. -DNA protein-encoding sequences can be "interrupted." -The interrupting sequences are removed from the primary (RNA) transcript during gene expression, generating an mRNA that includes a continuous base sequence corresponding to the polypeptide product as determined by the genetic code.

Plasmids

-are extrachromosomal, double-stranded circular DNA molecules. -Contain wide variety of genes, such as antibiotic resistance. -Can be used as tools to "grow" genes = vectors. -Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels.‏

DNA fingerprinting

-is the technique of using DNA fragment lengths Treat DNA segment with restriction enzymes: -A unique collection of different fragments is produced -Gel electrophoresis separates the fragments according to their charge/size -Produces distinctive banding pattern

Generation of the DNA Construct steps 1-2

1. Generation of the DNA Construct A) Milk Protein Promoter DNAL Allow for expression only in goat mammary glands. B) Therapeutic Protein Gene: Encodesa protein known to treat disease in people. C) Terminator Sequence: Assures that only the gene of interest is controlled by A. D) Other DNA Sequences: Helps with the introduction of the new combination DNA strand. 2. The DNA construct is creating by combining A, B, C, and D.

Polymerase Chain Reaction (PCR) steps 1-5

1. Incubate target DNA at 94 degrees C for 1 minute to separate the strands. 2. Add primers, nucleotides (deoxynucleotides), and DNA polymerase. 3. Primers attach to single-stranded DNA during incubation at 60 degrees C for 1 minute. 4. Incubate at 72 degress C for 1 minute; during this time, two copies of target DNA are formed. (First cycle 1-4) 5. Repeat the cycle of heating and cooling to make two more copies of target DNA. (Second cycle)

Blue-White Screening: One method for selecting recombinant bacteria steps1-3

1. Plasmid DNA and foreign DNA are both cut with the same restriction enzyme. The plasma has the genes for lactose hydrolysis (the lacZ gene encodes the enzyme B-galactosidase) and ampicillin resistance. 2. Foreign DNA will insert into the lacZ gene. The bacterium receiving the plasmid vector will not produce the enzyme B-galactosidase if foreign DNA has been inserted into the plasmid. 3. The recombinant plasmid is introduced into a bacterium, which becomes ampicillin resistant.

Restriction Enzyme & Recombinant DNA steps 1-3

1. Restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites. 2.These cuts produce a DNA fragment with two sticky ends. DNA from another source, perhaps a plasmid, cut with the same restriction enzyme. 3.When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing.

Blue-White Screening Explained 1-3

1. The plasmid vector and foreign DNA are digested with the same restriction enzyme. 2. The foreign DNA will insert into the gene for B-galactosidase. Therefore, the bacterium receiving the plasmid vector will not produce the enzyme B-galactosidase if foreign DNA has been inserted into the plasmid. 3. The recombinant plasmid is introduced into a culture of ampicillin-sensitive bacteria by transformation.

Bacterial Transformation with Recombinant DNA steps1-4

1. vector such as a plasmid is isolated 2. DNA is cleaved by an enzyme into fragments/DNA containing gene of interest 3. Gene is inserted into plasma (recombinant DNA plasmid) 4. Plasmid is taken up by a cell such as a bacterium (recombinant bacterium)

Generation of the DNA Construct steps 3-6

3. The new DNA strand is then introduced by any of a number of methods into an animal cell, such as an egg, that is then used to produce a genetically engineered animal. 4. The first genetically engineered goat is produced. 5. The offspring of the first genetically engineered goats, referred to as production animals, are milked. The milk is transferred to a purification facility. 6.The drug to be used to treat human disease is purified from the goats milk.

Blue-White Screening: One method for selecting recombinant bacteria steps 4-5

4. All treated bacteria are spread on a nutrient agar plate containing ampicillin and a B-galactosidase substrate and incubated. The B-galactosidase substrate is called X-gal. 5. Only bacteria that picked up the plasmid will grow in the presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound. The indigo turns the colonies blue. Bacteria that cannot hydrolyze X-gal produce white colonies.

Blue-White Screening Explained 4-6

4. The bacteria is plated on agar containing ampicillin and X-gal. The ampicillin prevents the growth of any bacterium that has not successfully received the ampicillin resistance gene from the plasmid. X-gal is the substrate for the B-galactosidase enzyme. 5. Only bacteria that picked-up the plasmid will grow - because they are now ampicillin resistant. Bacteria that picked-up the plasmid in which the new gene was inserted into the B-galactosidase gene will not hydrolyze X-gal and will produce white colonies. If a bacterium picked-up the original plasmid containing the intact B-galactosidase gene, they will hydrolyze X-gal to produce blue-colored colonies. 6. Therefore, white colonies are the desirable ones.

RNAi

A "gene silencing" process built-in to cells Thought to be a primitive defense mechanism against viruses to prevent host cell translation of foreign (viral) genes Specific mRNAs can be degraded in vivo by treating with double-stranded small interfering RNAs (siRNAs) containing part of the sequence of the target mRNA. Cells treated with RNAi cannot make the protein encoded in the target mRNA. Libraries containing thousands of siRNAs are available for the study of gene function. Strategies for using RNAi to combat disease are being developed against cancer, viruses, and some genetic disorders Can be utilized to determine gene function by gene elimination

RNAi explained

A long time ago, before plants and animals existed on our planet, a system evolved in the most primitive single celled organisms to protect them from viruses. Only within the past few years have scientists discovered that this same system, called RNAi, still exists today in humans, as well as in plants and animals, and that is may have the potential to forever change the way doctors fight some diseases.

Mature transcript

A modified RNA transcript with the intron sequences removed.

Intron

A segment of DNA that is transcribed, but later removed from within the transcript by splicing together the sequences (exons) on either side of it.

Recombinant DNA (rDNA) contains DNA from two or more different sources

A vector Two enzymes are required to introduce foreign DNA into vector DNA

AquAdvantage fish (AKA "Frankenfish")

AAS contains a growth hormone gene from the Chinook salmon and a DNA fragment from the ocean pout that helps activate the gene. The gene has the effect of speeding up development in the first year, slashing time-to-full-growth in half.

An example of DNA cloning using bacterial plasmids

After bacteria are transformed, they should be grown in media that contains the antibiotic specific for the resistance gene on the original plasmid.This way, only bacteria that actually took up the plasmid will grow. But, you still have a problem: not all of the transformed bacteria will have your gene of interest in the plasmid.

Why Have Introns?

Although the presence of introns creates an added burden for cells, because they have to remove intervening sequences from their transcripts, introns may provide advantages to organisms.

Transgenic organism

An organism whose natural DNA has been modified to contain exogenous genes (usually transferred from another organism).

bioreactors

Bacteria are grown in large vats called bioreactors and product is harvested. -Products on the market include insulin, hepatitis B vaccine, and human growth hormone

RNA interference

Cells have a bult-in system called RNA interference, or RNAi, that can prevent viral RNA from replicating successfully. The first step of the RNAi process involves an enzyme that researches have nicknamed Dicer. It chops up any double-stranded RNA it finds (in this case, viral RNA) into short pieces about 22 chemical units in length so that it's easier to manage. Then a bunch of proteins known as RISC, short for RNA-Induced Silencing Complex, unwinds it, leaving only one strand, which the protein complex carries around.

Plasmid Vector Used for Cloning

Contains a ~30-base long piece of DNA containing RE sites, allowing foreign DNA to be inserted into the multiple cloning region (MCR), which interrupts the lac Z gene and prevents the formation of a functional β-galactosidase protein.

Messenger RNA

DNA uses RNA, its close chemical cousin, to ferry genetic information from the nucleus to other parts of the cell. Because of its role, RNA is often referred to as mRNA, or messenger RNA. Here, RNA polymerase, a protein, is transcribing a gene in DNA into mRNA, which is single-stranded. The mRNA takes chemical recipes from DNA and exports them out of the nucleus through a pore and into the cytoplasm that makes up most of the surrounding cell.

Genetically Engineered Animals

FDA has approved one application related to a genetically engineered animal. This is for a goat engineered to produce a human pharmaceutical in its milk. That pharmaceutical has also been approved by the European Medicines Agency (EMEA). In the United States, FDA's Center for Veterinary Medicine approved the recombinant DNA construct in the goat, and the Center for Biologics Evaluation and Research approved the pharmaceutical (recombinant human antithrombin III) for use in individuals with clotting disorders.

Virus

The RISC/RNA molecule looks around the cell for messenger RNA on which it recognizes matching sequences- other viral RNA that might be loose in the cell. When it finds a matching long piece of RNA on which any one segment has the same code as the small piece its carrying, the RISC/RNA complex binds to it. Binding to the target RNA leads to partial or sometimes complete blockage of the viral RNA's protein production, stopping the virus in its tracks. This process occurs over and over again, blocking any RNA that matches the virus.

Primary (RNA) transcript

The original unmodified RNA product corresponding to a transcription unit.

Since eukaryotes have interrupted genes, do you see any issues with this way of cloning the human insulin gene?

The problem is that the gene for human insulin contains introns, and if those introns are not removed, the bacteria will produce pre-mRNA that it cannot process into a mature transcript. So, unless the introns are removed before making the recombinant plasmid, you won't successfully produce insulin protein in transgenic bacteria.

How can you remove the introns? by making complementary DNA (cDNA).

cDNA is made from mRNA by the reverse transcriptase enzyme: cDNA of gene without introns can then be inserted into plasmid.

Gene cloning

is production of many identical copies of the same gene If the inserted gene is replicated and expressed, the cloned gene or protein product can be recovered and used for research/therapy

RNA interference of Bioluminescence

mRNA is translated resulting in "glowing protein" Adding dsRNA sharing the same sequence as the translated mRNA causes RNA interference (RNAi) This results in an inhibition of gene expression (loss of the glow)