6.1 - The Central Dogma

• The central dogma - coined by Francis Crick in 1957 to describe the flow of genetic information through a biological system.

• DNA ➔ RNA ➔ Proteins; the processes involved in making this flow of information possible - replication, transcription, and translation.

6.2 - Replication of DNA

• DNA replication copy DNA molecules from cell to cell.

• Occurs during the S-phase of the cell cycle to ensure that every cell produced during mitosis or meiosis receives the proper amount of DNA.

• The mechanism for DNA replication - debated in the mid-1900s.

• Conservative DNA replication - the original double helix of DNA does not change at all; an exact duplicate is made.

• Semiconservative DNA replication model - agrees that the original DNA molecule serves as the template but proposes that before it is copied, the DNA unzips, with each single strand serving as a template for the creation of a new double strand.

• Dispersive DNA replication model - suggested that every daughter strand contains some parental DNA, but it is dispersed among pieces of DNA not of parental origin.

• During the S-phase of the cell cycle, the double-stranded DNA unzips and prepares to replicate.

• Helicase - enzyme - unzips the DNA, breaking the hydrogen bonds between the nucleotides and producing the replication fork.

• Each strand then functions as a template for production of a new double-stranded DNA molecule.

• Specific regions along each DNA strand serve as primer sites that signal where replication should originate.

• Primase binds to the primer, and DNA polymerase, the superstar enzyme of this process, attaches to the primer region and adds nucleotides to the growing DNA chain in a 5′-to-3′ direction.

• DNA polymerase is restricted in that it can only add nucleotides to the 3′ end of a parent strand.

• This creates a problem because this means that only one of the strands can be produced in a continuous fashion.

• Continuous strand - leading strand.

• The other strand - lagging strand.

• The lagging strand consists of tiny pieces - Okazaki fragments - are later connected by an enzyme - DNA ligase to produce the completed double-stranded daughter DNA molecule.

• This is the semi-conservative model of DNA replication.

• A series of proofreading enzymes function to make sure that the DNA is properly replicated each time.

• During the first run-through - estimated that a nucleotide mismatch is made during replication in 1 out of every 10,000 basepairs.

• Mismatch error in replication occurs in only one out of every billion nucleotides replicated.

• Mismatch Repair - the polymerase removes the incorrect nucleotide, adds the proper one in its place.

• Excision repair - a section of DNA containing an error is cut out and the gap is filled in by DNA polymerase.

  Key Enzymes to Know for DNA Replication

• Helicase: enzyme that unzips DNA, breaking the hydrogen bonds between the nucleotides and producing the replication fork for replication

• Topoisomerase: enzyme that helps in the unwinding or rewinding of DNA.

• DNA polymerase: the main enzyme in DNA replication that attaches to primer proteins and adds nucleotides to the growing DNA chain in a 5′-to-3′ chain.

• Ligase: enzyme that connects two strands of DNA together by forming a bond between the phosphate group of one strand and the deoxyribose group of another.

• RNA polymerase: enzyme that runs transcription and adds the appropriate nucleotides to the 3′ end of the growing strand.

6.3 - Telomeres

• In eukaryotic chromosomes, DNA replication - issue for the structure of the chromosome.

• The leading strand is completely replicated, but the lagging strand is not able to be completed all the way to the end.

• Occurs because the primer used to start DNA replication on the lagging strand is not replaced.

• As a result, during each round of replication, the lagging strand template would produce a shorter chromosome.

• Telomeres - specialized structures composed of short repeated sequences of DNA that are made by telomerase.

• Found on the ends of eukaryotic chromosomes that protect the integrity and length of the chromosomes after each replication.

6.4 - Transcription of DNA

• DNA does not directly produce the proteins that it encodes.

• DNA must first be transcribed into an intermediary: mRNA.

• This process is called transcription because both DNA and RNA are built from nucleotides.

• DNA - acts as a template for mRNA, which then conveys to the ribosomes the blueprints for producing the protein of interest.

• Transcription occurs in the nucleus.

• Transcription - consists of three steps: initiation, elongation, and termination.

• Begins when RNA polymerase attaches to the promoter region of a DNA strand (initiation).

• A promoter region- a recognition site that shows the polymerase where transcription should begin.

• The promoter region contains a group of nucleotides - TATA box - that is important to the binding of RNA polymerase.

• Polymerase of transcription needs the assistance of helper proteins - transcription factors - to find and attach to the promoter region.

• Once bound, the RNA polymerase works its magic by adding the appropriate RNA nucleotide to the 3′ end of the growing strand (elongation).

• Like DNA polymerase of replication, RNA polymerase adds nucleotides 5′ to 3′.

• The growing mRNA strand separates from the DNA as it grows longer.

• Termination site tells the polymerase when transcription should conclude (termination).

• After reaching this site, the mRNA is released and set free.

6.5 - RNA Processing

• In bacteria, mRNA - ready immediately after it is released from the DNA.

• In eukaryotes - the mRNA produced after transcription must be modified before it can leave the nucleus and lead the formation of proteins on the ribosomes.

• The 5′ end is given a guanine cap - serves to protect the RNA, helps in attachment to the ribosome later on.

• The 3′ end is given a polyadenine tail - may help ease the movement from the nucleus to the cytoplasm.

• The introns (noncoding regions produced during transcription) are cut out of the mRNA, and the remaining exons (coding regions) are glued back together to produce the mRNA that is translated into a protein - RNA splicing.

• It is hypothesized that introns exist to provide flexibility to the genome.

• They could allow an organism to make different proteins from the same gene; the only difference is which introns get spliced out from one to the other.

• Also possible that this whole splicing process plays a role in allowing the movement of mRNA from the nucleus to the cytoplasm.

6.6 - Translation of RNA

• mRNA- escaped from the nucleus - it is ready to help direct the construction of proteins.

• Occurs in the cytoplasm, the site of protein synthesis is the ribosome.

• Each protein has a distinct and particular amino acid order.

• Therefore, there must be some system used by the cell to convert the sequences of nucleotides that make up an mRNA molecule into the sequence of amino acids that make up a particular protein.

• The cell - carries out this task from nucleotides to amino acids through the use of - genetic code.

• An mRNA molecule is divided into a series of codons that make up the code.

• Each codon a triplet of nucleotides that codes for a particular amino acid.

• 20 different amino acids, and 64 different combinations of codons.

• Some amino acids are coded for by more than one codon.

• Of these 64 possibilities, one is a start codon, AUG, which establishes the reading frame for protein formation.

• Also among these 64 codons are three stop codons: UGA, UAA, and UAG.

• When the protein formation machinery hits these codons, the production of a protein stops.

• Ribosomes - made up of a large and a small subunit.

• A huge percentage of a ribosome is built out of the second type of RNA mentioned earlier, rRNA.

• Two other important parts of a ribosome - A site and the P site - tRNA attachment sites.

• The job of tRNA - carry amino acids to the ribosomes.

• The mRNA molecule - consists of a series of codons.

• Each tRNA has, at its attachment site, a region - the anticodon - a three-nucleotide sequence that is perfectly complementary to a particular codon.

• For example, a codon that is AUU has an anticodon that reads UAA in the same direction.

• Each tRNA molecule carries an amino acid that is coded for by the codon that its anticodon matches up with.

• Once the tRNA’s amino acid has been incorporated into the growing protein, the tRNA leaves the site to pick up another amino acid just in case its services are needed again at the ribosome.

• An enzyme known as aminoacyl tRNA synthetase makes sure that each tRNA molecule picks up the appropriate amino acid for its anticodon.

• Wobble - a uracil in the third position of an anticodon can pair with A or G instead of just A.

• There are some tRNA molecules that have an altered form of adenine, called inosine (I), in the third position of the anticodon.

• This nitrogenous base is able to bind with U, C, or A.

• Wobble allows the 45 tRNA molecules to service all the different types of codons seen in mRNA molecules.

• The first codon for this process is always AUG.

• This attracts a tRNA molecule carrying methionine to attach to the AUG codon.

• When this occurs, the large subunit of the ribosome, containing the A site and the P site, binds to the complex.

• The elongation of the protein is ready to begin.

• The P site is the host for the tRNA carrying the growing protein, while the A site is where the tRNA carrying the next amino acid sits.

• AUG - the first codon bound, in the P site is the tRNA carrying the methionine.

• The next codon in the sequence determines which tRNA binds next, and that tRNA molecule sits in the A site of the ribosome.

• An enzyme helps a peptide bond form between the amino acid on the A site tRNA and the amino acid on the P site tRNA.

• After this happens, the amino acid from the P site moves to the A site, setting the stage for the tRNA in the P site to leave the ribosome.

• Translocation - the ribosome moves along the mRNA in such a way that the A site becomes the P site and the next tRNA comes into the new A site carrying the next amino acid.

6.7 - Gene Regulation

• Promoter region: a base sequence that signals the start site for gene transcription; this is where RNA polymerase binds to begin the process.

• Operator: a short sequence near the promoter that assists in transcription by interacting with regulatory proteins (transcription factors).

• Operon: a promoter/operator pair that services multiple genes; the lac operon is a well-known example.

• Repressor: protein that prevents the binding of RNA polymerase to the promoter site.

• Enhancer: DNA region, also known as a “regulator,” that is located thousands of bases away from the promoter; it influences transcription by interacting with specific transcription factors.

• Inducer: a molecule that binds to and inactivates a repressor (e.g., lactose for the lac operon).

  Prokaryotic Gene Regulation

• In bacteria, operons are a major method of gene expression control.

• The lactose operon services a series of three genes involved in the process of lactose metabolism.

• This contains the genes that help the bacteria digest lactose.

• In the absence of lactose, a repressor binds to the promoter region and prevents transcription from occurring.

• When lactose is present, there is a binding site on the repressor where lactose attaches, causing the repressor to let go of the promoter region.

• RNA polymerase is then free to bind to that site and initiate transcription of the genes.

• When the lactose is gone, the repressor again becomes free to bind to the promoter, halting the process.

  Eukaryotic Gene Regulation

• Because gene expression in eukaryotes involves more steps, there are more places where gene control can occur.

• Interactions of various factors within the transcription complex.

• All specific transcription factors bind to enhancer sequences that may be distant from the promoter.

• These proteins can then interact with the initiation complex by DNA looping to bring the factors into proximity with the initiation complex.

• Some transcription factors, called activators, can directly interact with the RNA polymerase II or the initiation complex, whereas others require additional coactivators.

• Histone modification affects chromatin structure

• DNA in eukaryotes is organized first into nucleosomes and then into higher-order chromatin structures.

• The histones that make up the nucleosome core have amino tails that protrude.

• These amino tails can be modified by the addition of acetyl groups.

• The acetylation alters the structure of chromatin, making it accessible to the transcription apparatus.

6.8 - Cell Specialization

• The differences in the expression of genes controlled by the cell lead to different phenotypes and cells in an organism.

• Transcription factors play a vital role in the regulation of transcription, determining which genes are active in each cell of your body.

• These factors are needed to assemble a transcription apparatus at the promoter region with RNA polymerase during transcription and can enhance the sequence leading to the transcription of a particular gene.

• While some transcription factors act as enhancers, others act as repressors by binding to the DNA and blocking transcription.

• The regulation of eukaryotic gene expression leading to cell specialization continues with the packaging of the DNA into chromatin.

• The methylation of DNA bases in the chromatin correlates with genes being “turned off” while the acetylation of DNA bases in the chromatin correlates with the genes being “turned on.”

  Cell Differentiation

• Not every cell receives the same amount of cytoplasm during the cleavage divisions.

• It is thought that this asymmetric distribution of cytoplasm plays a role in the differentiation of the daughter cells.

• Cells containing different organelles or other cytoplasmic components are able to perform different functions.

• Two other factors, induction and homeotic genes, contribute to cellular differentiation.

• Induction the influence of one group of cells on the development of another through physical contact or chemical signaling.

• German embryologist Hans Speman - his experiments revealed that the notochord induces cells of the dorsal ectoderm to develop into the neural plate.

• When cells from the notochord of an embryo are transplanted to a different place near the ectoderm, the neural plate will develop in the new location.

• The cells from the notochord region act as “project directors,” telling the ectoderm where to produce the neural tube and central nervous system.

• Homeotic genes regulate or “direct” the body plan of organisms.

• For example, a fly’s homeotic genes help determine how its segments will develop and which appendages should grow from each segment.

• Scientists interfering with the development of these creatures have found that mutations in these genes can lead to the production of too many wings, legs in the wrong place, and other unfortunate abnormalities.

• The DNA sequence of a homeotic gene that tells the cell where to put things is called the homeobox.

• It is similar from organism to organism and has been found to exist in a variety of organisms—birds, humans, fish, and frogs.

  Factors in Cellular Differentiation

• Cytoplasmic Distribution - Asymmetry contributes to differentiation, since different areas have different amounts of cytoplasm, and thus perhaps different organelles and cytoplasmic structures.

• Induction One group of cells influences another group of cells through physical contact or chemical signaling.

• Homoeotic Cells - Regulatory genes that determine how segments of an organism will develop.

6.9 - Mutations

• Mutation - a heritable change in the genes of an organism.

• Can result in changes to the phenotype of an organism or be silent and not affect the phenotype of an organism.

• These alterations of the DNA sequences in organisms contribute to variation in a population and can be subject to natural selection.

• Point mutations, which alter a single base, can be a substitution of another base, a deletion of a base(s) or an addition of a base(s).

• Frameshift mutations -

  • Deletion or addition of DNA nucleotides that does not add or remove a multiple of three nucleotides.

  • mRNA is produced on a DNA template and is read in bunches of three - codons, which tell the protein synthesis machinery which amino acid to add to the growing protein chain.

  • This kind of mutation usually produces a nonfunctional protein unless it occurs late in protein production.

• Missense mutation -

  • Substitution of the wrong nucleotides into the DNA sequence.

  • These substitutions still result in the addition of amino acids to the growing protein chain during translation, but they can sometimes lead to the addition of incorrect amino acids to the chain.

  • It could cause no problem at all, or it could cause a big problem as in sickle cell anemia, in which a single amino acid error caused by a substitution mutation leads to a disease that wreaks havoc on the body as a whole.

• Nonsense mutation -

  • Substitution of the wrong nucleotides into the DNA sequence.

  • These substitutions lead to premature stoppage of protein synthesis by the early placement of a stop codon, which tells the protein synthesis machinery to grind to a halt.

  • The stop codons are UAA, UAG, and UGA.

  • This type of mutation usually leads to a nonfunctional protein.

• Thymine dimers -

  • **Result of too much exposure to UV (ultraviolet) light.

  • Thymine nucleotides located adjacent to one another on the DNA strand bind together when this exposure occurs.

  • This can negatively affect replication of DNA and help cause further mutations.

• Sickle cell anemia represents a prime example of a point mutation leading to a phenotypic change in humans.

• Sickle cell is caused by a mutation of the fourth codon in the gene for hemoglobin.

• The substitution of glycine (a polar amino acid) to valine (a nonpolar amino acid) causes the shape of the hemoglobin protein to change, distorting the shape of the red blood cells.

• Chromosomal mutations can change the structure of chromosomes and lead to many -different disorders found in humans.

• Four types of chromosomal mutations: deletion, duplication, inversion, and reciprocal translocation.

6.10 - The Genetics of Viruses

• Virus a parasitic infectious agent that is unable to survive outside of a host organism.

• Viruses do not contain enzymes for metabolism, and they do not contain ribosomes for protein synthesis.

• They are completely dependent on their host.

• Once a virus infects a cell, it takes over the cell’s machinery and uses it to produce whatever it needs to survive and reproduce.

• All viruses have a genome (DNA or RNA) and a protein coat (capsid).

• Capsid a protein shell that surrounds the genetic material.

• Some viruses are surrounded by a structure - a viral envelope, which not only protects the virus but also helps the virus attach to the cells that it prefers to infect.

• The viral envelope is produced in the endoplasmic reticulum (ER) of the infected cell and contains some elements from the host cell and some from the virus.

• Each virus has a host range, which is the range of cells that the virus is able to infect.

• For example, HIV infects the T cells of our body, and bacteriophages infect only bacteria.

• Retroviru - an RNA virus that carries an enzyme called reverse transcriptase.

• Once in the cytoplasm of the cell, the RNA virus uses this enzyme and “reverse transcribes” its genetic information from RNA into DNA, which then enters the nucleus of the cell.

• In the nucleus, the newly transcribed DNA incorporates into the host DNA and is transcribed into RNA when the host cell undergoes normal transcription.

• The mRNA produced from this process gives rise to new retrovirus offspring, which can then leave the cell in a lytic pathway.

• A well-known example of a retrovirus is the HIV virus of AIDS.

• Once inside the cell, a DNA virus can take one of two pathways—a lytic or a lysogenic pathway.

• Lytic cycle - the cell produces many viral offspring, which are released from the cell—killing the host cell in the process.

• Lysogenic cycle, the virus falls dormant and incorporates its DNA into the host DNA as an entity - provirus.

• The viral DNA is quietly reproduced by the cell every time the cell reproduces itself, and this allows the virus to stay alive from generation to generation without killing the host cell.

• Viruses in the lysogenic cycle can sometimes separate out from the host DNA and enter the lytic cycle.

• Viroids plant viruses that are only a few hundred nucleotides in length.

• Prion - an incorrectly folded form of a brain cell protein that works by converting other normal host proteins into misshapen proteins.

• An example of a prion disease - “mad cow” disease.

• Prion diseases are degenerative diseases that tend to cause brain dysfunction - dementia, muscular control problems, and loss of balance.

6.11 - The Genetics of Bacteria

• Bacteria - prokaryotic cells that consist of one double-stranded circular DNA molecule.

• Present in the cells of many bacteria are extra circles of DNA - plasmids, which contain just a few genes and have been useful in genetic engineering.

• Plasmids replicate independently of the main chromosome.

• Bacterial cells reproduce in an asexual fashion, undergoing binary fission.

• The cell replicates its DNA and then physically pinches in half, producing a daughter cell that is identical to the parent cell.

• As in humans, DNA mutation in bacteria occurs very rarely, but some bacteria replicate so quickly that these mutations can have a pronounced effect on their variability.

  Transformation

• An experiment performed by Griffith in 1928 - transformation - the uptake of foreign DNA from the surrounding environment.

• Transformation occurs through the use of proteins on the surface of cells that snag pieces of DNA from around the cell that are from closely related species.

• This particular experiment involved a bacteria known as Streptococcus pneumoniae, which existed as either a rough strain (R), which is nonvirulent, or as a smooth strain (S), which is virulent.

• A virulent strain is one that can lead to contraction of an illness.

• The experimenters exposed mice to different forms of the bacteria.

  • Mice given live S bacteria died.

  • Mice given live R bacteria survived.

  • Mice given heat-killed S bacteria survived.

  • Mice given heat-killed S bacteria combined with live R bacteria died.

  • Those exposed to heat-killed S combined with live R bacteria contracted the disease because the live R bacteria underwent transformation.

  • Some of the R bacteria picked up the portion of the heat-killed S bacteria’s DNA, which contained the instructions on how to make the vital component necessary for successful disease transmission. These R bacteria became virulent.

• Phage - a virus that infects bacteria.

  • A phage contains within its capsid the DNA that it is attempting to deliver.

  • A phage latches onto the surface of a cell and fires its DNA through the membrane and into the cell.

• Transduction the movement of genes from one cell to another by phages.

  Generalized Transduction

• Take a phage virus infects and takes over a bacterial cell that contains a functional gene for resistance to penicillin.

• Occasionally during the creation of new phage viruses, pieces of host DNA instead of viral DNA are accidentally put into a phage.

• When the cell lyses, expelling the newly formed viral particles, the phage containing the host DNA may latch onto another cell, injecting the host DNA from one cell into another bacterial cell.

• If the phage attaches to a cell that contains a nonfunctional gene for resistance to penicillin, the effects of this transduction process can be observed.

• After injecting the host DNA containing the functional penicillin resistance gene, crossover could occur between the comparable gene regions, switching the nonfunctional gene with the functional gene.

• This would create a new cell that is resistant to penicillin.

  Specialized Transduction

• Involves a virus that is in the lysogenic cycle, resting quietly along with the other DNA of the host cell.

• Occasionally when a lysogenic virus switches cycles and becomes lytic, it may bring with it a piece of the host DNA as it pulls out of the host chromosome.

• Imagine that the host DNA it brought with it contains a functional gene for resistance to penicillin.

• This virus, now in the lytic cycle, will produce numerous copies of new viral offspring that contain this resistance gene from the host cell.

• If the new phage offspring attaches to a cell that is not penicillin resistant and injects its DNA and crossover occurs, specialized transduction will have occurred.

  Conjugation

• The transfer of DNA between two bacterial cells connected by appendages called sex pili.

• Movement of DNA between two cells occurs across a cytoplasmic connection between the two cells and requires the presence of an F-plasmid, which contains the genes necessary for the production of a sex pilus.

6.12 - Biotechnology

• Restriction enzymes enzymes that cut DNA at specific nucleotide sequences.

• When added to a solution containing DNA, the enzymes cut the DNA wherever the enzyme’s particular sequence appears.

• This creates DNA fragments with single-stranded ends - “sticky-ends,” - find and reconnect with other DNA fragments containing the same ends (with the assistance of DNA ligase).

• Sticky ends allow DNA pieces from different sources to be connected, creating recombinant DNA.

• Vector - moves DNA from one source to another.

• Plasmids can be removed from bacterial cells and used as vectors by cutting the DNA of interest and the DNA of the plasmid with the same restriction enzyme to create DNA with similar sticky ends.

• The DNA can be attached to the plasmid, creating a vector that can be used to transport DNA.

  Gel Electrophoresis

• This technique is used to separate and examine DNA fragments

• The DNA is cut with the restriction enzymes, and then separated by electrophoresis.

• The pieces of DNA are separated on the basis of size with the help of an electric charge.

• DNA is added to the wells at the negative end of the gel.

• When the electric current is turned on, the migration begins.

• Smaller pieces travel farther along the gel, and larger pieces do not travel as far.

• This technique can be used to sequence DNA and determine the order in which the nucleotides appear.

• It can be used in a procedure known as Southern blotting to determine if a particular sequence of nucleotides is present in a sample of DNA.

• Electrophoresis is used in forensics to match DNA found at the crime scene with DNA of suspects.

• This requires the use of pieces of DNA called restriction fragment length polymorphisms (RFLPs).

• DNA is specific to each individual, and when it is mixed with restriction enzymes, different combinations of RFLPs will be obtained from person to person.

• Electrophoresis separates DNA samples from the suspect and whatever sample is found at the scene of the crime.

• The two are compared, and if the RFLPs match, there is a high degree of certainty that the DNA sample came from the suspect.

  Cloning

• Sometimes it is desirable to obtain large quantities of a gene of interest, such as insulin for the treatment of diabetes.

• Plasmids used for cloning often contain two important genes—one that provides resistance to an antibiotic, and one that gives the bacteria the ability to metabolize some sugar.

• In this case, we will use a galactose hydrolyzing gene and a gene for ampicillin resistance.

• The plasmid and DNA of interest are both cut with the same restriction enzyme.

• The restriction site for this enzyme is right in the middle of the galactose gene of the plasmid.

• When the sticky ends are created, the DNA of interest and the plasmid molecules are mixed and join together.

• The recombinant plasmids produced are transformed into bacterial cells.

• The transformed cells are allowed to reproduce and are placed on a medium containing ampicillin.

• Cells that have taken in the ampicillin resistance gene will survive, while those that have not will perish.

• The medium also contains a special sugar that is broken down by the galactose enzyme present in the vector to form a colored product.

• The cells containing the gene of interest will remain white since the galactose gene has been interrupted and rendered nonfunctional.

• This allows the experimenter to isolate cells that contain the desired product.

  Polymerase Chain Reaction

• It is used to produce large quantities of a particular sequence of DNA in a very short amount of time.

• This process begins with double- stranded DNA containing the gene of interest.

• DNA polymerase is added to the mixture along with a huge number of nucleotides and primers specific for the sequence of interest, which help initiate the synthesis of DNA.

• PCR begins by heating the DNA to split the strands, followed by the cooling of the strands to allow the primers to bind to the sequence of interest.

• DNA polymerase then produces the rest of the DNA molecule by adding the nucleotides to the growing DNA strand.

• Each cycle concludes having doubled the amount of DNA present at the beginning of the cycle.

• The cycle is repeated over and over, every few minutes, until a huge amount of DNA has been created.

• PCR is used in many ways, such as to detect the presence of viruses like HIV in cells, diagnose genetic disorders, and amplify trace amounts of DNA found at crime scenes.

  DNA Sequencing

• In 2003, the international science community completed sequencing of the human genome.

• Using cutting edge techniques, scientists were able to determine the sequence of nucleotide bases for a human’s DNA.

• This discovery has led to new techniques and technologies that allow for the sequencing of small pieces of DNA to entire genomes of organisms.