MMBIO 240 Learning Objectives #2

•Be able to draw a diagram of a Holliday junction, and explain the 2 possible ways to resolve the junction

-1) The branch migrates all the way to the end of the linear molecule -2) A double-strand break occurs before reaching the end of the molecule (then repaired by DNA ligase), 2 possible ways to perform the break shown below:

•Be able to diagram how microarrays work and be able to interpret data

1. Isolate mRNA's from cells at two stages of development 2. Convert the mRNA's to cDNA's by reverse transcriptase by using fluoresently labeled deoxyribonucleotide triphosphates. 3. Add the cDNA's to a microarray. the fluorescent cDNA's will anneal to the complimentary sequences on the microarray. 4. Each fluorescent spot represents a gene expressed in the cells. 5. If the spot is more green, the gene is expressed more in sample 1, which is the control, if the spot is more red, the gene is expressed more in sample 2, which is the experiment. If the spot is yellow, it means equal expression between control vs. experimental conditions.

•Be able to list 3 ways in which DNA recombination can play a role in repairing DNA damage

1. Recombination can repair double-stranded breaks 2. Homologous recombination for DNA repair: sometimes DNA polymerase skips past a lesion 3. DNA polymerization stops at a DNA lesion

•Be able to list the molecular events required for homologous recombination

1. Sister chromatids align during meiosis (or mitosis) 2. "Nicks" in the DNA are required for dsDNA recombination 3. DNA strands can "breathe" apart (de-hybridize) and re-anneal with sister strand due to similar sequences 4. DNA strands can displace each other for varying distances

How are cDNA's produced?

1. Start with mRNA 2. Attach a primer like Poly-dt to the end 3. Reverse transcriptase comes in and forms CDNA strand leaving you with a mRNA and cDNA hybrid. 4. Degrade the mRNA strand with alkali 5. Use a gene specific primer then use PCR to amplify the # of copies of the cDNA.

•Be able to describe how the ChIP assay works and how to interpret the results; including the various ways the DNA can be analyzed

1. You have cells, break them open. 2. Sonicate the DNA so it breaks up into smaller pieces 3. Mix broken DNA fragments with the antibodies so proteins attached to the DNA will bind to the beads with antibodies. 4. You can then separate the protein from the DNA by stripping the protein with the antibodies then using DNA purification. DNA will stick to the proteins that are being pulled out by the antibodies. 5. You then use PCR to amplify the DNA that is present in the sample. 6. After PCR, you can analyze the DNA present through gel electrophoresis, microarray, or DNA sequencing.

deletion

A change to a chromosome in which a fragment of the chromosome is removed.

mutagen

A chemical or physical agent that interacts with DNA and causes a mutation.

substitution

A mutation in which a nucleotide or a codon in DNA is replaced with a different nucleotide

insertion

A mutation involving the addition of one or more nucleotide pairs to a gene.

silent mutation

A mutation that changes a single nucleotide, but does not change the amino acid created.

restriction fragment length polymorphism (RFLP)

A single nucleotide polymorphism (SNP) that exists in the restriction site for a particular enzyme, thus making the site unrecognizable by that enzyme and changing the lengths of the restriction fragments formed by digestion with that enzyme. A RFLP can be in coding or noncoding DNA.

RT-PCR

A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction.

pulse-chase experiment

A type of experiment in which a population of cells or molecules at a particular moment in time is marked by means of a labeled molecule (pulse) and then their fate is followed over time (chase).

•Be able to explain how the Ames test works to identify potential DNA mutagens, and why this method is superior to monitoring for loss-of-function mutations

Ames test uses several strains of bacteria (Salmonella, E.coli) that carry a particular mutation. Point mutations are made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied. But culturing His- Salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine (His+). This is to say that when a mutagenic event occurs, base substitutions or frameshifts within the gene can cause a reversion to amino acid prototrophy. This is the reverse mutation. These reverted bacteria will then grow in histidine- or tryptophan-deficient media, respectively.

•retrovirus

An RNA virus that reproduces by transcribing its RNA into DNA and then inserting the DNA into a cellular chromosome; an important class of cancer-causing viruses.

Reverse Transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

reverse transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

telomerase

An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.

ligase

An enzyme that connects two fragments of DNA to make a single fragment

primase

An enzyme that joins RNA nucleotides to make the primer.

helicase

An enzyme that untwists the double helix of DNA at the replication forks.

-Including the specific roles played by histone cores and histone tails

C-Terminus-globular (DNA wrapping) positively charged. N-Terminus- linear tails (available for modification) Negatively charged Histone proteins have a net overall positive charge. HISTONE CORES FOR DNA COMPACTION HISTONE TAILS FOR GENE EXPRESSION

ChIP assay

Chromatin Immunoprecipitation. Grab proteins with antibodies, centrifuge them, separate associated DNA sequences, analyze them. Looking for specific proteins stuck to specific DNA sequences.

chromatin

Clusters of DNA, RNA, and proteins in the nucleus of a cell

cDNA

Complementary DNA. DNA produced synthetically by reverse trascribing mRNA. Because of eukaryotic mRNA splicing, cDNA contains no inrons.

Be able to explain the contributions of the various enzymes, proteins, and DNA elements to DNA replication (Topic 14 also)

DNA origin of replication (ori): an A:T rich region of DNA where DNA replication begins Initiator (DnaA protein): recognizes ori sequences and bind to them; Starts to unwind DNA; Recruits other proteins to the site •Helicase (DnaB protein): break hydrogen bonds to open DNA template (make 2 single strands from one double-stranded) •Single-stranded DNA (aka ssDNA) binding proteins (SSBs)- •Keeping DNA as a suitable template molecule (ssDNA) •Primase (DnaG): synthesize small primers made of RNA to start the process •DNA Polymerase: adds nucleotides (pre-existing 3' -OH required)

nucleosome

DNA+histone octomer

•Be able to describe how restriction fragment length polymorphisms (RFLPs) are generated and used to identify diseases, crime victims, and criminals

Extract DNA from blood semen, etc, then use restriction enzyme to cut at specific sites of the DNA, use radioactive probes to bind to specific DNA fragments, transfer DNA fragments to a membrane (a.k.a southern blot), separate DNA fragments by electrophoresis and compare DNA pattern to patterns of known suspects. If DNA matches, then the subject is guilty.

•Be able to explain the contributions of the various enzymes, proteins, and DNA elements to DNA replication (Topic 13 also)

Helicase: break hydrogen bonds to open DNA template (make 2 single strands from one double-stranded) •Single-stranded DNA (aka ssDNA) binding proteins (SSBs) •Keeping DNA as a suitable template molecule (ssDNA) •Primase (DnaG): synthesize small primers made of RNA to start the process •Provide 3' -OH ends for extension (no pre-existing 3' -OH needed for primase, just for DNA polymerase) DNA polymerase III:One copy does essentially all of the DNA synthesis on the leading strand One copy does most of the DNA synthesis on the lagging strand Proofreading capacity: detects mistakes (a.k.a. mismatched base pairs), cuts them out, fixes them •DNA Polymerase I: •Primary job is to fill in the gaps created as RNA primers are removed from Okazaki fragments on the lagging strand •Synthesizes only a minor amount of DNA •Same proofreading capability as DNA pol III- removes last nucleotide •Topoisomerase I: As the DNA gets unwound in one stretch, it has to get overwound in another stretch. -Eventually, the tension would get too high and DNA replication would cease -Topoisomerases make a nick in one strand of DNA, which is all it takes to relieve the pressure -Topoisomerases are upstream of the replication fork, where tension builds •Ligase: DNA ligase makes phosphodiester bonds to seal DNA fragments together •Telomerase:Telomerase extends the ends of newly replicated linear chromosomes; Telomerase extends leading strand without a template strand by using an internal RNA as template to synthesize new cDNA Rnase H: Finds the RNA primers (now part of larger DNA molecules) and removes nearly all of the RNA nucleotides except the last nucleotide

-Know what frameshift mutations are and why these have dramatic phenotypes

Insertion or deletion of a nucleotide that is not 3. These are dramatic because it changes the codon sequence of amino acids and can even lead to stop codons, which are especially bad.

-Also, be able to predict the result of attempted DNA replication of some if the machinery were lacking (e.g., gene was mutated)

LOYO

•Be able to explain how DNA can be compacted (via histones and other interactions)

Level 1: naked DNA, no compaction. 2nm Level 2: DNA associates with histone octomers Nucleosome: DNA + histone octomer. 11nm Level 3: Interactions between histone octomers (and likely involving histone H1 protein) result in greater compaction. Level 4: 30nm fiber associates with a protein scaffold for further compaction. Histone protein H1 is involved in forming the structure.

•Be able to explain how modification of histone proteins can lead to changes in chromatin structure and gene expression

Low Acetylation means more methylation. More methylation means closed chromatin leading to transcriptional repression. High acetylation leads to an open chromatin, which allows for transcriptional activation. •Acetylated histones usually indicate active genes Histone acetyltransferases (HATs) add acetyl groups and Histone deacetylases (HDACs) remove them • •Deacetylated histones usually indicate deactivated genes

•Be able to explain/diagram the 5 steps in the process of making a knockout/knockin mouse

Making a knockout mouse: •Clone the DNA for the gene of interest, plus some additional surrounding DNA, into a plasmid vector •Use restriction enzymes and DNA ligase to specifically cut out a particular region of the gene (usually the entire gene), but need to retain some surrounding DNA, too Introduce a selectable marker gene into the space that you just deleted (again, restriction enzymes and ligase used).. If NeoR gene is present, neo protein is produced which destroys geneticin and cells can survive. If NeoR gene is not present and you expose cells to geneticin, they will die. Allows for selection of cells with desired changes in the genome. Introduce the recombinant DNA molecule you produced (#2) into mouse cells; Embryonic stem cells. Homologous recombination will swap new sequences into the mouse genome (#1) at that specific location at a low frequency. Note that swapping NeoR in results in loss of Exons 1-3. --This is a rare event, so you have to try this in many cells. However, it is easy to find the cells with a successful Implant knockout cells into female mice and pups born should have copies of gene of interest knocked out. Breed together to get to homozygotes

•Be able to differentiate between the methods used for PCR, RT-PCR, Q-PCR, and Q-RT-PCR

PCR- Uses DNA RT-PCR- Uses RNA instead of DNA. Very sensitive, less expensive. Only semi quantative, only one gene at a time Q-PCR- Q-RT-PCR- Used to get a specific number of dna that has been replicated. Only does a few genes at a time. Most sensitive, Highly quantitative, highly expensive.

-Also, how DNA microarrays are the current technique of choice

Perform a DNA based microarray that focuses on SNP's only. -Isolate genomic DNA from blood, label all DNA molecules with a single color of fluorophore, then hybridize to a microchip. -If the DNA sample has a SNP that matches with the sequence on the chip, it will hybridize. •Rapid screening of many, many SNPs in hours. FASTER THAN RFLP's and more confident.

•Be able to explain the cellular consequences of different types of DNA damage

Pyrimidine Dimers: intra strand cross links across two bases on the same strand due to UV light exposure. Consequence: Hydrogen binding between adjacent pairs is disrupted leading to a bubble like structure. Photoproducts: Similar to pyrimidine dimers but only one bond is formed due to UV light. Consequence, DNA isn't able to be transcribed correctly Hydrolytic Cleavage- 3 types of bonds susceptible to cleavage. 1. Phosphodiester bonds, leads to single strand breaks. It is rare and easily fixed by DNA ligase. 2. N-glycosyl bonds, leads to depurination meaning the base is taken out. 3) Bonds linking exocyclic amine groups to bases. Leads to deamination, meaning bases can potentially change to a different base. Alkylation- Refers to adding an alkyl group such as CH3. Since bases are close to the same, added CH3 can make it look like another base, which can drastically change the DNA. ROS- Extra OH and =O groups added onto bases. Different bases can hydrogen bond resulting in potentially permanent mutations. Interstrand cross links- Makes DNA replication impossible Ionizing radiation- induces double strand DNA breaks. Random joining can occur

Q-RT-PCR

Quantitative RT-PCR. Uses reverse transcription to produce cDNA then you amplify that strand which has a quencher and fluorescent dye attached.

•Be able to outline a generic pulse-chase experiment

Question: How quickly are IRP2 proteins degraded in the presence or absence of the drug FAC? 1.Grow cells that make these proteins in the presence (+FAC) or absence (-FAC) of FAC 2.Pulse-label ALL proteins with S35 for 2hr 3.Chase by removing S35 medium 4.Purify IRP2 proteins by affinity chromatography 5.Run purified proteins on SDS-PAGE 6.Detect radioactivity with film Quantify amount of radioactivity per band

DNA Polymerase I

Removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides

transposable element

Segment of DNA that can move spontaneously within or between chromosomes.

•Be able to explain the problems associated with having a linear DNA genome (like the human genome)

Since the lagging strand has its RNA primer removed, there is a space left in the lagging strand, which shortens the length of the DNA.

•Be able to explain why polymorphisms may or may not have an impact on the phenotype of an organism

Some mutations will keep the same amino acid which doesn't affect the phenotype of an organism very much.

•Be able to draw a diagram depicting the 4 steps of base excision repair

Step 1: damaged base removed; sugar flips to outside of DNA Step 2: cleave DNA backbone on 5' side of damaged base Step 3: fill in the missing nucleotide (short patch) or nucleotides (long patch) Step 4: DNA ligase seals the remaining nick

pyrimidine dimer

Structure in which a bond forms between two adjacent pyrimidine molecules on the same strand of DNA; disrupts normal hydrogen bonding between complementary bases and distorts the normal configuration of the DNA molecule

•Be able to explain and/or diagram how human cells deal with the problem of preserving the ends of the chromosomes

Telomerase uses exonuclease to degrade lagging strand. Then it extends the leading strand without a template strand by using internal RNA. Primase and then DNA polymerase comes in to finish lagging strand synthesis. End product will still not have equal end lengths which is ok.

•Be able to explain how cre recombinase works to make a conditional knockout mouse

The cre-lox system: intervening sequences are deleted between the loxP sites. Cre is a recombinase that looks for loxP sites, lines them up together, and forces homologous recombination between them.

genetic polymorphism

The existence of two or more distinct alleles at a given locus in a population's gene pool.

alkylation

The transfer of an alkyl group from one molecule to another

dimer

a compound whose molecules are composed of two identical monomers

Chromosome

a threadlike structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes.

DNA Polymerase III

adding bases to the new DNA chain; proofreading the chain for mistakes

•Be able to explain why synthesis of the 2 different DNA strands must be fundamentally different from each other

all DNAs and RNAs are polymerized in the 5' to 3' direction (you can only add onto the 3' OH and not onto a 5' P)

micrococcal nuclease

an enzyme that degrades any DNA that is not protected by being wrapped around proteins such as nucleosomes. It is an exo/endo nuclease, meaning it universally degrade unprotected DNA

linker regions

areas of DNA between histone octomers

topoisomerase

corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands

reactive oxygen species

highly reactive forms of oxygen

semi-conservative

in each new DNA double helix, one strand is from the original molecule, and one strand is new

frameshift mutation

mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide

acetylation

of DNA and histones causes nucleosomes to loosen and spread apart

Gene Expression

process by which a gene produces its product and the product carries out its function

Histones

protein molecules around which DNA is tightly coiled in chromatin

semi-discontinuous

refers to how DNA must use okazaki fragments to synthesize lagging strand

Microarray

shows which genes are being actively transcribed in a sample from a cell

Rnase H

the enzyme that helps to remove the RNA primer during DNA replication

•Be able to list the 3 main mechanisms by which DNA damage is detected

•1) DNA damage often results in changes in DNA structure -DNA repair enzymes scan the surface of DNA for bulges, kinks, holes, etc. •2) DNA replication 3) RNA transcription enzymes sometimes discover the damage as they use the DNA as a template molecule

•Be able to differentiate between the types of mutations repaired, and the mechanisms used, in the 3 main types of DNA repair

•1) Direct repair: no DNA (no bases, backbone, etc.) is removed and replaced, the damage is simply reversed -Only for minor damage •2) Excision repair: bulky additions to nucleotides are removed and replaced with correct nucleotides (base repair for a single base or nucleotide repair for stretches of mistakes) •3) Mismatch repair: base-pairing mismatches, short insertions/deletions following DNA replication are repaired using similar mechanisms to excision repair

•Be able to explain the goals of a cell when performing DNA replication

•Make complete new copies of genetic material in preparation for cell division so that each daughter cell has same genes/traits as parent cells •Maintain genetic information with accuracy •SOME cells have to perform extra work to keep the ends of the DNA molecules from getting shorter (organisms with linear DNA genomes, doesn't apply to circular genomes)

•Be able to explain how cDNAs are used as tools in molecular biology, and why they are often preferred to working with entire genes (containing introns)

•Many genes are large due to presence of introns: nearly impossible to work with!•Using reverse transcriptase, you can readily clone the mRNA after splicing is complete. •If you clone a cDNA into an expression vector, you can make the recombinant protein for analysis

Compare and contrast priming methods for CDNA production

•Poly-dT (Oligo-dT) 5' TTTTTTTTT 3' To make cDNA of ALL MATURE mRNAs (eukaryotes have poly-A tails on mature mRNAs) •Random hexamers Random 6-nucleotide primers Make cDNA of ALL RNAs (random primer mix will bind to everything) •Gene-specific primers •Primers specific to a certain gene •To make cDNA of very specific sequences

•e able to explain how DNA mutations can be beneficial to organisms (topics 15/16) and how we can induce DNA mutations for a therapeutic benefit or to discover gene functions

•Randomly disrupt genes in a cohort of organisms, then examine phenotype of those organisms in order to find those rare individuals that have lost a particular trait -E.g., knockout mice to be discussed in topic 18 • •Search the genome to find out where insertion took place, and you may find a gene that contributes to that phenotype

•Be able to explain why one would want to make these types of mice

•To study the function of a particular protein by producing an organism that is incapable of making that protein •To produce animal models of human diseases -Can perform experimental treatments on animals in order to test it out before attempting on humans •Let's say that gene X is thought to contribute to disease in humans -Perform a gene X knockin into mice and then see if mice develop the same disease

Reverse Transcription

•Uses the enzyme reverse transcriptase (isolated from retroviruses) to convert RNA to cDNA