Cell Biology Exam 2 Study Guide

What are four ways of introducing DNA into mammalian cells? T/F: Biosaftey committees have no problem with researchers using Lentivirus (Fig. 6-30).

1. Ca++-DNA precipitate: DNA enters by pinocytosis 2. Liposome fusion: DNA surrounded by lipid micelle 3. Retrovirus delivery (potential risk) 4. Electrophoresis: brief shock makes cells permeable to external DNA

What do we mean when we say "this is a Cre mouse?" What's a Cre mouse good for?

Cre is a mutant. You cross it with LoxP to get loxP/X+ and Cre/+. This makes Cre only expressed in liver cells and normal.- Cre gene encodes protein that allows recombination for lox elements

DNA repair mechanisms (Figs. 5-34, 5-35, 5-36, 5-37, 5-38). What's no-homologous end joining (5-39). Do you really want this mechanism repairing you broken chromosomes? I'd prefer homologous recombination myself (5-41).

DNA Repair: · 5-Methylcytosine can be deaminated to form thymine · can deaminate normal dC to yield dU. A) base excision repair (prior to DNA replication) ○ recognizes G-T base mismatch or dU. 1. a DNA glycolase specific for dG:dT (or dG:dU) mismatches removes T (U). 2. Apurinic endonuclease 1 (APE1) then cuts the DNA backbone. 3. Apurinic lyase removes the deoxyribose-PO4, 4. while DNA pol B and ligase fill in the gap with normal dC. - not methylated can occur later Can be in G1 of cell cycle before DNA replication. DONOT want this in the S phase. B) mismatch excision repair (just after DNA replication): ○ S Phase is finished. Replicated genome and DNA pol proofread. After DNA replication ○ errors in the newly synthesized daughter strand are repaired. ○ MSH2 and MSH6 are proteins that recognize the mismatch base pairs. i) Open DNA and cleave away a portion of the brand new strand that was laid down. Using MLH1 endonuclease, PMS2; DNA helicase and DNA exonuclease. ○ mutations in genes encoding MSH2 and MLH1 are linked to colorectal cancers. ○ Fill in the region with DNA pol and DNA ligase seals the ends and mismatch repaired. ○ Question: how does the cell know which is the daughter strand vs. the old parental strand? C) Nucleotide excision repair: ○ recognizes perturbations (bulges) in normal B-form DNA i) can be caused by chemicals binding to the bases (chemical adducts), ii) or by UV irradiation which causes Thymine dimer formation. ○ Ex. Sunburn: UV light crosslinks thymine nitrogenous bases (thymine dimer). Covalently crosslinked. Causes perturbation. ○ XP-C and 23B proteins recognize DNA perturbation in B form DNA. ○ Protein TFIIH then unwinds helix. i) Transcription factor IIH ii) Moonlights in DNA repair - normally used in transcription. ○ XP-G and RPA (same as in replication) further unwind and stabilize. ○ XP-F with XP-G cut out damaged DNA. ○ Gap filled in by DNA Pol and ligase seals the ends. Disease: ○ XP: Xeroderma pigmentosum: a hereditary disease linked to skin cancers: melanomas and squamous cell carcinomas. D) Repairing broken ends (non-homologous end joining, NHEJ)of chromosomes. ○ Do not want this if you have a broken chromosome. Cell uses proteins pol and ligase - you lose nucleotides from the cut site. Can insert wrong nucleotides. ○ could be within a gene: - usually a loss (trimming) of base pairs = mutation i) shift reading frame; mRNA is garbage = non-functional protein ○ could be broken ends of separate chromosomes = translocation ○ NHEJ important for later discussions on telomeres and CRISPR E) Homologous recombination: To repair double-strand breaks (broken chromosomes) ○ blue father; pink mother. ○ Paternal chromosome serves as a template to repair broken maternal chromosome (homo repair) ○ Maternal strand invades paternal strand. Chromosome repaired. Left hand is still maternal but the right side is paternal. Recombined the 2 chromosomes in this mechanism ○ Using intact paternal chromosome to repair broken maternal chromosome it is perfect down to nucleotide. ○ Recombination between sister chromatids ○ Should be error free (what you want!!!!!)

OK, the big question: what's the difference between genetic complementation (Fig. 6-7) and functional (or molecular) complementation (Fig. 6-16)? Thinking beyond: We knocked out a gene in Drosophila whose protein product is needed for ribosome production. How do we know for certain that the phenotypes we see in the mutant fly are directly related to the gene we knocked out rather than to some second site mutation(s) that we could have created inadvertently? CAUTION - Answering this question correctly makes you a Cell Biologist!!

Genetic Complementation: · Used by Leland Hartwell - a yeast geneticist o Making a bunch of temperature sensitive conditional mutations that block cell cycle at different points o Called cdc = cell division cycle mutants · Lots of cdc mutants and wants to categorize them. · Taking X mutation and Y mutation - temperature dependent cells = can grow at 23oC not at 36oC o 2 haploid cells are mated § Mate a and α o Product is the diploid cell which grows at 23oC o Take the master plate and grow replica plates at 36oC § Diploid cells are growing at this temperature. Haploid would not grow. o X and Y are recessive mutations o Wild type is complementing(rescuing) the mutant X and Y in the diploid cell to grow at non-permissive temperature. · Far Right side o X mutation and Z mutation mated making diploid cell o Grow at 23oC and will not grow at 36oC o X and Z gene are exactly the same they are alleles of one another. NO complementation - so no growth at 36oC. · Genetic complementation analysis: Determines allelism: mutations either complement (not allelic), or they fail to compliment (are allelic). Functional Complementation in yeast: · One cdc mutants and grows at 23oC but cannot grow at 36oC o It also is a temperature sensitive conditional mutation · The strain of yeast started with is URa3- o Uracil needs to be added to get the cell to grow at permissive temperature. o At permissive temperature, they can get by that point and grow o At non-permissive temperature, they stop at a point in the cell cycle. · Takes genomic library just formed in a shuttle vector and transforms library into the yeast cells · URA3+ gene on plasmids o Picked up they can grow in absence of uracil · Just a selectable marker to select for cells who picked up the URA3+ plasmid. · He complemented the original cdc- mutant gene with the wild-type gene o Similar to Hartwell but a plasmid is used instead of a genetic cross o A plasmid containing URA3+ and cdc+ is introduced into the cell and they can complement or rescue the mutants.· Once Nurse complemented (rescue) the genomic cdc mutation, He o Re-isolated the "red" plasmid from the rescued yeast cells o Sequenced the "red" gene that was ligated into the plasmid o Deduced the amino acid sequence to identify the protein product. § Now he knows the protein product coming from the cell regulating genes o Proteins are highly conserved These yeast proteins control the cell cycle; his and other labs determined how these conserved regulatory proteins work

How do most proteins bind DNA? (Fig. 5-5). What groove is usually involved? What interactions/bonds occur between DNA and protein?

Proteins make contact with bases via H-bonds and van der Waals, usually within the major groove. · TATA-binding protein (TBP) is one exception. o TBP binds the minor groove to bend the DNA thus separating the two strands. · DNA does change shape upon protein binding

The 5' cap (Fig. 5-14). Which eukaryotic polymerase is always associated with the 5' cap? Is this 5' cap found on prokaryotic mRNAs?

RNA Pol II (eukaryotes) protects the nascent mRNA prokaryotic mRNAs do not have. a5' cap, since prokaryote RNA does not need ot undergo further processing. everything happens in one step in nucleoside

1) Fred Sanger's DNA sequencing technique (lecture slides). Ingenious!! Give him a Nobel!!

So, what's a dideoxy ribonuclease triphosphate? · no 3' OH group to extend the chain o chain termination once its incorporated · H on both 2' and 3' of the sugar · once the dideoxy is incorporated, synthesis stops (chain termination) technique: · use a primer specific for sequence you want to determine. The primer anneals to target DNA. · Primer labeled with say 32P. · All 4 regular nucleotides present in 100microM · Add DNA polymerase which extends the primer · Spit rxn into 4 separate tubes - each containing a different dideoxy nucleotide. o If polymerase incorporates a dideoxy into a growing chain it's going to stop. o The dideoxy are at 1/100 concentration of the normal deoxynucleotides § Only 1 out of 100 will incorporate the dideoxy. · The "g" reaction yields thousands of fragments, all ending in ddG, but of different sizes. o Put on an acrylamide gel to get sequence. · Now automated; once used to sequence whole genomes. o Fluorescently-tagged nucleotides were used and detected in real time as DNA fragments move past a fluorescence detector o Vast sequence information was acquired. · Led to a new field called bioinformatics: o Resolution in computer technology to handle the vast sequence information o Compare nucleotide and deduced AA sequences. · Automated machines (sequencers) detect the fluorescent nucleotides in real time o With automation, the entire human genome was sequenced. o Many other genomes as well: drosophila, yeast, C. elegans, etc. Molecular evolution

Transmission electron microscopy (TEM) (Fig. 4-28, lecture slides). Why do you use heavy metal salts like lead citrate and uranium acetate to "stain" thin sections of cells for TEM? Resolution for TEM is ______. What kind of lenses is used in a transmission electron microscope? What is immuno-electron microscopy? Why gold? (Fig. 4-33)

TEM:· Electron wavelength = 0.005 nm · Resolution in theory should be 40,000x greater than light microscopy. o ITS NOT - only about 2000x better than a light microscope · Effective resolution of TEM down to about 0.1nm or 1.0 Angstrom. o Can start seeing atoms What was Super-Res Confocal? gets the resolution lower than a light microscope does.· Resolution about 10 nm at best Probably 20-50nm in x and y axis but about 120 in z axis. Using laser scanning confocal microscope to scan in one orientation and keep scanning in different orientations.§ Pile up the scans and then they will give a much sharper image. o So, TEM is 200x better in resolution than super confocal microscopy Electromagnet lenses!! · Special filament glowing red at top and below it is an anode. The voltage difference is 120kV. Want to get a 200-300kV. Greater accelerating voltage the better the resolution. Electrons come off filament and accelerated by anode. Electromagnets focus electron beam on specimen. Image generated on phosphorescent screen or camera down below. · Samples are placed on copper, gold or nickel grids coated with plastic and carbon o the grid is about 3mm in diameter, and placed midway of column o Column is perfect vacuum, electrons come straight down o Electrons deflect off sample because stained with heavy metals. (Uranyl acetate or lead citrate) o Prevents the electrons from hitting phosphorescent screen. o Electrons hit the phosphorescent screen and glows light. o Dark structures on the screen are where the heavy metals have deposited on sample or grid itself. · Instead of adding particles to the grid you can add thin sections of cells on the grid. o Can see very clear images. · Use an ultra-microtome to cut sections of 20nm thickness. o Stain with heavy metal solutions o Dark structures took up heavy metals. · Lead and uranium salts as "stains" to deflect electrons and thus provide a contrasting image. Immuno-Electron Microscopy: · Immuno-Gold labeling using TEM o Look at Catalase - enzyme found in peroxisomes. o Antibody against catalase and recognizes it specifically. o Take this gold particle and its coated with protein A and has a high affinity for conserved regions for IG type antibodies. o Gold deflects the electrons from the column o It appears dark o picture or in camera · Black specs in a picture represent gold particles in peroxisome. · Protein A from Staphylococcus aureus The gold particles are electron dense, so they deflect electrons.

tRNAs are good to know!! (Figs. 5-20a and b).

Transfer RNAs (tRNAs): · 70-80 nucleotides in length o fold back on themselves in secondary structures (stem and loop) · many of the nucleotides have been modified after they've been transcribed. Many nucleotides can be methylated. · 3' end of tRNA has been processed; they all end in -CCA 3' · the anticodon binds to the codon in mRNA · codon and anti-codon interaction is antiparallel · modifications to uridine occur after the tRNA is synthesized. · Inosine: in 5' end of the anticodon A tRNA has a 3D structure: · Anticodon loop and -CCA end are well exposed tRNAs in Bacteria: · 30-40 different tRNAs in bacteria · remember: there are 61 functional codons o Problem: - looks like bacteria produce fewer tRNAs than are required. · In these organisms, a single tRNA may recognize more than one codon in the mRNA. · How? o By the Wobble base position: (mostly bacteria) § The first base in the tRNAs anti-codon, 5' end § But the third base in the corresponding mRNA codon. 3' end · Recall: codon and anti-codon are antiparallel. o *Non-standard base-pairing between G and U can occur, but in the wobble position only. o So, this one in particular tRNA can bind two codons, UUC or UUU - both encode Phe. FYI: base pairing rules for the wobble position: · Inosine in the wobble position can pair with C, A, or U. The point is: one tRNA can base pair with 2 or 3 codons. · Inosine is the deaminated version of adenine; occurring after tRNA is synthesized. Still talking bacteria: · Bacteria have fewer tRNAs than they do functional codons. · Because certain tRNAs can bind to more than one codon, bacteria actually have fewer tRNAs than codons. tRNAs in eukaryotes: · Opposite problem than in prokaryotes · 50 -100 different tRNAs in eukaryotes (more than they need) · remember: only 20 AA's, but 61 "functional" codons · so, in eukaryotes, there are more tRNAs than codons o problem? · Ans: more than one tRNA can attach a certain AA to one codon. o E.g. there are multiple tRNAs for a single AA (codon) · One specific AA is said to have a few "cognate" tRNAs. o One amino acid can have multiple tRNAs carry it. Is the wobble position used in eukaryotes? NO, however yeast have some use for it.

What is a cDNA (Fig. 6-17), and why is reverse transcriptase so important in making cDNAs?

cDNAs (complementary DNAs) · Made from tissue-specific or cell- specific mRNAs (liver vs. brain) · Or perhaps from a particular stage of embryogenesis (day 2 vs day 24) · TEST QUESTION: o Since cDNAs are made from mature, cytoplasmic mRNAs, cDNAs have no introns, no promoters (turns transcription on/off), no intergenic (genes in between our DNA) DNA. · cDNA sequence indirectly provides the protein sequence. o i.e. the protein sequence is "deduced" from cDNA sequence · cDNA libraries are specific for a given tissue or cell type o contains about 10,000 different cDNAs. § About 10,000 mRNAs expressed in one tissue type; some are rare, some are abundant

Why do you think eukaryotic cells use alternative splicing of their pre-mRNAs? (Fig. 5-16) How many genes are there in humans? What if one gene alternatively splices its pre-mRNA? So could humans produce more different kinds of proteins than the gene number indicates?

depending on where the protein is going or its designated future function, they splice out the genes that are not needed. · looking at the fibronectin gene which is active in both hepatocytes and fibroblasts. o Fibroblasts and hepatocytes each have different splicing patterns and include some genes the other one doesn't. humans have about 20000 protein coding genes. alternatively splice these, eukaryotic cells can produce far more proteins than just those 20,000 protein coding genes. Very powerful method of gene expression and protein regulation in eukaryotes: Ability to differentially slice pre-mRNa into different protein products, from the same gene, but in different cell types. humans could produce more proteins if alternatively spliced.

What's an intron, and do prokaryotes have introns in general? (Fig. 5-15).

introns are the coding parts of the genome that have no function. prokaryotes lack introns

Reading frames (Fig. 5-18). What sets the correct reading frame in an mRNA?

o which is the correct reading frame for particular mRNA? The initial start codon AUG starts.

cDNA libraries and Colony Lift Hybridizations to screen these libraries. In autoradiography, what is reducing the silver bromide crystal?

the beta particle from tritium atom

Base pairing rules: (Fig. 5-3b).

· Based on size shape and composition of the bases · G-C with 3 H-bonds · A-T with 2 H-bonds · Sugar phosphate backbones should be outside with bases projected inward. · Anti-parallel strands in any double stranded nucleic acid sequence.

Gene knockout in mouse, and the construction of a transgenic mouse (from DNA cutting and pasting, to ES cells, to blastocysts, to Mendelian genetics). (Fig. 6-37 and 6-38) NOTE: Keep in mind that the Herpes simplex viral thymidine kinase enzyme converts ganciclovir into a toxic compound that kills the cells that still retain the TKhsv gene.Cells that lack the TKhsvgene due to homologous recombination survive in the presence of ganciclovir

- Embryonic Stem (ES) cells from blastocyst mouse embryos with coat color marker genes: brown (A) is dominant to black (a)- totipotent- easily cultured and transfected by electrophoresis- homologous recomb disrupts/replaces endogenous gene (normal gene in chr.)- can select for homologous recomb. (+/- selections)- yields transgenic knockout animal

Cryo-EM/Tomography: why do you need a good camera and sophisticated computers?

Look at surface of your protein or surface of a structure. · Samples are frozen in liquid nitrogen (helium) · In their native, hydrated state · Tilting with compilation of multiple high-resolution images provides 3D structures · Need a microscope in 200-300kv o Sample is on grid o Rotate stage 70 degrees one way and then the other. Computer takes pics of every particle in the grid and all slightly different which helps get a 3D structure of the particle. Because you cannot effectively get clear images.

Scanning EM: what's the resolution, and what do you use SEM for?

Sample is at the bottom of the SEM. · Surface features of the sample · 10nm resolution - not as good as TEM · tissue: fixed, critical-point dried (freeze-dried), then coated with a thin layer or films of gold or platinum. · Electron beam scans coated specimen. o Electrons deflected off specimen · Secondary electrons are collected by a detector · Forms 3D images Electron gun at top of microscope; electromagnets focus beam on top of sample coated in gold or platinum and a detector picks up the deflected electrons from the sample; the computer then generates a 3D image

Basic cloning strategy. Fig. 6-14. What do we mean by the term Transformation?

Recombinant DNA Technology: ("cloning" DNA) · 1970's: molecular biology started o started with discovery of restriction enzymes § cutting and pasting DNA o DNA Ligase from bacteria § Cutting and pasting DNA o DNA sequencing (Sanger's method) · DNA Vectors: (to clone DNA) o DNAs that replicate autonomously § Like circular plasmid DNAs in bacteria § Or viral genomes: Bacteriophage lambda (l) vectors · Recombinant DNA: o Insert (ligate) a DNA sequence of interest into a vector. o This "insert" will be replicated along with the vector · Plasmids: o Circular, dsDNA in bacteria, yeast, a few higher organisms. o Small compared to the main E. coli chromosome o Few kb pairs to over 100 kb pairs o Symbiotic relationships: antibiotic resistance gene. § Bacteria cell and plasmid are surviving because of abx resistance. A typical E. coli plasmid used for cloning: · has an origin of DNA replication - they replicate autonomously on their own · origin of replication is AT rich sequence. · Replication forks will start at both ends of the bubble and go around the plasmid. · Abx resistsance gene os part of plasmid. o Ampicillin resistance gene ampr o Polylinker region is what were interested in - has several restriction enzyme sites for us to insert your DNA of interest using DNA ligase § Part of DNA plasmid which replicates inside E. coli cell The Polylinker region of a typical cloning vector: · Heavily engineered by biotech companies · All restriction enzyme sites in DNA · Notice the overlap in some restriction site sequences Restriction Enzymes: · Cut DNA at very specific DNA sequences (restriction sites) · EcoR1 - E. coli Restriction enzyme 1 o reads a palindromic sequence - the same forward and backward. o Cuts each of 2 genes and the phosphodiester linkage. Makes sticky ends. o Many enzymes leave the stick ends by not cutting properly · Note: many of the restriction sites are palindromic o Some enzymes cut in same position (non staggered) - blunt-ends · Ligation is "pasting" two DNA molecules together: · can be finicky*NOTE: there are 5' phosphates and 3' hydroxyls on the DNAs you want to ligate - need to match. · Say DNA1 is our vector and we want to ligate your DNA of interest into a vector. o Many different DNAs but only one has sticky ends that can H-bond or anneal to DNA1. o Transient interaction between DNA1 and vector you want to add it to. § DNA ligase seals the phosphodiester linkages between vector and your DNA using ATP § Forms a recombinant plasmid DNA molecule § Blunt end needs more DNA ligase to do it. (e.g. Sma I in table 6.1) Transformation: · Once you have your recombinant plasmid, now introduce it into E. coli. o Uptake and expression of foreign DNA o First discovered by Frederick Griffith, 1928 § In Streptococcus pneumoniae - dangerous o Avery, McCarty and McLeod later showed that DNA is the transforming material (1944). o Today we transform E. coli with recombinant DNA every day in lab. Summary: · cut and paste plasmid to build add a DNA fragment = recombinant plasmid. · Introduce plasmid into E. coli by transformation. · Antibiotic resistance gene allows only transformed E. coli to survive and replicate. (Non-transformed cells can't grow in the presence of ampicillin.) Abx: Ampicillin, Tetracycline, Chloramphenicol, Kanamycin

We once sent some mammalian cells to the National Cancer Institute in Frederick, MD. How did we send them (thawed? frozen?), and what solution were the cells in?

They would be frozen using DMSO and protein-rich fetal calf serum, which prevents the formation of ice crystals, and stored in liquid nitrogen

What is stable transfection (Fig. 6-29b)? What does G418 allow you to do?

- Now have neomycin resistant gene on the plasmid, this allows you to select for those cells that have taken up the plasmid. Treating cells with this drug, G418. Over time got colonies growing, all resistant to G418, that means they have the plasmid. (transfect cultured cells with lipid treatment). Random insertion onto the cells chromosome. Eventually DNA in plasmid will integrate into the genome, into one of the chromosomes and is stable. Will be there for a while. It allows the cell to be resistant to neomycin - a polypeptide synthesis blocker. - cDNA plasmid integrates into chromosomes of a mammalian cell- permanent transformation of genome; continuous selection so 100% cells express the change- G418 (neomycin) kills all euks that failed to take up exogenous DNA; allows us to select for resistant colonies- neoR resistance to G418- random insertion

Gene knockout in yeast (Fig. 6-36)

- Target gene in blue. Sequences in front of and behind that you know. Entire yeast genome has been sequenced. With the sequence known, you design oligonucleotides, this is the forward and the reverse primer, the ends of your primers are homologous to sequences flanking target gene. Color codings indicate what sequences are identical. Second half of primers are identical to KanMX gene. In yeast, the KanMX gene is encoding neomycin resistance. Use forward and reverse primers to PCR amplify kanMx gene. You are using only the portions of the primers that are homologous to the KanMX gene. But, your PCR product now will have flanking sequences that are now homologous to the target gene Transfect into diploid yeast cells your PCR product made on previous slide. The very flanking sequences of your PCR product are homologous to the sequences flanking your target gene. The PCR product can undergo double homologous recombination with the flanking sequences of your target gene. When that happens you are essentially replacing normal endogenous gene on this one chromosome with your PCR product. PCR product is the KanMX gene. Cell is now resistant to G418. Cell is heterozygous, you still have wild type copy of your target gene, but the other copy of the gene is simply replaced by that KanMX. You can force cell into meiosis, by starving it, and can hand isolate these 4 haploid cells in tetrad analysis. Determine if these haploid cells with the gene knockout are viable, can they survive with this gene being replaced by kanMX gene?

What specific DNA sequence would you look for to determine if your mouse has the knockout gene, and what techniques would you use to detect this DNA sequence?

- The DNA sequence would be homologous to the X gene.- You would use genomic PCR with Forward primer and both reverse primers to test if the mouse is homo or hetero for the disrupted gene

What do we mean when we say "this is a flox'ed mouse?" What is a flox'ed mouse good for?

- This mouse carries conditional (floxed) alleles of gene Y- Gene Y is flanked by lox elements in all tissues - Your mouse is floxed mouse. Your gene of interest, Gene Y, putting lox elements on either side of the entire gene. Not just putting them on either side of one exon, the entire gene. Homozygous. Lox elements are not going to cause any phenotype or damage. Cross this mouse to cre mouse and in particular tissue of your choice, lox elements will undergo homologous recombination taking out both copies of the genes in one swoop

Review Mendel's Law of Segregation. We didn't talk about his Law of Segregation, but do you recall what that involves? Who's Thomas Hunt Morgan, and what did he discover (hint: Fig. 6-10)?

- states that allele pairs separate or segregate during gamete formation and randomly unite at fertilization. classic 3:1 phenotypic ration in F1. - T.H. Morgan discovered crossing over in drosophila. chromosome theory of inheritance. Discovered genes lie on chromosomes and they can be recombinant or cross over.

5' phosphates and 3' hydroxyl groups (Fig. 5-2a).

A single polynucleotide DNA chain: · 5' phosphate · 3' hydroxyl · phosphodiester bond holds nucleotides together. · synthesis always in 5' to 3' direction - add nucleotide at 3' end · why? You need the hydroxyl group for condensation synthesis. Chain written as 5'CAG3'

Prokaryotic genome organization versus eukaryotic genome organization (Fig. 5-13). The lac operon: negative and positive control.

Baceria: contain operans: ex. Trp, Lac Trp is 5 genes tightly linked together E-D-C-B-A One promoter drives the transcription of these 5 genes. Get transcription on first gene, then RNA polymerase will just keep going down the operon. Product is 1 long mRNA from one promoter upstream of gene E - called polycistronic mRNA, found in bacteria, prokaryotes (NOT found in Eukaryotes) polycistronic mRNA will begin translation at 5 different spots. you will get 5 different enzymes needed for biosynthesis from this Trp operon. eukaryotic · Separate genes for the 5 enzymes necessary for Trp biosynthesis (in yeast). o An essential Amino Acid in humans · Note: o no poly-cistronic mRNAs o All 5 TRP genes coordinately regulated using their own promoters. · Pre-mRNA processing in Eukaryotes · Notice the 5' Cap specifically m7Gppp · Notice the poly(A) tial - n=250-300 A's essentially · Poly(A)-binding protein - called PABP1 · Splicing in eukaryotes: o Extremely rare in prokaryotes o Notice 5' and 3' UTRs (in gray) · The 5' cap on mRNAs and some small nuclear RNAs (snRNAs) in eukaryotes (not prokaryotes) · 5' cap found on any RNA transcribed by RNA polymerase II (eukaryotes)(responsible for mRNA) · Note: (parts of 5' cap) o 7-methylguanylate o 5'-5' tri-phosphate linkage o methyl groups on the first two nucleotides encoded by the gene · an aside: NAD+ (nicotinamide adenine dinucleotide) also has 5'-5' linkage using two phosphates - negative control. Repressor is bound to keep operon silent - positive control, where CAP proteins recruit RNA polymerase to get transcription rates going up.

Translation initiation in eukaryotes: know the functions of IF's (in general), SSU, m7G, Kozak's AUG, LSU, ATP and GTP hydrolysis (Fig. 5-24).

COME BACK!!! ● As in prokaryotes, eukaryotic LSU and SSU are kept separate prior to translation by Eukaryotic Initiation Factors (eIFs) 1. mRNA needs to be translated with 5' untranslated region. 7 methyl G Cap unique to eukaryotes. Stem loop secondary structures can be a problem. 2. AUG start codon further downstream a. Most mRNA in eukaryotic systems or polyadenylated. PABP binds here. 3. eIF4 complex - binds to 5' cap. a. PABP binds to one of the subunits of the eIF4. Forming a circle 4. eIF bind to SSU. GNSP has GTP bound as it associates with SSU. tRNAiMet binds to middle region of SSU. eIF4 complex recruits the SSU. Starts scanning 5' UTR for AUG codon in Kozacs sequence. a. No shine delgarno 5. eIF4 subunit a is a helicase and dissolves any stem/loop structures formed in 5'UTR so SSU can scan freely. 6. Once SSU binds to Kozacs sequence eIF2 with its GTP hydrolyzes GTP and GNAP turns off a subunit eIF5. 7. LSU joins SSU. In the intact ribosome, subunits 1A and 5B-GTP block A site. They pop out once GTP is hydrolyzed. Now A site open and elongation starts.

Classic genetics versus reverse genetics (Fig. 6-1). Which one do most cell biologists now use?

Classic genetics approach: · Identifying a particular phenotype then eventually identifying the gene and what it encodes = protein product Reverse genetics approach: Got the gene and think we know the function; can mutate and knockout gene. Start at DNA level and work back to what phenotypes are caused by the gene knockout. Most cell Biologists use reverse genetics now.

What is supercoiling? What do the topoisomerases do? What introduces supercoils in bacteria? (Fig. 5-8)? Any preliminary idea on how eukaryotic DNA is negatively super-coiled?

DNA is supercoiled in living cells. Supercoiled DNA: · Localized unwinding of the helix occurs at sites of DNA replication, transcription, repair, and recombination. · Unwinding causes stress within the rest of the helix · Stress relieved by supercoiling · Degree of supercoiling is regulated by Topoisomerases. o Topo I: nicks one strand, then reseals o Topo II: cuts both strands, then reseals. · Topoisomerases relieve supercoils (that are in excess) · DNA in all living cells is negatively supercoiled o Gyrases in bacteria introduced these negative supercoils No gyrases in eukaryotic cells DNA is negatively supercoiled around histone proteins.

Melting dsDNA: What is Tm? What does the G-C content of DNA do to the Tm? (Fig. 5-7).

Denaturation/Renaturation: · Strands must separate normally at: o 1. Replication o 2. transcription o 3. recombination o 4. DNA repair · blue strands are complementary to each other, so they anneal · red strands are complementary to each other, so they anneal · the native state of DNA can be reacted with heat,OH- which denatures or melts the DNA into single strands. Renaturation requires special conditions. As DNA melts its absorbance at 260nm essentially doubles: · Tm is dependent on the base composition. (important for Polymerase Chain reactions (PCRs) Tm is the melting point for double-stranded nucleic acid. this is defined as the temperature at which 50% of the strands are in double-stranded form and 50% are single-stranded, i.e. midway in the melting curve. · The more G-C pairs, the higher the Tm - meaning more energy (higher temperature) is needed.

Translation elongation: P site versus A site versus E site, and the mechanism of elongation. Fig. 5-25. Translation Termination: Fig. 5-26.

Elongation: ● Elongation factors (EFs) facilitate: ● How does the ribosome know to bind aminoacyl-tRNA #2 instead of #3 or #4 in the A site? o The codon selects which tRNA can fit into the A site. Ribosome does have a proofreading function. ● EF1aGTP - GNSP o Hydrolyzes GTP, allows for tRNA to fill the A site. Steps: Peptidyl-tRNA already in P site. Aminoacyl tRNA arrives in the A site complexed with EF1a-GTP Peptidyl transfer by Peptidyl transferase activity - is a function of the 28S rRNA, Recall "ribozyme" in eukaryotes. Note: the peptide chain is briefly attached to the tRNA in the A site. i. Transiently in A site. ● Formation of a peptide bond: o Amino group of incoming AA attacks bond of the peptidyl-tRNA in the P site. (high energy ester linkage) ● Note: the peptide chain has been temporarily transferred to the A site. EF2GTP hydrolyzes its GTP and the energy released helps the Ribosome shift to the right by one codon, while the tRNAs remain bound to their respective codons. o Free tRNA in E site is now ejected. A site open. · Continues until a stop codon is reached and recognized by a release factor. Termination: ● Peptide release factors (RF1 and RF2) look like tRNAs (mimicry). Enter A site. (protein assumes structure of tRNA) o In bacteria RFI and RF2 RF1 - UAG, UAA RF2 - UAA, UGA o Eukaryotes eRF1+eRF3-GTP work together does all 3 stop codons ● Once RF1 (or RF2) binds A site, RF3-GTP provides energy to cut the last peptidyl-tRNA bond by GTP hydrolysis. ● ABCE1 breaks SSU and LSU apart. o Complex of proteins destroys ribosome. ● eIF1, 1A and 3 subunits rejoin SSU - to prepare for next translation Original initiation factors

T/F: RNA is always linear. Name two examples of RNAs with higher order structures (Fig. 5-9).

FALSE § Can be linear or circular; single or double stranded § Double stranded RNA provides secondary structure Secondary structures can fold into tertiary structures 2 examples: stem-loop and hairpin

FACS (Fig. 4-2). So, can FACS distinguish between G1 cells, versus cells in S phase, versus G2 cells?

Fluorescence Activated Cell Sorter (FACS) · Device for isolating certain cells grown in culture or primary cells isolated form tissues. · Still dealing with cells grown in culture · Technique: Can separate out individual cell types from other cell types. provided they have specific cell surface markers that identify them · Send individual cells down a column and are differentially fluorescent. Antibody can recognize a cell surface antigen on cell you want to isolate. Let's say red. Isolate cells fluorescing red by the device. The cells get a differential charge, negative charge and cells you really want get a double negative charge and other one negative charges move to another bin. o Sort cells based on differential fluorescence. You dial it into the machine. Yes, it can. It can isolate cells between 1 and 2 in S phase

The PCR (Fig. 6-18). Why is this technique so powerful? How would you ligate a PCR-amplified DNA product into a plasmid (Fig. 6-19)? Pretty slick, hey!!?

Polymerase Chain Reaction (PCR): · Probably the most powerful molecular biology technique ever devised (CRISPR is rapidly superseding) · Primers used are synthesized in labs - synthetic · Target DNA is on the top · Design an oligonucleotide or reverse primer that anneals to the top strand with the target DNA in a 5' to 3' orientation o 3' OH to extend primer to synthesize complementary strand · the forward primer anneals to the other strand of DNA · heat or denature template / target DNA · drop temperature to the melting temperature of the 2 oligonucleotides · Tm dependent on sequence of primers.*** · Anneal 2 primers to 2 template strands of DNA · Raise temperature back to 72oC · Special DNA Pol isolated from Archaea o Works best at 72o o Extends the 3'OH group for both the forward and reverse primers o Get replicated DNA · Primers in vast excess so get temp back up to 98oC o Melt DNA and make single strand and anneal more primers to target sites and extend · PCR developed by Kery Mullis, 1983. · Amplifies (replicates) specific DNA sequences, but any that you choose. · Extremely sensitive: o Basic cell bio research o Human genetics and health o Criminal forensics, and archaeology/anthropology (primate evolution) Design your primers to include restriction enzyme site. · Design a forward primer to be complementary to one of the strands of your DNA · 5' to that complementary sequence we included a restriction enzyme complex o BAMH1 site · Reverse oligo site we design in the 3' HindIII site · RE cleave and form sticky ends we can ligate the vector into a plasmid. A new method for producing recombinant plasmids: · Gibson assembly o Uses PCR products o Take 5 different PCR products all with different forward and reverse oligos o In 60-80 minutes, they assemble in the proper order and ligate into cloning vector. 20-40 base pairs

The proof-reading function of DNA polymerase (Fig. 5-33).

Polymerase has proofreading function - 3' end of growing strand has incorrect nucleotide added. Signals to flip strand to exonuclease domain which trims off the incorrectly placed nucleotide then flips it back up into polymerase domain to continue on.

RNA versus DNA structures and chemical differences. Which is more stable, DNA or RNA? Why?

RNA: · Differs from DNA: o Ribose instead of deoxyribose § RNA has that 2'-OH group which makes it unstable o Uracil instead of thymine § Can be linear or circular; single or double stranded § Double stranded RNA provides secondary structure § Secondary structures can fold into tertiary structures. · SOUNDS FAMILIAR · RNA is exceedingly sensitive to ribonuclease activity and base-catalyzed hydrolysis RNA secondary structures: helical regions parallel or antiparallel? - exist naturally in antiparallel orientation; however fig. 59a the double helical stem region is parallel. DNA is more stable than RNA because RNA is susceptible to alkaline hydrolysis because the ribose sugar in RNA has a hydroxyl group at the 2' position, which makes RNA chemically unstable compared to DNA (DNA has hydrogen at the 2' position). DNA is stable in alkaline conditions.

Overview of viruses. The retroviruses are most important here. What's reverse transcriptase?

Retroviruses: (super nasty) · Enveloped, membrane form previously infected cell. · Contain two copies of genomic RNA · RNAs are templates for DNA synthesis · Reverse Transcriptase reads viral RNA as it synthesizes single-stranded DNA o Then reads the single-stranded DNA to make a complementary DNA strand to yield double-stranded DNA by same enzyme. = dsViral-DNA · Double-stranded DNA then integrates into chromosomes o Called a provirus · Provirus expresses more viral RNA - a population of new viruses to go infect more healthy cells. · Some retroviruses contain oncogenes (mainly=mice and birds) · Human T-cell lymphotropic Virus (HTLV) = a leukemia · Human Immunodeficiency Virus = AIDS Life cycle of a retrovirus: · Virus has a membrane envelope with proteins that bind to target receptor proteins. · Virus contains: 2 copies of RNA genome and 2 RT enzymes · Gets into cell by endocytosis - RT copies RNA into dsDNA - dsDNA sent to chromosomes o Provirus here in multiple copies - cell is forever infected. - no way to get provirus out of cell. § Provirus gives rise to new generations of provirus and proteins. · Is there a way to inactivate the retrovirus? By CRISPR

Southern blots (Fig. 6-24) versus Northern blots (versus Western blots).

Southern Blots: · Take genomic DNA; cut with particular RE like EcoR1 o Generates thousands of genomic fragments - different sizes · Interested in just one fragment. · Algarose gel laid on top glass plate on top of paper. · Alkaline solution wicking through to nitrocellulose paper. · As alkaline runs through gel by diffusion it gets DNA to move from gel to paper and denatures it. Makes it ssDNA and sticks to nitro paper. o Can now probe paper with a nucleic acid probe that's single stranded and is complementary to your DNA and if it present it will anneal by H-binding. o Probe can be ssRNA or ssDNA. § Complementary to gene of interest it will light up by radioactive probes with 32P § X-ray film can be used to detect the hybridized probe or a machine called phospho-imager to look for radioactivity. § # of genome copies will make that number of bands. Northern blot: · RNA is blotted (transferred) to the nitrocellulose o know cell type produces the mRNA and how much is there relative to other things. · Un-induced cell there's nothing on blot but, as time goes on you can see more of the mRNA produced from induction. · Shows induction profile · Differentiation of Erythro-leukemia Cells Southern Blots: · Can characterize the gene in the genome · Example o # of copies of the gene are there in the genome? o Determine the restriction or physical map of the gene. § Cut with RE to determine this Northern Blots: · Similar blotting technology as used in southern blots (neutral pH) · RNA is blotted to paper instead of DNA · To detect a particular mRNA within a cell type o Its relative abundance (compared to like actin mRNA) o Its tissue type expression The induction profile of the gene encoding the mRNA. Western Blots: denature protein run an agarose gel or plyacrylamide gel. get nitrocellulose paper incubate with primary anitbody then incubate with a second antibody and observe for fluorescence (2 antibody is tagged) analyze bands on the gels.

B form helix versus A form versus Z form helix. Which form of DNA is most prevalent in our chromosomes? What are its dimensions, structural properties? (Fig. 5-3a). triple helix: natural? Exists in vivo?

Watson and crick, 1953: · Solved structure for dsDNA · Base pairs are not completely perpendicular to the axis of helix (actually a 6 degree tilt) · Base pairs stack on top of each other allowing for van der waals interactions that help stabilize the helix. · Standard B form DNA o Most prevalent form in the cell in chromosomes. · Helix is right handed o About 10 base pairs per turn · Diameter is 2nm =20 angstroms B-Form DNA: · Major and minor grooves · Bases are exposed in the major groove (easy for things to bind), less in the minor groove. o Proteins bind in major groove · This is critical for gene expression A-Form DNA: made in lab · B-form can be converted to A-form in 70% EtOH o Very low humidity, dehydrating the helix · DNA-anneal to RNA hybrid helices are more A-form than B-form - normal inside cell. · RNA-RNA helices have A-form structure. · Base stacking is more compressed (11 bp per turn) · Base-pairs are severely tilted and off center Z-Form DNA: gene sequence altered in lab · Artificially created in the lab · Exists in vivo? Many gene promoters are G-C rich - are they more helical or smashed (Z form or B form) · Alternating G's and C's in the two strands Sugar-phosphate backbone appears to zigzag; triple helix - artificially created in lab possibly could exist in vivo with an RNA.

Restriction enzymes and DNA ligase, what do they do? Fig. 6-11 and 6-12. Why does Eco R1 not chop up the E. coli genome?

· Cut DNA at very specific DNA sequences (restriction sites) · EcoR1 - E. coli Restriction enzyme 1 o reads a palindromic sequence - the same forward and backward. o Cuts each of 2 genes and the phosphodiester linkage. Makes sticky ends. o Many enzymes leave the stick ends by not cutting properly · Note: many of the restriction sites are palindromic o Some enzymes cut in same position (non staggered) - blunt-ends · Ligation is "pasting" two DNA molecules together: · can be finicky*NOTE: there are 5' phosphates and 3' hydroxyls on the DNAs you want to ligate - need to match. · Say DNA1 is our vector and we want to ligate your DNA of interest into a vector. o Many different DNAs but only one has sticky ends that can H-bond or anneal to DNA1. o Transient interaction between DNA1 and vector you want to add it to. § DNA ligase seals the phosphodiester linkages between vector and your DNA using ATP § Forms a recombinant plasmid DNA molecule Blunt end needs more DNA ligase to do it. (e.g. Sma I in table 6.1) Why does Eco RI not chop up E. coli's own genomic DNA? · Just as you got restriction enzyme called EcoR1; another enzyme called a EcoR1methylase recognizes the same site. Methylates the 2 end A's so EcoR1 cannot bind So E. coli chromosome is resistant to the RE the cell made. All EcoR1 sites in E. coli are methylated.

Basic genetics definitions given in lecture.

· Genotype - genetic constitution of an individual · Phenotype - function and physical appearance due to genotype · Haploid - a single set of chromosomes (i.e. maternal) · Diploid - two sets of chromosomes (maternal and paternal) · Alleles - different forms of a given gene (WT vs. Mutant) o Can have multiple alleles of that gene in a population. · Homozygous - a diploid organism has two identical alleles · Heterozygous - a diploid organism has two different alleles of the same gene. · Recessive - both copies of a gene must be mutant to see a phenotype. Must be homozygous for the mutant recessive allele to show a phenotype. · Dominant - mutant phenotype is observed when an individual contains a WT allele and a mutant allele. · Cancer: a multi-hit phenomenon: o Loss-of-function: associated with recessive mutant alleles. § Genes are mutated and cell is losing function from mutation § Most are recessive; WT still there; mutant is there; WT produces enough product to keep cell healthy (lose both WT genes and function if both become mutated). § E.g. tumor suppressor genes (loss of function, recessive alleles.) · Loss of front and back brakes on your car o Gain of function - associated with dominant mutant alleles § 2 alleles and one goes bad; cell acquires a phenotype to bad allele; usually tell cell to speed up cell cycle out of control. § E.g. oncogenes = cancer causing genes · Gas pedal stuck wide open · Haplo-insufficient - loss of one WT gene shows an adverse effect. Remaining WT gene is insufficient for cell. · Dominant-negative: mutant gene product adversely affects remaining WT gene product. One bad allele is producing product which adversely affects the good protein phenotype

Making Monoclonal antibodies (Fig. 4-6). What is a hybridoma clone? Why do we call the antibody, monoclonal?

· Immunize a mouse with any antigen you want to make an antibody for. · Each red spleen cell makes its own unique antibody against one of the many epitopes on the antigen. o Another cell makes a different antibody for a different epitope. o Goal is to get many antibodies for one antigen. o Not all spleen cells produce antibodies. o Isolate all spleen cells. · All antibodies coming from one B cell and its mitotic descendants are identical (monoclonal). · Cannot culture B cells long because they senescence. · So, fuse all the spleen cells to immortal mouse myeloma (cancer) cells that cannot grow themselves in a selection medium. · Only the fused cells (hybridomas) grow in the selection medium. o They are immortal § Spleen cell still produces antibodies. o An individual hybridoma clone makes and secretes a unique monoclonal antibody § The unfused cells will die away o Collect the culture medium which contains the monoclonal antibody. § Isolate colonies into 96 well plates - separate colonies in different wells. Screen each colony since its unknown which are producing antibodies. - screen with western blocks. · Monoclonal - clone of cells from original spleen cells, single antibody against one epitope of the antigen.

The wobble base position (Fig. 5-21): how does this relate to the genetic code being redundant (degenerate)? What's inosine doing in a tRNA? Why is inosine "promiscuous"? A codon bound to an anticodon: is it parallel or anti-parallel?

· In these organisms, a single tRNA may recognize more than one codon in the mRNA. · How? o By the Wobble base position: (mostly bacteria) § The first base in the tRNAs anti-codon, 5' end § But the third base in the corresponding mRNA codon. 3' end · Recall: codon and anti-codon are antiparallel. o *Non-standard base-pairing between G and U can occur, but in the wobble position only. o So, this one in particular tRNA can bind two codons, UUC or UUU - both encode Phe. Base pairing rules for the wobble position: · Inosine in the wobble position can pair with C, A, or U. The point is: one tRNA can base pair with 2 or 3 codons. · Inosine is the deaminated version of adenine; occurring after tRNA is synthesized. · Bacteria have fewer tRNAs than they do functional codons. Because certain tRNAs can bind to more than one codon, bacteria actually have fewer tRNAs than codons. codon and anti-codon are antiparallel.

When making a genomic library, why do you want a partial digestion (Fig. 6-15) (hint: see Fig. 6-35).

· Includes everything found in chromosomes · Genomic DNA is cut using Sau3A at small concentrations · Enzyme cuts genome but not at all Sau3A sites. · You want to generate genomic fragments that overlap · Sau3A leaves sticky ends like BAMH1 · Ligate the fragments into the shuttle vector = genomic library o Do not need to use an E. coli plasmid o Can use lambda phage Why a partial digestion with very low concentrations of Sau3A? Sau3A restriction site: ...GATC... A "four" -cutter ...CTAG... · four cutter sites are far more prevalent in the genome than are six-cutter sites (EcoRI, BamHI, etc.) · why partial? o Important to have the genomic library contain overlapping fragments. § For sequencing or linkage studies § Called Chromosome walking Sau3A and BamHI leave the same compatible sticky ends.

RNA synthesis (aka transcription: Fig. 5-10, 5-11, and 5-12).

· Incoming rNTP base-pairs with the base in the DNA template strand · the DNA 3' -OH attacks the alpha phosphate of rNTP · synthesis is 5' to 3' (thick tan arrow) · RNA polymerase reads the DNA template strand 3' to 5' · Alpha- or gamma- [32P] labeled rNTPs? - used for labeling and tracking I guess. · AUG is the start codon also codes for methionine. - pol binds to promoter sequence in duplex DNA "closed complex" - pol melts DNA near transcription start site, forming a transcription bubble. "open complex" - pol catalyzes phosphodiester linkage of 2 initial rNTPs - pol advances 3' to 5' down template strand, melting DNA and adding rNTPs to growing RNA the RNA/DNA helix is more A form than B form. - at transcription stop site, pol releases completed RNA and dissociates from DNA

In situ hybridization; old school (Joe Gall's original method) vs. modern techniques (Fig. 6-25).

· Invented by Jo Gall and grad student Mary Pardue o Single stranded RNA nucleic acid probe labeled (3H), a radioactive isotope of hydrogen. § Tritium labeled RNA probe specifically hybridizes (anneals) with target mRNAs inside the cell (in situ) that have complementary sequences. § Radioactivity localized by a photographic emulsion (at that time). · Appears as silver grains in the light microscope (that was "in the day") RNA still in cell but where? · Use photographic emulsions to coat microscope slide cells are fixed to and radioactivity coming off probe reduce the silver bromide particles. o Silver grains exposed indicates location of RNA in cell Today's uses: (Modern) o Probes tagged with specialized nucleotides and there are antibodies against the modified nucleotides § Actually detecting antibodies o Locating a particular mRNA in both space and time (development) o Single-stranded DNA or RNA probes containing modified nucleotides anneal to the target mRNA. o Antibodies directed against the modified nucleotides are tagged with a reporter enzyme like peroxidase: yields a purple product. antibodies are what your e actually detecting

Ribosomes!! (Similar to Table 5-3). In humans, what constitutes the LSU, and what constitutes the SSU? Can you crystallize ribosomes?

· LSU assembly in the nucleolus (eukaryotes): o LSU rRNAs: 28S, 5.8S, and 5S o LSU proteins: 47 large subunit proteins, called L1, L2, L3... · SSU assembly also in the nucleolus (eukaryotes) o SSU rRNA: 18S o SSU proteins: 33 small subunit proteins, called S1, S2, S3.... § 80S ribosome = LSU:SSU ribosomes can be crystallized and seen under Cryo-TEM microscopy

What's the difference between a nucleotide versus a nucleoside (Fig. 2-16a)? What's the N-glycosidic bond? Memorize your purines and pyrimidines!!

· Nucleotide consists of o 1. Nitrogenous base o 2. Ribose (or deoxyribose) 2' o 3. 1-3 phosphates linked to 5' C · what's a nucleoside? No phosphate group attached N-glycosidic bond connects the ribose or deoxyribose to a nitrogenous base.

Cognate tRNAs in eukaryotes - is that a problem?

· Opposite problem than in prokaryotes · 50 -100 different tRNAs in eukaryotes (more than they need) · remember: only 20 AA's, but 61 "functional" codons · so, in eukaryotes, there are more tRNAs than codons o problem? · Ans: more than one tRNA can attach a certain AA to one codon. o E.g. there are multiple tRNAs for a single AA (codon) · One specific AA is said to have a few "cognate" tRNAs. - (multiple versions of it). o One amino acid can have multiple tRNAs carry it. · Is the wobble position used in eukaryotes? NO, however yeast have some use for it.

What properties of a shuttle vector are needed for propagation in a bacterial cell, and what properties of a shuttle vector are common to a eukaryotic chromosome? (Fig. 6-15a). How does the URA3+ gene on the plasmid act as a selectable marker gene for the plasmid?

· Origins of replication: o ARS for eukaryotes o ORI for prokaryotes · Take recombinant plasmid and introduce it into yeast a eukaryotic cell o We need a region that will initiate DNA replication § ARS for eukaryotes o Centromere separates daughter cells · URA3+ is a selectable marker gene ("analogous" to ampr) o Wild type gene · Take plasmid with URA3+ gene and transform it into yeast cells that are defective for the URA3+ gene · URA3- yeast cells require uracil (auxotrophic for uracil) · the cells that pick up the plasmid they can manufacture uracil · if they do not pick up the plasmid they cannot produce uracil. You need to add uracil to the medium.

Suppression and Synthetic Lethality (Fig. 6-9)

· Suppression o 2 proteins A and B make a protein complex, AB. Has a Kd of e-9 o in Mutation aB or Ab § say Kd goes up to e-6 (wimpy Kd) o say 2 mutant alleles in complex, ab. § Kd goes back to e-9 § Both mutations suppress each other; one affects (suppresses). the other and functionality is restored. Synthetic lethality: Two definitions (examples) SL 1: · A and B form a complex. o Example aB (a mutant) or Ab (b mutant) (complex formed - doesn't work right) § Maybe due to slow growth; opposite to suppressor mutations § Ex. Dominant-negative o Both mutations in cell (ab) - yeast cell dies; phenotype is worse than just one mutation SL2: · AB genotype (a, b - mutant alleles) - production of product for cell to live? · aB - B pathway produces enough product = cell viable · Ab - A pathway produces enough product = cell viable ab - phenotype is worse - cell dies, non-viable

Next Generation DNA Sequencing (Fig. 6-21 and 6-22).

· Take entire genome and chop into tiny tiny fragments. Billions of fragments about 100bp in length. · Billions of sequencing reactions all at the same time on a solid support Looking at just one fragment: · Ligate linkers to ends of genomic DNA fragments (very short about 100 bps) - don't know sequence of genomic fragment o Primers allow to anneal the fragment to a matrix or substratum o Many primers positioned on the spot and DNA anneals to its complementary primer and DNA synthesis gives you the other strand back. o Denature to make single-stranded again. This end anneals to the other primer o PCR amplify this 100 bp fragment of genomic DNA on this one spot of the substratum. § You have a billion fragments mounted to substratum. Each fragment is PCR amplified using same linkers (oligo's) o By PCR amplification you're cloning this one fragment of DNA on this one spot. But there's billions of spots on this substratum. · How do you sequence 1 billion fragments each 100bp o Cut DNA make single-stranded and put on another primer o Extend primer using fluorescently tagged nucleotides. o 1 nucleotide at a time = 100 cycles. o First nucleotide to be incorporated is a C - its fluorescently read o The entire substratum with fragments are monitored by a microscope. § Pop off fluorescent on C § Next spot is another C § Repeat and next nucleotide is a T = green · Sequencing the fragment of DNA. o Computer takes sequence from all fragments and overlaps them to give back entire genome sequence of the organism. NOT used on cDNA ONLY GENOMIC DNA

Aminoacyl tRNA synthetases must work flawlessly. Why? (Figs. 5-19).

· This enzyme links a specific AA to its cognate tRNAs o Ex. Phe to tRNAphe by a high energy ester bond · Recall ... CCA-3' end of the tRNA: it has a hydroxyl group · 20 different amino-acyl tRNA synthetases. · Each enzyme is specific for only one of the 20 AA's. · Once coupled to the appropriate AA, the tRNA is said to be "activated" or "charge". · Synthetases have a "proof-reading" function/ - knows the right cognate tRNA it needs to bind.1 o Meaning... enzyme picks up the wrong amino acid. The enzyme knows it made a mistake and ejects the wrong AA and waits for the correct one. · One synthetase in eukaryotes can recognize the various cognate tRNAs for a given AA. o Means that an enzyme recognizes some "identity site" on the cognate tRNAs o Identity site = anti-codon (in part) o Other parts of the tRNA also contribute to recognition. separate regions of the particular tRNA are recognized by the appropriate Aminoacyl-tRNA synthetase.

Genetic Complementation analysis (Fig. 6-7). This is absolutely critical. What would happen in the complementation assay if instead of being a recessive conditional mutation, one of the cdc mutations actually turned out to be a dominant negative? Think beyond what we covered!!!

· Used by Leland Hartwell - a yeast geneticist o Making a bunch of temperature sensitive conditional mutations that block cell cycle at different points o Called cdc = cell division cycle mutants · Lots of cdc mutants and wants to categorize them. · Taking X mutation and Y mutation - temperature dependent cells = can grow at 23oC not at 36oC o 2 haploid cells are mated § Mate a and α o Product is the diploid cell which grows at 23oC o Take the master plate and grow replica plates at 36oC § Diploid cells are growing at this temperature. Haploid would not grow. o X and Y are recessive mutations o Wild type is complementing(rescuing) the mutant X and Y in the diploid cell to grow at non-permissive temperature. · Far Right side o X mutation and Z mutation mated making diploid cell o Grow at 23oC and will not grow at 36oC o X and Z gene are exactly the same they are alleles of one another. NO complementation - so no growth at 36oC. · Genetic complementation analysis: Determines allelism: mutations either complement (not allelic), or they fail to compliment (are allelic).

Classes of RNA in the cell and their relative abundances.

· mRNA carries genetic code from gene in nucleus to cytoplasm for translation · tRNAs recognize codons within mRNA and deliver a particular amino acid to match the codon · rRNA - goes to build ribosome itself. o LSU and SSU o mRNA = ~ 2% o tRNA = ~ 3% o rRNA = ~ 95%

Poly-ribosomes, PABPI, recycling of ribosomes (Fig. 5-27), and polysome profiles using sucrose gradients. My grad student knocked out a nucleolar protein involved with ribosome subunit assembly. What do you predict the polysome profile would look like for the 40S SSU, the 60S LSU, and the 80S intact ribosome? What about the polysomes?

· mRNA forms a circle ● PABP1 interacts with eIF4G (bound together in circle): for efficient re-use of ribosomes. ● But the mRNA was linear during elongation in the previous model. - still going back and forth between circular and linear model of RNA ● Atomic force microscopy: can see circular mRNAs better Rate zonal centrifugation using sucrose gradients: ● To analyze "polysome" profiles (polysome - several ribosomes on mRNA is polyribosomal) o All translating the same protein. ● Separates the polysomes by rate zonal centrifugation - using sucrose gradients - thin on top and heavy on the bottom o Polysomes can be separated on these gradients. o Can isolate the SSU - high up in gradient o LSU is heavier o Intact ribosome is even more dense. o Then each peak after the intact ribosome is 1 additional ribosome on mRNA. ● Thick black line: Normal protein synthesis ● Thin black line: protein synthesis inhibited ● Poison a cell using drugs that block ribosome function - The polysome profile just drops off the table. Very low frequency of ribosomes. Go from a nice profile of ribosomes to nothing. ● What would the profile look like if we knocked out a nuclear protein required for normal ribosome assembly? Series of peaks diminish, not as many ribosomes on mRNA for translation.

What are conditional mutations (Fig. 6-6)?

· starting with haploid yeast strain grown in large flask · add a mutagen, to mutate genes in haploid strain · subdivide the large culture into smaller aliquots and plate each aliquot on the agar plate. · Grow first plate at 23 degrees - called permissive temperature. o Lots of colonies grown at 23oC o 2 Replica plates from master plate o 1 at 23oC - colonies grow o 1 at 36oC - colonies do not grow - has a mutation in it not allowing cell to grow at that temperature. But grows fine at 23oC - called non-permissive temperature. o Mutation is temperature sensitive which is a conditional mutation. o Condition is temperature!!! · Conditional mutations does not have to be temperature sensitive, can be other things · Auxotrophy - a conditional mutation where something needs to be added to get the cells to grow Uracil, leucine

Fig. 5-17 is a good review for protein translation.

· three RNAs in protein synthesis · mRNA carries genetic code from gene in nucleus to cytoplasm for translation · tRNAs recognize codons within mRNA and deliver a particular amino acid to match the codon · rRNA - goes to build ribosome itself. LSU and SSU

Semi-conservative DNA replication (Fig. 5-28, the little figure on page 198, Figs. 5-29, 5-30, and 5-32). Why is the Meselson and Stahl experiment a Pulse-Chase experiment?

● Meselson and Stahl ○ a classic experiment (a pulse chase experiment) ○ pulse: using 15N (nonradioactive). 1 extra neutron makes it heavier. All the DNA will start heavy. Grow bacteria for many generations. ○ Chase: 15N chased with 14N in first generation. 14N if conservative was correct then all new DNA should be light and old DNA should remain heavy and no intermediate of mixing. Semiconservative model - the original 15N strands come apart and are templates for new DNA which has 14N in it. ○ Chase went for a second round of division. ● Meselson and Stahl's classic 1958 experiment actual results: ○ determined the true model for DNA replication - semiconservative ○ start with 100% heavy DNA, then centrifuge DNA in cesium chloride gradients. Its equilibrium or zonal centrifugation. Now chase with 14N then look at new DNA made. Its mixed with light and dark DNA. No heavy DNA left. Now another round and then completely light DNA is made. Mechanism of DNA synthesis starts with an RNA primer ● The primer started with is RNA, laid down by Primase! ○ 3' OH on the last nucleotide is needed for condensation synthesis (nucleophilic attack on alpha phosphate of the in-coming dNTP) ● DNA polymerase reads the template strand 3' to 5' to synthesize the daughter strand 5'to 3'. Leading versus lagging strand synthesis: · Leading strand is started with an RNA primer and DNA pol picks up off the primer and lays down a brand-new strand of DNA. Its read 3' to 5' right to left. o The polymerases must obey the rules · Lagging strand (Okazaki fragments) in opposite direction the polymerase wants to go; begins with RNA primers. DNA laid down in short bits and pieces while DNA Pol sits at replication fork. Only has time to lay down a short amount of DNA. · Okazaki fragments are ligated together after RNA primers are digested away called ligation by DNA polymerase. ● topoisomerases relieve torsional stress (excessive supercoils) introduced ahead of the replication fork. ● both strands of the Parental Helix must serve as templates. ● RNA primers removed by RNAase H and FEN I; and ligated together.

Translation initiation in prokaryotes, fMet, Shine-Dalgarno sequence, IFs.

● usually, the first AUG codon is the start codon. It sets the reading frame. (Met) o Doesn't have to be must AUG to start translation. ● Two different tRNAs for Met*: ● Cognate tRNAs: o 1. tRNAiMet for initiation - binds to start codon AUG ▪ in eukaryotes and archaea - binds to start codon ▪ bacteria - add a formyl group (CHO) to bind to start codon o 2. tRNAMet for elongation - AUG codon further down the strand - (for all cells) o *only one amino-acyl tRNA synthetase for Met 1. 30S SSU plus initiation factors (IFs) 1,2-GTP, and 3 form a pre-initiation complex. 2. Complex binds fMet-tRNAiMet and the mRNA - scans for AUG 3. Shine-delgarno sequence helps position 30S complex. a. Unique to bacteria 4. 50S LSU joins. IF2-GDP pops off 5. fMet-tRNAiMet in the P site. Ready for elongation ● only (f)Met-tRNAiMet can bind the P site at initiation; ● Met-tRNAMet cannot bind P site at initiation, only during elongation ● Note -CHO = formyl group (f)