Molecular Genetics Exam 2

Centromeres

Segration of condensed chromosomes depends on centromeres -centromeres appear as constrictions on chromosomes- contained within blocks of repetitive noncoding sequences called satellite DNA, satellite DNA consists of short sequences 5-300 bases in length -centromeres have two functions- hold siter chromatids together, kinetochore- (protein complex) structure composed of DNA and protein that help power chromosome movement -physically visible part of chromosome -role is to attach to microtubles so during mitosis pulled apart forming 2 sister chromatids

How to assay apoptosis?

Tunel assay: TdT-mediated BrdUTP nick end labeling, terminal deoxynucleotidyl transferase is a DNA polymerase distinct from other DNA polymerases in that it can add nucleotides to the 3' end of gene segments without the use of a template strand -when cell is dying it starts to fragment its DNA, exposes 3' OH group. in tunel assay can add labeled DUTP in presence of enzyme TdT which doesnt need template to work can add to 3'OH creates chain. labeled bromide to dUTP so can be recognized by antibody and bind to tail and linked to enzyme to add color change, can see when antibody binds knowing apoptosis is happening in that cell

Mouse and Homology-based gene knockout

Can use mouse as an example to illustrate homology-based gene knockout 1. Using recombinant DNA techniques, a gene specifying resistance to the drug neomycin (neo') is inserted into an exon of the gene of interest (done in test tube) 2. purebred agouti mice are mated to produce an early ambryo (a blastocyst). embryonic stem (ES) cells from the inner cell mass of the blastocyst are cultured to increase their number 3. Cultured ES cells are treated with the cloned exon disrupted by neo'. In a small number of these cells, homologous recombination swaps a WT exon on a mouse chromosome in the ES cells with the disrupted version of the exon (a knockout allele). the cells with knockouts are selected by exposing the ES culture to neomycin, and they are grown into colonies

Heteochromatin vs euchromatin

Heterochromatin-highly condensed DNA unavailable for transcription -darkly stained region of chromosome -highly compacted even during interphase- compacted becaused of repeated sequences -using found in regions near centromere -constitutive heterochromatin remains condensed most of time in all cells (eg Y chromosomes in flied and humans) -genes in the region are silenced, contains lots of transposons- dont want them jumping around interfering Euchromatin -lightly stained regions of chromosomes -contains msot genes -site of active transcription

what type of instruments are used with real-time PCR?

real time PCR instruments consist of 3 main components: 1. thermal cycler (PCR machine) 2. optical module (to detect fluorescence in the tubes during the run) 3. computer (to translate the fluorescene data into meaningful results) -detects fluorescence at each cycle -the fluorescence data collected during PCR tells us "how much" but there is another type of analysis we can do that tells us "what"- melt curves- tells what products are in a reaction, based on the principle that as DNA melts (becomes single stranded) intercalating dyes will no longer bind and fluoresce, can het up sample, SYBR green comes off and denatures, diff amplicons will have diff melt peaks, primer-dimers will have a very diff melt peak

Introducing subtle mutations with minimal footprints

two steps: 1. target gene by homologous recombination 2. remove or replace selection marker gene by counter selection (eg TK gene is lethal in the presence of toxic thymidine analogs like ganciclovir) -select for TK in first step, select against it in second step -tag exchange strategy- first transformation, select for neo, counter selection:select against TK gene by adding ganciclovir (lethal nuleotide only incorporated into the cell in the presence of Tk)- very clean strategy no markers are introduced but still made mutation

General features of enhancers (summary)

-major determinant of differential transcription in space (cell type) and time, critical for development -able to function far from the promoter so each gene can have multiple enahncer elements (that respond to diff signals) to determine whether a given gene is transcribed, adds complexity -modular: various DNA elements that regulate temporal and spatial gene expression and these can be mixed and matched -the modularity of enhancers makes them good targets for evolution as individual functions of a gene can be gained or lost without affecting other functions -some enhancers inhinbit transcription, negative enhancers are also called silencers

Transfection by DNA/calcium phosphate co-precipitate

-mammalian cells will take up DNA with this method-endocytosis of the precipitate? -only suitable for cell monolayers not cell suspensions -up to 20% of cells take up DNA

Regulators and effectors of apoptosis

-many cell death signals induce apoptosis via a conserved pathway of regulators, adaptors and caspases -in c. elegans, the negative regulator Ced9 inhibits apoptosis by binding to the adaptor ced4. in the absence of inhibition by ced9, ced4 binds two molecules of the caspase ced3, resulting in autocleavage and caspase activation. -in mammals regulators of the BCi-2 family (ced9 homologs) act at the mitochondria to control release of cytochrome c, which is required for the binding of caspase-9 to the adaptor Apaf-1 (the ced4 homolog) -release od cytochrome c from mitochondria thurs signals the activation of caspase-9 which then activates downstream caspases to induce apoptosis

Developmental genes regulated

-many developmental genes and switches are epigenetically regulated -Toadflax flowers- the peloric phenotype is due to gene silencing via DNA methylation of CYCLOIDEA gene, leading to radial flower symmetry. the peloric phenotype is heritable but occasionally reverts to WT due to DNA demthylation of CYC gene -nonsymmetrical flowers found normally in nature but also find symmetrical. cyc gene is responsible- when methylated creates symmetrical flower, can disrupt this by disrupting MET1 by RNAi- chemicals can also inhibit methylation

Basal/general transcription facotrs position RNA pol II

-many eukaryotic genes have TATA box- 30bp upstream -TBP binds TATA -TBP binds additional proteins TBP-associated factors (TAFs) -complex of basal factors recruit RNA pol II to promoter -directs RNA pol 2 to correct place to make mRNA -huge complex required for correct initiation- entire initiation complex contains 60-70 polypeptides with mass similar to ribosome -basal factors + RNA pol II, basal levels of transcription- if just have basal factors- low levels of transcription

Map-based cloning (poitional cloning) of ced 7 gene

-map to chromosome, marrkers flanking gene to code out contigs -can use ends to walk to build up contig. inject back into which can rescue worm -once find gene can seq it found ced3 is a protease- can chop up proteins -ced4 cofactor of protease -human homologs exist same genes program cell death in humans

H3k27me3 methylation is conferred by polycomb repressive complex 2 (PRC2)

-methylation at lysine at positin 27 at histone 3 protein -methylation at this lysine signals silencing of this region -catalyzed by polycomb repressive complex 2 adds this methylation -once methylated, condenses -active genes --> silenced genes -H3K27me3 is a repressive mark

Direct DNA transfer

-microinjection- for large cells like eggs, can only transform a few cells at a time, can inject right into male pro nucleus -transformation with microprojectiles- works well on plant cells, worms, tissues, organelles- the direct DNA transfer methods are used when other easier methods fail, can put dye into cells using same method as well, helium pusles particles coated with dna through

Epigenetic marks: DNA and histone methylation

-modification of primary sequence: CG, CHG, CHH methylations (H: not G)- DNA methylation occurs at 5MC within CpG dinucleotides. 5MC constitutes <1% of nucleotides -modification of amino terminal of histones: methylation of lysines/arginines, acetylation/ubiquitation of lysines, serine phosphorolation

Histone code hypothesis

-modifications of the hsitone tails act as marks that can be read by other proteins to control the expression or replication of chromosomal regions. the coding in the histones may be heritable -not limited to just methylation -serves as a code whether this region of DNA should be silenced or not

What controls gene expression?

-much has been learned from prokaryotic systems -some differences in eukaryotess but basic principles are very similar -a lot learned from bacteria

Chromatin organization

-multiple levels of packaging are required to fit the DNA into the cell nucleus -short region of DNA double helix -"beads on a string" form of chromatin -30nm chromatin fiber of packed nucleosomes -section of chromosome in extended form -condensed section of chromosome -entire mitotic chromosome -net result:each DNA molecule has been packaged into a mitotic chromsome that is 10,000 fold shorter than its extended length

Genetic approaches to identify trans-acting factors

-mutations in a gene encoding an activator reduce expression of the reporter -mutations in a gene encoding a repressor inc expression of the reporter

table of different enzymes used in recimbinant DNA technology

-nucleases- restriction endonucleases, restriction exonucleoases, ribonuclease H- useful when want to work with RNA turns it to DNA so can manipulate -DNA modifiers- DNA polymerase, reverse transcriptase, alkaline phosphatase, polynucleotide kinase, terminal nucleotidyl transferase, methyl transferase -DNA ligases- DNA ligase wont ask you to come up with name but may be in multiple choice for exam so recognize

Michael smiths experiment

-oligonucleotide based site directed mutagenesis -study protein wanted to make mutations to see how its important -isolated DNA molecule with mutation -site detected mutagenesis

enhancer-promoter interactions

-once have enhancer- bound by activator protein to DNA arrives expression higher levels

Discovering RNAi

-originally called co-supression -phenomenon in plants in 1990 by Richard Jorgensen -over-expressing purple gene resulted in white flowers

Repressor proteins can act through competition with an activator protein

-other enhancer binding proteins act as repressors- recognizes seq and repress expression of gene- ex. competition for binding between repressor and activator proteins- binding of repressor to enhancer blocks binding of activator -an indirect repressor interferes with the function of an activator- competition due to overlapping binding sites, repressor binds to activation domain (quenching), binding to activator and keeping it in cytoplasm, binding to activator and preventing homodimerization

modular enhancers provide explanation of pleiotropy for some genes

-pax6 expressed in embryonic neural tube -pax6 expressed in eye -independent enhancers direct expression in these diff tissues -changes in one enhancer will not afect function of the other -thus cis-regulatory chnages can impact expression in one tissue but not another

Example of plasmid

-polylinkers cloning site interupts lacZ, if cloned gene-disrupts lacZ wont get blue bacteria know gene is there -is lacZ is present- plasmid confers resistance to ampicillin and can make functional beta-galactosidase -cut with same restriction enzyme- restriction cut in polylinker -DNA ligase- DNA insertion in disrupted lacZ gene- plasmid confers ampicillin resistance but cant make functional beta-galactosidase

Position of genes and heterochromatin example

-position effects variegation in drosophila: moving a gene near heterochromatin prvents its expression -facultative heterochromatin- moving a gene near heterochromatin silences its activity in some cells and not others -can visualize this in eye pigment- gene activity -inversion (x-rau induced maybe) breaks and flips, fuses -genes for eye color and texture close to heterochromatin region so becomes silenced- results in variegated eye vs WT red eye -a model for position-effect varigation- heterochromatin can spread different distnces in diff cells -Red smooth sectors- rearrangement brings w+ and rst+ close to heterochromatin near centromere. heterochromatin does not invade gene- both genes active -white smoth sectors- w+ gene inactivated by spread of heterochomatin. rst+ gene is active- white gene silenced -white rough sectors- both w+ and rst+ genes inactivated by spread- both silenced -red rough sectors- never observed because can skip genes, heterochromatin spreads linearly without skipping genes , cant jump over w+

Endogenous control gene

-present in all experimental samples -expression does not vary between treatments, tissues, age etc ie constant expression levels -by using an andogenous control as an active reference you can normalize quantification of mRNA target for differences in the amount of total RNA added to each sample ie loading control

Telomeres

-preserve the integrity of linear chromosomes -telomeres are protective caps on eukaryotic chromosomes- rpevent fusion with other chromosomes -DNA polymerase cant reconstruct 5' end of a DNA strand- ends get shorter as you age, exposing ends can cause issues -DNA replication requires RNA primer that needs to be cleaved and leaves gap- removes RNA primers by ribonucleases- newly synthesized strands are shorter by the length of the RNA primer -telomerase is a protein-RNA complex the RNA serves as template for telomere elongation- binding of telomerase to region and addition of RNA extends the ends- generates many repeats of specific seq

Gene expression is controlled by cis-regulatory elements

-promoter necessary for gene expression in both prokaryotes and eukaryotes -promoters bind/position RNA polymerase, allowing basal levels of transcription -enhancers modulate expression levels, usually in cell type-specific fashion

considerations in homologous recombination strategies

-random insertion of DNA often occurs- how to get around this problem? 1. add a negative selection gene to the DNA outside of the region of homology (ensure that the cells containing this gene via non-specific integration will die) 2. screen transformants by PCR for correct position of recombinant DNA insertion -targeting specific spot- some will go to that spot but some will go to other spots, if other spots- will die

X chromosome inactivation

-randomly choose to inactivate 1 of the 2 X chromosomes maternal or paternal -all mosaic- some cels only express maternal X chromo genes and some cells only express paternal X chromo genes -this is inherited -diseases- some diseases result from multiple X chromosomes active- can diagnose by looking at how many barr bodies know it has to silence 1

What do you create by cloning?

-recombinant DNA libaries (2 major types): 1. genomic/chromosomal library, collection of cloned restriction enzyme digested DNAs containing at least one copy of every DNA sequence in a genome or chromosome 2. complementary DNA (cDNA) library, collection of clones of DNA copies made from mRNA isolated from cells -reverse transcriptise (RNA dependent DNA polymerase) -synthesizes DNA from an RNA template -cDNA libraries reflect what is being expressed in cells -number of clones required for a complete library can be calculated from the size of the genome and average size of overlapping fragments cut by restriction enzymes -library should contain many times more clones than the calculated minimum number of clones -know what library is! collection of diff clones so can used to find specific gene your interested in

Knocking

-recombination where host starts out normal then gets knocked in from recombination

Epigenetics

-refers to heritable changes in gene expression (active vs inactive genes) that does not involve changes to the underlying DNA seq, a change in phenotype without a change in genotype -not permenant -how to modifiy expression that is inherited but not permanent, regulating gene expression

Reporter gene constructs

-replace coding region with innocuous gene encoding easily dtectable product -express in cells or organisms -assay expression- easy to assay where expressed -reporter gene contrusts test for enhancer function-add DNA encoding putative enhancer -ask if gene expression levels are changed -replace coding with lacZ gene (for ex) see where expressed -add enhancer infront and see where expressed

Gene therapy with viral vectors

-retroviruses- integrate into genome, can cause mutations- if insert into wrong place can cause mutations, can make WT and rescue defect of host -AAV vector- doesnt integrate into genome, therapeutic vector eventully degrades, doesnt insert itself but cna be transcribed or translated but need to add again later

TFs corrdinately regulate SETS of target genes

-same TFs can bind similar enhancers regulating different genes in same pattern -diff combos of TFs can bind to diff sets of DNA seq, activating the target genes in slightly diff patterns -together, TFs can regulate SETS of genes in a coordinated fashion. master regulators -similar to coordinate regulation in prokaryotes but diff mechanism -might bind enhancers in diff combos- diff expression patterns

functions of apoptosis

-sculpt body structure eg hand digi- between digit cells habe to die to make separate digit -serve some function but no longer needed eg tadpole tail of frog -prevalent during development

Enhancer binding proteins

-sequence specific DNA binding proteins -transcription activation domains activate transcription -modular structures similar to lac repressor- independent domains within the TFs have independent function -some TFs have an additional domain that responds to signals- once binds to receptor changes conformation so can activate gene expression ex steroid hormone receptors- without steroid hormone the receptor cant bind to enhancer, binding of SH induces allosteric change in receptor, receptor can now bind to enhancer

Site specific recombination

-specialized machinery governs process -recombination occurs at short specific recognition sites

CRISPs: Clusters of regularly interspersed short palindromic repeats

-TracrRNA: trans-activating CRISPR RNA -crRNA: CRISPR RNA -sgRNA: single-guide RNA -PAM: protospacer-adjacent motif -first dicovered in bacteria -can apply to engineering genome -repeats are all same -genes encode nucleases (Cas9) transacting CRISPR RNA, another gene made -RNase III will cleave and pair -Cas9 can grab hairpin and double strand area and guided by red peice to find target- new phages DNA

eukaryotic TF function (summary)

-WT TF function ensures proper development formation of distinct cell types and expression of diff genes, maintains homeostasis -TFs regulate batteries of genes -mutations in one of these downstream genes may have minor impact -when TF function is altered normal development or homeostasis can be perturbed -muts in many TFs will cause early embryonic lethality- kills expression of a lot of genes

In situ hybridization

-a method of detecting RNA in intact cells/tissues -uses probe -use whole organism, probe labeled with dye goes in and finds pairing with corresponding RNA

Stable transformation

-a small fraction of the DNA may be integrated into the genome-these events lead to stable transformation -homologous recombination can be exploited for genome engineering -results in formation of a cell line that carries and expresses the transgene indefinitely -selectable markers greatly assist in isolating these rare events -takes time to get cell line with dna stabling transformed in it, similar to plasmids in bacteria same idea selectable markers that help cell line contain stable DNA -mysteries of stable transfection/transformation- mechanism of transport of DNA is not known: some DNA is transported to the nucleus, non-homologous intermolecular ligation events may occur, large concatameric rDNA structure may eventually integrate usually by non-homologous recombination, best case scenario- 1 in 1000 transfected cells will carry the transfected gene in a stable fashion

why study programmed cell death?

-abnormalities in programmed cell death are associated with many human diseases

CHIP (chromatin immunoprecipitation)

-an assay that detects histone modification at specific genomic regions -antibody can specifically recognize position and modification -cell -> crosslink protein to DNA extract DNA out of cell, break up into smll pieces, use specific antibody, add to mix only pull down what its looking for, seq DNA, map to gemone, see peaks- thats DNA seq correlated to those genes -can look at all genes in genome which ones silenced which active

DNase I foootprint

-an assay, a way to look on DNA where nucleosome is located -nucleosome protect nucleoase so cant access that region so no band in area 1. genomic DNA PCR amplify creates labeled template DNA containing protein binding site 2. add protein of interest 3. cleave DNA 4. run on denaturing polyacrylamide gel -protected footprint- region where see no bands meaning unable to cut there

gene transfer to animal cells- why?

-animal cell culture useful for production of recombinant animal proteins: accurate post-translational modifications -excellent tool for studying the cell biology of complex eukaryotes- isolated cells, simplifies analysis, human cell lines: a way of studying human cell biology without ethical problems -establish conditions for gene therapy-treatment of genetic disorders by restoration of gene function

plasmid cloning vectors

-bacterial plasmids naturally occurring small satellite chromosome circular double-stranded extrachromosomal DNA elements capable of replicating autonomously- can grow on own -plasmid vectors engineered from bacterial plasmids for use in cloning -features (eg E.coli plasmid vectors): 1. origin sequence (ori) required for replication- can initiate rep and make more copies of itself 2. selectable trait that enables E.coli that carry the plasmid to be separated from Ecoli that dont (eg. antibiotic resistance grow cells on antibiotic only those cells with the antibiotic resistance gene grow in colony)- can tell whether or not plasmid is there often use resistance 3. unique restriction site such that an enzyme cuts the plasmid DNA in only one palce. a fragment of DNA cut with same enzyme can then be inserted into the plasmid restriction site- engineered restriction sits 4. simple marker that allows you to distinguish plasmids that contain inserts from those that dont (eg lacZ+ gene)- identify which have inserts which has gene want to clone, and which dont

X-inactivation in cats

-barr body= inactive X chromo regulate gene dosage in females during embryonic development -because of this only female cats can be tortoiseshell or calico -calico cat- patches of prange and black -rainbow- clone of mothers nucleases- genome identical but dont look same because copy cat its owen cells have undergone its own random activation

Effect of SWI/SNF

-can be obsered using DNASEI footprint -how nucelosome changes during gene expression -the SWI/SNF chromosome remodeling complex binds to chromatin and using the energy generated by ATP hydrolysis, mobilizes nucleosomes. this chromatin remodeling regulates the accessibility of DNA to the transcription machinery

LacZ reporter constructs to identify enhancers in transgenic mice

-specific enhancers direct expression of lacZ in specific regions of the embryo -incubation with X-gal produces blue color -can look at specific enhancers and will light up diff parts of mouse embryo -reporter constructs used to discover enhancers-want to know where expressed, can look at upstream and close DNA in front of lacZ gene might make deletions of it less and less in front of lacZ can stain it and see which still express gene to know where located -enhancer now subject to further analysis-enhancer must be in this region were X-gail staining is -subdivide region to identify modules that diret expression in diff cell types-also look at central nervous system so know its not it same place for CNS

The same transcription factor can plau different roles in different cells

-drosophila dorsal protein is an activator of gene 1 -the combo of dorsal and another transcription factor causes dorsal to act as a repressor -dorsal binds to enhancer- can either be activator or repressor depends what decides to interact with whats around it

Structure of yeast centromere

-easiest to study -2 repeat sequences- with conserved elements, middle are the A-T rich element

How do cell-type specific enhancers function?

-enhancer DNA sequence present in all cells -active only in specific cells -enhancers are bound by sequence specific DNA binding proteins that are expressed in specific cell types -often it is the specific combo of proteins that generates cell type specificity

Reporter genes identify enhancers

-enhancers can be identified by constructing a recombinant DNA molecule that has a putative enahancer seq fused to a reporter gene such as the green fluorescent protein(GFP) -generating a transgenic organism that has the recombinant DNA in its genome

Mechanisms of activator effects on transcription

-stimulate recruitment of basal factors and RNA pol II to promoters -recruit coactivators to open chromatin structure -coactivator with activator upwraps chromatin to open so can get expressed

Histone octamers assemble from pairs of dimers

-the N-terminal tails of histones are subject to modification -8 histone proteins each appear twice (dimers)

Nucleosome

-the fundamental unit of chomosomal packaging rises from DNAs associated with histones -bead is a nucleosome with about 160bp of DNA wrapped twice around a core of 8 histones -40bp of DNA link together nucleosomes -models of higher level compaction seek to explain extreme compaction of chromosomes at mitosis

Gene therapy

-the use of DNA to cure disease -the idea is simple: introduce a therapeutic gene into the somatic cells of patients -can be used to knockout hyperactive genes like oncogenes -Retinoblastoma and tumor supressor so can restore this protein to stop this cancer Different strategies are required for diff diseases -disease caused by loss of function- add WT copy of gene -disease caused by gain of function- therapeutic gene must inactivate disease gene or protein product -diseases with complex origin- target gene for a genetic pathway involved

Cell death can be studied at a single cell resolution

-there are 2 cells, undergo programmed cell death during embryonic development, dead cell gets engulfed

threshold is important

-threshold deterimes Ct values -Ct values are used to calculate relative expression, presence/absence etc. its all about Ct values -threshold should be set in the linear portion in log view. always check -dont use Ct values of 35 or higher, repeat using more cDNA/DNA

programmed cell death and cancer

-took human bci-2 into ced9 mutant worm, can rescure affect of ced9 in worm- figured out they were homologs -bci-2 proto-oncogene- if DNA gets damaged tries to repair, if beyond repair those mut will persist in cell and become cancer. if cant repair activate pathway through p53 to activate bci-2 to activate promgrammed cell death -bci2 normally acts in mitochondria to release cytochrome (used in cell death). if abnormalities in bci2 (hyperactive for ex) will inhibit release of cytochrome c and inhibit cell death --> cancer

strategies for gene transfer

-transfection-cells take up DNA from medium -direct transfer- microinjection into nucleus(injecting DNA into cell), gene gun: particles coated with DNA bombarding cells (shooting DNA into cell) -transduction- viral mechanism for transfer of DNA to cells, uses a virus

different eukaryotic TFs interact to regulate transcription

-turn gene on at right time, off at right time, on in right place and off in right place -turn gene on or off in response to signal -basal transcription factors position RNA pol II at TSS -enhancer binding proteins activate or repress transcritpion often vis co-regulators -these TFs are modular: DNA binding domains and activation domains -interactions between TFs can add to types of responses (homodimers, heterodimers, corepressors) -all this function in the context of repressive chromatin -interact with basal to actually drive expression

reference sample

-used in comparative CT and relative standard curve experiments -sample used as the basics for relative quantitation results ie everything gets expressed and compared relative to this sample- also called calibrator -it doesmt matter which sample is used however normally the negative control is used -like a baseline

How do we measure DNA in a PCR reaction?

-we use reagents that fluorece in the presence of amplified DNA (double stranded DNA) -ethidium bromide and SYBR green I dye (less toxic than other) are two such reagents -they bind to double stranded DNA and emit light when illuminated with specific wavelength -SYBR green I dye fluoresces much more brightly than ethidium

Screening a genomic library

-why screen a genomic library?***** 1. transform Ecoli with a genomic library 2. velveteen surface (sterilized) prssed on plate, plate on selective growth medium, colonies grow 3. velveteen with cells from original colonies is pressed to selective medium plate with filter on its surface, replica plate colonies onto new selective medium plate with membrane fiter on surface colonies grow on filter 4. filter removed from culture dish bacteria lysed DNA denatured and bound to filter 5. probe DNA hybridized to DNA on filter, labeled probe solution added to filter in host-seelable bag 6. wash filter free of unbound probe, detect hybridization by autoradiography for radioactively labled probes or by chemiluminescent detection for nonradioactively labeled probe. dark spots indicate clones detected by probe

Counter-selectable markers

-you can select against thymidine kinase, by treating Tk+ cells with toxic nucleotide analogues that are only incorporated into DNA by thymidine kinase -ex: 5 bromo-deoxyuridine, ganciclovir -cells with TK die in the presence of these compounds, cells that lose the Tk gene survive

How environment can affect your epigenome and health?

-epigenetics can be reversed so if health problem due to epigenetics can cure more easily -tale of 2 mice- mother eats normal diet produced mostly yellow fat offspring, few brown skinny, genome of fat yellow offspring has transposon and agouti gene -mother eats modified diet- adding vitamin B12, folic acid choline and betaine- produces mostly brown skinny offspring few fat yellow, genome of brown skinny offspring contains methyl group, transposon and agouti gene switched off

Bar bodies

-example of heterochromatin decreasing gene activity -barr bodies- inactivation of one X chromosome to control for dosage compensation in female mammals. one X chromosome appears in interphase cells as darkly stained heterochromatin mass

Viral transduction

-exploiting viral lifestyle (attachment to cells and intro of genomic DNA) to introduce recombinant DNA -transfer genes to cultured cells or to living animals -potentially useful in gene therapy- retrovirus, adenovirus, herpes virus have all been approved for clinical trials -can get transient or stable

Polymerase chain reaction

-exponential amplification of DNA- can amplify DNA from trace samples, be done in absence of bacteria -uses primers, heat stable DNA pol, dNTPs -variations include: reverse transcriptase firdst to convert RNA to cDNA before PCR, introducing mutations or linkers with primers, quantitative real time PCR -denaturem add primers (need to know some of the seq), polymerase that will make copies -exponential growth -can induce mutations

Paul Berg's experiment

-first to generate recombinant DNA in vitro -enzymes discovered from bacteria were used -used for replication or transcription -allows to manipulate DNA in test tube made first recombinant DNA

Epistatic analysis of apoptotic genes

-form pathway- ced1 and ced2 involved in engulfing dead cell -ced3 and ced4 involved in execution of cell --> act upstream of other 2 -ced9- loss of function mut of ced9 lethal-all cells die -ced4 and ced3 espistatic to ced9. ced9 makes decision. if turned on cell survives, turn off cell death starts -next step-find gene map

How do we identify genes that regulate programmed cell death?

-forward genetic approach -some genes need to be turned on to program cell death uses forward genetics -in 1980, hedgecock isolated 2 cell death mutants (ced-1 and ced-2) which are pivotal for identification of the other cell death genes -looked for mutants, looking for 2 opposite phenotypes- extra cells surviving that cant undergo cell death or massive cell death-cells that should die, die, fewer cells -ced-1 and ced-2 genes are easy to spot because dont shrink and get engulfed, floats around in mutants, WT will get cleaned up -they look for mutants that fail to fie after mutagenizing, wont see dead bodies floating around -supressor screens: ced-3 and ced-4 - 2 more mutations, extra live cells

Features of eukaryotic enhancers

-found first in eukaryotic viruses -increase levels of gene expression -generally function in cell-type specific fashion -can be located upstream, downstream or within gene (intron) -can be located at any distance from the TSS- make it hard to determine which gene is being regulated -can function in either orientation

Enhancers

-function in position- and orientation-independent fashion -all work to drive expression regardless of orientation -do enhancers determine cell type specific of a gene? correct spatial, correct temporal expression, spatiotemporal pattern of expression -which enhancer elements are required where are they located in the genome and how large are they? -are there silencing elements? yes do opposite, block expression

Regulation of gene expression

-gene must be expressed in correct time and place- cell type specificity is determined by RNAs and proteins expressed in that cell, control is primarily at the level of transcription- by mRNA in cells -mutations that affect no gene are innocuous -mutations may affect the coding portion of gene (ORF) or cis-regulatory regions -mutations may affect gene regulation at different levels (loss of function or down regulation is more common)

cloning technology

-generation of many copies of DNA template (eg recombinant DNA molecule) that is replicated in a host -developed in the 1970s -prior to development of PCR technology -first method to generate an unlimited supply of gene copies -made molecular biology possible -many applications that complement genetic approaches: genetic mapping, DNA sequencing, mut studies, transformation, genetic engineering, genome sequencing -prior to the development of this technology, DNA was difficult to work with because it was hard to obtain sufficient copy number to visualize or manipulate -allows to manipulate and change DNA not poss without cloning, prior to 70s DNA was hard to work with because didnt have enough

DNA cloning in practice

-goal is to generate large amounts of pure DNA that can be manipulated and studied -DNA is cloned by the following steps: 1. isolate DNA from organism (eg extract DNA) 2. cut DNA with restriction enzymes to a desired size 3. splice (or ligate) each piece of DNA into a cloning vector to create a recombinant DNA molecule, cloning genes into plasmids- cloning vector= engineered DNA molecule capable of replicating in a host organism (eg bacteria) 4. transform recombinant DNA (cloning vector + DNA fragment) into a competent host that will replicate and make copies- get DNA inyo organism 5. E. coli is the most common host. grows quickly with doubling time of 30 min- normally bacteria would chew up foreign DNA use ecoli that is mutated to not do this

eukaryotic genes

-have promoters, similar to prokaryotes -promoter is close to a genes initiation site -typical eukaryotic genes have enhancers- elements that enhance gene expression

Histone code hypothesis and tails

-histone tails are subject to a variety of covalent modificiations -additional code -extension of protein coding if methylated it means repression of acetylation in some cases -open configuration (active protein)- H3K4me, H3S10P, H3K14,Ac -closed configuration (repressive code)- H3K9Me, H3K27Me, H3S28P -histone code different from genetic code because its reversible- less permanent

Homologous recombination

-homologous recombination is only limited to certain organisms -occurs rather rare, require screening a large number of ES cells -labor intensive -knockout mice is valuable for studying human diseases

applications of gene targeting

-homozygous, null mutants (knockout mice): what is the effect on the organism? if put WT in and organism is cured, know thats the gene -correction of mutated genes: recue experiment (confirming genetic origin of a disease) -exchange of one gene for another (gene knock in)- ex: exchange parts of mouse immune system with human immune systems, ex: insert a reporter gene -if have gene and what to know when or how its expressed can put in reporter genes, mice as models for human diseases

Cis regulatory elements

-how do these cis-regulatory elements work-bind trans-acting factors, transcription factors

DNA methylation

-how does the prescence of 5-methylcytosine lead to the silencing of genes in local areas of chromosome? -MT= DNA methyltransferase, MeCP2= methyl-CpG-binding protein, HDAC= histone deacetylase -when methylated, sends signal (MeCP2) bind to methylated cytosone once protein gets attracted to location results additional proteins (HDAC)- shut genes down

Imaging Real-time PCR

-if amplifying DNA, at some point amount of DNA has amplified to detectable level -imagine you start with 4 times as much DBA as i do, youd reach 1,000,000 copies exactly two cycles earlier than i would -we describe the position of the lines with a value that represents the cycle number where the trace crosses an arbitrary threshold- Ct value, ct values are directly related to starting quantity of DNA -hit threshold points at diff cycles, cycle it hits it at is the ct value- has to do with how much you start with -need control- good variation in endogenous control Ct values

Systematic RNAi screens in C. elegans and mammalian cells

-in the nematode, C. elegans, RNAi is easy to do- inject dsRNA, feed bacteria expressing dsRNA, or soak in solution of dsRNA -makes systematic RNAi screens possible

Methods of delivery

-in vivo gene therapy- therapy delivered to somatic cells in the body, ex: injected into retinal cells or inhaled into lungs -ex vivo gene therapy- cells removed from body, trwated, then put back in, ex: bone marrow cells -two types of viral vectors are commonly used to introduce foreign DNA into human cells 1. retroviral vectors- act like retrovirus but genome is engineered to not harm and to have foreign seq- some issue since injects into genome 2. Adeno-associated viral vectors- doesnt insert into genome have to continuously inject new

Prokaryotic gene expression: where does regulation occur?

-initiation of transcription -elongation -termination -RNA stability -translation- coupled transcription/translation in prokaryotes -bacterial genes in same pathway are organized in clusters called operons (eg lac operon) and can be regulated together -no organelles- transcription and translation occurs together

Prokaryotic gene expression: basic steps

-initiation of transcription- primary regulatory point- most energy efficient to control at this point -elongation -termination -translation

How does an enhancer know which genes to regulate?

-insulators are sequences located between an enhancer and a promoter. they block access to the promoter -can work really far away -insulaors organize genomic DNA into loops- enhancers activate promoters located in the same loop -looks keep enhancer away from gene on other side of insulator

How do cis-regulatory elements work?

-interact with/bind regulatory proteins= trans-actingfactors=transcription factors) -RNA polymerase -basal transcription factors -enhancer binding proteins -co-regulator proteins -all transacting facotrs all directed to right spot

RNA interference

-intially characterized in: 1. C. elegans- dsRNA injection-named RNAi 2. plants- resistance to spread of virus, suppression of transgene expression -function of RNAi likely used to detect- genome-invading transposable genetic elements and dsRNA viruses, and other abnormal gene expression

Sigma subunit

-is an initiation factor -binds specific sequences upstream of TSS -positions core polymerase -dissociates after initiation -specific sequence upstream, positions RNA pol comes off after about 30 nucleotides -what determines sigma binding specificity? look at transcription start site align sequence -how frequendt it can initiate transcription - the strength of the promoter- affinity of sigma to sequence -mutations in recognition sequences dec transcription- mutations dec affinitity for sigma binding, usually will not completely abolish binding but dec frequency of stable binding -smal, still can bind just not as tightly

Targeted Mutagenesis

-is key to functional genomics- to identify the function of each and every gene in a genome -the method varies in diff organisms- 1. Homologous recombination (yeast, moss, mouse) 2. Transposon insertion (flies, zebrafish, arabidopsis)- older method, limited, need to know what type of transposon to use

Liposome-mediated transformation

-liposomes-artificial phospholipid vesicles -cationic/neutral lipid mixtures spontaneously form stable complexes with DNA -liposomes interact with negatively charged cell membranes and the DNA is taken up by endocytosis -low toxicity works for most cell types works with cells in suspension -up to 90% of cells can be transfected -if have lipids often times it will self assemble in tha can put plasma DNA so can be taken up by cells

Measuring gene expression, why?

-low to midplex: northern blots- transcript size, alternate aplice forms, reporter genes- enhancer + reporter (lacZ, GFP), spatial, transgenic, in situ hybridization-spatial expression, qRT-PCR- sensitive (small samples) -multiplex: microarrays- need to know seq, oligos or cDNAs spottedm RNA seq- dont need to know seq ahead of time, expensive why would you want to make gene expression****

Transgenic technology

- next step after manipulating DNA is get into cells of organisms, foreign gene into an org, put in markers so can easily see like fluorescence

knock down

-100% function to lower than 100% of function but dont completely get ride of function

Cut, paste and copy

-1973-1976 -stanley cohen and herbert boyer perfect genetic engineering techniques to cut and paste DNA using retriction enzymes -sees the first expression of a human gene in bacteria -use of growing DNA (letting it replicate) in bacteria -used restriction enzymes

Human gene therapy holds promise for furture

-SCID-X1 (severe combined immune deficiency)- treated with retroviral gene therapy using ex vivo modification of bone marrow cells, patients regained immune system function, mutation resulted in four children developing leukemia -congenital blindness- recombinant AAV vectors injected into retinal cells -patients regained sight -patients may require additional treatments when vector degrades

Genomic library

-3 ways to cut the DNA for a genomic library: 1. complete digestion (at all relevant restriction sites)- choice of restriction enzyme determines size of fragments, produces a large number of non-overlapping DNA clones, genes containing two or more restriction sites may be cloned in two or more pieces -valuable for human genome project 2. partial digestion- limiting the amount and time the enzyme is active results in a pop of overlapping fragments, fragments can be size selected by agarose electrophoresis, fragments have sticky ends and can be cloned directly -want larger fragments doesnt cut at all sites -range of overlapping fragments similar to shotgun sequencing 3. mechanical shearing- produces longer overlapping DNA fragments, ends are not uniform requires enzymativ modification before fragments can be inserted into cloning vector

How is the DNA methylation inherited?

-CG methylation can be propagated during DNA replication -also heritable, once get gene silenced all progeny cells, same gene will be silenced -enzyme in progeny cells (Met1) maintenance enzyme, recognizes complementary strand should be methylated as well so methylates C on comp. strand so inherited pattern

Different families of DNA-binding TFs

-DNA binding domains within TFs directly contact DNA -different DBDs recognize diff specific DNA sequences -many TFs are members of large families, all of which bind similar sequences -transcriptional activators are DNA binding proteins- DNA binding alone will not activate transcription-activators bind enhancers -transcriptional activators also have transcription activation domains- may interact with another protein with activation domain or DNA binding domain -transcriptional activation domains contact other proteins to increase rate of transcription: activators bind basal factors or coactivators

Transient transformation (transfection)

-DNA maintained in nucleus for short time, extra chromosomal no replicon, no selection is required -its there but not trying to select for it -how is transient transformation useful? testing platform prior to rime-consuming and difficult cell-line construction, experiments: eg investigating gene regulatory regions- clone regulatory elements upstream of a eporter gene on plasmid, chloramphenicol acetyl transferase gene activity varying depending on the levels of transcription directed by regulatory elements, faster than selecting for stable transformation

selectable markers for transformation

-Dominant selectable markers- confer resistance to some toxin eg the neo marker confers survival in prescnece of aminoglycoside antibiotics -these selectable markers dont require a specific genotype in the transfected cell line, any cell line-dominant selectable marker confer resistence often times -endogenous markers- confer a property that is normally present in cells eg thymidine kinase (TK) (required for salvage pathway of nucleotide biosythesis)- can select for it can select against it- grow thymidine kinase knockout cells in HAT medium: pathways incolced in nucleotide synthesis in mammalian cell, aminoptoerin blacks de novo synthesis of TMP and A/GMP restore A/GMP synthesis with hypoxanthine, thymidine for salvage pathway (requires TK) -add thymidine in order to get dTDP -these markers may only be used with cell lines that already contain mutations in the marker genes

Initiation of transcription

-RNA polymerase contains multiple subunits -core polymerase includes alpha, beta subunits -holoenzyme includes alpha, beta and sigma subunits -alpha binds promoter, bringing the RNA polymerase enzyme to the correct position of the DNA -sigma factors give specificity, recognizes specific promoers etc lots of types of sigma factors

RNAi

-RNAi was a revolutionary gene silencing technology that can be applied to any organisms

TALENS

-can be used to modify the genome of any organism -TALEN-TALE (transcription activator-like effector) nuclease -composed of 33-35 amino acid repeats and a nuclease that cleaves one strand of DNA (at specific place)- each repeat recognizes and binds to a specific nucleotide -originally discovered in fungus -contains repeats each binds to one type of nucleotide only differences is the 2 amino acids -can make artifical nuclease if know sequence can stitch together the different domains with only difference is the 2 aa, nuclease then cuts -combination of 2 TALENs function as a restriction enzyme- repair of the double stranded break results in either a knockout or a knockin -compementary facing it, this cuts each and creates double stranded break, connect together and repear them by end joining- usually use few nucleotides --> causes deletions and then frameshifts -if provide donor template flanked by homology (has homology with host) break triggers recombination with donor and replaces, knocks in

Double stranded RNA-induced RNAi

-causes destruction of a specific mRNA in C. elegans -look at early embryo development- more strongly expressed in anterior 2 cells -(c) after probe and hybridizated RNA level is lower double strand (RNAi) more affective than antisense RNA -advantage- very specialized- only knocking down gene with exact homology to exact RNA put in -the immune system can recognize dsRNA (viruses tend to have this) -dicer chops up in 20bp sequence, loaded into RISC complex, slice mRNA then exposes the free end and exonucleases chew up RNA -dsRNA can use RDRP and facilitate reverse transcriptase and can repear itself and amplify itself

Histone methylation causes chromatin condensation

-chemical modifictions- acetylation (Ac) or methylation (me)- of histone proteins determine whether genes on the surrounding DNA are active or silenced. HP1 is a transcription-inhibiting protein -PRC2- responsible for methylation

Kinetochores assemble at centromeres

-chromosomes attach to microtubles of the mitotic spindle at the kinetichore -chromatin is packaged differently at centromere- H3 is replaced by the histone variant CENP-A, CENP-A nucleosomes act as scaffolds for many kinetochore proteins

Action of cohesion during mitosis

-cohesion is a protein complex that holds sister chromatids during metaphase -at anaphase, cohesion is enzymatically cleaved and sister chromatids are released from each other, separas- involved in degradation

Eukaryotes: controlling gene expression

-compared to prokaryotes, eukarytoes have additional levels of complexity for controlling gene expression- probably because of multicellularity-expressed in diff cells -in eukaryotes, transcription takes place in the nucleus and translation takes place in cytoplasm -additional RNA processing events occur in eukaryotes (splicing, polyA addition, 5'cap) -chromatin structure makes DNA unavailable to transcription machinery -gene regulation controls cellular differentiation into hundreds of specialized cell types

Knockout

-completely removes function of gene, 100% to 0%, no matter what approach

Chromatin

-consists of 1/3 DNA, 1/3 histone proteins, 1/3 nonhistone proteins -5 different kinds of histones -not 100% DNA

Applications of RNAi

-control virus infection- main function -analysis of gene function by silencing specific gene -target validation for drug development- good because only interferes with gene its silencing unlike chemotherapy which interferes with all genes -potentially new therapeutic approaches to treating diseases- a new approach to antisense and new possibilities for gene therapy (liver diseases)

Repressor proteins supress transcription initiation by recruiting corepressors

-corepressors have two alternative functions: prevent RNA pol II complex from binding the promoter and modify histones to close chromatin structure -happens after recruit corepressor to inhibit

biochemical methods to identify transcription factor binding sites: direct regulation

-crosslink DNA and protein component of chromatin -fragment DNA -allow an antibody specific to the protein of interest to bind -purify complexes with antibody protein of interest and DNA fragments -seq DNA

apoptosis is suicide not murder

-cytoplasm shrinks, chromosome condense and fragmented, nuclear membrane breaks down, apoptotic body formation, engulfment of cell corpse -observed cell death while watching cell divide -if gene involved in cell death is expressed it will die

Gene expression

-determine phenotype -gene must be expressed to have an effect -gene must be expressed in the correct time and place to function properly -a mut in a gene will affect function of a gene only in tissues that express that gene -different cells in body with diff function- what makes them different? gene expression -mut in one of the cells will only affect those cells -brain cells and pancreas cells-both carry insulin gene and neurotransmitter gene- what differs is which are expressed, mutations in neurotransmitter genes only affect brain cells

Dosage compensation

-difference in males and females -Y chromosome doesnt encode much besides being male, genes on X chromosome very important for development -different amount of gene products in male and females because females have 2 Xs -animals have to deal with this by dosage differences -worms: male vs hermaphrodites- they down regulate genes on X chromosome in hermaphrodite so only one expressed -fly: male have X chromosome genes expressed twice as much- upregulate in male fly -mammals- female- one X chromosome is silenced and condensed only 1 X is active in female, condensed into barr bodies

Mechanisms of X inactivation

-dont worry about details- lnow on both X chromosomes Xa- active, Xi-inactive, in both of these regions Xic- X chromosome inactivation center- encodes several genes that dont code for proteins on expressed RNA -Xist and Tsix- 2 genes well studied in this area, which chromo gets inactivated depends which gene its expressing if Xist is highly expressed condenses and forms barr body- histone methylation complex surrounds it and condenses -initiation of XCI depends on Xist (the 17 kb X-inactive specific transcript) that targets and tethers PRCs to the X chromo in cis. Xist is dispensable once the Xi is established -Xi-> inc Xist expression, dec Tsix expression -Xa-> dec Xist expression, inc Tsix expression

Hosts for DNA recombinant technology

1. bacteria- ecoli used because is easily grown and its genomics are well understood- gene product is purified from host cells 2. yeasts - saccharomyces cerevisiae- used because it is easily grown and its genomics are known, may express eukaryotic genes easily, continuously secrete the gene product, easily collected and purified 3. plant cells and whole plants- may express eukaryotic genes easily, plants are easily grown-produce plants with new properties 4. mammalian cells- may express eukaryotic genes easily, harder to grow, medical use

How to detect DNA methylation?

1. busulfite sequencing- if we know seq but dont know which CG is methykated- first denture with chemical, sodium bisulfite, if C isnt protected by methylation it will be converted to U. can do PCR to amplify DNA -seq reads GCAC so know first C is methylated and second C isnt 2. methylation sensitive restriction enzymes- certain restriction enzymes are sensitive to methylation. recognizes CCGG if C is methylated it cant cut. MSP1 insensitive to metylation. if see diff then methylation is there, use as pair, one is control (Msp1)

cDNA library

1. cDNA is derived from mature mRNA, does not include introns (1-2% of genome) 2. cDNA may contain less info than the coding region 3. cDNA library reflects gene activity of a cell at the time mRNAs are isolated (varies from tissue to tissue an with time) 4. mRNA degrades quickly after cell death, and typically requires immediate isolation 5. creating cDNA library: isolate mRNA, synthesis cDNA, clone cDNA -similar approach to genomic but have to make cDNA first -why-more accurate representation of what gene is like doesnt have introns, includes splicing, hard to tell what splices are used- allows to see that -can compare to genomic to see whats used

Screening a cDNA library

1. cDNA libraries are used to detect or sequence genes for proteins becayse cDNAs are generated for genes that are transcribed 2. if you know the DNA seq for the protein coding gene you want to find, a homologous DNA probe can be used (heterologous probe=know seq from other species) 3. or the cDNA loibrary can be sequenced directly using the universal M13 primers in the plasmid vector Applications -isolate and seq the genes for proteins -identify and compare homologous sequences in various types of organisms -quantify amount of mRNA synthesized from a gene or all genes and measure the level of gene expression -finally, if no homologous DNA seq is available, expression vector cDNA library can be probed with an antibody that recognize the protein produced by a cDNA

Creating an expression vector

1. cloned cDNA is inserted into a specifal type of plasmid vector possessing a promotor and transcription terminator before it is transformed 2. mRNA is transcribed from the cDNA and translated by Ecoli producing the protein of interest -low copy- want to express well

Forms of information in genomes of the cell

1. genetic information: provide the building block for the manufacture of all proteins needed for the cell functional activity 2. epigenetic info: provides additional instruction on how, when and where these info should be used-

Eukaryotic cell transformation

1. getting DNA in: method depends on the type of cells 2. transient transformation: no selection 3. stable transformation: selection is required (also counter-selection can be useful)

some features of pUC19 plasmid vector

1. high copy number in Ecoli- 100 copies/cell provides high yield 2. selectable marker is amp. ampicillin in growth medium prevents growth of all other ecoli that dont contain plasmid 3.cluster of several diff restriction sites called a polylinker occurs in the lacZ (beta-galactosidase) gene 4. cloned DNA disrupts reading frame and beta-galactosidase production 5. add X-gal to medium turns blue in presence of beta-galactosidase 6. plaque growth: blue= no inserted DNA and white= inserted DNA 7. some percent of digested vectors wll reaaneal with no insert. remove 5' phosphates with alkaline phosphatase to prevent recircularization (this also eliminates some blue plaques)- when have vector digested and linear want to recirculize without gene so add alkaline phosphate- prevents resealing without gene in it 8. finally, plasmids are transformed into Ecoli by chemical incubation or electroporation (electrical shock disrupts the cell membrane) 9. good for <10kb, cloned inserts >10kb typically are unstable- might add 10 fold so drives rxn to include gene

2nd part: after introducing the foregin gene into ES cells homologous recombination replaces WT allele with foreign mutant allele in ES cells

4. individual neomycin-resistent cell clones are tested to identify a clone where integration occured by homologous recombination 5. purebred black mice are mated to produce a blastocyst that is removed from the female

3rd part: knock out mice are raised

6. About 10 ES cells with the knockout allele from the same colony are infected into the blastocyst from the black mice. this blastocyst is then placed in the uterus of another black female, where it can develop into a live-born mouse 7/ some of the mice tht develop from the injected blastocysts will be chimeras with some cells derived from the black parents and other cells derived from the agouti S cell culture. chimeric animals are mated with purebred black mice. if the germ line of the chimera contains agouti-derived cells, the some of the offspring of this mating will be agouti (which is dominant to black). these agouti progeny should be heterozygous for the knockout allele, this can be checked by analyzing DNA from the tail. agouti brothers and sisters with the knockout allele can subsequently be mated with each other to produce mice homozygous for the knockout allele.

Histone Acetylation

HATs: histone acetyl transferase, HDACs: histone deacetylase -histone acetylation (preformed by HATs) is associated with transcriptionally active genes -deactylation (performed by HDACs) is associated with inactive genes -lysine is positively charged, allows to neutralize phosphate which is negatively charged, result: DNA neg charge will be neutralized which wont repel each other so can condense -but if acetylation occurs- no longer pos charge on lysine cant neutralize phosphate or DNA so DNA repels and more opne -deacytlation- chromatin condensation -bu removing aceytalation also leaves histone tails might be methylated leading to more condensation

creating a cDNA library- step 1- isolate mRNA and step 3 cloning ofDNA

Step 1-mature eukaryote mRNA has a poly A tail at the 3' end -mRNA is isolated by passing cell lysate over a ployT column composed of oligodTs -polyA tails stick to the oligo dTs and mRNAs are retained all other molecules pass through column -polyA tails stick to polydTs Step 2 1. cDNA has blunt ends thus need to add restriction site linkers or adapters to make them sticky 2. use T4 DNA ligase and blunt end ligation to add restriction site linkers (or adapteers) to each end of the cDNA 3. next, digest the linkers with the same restriction enzyme used to cleave 4. mix cDNA with cut vecotr DNA in the presence of DNA ligase 5. transform into an Ecoli host for cloning 6. if cDNA has the same restriction site as the linkers DNA will be cloned in pieces 7. solution use adapters with single stranded overhangs that match the restriction site on the vector (eg skip #3 and proceed to 4)

The Era of genome editing

TALENs and CRISPR/Cas9 Distinctions with RNAi: -acts on DNA (not on RNA) -cause complete knockout (RNAi only knockdown) -effect is permanent (RNAi may loose its effect after several generations) -modify, inc or dec function (RNA only reduce) Similarity with RNAi: -directly targeting a gene based on homology -works in any organisms -require ways to introduce the foreign DNA into a cell

step 2- cut DNA with restriction enzymes

restriction enzymes- recognize specific bases pair sequences in DNA called restriction sites and cleave the DNA by hydrolyzing the phosphodiester bond -cut occurs between the 3' carbon of the first nucleotide and the phosphate of the next nuceotide -restriction fragment ends have 5' phosphates and 3' hydroxyls -most restriction enzymes occur naturally in bacteria -prtoect bacteria against viruses (bacteriophages) by cutting up viral DNA -bacteria protet their DNA by modifying possible restriction sites (methylation) -more than 400 restriction enzymes have been isolated -names typically begin with 3 italicized letters- EcoRI, HindIII, BamHI -many restriction sites are palindromes of 4,6 or 8 base pairs -short restriction site seq occur more frequently in the genome than longer restriction site seq -their frequency is a function of (1/4)^n -if recognize 4 bp seq it will occur more frequently than 8bp seq -some restriction enzymes produce blut ends, wheras other produce sticky (overhanding staggered) ends -sticky ends are useful for DNA cloning because complementary sequences anneal and can be joined directly by DNA ligase without using adapters- if doesnt have the end you ant you can use adapters to stick in ones you want, dont still have sma1 site becayse gets disrupted

steo 3-splice (or ligate) DNA into some kind of cloning vector to create a recombinant DNA molecule

six diff types of cloning vectors: 1. plasmid cloning vector, phage lambda cloning vector 2. cosmid cloning vetor 3. shuttle vectors 4. yeast artifical chromosome (YAC) 5. bacterial artificial chromosome (BAC) 6. fosmid cloning vector