Genetics final exam

chromatin remodeling complexes alter the positions and compositions of nucleosomes

- for B-globin nucleosome positioning changes in the promoter region as part of gene activation -nucleosome positioning between two different types of cells will determine whether or not the B-globin gene is transcribed in that cell or not -->B-globin gene in RBCs are actively transcribed-nucleosome positioning allows for it vs -->B-globin in pancreatic cells are not actively transcribed ---->can run a Northern blot-isolate RBC mRNA and pancreatic mRNA-and see if B-globin is highly expressed in RBCs or pancreatic cells-nucleosomes are positioned differently on the B-globin gene -transcriptional activators orchestrate changes in chromatin structure -> through ATP-dependent chromatin remodeling -->energy of ATP hydrolysis is used to drive change in location-can bind ATP-break-use the energy to move nucleosomes -change in location or composition ---> makes DNA more or less ready for transcription

trans-epigenetic changes

--changes are maintained by diffusible factors such as transcription factors=affects both copies of a gene -gene B paternal chrm is actively expressed with the presence of a transcription factor (diffusible) -maternal chrm-off-does not have transcription factors -cell fusion occurs-the two cells fuse together and the transcription factor diffused across into the maternal chrm and turn it on-both chrms are actively expressed now -due to the presence of a transcription factor-both copies of gene b are expressed in the fused cell expression factors are maintained after cell fusion

Molecular mechanisms that underlie epigenetic gene regulation:

-DNA methylation --->changes whether genes are expressed ex. CpG islands (DNA methyltransferase marks cytosine) -Chromatin remodeling --->DNA translocases are ATP dependent-evict or move nucleosomes -covalent histone --->N-terminal of histone was target for post translational modification-can leave marks (methylation-of lysine or arginine R groups; acetylation; ubiquitination-small addition of protein to histone-covalent linkage through lysine) -localization of histone variables --->certain histones have variants that cause gene activation/repression (H1*of H1=gene repression) -feedback loops --->and feedforward- communication-allow for info to be exchanged with organism & environment

abnormalities in chromatin modification are common in cancer cells

-DNA methylation -->hypermethylation=abnormally high level of methylation-typically at CpG islands-promote cancer inhibiting expression of tumor suppressing genes/p53 -covalent modification of histones -->H3K27Me3->signal repression -->increase the expression of oncogenes or inhibit the expression of tumor-suppressor genes -chromatin remodeling -->move histones; abnormalities in the locations of nucleosomes = cancer

MacLeod and McCarty experiment

-DNA, RNA , proteins and carbohydrates are the major constituents-find out which are the genetic material -Prepared extracts from type S cells and purified each type --->only the extract that contained purified DNA was able to convert type R bacteria into type S --->treatment of the DNA extract with RNase or protease did not eliminate transformation-RNase breaks down RNA and protease breaks down proteins=proved that RNA and protein are not acting as genetic material --->treatment with DNase eliminated transformation; if no DNA extract was added no type S bacterial colonies were found on the petri dish ------->DNase breaks down the DNA needed to transform-degradation of DNA by DNase prevented conversion of type R to type S

glucocorticoid hormones

-GRE (glucocorticoid response elements) function as enhancers-located near dozens of diff genes so the hormone can activate many genes -->inside the nucleus-glucocorticoid response element-enhancer embedded in DNA regulatory switch) -->glucocorticoid receptor binding as a dimer-drives towards transcription -glucocorticoid-produced by a particular location in your body and travels though blood to target tissue -glucocorticoid receptors inside the cytoplasm are bound to HSP90 (blocking nuclear localization signal) -once glucocorticoid is bound HSP90 disassociates and exposes NLS-dimerize with another and enter the nucleus to drive transcription -once into the nucleus it binds to enhancer-glucocorticoid response element (GRE)

polycomb group complex

-PRC1 and PRC2 complexes -repression may begin by the binding of PRC2 to a polycomb response element-leading to the trimethylation of lysine 27 on histone H3=repression -PRC! is then recruited to the gene and may inhibit transcription in three ways: 1) chromatin compaction-PRC1 may cause nucleosomes in the target gene to form a knot-like structure 2)covalent modification of histones-PRC1 may covalently modify histone H2A by attaching ubiquitin molecules 3) direct interaction with a transcription factor-PRC1 may directly inhibit proteins involved with transcription like TFIID

double helical structure of DNA

-Pauling-regions of protein can fold into a secondary structure = alpha helix - built a ball and stick model

enhancers and silencers

-TF binding to an enhancer increases the rate of transcription=upregulation (10-1000 fold) -TF binding to a silencer decreases the rate of transcription=downregulation -orientation independent or bidirectional -can function at large distances from the promoter

cancer

-a disease characterized by uncontrolled cell division ->3 diff types-based on what cell type makes the mistake/mutation and grows out of control -two general types of abnormalities 1. some genes are overactive in cancer cells-high level of expression of these genes=oncogenes-causes cellular changes that promote cancer 2. other genes exhibit a decrease in expression in cancer cells=tumor suppressor genes-proteins encoded by tumor suppressor genes help to prevent cancer (a decrease in their expression may allow cancer to occur) -liver cells can transform into cancer cells bc of epigenetic modifications = hepatoma (cancer cell) -B-cell immune system cells (make antibodies)-in blood-leukemia -p53 gene=transcription factor -->controls genes that regulate whether the cell can go through the cell cycle or not (regulates cell cycle)=helps regulate growth of: 1. TF 2. protooncogene 3. tumor-suppressor gene-doesn't allow for tumors to form; p53 will not allow damaged cells to go into the cell cycle -protooncogene with p53-if p53 becomes mutated (doesn't work)-the p53 is broken/not translated=high level of expression of oncogene=cancer -if p53 is not mutated=normal translation-regulates growth -if promoter is heavily methylated=shut down p53 gene=lower shield

regulation of Translation

-after an mRNA is completely made-its expression can be regulated by -->regulation by small RNAs -->RNA binding proteins affect the translation and degradation of mRNAs-ex. Iron response protein

features of agouti genes

-agouti gene in mice promotes the synthesis of yellow fur pigment; in one strain of mice a transposable element carrying a promoter is inserted upstream from the agouti gene = Avy allele -agouti allele-responsible for yellow pigment-gene is transiently expressed in development ( if gene is shut off-default=black) --->yellow pigment->pheomelanin -AA homozygous dominant -rodent coat color-agouti (beige brown) -->black-yellow-black sandwich --->black pigment= eumelanin -A^vy allele (diff from agouti allele) -->v=viable -->y=yellow -->there is a transposon element (chunk of DNA) that is inserted into the promoter-the transposon element has CpG islands that are targets for methylation of DNA methyltransferase-this will shut off transcription of the agouti allele=rodent will be black ---->transposon elements can copy themselves and move at different regions of the genome (change allele to vy -not a lot of methylation=transcription takes place = more yellow pigment -a little bit of methylation = black --> increasing methylation of CpG islands in the transposable element= more black=less expression (less transcript) yellow-->pseudoagouti -diet of mother controls this-when pregnant mice were fed a diet that contains chemical tend to increase DNA methylation-the offspring has darker fur=DNA methylation inhibits agouti gene Diet where DNA Methylation occurs: -mom before and after pregnancy -S-adenosyl methionine (SAM)=methyl donor for methyltransferase -DNA methyltransferase uses SAM as a substrate, use DNA CpG islands, transfer methyl group off of SAM and onto CpG islands -->Supplemental diet compounds that increase SAM ----->betaine (tri amino glycine) ----->vitamin B12 ----->choline chloride -------->they all increase level of SAM=more methylation; supplemental =more pseudogouti Diet for lower/no methylation -no dietary supplements -SAM -no methylation = yellow -the darkness of the coat color correlated with the lev of DNA methylation of CpG islands within the transposable element

attenuation in trp operon

-another mechanism of regulation = attenuation-when attenuation occurs-the RNA is transcribed only to the attenuator sequence-and then transcription is terminated (transcription begins but stops at the attenuator sequence-stops before the entire mRNA is made) -->can occur in bacteria bc transcription and translation are coupled -the first gene in the trp operon is trpL-encodes leader peptide

process of X chrm inaction /barr body

-before x inactivation-pluripotency factors bind and stimulate transcription from Tsix and inhibit transcription of Xist (both chrms are active) -x chrm pairing-CTC factors bind to the Xic and the X chrm binds with each other at the Tsix gene ' -choosing the active and inactive chrm-pluripotency factors and CTCFs shift to one x chrm-this chrm expresses the Tsix and remain active; the other chrm can now express Xist and becomes inactivated -binding of the first Xist ncRNA to inactivated x chrm-a tethering protein is bound to Xic and the repeat Cs within the ncRNA=tethering the Xist RNA to Xic (many copies of Xist ncRNA are made); tethering protein recruits in hnRNP-u -spreading phase-the Xist RNAs binds to each other and to a DNA-binding protein called hnRNP-U-which binds to numerous AT-rich sequences within the Xi chrm-spreading continues in both direction to the ends of the inactivated x chrm; hnRNP-u recruits in a series of complexes: ------->polycomb repressor complex 2 (PRC2)-involved in changing chromatin structure (tighten) ------->MacroH2A-variance of histone involved in compaction; with DNA methyltransferase - repression -gene silencing and compaction-repeat A within the Xist ncRNA recruits protein to the Xi that silence gene expression and promote the compaction of Xi into a barr body

DNA methylation

-change in chromatin structure that silences gene expression -carried out by the enzyme DNA methyltransferase -adds in methyl group-marks cystine base-marking 5'C= 5-methylcytosine -->yeast and drosophila have little DNA methylation -->vertebrates and plants have abundant DNA methylation Methylation of Cytosine -DNA methyltransferase enzyme marks and puts methyl group on cytosine -->methylate CG islands(dinucleotide sequence within promoter)= CpG island in promoter -CpG near promoters -->contain high number of CpG sites -->in housekeeping genes-the CpG islands are unmethylated-genes will be expressed; housekeeping genes=unmethylated genes-so transcription always occurs (constitutively expressed) -->genes that are methylated are shut down=no transcription -->in tissue specific genes (genes that are expressed in some tissues but not others) ---->the expression of these genes may be silenced by the methylation of CpG islands ---->methylation may influence the binding of transcription factors ---->Methyl-CpG-binding proteins may recruit factors that lead to compaction of chromatin ex. pancreatic cells insulin gene -CpG islands is unmethylated =transcription-insulin is secreted into the blood vs. fat cells insulin gene -has the same insulin gene but it is not being transcribed -the promoter region that has the CpG islands are methylated = no transcription -hemimethylated-only one cytosine methylated in CG islands -fully methylated-both cytosine are methylated

Cis-epigenetic changes

-changes are maintained at a specific site; change may affect only one copy of gene but not the other copy -cis=epigenetic changes are maintained during cell division -subsequent cell divisions-methylated copy of gene B is always methylated whereas the other copy remains unmethylated -in a diploid cell-the maternal copy is methylated and it doesn't allow expression for only this chrm; the paternal chrm is not methylated so it is expressed ---> does not effect both chrms=cis

chromatin remodeling and histones

-chromatin structure plays a large part on whether genes are transcribed or not-->loose chromatin = transcription -ATP dependent chromatin remodeling refers to dynamic changes in chromatin structure -can remodel specific regions of a chrm-nucleosomes play a huge role bc DNA is wrapped around it for compaction; some areas may not have nucleosomes which allow for transcription -a lot of proteins are involved in remodeling-moving histones -three dimensional packing of chromatin-alternate between two conformations: 1. closed conformation-chromatin is very tightly packed-transcription does not occur 2. open conformation-chromatin is accessible to transcriptional factors-transcription will take place

role of epigenetics in cancer

-correlation coefficient-compares two variables to see if they are related to each other -association-suggests that changes in the two variables follow a pattern-an association between epigenetics and a human disease can be due to: 1. epigenetic change directly contribute to the disease symptoms 2. disease symptoms may arise first, and then they causes subsequent epigenetic changes to happen(epigenetic changes as a consequence of disease) -variants in nucleosomes can shut gene expression off or on -> can affect growth-cancer cells-growing too fast-mutation can accumulate (steal nutrients from body) -epigenome-sequencing entire genome-degree of methylation @ certain developmental stages ->gather DNA from people at diff. ages->DNA methylation increases with age (more methylated DNA); if you reach a certain level of methylation = death

genomic imprinting

-developmental -offspring expresses the copy of a gene from one parent but not both -in mammals only the IgF2 (insulin growth factor II-protein secreted that can affect growth) gene inherited from the father is expressed -->IgF2 gene is methylated during spermatogenesis -->methylation occurs at two sites: imprinting control region (ICR) and differentially methylated region (DMR) during spermatogenesis -------->ICR=imprinting control region -------->DMR= differentially methylated region -in mother (egg)-during oogenesis DNA methyltransferase does not methylate ICR or DMR (both unmethylated)=shuts allele off )not expressed) -fusion occurs (2n)-allelic pair can have exact same base sequence-but during spermatogenesis the chrm gets marked/methylated; the methylated/male gene gets expressed because the methylation is not with the CpG islands = different effect -during fertilization (inside mother)-the dad ensures that the cell begins to divide then form the embryo that digs into uterine lining by secreting IgF2; IgF2 promotes the formation of the placenta-makes sure the cell gets replicated and offspring is born (IgF2 is on and expressed after fertilization -imprinted=maintains methylation in descendants; all cells that derive from the initial cells will carry the methylation (all descendants are methylated from the paternal)=methylation maintenance

ATP dependent chromatin remodelers `

-enzymes/proteins that bind ATP (stored energy source) -can move nucleosomes that are on DNA -DNA translocases-enzyme that catalyze rxn of moving nucleosomes-require ATP -discovered in yeast (simple eukaryote-linear chrm) -eukaryotes have multiple families of chromatid remodelers: 1. SWI/SNF -->had a mutation within the SWI gene from yeast cell-lost the ability to switch between mating types because yeast do not have defined male and female sex-certain changes in chromatin structure did not occur between mating types -->yeast considered to be sucrose non-fermenters (cant break down sucrose bc of mutation breaks translocases) 2. ISWI 3.INO80 4. Mi-20 a) changes in nucleosome position -ATP dependent chromatin remodeling complex requires ATP to move nucleosomes -tails are targets for posttranslational modifications -octamer histones shown with radial loop -there can be a change in relative positions of a few nucleosomes-opens up space for a promoter and its basal transcription machinery or -change in spacing of nucleosomes over a long distance b)histone eviction-creates a gap with no nucleosome -histone octamer is removed from DNA to create open complex for transcription to occur (but it cannot remove all nucleosomes or else it wot be able to fit in the nucleus) c)replacement with histone variants -variant of H3 = H3.3-makes protein whose sequence will be slightly off/different -DNA translocases can replace H3 with H3.3 variant -there are multiple copies of H1, H2A, H2B, H3, and H4-other variants have different base sequences -the variant nucleosome signals that there is an open chromatin at that region=gene activation=transcription

Hershey and Chase evidence that DNA is genetic material

-evidence that DNA is the genetic material of T2 phage (composed of only DNA and proteins) -virus known as T2-infects E.coli bacterial cells = bacteriophage -during infection the phage coat remains attached to the outside(does not enter the cell)-only the genetic material of the phage enters -used radioisotopes to distinguish DNA from proteins -->32P labels DNA specifically-phosphorus atoms are found in DNA but not in the phage proteins -->35S labels protein specifically-sulfur atoms are found in protein but not in DNA -allowing sufficient time for infection-most of the 32P had entered the bacterial cells whereas most of the 25S remained outside the cells-consistent with the idea that the genetic material of the bacteriophage is DNA not protein

insulators

-gene regulation occurs over long distances-but must be limited to one particular gene-and not to neighboring genes -insulators are segments of DNA that insulate a gene from regulatory effects of other genes -->some act as barriers to chromatin remodeling -->others block the effects of enhancers-by chromosome looping -insulators recruit in DNA binding proteins to block/insulate gene B so it wont be transcribed and gene A can have a strong enhancer -insulators as a barrier to change in chromatin structure -->transcriptionally active but we don't want HAT from moving and acetylating neighboring genes so they don't become active -insulators that blocks the effects of a neighboring enhancer -->protein bound to an insulator-prevents the enhancer for gene A from activating the expression of gene B

regulation of gene expression: Transcription

-genetic regulatory proteins bind to the DNA and control the rate of transcription -attenuation-Trp operon-transcription terminates soon after it has begun dur to the formation of a transcriptional terminator -RNA polymerase allows for transcription -Regulatory site-activators, repressor, or inhibitor binds -includes promoter and terminator (Rho dependent or independent)

Epigenetic gene regulation in development (modifications of bases)

-genomic imprinting -x chrm inactivation -cell differentiation -development series of genetically programmed stages in a fertilized egg becomes an embryo then adult (impact whether DNA is methylated or not)

cells respond to steroid hormones in different ways

-glucocorticoids -involved in affecting whether the cell utilizes glucose, breaks down fat, or protein, etc -->influence nutrient metabolism-promote glucose utilization, fat mobilization, and protein breakdown -Gonadocorticoids -->include estrogen and testosterone-influence the growth and function of the gonads -->estrogen and testosterone are steroid hormones that are going to need specific receptors ----->glucocorticoid receptor highly specific-binds steroid hormone it is specific for ----->estrogen receptors=transcription factors bc it is in the cytoplasm-specific for estrogen ----->testosterone receptor specific for testosterone

regulation of the trp operon `

-has an extra layer of regulation =attenuation -involved in the biosynthesis of amino acid tryptophan -structural genes: trpE, trpD, trpB, and trp A encode enzymes involved in tryptophan biosynthesis=anabolic pathway -the genes are trpR and trpL are involved in regulation of the trp operon -->trpR-encodes the trp repressor protein -->trpL-encodes a short peptide=leader peptide-functions in attenuation -one transcriptional unit: constitutive promoter of trpR + trpR gene +terminator -trpR needs a corepressor to bind to the operon of the trp operon and blocks transcription --> if you have a lot of tryptophan the corepressor will bind to the repressor and the repressor will bind to the operon and block transcription-wont build more enzymes that make tryptophan bc you have enough already -within the trp operon-there is the trpL gene and attenuator + 5 structural genes -->attenuator= DNA sequence @ 3' of the trpL gene-plays a role in stopping transcription -at low tryptophan levels-transcription of the entire trp operon occurs (inactive trp repressor-corepressor is not present) -at high tryptophan levels-repression occurs; corepressor bind to the repressor-binds to the operon =no transcription of the 5 structural genes that give rise to the enzymes that make up tryptophan

structural motifs of transcription factors

-helix-turn-helix motif -->have interactions b/w the R groups of amino acids forming hydrogen bonds with bases -->binds to major groove of DNA -->alpha helical structure with turns (6aa) -->found mostly in bacterial cell transcription factors but in some eukaryotic -helix-loop-helix motif -->dimerization motif -->DNA binding motif ---->same domain serving 2 functions found in TFs (2 TFs come together) -->turn has more amino acids than six-has alpha helical structure -zinc finger motif -->Beta sheet structure (2B strands) --->alpha helix structure --->divalent cation-zinc 2+: important in holding structure together with the sheet structure and alpha helical structure to form zinc finger ------>side chain group of cysteine-only requires 4 of them to hold the zinc divalent cation ------>if you don't have zine-wont have TFs assembled -wont be able to bind to DNA ---> helps TFs bind to DNA through the major groove --->antiparallel Beta sheet structure-chain fully extended-secondary structure held together by hydrogen bonds that involve the backbone of the atoms; 2 beta strands coming to form a beta sheet -leucine/zipper finger motif -->dimerization motif -->2 protein structures with alpha helical structure-with leucine sticking out; leucine has a hydrophobic group-when the two chains face each other there is a hydrophobic interact (zips)-2 proteins will dimerize and stick together using leucine zipper motif -->coiled chord structure-2 diff. alpha helical structures from 2 different proteins twisted around and put together

steroid hormones and regulatory transcription factors

-hormones elicit changes in gene expression =mood changes; travel through blood and impact other tissue nd change gene expression -steroid receptors-regulatory transcription factors that respond to steroid hormones -->hormone actually binds to the TF -steroid hormones are produced by endocrine glands and are secreted into the bloodstream-then get taken up by cells that respond to the hormone -->structurally looks like cholesterol-has 4 fused rings -->nonpolar-can go through plasmid membrane -->steroid hormone signals tissue/cell to change its gene expression-change the rate of transcription -steroid hormone from the blood enter the cell easily through the lipid bilayer bc it is nonpolar -once inside the cell-there is a steroid hormone receptor in the cytoplasm -steroid hormone receptor is initially bound to heat shock (HSP90) protein which masks nuclear localization signal (NLS)-the signal helps TF know to go to the nucleus-bc the signal is hidden TF wont go into the nucleus until the hormone/steroid enters and binds to the hormone receptor -once the hormone binds to receptor HSP90 disassociates no longer masking the NLS-2 molecules of the receptor dimerize and form a dimer-move into the nucleus and attaches to DNA-go into response element binding to DNA and help drive transcription

transcription activation

-how an area free of nucleosomes can drive transcription -NFR-transcriptional start site at the core promoter-can recruit in an enhancer binding protein -the enhancer recruits in an activator-which gives rise to transcription -binding of activators: -->activation protein bind to enhancer sequences -->the enhancer may be close to the transcriptional start site or far away -chromatin remodeling and histone modification: --> an activator recruits a chromatin remodeling complex -ex. SWI/SNF and a histone modification enzyme-ex. histone acetyltransferase -->nucleosomes may be moved, evicted, or replaced with variants; some are subjects of covalent modification such as acetylation

histone code

-identified 50 amino acids that selectively modify the amino terminal tails of histones -->modifications include: acetylation, methylation, and phosphorylation -these modifications affect level of transcription -->influence interactions between nucleosomes -->occur in patterns that are recognized by proteins=histone code ---->pattern of modifications provide binding sites for proteins that specify alterations be made to chromatin; proteins bind based on code -3 modifications 1. p=phosphate 2. ac=acetyl group 3. m=methyl group -histone protein's N terminal tail are targets for enzymes to make modification to the amino acid residues --> can add a phosphate group-need an amino acid present with an alcohol group =post translational modification -->can also add an acetyl group-added to either lysine or arginine ----->lysine loses charge K17me or K17ac K=lysine 17=17th amino acid in the sequence (position) me=undergone methylation ac=undergone acetylation

regulation of gene expression: protein post-translational

-in feedback inhibition-the product of a metabolic pathway inhibits the first enzyme in the pathway -covalent modifications to the structure of a protein can alter its function

barr body

-in human female cell -x chromosomal deactivation-not changing base sequence-but preventing genes from being expressed -goes under a severe amount of compaction=not able to be transcribed

iron uptake by mammalian cells

-iron does not run freely through the blood -transferrin protein carries iron through the bloodstream to protect the body -->transported into the cytosol by endocytosis -excess iron is stored within a hollow spherical protein = ferritin 1. diet Fe3+ iron-into body 2. transferrin protein carried iron to the cell 3. binds to transferrin receptor on the membrane 4. endocytosis 5. once inside the cell it will be stored inside a vesicle 6. transferrin protein will be released from iron and stuck to its receptor 7. iron is released into the cytosol-some iron binds to cellular enzymes; excess iron is stored within ferritin -how ferratin mRNA and transferrin receptor mRNA is regulated translationally depends on how much iron is around --> low iron: ----->Ferratin: no translation ----->Transferrin receptor: translation-want more iron transferred into the cell -->high iron: ----->ferratin: translation-excess iron is stored ----->transferrin receptor-no translation -excess iron can be toxic so levels must be regulated -the two mRNAs that encode ferritin and the transferrin receptor are both influenced by an RNA-binding protein = iron regulatory protein (IRP) --> IRP binds to a regulatory element within these mRNAs known as iron response element (IRE) -->IRE in ferritin mRNA is in 5'UTR -->IRE in transferrin receptor mRNA is in 3' UTR Ferritin mRNA -low iron: IRP binds to IRE prevents translation of the ferritin mRNA; IRP binding blocks translation -high iron: Iron binds directly to IRP and prevents IRP from binding to the IRE-so the ferritin mRNA is translated to make more ferritin protein; iron binds to IRP and causes it to disassociate =translation Transferrin receptor mRNA -low iron: mRNA is translated to make more transferrin receptor protein -high iron: iron binds to IRP-causes IRP to disassociate from transferrin receptor mRNA-exposing sites that are recognized by endonucleases and the mRNA is degraded

epigenetics

-is the study of mechanisms that lead to changes in gene expression that can be passed from cell to cell and are reversible, but do not involve a change in the sequence of DNA -epigenetic inheritance involves epigenetic changes that are passed from parent to offspring--> genomic imprinting

the lacI gene encodes a repressor protein

-making a repressor that can diffuse across in a trans acting fashion and bind to regulatory circuit of the lac operon and control transcription -rare mutant strains of bacteria with abnormal lactose adaptation --->produced large amounts of enzymes and didn't matter whether or not lactose was present (substrate enzyme adaptation) -one type of mutant involved a defect in the LacI gene --->LacI-(negative) ----->resulted in constitutive expression of lac operon even in the absence of lactose (get transcription of lac operon all the time bc you broke repressor) ---->correct explanation: encodes a repressor protein (diffusible protein); the lacI- eliminates the function of the lac repressor (breaks repressor and cant bind to operator)-then make polycistronic mRNA ---->internal activator hypothesis-mutation caused an appearance of activator-internal activator-which drives transcription-the lacI- mutation results in the synthesis of an internal activator

epigenetics and environmental agents `

-many environmental agents have been shown to cause epigenetic changes -include dietary effects as well as toxins -->dietary effects on the agouti gene in mice (how diet can affect methylation status of promoter) -->toxins contribute to cancer

DNA methylation is heritable

-methylated DNA sequences are inherited during cell division -explains genomic imprinting -->specific genes are methylated in gametes from mother or father -->pattern of one copy of the gene being methylated and the other not is maintained in the offspring/daughter cells -De novo methylation is an infrequent and highly regulated event-marks are being made from scratch

genomic imprinting occurs during gamete formation

-methylation inhibits the binding of a protein = CTC-binding factor-which allows the IgF2 gene to be stimulated by a near enhancer -CTC-binding factor binds to the unmethylated gene and inhibits transcription by forming a loop -->after fertilization-non methylated maternal DMR recruits in transcription factor CTC-binds prevents it from being transcribed through the formation of a DNA loop structure; IgF2 is not stimulated by the enhancer --->through methylation of the paternal chrm CTC factors cannot bind so there is no DNA looping and transcription occurs; methylation prevents the binding of CTC factors and IgF2 can be stimulated by the enhancer

basal transcription machinery: TFIID and Mediator

-most regulatory TFs do not bind directly to RNA pol 1. TFIID-direct or through coactivators 2. mediator 3. recruiting proteins that affect nucleosome composition-recruitment of proteins that can affect nucleosome

effect of acetylation

-normally the amino acid R group of both lysine and arginine have a positive charge -positive tails-closed compaction state =no transcription -Histone acetyltransferase (HAT) enzyme transfers and adds an acetyl group to lysine of arginine -once acetylated DNA is less tightly bound to the histone proteins-you lose the positive charge so there is no more electrostatic interaction so it loosens=transcription

nucleic acid units

-nucleoside = base + sugar (no phosphate) --> adenine + ribose = adenosine -->guanine + ribose = guanosine -->cytosine + ribose = cytidine -->uracil + ribose = uridine --> adenine + deoxyribose = deoxyadenosine -->guanine + deoxyribose = deoxyguanosine --> thymine + deoxy= deoxythymidine -->cytosine + deoxy= deoxycytidine -nucleotide = base + sugar +phosphate(s) -->adenosine monophosphate/diphosphate/triphosphate

nucleotide structure

-nucleotide-repeating structural unit of DNA and RNA -three components: 1. phosphate group 2. pentose sugar -->ribose in RNA -->deoxyribose in DNA 3. nitrogenous base -->purines=double ring structure -adenine & guanine -->pyrimidine = single-ring structure = thymine, cytosine, and uracil -2' H = DNA -2' OH = RNA -3'OH group is important for allowing nucleotides to form covalent linkages with each other -5'C-phosphate attachment -1'C= base attachment -cytosine can undergo methylation-add 5' Ch3 (5-methyl cytosine)-deamination = thymine

structure of DNA strand

-nucleotides are covalently linked together by phosphodiester bonds-phosphate connected the 5' carbon of one nucleotide to the 3' carbon of another -->strand has directionality = 5' to 3' -the phosphates and sugar molecules form the backbone of the nucleic acid strand-bases project from the backbone (negatively charged due to negative charge on each phosphate) -phosphate group connects two sugar molecules via ester bonds

DNA and RNA structure

-nucleotides form the repeating unit of nucleic acids -nucleotides are linked to form a linear strand of RNA or DNA -Two strands (either DNA or RNA) can interact to form a double helix the 3-D structure of DNA results from folding and bending of the double helix-interaction of DNA with proteins produces chrms within living cells

x chromosome inactivation (XCI)

-occurs during embryogenesis in female mammals (entire chrm gets shut down -sperm with (X) and egg (X) fertilize and form a female-undergoes mitosis-embryonic stage =X inactivation in either the X chrm from the mom or father --->inactivates mom chrm and expresses genes on dad chrm-all descendants have inactivated maternal x chrm -in a specific part of the female body-ex. arm --->inactivates dad chrm and expresses gene on mom chrm-all descendants will have wadded up paternal x chrms in another part of the female's body -ex. leg ------>heterozygous = mosaic -all chrms have a X chrm inactivation center (XIC)-a piece of gDNA on the chrm-in this region there are two types of genes: 1)Xist 2)Tsix-early on before there is an x chrm inactivation it is being expressed/and making a transcript=ncRNA --->both run in opposite directions

regulation of the lac operon

-operon-cluster of genes(grouped together)-under the control of a single promoter-in bacteria only (eukaryotes don't have operons-gives every gene its own promoter) -treat bacterial cell with a specific substrate-enzyme appeared -enzyme appearing would activate gene transcription = enzyme adaptation -sense presence of substrate-cells were adapting to the presence of substrate causing enzyme levels to up -lac operon-lactose -dissacharide-2 individual sugars linked together-harness energy -operon-regulatory unit consisting of a few structural genes under the control of one promoter -->an operon encodes a polycistronic mRNA-contains the coding sequence for two or more structural genes -->allows bacterium to coordinately regulate a group of genes that encode proteins with a common functional goal -3 genes under the control of the same promoter-makes 3 different proteins involved in the same function

silencing by polycomb group complexes

-polycomb repressor element (PRE) on the double stranded DNA free of nucleosomes-for the protein complex to bind -the PRE binding protein binds to the PRE and recruits PRC2 to the site -PRC2 catalyzes the attachment of three methyl groups onto lysine 27 of histone H3 (in some cases the PRE and target gene may be far away and interact via DNA looping) -the trimethylation of lysine 27 (H#K27Me3) directly inhibit transcription by preventing the binding of RNA polymerase; trimethylation may recruit PRC1 to the target gene (trimethylation of N-terminus of histones-recruit in PRC1) -PRC2 gets recruited and does modification to the H2K27Me3-recruits PRC1-recruits chromatin remodelers, block TFIID from binding to promoter, DNA methyltransferase

regulatory transcription factors

-proteins that influence the ability of RNA polymerase to transcribe a given gene (influence RNA polymerase II to transcribe or not) -two main types: 1. general transcription factors/basal -helps recruit RNA polymerase II so it can find the promoters so that we can assemble an initiation complex for transcription -closed to open complex -required for the binding of the RNA pol to the core promoter and its progression to the elongation stage; necessary for basal transcription -ex. TFIID (have TATA box binding protein TBP as a subunit), TFIIF, TFIIH(kinase and helicase activity-involved in hyperphosphorylation of RNA pol II 2. regulatory transcription factors -enhancer binding proteins; proteins that could bind to silencers-regulating process of transcription -serves to regulate the rate of transcription of target genes -influence the ability for RNA pol II to begin transcription

non structural genes

-rRNA genes (ribosomal RNA-give rise to ribosomal RNA) -tRNA genes -microRNA genes-regulate gene expression -SnRNA-RNAs put into SNRPS making spliceosomes

regulatory transcription factors

-recognize cis regulatory elements (enhancers, promoters, silencers that are on the same chromosomal strand that will be transcribed) -activator-increases the rate of transcription-binds to enhancer -->enhancers --->can be upstream or downstream of gene/promoter --->function at large distances from the promoter --->orientation independent --->boost rate of transcriptional initiation by 1000 fold --->cell type specific --->bind a regulatory protein (enhancer binding protein-activator) -gene activation-RNA transcription is increased -repressor-decreases rate of transcription-binds to silencer -->gene repression-don't have basal transcription machinery-RNA transcription is inhibited -methods to look at DNA binding --> Gel-shift assay -->DNase I footprinting

mediator

-regulator sequence -works along side RNA polymerase II to help elevate transcription -has kinase activity-phosphorylate RNA pol II in the CTD region= boost in transcription -assist TFIIH in phosphorylation

combinatorial control

-regulatory proteins may alter nucleosomes near the promoter -DNA methylation may inhibit transcription -->prevent binding of an activator protein -->recruiting proteins that compact chromatin -various combinations of these factors can contribute to the regulation of a single gene -transcription complexes put together in different combinations to regulate more genes

effector molecules

-small molecules that can bind to repressors or activators to modulate its activity -->bind to regulatory proteins but not to DNA directly -inducers=small effector molecules that increase transcription -->bind to activators and cause them to bind to DNA; without the inducer the activator would not have affinity to bind to the DNA -->bind to repressors and prevent them from binding to DNA; binds to repressor and causes a conformational change-causes the repressor to no longer have affinity for DNA-allows for transcription to occur -genes regulated by this = inducible genes -small effector molecules may inhibit transcription -->corepressors bind to repressors and cause them to bind to DNA -->inhibitors bind to activators and prevent them from binding to DNA -genes regulated by this = repressible genes -Inducible system (off to on): in the absence of the inducer-the repressor protein blocks transcription; in the presence of the inducer causes a conformational change that inhibits the ability of the repressor protein to bind to the DNA-transcription occurs -Inducible system: the activator protein cannot bind to the DNA without an inducer present-when bound activator binds to the DNA and transcription occurs -repressible system (on to off):in the absence of a corepressor-the repressor protein will not bind to the DNA-transcription occurs; when corepressor is bound to the repressor there is a conformational change that allows the repressor to bind to the DNA and inhibit transcription -repressible: the activator protein will bind to the DNA without the aid of an effector molecule;' the presence of an inhibitor causes a conformational change that inhibits the ability of protein to bind to DNA

transformation

-something from the dead type S bacteria was transforming type R bacteria into type S -the substance that allowed this to happen is the transforming principle

histone variants

-standard histones: H1, H2, H2B, H3, and H4 -there are 70 histone genes and a few of them have accumulated mutations that have altered the base sequence=histone variants -DNA translocases insert variants Variant-H3.3; Standard H3 -->open chromatin state=gene activation/transcription Variant-H1*; standard-H1 -->involved in compaction-closed chromatin structure=gene repression=no transcription Variant- macroH2A; standard-H2A -->found in inactivated X chrm in females=closed configuration variant-H2A.Bbd; standard-H2A -->promotes open complex=gene activation

CREB protein

-steroid hormones using peptides that travel in the blood cant freely cross the membrane-have to signal the cell in a different way -CREB (cAMP response element binding)-another regulatory TF -CREB becomes activated in response to cell-signaling molecules that cause an increase in the cytoplasmic concentration of cAMP (cyclic adenosine monophosphate) --> CREB protein recognizes a response element with the sequence 5'TGACGTCA-3'=cAMP response element (CRE) -1st messenger-peptide cant freely cross the membrane -it will need to bind to a receptor embedded in the membrane -activates a G protein that is embedded in the membrane-has a GTP bound to make it active -then it binds to adenylyl cyclase-uses ATP and converts it into cAMP that serves as a second messenger -cAMP binds to protein kinase A that is dependent of cAMP to become active -this activates kinase activity-phosphorylating CREB once inside the nucleus -once phosphorylated it recruits in coactivator and helps drive transcription --->CBP protein dimer=coactivator binds to phosphorylated CREB protein dimer that is sitting on CRE -cAMP acts as a second messenger-activates protein kinase A -phosphorylated CREB binds to DNA and stimulates transcription -->unphosphorylated CREB can bind to DNA but cannot activate RNA pol -

effects of regulatory transcription factors on TFIID

-the activator/coactivator complex recruits TFIID to the core promoter and/or activates its function-transcription will be enhanced; transcriptional activation via TFIID -transcriptional activation via TFIID -coactivator doesn't contact DNA-helps in activation but does not directly contact DNA-but they do interact with regulatory proteins and also general transcription factors (TFIID)-have strong transactivation domain-helping in activation of transcription -the repressor protein inhabits the binding of TFIID to the core promoter or inhabits its function-transcription is silenced-transcription repression via TFIID -repressor protein binding to the silencer and it is blocking the TATA box-so TFIID cannot bind-and cant recruit the rest of the machinery to have transcription occur

formation of stem loops

-the first gene in the trp operon is trpL-encodes leader peptide -the formation of 3-4 stem loop causes RNA pol to pause-allowing the U-rich sequence to dissociate from DNA -->conditions that favor the formation of the 3-4 stem loop rely on the translation of the trpL mRNA 1. no translation 2. low levels of tryptophan 3. high levels of tryptophan

Features of regulatory TFs: domains and motif

-transcription factor proteins contain regions called domains-have specific functions -->one domain could be for DNA-binding -->another could provide a binding site for effector molecules -->allow for protein to protein interaction/dimerization -->activation of transcription=activation domain-interacts with basal transcription machinery -motif is a domain-or a portion of a domain-very similar structure in many different proteins

the lac operon is also regulated by an activator protein

-transcriptionally regulated in a second way-catabolite repression -the bacterial cell will use glucose first and prevent the use of lactose; once glucose is removed catabolite repression no longer occurs and the lac operon is expressed -sequential use of two sugars by a bacterium=diauxic growth -small effector molecule in catabolite repression is cyclic AMP (cAMP) -->it is produced from ATP via the enzyme adenylyl cyclase -->cAMP binds an activator protein = Catabolite Activator Protein (CAP) glucose sugar-> taken up by bacterial cell->adenylyl cyclase enzyme uses ATP to covert it into cAMP; if glucose levels are high it inhibits adenylyl cyclase -high glucose = inhibited adenylyl cyclase (cAMP) -low glucose = increase of cAMP-effects CAP -lactose, no glucose (high cAMP) -->repressor inactive --> cAMP-CAP complex binds to the CAP site near the lac promoter and increases transcription -no lactose or glucose (high cAMP) -->no inducer-with no lactose the repressor is still bound to the operator - transcription is very low due to binding of repressor --->cAMP-CAP complex still bound -lactose and cAMP(low cAMP) -->cells will choose to use glucose-transcription is very low-shouldn't be happening; CAP is nt bound -glucose no lactose (low cAMP) -->CAP protein wont bind -->repressor bound -->transcription is very low due to the lack of CAP binding and by the binding of the repressor

regulation of gene expression: mRNA translation

-translational repressor proteins can bind to the mRNA UTR Shine Dalgarno sequence-and prevent translation from starting -->Shine Dalgarno sequence is important in recruiting the 30S ribosomal subunit-driven by 3' end of 16S RBS -->protein that binds-prevents 30S subunit from binding and blocks translation (prevents ribosome from docking) -riboswitches-special sequences that form in the RNA that block or allow for translation to take place-can produce an mRNA conformation that prevents translation from starting -->stops or makes translation occur faster -->stem loop structure recruits protein-doesn't have shine Dalgarno sequence -antisense RNA can bind to the mRNA and prevent translation from starting -->antisense is complementary-binds to the mRNA and blocks translation

possible stem loop structures formed from trpL mRNA

-when translation is not coupled with transcription, region 1 hydrogen bonds to region 2 and region 3 hydrogen bonds with 4; because of the 3-4 terminator stem loop forms-transcription will be terminated at the U-rich attenuator

transcriptional activation

1) activator 2)activator recruits in DNA translocase (SWI/SNF) 3) DNA translocase recruits in HAT (histone acetyltransferase)-which adds acetyl groups to the N-terminal of histone tail 4)formation of preinitiation complex -->general transcription factors and RNA polymerase II are able to bind to the core promoter and form a preinitiation complex -acetylation spreads-loosens chromatin structure 5) elongation -->histones ahead of the open complex are covalently modified by acetylation and evicted -->behind the open complex-histones are deacetylated and become tightly bound to the DNA (after transcription they are deacetylated

lac operon has three operator sites for lac repressor

1. O1 next to the promoter 2. O2-downstream in the lacZ coding region 3. O3-slightly upstream of promoter -each operator will bind a dimer-need two operator sequences to be intact-so you can form 2 dimers or a tetramer -the lac repressor must bind to two of the three operators to cause repression -->it can bind to O1 &O2 or to O1 & O3 ---->but not O2 &O3 ------->if either O2 or O3 are missing-maximal repression is not achieved -binding of the lac repressor to two operator sites requires the DNA to form a loop -->a loop in the DNA brings the operator sites closer together-facilitates the binding of the repressor protein -if you have all 3 operators=maximum repression=1300 fold

basal transcription factors

1. TFIID: -works well with RNA polymerase II -complex capable of recognizing the TATA box -composed of TATA-binding protein (TBP) 2. TFIIB: -binds to TFIID and enables RNA polymerase II to bind to the core promoter -promotes TFIIF binding 3. TFIIF: -binds to RNA polymerase II and plays a role in its ability to bind to TFIIB and the core promoter -plays a role in the ability of TFIIE and TFIIH to bind to RNA polymerase II 4. TFIIE: -plays a role in the formation/maintenance of the open complex -facilitates the binding of TFIIH to RNA polymerase II and regulating its activity 5. TFIIH: -multiple subunit -certain subunits act as helicases and promote the formation of the open complex -other subunits phosphorylate the carboxyl terminal domain (CTD) of RNA pol II which releases its interaction with TFIIB-allowing RNA polymerase II to proceed to elongation

two types of competing complexes are key regulators of epigenetic changes during development that produce specific cell types and tissues`

1. Trithorax group (TrxG)-involved in gene activation 2. Polycomb group (PcG)-involved with gene repression -target H3 histone-modification of H3-there is a lysine residue @ position 4 = H3K4Mc3 (trimethylated)=activation/transcription -H3K27Mc3=repression-H3 being marked at different locations control whether it is activated or not -hawk genes are shut down in polycomb group -repressors control anterior/posterior-structure of body plan

Xic: X inactivation center

1. Tsix expresses a transcript ncRNA that docks to both x chrms 2. ncRNA recruits pluripotency TFs on both chrms 3. pluripotency TFs recruit CTC factors -initially they are both expressing the Tsix gene-making Tsix ncRNA -nothing is inactivated yet but when the two x chrms come together -there is a symmetry break -->the two chrms come together with both pluripotency TFs and CTCs assembled -->Xm- the pluripotency TF and CTCs from one chrm jumps onto the other (ex. maternal chrm)=active/expressed --->Xp-left with nothing allows for the expression of Xist-Xist ncRNA will drive compaction and inactivation=targeted for being condensed (barr body) or is inactivated -Xist ncRNA-17000 nucleotides long-incudes repetitive sequences -A,B,C,D,E,F -->most important segment = C -ncRNA docks itself to the chrm using the C segment -during embryogenesis-the X chrms pair up and a symmetry break causes the pluripotency factors to move to one X chrm-which remains active ; the other X chrm expresses the Xist gene -the Xist RNA binds to XIC and then spreads to both ends of the X chrm -Xist RNA recruits proteins to the X chrm that cause it to become more compact and be inactive-some genes on this chrm can be expressed to some degree

how function of regulatory factors can be modulated

1. binding of a small effector molecule -hormone-steroid-binds to TF-causes TF to bind to an enhancer and drive transcription 2. protein-protein interactions -most TFs don't bind as monomers-they bind as dimers or tetramers -if protein conc. within the cell is low=monomer-but as the protein conc. increases you get more molecules that will want to form a dimer (dimerization motif) -HLH loop helix domain-homo dimer-2 protein chains that have similar sequences and theyre coming together using this domain to form a dimer--> binds to DNA -high conc. of protein=dimer formation and then binding to DNA switch enhancer to drive transcription 3. covalent modification such as phosphorylation -TF complex bound to DNA must be modified-through a phosphorylation even on the R group of one of the amino acids -kinase enzyme responsible for adding the phosphate -after phosphorylation it might recruit another protein CBP which acts a coactivator-which helps recruit in another transcription factor -CRE protein/TF can bind to it but needs to first recruit CBP protein coactivator-coactivators don't bind directly but they are recruited in and have a strong trans activation domains that drive transcription -by phosphorylating-it brings in the coactivator CBP(cyclic AMP)-then creates this CREB binding protein=transcription --->CREB-cAMP response element binding protein TF

Gene regulation is necessary to ensure

1. expression of genes in an accurate pattern during the various developmental stages of the life cycle 2. differences among distinct cell types -->nerve and muscle cells look so different because of gene regulation rather than differences in DNA content -->46 chrms-genes scattered across the chrms-some genes are not expressed in diff. tissues-genes can be activated or repressed-environmental cues

testing/achieving the goal

1. grow mutant strain and merozygote strain separate 2. divide each strain into two tubes 3. to one of the two tubes add lactose 4. incubate the cells long enough to allow lac operon induction 5. lyse the cells with a sonicator-allows B-galactosidase to escape from the cells 6. add B-o-nitrophenylgalactoside (B-ONPG)-colorless compound -->of B-galactosidase is present-will cleave the compound to produce galactose and o-nitrophenol (O-NP) ---> O-nitrophenol has a yellow color-the deeper the yellow color the more B-galactosidase was produced 7. incubate the sonicated cells to allow B-galactosidase time to cleave B-o-nitrophenylgalactoside 8. measure the yellow color produced with a spectrophotometer -mutant (haploid)= I-P+O+Z+A+ -->if repressor or activator is broken it cannot bind to operator =will always transcribe=yellow -merazygote= I-P+O+Z+Y+A+/I+P+O+Z+Y+A+ --> in the absence of lactose-both lac operons are repressed <1% -->in the presence of lactose-both lac operons are induced-higher level of enzyme activity 220% interaction between regulatory proteins and DNA sequences have led to two def: 1. trans-effect -->genetic regulation that can occur even though DNA segments are not physically adjacent -->mediated by genes that encode regulatory proteins -->ex. the action of lac repressor on the lac operon 2. CIs-effect -->DNA sequence that must be adjacent to the so it regulates -->mediated by sequences that bind regulatory proteins -->ex. lac operator -------->Cis-acting- promoter, operator cant control the F factor -can only control same chrm ; Trans-acting-tetramer can diffuse across and effect expression on the F factor

identification of DNA as the genetic material criteria

1. information: it must contain the information necessary to make an entire organism-genetic material must contain the information necessary to construct an entire organism; provide the blueprint for determining the inherited traits of an organism 2. transmission: it must be passed from parent to offspring during reproduction 3. replication-it must be copied-in order to be passed from parent to offspring 4. variation-must be capable of changes-to account for the known phenotypic variation in each species

Targeting a gene for epigenetic modification by a noncoding RNA

1. ncRNA recognizes specific gene sequences (on double stranded) and binds to them -ncRNAs recruits DNA methyltransferase (signal) 2.ncRNA recruits other proteins such as histone modifying enzymes and DNA methyltransferase-this leads to changes in chromatin structure and/or DNA methylation 3. base sequence doesn't change but its gets modified (marked)-alter the expression of this gene and are maintained -ncRNA has the ability to hover over the double-stranded DNA-RNA gets in = triplex structure (docked)

epigenetic changes may be targeted to specific genes `

1. transcription factor recognizes specific gene sequences and binds to them 2. transcription factor=enhancer binds-the transcription factor recruits other proteins-such as histone modifying enzymes and DNA methyltransferase-this leads to changes in chromatin structure and/or DNA methylation 3. Undergoes methylation-prevents TF from binding-These changes alter the expression of this gene and are maintained in subsequent cell divisions --->DNA methyltransferase-enzymes that adds the methyl group on cytosine

merozygotes are partially diploid

1. two lacI genes in a merozygote may be different alleles --->lacI-(minus)-on the chrm --->lacI+: on the F' factor 2. genes on the F' factor are not physically connected to those on the bacterial chrm -if hypothesis 1 is correct-the inducer protein produced from the chrm can diffuse and activate the lac operon on the F' factor -if hypothesis 2 is correct-the repressor from the F' factor can diffuse and turn off the lac operon on the bacterial chrm -the lac I- (minus) either 1. results in the synthesis of an internal inducer 2. eliminates the function of a lac repressor that can diffuse throughout the cell

cycle of lac operon induction and repression

1. when lactose becomes available-small amount of it is taken up and converted to allolactose by B-galactosidase; the allolactose binds to the repressor-causing it to fall off the operator site 2. lac operon proteins are synthesized; this promotes the efficient uptake and metabolism of lactose 3. lactose is depleted; allolactose levels decrease; allolactose I released from the repressor-allowing it to bind to the operator site 4. most protein involved with lactose utilization are degraded -measure how much B-gal is present using western blot-taking cells after treatment with allolactose; use antibody against b-gal and run a western blot

Griffith experiments with streptococcus pneumoniae (bacterium)

S. pneumoniae comes in two strains -type S= Smooth -->secretes a polysaccharide sugar capsule-protects bacterium from the immune system of the animals -->when streaked onto petri plates containing a solid growth medium-capsule strains have a smooth colony-produce smooth colonies on solid media -type R= Rough --> unable to secrete a capsule -->produce colonies with a rough appearance -->none capsulated(rough)-destroyed the animal's immune system 1. inject mouse with live type S bacteria -->mouse died & type S bacteria was recovered from the mouse's blood 2. Inject mouse with live type R bacteria -->mouse survived & no living bacteria was isolated from the mouse's blood 3. inject mouse with heat-killed type S bacteria -->mouse survived and no living bacteria isolated from the mouse's blood 4. inject mouse with live type R + heat-killed type S cells -->mouse died and type S bacteria recovered from the mouse's blood; because living R bacteria alont cannot kill the mouse-something from the dead type S bacteria was transforming the type R bacteria into type S-transformation= substance causeing the transformation=transformation principle -------->the transformed bacteria got the info needed to make a capsule; variation exists in the ability to create a capsule& to kill the mouse; the genetic material necessary to create the capsule is replicated so it can be transmitted from mother to daughter

Chromatin Immunoprecipitation sequencing (ChiP-Seq)-DNA sequencing

allows determination of: -location of nucleosomes -where histone variants are found (look at specific regions for variants) -where covalent modification of histones occur -see if gene x has the variant H3.3 (indication of open chromatin state=active transcription) Take cell: 1. treat with chemical formaldehyde=chemical crosslinker-crosslinks proteins to DNA -->goes into nucleus-if there are nucleosomes found on the chrms it will form a covalent bond b/w the histone proteins and DNA-lock them onto the DNA so you don't get diffusion 2. break open cell with detergent 3. break gDNA into small pieces -->150bp pieces using sonication or enzyme micrococcal nuclease -->you have complete genome with pieces of gene x -->need to find pieces of DNA that has histone H3.3 4. use an antibody (immunoprecipitation) that is specific to protein : anti-H3.3 antibody -->mix antibody that recognizes H3.3 and will bind to it 5. add sugar bead that has protein A -->protein A captures all antibodies that have H3.3 bound 6. centrifuge -->pellet-sugar beads are heavy so they will sink to the bottom-have antibody and H3.3 bound ---->still don't know if the DNA fragments are from gene X -->supernatant-includes other nucleosomes that are not H3.3 7. wash beads -->reverse crosslinking step-removes the histones H3.3 off of the DNA --> keep the DNA that was bound to H3.3 and perform blunt end blunt end ligation ----->ligate linkers onto the ends of precipitated DNA molecule-know the DNA sequence-can amplify by docking F and R primer-then carrying out PCR to amplify the copy number of the CHIP pieces of DNA -------->then carry out DNA sequencing-search for pieces of DNA that correspond to gene x

regulation of gene expression

gene 1. transcription -regulatory transcription factor can activate or inhibit transcription -the arrangements and composition of nucleosomes influence transcription -DNA methylation inhibits transcription -insulators confine gene regulation to a specific gene pre-mRNA 2. RNA processing -alternative splicing alters exon choices -RNA editing alters the base sequence -control when pre-mRNA is converted into a mature mRNA mRNA 3. Translation -small RNAs (miRNAs & siRNAs)-silence the translation of mRNA=RNA interference -proteins that bind to the 5' end of the mRNA regulate translation -mRNA stability may be influenced by RNA-binding proteins protein 4. Post-translational modifications -feedback inhibition and covalent modifications regulate protein function -90% of genes are regulated-majority of regulation occurs at the first step-transcription-regulates mRNA production -after transcription mRNA is exported through nuclear pore and released into the cytoplasm-use the UTR region as a means to regulate translation -->repressor protein-translational repressor can bind and block translation -if translation occurs-proteins may not be active-need post translational modifications (amino acid needs to be modified in order for the protein to be activated) -->phosphorylation is a type of post-translational modification (protein/enzyme will be phosphorylated by a kinase (adds a phosphate group to the R group) ----->inactive protein-->kinase phosphorylates R group of amino acid=active protein -enhancers -silencers -nucleosomes (affect compaction) -->DNA translocases-enzymes that require ATP to move nucleosomes from DNA to open space for transcription to take place

bacterial conjugation

how can they maintain genetic diversity without sex? -2 haploid bacteria -->F-(minus) cell = female -->F+ cell=male ---->F+ cell has fertility plasmid which allows to make proteins involved in conjugation ---->plasmid F factor (fertility)-make proteins that carry out conjugation-transfer of DNA from one cell to another -Mating -->F-x F+ -->conjugation tube allows passage of DNA -->DNA is being transferred from the F+ strain to the F-(minus) (male to female) ----->TDNA (transfer)-special part of the plasmid F factor gets nicked and broken-DNA is unraveled/unfolds-feeds into the F-(minus) strain-rolling circle replication process (F-(minus) strain gets copied)--> now they are both F+ cells -can integrate the F+ into the host genome -->HFR Strain (high frequency of recombination) -->TDNA segment can get broken and conjugation can occur b/w an HFR strain and one the is F-(minus)-begin to transfer all genes into the F-cell-can undergo recombination into the host genome of the F-cell-recombination can occur at a higher frequency -can excise back out: --->perfectly --->imperfectly: there is host genome with lac open & F' + host genome -F' factor that carries the lacI gene -->bacteria will have two copies of the lacI gene (one on the chrm and the other on the F' factor) ------>merozygote-partially diploid-two copies of the lac operon but host genome only once

structural genes

protein coding genes

molecular genetics

study of DNA structure and function at the molecular level

effects of an activator and a repressor on mediator

the activator protein interacts with mediator-this results in the phosphorylation of the carboxyl-terminal domain of the RNA polymerase-some general transcription factors are released and RNA polymerase proceeds to the elongation phase of transcription = transcriptional activation via mediator -activator protein will be binding and the enhancer protein is further upstream-the mediator is contacting the activator=transcription occurs -through the interaction the mediator can cause the CTD (carboxyl-terminal domain) of RNA polymerase to become hyperphosphorylated -has protein subunits bc the mediator is a big protein complex-can facilitate hyperphosphorylation of CTD on RNA pol II and TFIIH to drive transcription the repressor protein interacts with mediator in a way that prevents the phosphorylation of RNA polymerase -blocks kinase activity=no hyperphosphorylation of CTD domain of RNA pol II -can also have a silencer binding to a repressor protein-stops kinase activity-not able to hyperphosphorylate=closed complex no transcription

the lac operon is regulated by a repressor protein

the lac operon can be transcriptionally regulated 1. by repressor protein 2. by an activator protein -inducible negative control mechanism -->involves lac repressor protein -->inducer is allolactose- it binds to the lac repressor and inactivates it a) no lactose -in the absence of the inducer allolactose-the repressor protein is tightly bound to the operator site-inhibiting the ability of RNA pol to transcribe the lac operon b)lactose present -when allolactose is available-it binds to the repressor-this alters the conformation of the repressor protein which prevents it from binding to operator site-RNA pol can transcribe the operon ---->lac Z-will transcribe B-galactosidase ---->lac Y-lactose permease ---->lac A-galactoside transacetylase --------->all help in the metabolism of lactose

lac operon

two distinct transcriptional units: 1. actual lac operon a) DNA elements: -->promoter-binds RNA polymerase -->operator-binds the repressor protein -->CAP site-binds Catabolite Activator Protein (CAP) ----->CAP protein needs a small effector molecule=cAMP to bind to the CAP side and help RNA polymerase to bind ----->cAMP bound to CAP-bound to the CAP site; RNA polymerase bound-DNA switches =cis acting-transcribe 3 structural genes "Z, Y, A" b)structural genes -->lac Z = encodes B-galactosidase --->enzymatically cleaves lactose and lactose analogues --->also converts lactose to allolactose ------>lactose -> B-gal->allolactose (inducer-small effector molecule that turns on gene) -->lac Y= encodes lactose permease --->membrane protein required for transport of lactose and analogues-transport lactose from outside of the cell to the inside=transport protein-when cell needs more lactose the protein embedded in the membrane helps with the transport -->lac A= encoded galactosidase transacetylase --->covalently modifies lactose and analogues-modifies lactose and adds an acetyl group if glucose is not available for energy you could use lactose instead -->break apart using enzymes: ----->glucose +galactose -------->glucose used in glycolysis for energy 2. the lacI gene -->not considered part of the lac operon -->has its own promoter -->constitutively expressed at fairly low levels -->encodes the lac repressor-functions as a tetramer -->only a small amount of protein is needed to repress the -->lac I gene has a constitutive promoter-always transcribed-translated into a homo tetramer repressor-binds to the operator sequence; when inducer-allolactose- binds to the repressor it comes off the operator and transcription can occur -if you had high levels of both glucose and lactose-you wont be transcribing the lac operon because it will use glucose over lactose; if glucose is around-transcription of the lac operon wouldn't take place; glucose sensing mechanism built in-preference of what sugar will be used -lactose operon=catabolic

epigenetic changes may arise because of mutations in genes that encode chromatin-modifying proteins

what causes chromatin modifications to become abnormal and promote cancer? 1. mutations may occur in genes that encode chromatin modifying proteins -->mutation of DNA methyltransferase 2. environmental agents alter functions of chromatin modifying proteins -ex. tobacco, automobile exhaust -DNA methyltransferases, covalent modification of histones, mutation in genes that encode DNA translocases (move nucleosomes)

dietary effects on the development of female bees

Worker bee vs queen bee -worker bees: -->live a few years -->sterile -->job=clean honeycomb -queen bee -->live several years -->produce of 2000 eggs/day Royal Bee Larva -early developmental stage -microinject with inhibitors that inhibit DNA methyltransferase-turns into queen bee ; want to know how much to inhibit DNA methyltransferase to activate certain gene --->larvae injected with DNA methyltransferase inhibitor became queen bees what makes a bee a worker or queen? -larval developmental stage (female) -soaked in royal jelly->continuously soaked in royal jelly->further fed by nurse bees = Queen ----->nurse bees produce a secretion called royal jelly -if you cut off royal jelly and feel pollen = worker

cancer treatment

inhibitors of DNA methyltransferases-treat certain forms of cancers; decrease in DNA methylation -improvement; lower level of DNA methylation has reversed the inhibition of tumor suppressor genes

Transcriptional Silencing by methylation

a) methylation inhibits the binding of an activator protein -pancreatic cell -->if the CpG island is not methylated the enhancer would recruit an activator and transcription takes place -fat cell -->methyl groups block the binding of an activator protein to an enhancer element ---->CG islands being methylated (putting marks)-prevents activator from binding; methylation inhibits the binding of activator = no transcription b) Methyl-CpG-binding protein recruits other proteins that change the chromatin to a closed conformation -methyl-CpG-binding protein binds to the methylated CpG island -the methyl-CpG-binding protein recruits other proteins-such as histone deacetylase (HDAC)-removes acetyl groups from histones=tightens chromatins structure=shuts down transcription

epigenetics

layer of regulation above regular standard DNA sequence -ways to regulate gene expression without altering base sequence -if you methylate you aren't changing base you are marking it (by methyl transferase enzyme)`

epigenetic regulation: epigenome

describes all modifications in the human genome -ex. methylation of DNA; describes all the marks occurring on the bases (looks at where bases are specifically methylated) -methylation can predict when you die

two general categories of epigenetic gene regulation

developmental change & environment

RNA polymerase II

in bacteria -multiple subununits=holoenzyme

sigma factor

in bacteria help RNA pol II bind to DNA -helix turn helix-recognizes groove helps dock promoter -when you pull the strands apart (closed to open)-and start transcribing-sigma factor disassociates = core enzyme=done with initiation and elongation

Nucleosome arrangements in protein encoding gene

nucleosome free region (NFR)-found at the beginning and end of genes -NFR in the beginning=transcriptional start site at the core promoter NFR at the end=transcriptional termination site

gene regulation

the level of gene expression can vary under different conditions -genes that are unregulated = constitutive -->they have constant levels of expression -->encode proteins that are continuously necessary for the survival of the organism 1. transcriptional regulation -->DNA to mRNA 2. translational regulation -->mRNA to protein 3. Post-translational regulation -->protein modified -->protein is there but needs modification to be active-phosphorylated to become active

transcriptional regulation

two main types of regulatory proteins: 1. repressors-bind to DNA and inhibit transcription 2. activators-bind to DNA and increase transcription -negative control-transcriptional regulation by repressor proteins -->tetramer repressor blocks transcription by binding to operator and blocks RNA pol ability to bind for transcription to occur -positive control-regulation by activator proteins -->protein activator binds with RNA pol to work together to drive transcription `