Bacterial and Organellar Genetics Exam Prep PDF

Lecture L: Bacterial and Organellar Genetics -​ Tree of Life image shown: what domain is missing? Archaea → three domains are Bacteria, Archaea, and Eukaryotes -​ Identifying (pre-molecular) -​ Eukaryotes: easily observable traits (in non microscopic ones) -​ Bacteria...

Lecture L: Bacterial and Organellar Genetics -​ Tree of Life image shown: what domain is missing? Archaea → three domains are Bacteria, Archaea, and Eukaryotes -​ Identifying (pre-molecular) -​ Eukaryotes: easily observable traits (in non microscopic ones) -​ Bacteria: mutations that affect growth -​ Chromosomes -​ Eukaryotes: multiple, paired (in diploids), linear -​ Bacteria: single, circular -​ Number of copies of each gene -​ Eukaryotes: 2 (in diploids) -​ Bacteria: 1 -​ Recombination: -​ Eukaryotes: sex, meiosis, segregation, independent assortment, crossing over -​ Bacteria: conjugation, transformation, transduction -​ Mapping: -​ Eukaryotes: frequency of crossovers -​ Bacteria: interrupted mating cotransformation, frequency -​ Bacteria are very successful -​ Highly variable: size/structure, habitat, metabolism -​ Bacteria differ from eukaryotes in several ways -​ Chromosomes, location of DNA, lack of membrane bound organelles, DNA tertiary structure, recombination, concept of “species” is more vague… individuals within the same species may show high variability in genes they possess -​ Bacteria provided key insights into genetics -​ DNA as genetic material, DNA replication/transcription/translation, gene regulation, recombinant DNA technology -​ Has common ancestor with eukaryotes so have much in common… but several details are diff -​ In the lab bacteria can be grown in liquid culture or colonies on agar plates -​ Is this how bacteria grow in the wild? → biofilms = groups of microorganisms that adhere to a surface usually embedded in a secreted matrix; heterogeneous population of cells -​ Escherichia coli is the standard bacterium used in many lab studies -​ Naturally found in the guts of mammals and birds -​ Usually harmless, but pathogenic strains -​ A pathogenic strain of E.coli -​ E.coli in our intestines: suppresses harmful bacteria, synthesizes vitamins -​ Strain O157:H7 causes gastroenteritis… why? -​ Wild-type v O157:H7 → share 3,574 genes -​ Standard lab E.coli has 528 genes not found in O157:H7 -​ O157:H7 has 1,387 genes not found in standard lab E.coli: some of those encode toxins (Shigs) that cause severe damage to the lining of the intestine -​ Genes in bacteria were identified by mutations that modify growth in some way -​ Identification of genes requires some variation in identifiable inherited traits -​ Colony morphology: visible phenotype: large/small, shiny/dull, round/regular -​ Antibiotic-resistance or bacteriophage resistant mutants can grow in the presence of a growth inhibitor or killer e.g. ampicillin, streptomycin -​ Nutritional mutants -​ Prototrophs: wild-type that can grow in the absence of chemical  -​ Auxotroph: cannot grow in the absence of a chemical, eg. methionine auxotroph need methionine -​ Carbon source mutants -​ Cannot grow on a particular course of carbon, usually a type of sugar -​ Conjugation: experiments demonstrate transfer of genetic material between bacteria involving physical contact (Lederberg and Tatum) -​ Transference of genetic material from one strain to another -​ B-M-P+T+: need biotin and methionine -​ B+M+P-T-: need proline and threonine -​ B+M+P+T+: grown on minimal plates -​ Davis U-tube experiment: is physical contact necessary for conjugation? -​ No cells grow on minimal media, physical contact is required for this transference -​ “Physical contact between bacterial cells is necessary for conjugation to occur because the genetic material could not cross the filter without direct contact.” -​ Conjugation is the process by which bacteria transfer genetic material to other bacteria by cell to cell contact -​ Genetics of conjugation: the F factor -​ Only some strains can act as donors - contain F factor -​ F+ strains have sex pili that mediate cell to cell contact -​ One cell acts as the donor, the other as recipient -​ F factor is a plasmid -​ Plasmids are minichromosomes that replicate independently of the main bacterial chromosome -​ Contain a variety of different genes, usually not required for normal growth -​ May contain genes required for growth in extreme conditions -​ Or antibiotic resistance genes -​ May be multiple copies inside a cell -​ Very useful in recombinant DNA technology -​ Transfer of the F factor -​ F factor plasmid contains: genes for making pili and transferring DNA, genes involved in replication, IS elements (transposable elements) 1.​ F pilus binds to F- cell wall 2.​ Pilus retracts and cells are drawn together 3.​ Gene transfer 4.​ After conjugation both cells are now F+ and can conjugate with other F- cells -​ Rolling circle replication of F plasmid, ssDNA transferred -​ F+ strains can only transfer the F plasmid and its genes to a recipient F- cell -​ F plasmid can integrate into the main bacterial chromosome creating Hfr strains -​ The E.coli chromosome contains IS transposable elements -​ Insertion sequence (IS) elements (IS1, IS2, IS3, etc.): similar to eukaryotic transposons: inverted repeat flanking transposase, number and position vary from strain to strain, number and position vary from strain to strain -​ Recombination between IS elements in F plasmid and the same element in main chromosome results in integration of plasmid -​ F plasmid can integrate at different sites and in either orientation -​ Hfr strains can transfer bacterial chromosome to an F- cell, maybe exchanging genes with the recipient -​ F pilus connects Hfr cell to F- cell -​ Single strand of integrated F plasmid is cut -​ Hfr chromosome replicates itself as transfer proceeds -​ F plasmid followed by chromosomal DNA passes into recipient  -​ Donor DNA is replicated by host -​ Donor DNA is integrated into host chromosome -​ Crossovers between homologous regions on donor and recipient DNA -​ Recipient carries part of F plasmid and some of Hfr genome plus its own F- genome -​ Recipient remains F- as not all of the F plasmid will be transferred -​ DNA not in main chromosomes is degraded by nucleases -​ Hfr strains can be used to map genes in bacteria using the interrupted mating technique -​ Wolman and Jacob: Hfr x F- -​ Hfr genotype: T+ L+ Azs T1s lac+ gal+ strs -​ F- genotype: T- L- Azr T1r lac- gal- strr -​ T+: able to synthesize threonine = minimal media -​ L+: able to synthesize leucine = minimal media -​ Azs: sensitive to killing by azide = presence of azide -​ T1s: sensitive to infection by phage T1 = presence of T1 phage -​ Lac+: able to metabolize lactose = lactose as only sugar -​ Gal+: able to metabolize galactose = galactose as only sugar -​ Strs: sensitive to killing by streptomycin = presence of streptomycin -​ Test whether allele from Hfr strain has replaced that in F- strain -​ Why do you need the donor to be strs and the recipient to be strr? → strs needed to select against Hfr cells -​ Interrupted mating involves initiating chromosomal transfer from Hfr strain and then stopping transfer at time after → slide 15 explain?? -​ Mapping using Hfr strains -​ Time of entry = estimated time gene is first transferred after conjugation is initiated -​ Order of genes on the chromosome -​ Genetic distance in minutes -​ Only part of the donor chromosome is transferred (conjugation does not last long enough for more) -​ Use different Hfr strains: combine maps -​ Allele exchange following transfer of DNA from Hfr strain to F- strain is through crossing over -​ Fragment transferred by conjugation onto host chromosome -​ Double crossover results in recombinant -​ A genetic map of E.coli chromosome → circular, 100 minutes -​ Hfr sequence location in different strains → first gene to be transferred is behind the arrowhead -​ TOPHAT QUESTION: In E.coli four Hfr strains donate the following markers, shown in the order donated= Strain 1: M Z X W C, Strain 2: L A N C W, Strain 3: A L B R U, Strain 4: Z M U R B. Draw a map of the E.coli chromosome containing these markers in the correct order and teh location and orientation of each F- factor -​ Gene transfer by transduction in bacteria involves accidental transfer in bacteriophage -​ Normal lytic cycle: cell lyses, releasing phage → phage attaches to host and injects DNA → phage DNA circularizes → New phage DNA and proteins are synthesized and assembled into phage, host DNA is hydrolyzed → cell lyses, releasing phage again -​ Transduction: incorporation of bacterial DNA into phage instead of phage chromosome -​ Donor is his+, IS infected by phage → occasionally, bacterial DNA fragments are packaged in a phage capsid → transducing phage infect new host cells (host is his-) → recombinant bacterium is his+ -​ Cotransduction frequency = transduction frequency of two genes together compared to each separately -​ Genes that are located close to each other will be cotransduced at a higher frequency than those located further apart -​ Genes far apart are never cotransduced, genes close together may be cotransduced but not always -​ Unrelated but there is a limit to how much DNA will fit into phage head -​ Genes can be mapped by cotransduction frequency -​ Cotransduction frequency = (# of transducing phage containing both genes)/(# of transducing phage containing one of the genes) -​ d=distance between genes, L=size of chromosome piece (minutes) in phage (2 for phage P1) -​ Cotransduction frequency = (1-d/L)^3 -​ d=2-2(cotransduction frequency)^(⅓) -​ Only genes that are close together can be mapped - within 2 mpa units for P1 (115kb) -​ Cotransduction frequency produces finer maps than interrupted mating with Hfr strains -​ TOPHAT: To map the genes arg1, trp2 and ser3 in E.coli by transduction, cells that were arg1+, trp2+, ser3+ were infected with P1 phage, the lysate was collected and used to infect cells that were mutant for all three genes. The infected cells were plated on minimal media containing tryptophan and serine. 100 colonies were recovered and each colony was then streaked separately on plates containing only tryptophan or only serine with the following results: Only tryptophan: 29 colonies, Only serine: 0 colonies -​ (a) What is the distance between the arg1 and the ser3 genes? -​ (b) What is the distance between the arg1 and the trp2 genes? -​ TOPHAT INFO CONT. Only tryptophan = cotransduced ser3 and arg1, only serine = did not cotransduce trp2 and arg1 -​ (a) 100 phage transduced the arg1 gene, 29 phage transduced the arg1 and ser3 genes -​ Cotransduction frequency equation : 29/100 = 0.29 -​ d= 2-2(0.29)^(⅓) = 2-2(0.66) = 0.68 minutes apart for the arg1 and the ser3 -​ (b) the trp2 gene must be more than 2 minutes away from the arg1 as no phage containing both genes were obtained -​ Genes can also be transferred by transformation: uptake of DNA from the environment derived from dead bacteria -​ Movement of donor DNA molecules across the cell membrane and into the cytoplasm of recipient bacteria is an active energy requiring process → occurs only in some bacteria -​ Evolution of bacteria through horizontal gene transfer -​ Vertical gene transfer: genes passed down from one generation to next through sexual or asexual reproduction, each gene can be traced back through a lineage to an ancestral species -​ Horizontal gene transfer: genes acquired from source other than common ancestor -​ Occurs how? By transformation? Transduction? Conjugation? -​ Very common in bacteria -​ Genetic analysis in Bacteria  -​ Forward genetics: from mutation to identifying the gene disrupted, rescue using plasmid libraries -​ Making forward genetics easier: tag mutations molecularly, mutagenesis with TEs -​ Reverse genetics: determining function of genes identified only by sequence, gene knockouts by direct targeting -​ Using genomic libraries to “clone” genes -​ Clone = isolate DNA corresponding to a gene previously identified only by a mutation = forward genetics; from mutation to DNA -​ Goal: to identify the gene mutated in arg- -​ Transform mutant with genomic library -​ Cells acquire a single clone = small region of the genome -​ If that region contains the arg+ gene, they will grow on minimal media -​ Today could possibly just sequence whole genome to identify point mutant -​ Molecularly ‘tagging’ mutants with transposons -​ Identifying point mutations is difficult -​ Large insertions such as transposon in genes are much easier to identify in a genome -​ Recombinant mariner transposon and mariner transposase gene introduced to E. coli chromosome on a plasmid lacking replication origin -​ Select for kanamycin resistance and mutant phenotype of interest -​ Recombinant mariner transposon hopped into E. coli chromosome in gene of interest -​ Isolate genomic region containing transposon. E.g. make genomic library, screen library with DNA from the Mariner transposon -​ Identify genes in region -​ Using homologous recombination to knock out a gene -​ Reverse genetics: generate a mutation by targeting a gene based on its sequence and then characterize the mutant phenotype to determine its function -​ Have DNA sequence of a gene but do not know its function -​ What function does gene X have -​ Transform plasmid: amp gene flanked by same sequences (L and R) that flank gene X -​ Homologous recombination: replies gene X with amp -​ Select for this event on amp plates -​ Characterize the phenotype of bacteria carrying this mutation in gene X -​ Mitochondria and chloroplasts originated from bacteria -​ So how do we know they are related to bacteria? -​ Singular circular chromosome -​ No chromatin/histones -​ Translation initiated with fMet -​ Translation blocked by bacterial inhibitors -​ Sequence comparison confirms relationship with bacteria -​ Genetics of mitochondria and chloroplasts -​ Modified genetic code -​ Some genes have moved to the nucleus -​ Are inherited maternally in many organisms (from large gamete… not sperm/pollen) -​ Non-mendelian inheritance of mutations in mitochondrial and chloroplast genes -​ Variable regions used to establish relatedness across generations (Richard III) -​ Several human diseases associated with mitochondrial mutations -​ Solution: remove nucleus from mother’s egg, remove nucleus from donor’s egg, insert mother’s nucleus into donor’s egg Lecture M: Regulation of Gene Expression in Bacteria -​ Differential gene expression  -​ A characteristic of all life forms is that in any particular cell at any given time only some of its genes are expressed: all genes are not expressed all of time -​ What do we mean by ‘expression’? -​ Context dependent… often means ‘ is the gene being transcribed’? -​ Or more correctly could mean ‘is the protein encoded by the gene present in the cell’? -​ Why regulate gene expression? -​ Cost: wasteful to make protein that is not required -​ Transcription and, in particular, translation use a lot of energy -​ Harmful/incongruous: presence of protein would impair cell function -​ E.g. a differentiated cell in a multicellular organism must: (a) express those genes required for it to carry out its activities (b) not express those would impair its ability to do this -​ When are genes turned on and off? -​ In response to the external environment -​ Food source: enzyme required to utilize it should be expressed only when it is present -​ Metabolite: enzymes required for synthesis should only be expressed when unavailable -​ Pathogens: proteins required to fight off infection are needed only when challenged -​ Temporally, to carry specific cellular or organismal programs -​ Mitosis and meiosis: require the precise expression of the correct proteins to orchestrate these activities (e.g. enzymes required for replication) -​ Time of day or year -​ Development of multicellular organisms -​ Generation of different cell types, each expresses a different subset of genes -​ Post-embryonic life: cell proliferation in most tissues is at maintenance levels -​ Deregulated = cancer -​ Response to internal environment -​ Genes expression is modified to carry out programs instigated by hormones -​ Homeostasis, etc. -​ Gene expression can be controlled at any level between transcription of DNA to presence of protein product -​ BACTERIA: gene —transcriptional→ mRNA —translational→ protein —posttranslational→ functional protein -​ EUKARYOTES: gene → pre-mRNA —RNA processing→ mRNA → Protein → functional protein -​ Just because a gene is being transcribed does not mean a protein encoded by it is present in a cell -​ Just because an mRNA is translated does not mean there are significant levels of protein in the cell -​ [Just because a protein is present in a cell does not mean it is active] -​ Gene expression can be constitutive, inducible, or repressible -​ Constitutive: expressed continually → ‘housekeeping’ genes such as tRNAs, rRNAs -​ ‘On and off’ genes: expressed only under certain conditions -​ Inducible: expression is turned on in response to a certain factor -​ Repressible: expression is turned off in response to a certain factor -​ Level of expression can also be modulated -​ Not just on and off, levels can be increased or decreased -​ Applies to constitutive genes as well, might be on, but how high may often be regulated -​ Transcription is regulated by transcription factors -​ Transcription factors (TF) -​ Sequence specific DNA binding proteins: binding site is defined sequence of bases  -​ Bind to genes they regulate…because those genes have binding site -​ Binding sites are usually…but not always…outside of the region transcribed -​ Activators positively influence the ability of RNA polymerase to transcribe the gene -​ Repressors negatively influence the ability of RNA polymerase to transcribe the gene -​ There are many mechanisms by which activators and pressors function -​ So transcription factors usually have several domains -​ DBD: DNA binding domain -​ RegD: Regulatory domain: control activity of protein, may be bound by regulator -​ AD: activation domain or RD: repression domain, or both, or neither -​ (Not every transcription factor will have a RegD) -​ The basics of transcriptional regulation in bacteria: -​ 1. Negative regulation: transcription in the absence of a repressor, no dedicated activator required -​ For some genes, a ‘good’ promoter alone is usually sufficient to recruit to recruit sigma factor/RNA Pol and result in transcription -​ Operator: binding site for repressor, found adjacent the promoter in some genes -​ Repressor bound to operator stops transcription -​ No repressor bound - transcription -​ Repressors function by interfering with sigma factor/RNA polymerase -​ 2. Positive regulation: RNA pol/sigma factor insufficient, dedicated activator required -​ Not all promoters are ‘good’, insufficient to efficiently recruit sigma factor/RNA Pol -​ Transcription from weak promoters can be boosted by activators -​ Activator binding sites are found next to promoters -​ Activators can function by binding to RNA polymerase or sigma factor and so help recruit them to the promoter -​ Transcription factor activity can be regulated -​ Why is an activator or repressor bound to a binding site next to a promoter? -​ Expression of TF is regulated -​ Activity of constitutively expressed TF is regulated -​ Binding of small molecules can cause conformational change and could promote or prevent DNA binding -​ conformational/activity can also modulated by posttranslational modification, e.g. phosphorylation -​ The lac operon is a model system for understanding transcriptional regulation in bacteria and eukaryotes -​ E.coli can utilize lactose - broken down to glucose and galactose by beta-galactosidase -​ Jacob and Monod (Nobel Prize 1965) -​ Identify cells expressing beta-gal using colorless lactose analogs hydrolyzed to colored breakdown products - easily measured -​ When is beta-gal expressed? -​ + lactose (- glucose)> beta-gal expressed -​ - (+ other carbon source) > beta-gal not expressed -​ +lactose/+glucose>beta-gal not expressed -​ Conclusion: beta-gal inducible (by lactose) and repressible (by glucose) -​ Why is beta-gal expressed (or active?) only when lactose is present (and no glucose)? -​ What are two main approaches to address a question like this? -​ Genetics -​ Uncover mutants that cannot utilize lactose, identify and characterize the genes  -​ Uncover mutants that have the enzyme(s) even when lactose is absent, identify and characterize the genes (....binding sites?) -​ Biochemistry -​ Identify what proteins are present only in cells exposed to lactose -​ Determine the function of those proteins and how they are regulated -​ Analyze the function of proteins encoded by genes identified in genetic screens -​ The lacZ and lacY genes were uncovered as mutants unable to hydrolyze lactose -​ lacY encodes Lac Permease required for transport into cell -​ lacZ encodes beta-galatosidase enzyme -​ Proteins (and mRNA) are at very low levels in the absence of lactose -​ If add lactose (low glucose) -1000 fold increase -​ lacP mutants can also not hydrolyze lactose -​ Promoter for lac operon - i.e. binding site for sigma factor, not protein encoding gene -​ lacI and lacO were identified as mutants which had enzymes that could hydrolyze lactose even in its absence -​ Constitutive mutants lacI and lacO -​ lacY and lacZ expressed at high levels even in the absence of lactose -​ Consistent with negative regulation -​ Jacob and Monod -​ lacI or lacO could encode a repressor… or could be the binding site for the repressor -​ Knocking out either would result in transcription of lacY and Z -​ How do you test if one encodes for a protein or for a DNA binding site? Cis/Trans test -​ A cis/trans test requires two copies of a gene -​ cis-acting elements: influence expression of genes only on the same chromosome -​ Binding sites for transcription factors, control expression of adjacent gene -​ Trans-acting factors: influence transcription on same and other chromosome -​ Encode transcription factors (or regulatory RNAs): diffusible, bind to any target in cell -​ Cis/trans test requires mutant and wild-type copies: can wild-type rescue mutant -​ Cis → mutation in binding site:wild-type operator (binding site) has no effect on gene expression on other chromosome -​ Trans → mutation in TF: wild-type repressor (TF) can bind to operator on both chromosomes -​ Cis and trans test can be done in E. coli by making partial diploids using modified F factor plasmid carrying lac operon -​ Wild type cells containing F lac are partial diploids -​ F lac plasmid has the lac operon -​ Construct strains with different lac mutations on either the chromosome or F lac -​ L- mutant: regulated, l encodes the repressor -​ O mutant: still constitutive, o=operator binding site for repressor -​ Binding of allolactose to lacL repressor prevents it binding to DNA -​ No lactose in the environment = repressor binds to operator, blocks RNA polymerase, no expression -​ Lactose in the environment: binding of allolactose to repressor, causes conformational change, cannot bind DNA -​ Some lactose is isomerized to allolactose -​ There is some transcription of the lac operon even with no lactose (but very low) -​ No lactose permase = lactose could not get into cell -​ Also, beta-galactosidase has a second function, isomerizes lactose to allolactose -​ No expression inundated wild-type -​ The lac operon consists of several elements and genes (Lec M Slide 19) -​ Which operon will produce ß-galactosidase even if lactose is absent? Answer either: yes (high levels), or no (includes low/basal levels) and provide an explanation -​ A. lacI+ P+ Oc lacZ- → No (lacZ gene encodes ß-galactosidase and is mutant here) -​ B. lacI+ P+ O+ lacZ+ → No (this is wild-type for all genes) -​ C. lacI- P- Oc lacZ+ → No (promoter is mutant, so no expression even if operator is mutant) -​ D. lacI- P+ O+ lacZ+ → Yes (no repressor so will get expression in the absence of lactose) -​ LacI repressor has multiple domains and this can explain dominant mutations -​ DNA-binding domain: dominant negative mutation = I^(-d) -​ Inducer-binding domain: gain of function mutations = l^s -​ Repressor oligomerizes as a tetramer and has two key domains: DNA binding domain and inducer binding domain -​ L+ wild-type repressor (yellow monomer with the three domains; DNA binding domain DBD, inducer binding domain IBD, multimerization domain -​ Tetramer bound to operator, then allolactose bound to repressor → cannot bind to DNA -​ Binding of allolactose causes a conformational change in the tetramer -​ Simple loss of function mutants in lacI repressor are recessive, but the I^(-d) mutant is dominant -​ Example below are with no lactose -​ 1. I- : simple loss of function mutant → constitutive lac Z expression -​ 2. I-/I+ : introduce wild-type I → lac Z expression regulated -​ 3. I^(-d) : I^(-d) mutant → consitutive lacZ expression -​ 4. I^(-d)/I+ : introduce wild-type I → constitutive lacZ exprsesion, I^(-d) mutant makes protein that interferes with wil-type = dominant negative -​ I^(-d) mutant has a defective DNA binding domain: repressor cannot bind DNA -​ I^(-d) haploid: full lacZ expression in absence of lactose -​ I^(-d)/I+ partial diploid→ wild-type protein will form tetramers with mutant: most mixed tetramers cannot bind DNA, so most wild-type protein are also inactive -​ LacI^s – superrepressed – is a hypermorphic (gain of function) mutant -​ Example is WITH lactose -​ 1. P- : promoter mutant → no lacZ expression -​ 2. P-/P+ : introduce wild-type operon → lacZ expression -​ 3. I^s : I^s mutant → no lacZ expression -​ 4. I^(-d)/I+ : introduce wild-type I → no lacZ expression, I^s mutant still active even in presence of lactose -​ LacI^s has a mutation in the Inducer Binding Domain -​ Mutant protein cannot bind to allolactose so remains bound to DNA even with lactose -​ TOPHAT: Below are some partial diploids for the lac operon. For each of these state whether ß- galactosidase will be detected in the cells (a) in the absence of lactose and (b) in the presence of lactose (assume glucose levels are low). Answer either: yes (high levels) or no (includes low/basal levels). Provide explanation. -​ (i) I+ O+ Z+ Y-/ I+ O+ Z+ Y- -​ (a) NO -​ (B) NO -​ Both copies of the operon are mutant for Y, so no lactose gets imported so repressor on all the time -​ (ii) I+ Oc Z- Y+/ I+ O+ Z+ Y+​ -​ (a) NO -​ (b) YES -​ There is a mutant operator on the left, but the Z is mutant there so no constitutive expression fro that and the operon on the right is normal  -​ (iii) I+ Oc Z+ Y+/ I+ O+ Z- Y+ -​ (a) yes -​ (b) yes -​ Mutant operator on the left, wild-type Z on left so constitutive expression -​ (vi) Is O+ Z+ Y+/ I+ O+ Z- Y+ -​ (a) no -​ (b) no -​ I^s on left, operator is wild-type, so I^s protein will keep that operon off all the time even though there is a wild-type I on the right that is recessive fot eh I^Ss (not a typo) -​ Lac repressor binding to DNA is sufficient to prevent RNA polymerase from binding -​ Actually 3 operators -​ O1 is essential but not sufficient, O2 and O3 are redundant -​ Each tetramer binds O1 and either O2 or O3 → induced a loop in DNA that prevents binding of sigma factor/RNA Pol -​ Repressor does not need a ‘repression domain’: binding alone is sufficient to repress -​ The lac operon is also activated by a positive regulator -​ Catabolite repression: glucose represses the lac operon -​ If E. coli is grown with glucose and lactosee, the lac operon is not transcribed -​ E.coli prefers to use glucose first if available, will only use lactose -​ Transcription of the lac operon requires an activator: CRP-cAMP -​ CRP = cAMP receptor protein (also called CAP) -​ CRP is ubiquitously expressed (expressed everywhere) -​ CRP activity is positively regulated by cAMP (only active in low glucose) -​ Glucose blocks the formation of cAMP -​ ATP can only convert into cAMP is glucose is there to inhibit adenylate cyclase -​ Low glucose - high cAMP -​ High glucose - low cAMP -​ Lac operon has binding site for CRP upstream of promoter -​ CRP bound ,operator free = transcription -​ The lac operon is controlled by lactose and glucose via a negative regulator (Lac repressor) and a positive regulator (CRP-cAMP) -​ Positive regulator (lactose) inhibits activity of repressor -​ Negative regulator (glucose) inhibits activity of activator -​ CRP-cAMP/CAP-cAMP helps RNA polymerase initiate transcription through direct protein-protein contact -​ CRP is needed because the lac promoter is ‘weak’ -​ The sequence at -10, TATGGT, does not match the consensus (TATAAT) -​ Mutate the promoter to consensus: high levels of expression with glucose/ no CRP -​ With both lactose and glucose present -​ Wild-type promoter: -10 = TATGTT -​ UV5 promoter: -10 = TATAAT → sigma factor binds to -10 and -35 region -​ UV5 promoter has RNA Pol -​ The lac operon established a clear understanding for how transcription is regulated in prokaryotes and eukaryotes -​ Transcription of genes can be regulated by specific cues -​ Transcription is regulated by sequence specific DNA binding transcription factors -​ Transcription factors can act as activators or repressors -​ The activity of transcription factors can be modulated (here by small molecules -​ Transcription factors act in trans on binding sites on any chromosome in cell  -​ Binding site - cis-elements - only work in cis on genes in the immediate vicinity -​ Transcription - presence of mRNA - can be detected in several ways -​ Specific genes → Northern, RT-PCR, in situ hybridization -​ Detect transcription of all or many genes → microarrays, RNA seq -​ RT-PCR → is your favorite gene expressed in the liver or the heart or the lung? -​ Extract mRNA from liver/heart/lung -​ Reverse transcribe into DNA -​ Use gene-specifc primers to amplify specific transcript by PCR -​ Run PCR product out on gel -​ Gene expressed in liver and lunch, differential splicing -​ Provides information on -​ Where gene is expressed (positive signal) -​ Detection of splicing is dependent on primers chosen -​ Quantitative - real-time PCR- no gel -​ In situ hybridization reveals where a transcript is located -​ Synthesize labeled complementary probe -​ Use nucleotide with label, e.g.. Digoxigenin -​ Probe must be complementary to the mRNA -​ Hybridize probe to fixed embryo -​ Add ab against Dig conjugated with alkaline phosphatase -​ Add color reagent, colorless but converted to colored product by AP -​ CHATGPT in situ hybridization explanation: -​ In situ hybridization is a technique used to locate specific RNA molecules in a sample, such as a fixed Drosophila embryo. Here’s a simple explanation based on the slide: -​ Synthesize a Probe: A single-stranded RNA probe is created to be complementary to the target mRNA. The probe is labeled with a marker, like digoxigenin (DIG), for detection. -​ Hybridize the Probe: The probe binds to the complementary mRNA in the fixed sample. -​ Detect the Probe: An antibody that recognizes the DIG label is added. This antibody is attached to an enzyme called alkaline phosphatase (AP). -​ Color Reaction: A colorless substrate (NBT/BCIP) is added. AP converts it into a blue or purple product, making the location of the mRNA visible. -​ The final result is a stained area in the embryo where the target mRNA is located, as shown by the blue/purple color. -​ RNA-seq provides direct analysis of transcripts present in an RNA sample -​ NGS technology is used to sequence cDNAs generated from an RNA sample directly without cloning into a library -​ Isolate RNA from tissue → convert to cDNA → illumina sequence → generates millions of sequence ‘reads’ → align all reads to genomic sequence → calculate how often a genomic region is included in a read -​ Repeat for all tissues, all times, etc. -​ Which technique to use? -​ RT-PCR = lower cost, needs PCR machine + gell apparatus, single gene, shows expression levels of genes with qPCR, relatively high reliability rate, identifies many different cells, analysis of data is simple -​ In situ hybridization = lower cost, needs a microscope, single gene, not great expression levels of genes, highly reliable, individual cells identified visually, simple analysis of data  -​Bulk (tissue) RNA seq = usually send to outside company, all genes, expression level of genes present/is the number of reads, not as reliable, many different cells identified, need annotated genome for analysis therefore might be complex -​ Single cell RNA sequence = usually send to a company, most highly expressed genes, expression level of genes present/is the number of reads, not as reliable, individual cells identified and sorted, need annotated genome for analysis therefore might be complex -​ Single cell RNA sequence involved fractionation tissue into individual cells, then possibly sorting to identify specific cells, then sequencing all the transcripts in individual cells or nuclei separately, only the more highly expressed genes will be reliably identified -​ LacI repressor is a DNA binding protein -​ Purified LacI protein shown to bind DNA containing operator sequences -​ Radioactively labeled repressor protein + DNA containing wild-type (lacO+) → mix and sediment by centrifugation in glycerol gradient = DNA + radioactivity (repressor protein) -​ Radioactively labeled repressor protein + DNA containing mutant operator (lacO^c) → mix and sediment by centrifugation in glycerol gradient = DNA (no radioactivity, i.e. protein, present) -​ Today, experiment would be done by EMSA (electrophoretic mobility shift assay) (slide 40) -​ DNA binding domain of LacI is a helix-turn-helix -​ Recognition helix: one helix inserts into the major groove and contacts bases: sequence specificity -​ There are several types of DNA binding motifs/domains found in transcription factors -​ Many use an alpha helix to insert into the major groove -​ Amino acids in this helix – the recognition helix– interact directly with bases, this provides the sequence specificity of transcription factors -​ DNase footprinting is used to characterize the sequence a transcription factor binds -​ Regulate DNaseI activity so that it leaves a series of fragments increasing in size by one base -​ Take this altered DNase I by LacI protein bound to DNA -​ Can determine the sequence of this region -​ The LacI repressor binding site is palindromic -​ Key bases in a binding site are also revealed by mutations that abolish binding -​ The repressor binds as a tetramer with one dimer binding to this palindormic sequence wile the other dimer binds to a second similar palindromic sequence Lecture N: Regulation of Gene Expression in Eukaryotes -​ Basic principles of trnascriptional regulation are similar in eukaryotes and prokaryotes -​ SImilarities -​ Some genes are constitutively expressed -​ Some geens are on in some situations and off in others -​ Level of expression of many genes is controlled even if they are on all the time -​ Transcription is controlled by sequence-specific DNA binding transcription factors -​ Transcription factors bind ‘adjacent’ to genes they regulate -​ Transcription factors can act as activators or repressors -​ The activity of some transcription factors can be modified by external signals -​ Differences in eukaryotes -​ An activator is usually required: a promoter is not sufficient -​ Transcription factor binding sites may be situated some distance from promoter -​ TFs actively activate and repress, DNA binding alone is usually insufficient -​ Chromatin gets in the way, bad and good -​ Anatomy of a eukaryotic gene -​ Promoter: similar for all genes  -​ Immediately upstream of coding region -​ Bound by general transcription factors (GTFs) and RNA Polymerase I -​ Enhancer (response elements, cis-regulatory element): unique for each gene -​ Bound by regulatory transcription factors (RTFs) – activators and repressors (Rep) -​ Activators and repressors are sequence specific DNA binding proteins -​ The binding sites of these factors are clustered in regions known as enhancers -​ Transcription of most genes requires a dedicated activator (unlike many bacterial genes) -​ May be more than one enhancer -​ Why is a promoter not sufficient, why is a gene not transcribed all the time, how do RTFs influence transcription positively and negatively? → chromatin -​ A promoter is necessary but usually not sufficient for transcription in eukaryotes -​ Nucleosomes inhibit binding of general transcription factors -​ Transcription requires repositioning or removal of nucleosomes over a promoter by chromatin remodeling enzymes such as SWI/SNF -​ Loss of nucleosomes can be demonstrated by sensitivity to digestion by DNase -​ The region around the promoter of some housekeeping genes may be inherently nucleosome free due to the DNA sequence – genes transcribed all the time -​ Enhancers in unicellular eukaryotes are positioned adjacent the promoter, in metazoans they can be found at a distance: upstream or downstream or even in introns -​ More ‘primitive’ eukaryotes have enhancer and promoter closer together -​ Metazoan have enhancer farther way from promoter -​ So what is the advantage of being able to have your enhancers situates away from the promoter? -​ Can now have more than one enhancer for single gene… the same gene can be expressed in different cell types being regulated by different RTFs in each -​ Similarities and differences between promoters and enhancers -​ Similarities -​ Both are essential for transcription of a gene -​ Both bind transcription factors -​ Characterized by loss of nucleosomes and modifications to adjacent chromatin -​ Differences -​ Directionality: promoters are directional/enhancers are not – can be inverted and will still function -​ What proteins bind: all promoters bind the same General Transcription -​ Factors/Enhanvers bind Regulatory Trnascription factors that are gene-specific -​ Specific chromatin modification: are different at Promoters and Enhancers -​ Enhancers can be identified by a number of approaches -​ Reporter gene assays -​ Identify clusters of binding sites for an RTF in the genome -​ CHiP: localize RTF or enhancer-specific chromatin modifications in chromosomes -​ ATAC-seq: identify accessible chromatin/DNA in vivo -​ Enhancers can be identified with reporter gene constructs (slide 9) -​ Reporter gene = a gene whose expression can be easily monitored -​ lacZ – beta-gal = colored breakdown products from colorless precursor (X-gal) -​ Reporter gene construct = fragment of genomic DNA potentially containing an enhancer fused to reporter gene with minimal promoter -​ Genes may have multiple enhancers driving expression in different cells or at different times -​ Even-skipped gene from drosophila -​ Can be multiple independent enhancers  -​ Enhancers can be situated: many kb away from the promoter, 3’ or 5’ of the promoter, or in introns -​ Unlike promoters, enhancers are not polarized, they will work identically if inverted -​ A gene will be transcribed if activators are present/active and repressors are nto at a particular enhancer -​ Why does this region of DNA drive expression of eve in this region of the early embryo? -​ Genetic studies identified transcriptional activators required for even expression and repressors required to limit its expression -​ Activators: Bicoid (Bic) and Hunchback (Hb) -​ Repressors: Giant (Gt) nad Kruppel (Kr) -​ Binding sites in stripe 2 enhancer → identified with EMSA, Footprinting, Mutation of enhancer/reporter construct -​ The black band on the repressor image is the only place where there are activators and no repressors -​ Q1 The stripe 2 enhancer of the eve gene drives expression in a single stripe in early embryos. What would expression of a reporter gene where eve 2 stripe is diving GFP expression look like in a Giant mutant? -​ B -​ Q2 The stripe 2 enhancer of the eve gene drives expression in a single stripe in early embryos. What would expression of a reporter gene where eve 2 stripe is diving GFP expression look like in a Kruppel mutant? -​ C -​ Enhancers can also be identified by characterizing where RTFs bind using bioinformatics -​ If you know the sequence your favorite RTF binds to you can search the genome for potential binding sites -​ Potential problems: -​ Consensus binding site short and loose -​ Many, if not most, putative binding sites are not real -​ Possible resolution: search for clusters of sites or combinations of sites for different RTFs, or even search for clusters of more than one RTF at the same time, e.g. Giant and Kruppel -​ Enhancers can also be identified by characterizing where RTF;s bind using ChIP-sequencing -​ ChIP = chromatin immunoprecipitation = a method to isolate DNA fragments to which a transcription factor has bound -​ Can also be used to identify where specific chromatin modifications occur -​ 1. TF bound to DNA/chromatin in cell → isolate chromatin from nucel, crosslink protein to each other and DNA using formaldehyde -​ 2. Shear DNA -​ 3. Add antibody against TF to imunoprecipitate fragments to which TF is bound -​ 4. Purify DNA -​ 5. Use NGS to sequence all of the DNA fragments isolated. Align sequence reads to the genomic sequence -​ Conclusion: Expression of gene x may be regulated by the TF -​ Peaks = lots of sequence reads = likely binding site for TF -​ Enhancers can also be identified by ATAC-seq -​ ATAC = Assay for Transposase accessible chromatin using sequencing data -​ ATAC seq identifies ‘open/accessible’chromatin, i.e. nucleosome free = promoters, enhancers  -​ Tn5 transposase is modified to be hyperactive and cut DNA at high frequency adn then insert sequencing primer -​ 2 cuts produce a DNA fragment that can be amplified and then sequenced by NGS -​ Line up reads to genome -​ Chromatin structure ismodified in two general ways -​ Remodeling = repositioning or removal of nucleosomes by chromatin remodeling enzymes -​ Inactive gene: promoter protected from digestion -​ Active gene: promoter seistive to digestion -​ Covelanet modification = addition or removal of chemical groups or small proteins on the N-terminal tails of histones by specific histone enzymes -​ The N-terminal tails of histones extend out from the nucleus -​ Chromatin structure can be modified by covalent modification of histones -​ Acetyl, methyl, phosphate groups and small proteins, including ubiquitin can be added or removed to histone tails -​ Done by specific enzymes, e.g. histone acetyl transferase (HAT), histone methyl transferase (HMT), histone deacetylase (HDAC) -​ Activity of these enzymes is controlled by transcription factors and RNA pol II which localize these activities to specific regions on chromosomes -​ Modifications influence chromatin structure and the ability to transcribe a region -​ Acetylation is associated with active chromatin -​ Addition of acetyl groups to histone tails associated with DNA in chromatin being actively transcribed and promotion of transcription -​ The negatively charged groups on adjacent nucleosomes are thought to repel each other leading to a more ‘open’ chromatin conformation, and looser association of the DNA with the histones (allowing DNA binding proteins easier access) -​ H3K27 acetylation: associated with active enhancers -​ H3= histone H3 -​ K27 = amino acid #27 which is lysine -​ Activators usually recruit histone acetyltransferasess -​ Repressors can recruit histone deacetylases -​ Deacetylated chromatin is usually silent -​ Methylation can be associated with inactive or active chromatin: it depends which sites on histones are modified -​ H3K27: repression → recruits proteins that facilitate compact chromatin -​ H3K4, H3K36: activation → facilitate acetylation of other residues and open chromatin -​ Chromatin is modified during transcription -​ Chromatin is modified during transcription -​ Chromatin is modified to facilitate removal of nucleosomes ahead of RNA pol -​ Histones are acetylated ahead of Pol II -​ Nucleosomes are repositioned after RNA pol has passed through -​ This chromatin is remodified – deacetylated – to ensure no elicit transcriptional initiation -​ Activation bind to enhancers at genes they regulate but need additional activities to promote transcription -​ Activators need at least two domains -​ DBD: DNA binding domain → sequence specific -​ AD: activatino domain → promotes transcriptional activation, may have intrinsic activity, often recruits coactivator which has this activity -​ Activation: activators can bring chromatin remodelers to a promoter to remove nucleosomes -​ Activators can bring chromatin modifying enzymes, e.g. histone acetyl transferase  -​ May directly help recruit Mediator activation complex and general transcription factors -​ All this can help to establish TADs -​ Topological Associating Domains (TADs) facilitate promoter-enhancer interactions -​ Originally thought that there must be some physical link between enhancers and promoters, eg facilitates by Mediator complex -​ But now – interior of cells may resemble and behave as an emulsion – liquid-liquid phase separation – like oil separating out from water – at enhancer the proteins may concentrate into such a phase-separated condensate -​ Enhancers/activators are often situated long distances from promoter… why do they interact with only one promoter? -​ Insulators – bound by CTCF – facilitate DNA looping and establishing TADs -​ Enhancer recruits many proteins – activators, coactivattors, chromatin modifiers, Mediator complex → forms a stable phase-separated condensate -​ Trancsriptional repression is more complex in eukaryotes than bacteria -​ DNA binding is not usually sufficient: need additional ‘repression’ domain which usually functions by modifying chromatin structure -​ Repressors have at least two domains: a DBD and repression domain (RD) -​ RDs may have autonomous activity but usually recruit additional proteins: corepressors -​ CoRs in turn recruit chromatin modifying enzymes (e.g. histone deacetylases) -​ Repression Mechanisms -​ Interfere with the basal transcriptional machinery -​ Modify chromatin structure to form closed conformation -​ Interfere directly with activator binding or activity -​ Some transcription factors can act as an activator or repressor – whether a coactivator or corepressor is bound -​ Notch pathway → delta = ligand, notch = receptor, Su(H) = transcription factor -​ Regulates neurogenesis: neuron is default state, noth signaling promotes non-neural fates (epidermis) -​ No Delta/pathway off = corepressor bound to Su(H) so acts as repressor -​ Delta present/pathway on = notch intracellular domain is cleaved off, binds to coactivator Mam and to Su(H), converting it to an activator -​ Regulating transcription factor activity -​ Many transcription factors are active all the time → so their activity is essentially controlled by controlling their expression -​ The activity of some transcription factors can be modified posttranslationally -​ Directly by small molecules (ex. steroid hormones) -​ By covalent modification, usually phosphorylation by a kinase, which can induce a conformational change modifying protein activity up or down -​ Indirectly through regulation of coactivator (or less commonly corepressor) availability (e.g. Su(H) in Notch pathway, TCF in Wnt pathway) -​ (iv) Indirectly through partial (e.g. Gli in Hh pathway) or complete degradation -​ Steroid hormones: direct modification of TF activity by external signal -​ Similar situation to bacterial TFs – activity modified by small molecule -​ Steroid receptor = transcription factor -​ Binding of steroid induced conformational change to a form that can bind DNA -​ No steroid → steroid receptor not bound to DNA -​ Steroid present → steroid receptor bound to DNA -​ Signaling proteins: indirectly modify TF activity, e.g. BMP signaling regulates transcription factor R-Smad  -​ BMP=bone morphogenetic protein -​ No BMP = receptors are monomers, R-Smad and Co-Smad in cytoplasm -​ Cell exposed to BMP = BMP binds to receptors, Receptors dimerize, R-Smad is phosphorylated by receptor, R-Smad/Co-Smad translocate to nucleus, R-Smad /Co-Smad bind to DNA and activate -​ DNA methylation is associated with transcriptional silencing -​ DNA methylation: a methyl group (-CH3) is added to the 5’ carbon of cytosine -​ (Don’t confuse with methylation of histones) -​ Occurs at CpG pairs (p=phosphate) -​ Catalyzed by DNA methyl transferases (DNMT) -​ Methylation can inhibit binding of transcriptional activators and results in silnecing of gene transcription -​ DNA methylation is important in vertebrates but generally not in other eukaryotes -​ Epigenetics: a heritable change in gene expression that does not involve a change in DNA sequence -​ Originally → essentially meant differential gene expression during development -​ Today: has come to mean self-perpetuating changes in chromatin/DNA (not sequence) -​ i.e. a change (which controls gene expression) that is maintained in daughter cells in the absence the initial signal that established it -​ Implies some kind of ‘memory’ -​ Does this mean during mitosis, gene expression patterns are maintained in daughters? -​..... or that new patterns established by an environmental conditional in an individual may even been remembered in their offspring? For example, if an individual experiences periods of famine, gene expression patterns are established to facilitate fat deposition when food is available...if these patterns are maintained in offspring not exposed to any famine it could result in obesity. Larmarkism? -​ DNA methylation= methylation patterns that can be inherited, possibly even in offspring? -​ DNMT functions at replication fork -​ Suggested modifications could be passed on to offspring… but methylation is wiped in germ cells… but may be mechanism to reestablish patterns from previous generation -​ So methylation patters can be maintained through mitosis -​ Histone modifications: Old nucleosomes are removed during replication...but some modified ones may be reused to maintain gene expression patterns, but eventually unmodified ones will be added after more cell division so patterns are not really heritable and not generationally -​ Some proteins involved in controlling histone modifications will reestablish patterns on new histones after replication -​ Gene expression can also be regulated post-transcriptionally: double-stranded RNAs can inhibit gene expression -​ RNA interference: expression of an mRNA is silenced by a complementary double-stranded RNA → first demonstrated in 1998 by Mello and Fire in C. elegans (Nobel Prize 2006) -​ Subsequently shown to work in other organisms -​ RNAi: results in degradation of mRNA (compelte complementarity with target) -​ Key feature: only very small quantities are required - what does this tell you? -​ Not just structural interference, infers it is acting like an enzyme -​ Micro-RNAs are naturally occurring double stranded RNAs that regulate gene expression -​ RNAi utilizes endogenous gene regulation mechanisms that uses dsRNAs -​ microRNAs are small double stranded RNAs complementary to part of the 3’UTR of some mRNAs and prevent translation of that mRNA -​ Transcribed as single stranded RNAs; complementary region within the RNA forms a hairpin (double stranded– ds– region)  -​The pri-miRNA is processes to leave the ds hairpin and one strand od this is included into a ribonucleoprotein complex, miRISC (miRNA-induced silencing complex), including an Argonaute protein -​ mRNAs with complementary 3’ UTRs to micro RNAs can be prevented from being translated or can be degraded -​ Most animal mRNAs show incomplete complementarity with their micro RNA and these are not degraded, but translation is inhibited -​ If an mRNA has good complementarity then it may be degraded...some plant miRNAs (artificial RNAi) -​ A single microRNA may regulate expression of several genes which all have a region partially complementary to it in their 3'UTR -​ Only some genes are regulated by miRNAs, not all eukaryotes use miRNAs -​ RNAi is an important tool to knock down expression of specific genes to phenocopy mutants -​ RNAi is the term used when experimenters introduce in vitro synthesized ds RNAs into a cell to knock down expression of a particular gene -​ Model organisms -​ Can be used to do genome-wide screens to knock down all the genes in the genome (separately) to determine which ones might be involved in a particular process -​ Can knock down expression in tissue culture cells -​ Non-model organisms -​ Genetics can be difficult, RNAi allows knock down of specific genes in many non- model species -​ Not perfect -​ Variable effect, rarely matches the effect of a null mutant, sometimes no knock down -​ Off-target effects - a dsRNA may knock down another gene with similar sequence -​ So why is a gene expressed or not expressed in any given cell? -​ No active activator present or enhancer has a binding site for repressor and active repressor is present -​ OR microRNA is present and gene has complementary region in 3’UTR (check if mRNA is present) -​ Gene is transcribed: has enhancer to which Activator can bind → activator ir present and active + repressor protein is not present or if present, it is not active -​ Gene is translated: microRNA specific to gene is not present -​ Why is protein X present in certain cells pathway: -​ Gene → pre-mRNA → mRNA → protein → functional protein Lecture O: Manipulating the Genomes of Eukaryotes -​ What is a transgenic organism? -​ Has cloned DNA (genomic/cDNA) from another species integrated into its genome -​ OR has cloned DNA (genomic or cDNA) from itself is integrated into its genome at a different location than the endogenous gene -​ Cloned integrated DNA = transgene - integrated into all its cells (the organism was derived from gamete carrying the transgene). -​ What is not a transgenic organism? -​ Simple mutations engineered by CRISPR/Cas9, (some CRISPR,Cas9 gene replacements with be transgenic) -​ Modifications of somatic cells only, so not heritable – all genetic modifications in humans are only to somatic cells, so we do not have transgenic humans… yet.. (although you could argue that modified somatic cells are transgenic cells.) -​ Why ‘manipulate’ genomes/generate transgenic organisms?  -​ Analysis of gene function, production of human proteins, modifications of agricultural plants and animals for favorable characteristics, generation of animal models of human diseases -​ Replace (or add) endogenous genes with a modified version -​ A good copy with a bad copy: generate mutation – investigate function -​ A bad copy with a good copy: gene therapy -​ Ethics: should we be manipulating genomes? -​ Transgenics in mammals -​ How you make transgenics varies from species to species -​ Mammals -​ Ectopic insertions (gene addition) -​ Injection of DNA into the prenucleus of eukaryotic eggs often results in random insertions into the genome (form, concatemers – several copies in the same region) -​ Several injected embryos are places into oviduct of receptive female → progeny are tested for presence of injected DNA (may carry marker gene) → check for mosaicism -​ In genetics, "mosaicism" refers to a condition where an individual has two or more distinct populations of cells within their body, meaning some cells have a different genetic makeup compared to others, arising from a mutation occurring during early embryonic development -​ Transgenics in flies 1: P-elements (transposable element) -​ Wild type P-element transposon has transposase gene flanked by P-element ends (inverted repeats) -​ For transgenics: use modified P-element, replace transposase with gene to be integrated and a market gene (in plasmid) -​ Inject into embryo along with the source of transposase -​ P-element jumps from plasmid into chromosome of germ cell progenitors of the G0 generation -​ Transformants only identified in next, G1, generation -​ Transgenic in flies 2: bacteriophage integrase -​ Lysogenic cycle: phage chromosome integrates into bacterial chromosome -​ Phage DNA integrates into bacterial chromosome becoming a prophage -​ Bacterium reproduces normally, copying the prophage and transmitting it into daughter cells -​ Integration occurs by recombination between attP site in phage chromosome and attB site in bacterial chromosome and is catalyzed by phage integrase enzyme -​ attP sites are inserted into the fly genome with P-elements. Plasmids with attB sites can then be integrated into these sites using integrase (BACTERIOPHAGE IS NOT USED) -​ Advantages: integration of all plasmids into same site, integration of larger plasmids -​ Transgenic in plants are made using Ti plasmid​ -​ Ti plasmid: from soil bacteria, Agrobacterium tumefaciens: can infect most flowering plants -​ During infection, part of the Ti plasmid – T-DNA – is transferred to host and integrates randomly into host genome by non-homologous recombination -​ Plant transformation vectors:most of T-DNA is replaced with gene of interest to be integrated plus marker -​ Take T-DNA plasmid and helper plasmid → transform agrobacterium with plasmids and spray transformed bacteria on plants → recombinant T-DNA transferred to plant cell and integrates into plant genome → grow embryos from single cells; add herbicide to select for transformants → transformed plant -​ Transgenics are used in many ways to study gene function -​ Rescue mutants with wild-type copy of a gene  -​ Confirm which gene is defective in a mutant strain (e.g. deletion mutates gene A and gene B, has defective eye: which gene is required for normal eye development?) -​ Make transgenics of each gene separately; introduce into deletion strain; if transgene rescues; eye phenotype in deletion is due to loss of gene B -​ Reporter gene constructs to identify and analyze enhancers -​ mRNA expression in early Drosophila embryo → transgenic embryo carrying reporter construct -​ Transgenics are used in many ways to study gene function -​ Ectopically express a gene to determine its function -​ Drive expression in cells in which it is not normally expressed and assess response -​ Drosophila: UAS/Gal4 system -​ Gal4 is yeast transcription factor (activator), binds to sequence known as UAS -​ Eyeless gene is expressed in the eye, is required for eye development: how important is it? -​ Drive expression of eyeless in other tissues -​ Get ectopic eyes developing, eyeless is ‘master-control’ gene for eye development -​ Transgenics are used to make specific proteins -​ Human proteins can be made in E.coli: make inducible -​ Human growth hormone gene + plasmid vector (what else is needed for the gene to be expressed?)→ ligate DNA fragment and vector → transform E.coli → growth hormone gene (next to lac control region) → induce lac expression → human growth hormone collects in E. coli cells -​ Also needs Shine-Delgarno, terminator -​ Human proteins can be made in farm animals -​ Not all proteins can be made in E.coli, many require post-translational modifications that do not happen in bacteria -​ Sheep can be engineered to secrete proteins such as blood clotting factors into their milk -​ Beta-lactoglobulin enhancer + human antithrombin III cDNA → DNA is injected into pronucleus (sheep ovum) → transgenic progeny are identified by PCR → expression of gene is restricted to mammary tissue → obtain milk containing Antithrombin III produced from transgenic sheep → fractionate milk proteins → get pure antithrombin III -​ Transgenics - genetically modified organisms (GMOs) - are used in agriculture -​ Resistance to herbicide glyphosate (sound-up) -​ Glyphosate inhibits enzyme EPSPS in plants, EPSPS required in amino acid synthesis -​ Transgenic crops have been engineered to carry bacterial EPSPS: resistant to glyphosate -​ GMOs: the solution to world hunger or the harbingers of doom? -​ What are the issues regarding genetically modified agricultural species? -​ Advantages: -​ Disease resistance (reduced antibiotic and fungicide use) -​ Longer shelf-life -​ Increased yield in particular from ability to kill off weeds -​ Pest-resistance (reduced pesticide use) e.g. plant produces insect toxin -​ Expand environmental range -​ More nutritious (e.g. add ability to make a vitamin) -​ Disadvantages: -​ Allergies to proteins encoded by transgenes -​ Transfer of transgenes to non-agricultural species in the wild (mating, horizontal)  -​ Domination of agriculture by large GMO companies -​ Negative impact on ecosystem of pesticides, on specific species, (e.g. monarch butterflies, amphibians) -​ Excessive use of pesticides results in resistance in weeds -​ Cancer risks from pesticides -​ Distinguish between methods that employ integration of novel genes and those that modify existing genes – latter being what has been done by man for 1000s of years -​ Transgenics can be made to model dominant human diseases -​ Gain of function - dominant - mutations in human genes can be introduced into model species -​ Loss of function mutants in mice can be made to model recessive traits -​ Mouse is the more convenient model, but monkeys provide a better system for studying brain-associated diseases such as neurodegeneration -​ Yeast, drosophila, C. elegans and zebrafish can also be used\ -​ Why make models? -​ Allow more detail analysis, e.g. of cell function -​ Test drugs for alleviating symptoms -​ Issues -​ Ethics of using animal models in particular primates -​ How close is a disease actually replicated in the model? -​ Organoids may be a way to go in the future -​ ‘Mini’ organs grown in vitro from stem cells can be used to test drugs -​ Human gene therapy: somatic cell ‘transgenics’ or gene replacement -​ Not allowed to make human transgenics or modify the human germline in other ways -​ But you can modify somatic cells of individuals with genetic disease -​ Ideal goal: to replace mutant gene with good copy (not actually ‘transgenic’) -​ Usual goal so far: to insert good gene somewhere in genome to produce good protein -​ But ideal goal may be more readily realized with CRISPR/Cas technology -​ Somatic cell therapy: cure patients with a genetic disease -​ Problem: how to get DNA in to cells -​ Modified viruses are usually used -​ Problem...viruses integrate into host genome...integrations can cause mutations -​ Some tissues are more easily manipulated - blood disorders, stem cells can be isolated and manipulated in tissue culture and then reinjected into patient, e.g. sickle cell disease -​ Introducing a good gene can overcome a loss of function mutation....but not a gain of function mutation....so gene editing is required -​ Reverse genetics: making targeted mutations/gene edits -​ How do you ‘knock out’ a gene? -​ Homologous recombination (see Lecture L) -​ Still used in some species, e.g. yeast -​ Was used to make KOs in mice: homologous recombination in embryonic stem (ES) cells, then introduce into embryos -​ CRISPR/Cas9 -​ Generate double-strand breaks in precise locations in genome: CRISPR/Cas -​ Used in many species -​ Why do you want to do it? -​ Analyze gene function: generate mutations in specific regions of a protein as well as make null mutant -​ Replace mutant gene with good copy -​ Gene targeting/gene editing by the creation of double strand breaks at a single precise location in a genome -​ New techniques – CRISPR/Cas9 - can cut a genome at one specific place, e.g. in a gene to be mutated -​ Double strand breaks (DSB) can lead to mutations or can be manipulated to generate gene replacement -​ Nuclease-induced double-strand break -​ Non-homologous end-joining (NHEJ) -​ Homology directed repair -​ More efficient at creating KOs and gene replacements than homologous recombination -​ The CRISPR/Cas system evolved to protect bacteria against bacteriophage -​ Clustered, Regularly, Interspaced, Short, Palindromic, Repeats = include fragments of phage DNA incorporated into the host bacterial genome after unsuccessful infection -​ CRISPR associated sequences = encode proteins involved in this process -​ Cas9 = double stranded nuclease -​ Phage DNA is degraded -​ Fragments are incorporated into the CRISPR locus (as ‘spacer’), separated by repeats -​ CRISPR locus is transcribed = crRNA -​ crRNA is processed -​ Spacer (phage sequence)/repeat incorporated into Cas9 nuclease -​ CRISPR/Cas9 targets and cleaves any DNA complementary to spacer, i.e. infecting phage DNA -​ CRISPR/Cas complex cleaves any DNA molecule that is complementary to the CRISPR RNA -​ Active complex needs: -​ Cas9 nuclease -​ crRNA = 5’ spacer + repeat -​ tracrRNA = base pairs with crRNA to help loading onto Cas9 -​ Spacer base pairs with complementary and the other strand = double strand break -​ CRISPR?cas system has been modified to make double strand breaks at targeted sites in eukaryotic genomes -​ Bacterial version: Emmanuelle Charpentier and Jennifer Doudna (Nobel Prize 2020) -​ Genome editing version: Guide RNA = spacer + repeat/tracrRNA in a single molecule, spencer = the genome sequence to target, Cas9 is modified with nuclear localization signal -​ Recognitions of CRISPR target also requires PAM sequence = NGG in the genome immediately downstream of spencer -​ To generate a DSB: need to get modified Cas9 and guide RNA into your favorite cell -​ Put sequences encoding both in a plasmid and inject that -​ Make protein and RNA in vitro, form complex, inject that -​ DSB generated by CRISPR/Cas can be used to generate mutations -​ The DSB may be repaired by non-homologous end-joining (NHEJ) -​ NHEJ is sometimes not perfect and one or more bases are inserted or deleted at the repair site -​ Indel = insertion OR deletion -​ CRISPR/Cas can also be used to edit genes via HDR -​ HDR = homology directed repair. A cell can repair a DSB using a homologous region -​ Repair will not be perfect if the homologous region is identical -​ …but not if the homologous region contains an insertion -​ Introduce DNA along with Cas9 complex before HDR proess -​ This approach can be used to repair mutations -​ Why is CRISPR/Cas so successful for genome editing?  -​Simplicity -​ Need just Cas9 and a modified RNA sequence -​ Efficiency -​ Incredibly efficient, orders of magnitude more efficient than most other methods -​ Specificity -​ Recognition sequence is 17-20 nucleotides -​ Versatility -​ Ability to do knockouts and gene modifications -​ Issue: off-target effects -​ Specificity is still somewhat of an issue, although it will target the sequences expected, other sequences may also be targeted. -​ Modifications -​ Other CRISPR systems are being developed that are even more specific. -​ DSBs are bad! CRISPR systems have been developed that generate only single-strand breaks. -​ Catalytically dead Cas9 will still be directed to a specific location in the genome by its gRNA and it can be modified, eg. HDAC could be fused to it so that that region in the genome will be deacetylated -​ Using CRISPR/Cas9 to ‘cure’ sickle cell disease and beta-thalassemia -​ Both diseases are associated with mutations in the gene encoding ß-globin -​ Cluster of ß-globin genes in the genome: epsilon, upsilon, sigma, beta -​ Differentially regulated during development epsilon, upsilon on in embryos, sigma, beta in adults. -​ Upsilon is silenced in adults by repressor BCL11A binding to region upstream -​ CRISPR/Cas9 used to mutate BCL11A binding site in hematopoietic stem cells derived from patients, then replace other stem cells with these -​ Making loss of function mutations with CRISPR/Cas9 is generally easier than doing gene replacement -​ How long before we start messing about with the human genome, i.e. Targeting the germline? -​ Somatic cell modification -​ CRISPR cell modifications will make editing somatic cells more efficient, repairing mutations, modifying cells to target cancers -​ Germline modification -​ UK – approved modification of viable embryos that must be destroyed after 14 days. -​ First reported humans with germline modifications reported in China in 2018 Lecture Q: Genetics of Cancer -​ Cancer is associated with excessive cell proliferation -​ Cancer cells proliferate excessively leading to a tumor -​ They are able to circumvent mechanisms that restrict their proliferation -​ Their behavior changes -​ Cells undergo metastasis: invade surrounding tissue and spread to other locations in the body -​ Properties of cancer cells -​ Self sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis, evading apoptosis -​ Cancer is caused by mutations that disrupt normal growth control -​ Cancer causing genes can be separated into two classes -​ Oncogenes: wild-type gene promotes growth  -​ Wild-type gene = a proto-oncogene, is required for cell proliferation but its expression and/or activity is normally tightly regulated to control the amount of proliferation -​ Oncogene = gain of function (hypomorphic or neomorphic) mutation in proto-oncogene -​ Dominant: one mutant copy is sufficient to promote cancer -​ Tumor suppressor genes: wild-type gene suppresses growth -​ Wild-type gene is required to negatively regulate cell proliferation. Proliferation is not correctly regulated in cells in which both copies of a tumor suppressor are mutated -​ Loss of function mutations in tumor suppressor genes result in cancer -​ Tumor suppressor = loss of function mutation -​ Recessive: both copies of gene must be inactivated to promote cancer -​ Mutations leading to cancer occur in somatic cells -​ Germ line =cells that give rise to the gametes (sperm, eggs, etc.) -​ Somatic cells = all the other cells -​ Mutation in gene in somatic cell → germ cells and gametes do not carry mutation -​ Oncogenic mutations -​ Dominant so only a mutation in only one copy of the gene is required - but mutation is usually quite specific - so rare -​ Tumor suppressor mutations -​ Recessive so a mutation in both copies is required - simple loss of function is sufficient, but knocking out both copies is rare -​.....unless you are already heterozygous and only have one good copy -​ Individuals with a high predisposition to a specific type of cancer - are usually heterozygous for a mutation in a tumor suppressor gene. -​ An embryo that was homozygous for a tumor suppressor mutation or heterozygous for an oncogenic mutation would die -​ Cancer is caused by mutations: intrinsic or extrinsic? -​ Are mutations spontaneous? -​ Replication errors, base tautomerization, base deamination -​ Unavoidable -​ Or are the mutations caused by mutagenic external agents? -​ Chemicals, UV, ionizing radiation, viruses -​ Avoidable -​ Support for intrinsic origin -​ Cancer occurs more often in some organs than others; this may correlate with the number of stem cell divisions in an organ -​ Cancer risk often correlates with age -​ Molecular signatures of some cancer-associated mutations appear replication-related -​ Support for extrinsic origin -​ Clear environmental cause: tobacco smoke, human papillomarivuses, sun -​ The risk for some types of cancer is increasing compared to the past -​ Molecular signatures of most cancer-associated mutations appear non-age related -​ Hereditary origin -​ Some mutations – tumor suppressor mutations – may be inherited from parent -​ Individuals with predisposition to cancer are heterozygotes for this type of mutation -​ Proliferation requires increase in cell size and activation of cell cycle genes  -​ The cell cycle: to maintain regular cell size there is a link between cell growth and when a cell decides to divide -​ G1: cells need to increase in size before dividing, prepared for replication -​ G0: if cells do not receive the correct signals or nutrients they will drop out of the cell cycle -​ G1 -to-S checkpoint: The point of no return. Usually if cells decide to initiate replication they will proceed through mitosis -​ Characterized by activation of TF, E2F -​ Cells that are stressed or damaged will stop here to repair -​ Phosphorylation regulated activity of cell cycle protein -​ Progression through the cell cycle requires the coordinated action of many genes in particular -​ CDK:cyclin-dependent kinases -​ Cyclins: regulate CDKs -​ Extracellular signals control growth and proliferation -​ Signals activate or repress genes required for increase in cell size and progression through G1 into cell cycle -​ Growth factors, anti-growth factors, local interactions -​ Growth factors: steroids/thyroxine/retinoic acid, secreted signaling proteins -​ Produced locally within tissue or systemic throughout body -​ Signal on neighboring cell or in ECM or environmental (e.g. pathogen) -​ Cell density (if too high may inhibit growth) -​ Two (three) ways proliferation control is lost leading to cancer -​ 1. Loss of protein negatively regulating entry into or progression through the cell cycle or inappropriate activation of positive regulator -​ 2. Inappropriate activation of signaling pathway promoting proliferation or loss of signaling pathway inhibiting proliferation -​ 3. Defect in DNA repair… more mutations…indirectly leading to one of the above -​ E2F activation marks the G1-to-S transition -​ E2F activates transcription of genes required for DNA synthesis -​ E2F activity is regulated through availability of its corepressor, Retinoblastoma, Rb -​ G1: Rb is available, E2F functions as a repressor -​ G1-to-S: Rb is phosphorylated by CDK2/4, conformational change and can no longer bind E2F -​ Their activity is regulated by cyclins D and E -​ Progression through the other stages of the cell cycle involved inactivation and inactivation of other CDKs by other cyclins in a coordinated fashion -​ Retinoblastoma is a negative regulator of the cell cycle and is a tumor suppressor -​ Retinoblastoma is a cancer of the eye found almost exclusively in children -​ Associated with loss of both copies of RB1 gene 1/3 of patients are heterozygous: only one mutation is required in remaining good copy -​ Families are susceptible to this disease 2/3 of patients have two good copies, but both have been mutated in a cell in the eye -​ Normal cell: E2F is only activated at the G1-to-S transition, repressed by Rb -​ Rb mutant: E2F will be active all the time -​ As with many types of cancer, a gene functioning in many tissues is associated with tumors in mainly one tissue -​ Susceptibility to cancer in tumor suppressor heterozygote: is a dominant, not fully penetrant trait. Cancer/overproliferation: is a cellular recessive trait. -​ P53 is a tumor suppressor protein that regulates the G1-to-S checkpoint  -​ P53 is a transcription factor required for response to stress, including DNA damage: cells need time to repair such damage before entering the cell cycle -​ P53 is activated by UC or ionizing radiation -​ Induces expression of CDK inhibitor, p21 -​ P21 inhibits activity of CDK4-cyclinD complexes -​ Rb remains unphosphorylated so it can inhibit E2F, preventing entry into S phase of the cell cycle -​ Induces expression of DNA repair genes -​ Induced expression of apoptosis genes -​ Cells may even be better off committing suicide - apoptosis - if the damage is severe -​ Arrests cell cycle at G1-to-S checkpoint -​ 50% of human cancers have mutations in both copies of TP53 (gene that encodes p53) -​ Premature entry into cell cycle - failure to repair DNA damage leading to mutations -​ Signals control proliferation via signal transduction pathways that activate expression of growth promoting genes -​ steroid/thyroxine/retinoic acid signaling is simple -​ Steroid → steroid receptor → nucleus → growth promoting gene on → protein that stimulates cell division -​ Extracellular protein signals require a more complicated pathway -​ Example of signaling pathway that influences growth: Wnt signaling pathway -​ Many of the genes identified in developmental screens encode signals or downstream components involved in transducing those signals, e.g. Wingless (a Wnt) -​ Wnt are a family of secreted proteins/growth factors that promote growth -​ Pathway modifies the activity of the transcription factor (TCF) -​ Absence of Wnt → beta-catenin degraded, TCF is a repressor, growth promoting genes OFF -​ Presence of Wnt → beta-catenin stabilized, displaces corepressor, acts as coactivator, growth promoting genes ON -​ Components of signaling pathways can be oncogenes or tumor suppressors depending on their activity and the activity of the pathway -​ Pathway that promoted growth -​ A gene that positively regulates the pathway could be a proto-oncogene -​ Mutation to oncogene could involve: -​ Expression at much higher levels (e.g. gene duplication) -​ Missense mutation in regulatory domain -constitutively active -​ Fusion with another gene following translocation or inversion that results in constitutively active protein -​ Expression in the wrong cells (usually a signal which shows restricted expression) or failure to turn off expression -​ A gene that negatively regulated the pathway could be a tumor suppressor -​ Homozygous simple loss of function mutation = pathway always being on -​ Pathway that suppresses growth -​ A gene that positively regulates the pathway could be a tumor suppressor -​ A gene that negatively regulates the pathway could be a proto-oncogene -​ If Wnts function to promote growth, which of the signaling components are potential proto-oncogenes? -​ Options: Wnt, Fx, Axin, APC, GSK3, beta-catenin -​ Absence of Wnt → beta-catenin degraded, TCF is a repressor, growth promoting genes OFF -​ Presence of Wnt → beta-catenin stabilized, displaces corepressor, acts as coactivator, growth promoting genes ON -​ TOPHAT (a) If Wnt functions to positively regulate cell proliferation, are the genes encoding the following components of the signaling pathway potentially proto-oncogenes or tumor suppressors? (b) if proto-oncogenes what specific mutation would you expect to convert the gene into an oncogene? -​ Wnt: oncogene; ectopic expression… moved next to enhancer from other gene, not human cancer -​ Fz: oncogene; gene amplification, stability mutation, not human cancer -​ Axin: tumor suppressor; loss of function, unknown if human cancer -​ APC: tumor suppressor; loss of function (but apparently not null in real cancers), human cancer -​ GSK3: tumor suppressor; loss of function, not human cancer -​ Beta-catenin: oncogene; KO of regulatory region that is required for degradation, human cancer -​ Beta-catenin is a positive regulator of the Wnt signaling pathway and is a proto-oncogene -​ Beta-catenin is targeted for degradation by phosphorylation of residues ina regulatory domain in the N-terminus -​ Oncogenic mutations in this region are found in beta-catenin in several types of cancer -​ In cells carrying these mutants, the Wnt pathway is on even when Wnt is absent -​ APC is a negative regulator of the Wnt signaling pathway and is tumor suppressor -​ APC = Adenomatous polyposis coli -​ APC mutation/no Wnt = destruction complex cannot form so beta-catenin is not degraded -​ In APC mutant Wnt is no longer required for activation of growth promoting genes -​ Mutations are associated with colorectal cancers (heterozygous individuals have a >95% chance risk of colorectal cancer by age 50) -​ Mutations are not simple loss of function.....they appear to reduce ability to act in destruction complex but APC has other functions that are required for cell viability -​ Mutations in Axin have been implicated in hepatocellular carcinomas -​ Understanding pathways involved in promoting or inhibiting cell proliferation is the basis for drugs to treat some cancers -​ Would a drug that targeted Fz be useful to treat individuals with APC mutations with cancer? NO -​ Would a drug that targeted APC be useful to treat individuals with APC mutations associated with cancer? NO -​ Would a drug that targeted ß-catenin be useful to treat individuals with APC mutations with cancer? YES -​ Small molecule inhibitors of some components of the Wnt pathway have been identified....But at present there don’t appear to be any good drugs to target an of these proteins -​ Not all pathways promote growth: Smad4 (co-Smad), a positive regulator of TGF-ß signaling, is a tumor suppressor -​ TGF-ß signal transduction is similar to BMP signaling -​ TGF-ßs negatively regulate cell proliferation in some tissues, including the gut -​ Cell exposed to TGF-beta -​ TGF-ß binds to receptors, Receptors dimerize, R-Smad is phosphorylated, R/Co-Smad translocate to nucleus, bind DNA, activate growth inhibiting gene ON -​ No TGF-beta -​ Receptors are monomers, R-Smad and Co-Smad in cytoplasm, Growth inhibiting gene OFF -​ TGF-beta/Smad4 mutant  -​ TGF-ß binds to receptors, Receptors dimerize, R-Smad is phosphorylated, Growth inhibiting gene OFF -​ Genes required to repair DNA damage are also tumor suppressors -​ Gene BS; fxn = DNA/RNA ligase involved in recombination repair -​ Gene XP; fxn = enzymes involved in excision of DNA damage -​ Gene hMSH2, hmLH1; fxn = enzymes required for correction of base-pair mismatches -​ Gene BRCA1, BRCA2; fxn = repair of DNA breaks -​ Tissue specificity -​ Curiously, many are only associated with tumors mainly in one or two tissues -​ e.g., BRCA1 and 2 in the breast and ovary -​ Often true of other tumor suppressors -​ e.g., APC in the gut -​ Cancer cells have unstable genomes -​ Due to defects in DNA repair mechanisms, loss of cell-cycle checkpoints and defects in proteins involved in mitosis -​ Have aberrant karyotypes -​ Chromosome complements such as this would obviously be lethal if all the cells were like this, but individual cells can survive...and grow aberrantly -​ Show gene amplification -​ Some genes may have hundreds of copies -​ May be visible under microscope -​ Double minutes = small chromosome-