Bio 330 Semester Notes
Bio 330 Semester Notes Bio 330, Genetics
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Date Created: 05/04/16
• strategies for regulating gene expression transcriptional control ‣ negative control- transcription always on unless neg reg protein binds to operator to block rna pol at promoter(promoter is strong) • if promoter looks like conserved consensus,will have neg control ‣ positive control- transcription always off unless pos reg protein binds to operator to help rna pol at promoter (promoter is weak) operon- series of genes co-regulated(have same promoter) by regulating synthesis of common mrna (all translated on same mrna) • only in proks ‣ lac operon- under neg control- methylation of lactose and sugar(betagalactisidase)...degradative operon • structural genes- z,y,a..code for things that do what operon is there for z- betagalactisidase-lactose will be broken down into simplier sugars:guclose and galactose(depends on concentration of lactose in cell.high concentration means reaction will occur.but if concentration is low,allolactose(an inducer) is created instead of g & g) y- permiase- brings in lactose a- transacetylase • promoter isnt regulation bc must be there for genes to be expressed.. rna pol binds to promoter to initiate transcrip • operator is next to promoter(for regulation) • i gene and its own promoter are there for regulation rna pol binds to i promoter and mrna for i gene is made and translated into lac i regulatory protein...then lac i reg protein binds to operator to block rna pol and causes transcrip to dec • inducer- no gene codes for it- comes from environment would bind to laci reg protein • partial diploid- add more genes in bacterial cell on plasmid(bc replicates),which makes some genes on bacteria diploid.add plasmid(circular) to the cells that carries the extra genes if you put in a functional gene into a mutant,phenotype should change • catabolite repression- effect of glucose on transcrip levels of multiple operons glucose turns down expression of operons by affecting cap binding site ‣ catabolite activator protein (cap protein) binds with cAMP and binds to cap binding site...when this happens,it helps rna pol bind to promoter • must happen for lac transcription to occur • glucose prevents creation of cAMP adenylate cyclase(creates cAMP) is blocked by glucose • trp operon- biodynthesis of trp (onAA) R codes for reg protein trp reg protien made,and in presence of trp,trp reg protein becomes active and binds to operator,which blocks rna pol from binding to promter(neg control) attentuation- folding of protein..162 nucleotide rna that can alternatively fold ‣ 600 nucleotide transcription units--> neg control--> 8-10 units--> attenuation--> 1 unit ‣ normal proteins have 1/100 trps ‣ other peptide has 2 adjacent trps(2/14).. ‣ availability of trp affects folding of protein by • if 3 and 4 bind together,its a rho-independent termination signal for transcription • enhancers- most have no conserved identiﬁed sequence,most can be up or downstream,or inside the genes they affect • UAS enhancers- have conserved sequences,must be upstream of gene they affect in galactose pathway • hormones- usually pos control...type of inducer bind to hormone receptor protein,which binds to dna • more than one bp changes loss of genetic info ‣ major deletions • loss of chunk of chromosome 5(in humans)...results in developmental problems of child(larynx particularly) -->"cri-du-chat" • fragile x- CGG repeating(middle of FMR1 gene) ....number of repetitions determines how fragile it is(the more repeats,the more fragile) mental retardation ‣ 1/2500 females ‣ 1/1250 males • monosomy- loss of an entire chromosome- usually lethal in-utero unless....individuals with single X chromosome can live...turner's syndrome (sterile females) no loss of dna ‣ inversions- sequence is inverted with respect to normal orientation of chromosome • affects homologous chromosomes pairing up during meiosis..affects production of gametes..affects offspring,not individual • could affect promoters,gene,anything, gaining dna ‣ transposition- region of dna that can make copy of itself and send that copy to another unrelated chrom • could change reading frame a lot,randomly inserts 100s or thousands of bps,disrupting fx ‣ translocation- recombination between nonhomologous chromosomes w/ exchange of genetic material that is not reciprocal • no loss of dna,no phenotype change(individual appears like wt) ‣ aneuploidy- get one extra chromosome • reason for about 95% of downsyndrome..usually when older women have kids • nondisjunuction- chromosomes dont seperate..they move together ‣ gene duplications • ex) one copy of gene for a certain trna,copy starts collecting mutations...if mutated gene acquires new function,then new one could provide beneﬁt ‣ polyploidy- gaining entire new set of chromosomes (become triploid instead of diploid) Make 3 peer wise questions and answer 25 before each exam • Genetics- study of heredity,control of an individuals traits • Prok- no nucleus..Bacteria and Cyanobacteria(acellular).Circular chromosomes with nucleic acids.Ribosomes:70s Infected by viruses called bacteriophages Can get 10^9 offspring per ml in 6-8 hrs • Euk- nucleus,other membrane bound organelles.All other oCellular organisms. linear chromosomes with nucleic acids and proteins.Ribosomes:80s with 70s in organelles Infected by viruses called euk.Viruses • Gene- unit of heredity Stable Mutation possible->variation Stored in common "language" Must be capable of replication (being copied) Correlation with a particular trait or phenotype. • What is the genetic material? 1903 beaver and sutton (seaurchins)...Number of chromosomes affects phenotype 1913-1915- stuatouant and drosophilia(fruit ﬂies)..X chromome--> eye color 1928 Grifﬁth- streptocAcus pneumoniae- mice will die ‣ Have smooth(regular..."Wild type") mucopolysaccharide coat which makes them virulent so they can kill the mice • Different types of coats on outer surface of cells like those above..(Ex: smooth type 1 produced S1 and rough type 1) • Odd shaped colony is called rough(mutants) Can produce revertants(smooth type 1) etc ‣ Mixed rough type 2 with heat killed smooth type 3 and injected into mice(die)...ﬁnds live s3 bacteria in them ‣ Injected just rough 2 into mice(live) ‣ Injected just heat killed smooth type 3 into mice(live) 1944Avery,McClead,McCarty...Agglutination assay.. ‣ Antibody: Allow speciﬁc interaction with speciﬁc mol • Pure r2 coat would be recognized by our bodies as foreign and we would produce antibodies to rid our bodies of r2 ‣ Repeated 1928 experiement with that logic,but puriﬁed components of s3 to get s3 nucleic acid,and added antibodies.Then did agglutination assay and found a pellet of r2 cells with r2 antibody,but in solution found live s3 ‣ With just r2 and anti r2,found pellet and antibodies,no s3 cells ‣ With just s3 nucleic acid and anti r2,found no s3 cells ‣ If add nuclear,get no s3 cells bc it destroys s3 nucleic acid ‣ ...nucleic acid is genetic material • if you mix dead and live bacteria.get live bacteria • immune response killed the mice • • 1953- Hershey & Chase..bacteriophage(T2) P32 phage + bacteria --> blend together and collect--> are bacteria radioactive? are there phage off spring and are they radioactive? ‣ get bacteria that are radioactive,and phage offspring,some of which are radioactive same thing with S35 phage..bacteria were infected by phage,there were phage offspring,but none of offspring were radioactive so can conclude to get phage offspring,have to transfer nucleic acid of phage into bacterial cells ‣ nucleic acid is genetic material for proks ‣ Dna is primary genetic material forT2...what about rna? • tobacco mosaic virus take rna from one(WT or HR mutant),and protein coat from another,mix with tobacco and infect it,and see what kind of offspring do you get ‣ WT rna + HR protein coat=WT ‣ HR rna +WT protein coat= HR mutant ‣ so..rna is the genetic material...so nucleic acid is genetic mat of euks too • chemistry of nucleic acids nucleotides have sugar & base(ctuag) components ‣ rna(ribonucleic acid)..ribose sugar can have CUA or G..but notT ‣ dna(deoyribose)..CTAG..no U triphosphates: ‣ • Chargaff's Rules [G]:[C] are equal;[A]:[T} are equal...for all species if you take (G+C)/(A+T)..ratio varies btwn all species ‣ tells that theres enough sequence variation in nucleotides to acct for variation btwn species • 1954..3D model for DNA..Watson & Crick model ‣ double helix ‣ base pairing btwn strands to hold them together ‣ antiparallel pairing...5' pairs with 3' evidence for model.. ‣ xray diffraction...crystallize something to force into uniform structure,so take crystals of nucleic acid and shoot xrays through them and xrays will pass through if space,but bounce off if hits something..so gives image of what was blocking the rays onto xray ﬁlm • supports double helix ‣ Chargaff's rule is consistent withA andTs of opposing strands pairing together,and Gs & Cs...so got slimy dna(mucus) gotten from breaking open cells and isolating dna. • can raise ph to 12 or temp to 95 deg.C as treatment to try and break H bonds...solution becomes clear and see single stranded chains of nucleotides joined together 5' to 3' from breaking H bonds and breaking strands of dbl helix apart into single strands • G has 3 H bonds btwn it and C • A has 2 H bonds btwn it andT ‣ only way strands ﬁt is 5' to 3' connections • 5' nearest neighbor exp..takes off phosphate of the nucleotide being looked at 5'p-G-p-C-p-A-p-C-p-T-p-G-p-G-p-A-p-T-p-C-p-A-p3' 3'p-C-p-G-p-T-p-G-p-A-p-C-p-C-p-T-p-A-p-G-p-T-p5' if parallel,freq that G is 5' nearest neighbor ofA will = the freq that C is the 5' nearest neighbor ofT if antiparallel,freq that G is 5' nearest neighbor ofA will = freq thatT is 5' nearest neighbor of C radioactive phosphate group btwn eachA and its nearest neighbor,so an enzyme cleaves the bond btwn every nucleotide and its 5' phosphate,so some nucleotides(all 5' nearest neighbors ofA) are radioactive and some aren't ‣ can make table with frequencies ofA's nearest 5' neighbors ‣ repeat with each other nucleotide phosphate group (p-P32-p-t,p-p32-p-c,& p-p32-p-g) ‣ results point to antiparallel because freqs of antiparallel conditions are equal • W & C's predictions from their model DNA could be genetic material right handed helix ‣ 36 degrees btwn base pairs ‣ 10 base pairs btwn turns ‣ ...A.Rich at MIT discovered that more homogeneous strands(crystals) prouduce better diffraction pictures...used zDNA • got zigzagging base pairs • 30 degrees btwn base pairs • 12 base pairs per turn • but..left handed helix ...can be multiple types of dna in chromosome,seperated by zDNA...typical in euks possible replication mechanism- separate the 2 strands(w/out breaking covalent bonds)...each strand becomes template for making a new complementary strand • 3 possible ways DNA could be replicated conservative replication- conserve original mol throughout rep dispersive replication- parts of original and parts of copy in each strand semi conservative • Meselsen & Stahl...e-coli CsCl density gradient to characterize DNA...spin at high speeds and creates gradient with increased density towards bottom..putting biological mol in tube will cause it to move to a point where it equals is buoyant density ‣ two diff types of nitrogen (14 & 15)..can grow on either...N15 dna will be lower in tube than N14 dna • general replication reqs DNA (template) place to start unwind dna way to keep strands unwound something to carry out replication • how to meet reqs origin of rep ‣ prok:often unique ‣ euk:lots helicase unwind dna single strand binding proteins(ssb) keep them apart dnapolymerase carries out rep • steps to rep: origin-where rep starts helicase unwinds towards 5' end single strand binding proteins keep unwound dna pol needs nucleotides to binds to,so primase(rna pol) goes and adds ﬁrst nucleotide (is ribonucleoside) "rna primer" is put on dna pol takes over and adds on to rna primer leading strand(unwinding towards 5' end) is synthesized continuously and lagging strand is synth noncontinuously (unwinding towards 3' end) ‣ dna pol III- does most of rep ‣ dna pol II- not essential ‣ dna pol I- exolnuclease(cuts out rna parts),proofreads strands dna pol I replaces rna primer with dna dna ligase goes through and adds 5' phosphate to join okazaki fragments ‣ ecoli ligase needs nadh to bind them ‣ t4 ligase needs atp to bind them • telomere- added to 3' end of euk chromosomes(linear)...repeated sequence(differs from species to species)..protects dna telomerase- ribonucleo protein (rna(template used by reverse transciptase protein) + protein<-- enzymatic part..reverse transcriptase) ‣ builds buffer it knows will be eaten away over time t loop and d loop fold back on itself at end of telomere • replicon- any piece of nucleic acid that speciﬁes its replication..at least 1 origin..all cellular organisms have one: ex: ‣ chromosomes (10^9 bp is big) • humans,yeast,and ecoli are large ‣ plasmids- replicons that exist independent of the main chromosome • bacteria,some euks(yeast,fungi,some protozoans) • 10^3 bp to 1/3 chromosome size ‣ episome- replicon that can exist as a plasmid or as part of another replicon ‣ organelle dna:mitochondrial dna or chloroplast dna • rolling circle replication(plasmids and some bacteriophages): have to cleave 5' tail hanging off from when original template was gotten..then need to connect it back to itself (if circular) by adding 3' hydroxyl end by adding new primer w/3' hydroxyl wnd so dna pol can add to that and then remove the primer and ﬁll in missing nucleotides with dna(dna pol 1) and then ligase comes in to complete the circle • homologous- nucleotide seq is similar but not necessarily identical • alleles- diff forms of the same gene • meiosis- crossing over in homo chroms to form gametes with random mixture of alleles..gametes diff from parents' • recombination- increases variation • bacteria have ability to take up genetic material from environment...rec a homologous recombination system • mismatch repair then occurs to ﬁx bps...some systems know which strand is original and which is new and will want the new material..so one will be all R11 and one will be all S111 • one chromosome can be used to check the other chromosome • bacteria dont do bc they only have 1 copy of each chrom so rec a is part of a dna damage repair system • transcription- changes dna into rna only for certain gene(subsection of dna) rna pol- 2 alpha polypep subunits,a beta,a beta prime...form core enzyme.then sigma(recognizes signal of where to start) subunit provides speciﬁcity..that pluscore enzyme=holoenzyme promoter- transcriptional start signal conserved sequence at -10...pribnow Box- recognized by sigma subunit conserved seq at -35... for initial binding by rna pol • transcription initiation holoenzyme directed by sigma to ﬁnd pribnow box sigma directs rna to bind to -35 region(of promoter) rna pol unwinds dna and keeps the strands apart ﬁrst(1+) rntp(ribonucleoside triphosphate) which is a purine,and the second(2+) depends on 2+ template make dinucleotide(covalently) join short 8-10 nucleotides..til sigma leaves • elongation carried out by core enzyme as dna recoils as rna pol moves along,the newly synth srna will be forces off and will be hanging ‣ since hanging off,translation could start or it could wind up on itself to form secondary/teriary stxs • secondary stx:stem & loop..recognied by NuSA(protein) and tells core enxyme to pause when it sees one • termination- two types of stem & loops: stem 1:rho-dependant signal stem 2:rho-independent signal • rho protein- binds towards 5' end goal:to ﬁnd rna pol,and kick it off rho is slow,so catches up bc NuSA on core enyme will make ore enzyme pause when it notices rho-dependent signal so rho can kick rna pol off rna falls off dna when NuSA stops(from recognizing rho) bc when its gone things are much less stable and there are only single H bonds,so dna,rna,and rna pol seperate • 3 diff rna pols messenger rna- rna pol 2 • transcription factors are proteins that recognize the nucleotide seq in promoter and bind to it in euks to recruit rna pol and begin transcription • euks have to get rna out of nucleus(where its made) and into cytoplasm(where its used) • in prok ribosome binds to 5' end of dna to begin transcription • in euks,ribosome doesnt know where to bind to transcribe mrna cap put on 5' end to serve as ribosome binding site serious ofA's put on 3' end- polyadenylation- by enzyme ‣ signal for transport out of the nucleus ‣ protect rna from degredation • translation occurs in cytolpasm for both euks and proks • overlapping code- use aug as ﬁrst codon,then next codon is ugg,then ggu • partionally overlapping- aug,then ggu • nonoverlapping- aug,guc,ccc.......(correct w/degenerate code) • codons coding for someAA only have 3rd nucleotide different ex:CCC and CCA will never ﬁnd 2 codons coding for sameAA with middle nucleotide being different • ribosomes= rrna+ribosomal proteins+space • trna pairing btwn anticodon/codon best btwn 1st two positions of codon ‣ third position wobbels(can degenerate) and can vary prok- 70s ribosome- composed of 50s and 30s subunit euk- 80s ribosome- composed of 60s and 40s subunit • proks: initiation reqs:mrna+30s subunit of ribosome+ 1st trna attached to ﬁrstAA 30s subunit must attach to mrna...by using piece of rna found in subunit that is complementary to seq found towards 5' end of mrna(shine-dalgrino seq is rna seq found in mrna before start codon) 16s rrna(3' end) is in 30s and is complementary to shine-dalgrino seq met+trna speciﬁc for met...then enzyme(methyanil trna synthetase) that will take met and attach it to trna ‣ transformylase chemically modiﬁed met to form formyl-met(allows it to be distinguished as1stAA that goes into protein) then 50s subunit comes in and joins onto 30s p(peptidyl) site- peptide produced by this site within the ribosome..area w/ aniticodon A(aminoacyl) site- where new trna with new amino acids come in and attach correct protein to mrna codon ‣ eventuallyAA2 will attach at a site and aa2 will form a peptide bond to ﬁrst AA(met) • elongation factors gate keeper factors peptidyl transferase reaction..met attaches toAA2,so trna that was attached to met before is released,and ribosome moves one codon down mrna,and trna that was attached to met moves to e(exit zone) while it waits to exit GTP hydrolysis + G factor cause the ribosome to move so..aa2 is now in p site,and a site is empty until matching trna with aa3 comes in and ﬁlls it,and peptidyl transferase bonds aa2 to aa3 and elongates chain process then repeats and chain length increases until stop codon is reached in a site,and no trna matches stop codon so termination occurs as release factors recognize it (gcc)gcc[R]ccAUG- kozak seq...start of euk translation ‣ only one start codon on each euk mrna,but proks can have many ‣ lowercase letters arent as conserved as upper case • bacteria mrna "1/2 life of 2 mins to make 6 proteins/rna" every 2 mins degrade 1/2 of the rna you made bc no proofreading and its not efﬁcient(competitive) • operon- only in proks...series of genes whos expression is coregulated by regulating synthesis of their common mrna • before promoter is pribnow box (TATAAT consensus) • promoter- "good"/"strong" (matches consensus) ‣ itranscirption is easy bc its always on and so needs to be controlled...negative control- way to turn transcription down or off "bad"/"weak"( doesnt math consensus) ‣ transcription always off..positive regulation..turns up or on • structural genes- code for speciﬁc proteins or enzymes in pathwat inducer- chemical signal from outside...binds to reg.protein and alters conformation to change way it binds to operator • synthetic operon- biosynthesis of a product for the cell vs • degradative operon- to metabolize or break down something in the cell • operon- functional unit of genomic dna with cluster of genes that transcribe together • lac operon- • c-constitutive- always on • x-gal- substrate for Bgalactasidase(but NOT inducer) when cleaved by Bgalactasidase,changes colors(blue green) reﬂects high enough level of transcription to cleave x-gal if low,stays white • IPTG- gratuitous inducer • plasmid carries gene that makes bacteria resistant to a certain antibiotic(amphicillin),so that way bacteria left all have plasmid can also put gene for part of inducer,such as regulatory gene,promoter, operon,and z gene. so what happens to transcription when we make bacteria partially diploid(for lac gene)? • cap binding site catabolite activation protein- must bind with cyclicAMP(camp),then binds to CAP binding site,which helps rna pol at promoter • catabolite repression- regulates multiple operons simultaneously • glucose prevents synthesis of camp...adenylate cyclase(creates camp) is blocked by glucose • Promoters(DNA) basal level(TF's bind) • Enhancers(DNA) regulate transcription (regulatoryTF's) can affect transcription of more than one gene(that each have own promoter) • helix turn helix common in prok regulatory proteins • homeodomain binding(2 turns)- used for developmental control "muscle development" • dimer formation domains-proteins that bind to DNA have DNA binding domains and area to form a dimer • transcription activation domain- 30 to 100 acidicAAs • galactose pathway- from seaccharomyous cerasusine (euk yeast) not galactose operon- euks dont hav operons and stx genes would have to be co-transcribed and that isnt possible in euks this is a metabolic pathway ‣ chrom II andVII have structural genes • gal I,II,VII.. gal II...allows for transport of galactose from outside cell to inside protein • gal IV,III(regulation) gal IV codes for conserved UAS seq (UAS is not enhancer...must be upstream of stx genes ‣ exerts positive control on UAS seq,so all genes on that chromosome related to this pathway will increase in level of transcription • gal 80- codes for gal 80 protein- wants to bind to gal 4 protein and interfere with its regulation and transcriptional activation • hormone "inducer" • dna methylation- usually decreases transcrip levels ex:control if genes are expressed at one developmental stage or another (fetus vs.adult) sickle cell anemia- adult hemoglobin has mutation..on changed base pair,led to dingleAA change with dramatic effect ‣ treat cells with cytosine...but has replacement of N where C is usually found..dna wouldnt be methylated so fetal genes would be expressed can use methylation to regulate cell differentiation ‣ ex:muscle liver cell- diff genes expressed x- inactivation:x chrom has genes that get turned off due to dna methylation ‣ male sperm methylates diff genes than female egg cell frogs:xenopus oocytes ‣ period of rapid development (10^12 ribosomes/60-80 days)... about 300,000 ribosomes per second • so need a lot of rRNA's • reversible dna ampliﬁcation- start w/ 2 rRNA and end with lots of copies..adults go back down to 2 copies • euks alternative splicing- allows for different exons to be kept to change speciﬁcity ‣ ex) mouse amylase- 2 diff types of amylase,so 2 diff types of exons,and which is expressed depends on what part of the body the mrna is in • exon 1 could be for salivary in when in salivery gland,but could code for something different and be liver speciﬁc when in liver ‣ spliceosome does splicing • proks ex) R17 phage ‣ has single stranded rna as genetic material ‣ 3 genes:A gene,coat protein gene,& replicase gene ‣ single stranded rna usually used for protein syth in bacteria bc ribsome recognizes it and will start translating it into proteins...getA protein,coat protein,and replicase enzyme(catalytic) made ‣ goal of phage:wants to be replicated ‣ R17 phage needs:1 mol of coat protein per phage,1 mol of SS RNA per phage • replicase replicates this rna...1 copy of replicase can replicate many(100) copies of SS RNA..will eventually need more coat protein, so need to stop replicase. • shindelgrino seq on ss rna is on replicase and when there's enough coat protein it will cover this seq up on replicase gene,which stops translation(when enough rna has been produced and enough genes have been translated)...nothing blocks coat protein • mutation- a change in nucleotide seq in the primary genetic material DNA in cellular organisms RNA inVirus/virus like organisms • mutant- result/product/outcome of a mutation ex) organism displaying/carrying mutation,phenotype,gene expression, protein,mRNA,tRNA,rRNA ex) when dna exposed to UV light,thymine-thymine dimer produced(bad) ‣ t's are supposed to be paired withA's on other side,but theT's next to each other bind together...dna damage,messes up replication when repairing dna damage,dna damage repair systems are error- prone and likely to put in wrong nucleotide,resulting in mutation. • mutagens- increase rate of mutation above what is normal mammalian cells..lose ~ 10^4 purines per cell per day ‣ bad bc it tends to make the strand more susceptible to breakage ‣ strand breakage is dna damage...so again ﬁxed by error-prone damage repair systems • selection- creating envir that selects for certain individuals can distinguish btwn trp- and trp+ on same dish • screen- for additional traits/phenotypes • replica plating- since colonies are supposed to represent many many identical cells in the colony,when transferred to sterile surface,the same pattern on the plate is transferred to that sterile surface and you can put diff plates on it to test for diff traits • for bacteria: xgal(blue green bacteria) or xgal+IPTG(blue green bacteria) ‣ can tell about bacteria: • reg protein not binding to operator( B-galactisidase present) constitutive mutant: ‣ lacO^c ‣ lacI^- • theres a mutation(notWT)- bacteria is a mutant • lac operon is affected(some mutation in lac operon) • point mutation- single bp/nucleotide change map to a single point on a genetic map revert to original phenotype(same rate as original mutation appeared) ‣ ex) lacZ+ bacteria....isolated(mutants 1/million cells) • lacZ- mutants(if due to point mutation) should be able to grow and get lacZ+ revertants from every 1/1million offspring)..if it didnt revert 1/1 million it probably wasnt a point mutation • lacZ+ mrna AUGGUCUCACCCACGUAG • vs all other mutations that have more than 1 bp change(typically dont revert to original phenotype) nonsense mutation- codon normally coding forAA now codes for stop codon due to mutation ‣ nonfunctional....loss of function silent "- no observable effect on phenotype ‣ no change ‣ could be • different codon but sameAA • diffAA but similar function • mutations in promoter or operator • changes in trna or rrna missense- codon forAA changed to codon for diffAA ‣ less functional ‣ improves fx,reduces fx,change fx,or could destory fx of protein(ex: sickle cell anemia) ‣ ex)auxotrophic- has a different nutritional req vs that of theWT ‣ ex)conditional lethal- permissive conditions for growth- bothWT & mutant can grow and reproduce restrictive conditions " "-WT can grow and reproduce but mutant cant • ex) temp sensitive mutant- both can grow at 37 degrees(permissive) but at 42 degrees,WWT grows,but mutant doesnt ‣ protein may denature at higher temp bc of missense mutation • suppressor- second mutation that masks phenotype of ﬁrst mutation must be caused by point mutation...revertant now has 2 mutations and is functional • genetic mapping why do we care where these genes are located on chroms? ‣ can predict what happens in terms of heredity ‣ A more likely to show up next to B rather than E or F • bacteriaphage ...what traits? life cycle:(lytic cycle) ‣ 1) attach to outer surface of host bacterial cell ‣ 2) inject genetic material(nucleic acid) into host ‣ 3) hijack host "machinery" ‣ 4) make phage "offspring" ‣ 5) release phage offspring..cell lyses and releases them LC:lysogenic cycle (extra steps not in lytic cycle)..temperate phage(90%) ‣ phage nucleic acid either: integrates(recombines) with bacterial chrom replicates as plasmid • then phage remains dormant..offspring not produced,but nucleic acid of phage is being maintained in infected cell • this can occur then go back to lytic cycle by "induction" threat to bacterial cell detected by phage may cause for this to happen phage cant metabolize lactose--> the phages metabolize bacteria(who metabolizes lactose) mutant phages- could lose ability to infect bacteria ‣ phage could look different(turbid/non turbid)(big/small) • genetic mapping in bacteriaphage T4 virulent phage ‣ r2 mutant(fromT4)...conditional lethal.. • permissive:WT & R2 can infect & reproduce ecoliB • restrictive:WT can infect & reproduce..R2 cant ecoliK • 1) distance- how far is mutation in each R2 mutant from the mutation in every other R2 mutant • 2) function..how many diff genes(if mutated) will give R2 phenotype? if we have 2 R2 mutants,are their mutations in the same or different gene? ‣ what happens if take R2(96) and R2(48) and infect ecoliB...permissive conditions • will get R2(96) offspring and R2(48) offspring • the 2 pieces of dna could undergo homologous recombo while in bacteria..produces recombinants: ‣ could get double mutant dna ‣ could getWT dna with no mutations(from recombo or revertants) freq of recombos has yo do w/how far apart mutations are ‣ the further apart the easier random recombo is ‣ the closer together they are the less recombo there is ‣ take R2(96) and R2(48) and infect ecoliK ..restrictive conditions..woul giveWT(recombinant) orWT(revertant from pt mutations) • map distace formula= )#WT under restrictive conditions...K )/(total # of phage offspring under permissive conditions..ecoliB)x100x2 multiply by 2 bc for everyWT produced there should be an = # of double mutants(under restrictive conditions double mutants dont produce offspring) ..counted indirectly (bc cant be seen directly bc wont repoduce) • freq of revertants(for any R2 mutant)=(#WT in K ) (Total # offspring in EcoliB(permissive)) if revertants occur,it is a point mutation that causes phenotype if no revertants,no point mutations • function- are the mutations same or different genes? infect K with R2(98) and R2(43) (restrictive conditions) ‣ yes..mutations are in diff genes bc... no offspring because mutations in same gene • distance 1500 map units= 10^4 base pairs ‣ so 1 map unit is (10^4)/1500 • bacteria- info transfer btwn bacteria cells conjugation- requires cell to cell conntact ‣ F episome- has genes:F pilus(protein)+ability to transfer a copt to another cell • episome- replicon(piece of dna that can replicate) that can exist as a plasmid or part of another replicon • when these two cells touch,F episome initiates rolling circle rep knick made in origin on one strand(provides 3" OH and 5" phsophate end).as nucleotides added,5' p group comes off plasmid, and is sent over into F- cell,so that by the time the beginning is reached,the outer strand has been sent into F- cell....creates copy of F episome in F- cell,so that when complete,F- cell is now F+ cell and devlops pilus • when F integrated into chrom,cell is called Hfr (high freq of recombo btwn chrom and whats sent over into F- cell) • dont stay together,so whole chrom and whole F is not fully transferred,so not capable of becoming F+ • in order for dna that came from F+ cell to be used,must be integrated into F- chrom(bc its homologous) so it undergoes recombo with F- chrom once integrated,allows us to see distance between genes and the order they are in • interrupted mating allow mating at selected time points:take an aliquot-->vortex(shake it up)--> stop transfer ‣ at 1 min,5 min,10 min,20 min,30 min,40 min ‣ select recombinants- must be strR(from F-),Lac+,Gal+..must have all 3 because these are the functional genes if thy are strR then they are not Hfr cells bc Hfr cells have str (Str is at the end of the chrom so it will be the last thing being transferred at 3' end) look for order and distance of genes on chrom..as time increases,you're going to get more genes transferred to F- • conjugation F w/ Hfr F w/ R (plasmid...carry drugR(resistance) genes) • transformation- transfer of naked dna(not in any cell/virus,etc...sitting in environment) ‣ ex) like avery mcleod mccarty exp:naturally "competent" to take up dna ‣ ex) treat cells with chemicals to make them competent--> so they will take up a plasmid • transduction- phage mediated specialized transduction ‣ ex) phage (temperate..lysogenic or lytic cycle) • enters bacterial cell,becomes circular...at speciﬁc region on chrom(att ,recombo will occur btwn dna and bacterial chrom, generalized "- P1 dna always seperate from chrom(even during lysogeny) ‣ can switch to lytic cycle,and when this happens,it chops up bacterial chrom ‣ 2/1000 P1 offspring will carry bacterial DNA instead of P1 DNA(if DNA less than 10^5 bps) • when they infect next cell they infect it with bacteria so they dont really infect it bc that piece is less than 10^5 bp and it has to recombine with bacterial chrom and it will be homologous and recombine(could mask mutations) • co-translation of bacterial gene freqs lac and gal are cotranslated 40% of the time (can be no further from 10^5 bps apart) gal and trp " " 5% of te time (less than 10^5 bps apart) • euk mapping 1865- Mendel • quantitative • easy identiﬁable traits and looking at 1 or 2 at a time ‣ pea plants...can be hermaphrodites(male and female parts) and can self fertilize • simple traits: tall x dwarf(monohybrid cross..get F1 gen..all tall) ‣ breed til all tall parents..Po..true breeders only breed F1 to get F2 Mendel's Rules ‣ one unit from each parent • so in each individual there is a pair same=homozygous different=heterozygous ‣ in herterozygotes,1 version dominant,other is recessive ‣ segregation of units at each generation- alleles of a gene separate in gamete formation • mitosis- diploid(2n) produces diploid • meiosis- diploid(2n) produces haploid • ***AMANDAS NOTES**** • blood types... H locus:H or h alleles ‣ if homo recessive,have bombay phenotype- affects what happens at locus(allows addition of fucose to H substance precursor)..dont have fucose, sodont have H substance,so arent even type o blood • hard to get transfusion • epistasis- not type o,so not type a,b,or ab...so h locus(when hh) phenotype overrides phenotype fromABO locus ‣ locus- close cluster of genes ex) mice color ‣ A- agouti ‣ aa- black ‣ B- goes back toA locus ‣ bb- albino • sex determination- what det sex of individuals in certain species • humans: ‣ XX- female ‣ XY- male ‣ XO- sterile female...turner's syndrome ‣ XXY- male..Kleinfelter syndrome ‣ XYY- male male: ‣ SRY locus- sex determining region ofY(only characterized gene on y) • testes det factor ‣ X: • androgen response gene(testosterone response) mutation in that gene means response to testosterone wont happen..so baby will look like a female..testicular feminization syndrome (in XY males) • sex linked traits- genes located on sex chrom X- color blindness and hemophilia Y- SRY • x linked recessive- criss cross pattern of inheritance...skips a gen • autosomal- not sex linked • stain cells- ‣ ex) individual is XX...stain chrom..get barr body(inactivated x chrom) ‣ ex) " " XY...don't get barr body ‣ ex) XXY..get barr body ‣ ex) XXX..get 2 barr bodies x chrom is methylated so clumps...get 1 for every inactive x chrom ‣ # barr bodies= #x chroms-1 Mary Lyon Hypothesis- ‣ if start w/ 2 x chroms in somatic cell,at some point during development, one x will become inactive..when offspring are made,the same chroms will remain active and inactive as they were in the parent cell • ex) calico cat- female must be XX and male XXY...cat coat color is C linked • • trait(looked at by mendel) chromosome traits found on seed color (Y/G) 1 seed coat (colored/white) 1 mature pods(smooth/wrinkled) 4 plant height(1m/.5m) 4 unripe pods(G/Y) 5 mature seeds(smooth/wrinked) 7 • how to use all that for mapping closer together,lower recombo freq farther apart,higher recombo freq • morgan & Sturtuant 3 traits in drosophila ‣ yellow- body color ‣ white- eye color ‣ miniature- wings recombo freq ‣ yellow to white .5% ‣ white to mini 34.5% ‣ yellow to mini 35.4% map distance= single crossover+double crossover single crossover most common
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