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Lecture 5 and 7: Bio 2

by: Bree Scalzo

Lecture 5 and 7: Bio 2 BIOSC

Marketplace > Biology > BIOSC > Lecture 5 and 7 Bio 2
Bree Scalzo
Foundations of Biology 2
Dr. Swiganova

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Foundations of Biology 2
Dr. Swiganova
Class Notes
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This 14 page Class Notes was uploaded by Bree Scalzo on Friday February 6, 2015. The Class Notes belongs to BIOSC at a university taught by Dr. Swiganova in Fall. Since its upload, it has received 168 views.


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Date Created: 02/06/15
Lecture 5 Gene Expression in Eukaryotes 1 Overview a Eukaryotes i Amoeba ii Yeast HL Sponges iv Fungi v Plants vi Animals b Multicellular organisms use i Differential gene expression signal transduction pathways c Unique to Eukaryotes i lntrons and exons ii Splisosomes iii mRNA stability 5 and 3 cap 2 Chromatin Remodeling a Experiments on blood cells treated with DNase proved chromatin remodeling b H1 proteins bring histones wrapped with DNA together to make Chromatin c Super condensation i If DNA is in condensed form gene expression is off so no free DNA for RNA polymerase default condition 1 Need stimulus to turn on 3 DNA Methylation alters chromatin mechanisms a DNA methyltransferasecatalyzes transfer of methyl group to cytosine residues on DNA i Results in condensation therefore no gene expression b Actively transcribed genes have low level of methylation 4 Histone modi cation can increase or decrease chromatin condensation i Functional groups added to histones 1 Methyl groups 2 Phosphate groups 3 Short polypeptide chains 4 Acetyl groups b Histone Acetylation 1 Histones are usually positive and DNA is usually negaUve ii Adding acetyl groups to positively charge lysine residues on histones iii Causes chromatin decondensation lV Introduces neutral charge that causes disassociation of negatively charged DNA from the histone v Promotes gene expression Vl Acetyl group serves as a binding site for other proteins 5 Chromatin remolding complexes causes nucleosomes to slide along DNA or histones to disintegrate from DNA thus promoting transcription a If histones are resistant to acetylation there will be low levels of transcription 6 Chromatin modi cation can be inherited a Epigenetic Inheritance i May not based on differences in genomic information ii Parents pass down gene expression regulation 1 Research study with mother mice fed low protein diet a Pups put in regular diet b Found that pups with moms on low protein diets had more condensed chromatin i Sig difference in chromatin remodeling 2 lnherited pattern of gene regulation 7 Transcription Regulation a Promoterproximal elements i Regulatory sequences close to promoter speci c for different sets of genes b 1970 s Yasuji Oshima experiment on yeast galactose metabolism i 5 enzymes located on different chromosomes ii Upregulated when galactose is present downregulated when galactose absent iii Regulatory protein activator common for all 5 genes binds to a common regulatory sequence shared by all 5 genes 839 Enhameer Fram t rr Sta Site Enhancer Enhanger lUpatrcianl Promoter Ei l39l lintr39ciri EIUI I lintrnn lEmn Bowmanearn 4 Can 5233 regulatory sequences be located far from the promoter In the Intron a How are the introns related to gene expression i Steps 1 lsolate genes 2 Remove sections of introns 3 Reconnect fragments 4 Look for transcription of genes a Found that regulatory sequences can be located far from the promoter even downstream of a promoter and in an intron i Called silencers like operators or enhancers depending on function 9 Enhancersregulatory sequences far from the promoter can be located introns or UTRs a Unique to eukaryotes b Bind with activator proteins c Can be located on either the 5 or 3 side of a gene 10 11 12 13 14 earbe goom Many types exist Most genes have more than one enhancer Usually bind more than one protein Can work even in ipped orientation 3 l 5 or if they move to a new location in the vicinity of the gene i Compared to active binding site of prokaryotes Transcription factors Activators i Bind to enhancers Repressors i Bind to silencers a Both are regulatory transcription factors Basal transcription factors i Bind to promoters During development cells differentiate into a large variety of tissues such as muscle tissue connective nervous epithelial i All have same genome ii What determines that a cell becomes a muscle cell and not a liver cell 1 Speci c extracellular signal l speci c signal transduction response activation of speci c transcription factors regulation of speci c gene sets How do transcription factors recognize where to bind to DNA Inside double helix will have particular shapes i Created by Hydrogen bonding They recognize a speci c sequences of bases in target DNA What is a gene in eukaryotes A gene is portions of DNA that codes for a functional polypeptide or RNA molecule Differential Gene expression in eukaryotes Cece communication Chromatin remodeling Regulatory proteins bind to enhancers and silencers Different sets of genes are expressed Transcription initiation in eukaryotes Marking on chromatin to get ready for transcriptionexpression chromatin remodeling occurs i Loosening up DNA from histones Exposer of promoter and regulatory sequences i TATA binding protein recognizes TATA box RNA polymerase and basal transcription factors bind around DNA i Aka Assembly of proteins DNA looping by mediator by binding the enhancers and promoterproximal elements to it i Transcription can nally occur 15 Compare and contrast basal transcription factors vs regulatory transcription factors 16 PostTranscriptional Control a Alternative splicing is the major regularity mechanism in multicellular eukaryotes i Human genome contains about 20000 coding genes and 90 undergo alternative splicing explains why we are so complex 17 RNA Interference In uences mRNA stability Short segments of RNA molecules that are either encoded in the nucleus or brought into cell from the outside exogenous 18 RNA Interference a Dicer i DoublestrandedRNAspecific ribonuclease ii Cleaves double stranded RNA to about 21nuclotide siRNA duplex b Argonaute i Binds the 21nuclotide duplex ii Selects one strand and binds it to mRNA guide strand iii Second strand gets degraded c RNAinducing silencing complex RISC i Consists of short siRNA argonaute protein other proteins ii Cleaves the mRNA 1 SiRNA has perfect match cutting mRNA 2 No more proteintranslation will be made from mRNA O39QJ d Seed i Region that is complementary to mRNA 1 Why Too many proteins mutation in mRNA and needs to be stopped 19 Other mechanisms to control translations a Phosphorylation of regulatory proteins i High temp fever ii Viral infection b Posttranslational control 1 Either needs to be activated or inactivated Protein foldingchaperones iii Glycosylationmarking own cells iv Phosphorylationadding phosphate groups v Ubiquitinationproteasomes attaching marker to protein to be destroyed 20 Cancer and Defects in Gene Regulation a Tumor suppressor genes i Genes coding for proteins that stop the cell cycle ii In 61 phase checks for quality of DNA b Protooncogenes i Genes that code for promoting cell division c Cancer results from mutations in genes coding for regulatory proteins of the cell cycle i P53 the quotguardian of the genomequotregulatory protein Directs signals to someone else that does the work If DNA damage promotes cell arrest DNA repair or apoptosis lf doesn t work cell replicates with mutation If not okay prevents cell division 21 Gene Expression a Bacteria vs Eukaryota Level of Regulation Bacteria Eukaryotes Chromatin remodeling Limited packaging Extensive of DNA Remodeling not a major issue in regulating gene expression packaging of DNA Chromatin must be opened for transcription to begin Transcription Positive and negative control by regulator proteins that act at sites close to the promoter Sigma interacts with promoter Positive and negative control by regulatory proteins that act at sites closefar from the promoter Large basal transcription complex interacts with promoter Mediator complex required RNA processing None documented Extensive processing alternative splicing of introns addition of 5 and 3 caps mRNA stability 0 Some RNA 0 For many genes inference RNA interference documents limits life span or translation rate Translation 0 Regulatory Regulatory proteins bind to mRNAs andor ribosomes and affect translation proteins bind to mRNAs andor ribosome and affect translation rate rate Posttranslational 0 Folding by 0 Folding by modi cation chaperone chaperone proteins proteins 0 Chemical 0 Chemical modi cations may modi cation change activity 0 Ubiqination targets proteins for destruction by proteasome One plausible explanation for why so many more different proteins must assemble on promoter regions to initiate transcription in eukaryotes vs prokaryotes The use of large numbers of proteins and regulatory sequences in DNA open many possibilities for the sophisticated gene regulation required in eukaryotes Lecture 7 Methods of genetic engineering Cloning Human Growth hormone Polymerase Chain Reaction PCR Sanger sequencing Application in medicine Recombinant DNA technology Human Growth Hormone a GH1 codes for a pituitary growth hormone b Normal versus GH1de cient normal 739 Normal 6H1 gene Defective 6H1 gene l l bd ulh INHIOWHMDJNIDQ l l l l Little or no 6H1 protein produced in pituitary gland l Pituitary dwar sm Normal amount 0 slower growth GH1 protein produced shorter stature 2315 Human Growth hormone Genomics sequence 2660 bp genomic sequences GAATTCAGCACTGAATCATGCCCAGAACCCCCGCAATCTATTGGCTGTGC39I39I39TGGCCCCTI39TI39CCCAACACACACATI39CTGTCTGGTGGG quot quot quotATGC quot quot quot quotTAGGATAGAGAGTGGGATGGGGTC GCTAGGGGTCTCAAGGACTGGCCTATCCTGACATCC39I39I39CTCCGCG39I39I39CAGGTI39GGCCACCATGGCCTGCTGCCAGAGGGCACCCACGTGACCCTTAAAGAGAGGACAAGTTGGGTGGTATCTCTGGCTGACATTCTGTGCACAACCCTC ACAACGCTGGTGATGGTGGGAAGGGAAAGATGACAAGTCAGGGGGCATGATCCCAGCATGTGTGGGAGGAGCTTCTAAATI39ATCCATTAGCACAAGCCCGTCAGTGGCCCCAGGCCTAAACATGCAGAGAAACAGGTGAGGAGAA GCAGCGAGAGAGAAGGGGCCAGGTATAAAAAGGGCCCACAAGAGACCAGCTCAAGGATCCCAAGGCCCAACTCCCCGAACCACTCAGGGTCCTGTGGACAGCTCACTAGCGGCAATGGCTGCAGGTAAGCGCCCCTAAAATCCCTT TGGCACAATGTGTCCTGAGGGGAGAGGCGGCGTCCTGTAGATGGGACGGGGGCACTAACCCTCAGGT39I39I39GGGGC39I39I39ATGAATGTTAGCTATCGCCATCTAAGCCCAGTATI39TGGCCAATCTCTGAATGTTCCTGGTCCCTGGAGGAGG C quot quot quot quot quot quot quot quot CCAUL I LL I tuAACAGGGAGAGCGCTGGCCTC39I39I39GCTCTCCAGCTCCCTCTG39ITGCCTCCGGT39ITCTCCCCAGGCTCCCGGACGTCCCTGCTCCTGGC39I39I39TI39GGCCTGCTCTGCCTGTCCT GGC39I39I39CAAGAGGGCAGTGCCTI39CCCAACCATTCCCTI39ATCCAGGC39I39I39T39I39I39GACAACGCTATGCTCCGCGCCCGTCGCCTGTACCAGCTGGCATATGACACCTATCAGGAGTTI39GTAAGCTC39I39I39GGGTAATGGGTGCGCTTCAGAGGTGG CAGGAAGGGGTGAAT39I39I39CCCCCGCTGGGAAGTAATGGGAGGAGACTAAGGAGCTCAGGGTTG39I39I39TTCTGAAGTGAAAATGCAGGCAGATGAGCATACGCTGAGTGAGGTTCCCAGAAAAGTAACAATGGGAGCAGGTCTCCAGCAT AGACCTTGGTGGGCGGTCCTTCTCCTAGGAAGAAGCCTATATCCTGAAGGAGCAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGCTTCTCAGAGTCTA39I39I39CCAACACCTTCCAACAGGGTGAAAACGCAGCAGAAATCTG TGAGTGGATGCC39ITCTCCCCAGGTGGGATGGGGTAGACCTGTGGTCAGACCCCCCGGGCAGCACACCCACTGCCGGTCC39I39I39CCCCTGCAGAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCATGGCTGGAGCCCGTGCA GCTCCTCAGGAGCGTCTTCGCCAACAG CCTGGTGTATGGCGCCTCGGACAG CAACGTCTATCGCCACCTGAAGGACCTAGAG GAAGGCATCCAAACGCTGATGTGGGTGAGGGTGGCACCAG GATCCAATCCTGGGGCCCCACTGGC 39I39I39CCAGGGACTGGGGAGAGAAACACTGCTGCCCTC39I39I39TI39TAGCAGTCAGGCGCTGACCCAAGAGAACTCACCGTA39I39I39C39I39I39CA39I39I39TCCCCTCGTGAATCCTCCAGGCCTI39TCTCTACAACCTGGAGGGGAGGGAGGAAAATGGATGAATG AGAGAGGGAGGGAACAGTGCCCAAGCGCTTGGCCTCTCC39I39I39CTCTI39CC39I39I39CACT39I39I39GCAGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAATCAGTCCTACAGCAAGTTI39GACACAAAATCGCACAACGATGACGCAC TGCTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGCTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCC TCTCCTGGTCGTG GAAGGTG CT ACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTG CATCA39I39I39TI39G39I39I39TGACTAG GTGTCCT TGTATAATA39I39I39ATGGGGTG GAGGCGGGTGGTATGGAGCAAGGGGCCAG GTTGGGAAGAC AACCTGTAGGGCCTTCAGGGTCTATTCGGGAACCAGGCTGGAGTGCAGTGGCAGTCTTGGCTCGCTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGTCTCCCGAATAGTTGGGA39ITCCAGGCATGCAAGACCAGGCT CAGCTAA39I39I39TI39TGTAT39I39I39TTGGTAGAGACGGGG39I39I39TCACCATA39I39I39GGCCAGTCTGGTCTCCATCTCCTGACCTCAGGTAATCCGCCCGCCTCGGCCTCCCAAATTGCTGGGA39ITACAGGTATGAGCCACTGGGCCCTTCCCTGTCCTGTGA 39I39I39TI39AAAATAA39I39I39ATACCAGCAGAAGGACGTCCAGACACAGCATGGGCTACCTGGCCATGCCCAGCCAG39I39I39GGACA39I39I39TGAGTTGTTI39GC39ITGGCACTGTCCTCTCATGCA39I39I39GGGTCCACTCAGTAGATGCTTGTTGAATI39C HGH mRNA 822 nts APPA39T f f f AAFFFFFAAF39T FFFFI AAFFAF39T FAFFF39T FF39T F39T FI AFAI F39T FAFF39T AFF39T FFAAII I I IAI AI I I II I I I I AI I II I I II I II I II I I I I I II I I I II I II II I I II I I I II I I I II AI II FF39F39T FFFAAFFA39F39T FFF39F39T A39T FFAFI I AAIIIIAIIIIIIIIIIIIAIIIIIIIIAIIAIIIIIIIIIIIAIAIIIAII IIA39T A39T FFF quot AIIII39T f f AFAAIIIIIAIAIIIIIIIIIIIIIIIIAIAIIIIAIIIIIAIAIIIIIIAAI AFAAFAFAAA39T FFAAIIIAIAIIIIIIIIIIAIIIIIIIIIIIIIIAIIIAIIIIIIIIIIIAIIIIIIIIAII 39T FI II I39T FI FFAAFAI FI I I I I I I II II AI AI I AAI I II IAII AI I II I quot I A39T FFAAAI I I AI I I FFFI I AF39T I FI FAFA 39T F39F39T f AAFI AFAFF39T AFAFI AAF39T W FI AFAFAAAF39T FAFAFAAII AII AIIIAI IAI II AAI AAI IAIIIII III II IAI III III AIII AI AAII III AI AI AI III IIIII AIII IIIAI IIIIII I I AIIIIIIIIIIIIAIIIIIIIIIIIIIIAIIIIIIIIAIIIIIIIIIAIIIIIIIIIIIIIIII IAF39T FFAF39T FFFFAFFAI FF39F39T F39T FF mquotquotquotquotquotquotquotquotquot A39T FA HGH protein 192 aa MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPV QFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLR IVQCRSVEGSCGF Explain the size disproportion between the sequences and the growth hormone Dot plot comparison of genomic sequence and mRNA 0 500 1000 1500 2000 2500 0 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 200 I 39 Ch I 400 3 39 0 Z 600 gt 800 HGrigenon csequenceGrH What can you conclude from the Dot Plot 2315 Growth Hormone Decline Early treatment 0 10 30 20 60 40 50 70 80 90 Cows amp pigs GH noneffective Ageless HGH from human cadavers associated with prion disease Drug rare and expensive 1984 usage of HGH from human cadavers banned Current treatment Genetic engineering cloning HGH into Ecoli Making cDNA from mRNA l Reverse transcriptase l gt dsDNA ss mRNA Reverse transcripts Doublestranded cDNA Reverse transcriptase Singlestranded cDNA or I Doublestranded cDNA PFFr39n er DNA polymerase How many di erent mRNAs are in a cell isolated from pituitary gland How a 1 many of those code for human growth hormone 2315 Inserting cDNA into plasmids cloning vectors PROCESS INSERTING GENES INTO PLASMIDS 539 3 m nc 5 anquot ounc 3 requot cnuoquot39 an M n 5 3 V 5 Th 3 9 am 0 9 I Idont y a palindromic 3 an an m quotcognition site Attach same 339 It 1 i m recognition site tothe CDNA F39r c J I I 9 quot I 391quot 39 n quot Iquot Ptaumd quotr Restriction ondonucluu IEcoMI outquot 2 Add rootnctbon y C 39 39 0 ondonoctoou f I l 71 I I i 5 Restriction endonucleases cut 397 39 39 o o r 4 t m DNA sequence at palindromic sequence forming sticky ends 0 AAquot u w39cgcrtu 1 M znu 5 39 r 3 Shaky and mum Sticky ends 2 Shcky and 1 1quot quotr lt Kquot J 3 Gone u c give 4 i Th 4 Insert gone into plasmid Rocon39bmam 1r I quot plasmid 3 Vquot TquotLquot rquot39quot M iiiquot I PROCESS CREATING A cDNA LIBRARY quot ti M mRNA M tlsolato mam New 0 transcriptase stranded CDNA 2 Syntheslu cDM mRNA Double 3 Make 00 doubleostranded stranded cDNA O Where is the gene coding for 0 3 human growth hormone cDNA library is a collection of cDNAs each inserted into a vector isolated from particular cell type or tissue 6 23Wc 39u I a 397 Recombinant quot plasmid 1 cDNA v t l 1quot L 4 Make recombinant plasmid 5 Transformation I A n t MNI t 040139quot I 2315 ssDNA probe is used to locate the gene of interest in a pool of single stranded sequences Hybridization between the DNA probe and ssDNA sequence based on nucleotide complementarity probe 2 Expose probe to singlestranded DNA 3 Isolate labeled DNA 1 Make labeled PROCESS USING A DNA PROBE Labeled probe single stranded I39mI39rm Screening cDNA library for GH mRNA PROCESS SCREENING A cDNA LIBRARY l Grow transformed cells 2 Sample colonles with filter 3 Make DNA slngle stranded Labeled probe sunI39d I o Labeled colony 39i 39 E can contalnlng growth hormono 4 Probe cDNAs 5 Find probe 8 Identify labeled colony 2315 What is the next step To produce large quantities of the HGH protein the gene has to be cloned into expression vector and transformed into E coli cells Expression vector contains bacterial promoter ORI origin of replication antibiotic resistance gene coding region of the HGH fusion protein lacZ or trpD Bacteria expressing HGH grown in large quantities HGH harvested and purified Polymerase chain reaction PCR Iquot l Limitations Amplification of target DNA sequence must know primers Size of ampli ed fragments 1000 1500bp fragments 2315 2315 a PCR primers must bind to sequences on either side of the target sequence on opposite strands Primer 539 f blndlngJEItG 3 3 y Y J V J 539 Primer Region of DNA to binding site be amplified by PCR b When target DNA is made single stranded primers bind and allow DNA polymerase to work 5 DNA polymerase 3 5 5 3 m 3 5 PCR reaction PC R O n To Each tube add in this order 20100ng of DNA template In vitro DNA replication 75W dHZO low of 10X Buffer 339 5 Taq polymerase will of 10mm dNTPs 39 2w primer F 39 Z 39 Zquot 39 Z ZptlprimerR f 39 E lul Taq polymerase V5 3 I dNTPs Primers PCR cycling 959C for 3 mins 959C for sec Denatura on 4563 for 30 sec 2530 Annealing 729C for 30 sec cycles Extension 729C for 7 min 49C forever 2315 Draw a graph depicting accumulation of ampli ed DNA during PCR reaction Ampli ed DNA cycles Sanger sequencing dideoxy DNA sequencing ddNTPs terminate DNA synthesis K R a B R ek 39 s39CHz Base 539CH2 Base 7 39 2 339 Q I 0 H gj No OH Normal dNTP ddNTP A I lt extends DNA strand terminates synthesis inntrim limllkmgll V V V V A C C 5 A A c A C


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