MOL GEN CROP IMPROVMT
MOL GEN CROP IMPROVMT AGR 5307
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This 52 page Class Notes was uploaded by Elaina Gorczany on Friday September 18, 2015. The Class Notes belongs to AGR 5307 at University of Florida taught by Fredy Altpeter in Fall. Since its upload, it has received 39 views. For similar materials see /class/206611/agr-5307-university-of-florida in Agricultural & Resource Econ at University of Florida.
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Date Created: 09/18/15
AGR 5307 10405 Genome Organization in Plants Transcription of the Eukaryotic Genome Fredy Altpeter 2191 McCarty Hall faltpetermailifasufedu Subcellular compartments for storage transcription and translation of gene c information Organization levels of genetic information in eukaryotes Genome Chromosomes Genes Nucleotides DNA in the Nucleus is Organized into Chromosomes Chromosomes One very long linear dsDNA moleculechromosome with Single copy Repetitive and Highly repetitive sequences Centromere sequences Two teleomere sequences Multiple origins of replication Proteins that fold and pack the long DNA strand into more compact chromatin Histones Nonhistone chromosomal proteins INTERPHASE I telomere T I q replication I n I origin 39 quotVJ centromere I p l39 Packing of DNA into the Nucleus Multiple Levels of Compaction A maize cell contains 10 m of DNA stretched end to end that must fit in a nucleus that is lt10 uM in diameter Compaction is 1000 fold for interphase chromosomes and 10000 fold between dsDNA and mitotic chromosomes short region of DNA double helix 3939 39 2 m quotbeadson astringquot 39 form of chromatin 3fold 30nm chromatin fiber of packed nucleosomes 27 fold section of chromosome in extended form 700 fold condensed section of chromosome 1000 fold 9 U centromere entire 39 139 lg mitotic gt chromosome NET RESULT EACH DNA MOLECULE HAS BEEN PACKAGED INTO A MITOTIC CHROMOSOME THAT IS 10000FOLD SHORTER THAN ITS EXTENDED LENGTH Figure 4 55 Molecular Biology of the Cell 4th Edition l 1400 nm i Nucleosome Core Structure Core is a histone octamer with 2 subunits of H2A HZB H3 and H4 with DNA wrapped around 165 turns in a left handed coil Histones are basic proteins rich in lysine and arginine that make salt bridges with the backbone phosphates Hydrophobic bonds and salt bridges hold the core together and the DNA The long amino terminal tails of each histone extend out Modification of Histone NH4 Terminal Tails Affect the Stability of 30nm Fiber and Higher Order Structures The NH4 tails of the histones in the nucleosomal core are reversibly Acetylated by histone acetyl transferases Deacetylated by histone deacetylases Phosphorylated by histone kinases Dephosphorylated histone phosphatases Methylated by methylases Demethylated by demethylases H4tail HB tail H2Atai 1 VA HStail H4 tail A H3 tail Figure 4A32 Molecular Biology of the Cell 4th Edition Histone Code Hypothesis Distinct markings of histone tails confers particular meanings by attracting those proteins involved with appropriate func ons Gene expression should not take place DNA has been recently replicated Nterminal tall 910 14 18 23 28 Ac Z Z l Me rHlSTONE H41 HISTONE H3 l 39 39 I e o E modification state margin liufvggiw unmodified gene silencing acetylated gene expression acetylated histone deposition methylated gene silencing heterochromatin phosphorylated mitosismeiosis phosphorylated acetylated gene expressmn higherorder 7 combinations unmodified gene silencing acetylated histone deposition acetylated gene expression Figure 4 35 part 2 of 2 Molecular Biology of the Cell 4th Edition Chromatin Remodeling Dynamic Repositioning of Nucleosomes remodeling complex A Chromatin remodeling standard complexes are multisubunit protein c complexes that hydrolyze ATP to change the structure of the nucleosome l core so that the DNA ds xcli zm additipnpf becomes less tightly Namignngdgixng 33239 associated quot Movement 01 the HZA AND 59325333ERAREEFEESEE SESSTo amp dimers in the DNAPACKAGED IN NUCLEOSOMES n u Figure 4 34 Molecular Biology of the Cell 4th Edition be the mechanism Genome organization A vast array of genes reside on any one Chromosome 39u39 u Genome organization A genetic map can be created for a chromosome by assigning genes to their relative position on a chromosome F If 39 39 Q w u Individual genes within the physical map can be analyzed as to their specific sequence of building blocks nucleotides Genome organization Euchromatin Euchromatin is threadlike delicate It is most abundant in active transcribing cells See Alben s et al Molecular Biology of the Cell pp 351 352 Model for Higher Order Euchromatin Structure 30nm fibers are folded into loops of 20000100000 bp that are attached to a scaffold through matrix attachment regions MARS Chromatin remodeling unfolds 30nm fibers to expose the regions for other proteins to access and perform functions such as transcription and DNA replication looped domain folded 30 nm fiber ii proteins forming chromosome scaffold histone modifying enzymes chromatin remodeling complexes RNA polymerase high level expression of genes in loop Figure 444 Molecular Biology of the Cell 4th Edition Heterochromatin Structure Heterochromatin contains few genes Heterochromatin is highly condensed and more compact than the loops of 30nm fibers Remains tightly condensed even in interphase Heterochromatin regions can spread and retract Histone tails are underacetylated and methylated in heterochromatin Heterochromatin represses gene expression Centromeres and telomeres are organized in heterochromatin lAl telomere telomere ADEZ gene at normal location on chromosome white colony of yeast cells ADE2 gene moved near telomere red colony of yeast cells with white sectors 8 white gene at normal location heterochromatin rare chromosome inversion white gene near heterochromatin Figure 4 45 Molecular Biology of the Cell 4th Edition Questions About Genomes amp Genome Organization Genome size of various organisms Does the size of the genome correlate with organism complexity How many genes are present Does the number of genes correlate with organism complexity What is the arrangement of genes on chromosomes Is this conserved within and across spp How much of the genome is composed of gene sequences How stable are genomes What changes occur within and between species Genome DNA Content The DNA content for large numbers of organisms has been measured by microanalytical techniques These amounts are usually presented as pgcell or nucleotidescell of the haploid genome The amount of DNA does not correlate with the complexity of the organism The number of genes does not correlate with the complexity of the organism A V A n V 7 Fl l39l39m39l39d l Amlzirilrlpsis Kiri U Snapdl agnn Wiral an L Mam Fl nls i quot 1 22 77 r y r Hambqu mnjlpnride pairs pux 1m plnid genome What Makes Some Species Have Larger Genomes Genome duplication Polyploidization Endoreduplication Segmental duplication More genes Larger introns More DNA in intragenic regions All of the above Genic and Nongenic Regions percentage El llIU 2390 3390 4390 5390 6390 7390 8390 9390 1 3930 45 53 92 LINES SINEe A I I Introns protein coding regions retroviraI like elements DNAonly transposon fossils segmental duplications genes simple sequence repeats hetemmmmatm quot u I REPEATS UNIQUE NOT YET SEQUENCED Figure 4 17 Molecular Biology of the Cell 41th Edition Full Genome Sequencing Genome Size amp Predicted Gene Content Organism DNA bp Genes H in uenzae 183 x106 1743 Synechocytis 357x106 3217 E Coli 46x106 4377 S Cerevisiae 12x107 5885 C Elegans 97x107 19000 D melanogaster 18x108 13600 A Thaliana 125x108 25556 0 Sativa 42x108 35000 H Sapiens 33x109 35000 Oryza sativa Genome Estimates Size is 420 japonica or 466 mb indica 29961 40884 japonica or 46022 55615 genes indica 1530 of the genes per chromosome arose through duplication Table 3 Local duplication of ngnezac39c and Mgencsam in Syd Gone from individual Eli ill cJntigs w quot compared to each other I TBLASTX and pairs with Eualues 23 were defined as paralogs representing locally duplicated gene BA 39 ci39agu n Ll mber Jigm ragc numtnzi39 t hi u m osu me cm as Lu ii I s P a l a lugs i 9 F i 9 393 4 4 6T 4 r 9 E 2 N 3 J l 1 41 I33 3 8399 3 1393 7 36 55 4 E l 2 679 44 l 39l 3 5 SE 2 42 6 3E Edi 6 ET 2342 35 72 7 69 2 50 36 E12 8 EE 2 35 T 5 9 47 LE 1 34 69 10 SE l724 31 ii 45 1834 38 116 12 49 180 38 10 1 Total T9 299615 6 38 83 39As a result of ganghe duplication some sequence contigs and genes therein are mapped to multiple iotations thus inflating the Dial number of mapped genes SCIENCE VOL 296 5 APRIL 2002 Grass Genomes SyntenyMacrocolinearity Comparative GRASS phySICal mappmg GENOMES shows that DNA markers are found in the same order amongst Different grasses with up to 40 fold an differences in ml genome size om 23 This suggests that similar gene orders Signquot exist and that size Sugarcane differences are due WWW to expansion in the R39 intergenic regions Proc Natl Acad Sci USA Vol 95 pp 1971 1974 March 1998 Genome organization i r g g mg L I lt 39 f l39 I t39IITIT hfll l ilquot i39ll3939 Functional evaluation of a gene and its regulatory elements are done at the level of transcription or translation mRNA or Protein DWI RNA Pmlrln IT39TJHI lrli 4 nI Ir l r 39wigqlrwl39ranln l pa umilr dEj iv nI i Lyralu39d39ilrhw ill rzlul illl Ii39a llifilquot J39I39L39JmfrII1Inl39m hula According to the MasonCrick model Watson and Crick 1953 a DNA molecule consists of two polynucleotide strands coiled around each other in a helical quottwisted ladder structure The sugarphosphate backbone is on the outside of the double helix and the bases are on the inside so that a base on one strand points directly toward a base on the second strand When using the twisted ladder analogy think of the phosphate backbones as the two sides of the ladder and the bases in the middle as the rungs of the ladder In effect each strand of DNA is one half of the double helix The two halves come together to form the double helix structure Dauxyribonucleic Acid DNA 5 HDEHE 0 DH 39 Nuclak Etc1 5 TH Kc H 52quot quotKCquot L One nucleotide nlzll 05 N H T O CH2 39 0 g 0 HCI SfH f39 W WH The L NH Phosphodiester I bond Jinks 395ch J H N C nuclearmics I I together III one c C strand 0 OJ 3 RH H The Q second T 0 7 D nurieonhde e a m e I am 0 H9 EH CH c OH H Building blocks of nucleic acids Nucleotide ribonucleoside phosphate E K Base Adenine in H 4quot H Nglycosidic bond n Pentose sugar Deoxyribose 7 HUSH 0 DH 39ulNauruquotax39muwlillzzlllulu Livquot quot39 twin 4 yquot quotfquot H i F l E Phosphate cm I The DNA code is determined by four bases Adenine Guanine Cytosine and Thymine ln RNA Uracil replaces Thymine NHg CI NH A N 2 H IZ Ade me mm H H CYTOSl39Ie Thymme Uracil This one39s in RNA39 Pyrimidine single ring bases You should notice that the purines have two ring structures while pyrimidines have only one ring structure The way I39ve always remembered the difference is that the LONGER word pyrimidine represents the SMALLER structure only one ring THERE ARE TWO HYDROGEN BONDS BETWEEN ADENINE AND THYMINE ng Thymine HKNH iC cH I K J mo ka H C SUGAR H cf H fNCN CH SUGAR Adenine T Cymsme HNxcceq x THERE ARE THREE HYDROGEN BONDS quot BETWEEN GUANINE AND CYTOSINE N Cf NC39hN N quot NfHquot f SUGAR H 00 I Z 2 H RC xquot N H 393 C Guanine What sequence of bases of one strand of DNA is complementary to the sequence TATGCAG Remembering that A and G bond to T and C respectively go through quotREADquot the original sequence and put in quotSYNTHESIZEquot the complementary bases each A by T each G by C each T by A and each C by G Original TATGCAG RNA Intermediary in Protein Synthesis Why would the cell want to have an intermediate between DNA and the proteins it encodes The DNA can then stay pristine and protected away from the caustic chemistry of the cytoplasm Gene information can be amplified by having many copies of an RNA made from one copy of DNA Requlation of qene expression can be effected by having specific controls at each element of the pathway between DNA and proteins The more elements there are in the pathway the more opportunities there are to control it in different circumstances DNA versus RNA They differ in composition The sugar in RNA is ribose not the deoxyribose in DNA The base uracil is present in RNA instead of thymine They also differ in size and structure RNA molecules are smaller shorter than DNA molecules RNA is singlestranded not doublestranded like DNA Amount of an RNA made from a particular eukaryotic gene depends on Chromatin structure assembly of machinery at site Frequency of transcription initiation at a site main determinant of transcriptional regulation All of the steps in the process of transcription can directly or indirectly influence the rate of transcription can be regulated and therefore affect how much RNA is made The steps leading to transcriptional activation Condensed chromatin transcription DOIVmerase Lodish et al M08 2004 fa CTO I S The proteins involved in transcription in eukaryotes 1 the basic transcription apparatus and associated factors also known as general or basal transcription factors 2 Multisubunit coactivators mediator and other cofactors 3 sequencespecific DNAbinding transcription factors and 4 chromatincondensation related proteins chromatin remodeling complex I ll mediator I histone acetylase THANSCHlPTlON BEGINS Figure 6 i9 Molecular Biologyr of the Cell 4th Edition The steps leading to transcriptional activation ge quot quotquot ei Upstream sequences are recognized by a tx factor accessing its targets site T TA hismneacewjmse despite the packing of the HAT mmi nfg fde39ing DNA into chromatin 4 This tx factor recruits the SWISNF complex and HAT Specific pattern of remodeled nucleosomes histone acetylation Resulting in the remodeling of S ge 39 r gfifl rfslmquot5 chromatin and localized MBC 4th edition holoenzyme TRANSCRIPTION ACTIVATION 39 This facilitates the access of additional transcription factors to cis regulatory sequences The secondary activators direct gene transcription through multiple interactions with cofactors and the core machinery recruiting the RNA polymerase complex to the transcription initiation site Chromatin remodeling complexes that hydrolize ATP for reorganizing chromatin structure such as SWISNF and ISWI complexes a Sliding SN lt1 b Conformational Change wmQ intermediate Cell 108 475 487 2002 Regulators HAT complexes and ATPdependent remodeling complexes can act in different orders Cell 108 475487 2002 pathway A B or C and still give the same end result a template competent for transcription Although not shown it is also possible that binding by the general transcription factors precedes the action and recruitment of HAT complexes and ATP dependent remodelers Histone tails in chromatin undergo modifications reversible acetylation phosphorylation monoubiquitination and irreversible methylation Creating a histone code which affects binding of histone modifying enzymes Euchromatin activeopen Acl Al c H3 AR TKQTARKSTGGKAPRKQL 910 14 Alic Nlle H3 AR TIEQTARK STGG1K4APRKQL Alia le H4 SGEGKGGKGLGKGGAKRHRK 5 Lodish et al M08 2004 Heterochromatin inactivecondensed H3 CENP A H3 H4 ARTKQT RKSTGGKAPRKQL 10 MGPRRRSRKPEAPR RRSPSP 7 Nlie Ac ARTKQTARI ST GKAPRKQL SGRGKGGKGLGKZGGAKRHRK 1 Dual role of activators recruiting chromatin modifying activities and inducing positioning of the basal transcription apparatus 3 quotxi vain r A C 39L V 739 IV TIBS Vol 24 12 46 49 1999 if u 9191 39 Deace l ylase Histone acetyltransferase activities are found in coactivators and deacetylase activities in corepressors Acetylation of lysine residues on the histone proteins neutralizes the positive charge weakens the interaction with DNA and allows transcription factors to act The steps leading to transcriptional activation DNA binding domain Mediator Activator quot domain TBP TFIIB Lodish et al M08 2004 RNA polymerases in the Eukaryotic cell 1 RNA pol I transcribes ribosomal genes accounts for most cellular RNA synthesis Resides in the nucleolus 2 RNA pol transcribes genes encoding proteins and snRNA s Pol II promoters are complex with many short cisacting sequence elements involved in identifying region for assembly activation initiation and ef ciency of RNA polymerase copying template Tissue or cell speci c inducible by environment conditions 3 RNA pol lll transcribes tRNA genes and other small RNAs All are multi subunit complexes 814 All can transcribe DNA nonspeci cally in vitro but promoter recognition depends on transcription factors 0 The following section will focus on transcription of mRNA which has the highest level of complexity The eukaryotic promoter for RNA pol II 39 core promoter 2i00 1 TATAA Initiator upstream enhancer cluster 30 bp upstream element 45 20 minimal promoter Minimal promoter is needed for basal activity and accurate initiation Minimal promoter is needed for the assembly of the transcription initiation complex at the correct site TATA box Wellconserved sequence centered about 25 bp 5 to start site TBP and TFllD bind Initiator Short segment around start site YANWYY where A is the start site YTorCWTorA Part of TFllD will bind here Use of sitedirected mutagenesis to define the promoter Use sitedirected mutations del nspt mutations in the DNA sequence to test promoter activity Ligate the mutated DNA fragments to the coding region of a reporter gene Where is the preme terferEMEE I I I I I U EDD 1311i 1513 ii ll Lueifereee E li39ii39t39j 1 1 r I Equot I I D 23in Lueifereee i i i r 112le I Lueifereee I r 10E J DU 5393 CI Lodish et al M08 2004 Ciselements in the promotor are recognized by specific or common transfactors endo16 cisregulatory system of sea urchin A The protein A 2300 m binding maps of 7 the 2300 bp sequence that is necessary and sufficient to B r g e n e ra 53iETclwmzc1occ LYEEJWJ FTCCJVMILLimKallblll 1 39WEENT39I CTT 139I39CAG l TGTI39A accurate Spatial and temporal l tlf jall 39 I pattern of 39 fl m expression Factors z l above the line bind W uniquely in a single 753734quot I 69 a 1 region of the r W quot l E ram I sequence those indicated below interact in multiple B Sequence of cisregulatory DNA beneath each in red regions is shown the target site mutations used to test function in vivo in the absence of that interaction 1 c raga LALJI IIl Development 128 6176292001 Transcription factors 1 Basal factors involved in initiation of RNA synthesis of all pol II promoters Form the preinitiation complex 2 Noninducible Upstream factors transactivators transfactors bind upstream elements speci c to different promoters 3 Inducible factors a type of upstream factor that controls highly regulated inducible transcription binds to a response element basal factors upstream RNA pol 11 f t TBP TFIID ac OI S TFIIB A F E amp H Modified from W Gurley I Types of Transcription I Basal low level background activity not regulated only requires a minimal promoter no involvement of upstream elements other ciselements or transactivator proteins 2 Activated requires upstream or other locations ciselements usually much higher levels of activity than basal transcription upstream factors Activated transcription requires upstream factors Modi ed from W Gurley Recruitment of basal factors I Preinitiation complex RNA 39 polymerase II TATAAA 1 gt transcription From William Gurley Basal factors Building the preinitiation complex PIC TFIID 1 Recognition of core promoter N800 kD 2 Target for recruitment by transactivator proteins TAFS 3 Binds other basal factors 9 to13 4 Enzymatic actIVItIes mRNA TBP TATAA binding protein TAF TBP associated factor modified from W Gurley TATAAA Basal factors IATA binding protein TBP 30 Kd TBP is one of the most conserved nuclear encoded proteins in eukaryotic cells TBP is one of the elements that determine the polarity of transcription It is based on the asymmetry in the TATAAA sequence itself he hydrophobic concave 5 underside of TBP makes 7 539 contacts with the39 minor l 80 bend 339 groove of DNA Figure 6 18 Molecular Biology of the Cell 4th Edition mpjwwwfhcrcoglabshahnchime paqestbp dnahtm Basal Factors TAF s and promoter recognition RNA polymerase II TATAbinding protein TBPassociated factors TAF s can direct promoter selectivity by RNA Pol II Core promoters featuring a TATA box require TBP for core promoter recognition and generally do not require TAFlls for basal tx in vitro TIBS 2611 655671 2001 TEES The in vivo requirements for TAFlls most likely depend on both core promoter elements and interactions with tx activators TAF s can directly recognize these core promoter elements which stabilizes TFIID promoter binding and mediates a higher level of tx TAF s serve as targets for recruitment acidic activation domains of upstream transactivator proteins bind TAF s Basal Factors TFIID Enzymatic activities of TAF250 kinase kinase TAND l BD1 TAND2 H AT ubac Zn BD2 Human TAFquot250 lZlWml C 1 Histone acetyltransferase HAT adds acetyl groups to amino tail of histones H3 and H4 loosens chromatin structure 2 Kinase phosphorylates TFllF RAP74 significance unclear 3 Ubiquitin activatingconjugation activity ubac monoubiquinates histone H1 significance is unclear From William Gurley
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