Class Note for MCB 411 with Professor Fares at UA 3
Class Note for MCB 411 with Professor Fares at UA 3
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Date Created: 02/06/15
September 12 Quiz and problem set review Methods summaryimportance Genome sequencing and the human genome proj eot Thursday DNA repair and mutationreview your amino acids Tuesday Sept 19 Midterm 1 everything through Thursday Sept 14 So What are all of these methods good for doublerstranded plasmid DNA expression vector promoter sequence I CUT DNA WITH RESTRICTION NUCLEASE INSERT PROTEIN CODING DNA SEQUENCE INTRODUCE RECOMBINANT DNA INTO CELLS overexp ressed m R NA Figure amp42 Molecular Biology oIthe Cell 4th Edition Recombinant proteins bacterial cell plant cell c osol ransgene selectable VI of interest marker gene nucleus recombinant plasmid in r I IZI gt Agrabacterium r TVDNA 257nucleoli deypair repeats plant chromosome 13 DNA 18 EXCISED FROM PLASMID AS A LINEAR MOLECULE AND IS TRANSFERRED DIRECTLY INTO THE PLANT CELL WHERE IT BECOMES INTEGRATED INTO THE PLANT CHROMOS Figure 8772 panz of 2 Molecular Biolugy onhe Cell4th Edition Value added or transgemc plants or ammals diSCS retrieved from leaf discs incubated with tobacco leaf genetically engineered g Agrabacreria for 24 h selection medium only callus allows plant cells that the bacteria to proliferate transfer grow up adult plant carrying shoot to root rooted quot159919 that we inducing seedling originally present in medium the bacteria Figure 8772 part1 of 2 Molecular Biology of the Cell 4th Edition Information for basic biology to help understand genetic diseases infectious diseases DNA sequencing of geneswhat it tells us m reading direction for sequence of top DNA strand 3 N ile leu phearg val ile arg prothr arg asnphe thrarg C readlng 2 Ntyr phe ile ser ser asn ser lhr leu asn ala lys leu his Ieu thr C frames 1 Neleu phe tyr pheglupheasp leu lys arg glu Ihr ser leu asn 7C 3 DNA 539 3 1 C lys ile glu leu leu glu val lys phe ala pheser lys val N reading 72 C le lys asn erg hr ile arg glyval arg phe lys val arng frames 73 Crasn lys ser thr asn ser erg leu arg ser val glu ser leu ser 7N reading direc on for sequence of bonom DNA strand Figure 8738 part 1 of 2 Molecular Bioiogv of the Cell 4th Edition With a DNA sequence we can compare sequences of DNA and proteins with all organisms whose sequences are in the database Score 399 hits 1025 Expect e lll Identities 198290 68 Positives 241290 82 Gaps 1290 Query 57 HEEPQKVBKIGEGTYGWYWETGBWALKKIRLMEHEGVPSTAIREISLLRBLNE 115 5 mu 3 I I w 7 m Sbjct 1 Hamsxvax1cacryswyu nxxmemmnInzumzswsmmmsmxsm 60 Query 1 1 1 7 mszxamamynvmmunx nm Ixsnmnmemcas 1 7 5 m m an Sbjctx 61 auzvmauvvnszxmnvnmmxxmnsmIxsyLYQIL Gvucas 12 Query 177 HRVLHRDLKPQNLLmm urxmnpmmvmmammmmm 235 quot km I Sbjct 121 HmRDproNLL1DmmxmmnmraIwmmaxvvnwmrmnmm 150 Queryx 236 xsmvnzwsnccwww12meenszmomnrmmpnmmmpms 295 II I I u m I I Sbjctx 191 ysmnvwsvcciPummnzsnssIDELEKIPmGpr wpsvsmpunm 240 Query 296 Pyxmon sivwmDWLSQMLUDPMRISMFFDDV 345 I I I II I l II a Sbjct 241 PPRWEKQDWPMDEGHDILSRH LRYBPSKRITARQELEHKYFKDL 290 Figure 8747 Motecular Bialogv 039 the Cell 4th Edition collection of generspeci c DNA molecules PCR amplification l robotic printing onto glass slide We can test under What conditions genes are expressed Microarrays mRNAfrom mRNA from sample 1 labeled sampleZ labeled with red with green fluorchrome fluorchrome HYBRIDle l WASH l SCAN RED AND GREEN SIGNALS AND COMBINE WIAGES small region of microarray representing expression of 110 genes from yeast Figure 8762 Molecular Biology ofthe Cell 4th Edition wound healing celrycle cholesterol gene genes biosynthesis genes Figure 63 Molecular Biology of the Ca 41h Edition normal A epithelium 1 loss of APC hyperproliferative epithelium increased genetic l instability loss I of p53 early adenoma 1 activation of 19 intermediate adenoma loss of Smad4 and other tumor suppressors late adenuma 1 loss of p53 carcinoma 1 other unknown alterations metastasis Figure Z3741 Molecular Biology olthe Cell 41h Edltion Cancer is a genetic disease Activation and inactivation of specific genes can increase the likelihood of an invasive tumor C activating phosphake substrate protein m lwxgmwmm h h EcrAbl Gleevec Figure 13745 pan 2 of 2 Molecular Bialogy of the Cell 4th Editmn Bergene on chromosome 22 Ablgene on chromosome 9 539 3 5 339 A A breakpoim breakpoinl TRANSLOCATION l 539 339fused BurAb gene I TRANSCRIPTION 5 lpoly A fused BcrAblmRNA TRANSLATION l E BcriAbl fusion protein Figure 2344 Molecular Bioiogv ollhe Cell 41h Edition Genome sequencing How it is done Why we are sequencing so many genomes What it tells us What we can use the information for What we would like to know What is the basic toolkit that we as humans contain and how does it all work What are the shared genes that are the basis of life What genes play roles in the differences between organisms What genes contribute to diseases and disorders Human genome project Goalunderstand the networks of molecular interactions that bring each person to life in a unique way An understanding of human genetic disease Began with a meeting in Santa Cruz 1985 12 biologists it is possible But the scientists split 5050 about whether it was a good idea Discovery science versus hypothesis driven science development of technology Arguments against 2 of genes is coding 98 junk why bother It will inhibit hypothesis driven funding 1988National Academy of Sciences Committee half for and half against One year of debate the committee endorsed the project 3 billion dollar budget 15 year time scale Headed by James Watson initially Goals Physical genetic and sequence maps of the human genome map first sequence later Model organism sequences Computational tools for analyzing sequence EST fragments and cDNA analysis to identify expressed mRNAs in different cell types Consideration of ethical social and legal challenges posed by genetic information genetic length in centimorgans CM physical length in kilobaseypairs 0 3957 011024 e Cd019 cch4 15quot cdciQ Cy53 maklb spo7 Cysa 0 centromere A 3P07 e Cd075 d 75 c c B FL0139 a FLOi pha d pm 7240 51 By measuring the reciprocal exchanges in meiosis a genetic map can be constructed Genetic distance is roughly correlated with physical distance Genetic and physical maps help identify genes responsible for specific processes Figure 20714 Molecular Biology oilhe Cell 4th Edition Goals Physical genetic and sequence maps of the human genome map first sequence later Model organism sequences Computational tools for analyzing sequence EST fragments and cDNA analysis to identify expressed mRNAs in different cell types Consideration of ethical social and legal challenges posed by genetic information Two strategies for sequencing a genome Hierarchical Shotgun Francis S Collins Director J Craig Venter CEO National Human Genome Celera Genomics Research Institute 1 Whole genome shotgun method Total DNA is sheared and cloned into a vector eg pUC M13 Sequence to 510fold coverage and in theory every nucleotide is obtained Assemble a complete contiguous sequence in silico Shotgun sequencing Fragment and sequence entire genome Ways of finding genes in DNA ORFsopen reading frames ESTsExpressed sequence tags Sequence of a cDNA from a library 4 million available sequences for humans Use BLAST asic Local Alignment earch Iool Yet other ways of finding genes Genefinder starttermination sites intronexon boundaries codon differences Does the ORF encode domains that resemble known domains or proteins with known biochemical function Compare to complete genome sequence of other organisms BLAST BLAST searches blastnnucleotide nucleotide blastpproteinprotein blastxnucleotide protein tblastnproteintranslated nucleotide tblastxtranslated nucleotidetranslated nucleotide Score 399 hits 1025 Expect e111 Identities 195290 681 Positives 241290 52 Gaps 1290 Qunxyx 57 142mm cacmcwrxmmmgzwnxnnmmcvrsmmsxsumna 115 E m IE 6 E m E m Sbjct 1 MumswanGEG39MGWYmummmmxnnmzbzswsmmaxsnnxsm so Quexy 117 nxvxmnwummnvmmnnxxrm mnxsnmnmsmcns 17s I l I Sbjctx 61 SnIvRIEDW SERXHLVFEYLB IKDLKKFHDSWIKSILYOILHGVAYCHS 120 Quexyx 177 ERVLHRDLKPQN LLINl g AIKLADPGLAmGVMTYTEEVVTLmAPEILLm 235 m 1 a I Sbjct 121 HRVLHRDLKPONILIDRE I NALKLADFGLARAFGIPVRTFTERWTLWYRAPEILLG RU 150 Querjx 236 ysuvnmsmcinmv mwensmDmvnxm ewpnmaw rvms 295 H m I m u n I Sbjctx 191 vsmzvnvwsvccIEmmmsysnsEmnmnmmnsaswmvs unrxm 240 Query 296 PPmongswiuDmmnsomnnpuxmsmmnobv 345 m Ii 6 IBVI shjct 241 FPRWWEQDWELDEEGMLSWRYEPSKRITMQALEHBYFKDL 290 Figure 847 Molecular Bialagv of the Cell 4th Edition With a DNA sequence We can compare sequences ofDNA and proteins with all organisms Whose sequences are in the database origin of replication A M 1A Escherichia coli K712 4539221 nucleotide pairs terminus of replication Figure 1730 Molecular Biology oflhe Cell 4th Edition Bi What can we learn by sequencing genomes the basic genes required for life a list of parts the genes that differentiate plants animals bacteria and fungi how genes change through evolution identify candidate genes for diseases oncogenes What can we learn by sequencing genomes Humans32000 genes Now around 25000 Classes of genes present Metabolism DNARNA Protein synthesis Transport and secretion Cytoskeleton Cell adhesion Protein folding and degradation Signal transduction Transcription factors Interesting findings from the genome sequences The human genome has more gene families and larger gene families than the invertebrates 1200 gene families 2 or more members Protein coding genesthe human genome contains 61 of the genes in the fruit fly genome 43 of C elegans 46 of yeast 7 are vertebrate specific Immune system proteins Nervous system More than 50 of the human genome is composed of repeated sequences One exampleretroviruses 8 of genome INTEGRATION OF DNA COPY INTO HOST DNA CHROMDSOME DNA REVEFISE TRANSCRIPTASE I RNA MAKES DNARNA AND THEN DNADNA DNA DOUELE HELIX t RNA F RNA envelope reverse transcriptase capsld ENTRY INTO CELL AND LOSS OF ENVELOPE Figure 5773 part1 QIZ Molecular Biology ofthe Cell Alh Edition viral DNA V A integraseO mtegrase cuts viral 03 5 V 3IOH p I 3 5 I I I target 5 V 3 attack of viral 5 3 3 5 chromosome 339 A 5 quot DNA on target 5 5 V 31 3 gap filling by5 V DNA repair I I I I 5 integrated viral DNA 339 5 3 3 5 3 5 short direct repeats of target DNA sequence Figure 575 Molecular Biology of the Cell 4th Edition The big questions Roles of noncoding DNA 987 in humans Functions of genes with no known domains or roles 43 in humans Model systems We can inactivate single or multiple genes and ask What the function of those genes arethis is called reverse genetics Mice zebra sh fruit ies nematodes yeast Arabidopsis
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