Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31The Graduate School Experience: Perspectives From Current UIUC Graduate studentsRSI invites you to attend a Graduate Student Panel about life as a graduate student conducting research here at University of Illinois. Grad students from a diverse array of backgrounds will be answering questions on life in grad school and the process of research. This is a great opportunity for anyone interested in continuing education to obtain advice directly from students with higher education experience.Sponsored by Research as Students at IllinoisWednesday, October 22, 7 PM165 Noyes LaboratoryPlease contact [email protected] for more information or find us on Facebook: www.facebook.com/ResearchStudentsIllinoisMolecular analysis of gene expressionReverse geneticsATGCGACATGCATGCATGCATCATCGCTACAGCAGCATCAGCAGTCGACGCCAAGAAGCTGTACAGTCAGCTAGCAAAGCTAGCTGGGGACGCTAGCAGCATAGCATGCATGCAGCATGCATGCATGCATGCAGGGSequence the genomeFind the geneDiscover phenotypeproduced by loss-of-function mutations.Given the vast amount of DNA sequence now available in the 21st century, reverse genetics has become the standard paradigm for studying new genes:•Genes are first identified by analysis of their DNA sequence.•This DNA sequence information is then used to study gene function [see Lectures 32 & 37].Lecture outline•DNA sequence analysis-Identifying genes in genome sequences.-Characterizing gene products by sequence analysis of cDNAs.•Measuring RNA expression-Northern blots-Microarrays-In situ hybridization•Measuring protein expression-Western blots-ImmunohistochemistryHow does one identify a gene when looking at a genome sequence?TTGACA TATAAT ATG .............................................................................. TAG+1Bacterial promoter-35 -10Start codonStop codonSome sequences are invariant. But they are short, and occur in the genome frequently by chance.Other sequences are variable, and so of limited diag-nostic value.5'TTCAACGCCCGGTGTTTCAATGTTTATGATGGTACCGTCATACGGCGATGCGAATCAGAACTTTAAGTTAATGCTGCAATCAGCCCAACGTATTGCCGACGATGTCGGTGGTGTGGTGTTAGATGATGAACGCCGCATGATGACCCCGCAGAAATTGGAATCATATAAGGCGCGTATCCGTGAAGTGCTGGATGCGAATACAATTGCTTGATAATCTTTTTTATAAAGCGATTGCCTCGTAATAAATCGAATTAACCCGAACCCCCGCATGCCGGGGGTTTTTTATCTTTGGTGATGAGCTATGGAATCGATAATCCAACAAATTAATCAACTAAGAACTTCATTGCGCCATCATGAACATCAATACCATGTGCTGGATGCCCCCGAAATCCCTGATGCTGAATACGACCGTATGATGCAACAACTGCGTGATTTAGAAGCACAACATCCTGAACTGGTGACTAATGACTCACCGACGCAACGGGTTGGTGCCGCGCCTCTTGATGCCTTTGAACAGGTGAAGCACGAAGTACCGATGCTGTCTCTTGATAATGTGTTTGATGAAGAAAGTTATCTGGCCTTCGATAAACGGGTACATGACCGGCTTAAAACGGCAGAACCACTGACATTTTGTTGTGAACTGAAGCTAGATGGTCTGGCGGTCAGTTTATTGTATGAAAATGGTGAATTGGTGAGAGCCGCGACACGTGGTGATGGCACTACGGGAGAAAATATTACCGCTAATGTGCGTACTATTCGTGCCATTCCACTGCGCCTACATGGGGATAATGTCCCGCGGCGCGTAGAAGTCCGTGGCGAGGTTTTTATGCCACAAGCCGGTTTTGAACAGCTTAACGAAGAGGCTCGTCGCAAAGGAGGGAAGGTCTTTGCCAACCCACGGAATGCGGCGGCGGGATCGTTACGCCAACTTGATCCCCGTATTACAGCCAAACGGCCACTCACCTTCTTCTGCTATGGTGTCGGTCTGCTGGATGGCGGCGAGCTACCCCGTAGCCATATTCAGTGTCTGATGCAATTTAAGGCATGGGGGTTACCCGTGAGTGAGCGGGTGAAACTCTGCACTGGCAGTGATCAGGTTATTGCTTTTTATCGTCAAATAGAGCAAGACCGCGCAGGCTTGGGCTTTGATATTGATGGTGTGGTGATCAAAGTTGATGATCTTGCTTTGCAGGAGCAATTGGGGTTTGTTGCGCGTGCCCCTCGCTGGGCGACGGCATTTAAATTCCCGGCGCAGGAACAGATCACACAGGTCCGTGAAGTGGAATTTCAAGTGGGTCGTACCGGTGCCATTACTCCCGTAGCCCGTCTGGAGCCGGTTCAGGTGGCTGGCGTGATTGTCAGTAATGCGACATTGCACAATGCCGATGAAATCGAACGCCTTGGTTTGCGTATTGGCGATACGGTCATTGTGCGCCGTGCGGGTGATGTGATCCCACAAGTTGTTGGTGTGGTCATGGAACAACGCCCTGATGATACGAAGGAGATCACTTTCCCAAGCCAATGCCCAGTGTGTGGCTCCGATATTGAACGGGTGGAAGGGGAGGCTGTTGCCCGCTGCACTGGCGGCTTGTTCTGTGCTGCACAGCGTAAAGAGGCACTTAAACATTTTGTCTCCCGGCGAGCACTGGATGTAGACGGTATGGGCGATAAAATTATCGAACAACTGGTCGAGAAACAGTATGTTGAAAATCCAGCTGATTTATTCCAACTGACCGCAGGTAAGTTGACGGGGTTAGATCGGATGGGGCCAAAGTCTGCGCAAAATCTGATTGCGGCGCTGGAGAAGGCCAAACAGACCACCTTTGCACGCTTCTTGTATGCCTTGGGGATCCGCGAAGTGGGCGAAGCGACGGCCGCAAATTTAGCCGCTCACTTCCGCACTCTGGATAATCTGCGTGCAGCAGATATTGAGACGTTAAAAAGTGTGCCGGATGTCGGCGAGGTGGTGGCTAAACATGTGATGAACTTCCTCAGTGAGGAACATAATCAGAAAGTCATCGAAGAACTGGAGAAAGTGGTCAGTTGGCCTGAGCCGCAGCAAATTGTAGTCGAGGAGAGCGATAGCCCCTTCGCTGGAAAAACGGTGGTCCTGACGGGGTCATTGACGATCCTTTCACGGGATGAAGCGAAAGACCGCCTGACAGCATTAGGCGCAAAAGTGAGTGGCAGTGTCTCTAAAAAAACGCATCTGGTGATCGCTGGCGAGGCGGCTGGTTCTAAACTGGCGAAAGCGCAGGAACTCGGAATTAAAGTTATTGATGAAGCTGAAATGATCCGTTTGCTGGGTGAATAATCCTAACGCCGGGGGCGATATGGAAAAAGAGAATTTGATTGAGATTGCTAATACCCGAATGCCTTTTGGTAAATACCAAGGGCGGGTGTTGATCGACTTGCCAGAAGAGTACCTATTATGGTTTGCGCGCAAAGGTGAATTTCCGCAGGGCAAACTGGGGATGCTGATGGAGATGACATTAGCGATCAAGATAGAAGGATTAGACGGCTTAGTGAAGCCGCTGAAGACCGGCTAAATCTGGCTAACAAATGGCGATGGAAAGACCCACTCGGAAAATATGATCTCTAGTCGGCCAACCAT -3'DNA sequence of a bacterial gene (1 strand)-How does one identify a gene when looking at a genome sequence?To find genes, genome sequences are analyzed by computer programs that identify long open-reading frames (ORFs). The amino acid sequences encoded by these ORFs are then compared to a catalog of known proteins.DNA ligaseIn E. coli, the majority of the chromosomal DNA consists of genes (GREEN) that encode functional RNAs or proteins. There is very little ‘intergenic’ DNA.Note: individual genes can be transcribed in either the same or opposite orientations (arrowheads).Watson, Fig. 8-2Representative portion of the E. coli genome>>>>>>>>>>>>>>>>>> > >>>>>> <<<<<<<<<<<< <Identifying genes from genome sequence analysis can be considerably more difficult in eukaryotes.Less than 2% of the DNA in the human genome encodes RNAs or proteins.In addition, many eukaryotic genes contain introns interspersed with exons.Hence, protein-coding regions are not necessarily continuous within the genome.In this fruit fly gene, the protein coding sequence [ ] is separated into three pieces by introns [ ].Watson, Fig. 7-21DNA is an information storage molecule. It has effectively no enzymatic activity of its own. It is the gene products (proteins & RNAs) that are the machines which make cells work.A cDNA (or 'complementary' DNA) is a synthetic DNA molecule generated by reverse transcription* using a single-stranded RNA (often an mRNA) as the template