Fine Structure and Analysis of Eukaryotic GenesSplit genes and intronsR-loops can reveal intronsExamples of R-loops in mammalian hemoglobin genesTypes of exonsFinding exons with computersFind exons for HBBGenscan analysis of HBB geneBLAST2: HBB gene vs. cDNAIntrons are removed by splicing RNA precursorsAlternative splicing can generate multiple polypeptides from a single geneAlternative splicing can generate multiple polypeptides from a single gene, part 2Multigene families, e.g. encoding hemoglobinBlot-hybridization analysis showing multiple beta-like globin genes in mammalsFunctional analysis of isolated genesGene Expression: where and how much?RNA blot-hybridizations = NorthernsRNA blot-hybridization: Stage specificityRT-PCR to detect RNAIn situ hybrid-ization and immuno-reactionsSequence everything, find function laterMassively parallel screening of high-density chip arraysExpression profiling using microarraysFind clusters of co-regulated genesSearch the databasesDatabases, cont’dExpress the protein productPhenotype of directed mutationLocalization on a gene mapFine Structure and Analysis of Eukaryotic GenesSplit genesMultigene familiesFunctional analysis of eukaryotic genesSplit genes and introns•The mRNA-coding portion of a gene can be split by DNA sequences that do not encode mature mRNA•Exons code for mRNA, introns are segments of genes that do not encode mRNA.•Introns are found in most genes in eukaryotes •Also found in some bacteriophage genes and in some genes in archaeR-loops can reveal intronsmRNA coding regions (exons) separated (by introns) on the chromosome:Restriction fragment of DNAmRNAAAAA+AAAAexon1intron1exon2AAAAintron1exon1exon2Examples of R-loops in mammalian hemoglobin genesTypes of exons5’3’StartStopTranscription startTranslationStoppolyA5’ untranslatedregion3’ untranslatedregion5’3’Proteincoding regionpromoterGTAGGTAGGTAGGTAGOpen reading frameGenemRNATranslationInitial exonInternal exonInternal coding exonTerminal exonFinding exons with computers•Ab initio computation–E.g. Genscan: http://genes.mit.edu/GENSCAN.html–Uses an explicit, sophisticated model of gene structure, splice site properties, etc to predict exons•Compare cDNA sequence with genomic sequence–BLAST2 alignments between cDNA and genomic sequences–http://www.ncbi.nlm.nih.gov/blast/–Better: Use sim4•Takes into account terminal redundancy at ends of introns•http://bio.cse.psu.edu•Follow link to “sim4 server in France”Find exons for HBB•Sequence for human beta-globin gene (HBB):–Accession number L48217–Thalassemia variant•Sequence for HBB mRNA–NM_000518•Retrieve those from GenBank at NCBI (or the course website)–http://www.ncbi.nlm.nih.gov–Get the files in FASTA format•Run Genscan and BLAST2 sequencesGenscan analysis of HBB geneGENSCAN 1.0 Date run: 8-Sep-100 Time: 11:29:36Sequence gi : 1827 bp : 41.54% C+G : Isochore 1 ( 0 - 43 C+G%)Parameter matrix: HumanIso.smatPredicted genes/exons:Gn.Ex Type S .Begin ...End .Len Fr Ph I/Ac Do/T CodRg P.... Tscr..----- ---- - ------ ------ ---- -- -- ---- ---- ----- ----- ------ 1.01 Init + 217 308 92 0 2 103 77 136 0.987 14.01 1.02 Intr + 439 661 223 1 1 100 96 217 0.999 20.91 1.03 Term + 1512 1640 129 2 0 116 43 119 0.862 7.40 1.04 PlyA + 1667 1672 6 -1.95Predicted peptide sequence(s):>gi|GENSCAN_predicted_peptide_1|147_aaMVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYHBLAST2: HBB gene vs. cDNAScore = 275 bits (143), Expect = 1e-71Identities = 143/143 (100%), Positives = 143/143 (100%) Query: 167 acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcacc 226 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 1 acatttgcttctgacacaactgtgttcactagcaacctcaaacagacaccatggtgcacc 60hemoglobin, beta 1 M V H Query: 227 tgactcctgaggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaag 286 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||Sbjct: 61 tgactcctgaggagaagtctgccgttactgccctgtggggcaaggtgaacgtggatgaag 120hemoglobin, beta 4 L T P E E K S A V T A L W G K V N V D E Query: 287 ttggtggtgaggccctgggcagg 309 |||||||||||||||||||||||Sbjct: 121 ttggtggtgaggccctgggcagg 143hemoglobin, beta 24 V G G E A L G R genecDNAIntrons are removed by splicing RNA precursorsexon1intron1exon2exon3intron2AAAAAAAAcapcapGene:duplex DNAPrimary transcript:single stranded RNAPrecursor to mRNAmRNAProteintranscription5' and 3' end processingsplicingtranslationAlternative splicing can generate multiple polypeptides from a single geneAAAAAAAAcapcapPrimary transcript:single stranded RNAPrecursor to mRNAmRNAProtein A5' and 3' end processingsplicingtranslation123exon1intron1exon2exon3intron2123The mRNA for Protein A is made by splicing together exons 1, 2 and 3:AAAAAAAAcapcapPrecursor to mRNAmRNAProtein Bsplicingtranslationexon1intron1exon2exon3intron2Or, by an alternative pathway of splicing that skips over exon2, Protein B can be made:3131Alternative splicing can generate multiple polypeptides from a single gene, part 2Multigene families, e.g. encoding hemoglobinHuman β-globinHuman α-globinεγγψηδβGAζ2ζ1α2ψα1α1θEmbryonicFetal Adult020406080kbChromosome 16Chromosome 11LCRHS-40Hb Gower-1 ζ ε22Hb Gower-2 α ε22Hb Portland ζ γ22HbF α γ22HbA2 α δ 22HbA α β22Blot-hybridization analysis showing multiple beta-like globin genes in mammalsHBE HBG HBD HBBRabbitClonesGenomic DNA3.3 2.8 6.3 2.6Size of EcoRIfragments thathybridize to globincDNA, in kbA: clones, gelB: clones, blot-HybridizationC: genomicDNA, blot-hybridizationFunctional analysis of isolated genesGene Expression: where and how much?•A gene is expressed when a functional product is made from it.•One wants to know many things about how a gene is expressed, e.g. –In which tissues?–At what developmental stages?–In response to which environmental conditions?–At which stages of the cell cycle?–How much product is made?RNA blot-hybridizations = Northerns28S rRNA18S rRNATotal RNA from mouse