Università degli Studi di Torino Dipartimento di Genetica Biologia e Biochimica Mario De Marchi Alfredo Brusco Silvia Saviozzi Alessandro Saluto Nicola Migone Via Santena 19, 10126 Torino (Tel. 011.6706662) Torta Margherita 200 g fecola di patate 200 g zucchero 4 uova succo di mezzo limone 1 bustina zucchero vanigliato 1 bustina lievito 1 pizzico sale Sbattere bene i tuorli d'uovo con lo zucchero e, sempre mescolando piano piano, aggiungere gradatamente la fecola di patate, il limone, lo zucchero vanigliato, gli albumi montati a neve e, da ultimo, il lievito. Disporre l'impasto in una teglia imburrata e passare in forno appena acceso per circa 25 minuti a temperatura moderata. Il DNA è un codice per la sintesi di proteine La “ricetta” (=sequenza) del gene ATM GCGAGAGGAGTCGGGATCTGCGCTGCAGCCACCGCCGCGGTTGA TACTACTTTGACCTTCCGAGTGCAGTGAGGCATACATCACAATTTGG AATTATGCATTGGTTTATCAATTTACTTGTTTATTGTCACCCTGCTGCCCAGAT ATGACTTCATGAGGACAGTGATGTGTGTTCTGAAATTGTGAACCATGAGTCTAGTACTTAATGATCT GCTTATCTGCTGCCGTCAACTAGAACATGATAGAGCTACAGAACGAAAGAAAGAAGTTGAGAAATTTAAGCGCCTGATTCGAGATCCT GAAACAATTAAACATCTAGAATTGGGATGCTGTTTTTAGATTTTTACAGAAATATATTCAGAAAGAAACAGAATGTCTGAGAATAGCAAAACCAAATGTATCAGCCTCAACACATCGGCATTCA GATTCCAAACAAGGAAAATATTTGAAGCCTCCAGGCAGAAAAAGATGCAGGAAATCAGTAGTTTGGTCAAATACTTCATCAAATGTGCAAACAGAAGAGCACCTAGGCTAAAATGTCAAGAACTCTTAAATTATATCATGGATACAGTGAAAGATTCATCTAATGGT GCTATTTACGGAGCTGATTGTAGCAACATACTACTCAAAGACATTCTTTCTGTGAGAAAATACTGGTGTGAAATATCTCAGCAACAGTGGTTAGAATTGTTCTCTGTGTACTTCAGGCTCTATCTGAAACCTTCACAAGATGTTCATAGAGTTTTAGTGGCTAGAATAATTCATGCTG TTACCAAAGGATGCTGTTCTCAGACTGACGGATTAAATTCCAAATTTTTGGACTTTTTTTCCAAGGCTATTCAGTGTGCGAGACAAGAAAAGAGCTCTTCAGGTCTAAATCATATCTTAGCAGCTCTTACTATCTTCCTCAAGACTTTGGCTGTCAACTTTCGAATTCGAGTGTGTGA ATTAGGAGATGAAATTCTTCCCACTTTGCTTTATATTTGGACTCAACATAGGCTTAATGATTCTTTAAAAGAAGTCATTATTGAATTATTTCAACTGCAAATTTATATCCATCATCCGAAAGGAGCCAAAACCCAAGAAAAAGGTGCTTATGAATCAACAAAATGGAGAAGTATTTTA TACAACTTATATGATCTGCTAGTGAATGAGATAAGTCATATAGGAAGTAGAGGAAAGTATTCTTCAGGATTTCGTAATATTGCCGTCAAAGAAAATTTGATTGAATTGATGGCAGATATCTGTCACCAGGTTTTTAATGAAGATACCAGATCCTTGGAGATTTCTCAATCTTACACTA CTACACAAAGAGAATCTAGTGATTACAGTGTCCCTTGCAAAAGGAAGAAAATAGAACTAGGCTGGGAAGTAATAAAAGATCACCTTCAGAAGTCACAGAATGATTTTGATCTTGTGCCTTGGCTACAGATTGCAACCCAATTAATATCAAAGTATCCTGCAAGTTTACCTAACTGTGA GCTGTCTCCATTACTGATGATACTATCTCAGCTTCTACCCCAACAGCGACATGGGGAACGTACACCATATGTGTTACGATGCCTTACGGAAGTTGCATTGTGTCAAGACAAGAGGTCAAACCTAGAAAGCTCACAAAAGTCAGATTTATTAAAACTCTGGAATAAAATTTGGTGTATT ACCTTTCGTGGTATAAGTTCTGAGCAAATACAAGCTGAAAACTTTGGCTTACTTGGAGCCATAATTCAGGGTAGTTTAGTTGAGGTTGACAGAGAATTCTGGAAGTTATTTACTGGGTCAGCCTGCAGACCTTCATGTCCTGCAGTATGCTGTTTGACTTTGGCACTGACCACCAGTA TAGTTCCAGGAGCGGTAAAAATGGGAATAGAGCAAAATATGTGTGAAGTAAATAGAAGCTTTTCTTTAAAGGAATCAATAATGAAATGGCTCTTATTCTATCAGTTAGAGGGTGACTTAGAAAATAGCACAGAAGTGCCTCCAATTCTTCACAGTAATTTTCCTCATCTTGTACTGGA GAAAATTCTTGTGAGTCTCACTATGAAAAACTGTAAAGCTGCAATGAATTTTTTCCAAAGCGTGCCAGAATGTGAACACCACCAAAAAGATAAAGAAGAACTTTCATTCTCAGAAGTAGAAGAACTATTTCTTCAGACAACTTTTGACAAGATGGACTTTTTAACCATTGTGAGAGAA TGTGGTATAGAAAAGCACCAGTCCAGTATTGGCTTCTCTGTCCACCAGAATCTCAAGGAATCACTGGATCGCTGTCTTCTGGGATTATCAGAACAGCTTCTGAATAATTACTCATCTGAGATTACAAATTCAGAAACTCTTGTCCGGTGTTCACGTCTTTTGGTGGGTGTCCTTGGCT GCTACTGTTACATGGGTGTAATAGCTGAAGAGGAAGCATATAAGTCAGAATTATTCCAGAAAGCCAACTCTCTAATGCAATGTGCAGGAGAAAGTATCACTCTGTTTAAAAATAAGACAAATGAGGAATTCAGAATTGGTTCCTTGAGAAATATGATGCAGCTATGTACACGTTGCTT GAGCAACTGTACCAAGAAGAGTCCAAATAAGATTGCATCTGGCTTTTTCCTGCGATTGTTAACATCAAAGCTAATGAATGACATTGCAGATATTTGTAAAAGTTTAGCATCCTTCATCAAAAAGCCATTTGACCGTGGAGAAGTAGAATCAATGGAAGATGATACTAATGGAAATCTA ATGGAGGTGGAGGATCAGTCATCCATGAATCTATTTAACGATTACCCTGATAGTAGTGTTAGTGATGCAAACGAACCTGGAGAGAGCCAAAGTACCATAGGTGCCATTAATCCTTTAGCTGAAGAATATCTGTCAAAGCAAGATCTACTTTTCTTAGACATGCTCAAGTTCTTGTGTT TGTGTGTAACTACTGCTCAGACCAATACTGTGTCCTTTAGGGCAGCTGATATTCGGAGGAAATTGTTAATGTTAATTGATTCTAGCACGCTAGAACCTACCAAATCCCTCCACCTGCATATGTATCTAATGCTTTTAAAGGAGCTTCCTGGAGAAGAGTACCCCTTGCCAATGGAAGA TGTTCTTGAACTTCTGAAACCACTATCCAATGTGTGTTCTTTGTATCGTCGTGACCAAGATGTTTGTAAAACTATTTTAAACCATGTCCTTCATGTAGTGAAAAACCTAGGTCAAAGCAATATGGACTCTGAGAACACAAGGGATGCTCAAGGACAGTTTCTTACAGTAATTGGAGCA TTTTGGCATCTAACAAAGGAGAGGAAATATATATTCTCTGTAAGAATGGCCCTAGTAAATTGCCTTAAAACTTTGCTTGAGGCTGATCCTTATTCAAAATGGGCCATTCTTAATGTAATGGGAAAAGACTTTCCTGTAAATGAAGTATTTACACAATTTCTTGCTGACAATCATCACC AAGTTCGCATGTTGGCTGCAGAGTCAATCAATAGATTGTTCCAGGACACGAAGGGAGATTCTTCCAGGTTACTGAAAGCACTTCCTTTGAAGCTTCAGCAAACAGCTTTTGAAAATGCATACTTGAAAGCTCAGGAAGGAATGAGAGAAATGTCCCATAGTGCTGAGAACCCTGAAAC TTTGGATGAAATTTATAATAGAAAATCTGTTTTACTGACGTTGATAGCTGTGGTTTTATCCTGTAGCCCTATCTGCGAAAAACAGGCTTTGTTTGCCCTGTGTAAATCTGTGAAAGAGAATGGATTAGAACCTCACCTTGTGAAAAAGGTTTTAGAGAAAGTTTCTGAAACTTTTGGA TATAGACGTTTAGAAGACTTTATGGCATCTCATTTAGATTATCTGGTTTTGGAATGGCTAAATCTTCAAGATACTGAATACAACTTATCTTCTTTTCCTTTTATTTTATTAAACTACACAAATATTGAGGATTTCTATAGATCTTGTTATAAGGTTTTGATTCCACATCTGGTGATTA GAAGTCATTTTGATGAGGTGAAGTCCATTGCTAATCAGATTCAAGAGGACTGGAAAAGTCTTCTAACAGACTGCTTTCCAAAGATTCTTGTAAATATTCTTCCTTATTTTGCCTATGAGGGTACCAGAGACAGTGGGATGGCACAGCAAAGAGAGACTGCTACCAAGGTCTATGATAT GCTTAAAAGTGAAAACTTATTGGGAAAACAGATTGATCACTTATTCATTAGTAATTTACCAGAGATTGTGGTGGAGTTATTGATGACGTTACATGAGCCAGCAAATTCTAGTGCCAGTCAGAGCACTGACCTCTGTGACTTTTCAGGGGATTTGGATCCTGCTCCTAATCCACCTCAT TTTCCATCGCATGTGATTAAAGCAACATTTGCCTATATCAGCAATTGTCATAAAACCAAGTTAAAAAGCATTTTAGAAATTCTTTCCAAAAGCCCTGATTCCTATCAGAAAATTCTTCTTGCCATATGTGAGCAAGCAGCTGAAACAAATAATGTTTATAAGAAGCACAGAATTCTTA AAATATATCACCTGTTTGTTAGTTTATTACTGAAAGATATAAAAAGTGGCTTAGGAGGAGCTTGGGCCTTTGTTCTTCGAGACGTTATTTATACTTTGATTCACTATATCAACCAAAGGCCTTCTTGTATCATGGATGTGTCATTACGTAGCTTCTCCCTTTGTTGTGACTTATTAAG TCAGGTTTGCCAGACAGCCGTGACTTACTGTAAGGATGCTCTAGAAAACCATCTTCATGTTATTGTTGGTACACTTATACCCCTTGTGTATGAGCAGGTGGAGGTTCAGAAACAGGTATTGGACTTGTTGAAATACTTAGTGATAGATAACAAGGATAATGAAAACCTCTATATCACG ATTAAGCTTTTAGATCCTTTTCCTGACCATGTTGTTTTTAAGGATTTGCGTATTACTCAGCAAAAAATCAAATACAGTAGAGGACCCTTTTCACTCTTGGAGGAAATTAACCATTTTCTCTCAGTAAGTGTTTATGATGCACTTCCATTGACAAGACTTGAAGGACTAAAGGATCTTC GAAGACAACTGGAACTACATAAAGATCAGATGGTGGACATTATGAGAGCTTCTCAGGATAATCCGCAAGATGGGATTATGGTGAAACTAGTTGTCAATTTGTTGCAGTTATCCAAGATGGCAATAAACCACACTGGTGAAAAAGAAGTTCTAGAGGCTGTTGGAAGCTGCTTGGGAGA AGTGGGTCCTATAGATTTCTCTACCATAGCTATACAACATAGTAAAGATGCATCTTATACCAAGGCCCTTAAGTTATTTGAAGATAAAGAACTTCAGTGGACCTTCATAATGCTGACCTACCTGAATAACACACTGGTAGAAGATTGTGTCAAAGTTCGATCAGCAGCTGTTACCTGT TTGAAAAACATTTTAGCCACAAAGACTGGACATAGTTTCTGGGAGATTTATAAGATGACAACAGATCCAATGCTGGCCTATCTACAGCCTTTTAGAACATCAAGAAAAAAGTTTTTAGAAGTACCCAGATTTGACAAAGAAAACCCTTTTGAAGGCCTGGATGATATAAATCTGTGGA TTCCTCTAAGTGAAAATCATGACATTTGGATAAAGACACTGACTTGTGCTTTTTTGGACAGTGGAGGCACAAAATGTGAAATTCTTCAATTATTAAAGCCAATGTGTGAAGTGAAAACTGACTTTTGTCAGACTGTACTTCCATACTTGATTCATGATATTTTACTCCAAGATACAAA TGAATCATGGAGAAATCTGCTTTCTACACATGTTCAGGGATTTTTCACCAGCTGTCTTCGACACTTCTCGCAAACGAGCCGATCCACAACCCCTGCAAACTTGGATTCAGAGTCAGAGCACTTTTTCCGATGCTGTTTGGATAAAAAATCACAAAGAACAATGCTTGCTGTTGTGGAC TACATGAGAAGACAAAAGAGACCTTCTTCAGGAACAATTTTTAATGATGCTTTCTGGCTGGATTTAAATTATCTAGAAGTTGCCAAGGTAGCTCAGTCTTGTGCTGCTCACTTTACAGCTTTACTCTATGCAGAAATCTATGCAGATAAGAAAAGTATGGATGATCAAGAGAAAAGAA GTCTTGCATTTGAAGAAGGAAGCCAGAGTACAACTATTTCTAGCTTGAGTGAAAAAAGTAAAGAAGAAACTGGAATAAGTTTACAGGATCTTCTCTTAGAAATCTACAGAAGTATAGGGGAGCCAGATAGTTTGTATGGCTGTGGTGGAGGGAAGATGTTACAACCCATTACTAGACT ACGAACATATGAACACGAAGCAATGTGGGGCAAAGCCCTAGTAACATATGACCTCGAAACAGCAATCCCCTCATCAACACGCCAGGCAGGAATCATTCAGGCCTTGCAGAATTTGGGACTCTGCCATATTCTTTCCGTCTATTTAAAAGGATTGGATTATGAAAATAAAGACTGGTGT CCTGAACTAGAAGAACTTCATTACCAAGCAGCATGGAGGAATATGCAGTGGGACCATTGCACTTCCGTCAGCAAAGAAGTAGAAGGAACCAGTTACCATGAATCATTGTACAATGCTCTACAATCTCTAAGAGACAGAGAATTCTCTACATTTTATGAAAGTCTCAAATATGCCAGAG TAAAAGAAGTGGAAGAGATGTGTAAGCGCAGCCTTGAGTCTGTGTATTCGCTCTATCCCACACTTAGCAGGTTGCAGGCCATTGGAGAGCTGGAAAGCATTGGGGAGCTTTTCTCAAGATCAGTCACACATAGACAACTCTCTGAAGTATATATTAAGTGGCAGAAACACTCCCAGCT TCTCAAGGACAGTGATTTTAGTTTTCAGGAGCCTATCATGGCTCTACGCACAGTCATTTTGGAGATCCTGATGGAAAAGGAAATGGACAACTCACAAAGAGAATGTATTAAGGACATTCTCACCAAACACCTTGTAGAACTCTCTATACTGGCCAGAACTTTCAAGAACACTCAGCTC CCTGAAAGGGCAATATTTCAAATTAAACAGTACAATTCAGTTAGCTGTGGAGTCTCTGAGTGGCAGCTGGAAGAAGCACAAGTATTCTGGGCAAAAAAGGAGCAGAGTCTTGCCCTGAGTATTCTCAAGCAAATGATCAAGAAGTTGGATGCCAGCTGTGCAGCGAACAATCCCAGCC TAAAACTTACATACACAGAATGTCTGAGGGTTTGTGGCAACTGGTTAGCAGAAACGTGCTTAGAAAATCCTGCGGTCATCATGCAGACCTATCTAGAAAAGGCAGTAGAAGTTGCTGGAAATTATGATGGAGAAAGTAGTGATGAGCTAAGAAATGGAAAAATGAAGGCATTTCTCTC ATTAGCCCGGTTTTCAGATACTCAATACCAAAGAATTGAAAACTACATGAAATCATCGGAATTTGAAAACAAGCAAGCTCTCCTGAAAAGAGCCAAAGAGGAAGTAGGTCTCCTTAGGGAACATAAAATTCAGACAAACAGATACACAGTAAAGGTTCAGCGAGAGCTGGAGTTGGAT GAATTAGCCCTGCGTGCACTGAAAGAGGATCGTAAACGCTTCTTATGTAAAGCAGTTGAAAATTATATCAACTGCTTATTAAGTGGAGAAGAACATGATATGTGGGTATTCCGGCTTTGTTCCCTCTGGCTTGAAAATTCTGGAGTTTCTGAAGTCAATGGCATGATGAAGAGAGACG GAATGAAGATTCCAACATATAAATTTTTGCCTCTTATGTACCAATTGGCTGCTAGAATGGGGACCAAGATGATGGGAGGCCTAGGATTTCATGAAGTCCTCAATAATCTAATCTCTAGAATTTCAATGGATCACCCCCATCACACTTTGTTTATTATACTGGCCTTAGCAAATGCAAA CAGAGATGAATTTCTGACTAAACCAGAGGTAGCCAGAAGAAGCAGAATAACTAAAAATGTGCCTAAACAAAGCTCTCAGCTTGATGAGGATCGAACAGAGGCTGCAAATAGAATAATATGTACTATCAGAAGTAGGAGACCTCAGATGGTCAGAAGTGTTGAGGCACTTTGTGATGCT TATATTATATTAGCAAACTTAGATGCCACTCAGTGGAAGACTCAGAGAAAAGGCATAAATATTCCAGCAGACCAGCCAATTACTAAACTTAAGAATTTAGAAGATGTTGTTGTCCCTACTATGGAAATTAAGGTGGACCACACAGGAGAATATGGAAATCTGGTGACTATACAGTCAT TTAAAGCAGAATTTCGCTTAGCAGGAGGTGTAAATTTACCAAAAATAATAGATTGTGTAGGTTCCGATGGCAAGGAGAGGAGACAGCTTGTTAAGGGCCGTGATGACCTGAGACAAGATGCTGTCATGCAACAGGTCTTCCAGATGTGTAATACATTACTGCAGAGAAACACGGAAAC TAGGAAGAGGAAATTAACTATCTGTACTTATAAGGTGGTTCCCCTCTCTCAGCGAAGTGGTGTTCTTGAATGGTGCACAGGAACTGTCCCCATTGGTGAATTTCTTGTTAACAATGAAGATGGTGCTCATAAAAGATACAGGCCAAATGATTTCAGTGCCTTTCAGTGCCAAAAGAAA ATGATGGAGGTGCAAAAAAAGTCTTTTGAAGAGAAATATGAAGTCTTCATGGATGTTTGCCAAAATTTTCAACCAGTTTTCCGTTACTTCTGCATGGAAAAATTCTTGGATCCAGCTATTTGGTTTGAGAAGCGATTGGCTTATACGCGCAGTGTAGCTACTTCTTCTATTGTTGGTT ACATACTTGGACTTGGTGATAGACATGTACAGAATATCTTGATAAATGAGCAGTCAGCAGAACTTGTACATATAGATCTAGGTGTTGCTTTTGAACAGGGCAAAATCCTTCCTACTCCTGAGACAGTTCCTTTTAGACTCACCAGAGATATTGTGGATGGCATGGGCATTACGGGTGT TGAAGGTGTCTTCAGAAGATGCTGTGAGAAAACCATGGAAGTGATGAGAAACTCTCAGGAAACTCTGTTAACCATTGTAGAGGTCCTTCTATATGATCCACTCTTTGACTGGACCATGAATCCTTTGAAAGCTTTGTATTTACAGCAGAGGCCGGAAGATGAAACTGAGCTTCACCCT ACTCTGAATGCAGATGACCAAGAATGCAAACGAAATCTCAGTGATATTGACCAGAGTTTCGACAAAGTAGCTGAACGTGTCTTAATGAGACTACAAGAGAAACTGAAAGGAGTGGAAGAAGGCACTGTGCTCAGTGTTGGTGGACAGGTGAATTTGCTCATACAGCAGGCCATAGACC CCAAAAATCTCAGCCGACTTTTCCCAGGATGGAAAGCTTGGGTGTGATCTTCAGTATATGAATTACCCTTTC e la proteina ATM ATM • E’ presente in tutti i tessuti nei soggetti sani • E’ assente o fortemente ridotta nei malati Che cos’è una mutazione: un cambiamento nella sequenza del DNA •Le mutazioni sono alla base della variabilità genetica, ad es. l’altezza, la pressione sanguigna, il colore della pelle. • In alcuni casi la variabilità genetica porta ad una malattia genetica Tipi di mutazione Cambia il senso Interrompe il codice ATM 20-40% 60-80% Il gene ATM è uno dei più grandi geni noti ATM Gene medio 10.000 basi 2500 basi Come si cercano le mutazioni • Prelievo di sangue • Estrazione del DNA • Analisi con tecniche di biologia molecolare (individuano la regione colpita) • Lettura della “sequenza” del DNA Perché si cercano le mutazioni • Diagnosi certa • Identificazione dei portatori • Diagnosi prenatale • Scopi di ricerca I nostri risultati Registro italiano pazienti A-T (RIAT)- Roma-Prof. L.Chessa Pazienti analizzati: 29 Analisi di mutazioni di ATM mediante PTT T7-eu-ATG 4-13 cDNA 12-24 22-32 cDNA 31-44 Reverse transcription and nested PCR PCR fragments 41-53 52-66 cCC 1 2 in vitro transcription mRNA in vitro translation protein PAGE-SDS 3 Alcune mutazioni di ATM Exon 1 10 20 30 Interrompono il codice 1240 C T 2413 C T 5229 A T 5574 A T 6913 C T 7327 C T 7792 C T 8545 C T 8977 C T 9139 C T Q414X Gilad 1996 R805X K1743X Gatti W1858XGilad 1998 Q2304X R245X R2598X Wright 1996 K2848X Gatti el al. R2993X R3065X Gilad 1998 40 50 60 Cambiano il senso 128 T C L43P Shiloh 7408 TG Y2470D 8708 C T P2903L 8711 A G E2904G Gilad Distribuzione delle mutazioni ATM * Exon 1 10 20 * 30 40 50 Inserzioni Non senso Difetti di splicing Interrompono il codice Delezioni Cambiano il senso * Mutazioni presenti in più famiglie 60 Conclusioni • Sono state individuate il 70% delle mutazioni • La proteina può essere colpita in qualunque posizione • Sono rare le mutazioni che ricorrono in più famiglie • Non è possibile predire l’andamento della patologia sulla base delle mutazioni Nuovo progetto Messa a punto di sistemi di analisi di mutazioni mediante DHPLC Analisi di ATM mediante DHPLC Per ogni paziente si eseguono 66 reazioni indipendenti che la macchina analizza in automatico. Se in una reazione si ottiene una possibile mutazione si sequenzia il DNA corrispondente Prospettive • Sistema rapido per analisi di mutazioni • Ricerca di tutti i tipi di mutazione • Fornire un test per le famiglie che lo richiedano Ringraziamenti Associazione ONLUS Gli Amici di Valentina
