MED-BIO 04-DNA-genoma
annuncio pubblicitario
Sintesi del DNA REPLICAZIONE Sintesi RNA TRASCRIZIONE Sintesi delle proteine TRADUZIONE Crick’s Central Dogma La relazione tra acidi nucleici ed informazione genetica è sintetizzata dal dogma centrale della biologia Un gene una proteina (le eccezioni confermano la regola) DNA RNA 5’ 3’ 5’ 3’ AGCT TCGA trascrizione 3’ AGCU traduzione Proteina C-ter N-ter 5’ • Il DNA è la molecola che consente il trasferimento dell’informazione genetica da una cellula alle cellule figlie, da una generazione alla successiva e nella popolazione • Il RNA è la molecola che trasferisce, all’interno della cellula, l’informazione genetica all’apparato che converte questa in proteine, le vere molecole effettrici dei caratteri fenotipici. LA CELLULA ANIMALE membrana nuleare membrana plasmatica Golgi mitocondrio perossisomi lisosoma nucleo vescicole secretorie reticolo endoplasmico Golgi nucleo lisosoma mitocondrio reticolo endoplasmico Geni e Genoma L’informazione genetica presente sul DNA viene utilizzata per dirigere la sintesi di proteine. L’insieme delle sequenze nucleotidiche presenti nel DNA cellulare formano il genoma umano. La sequenza di nucleotidi sufficiente per la sintesi di una data proteina costituisce il gene per la proteina. Le sequenze nucleotidiche che codificano per proteine rappresentano solo il 1-2% dell’intero genoma. Il genoma umano è diploide – esistono due copie di DNA doppia elica con sequenze nucleotidiche tra loro omologhe. 8 3 Il genoma umano è diploide, è organizzato in 23 coppie di cromosomi (46 cromosomi totali). I cromosomi umani - cariotipo E 8-2; 4th 4-10 4th 4-12 Cromosomi Cromosomi Struttura di un cromosoma eucariotico Festival di San Remo 1999 Renato Dulbecco Premio Nobel 1975 Promotore del progetto GENOMA UMANO Bold Words from Francis Collins: “The history of biology was forever altered a decade ago by the bold decision to launch a research program that would characterize in ultimate detail the complete set of genetic instructions of the human being.” Francis S. Collins Director of the National Human Genome Research Institute N Engl J Med 1999 882:42-65 2001 la prima sequenza “completa” del nostro genoma Genoma umano: 6.4 x109 paia di basi Che cosa significa avere 6.4x109 bp di informazione nel genoma ? 1 pagina di libro = 1000 lettere 1 libro = 1000 pagine x 1000 lettere/pagina = 1.000.000 (106) lettere 6400 libri x 106 lettere/libro 6.4x109 lettere (genoma umano) AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGA CAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGAC TAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTA CGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTAC GACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATG CGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACG ACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGAC GACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCC GGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGA ATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTCTAGCAGATACGCATACAGACTAACGCATACGACATACGCAGCTTTTGC AAAAGCATAGACTACGACTCAGACGCATCAGCACAAAGACGATGATTGACGTAGCATGACGATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTAC GACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCACAAGTACGATACAGATACGACAACAACAAT CAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGAC TACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGA CGACTACACAGCGACTAACGACGCATATTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACA GCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAAT GCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACA GACTACACCCCCCCCCCCCGGGGGAGTAGGGTAGGCACGACGACTAGCAGACGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATA CGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAGCTACGACATACGCCAGACATACA GACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAG ATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACG ACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAAC TCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACA GACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAG ATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACG ACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATA CGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAGCTACGACATACGCCAGACATACAGACTAC GCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGA CAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGAC TACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACT CAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATA CAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATAC AGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTA CGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATAC GACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGCCCCCCCATAGCATGGGGGGGACGACTACGACATACCATGACTACCAGCATACGACATCAT CAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGG CATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAATACATGACGACTGATACTGACAAT CAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGC ATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACATACAGACTACGCTAC AGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCATAGCATGGGGGGGACGACTACGAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACG CTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGCATGACGACTACGACATACAAGTGCGCGGCGCGTTGTG TGTCGCGCGCGCTGGTAACGACAGCGCAGCAGCGCAGACACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGT ACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTA CGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTCAGCACAAAAGGGACGACTACGACATACCATGACTACCAGCATA CGACATCATCAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACTACATGACGACTGATACTGAAGACGACTACGACAGACTACGACAGACTA CAGACTACACCCCCCCCCCCCCCCCCCCCGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGA CTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTT TTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCC CCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTT TTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCC CCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAG ACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTT ACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATA GACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGGACTACGACAGACTACAGACTACACC PAGINA 1 AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGA CAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGAC TAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCC CGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACG AATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTCTAGCAGATACGCATACAGACTAACGCATACGACATACGCAGCTTTTG CAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAGACGATGATTGACGTAGCATGACGATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTA CGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCACAAGTACGATACAGATACGACAACAACAA TCAATGCGATAGACAGACGACTACGACAGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGACGACGACGAC TACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACACAGACGACTACGACAGACTACGACAGA CTACAGACTACACCCCCCCCCCCCCCCCCCCCGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGA CAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGAC GACGACGACTACACAGCGACTAACGACGCATATTTTTTGCAAAAGCATAGACTACGACTCCTACGACAGACTACGACAGACTACAGACTACACCCCCCCATCAATGCGATAGACAGACG AAAAATACATGACGACTGATACTGACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGA CTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCACGACTACGACAG ACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCGGGG GAGTAGGGTAGGCACGACGACTAGCAGACGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTAC ACAGCGACTAACGACGCATATTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCAT ACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCG ATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGAC TACACCCCCCCCCCCCCCCCCGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGC GACTAACGACGCATATTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCAT ACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCG ATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGAC TACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTAC ACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACA TACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGAC AGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACC CCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCG ACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCA TACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGC GATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGA CTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATA CGACATACCCCCCCCCCCCCATAGCATGGGGGGCCCCCCCATAGCATGGGGGGGACGACTACGACATACCATGACTACCAGCATACGACATCATCAGCAGACGACGACGACAGCAGC AGCAGCAGACTTCGACAGCATTTGATACGACTACGACGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGA CTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTA CGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACG ACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGAAAAATACATGA CGACTGATACTGACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACG AATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAG CTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGAC TACGACATACAAGTACGATACAGATACGACACAAAAAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATA CCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTA CGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATA CGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAG ACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACATTTTGCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACA TACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCATAGCATGGGGGGGACGACTACGAAAAAAAAAAGCTACGACATACGCCAG ACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGCATGACGACTACGACATACAAGTGC GCGGCGCGTTGTGTGTCGCGCGCGCTGGTAACGACAGCGCAGCAGCGCAGACACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGAC TACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATA CGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTCAGCACAAAAGGGACGACTACGACATACCAT GACTACCAGCATACGACATCATCAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACTACATGACGACTGATACTGAAGACGACTACGACAGA CTACGACAGACTACAGACTACCATCAGCACAAAAAAACGGGCATACGAACTCAGGACTACGACAGACTACAGACTACACC PAGINA 2 AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGA CAGACGACTACGACAGACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACG ACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGC GATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGA CATACCCCCCCCCGATGACATACATCAGCACAAAGACGATGATTGACGTAGCATGACGATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGAC ATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCACAAGTACGATACAGATACGACAACAACAATCAAT GCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACATACAAGTACGATACAGATACGACAACAACA ATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAG ACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGA CTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTGGGACAGACGACTACGACAGACTACGACAGACTAC AGACTACACCCCCCCCCCCCCCCCCCCCAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGA CATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAG ACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACA CCCCCCCCCCCCGGGGGAGTAGGGTAGGCACGACGACTAGCAGACGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTC AGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTAC GCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGA CAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGAC TACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGAC GACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTAC GCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGA CAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGAC TACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACT CAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACA GCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAA CAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGAC AGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACG ACGACGACTACACAGCGACTAACGACGCATATTTCTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACT ACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCAC AAAAAAAAAAAAAGCTACGACATACGCCAGACATATTTTACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAATACATGACGACTGATACTGACAA TCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGG CATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACATACAGACTACGCTA CAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGAGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACA ACAACAATCAATGCGATAGACAGACGACTACGACAGACTATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGA CTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGAT ACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGAC AGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACA TACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGCCCCCCCATAGCATGGGGGGGACGACTACGACATACCATGACTACCAGCATACGACATCATCAG CAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCAT ACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAATACATGACGACTGATACTGACAATCAA TGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATA CGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGC ATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGAGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGGGGGGGGGGGGTTACAGATACGCATACGAC TACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTGCAATTTTTTTCTAGCAGATACGCATACAGACTA ACGCATACGACATACGCAGCTTTTGCAAAAGCATAGACTACGACTCAGACGCCATACCATAGCATGGGGGGGACGACTACGAAAAAAAAAAGCTACGACATACGCCAGACATACAGACT ACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGCATGACGACTACGACATACAAGTGCGCGGCGCGTT GTGTGTCGCGCGCGCTGGTAACGACAGCGCAGCAGCGCAGACACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACA AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGA CTACGACTACGAATACGACTAAAAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATAT TTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCC CCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACA GACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGT TACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCAT AGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGGACTACGACAGACTACAGACTACACC PAGINA 1000 AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGA CAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGAC TAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTA CGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTAC GACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATG CGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACG ACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGAC GACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCC GGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGA ATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTCTAGCAGATACGCATACAGACTAACGCATACGACATACGCAGCTTTTGC AAAAGCATAGACTACGACTCAGACGCATCAGCACAAAGACGATGATTGACGTAGCATGACGATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTAC GACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCACAAGTACGATACAGATACGACAACAACAAT CAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGAC TACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGA CGACTACACAGCGACTAACGACGCATATTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACA GCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAAT GCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACA GACTACACCCCCCCCCCCCGGGGGAGTAGGGTAGGCACGACGACTAGCAGACGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATA CGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAGCTACGACATACGCCAGACATACA GACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAG ATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACG ACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAAC TCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACA GACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAG ATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACG ACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATA CGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAGCTACGACATACGCCAGACATACAGACTAC GCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGA CAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGAC TACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACT CAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATA CAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATAC AGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTA CGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATAC GACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGCCCCCCCATAGCATGGGGGGGACGACTACGACATACCATGACTACCAGCATACGACATCAT CAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGG CATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAATACATGACGACTGATACTGACAAT CAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGC ATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTGCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACATACAGACTACGCTAC AGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCATAGCATGGGGGGGACGACTACGAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACG CTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGCATGACGACTACGACATACAAGTGCGCGGCGCGTTGTG TGTCGCGCGCGCTGGTAACGACAGCGCAGCAGCGCAGACACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGT ACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTA CGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTCAGCACAAAAGGGACGACTACGACATACCATGACTACCAGCATA CGACATCATCAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATACGACTACGACTACATGACGACTGATACTGAAGACGACTACGACAGACTACGACAGACTA CAGACTACACCCCCCCCCCCCCCCCCCCCGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGA CTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTT TTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCC CCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTT TTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCC CCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAG ACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTT ACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATA GACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATCAGACGCATCAGCACAAAAAAACGGGCATACGAACTCAGGACTACGACAGACTACAGACTACACC PAGINA 1 MILIONE AGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGA CAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGAC TAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAAT ACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTAC GACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCT ACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTA CGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAAT GCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATAC GACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGA CGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCC CGGGGGGGGCCCCCCCCCCGGGGGAGTAGGGTAGGCACGACGACTAGCAGACGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCAT ACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAGCTACGACATACGCCAGACATAC AGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACA GATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTAC GACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAA CTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATAC AGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACA GATACGACAACAACAATCAATGCGATAGACAGACGACTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGAC ATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACA GACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGGGG GGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAA GCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGA CATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGCTAAACGGGCATACGAACTCAGACG ACCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGAT GACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACGACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACG ACAGACTACAGACTACCCCGGGGGGGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGC GACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGA CATACGACATACCCCCCAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCGG GGGGGGAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCAT GGGGGGCCCCCCCATAGCATGGGGGGGACGACTACGACATACCATGACTACCAGCATACGACATCATCAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAGCATTTGATA CGACTACGACGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTT TGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAATACATGACGACTGATACTGACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTAC ACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTT GCAAAAGCATAGACTACGACTCAGACGCATAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACGACA TACCATAGCATGGGGGGGACGACTACGAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCATACGACATACGACATACCCCCCCCCGATGACATACG ACATACCCCCCCCCCCCCATAGCATGGGGCATGACGACTACGACATACAAGTGCGCGGCGCGTTGTGTGTCGCGCGCGCTGGTAACGACAGCGCAGCAGCGCAGACACGATACAGAT ACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGAC AGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTAC ACAGCGACTAACGACGCATATTTTCAGCACAAAAGGGACGACTACGACATACCATGACTACCAGCATACGACATCATCAGCAGACGACGACGACAGCAGCAGCAGCAGACTTCGACAG CATTTGATACGACTACGACTACATGACGACTGATACTGAAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGTTACAGATACGCATACGAC TACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGC ACAAAAAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGA CGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGCGCGTGAGATCGTAGATCGTAGGGCGCGTTATTAAGGACGCTGAGGAGCGCTC GCGTCGAGCGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTT TGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAGCTACGACACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAGACGACTAC ACAGCGACTAACGACGCATATTTTTTTTTCTAGCAGATACGCATACAGACTAACGCATACGACATACGCAGCTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAGACGA TGATTGACGTAGCATGACGATTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATACGCCAGACATACAGACTACGCTACAGCATACAGCAT ACGACATACGACATACCCCCCCCCGATGACATACGACATACCCCCCACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTA CAGACTACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGGGGGGGGG GGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGCATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTGAAAAAAAA AAAGCTACGACACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCCCCCCCCCCCCCCCGATGACATACGACATACCCCCCCCCCCCCATAGCATGGGGGGGACG ACTACGACATACAAGTACGATACAGATACGACAACAACAATCAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACAAGTACGATACAGATACGACAACAACAAT CAATGCGATAGACAGACGACTACGACAGACTACGACAGACTACAGACTACACCCCCCGGGGGGGGTTACAGATACGCATACGACTACAGACTACGACTACGAATACGACTAAACGGGC ATACGAACTCAGACGACGACGACTACACAGCGACTAACGACGCATATTTTTTTTTTTTGCAAAAGCATAGACTACGACTCAGACGCATCAGCACAAAAAAAAAAAAAGCTACGACATCAG ACGCATCAGCACAAAAAAACGGGCATACGAACTCAGGACTACGACAGACTACAGACTACACC PAGINA 6 MILIONI 1.000 basi al giorno 1 giorno tutto il genoma Quanto siamo diversi ? Genoma umano: 6.4 x109 paia di basi the cost to generate a high-quality 'draft' whole human genome sequence in mid-2015 was just above $4,000; by late in 2015, that figure had fallen below $1,500. The cost to generate a whole-exome sequence was generally below $1,000. I cromosomi umani - cariotipo E 8-2; 4th 4-10 4th 4-12 Cromosomi Personalized medicine takes into account individual genetic differences • Traditionally, doctors used: – Family history – Socioeconomic circumstances – Environmental factors • Now: – genomic/genetic testing – proteomic profiling – metabolomic analysis (study metabolites) Effectiveness of drugs: Old Paradigm: New Paradigm: Medicina personalizzata Quanto siamo diversi ? GENOMI Genoma umano: 6.4 x109 paia di basi Genomic sequencing Saccharomyces cerevisiae [Fungus] C. elegans [Worm] Arabidopsis [Plant] Escherichia coli [Bacterium] Drosophila [Insect] Genomic sequencing Staph. aureus [MSRA] Vibrio cholerae Bacillus anthracis [cholera] Yersinia pestis [Anthrax] [Plague] Plasmodium falciparum [Malaria] Salmonella Neisseria meningitidis [Food poisoning] [Meningitis] Cromosomi Helicobacter pylori 03/10/2005 13.22.00 Assegnato il Premio Nobel per la Medicina 2005, che è andato agli australiani Barry Marshall e Robin Warren per le loro ricerche sull'Helicobacter pylori. Lo ha reso noto la Nobel Assembly del Karolinska Institute di Stoccolma. La scoperta dei due ricercatori australiani, avvenuta nel 1982, ha trasformato l'ulcera peptica da infermità cronica a affezione curabile con un breve regime di antibiotici e altri farmaci, ha valutato il comitato del Premio Nobel. Il premio Nobel per la Medicina include un assegno di 1,3 milioni di dollari (oltre un milione di euro), un diploma e una medaglia d'oro. La cerimonia di consegna avverrà in presenza del re di Svezia il 10 dicembre prossimo. Il genoma umano Studio del DNA ripetitivo attraverso la denaturazione e rinaturazione del DNA Curva di DenaturazioneRinaturazione del DNA eucariotico Il genoma umano Tipi di sequenze del genoma Sequenze ripetute semplici (Alu) moderatamente ripetute trasposoni retrotrasposoni virali LINES (long interspersed elements) SINES (short interspersed elements) Sequenze ripetute in tandem codificanti per rRNAs rRNA 5S tRNAs proteine istoniche Sequenze codificanti per proteine geni singoli famiglie geniche pseudogeni non funzionali Tipi di sequenze del genoma Sequenze ripetute semplici (Alu) moderatamente ripetute trasposoni retrotrasposoni virali LINES (long interspersed elements) SINES (short interspersed elements) Sequenze ripetute in tandem codificanti per rRNAs rRNA 5S tRNAs proteine istoniche Sequenze codificanti per proteine geni singoli famiglie geniche pseudogeni non funzionali DNA moderatamente ripetuto in tandem. I geni per gli istoni DNA moderatamente ripetuto in tandem. I geni per l’RNA ribosomale La maggior parte del genoma umano è costituito da sequenze non codificanti Distribuzione dei geni in diversi organismi (da genomi compatti a genomi con ampi spazi intergenici) Struttura dei geni e del genoma umano Le sequenze codificanti per proteine sono una minima porzione delle sequenze genomiche DNA RNA 5’ 3’ 5’ 3’ AGCT TCGA trascrizione 3’ AGCU traduzione Proteina C-ter N-ter 5’ Un gene una proteina (le eccezioni confermano la regola) DNA gene della -globina segnale di poli-adenilazione Sito di inizio trascrizione hnRNA poli-A Splicing mRNA Organization of eukaryotic genes Regulatory sequence Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Organization of genes on a human chromosome Scale of human genome If 1bp is 1mm: The human genome would extend 3200 km. 1 protein coding gene every 300 m. An average gene would extend for 30 m (coding sequence: about 1 m) How the genome is packaged in the nucleus? Quanto è lungo il nostro DNA ? Genoma aploide 3.2 x 109 bp Genoma diploide 6.4 x 109 0,34 nm x 6.4 x 109 ??? Se svolgiamo tutto il dna le sue dimensioni lineari sono di circa 2 metri - cromosomi sono lunghi circa 1 - 2 cm. Spessore 2 nm - Il nucleo cellulare è lungo 5 mm Rapporto con le dimensioni “normali” Genoma 2 metri x 104 spessore 2 nm x 104 Nucleo 5 mM x 104 20 Km di filamento di DNA 20 mm 5 centimentri Il DNA del genoma umano è costituito da circa 6x109 COPPIE DI nucleotidi. Questa lunghissima molecola (metri) è ospitata nel nucleo delle cellule (mm) in virtù di una raffinato ed organizzato impacchettamento. Il DNA è associato a proteine e forma la CROMATINA IL DNA nei cromosomi è associato a proteine Gli ISTONI Istoni >H2b mtdkitkkkrnetysiyiykvlrqvhpkigvsskamnimnsfvndlferlvsesynlsnssrskt ltareiqtsvrlvipgelakhsvsegtkavakyrssi >H2a msgrgksgkartkaksrssraglqfpvgrvhrflrkgnyakrvgggapvymaavleyltaeilel agnaardnkksriiprhlqlavrndeelnkllggvtiaqggvlpniqavllpkktgks >H3 martkqtarkstggkaprkqlatkaarksapatggvkkphrfrpgtvalreirkyqkstellirk lpfqrlvreiaqdfktdlrfqssavaalqeaaeaylvglfedtnlcaihakrvtimpkdiqlarr irgera >H4 msgrgkggkglgkggakrhrkvlrdniqgitkpairrlarrggvkrisgliyeetrgvlkiflen virdavtyteharrktvtamdvvyalkrqgrtlygfgg K = lisina R=arginina H= istidina Nucleosoma: Ottamero istonico + 146 bp di dna 1.65 giri Istone H1 Nucleosoma H2B H4 05_23_histone core.jpg H3 DNA H2A “Nucleosome” • The nucleosome core particle consists of approximately 147 base pairs of DNA wrapped in 1.67 left-handed superhelical turns around a histone octamer • Octamer: 2 copies each of the core histones H2A, H2B, H3, and H4 • Core particles are connected by stretches of "linker DNA", which can be up to about 80 bp long ELECTRON MICROGRAPHS NUCLEOSOMES INTERPHASE CHROMATIN MITOTIC CHROMATIN Cromatina e Nucleosoma cromosoma metafasico telaio molecolare “scaffold” scaffold fibre cromatiniche 30 nm cromatina “filo di perle” DNA nudo Video assemblaggio cromosomi Epigenetics • Modern experimental techniques and technologies allow for the genome-wide study of different types of histone modifications, shedding light on the role of each one The histone code • Example H3K4me3 • H3 is the histone • K4 is the residue that is modified and its position (K lysine in position 4 of the sequence) • me3 is the modification (three-methyl groups attached to K4) • If no number at the end like in H3K9ac means only one group Different chromatin states Chromatin structure (and thus, gene expression) depend also on the post-translational modifications associated with histones forming nuclesomes
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