BIOLOGIA MOLECULAR 2011/1 Trabalho Introduo Bioinformtica Valor: 20% Prof. Bruno Xavier bmxavier@ufsj.edu.

du.br CAP Bloco 2 Sala 206

1. Cole a sequncia gnica do operon em estudo, em formato fasta.

Operon escolhido: Lactococcus lactis nisin A and nisB, nisC, nisT, and nisI genes, complete cds GAAACATTAACAAATCTAAAACAGTCTTAATTCTATCTTGAGAAAGTATTGGTAATAATATTATTGTCGA TAACGCGAGCATAATAAACGGCTCTGATTAAATTCTGAAGTTTGTTAGATACAATGATTTCGTTCGAAGG AACTACAAAATAAATTATAAGGAGGCACTCAAAATGAGTACAAAAGATTTTAACTTGGATTTGGTATCTG TTTCGAAGAAAGATTCAGGTGCATCACCACGCATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAAC AGGAGCTCTGATGGGTTGTAACATGAAAACAGCAACTTGTCATTGTAGTATTCACGTAAGCAAATAACCA AATCAAAGGATAGTATTTTGTTAGTTCAGACATGGATACTATCCTATTTTTATAAGTTATTTAGGGTTGC TAAATAGCTTATAAAAATAAAGAGAGGAAAAAACATGATAAAAAGTTCATTTAAAGCTCAACCGTTTTTA GTAAGAAATACAATTTTATCTCCAAACGATAAACGGAGTTTTACTGAATATACTCAAGTCATTGAGACTG TAAGTAAAAATAAAGTTTTTTTGGAACAGTTACTACTAGCTAATCCTAAACTCTATGATGTTATGCAGAA ATATAATGCTGGTCTGTTAAAGAAGAAAAGGGTTAAAAAATTATTTGAATCTATTTACAAGTATTATAAG AGAAGTTATTTACGATCAACTCCATTTGGATTATTTAGTGAAACTTCAATTGGTGTTTTTTCGAAAAGTT CACAGTACAAGTTAATGGGAAAGACTACAAAGGGTATAAGATTGGATACTCAGTGGTTGATTCGCCTAGT TCATAAAATGGAAGTAGATTTCTCAAAAAAGTTATCATTTACTAGAAATAATGCAAATTATAAGTTTGGA GATCGAGTTTTTCAAGTTTATACCATAAATAGTAGTGAGCTTGAAGAAGTAAATATTAAATATACGAATG TTTATCAAATTATTTCTGAATTTTGTGAGAATGACTATCAAAAATATGAAGATATTTGTGAAACTGTAAC GCTTTGCTATGGAGACGAATATAGAGAACTATCGGAACAATATCTTGGCAGTCTGATAGTTAATCATTAT TTGATCTCTAATTTACAAAAAGATTTGTTGTCAGATTTTTCTTGGAACACTTTTTTGACTAAAGTTGAAG CAATAGATGAAGATAAAAAATATATAATTCCTCTGAAAAAAGTTCAAAAGTTTATTCAAGAATACTCAGA AATAGAAATTGGTGAAGGTATTGAGAAACTGAAAGAAATATATCAGGAAATGTCACAAATTCTTGAGAAT GATAATTATATTCAAATTGATTTAATTAGTGATAGTGAAATAAATTTTGATGTTAAACAAAAGCAACAAT TAGAACATTTAGCTGAGTTTTTAGGAAATACGACAAAATCTGTAAGAAGAACATATTTGGATGACTATAA GGATAAATTTATCGAAAAATATGGTGTAGATCAAGAAGTACAAATAACAGAATTATTTGATTCTACATTT GGCATAGGAGCTCCATATAATTATAATCATCCTCGAAATGACTTTTATGAGTCCGAACCGAGTACTCTAT ACTATTCAGAAGAGGAGAGAGAAAAGTACCTCAGCATGTATGTAGAAGCCGTTAAAAATCATAATGTAAT TAATCTTGACGACTTAGAGTCTCATTATCAAAAAATGGACTTAGAAAAGAAAAGTGAACTTCAAGGGTTA GAATTATTTTTGAATTTGGCAAAGGAGTATGAAAAAGATATTTTTATTTTAGGGGATATCGTTGGAAATA ATAATTTGGGAGGGGCATCAGGTAGATTTTCTGCACTCTCTCCGGAGTTAACAAGTTATCATAGAACGAT AGTAGATTCTGTCGAAAGAGAAAATGAGAATAAAGAAATTACATCGTGTGAAATAGTATTTCTTCCAGAA AATATCAGACATGCTAACGTTATGCATACATCAATTATGAGGAGGAAAGTACTTCCATTTTTTACAAGTA CAAGTCACAATGAAGTTCTGTTAACTAATATCTATATTGGAATAGACGAAAAAGAAAAATTTTATGCACG AGACATTTCAACTCAAGAGGTATTGAAATTCTACATTACAAGCATGTACAATAAAACGTTATTCAGTAAT GAGCTAAGATTTCTTTACGAAATTTCATTAGATGACAAGTTTGGTAATTTACCTTGGGAACTTATTTACA GAGACTTTGATTATATTCCACGTTTAGTATTTGACGAAATAGTAATATCTCCTGCTAAATGGAAAATTTG GGGAAGGGATGTAAATAGTAAGATGACAATAAGAGAACTTATTCAAAGCAAAGAAATTCCCAAAGAGTTT TATATTGTCAATGGAGATAATAAAGTTTATTTATCACAGGAAAACCCATTGGATATGGAAATTTTAGAGT CGGCGATAAAGAAGAGCTCAAAAAGAAAAGATTTTATAGAGCTACAAGAATATTTTGAAGATGAAAATAT CATAAATAAAGGAGAAAAGGGGAGAGTTGCCGATGTTGTAGTGCCTTTTATTAGAACGAGAGCATTAGGT AATGAAGGGAGAGCATTTATAAGAGAGAAAAGAGTTTCGGTTGAACGGCGTGAAAAATTGCCCTTTAACG AGTGGCTTTATCTAAAGTTGTACATTTCTATAAATCGTCAAAATGAATTTTTACTGTCGTATCTTCCAGA TATTCAGAAAATAGTAGCAAACCTGGGTGGAAATCTATTCTTCCTAAGATATACTGATCCTAAACCACAT ATTAGATTGCGTATAAAATGTTCAGATTTATTTTTAGCTTACGGATCTATTCTTGAAATCTTAAAAAGGA GTCGGAAAAATAGGATAATGTCAACTTTTGATATTTCTATTTATGATCAAGAAGTAGAAAGATATGGTGG

ATTTGATACTTTAGAGTTATCCGAAGCAATATTTTGTGCCGATTCTAAAATTATTCCAAATTTGCTTACA TTGATAAAAGATACTAATAATGATTGGAAAGTCGATGATGTATCAATCTTGGTGAATTATTTATATCTGA AATGCTTCTTTCAGAATGATAACAAAAAGATTCTTAATTTTTTGAATTTAGTTAGTCCTAAAAAGGTTAA AGAAAATGTCAATGAAAAGATTGAACATTATCTTAAGCTTCTGAAAGTTAATAATCTAGGTGACCAAATT TTTTATGACAAGAATTTTAAAGAATTAAAGCATGCCATAAAAAATTTATTTTTAAAAATGATAGCTCAAG ATTTTGAACTTCAGAAAGTTTATTCAATTATTGACAGTATCATTCATGTCCATAATAACCGACTAATTGG TATTGAACGAGATAAAGAGAAATTAATTTATTACACACTTCAAAGGTTGTTTGTTTCGGAAGAATACATG AAATGAGGACTAATAGATGGATGAAGTGAAAGAATTCACATCAAAACAATTTTTTAATACTTTACTTACT CTTCCAAGCACCTTGAAGTTAATTTTTCAGTTGGAAAAACGTTATGCAATTTATTTAATTGTGCTAAATG CTATCACAGCTTTTGTTCCGTTGGCTAGTCTTTTTATTTATCAAGATTTAATAAACTCTGTGCTAGGTTC AGGGAGACATCTTATCAATATTATTATCATCTATTTTATTGTTCAAGTGATAACAACAGTTCTGGGACAG CTGGAAAGTTATGTTAGTGGAAAATTTGATATGCGACTTTCTTACAGTATCAATATGCGCCTCATGAGGA CTACCTCATCTCTTGAATTAAGTGATTATGAGCAGGCTGATATGTATAATATCATAGAAAAAGTTACTCA AGACAGCACTTACAAGCCTTTTCAGCTATTTAATGCTATCATTGTTGTGCTTTCATCGTTTATCTCATTG TTATCTAGTCTATTTTTTATTGGAACATGGAACATTGGGGTAGCAATTTTACTCCTTATTGTTCCAGTAT TATCTTTGGTACTTTTTCTCAGAGTGGGACAATTAGAGTTTTTAATCCAGTGGCAGAGAGCAAGTTCTGA AAGAGAAACATGGTATATTGTATATTTATTGACTCATGATTTTTCATTTAAAGAAATCAAGTTAAATAAT ATTAGCAATTACTTCATTCATAAATTTGGAAAATTAAAGAAAGGATTTATCAACCAAGATTTAGCTATTG CTCGTAAGAAGACATATTTCAATATTTTTCTTGATTTCATTTTGAATTTGATAAATATTCTTACGATATT TGCTATGATCCTTTCGGTAAGAGCAGGAAAACTTCTTATAGGTAATTTGGTAAGTCTCATACAAGCTATT TCTAAAATCAATACTTATTCTCAAACAATGATTCAAAATATTTACATCATTTATAATACTAGTTTGTTTA TGGAACAACTTTTTGAGTTTTTAAAGAGAGAAAGTGTAGTTCACAAAAAAATAGAAGATACTGAAATATG CAATCAACATATAGGAACTGTTAAAGTAATTAATTTATCATATGTTTACCCTAATTCGAATGCCTTTGCA CTAAAGAATATCAATTTATCCTTTGAAAAAGGAGAATTAACTGCTATTGTAGGAAAAAATGGTTCAGGGA AAAGTACACTAGTAAAGATAATTTCAGGATTATATCAACCAACTATGGGAATAATCCAATACGACAAAAT GAGAAGTAGTTTGATGCCTGAGGAGTTTTATCAGAAAAACATATCGGTGCTGTTCCAAGATTTTGTGAAG TATGAGTTAACGATAAGAGAGAATATAGGATTGAGTGATTTGTCTTCTCAATGGGAAGATGAGAAAATTA TTAAAGTACTAGATAATTTAGGACTCGATTTTTTGAAAACTAATAATCAATATGTACTTGATACGCAGTT AGGAAATTGGTTTCAAGAAGGGCATCAACTTTCAGGAGGTCAGTGGCAAAAAATTGCATTAGCAAGGACA TTCTTTAAGAAAGCTTCAATTTATATTTTAGATGAACCAAGTGCTGCACTCGATCCTGTAGCTGAAAAAG AAATATTTGATTATTTTGTTGCTCTTTCGGAAAATAATATTTCAATTTTCATTTCTCATAGTTTGAATGC TGCCAGAAAAGCAAATAAAATCGTGGTTATGAAAGATGGACAGGTCGAAGATGTTGGAAGTCATGATGTC CTTCTGAGAAGATGTCAATACTATCAAGAACTTTATTATTCAGAGCAATATGAGGATAATGATGAATAAA AAAAATATAAAAAGAAATGTTGAAAAAATTATTGCTCAATGGGATGAGAGAACTAGAAAAAATAAAGAAA ACTTCGATTTCGGAGAGTTGACTCTCTCTACAGGATTGCCTGGTATAATTTTAATGTTAGCGGAGTTAAA AAATAAAGATAACTCAAAGATATATCAGAAAAAGATAGACAATTATATTGAATATATTGTTAGCAAACTT TCAACATATGGGCTTTTAACAGGATCACTTTATTCGGGAGCAGCTGGCATTGCATTAAGTATCCTACATT TACGAGAAGATGACGAAAAATATAAGAATCTTCTTGATAGCCTAAATAGATATATCGAATATTTCGTCAG AGAAAAAATTGAAGGATTTAATTTGGAAAACATTACTCCTCCTGATTATGACGTGATTGAAGGTTTATCT GGGATACTTTCCTATCTATTATTAATCAACGACGAGCAATATGATGATTTGAAAATACTCATTATCAATT TTTTATCAAATCTGACTAAAGAAAACAATGGACTAATATCGCTTTACATCAAATCGGAGAATCAGATGTC TCAATCAGAAAGTGAGATGTATCCACTAGGCTGTTTGAATATGGGATTAGCACATGGACTTGCTGGAGTG GGCTGTATCTTAGCTTATGCCCACATAAAAGGATATAGTAATGAAGCCTCGTTGTCAGCTTTGCAAAAAA TTATTTTTATTTATGAAAAGTTTGAACTTGAAAGGAAAAAACAGTTTCTATGGAAAGATGGACTTGTAGC AGATGAATTAAAAAAAGAGAAAGTAATTAGGGAAGCAAGTTTCATTAGAGATGCATGGTGCTATGGAGGT CCAGGTATTAGTCTGCTATACTTATACGGAGGATTAGCACTGGATAATGACTATTTTGTAGATAAAGCAG AAAAAATATTAGAGTCAGCTATGCAAAGGAAACTTGGTATTGATTCATATATGATTTGCCATGGCTATTC TGGTTTAATAGAAATTTGTTCTTTATTTAAGCGGCTATTAAATACAAAAAAGTTTGATTCATACATGGAA GAATTTAATGTTAATAGTGAGCAAATTCTTGAAGAATACGGAGATGAAAGTGGCACGGGTTTTCTTGAAG GAATAAGTGGCTGTATACTGGTATTATCGAAATTTGAATATTCAATCAATTTTACTTATTGGAGACAAGC ACTGTTACTTTTTGACGATTTTTTGAAAGGAGGGAAGAGGAAATGAGAAGATATTTAATACTTATTGTGG CCTTAATAGGGATAACAGGTTTATCAGGGTGTTATCAAACAAGTCATAAAAAGGTGAGGTTTGACGAAGG AAGTTATACTAATTTTATTTATGATAATAAATCGTATTTCGTAACTGATAAGGAGATTCCTCAGGAGAAC GTTAACAATTCCAAAGTAAAATTTTATAAGCTGTTGATTGTTGACATGAAAAGTGAGAAACTTTTATCAA GTAGCAACAAAAATAGTGTGACTTTGGTCTTAAATAATATTTATGAGGCTTCTGACAAGTCGCTATGTAT

GGGTATTAACGACAGATACTATAAGATACTTCCAGAAAGTGATAAGGGGGCGGTCAAAGCTTTGAGATTA CAAAACTTTGATGTGACAAGCGATATTTCTGATGATAATTTTGTTATTGATAAAAATGATTCACGAAAAA TTGACTATATGGGAAATATTTACAGTATATCGGACACCACCGTATCTGATGAAGAATTGGGAGAATATCA GGATGTTTTAGCTGAAGTACGTGTGTTTGATTCAGTTAGTGGCAAAAGTATCCCGAGGTCTGAATGGGGG AGAATTGATAAGGATGGTTCAAATTCCAAACAGAGTAGGACGGAATGGGATTATGGCGAAATCCATTCTA TTAGAGGAAAATCTCTTACTGAAGCATTTGCCGTTGAGATAAATGATGATTTTAAGCTTGCAACGAAGGT AGGAAACTAGAGTGAAAAAAATACTAGGTTTCCTTTTTATCGTTTGTTCGTTGGGTTTATCAGCAACTGT GCATGGGGAGACAACAAATTCACAACAGTTACTCTCAAATAATATTAATACGGAATTAATTAATCATAAT TCTAATGCAATTTTATCTTCAACAGAGGGATCAACGACTGATTCGATTAATCTAGGGGAGCAGTCACCTG CAG

2. Identificar as ORFs da sequncia maiores que 50 nucleotdeos.

A sequncia apresenta 16 ORFs com sequencias maiores que 50 nucleotdeos.

3. Identificar as ORFS que possuem regies promotoras.

Todas exceto a 3 e a 16. A orf 3 no apresenta cdon de iniciao e nem cdon de terminao. A orf 16 no possui a regio tata Box.

4. Anotar as informaes das questes 2 a 4 na tabela abaixo:

# ORF 1

Localizao Tamanho (nucleotdeos) 5230 6486 1257

Tamanho (AA) 418

-10 (6) tattca

-35 (7) tgtcaat

6892 7011

120 (cdon start e stop) 162 (no tem atg) 2982

39

aagcgatat

agattaca

3 4

7261 7422 455 3436

53 993 ataaaaataa ctaaata

3947 4060

114

37

ttttat

ttatct

174 347

174

57

ttataagga

gaaggaacta

3447 5249

1803

600

aaatga

tgtttc

6483 -

738

245

aaagga

acttttt

7220 910 11 12 13 14 15 16 155 304 1868 1987 4148 4330 4870 4971 2407 2523 258 383 5490 5633 5961 6065 105 34 gcatggg (no uma orf) tattagt 126 144 41 47 tattttata attgatt taaata tttatct 117 38 gagagaaaa ttgtagt 102 33 agaaaa ttttatta 183 60 tatttcta aaacaatg 150 120 49 39 aacttgt aattat caaataa atttttta

OBS: A localizao da orf dever ser notada de acordo com a direo de leitura pela RNA polimerase, usando como padro a direo 5-3 usada na questo 1.
5. Identificar as ORFS contguas, listando-as na tabela abaixo:

No foi identificada nenhuma orf contgua na qual uma orf inicia logo em seguida que outra termina. Identificou-se duas orfs muito prximas, com uma diferena de 11 bases, mostrada no quadro abaixo. Contig 4 7 Localizao ORFs

6. Procure por regies conservadas nas ORFs listadas na questo 4. Anote os resultados

conforme o modelo abaixo. # ORF 4 Motivo -Lant_dehyd_C super family - thiopep_ocin super family Localizao 455..3436 Descrio breve Lantibiotic dehydratase, C terminus thiopeptide-type bacteriocin biosynthesis domain Lantibiotic dehydratase, N terminus

-Lant_dehyd_N super family

1

LanC

5242- 6486

LanC is the cyclase enzyme of the lanthionine synthetase. Lanthionine is a lantibiotic, a unique class of peptide antibiotics

No possui regio conservadas

Gallidermin

174- 347

Membrane lipids determine the antibiotic activity of the lantibiotic gallidermin.

OBS: Uma ORF pode ter mais de um motivo. Nesse caso, liste-os separadamente.
7. Dentre as ORFs da questo 6, escolha a que mais lhe parece mais simptica por qualquer

razo. Traduza a sequncia dessa ORF, postando o resultado usando o cdigo de uma letra para cada aminocido.
Orf NisC
MNKKNIKRNVEKIIAQWDERTRKNKENFDFGELTLSTGLPGIIL MLAELKNKDNSKIYQKKIDNYIEYIVSKLSTYGLLTGSLYSGAAGIALSILHLREDDE KYKNLLDSLNRYIEYFVREKIEGFNLENITPPDYDVIEGLSGILSYLLLINDEQYDDL KILIINFLSNLTKENNGLISLYIKSENQMSQSESEMYPLGCLNMGLAHGLAGVGCILA YAHIKGYSNEASLSALQKIIFIYEKFELERKKQFLWKDGLVADELKKEKVIREASFIR DAWCYGGPGISLLYLYGGLALDNDYFVDKAEKILESAMQRKLGIDSYMICHGYSGLIE ICSLFKRLLNTKKFDSYMEEFNVNSEQILEEYGDESGTGFLEGISGCILVLSKFEYSI NFTYWRQALLLFDDFLKGGKRK

8. Verifique se a ORF que voc escolheu possui domnios transmembrana:

O domnio transmembrana da orf NisC est mostrada no grfico abaixo.

9. Verifique se a ORF que voc escolheu possui stios de clivagem:

A chance de existir um stio de clivagem muito pequena. Isso mostrado no grfico abaixo.

10. Procure por 10 organismos que possuam motivos similares aos encontrados na questo 6. Lactococcurs Lattis, Lactococcus, Tberis, Streptococcus, Pasteurianus, Bacillus , Kaustophilus, Geobacilus Staohytococcus, Planomosnospora.

11. Construa duas rvores filogenticas dos organismos identificados, uma baseada no gene

escolhido e outra baseada no gene 16s.