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Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk

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Presentation on theme: "Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk"— Presentation transcript:

1 Department of Microbiology Faculty of Biology, Geography and Oceanology University of Gdańsk

2 1. Biology of bacterial DNA restriction and modification systems. 2. New approaches for DNA sequencing and analysis. Main fields of interest:

3 Restriction-modification systems. What are they for?

4 Table 1. Restriction-modification systems isospecific to HindIII Gram-positive bacteriaGram-negative bacteria BseHIBacillus stearothermophilusCPW5 Asp3065IAlcaligenes sp.3065 BsmGIIBacillus stearothermophilusG170 Asp52IAlcaligenes sp.52 BspKT8IBacillus sp.KT8 BbrAIBordetella bronchiseptica BspLAIIIBacillus sp.LAIII BbrIBordetella bronchiseptica 4994 Bst170IIBacillus stearothermophilusD170 BpeIBordetella pertusis BstFIBacillus stearothermophilusFH58 Cfr 32I Citrobacter freundii RFL 32 Bst Z1I Bacillus stearothermophilus 14P Cfr 83I Citrobacter freundii RFL 83 Lla CI Lactococcus lactis subs. cremoris CI Cfr 231I Citrobacter freundii RFL 231 ChuICorynebacterium humiferum Eco VIII Escherichia coli SsbIStreptomyces scabies Eco 1219I Escherichia coli RFL1219 Eco 98I Escherichia coli RFL98 Hin1076IIIHaemophilus influenzaeserotype b, 1076 Hin173IHaemophilus influenzae173 Hin5IIIHaemophilus influenzaeRFL5 HinBIIIHaemophilus influenzaeRb Hin dIII Haemophilus influenzae Rd HinfIIHaemophilus influenzaeRf HinJCIIHaemophilus influenzaeJC9 HinSAFIHaemophilus influenzaeserotype b, SAF6 HsuIHaemophilus suis MkiIMoraxella kingae SpaPIVSpirulina platensis strainpacifica REBASEhttp://rebase.neb.com

5 The HindIII restriction-modification system is composed of two enzymes. R.HindIII 5 A A G C T T 3 3 T T C G A A 5 CH 3 5 A A G C T T 3 3 T T C G A A 5 CH 3 M.HindIII toxin antytoxin toxin antytoxin

6 1. how similar are genes encoding isospecific enzymes ? 2. is it possible to map their functional domains ? 3. do they recognize cognate sequence in the same way ? 4. what is their mode of action ? Objectives:

7 R-M System HindIII ( Haemophilus influenzae Rd) Nwankwo et al., Gene (1994) R-M System LlaCI (Lactococcus lactis subs. cremoris CI) Madsen i Josephsen, Biol.Chem. (1998) R-M System EcoVIII (Escherichia coli E ) Mruk i Kaczorowski, Appl. Environ. Microbiol. (2003) Plasmid (2001) ecoVIIIR ecoVIIIM llaCIR llaCIM hindIIIR hindIIIM pHindIIIR pHindIIIM pLlaCIR pEcoVIIIRpEcoVIIIM R-M System Csp231I (Citrobacter sp. RFL231) Mruk i Kaczorowski, unpublished csp231IC csp231IR csp231IM pCsp231IR pCsp231IM pCsp231IC R-M System BstZ1II (Bacillus stearothermophilus 14 P) Mruk i Kaczorowski, unpublished bstZ1IIR bstZ1IIM pBstZ1IIR pBstZ1IIM

8 R.HindIII MKKSALEKLLSLIENLTNQEFKQATNSLISFIYKLNRNEVIELVRS 46 R.LlaCI MNSEFEKLKGTGLQTVDTEKVTLDFFKMLKKISDKEFINILIT 43 R.EcoVIII MDLTNIEFINDAAINRRRYWTNEIVKLSGHFVNDSSRVEEEIIYEVSKSGSQALLDHLRL 60 R.Csp231I MMKVVKMVKIPPLAQDCDLDSSELSEFTPEEHFNKSITRWFSDHYASYSNRFESYEYIQNYIQNEHFNWSVAPNTNVIATKFSAHLRTLSLKDFSFLLCH 100 R.BstZ1I MNMVDAFYLMNYVDDQFKVNAQLTEEHERIANEFLKDIKKFDDKTFNKLLVS 52 R.BbrI MPVARHGVGERVVQCPPMLTTSDPLPHPLLHDGVVAAVVRLCADATLDFPARCARLEAAIRACTRDQIIEHLQH 74 : : :.. : P D/E D D-K R--R R.HindIII IGILPEAIKPSSTQEKLFSKAGDIVLAKAFQLLNLN-SKPLEQRGNAGDVIALSKEFNYGLVADAKSFRLSRT-AKNQKDF-KVKALSEWRED R.LlaCI SGYIPDLYVADSKEETLFTKLCEALEVDWASRMGFE-ANAVTQKSSYEDVVIKIN--NKIIVSDTKSFRLGRSQQAPNVKDFVKPEDYSKWANRHSGQ R.EcoVIII CTAIPESYEHDSSEEKLYSKYTDALISECFKFFGLN-SIVLTERADAADVEVVCD-SYSFVADAKVFRLSRT-AKNQKDF-KVQAMDGWRNT R.Csp231I TGYIPEIYKADSSQETLYSKLVEAMVNEWALRMKFTYSLLPTQKSSKEDITISDG--ENIIVADAKSYRLGRSQAAPNVKDALKKGDITKWLAYYDQHKY 198 R.BstZ1I ATFIPDFYEPDSSRETLFSKLVEAMVTEWAIRMGYE-AAMQKEKASYEDVRIAIK--DKLIVVDAKTFRLGRSQAAPNVKDFLKLEDIRKWCSRYKN R.BbrI GGVISECIGHDSSEEKLLAKYSDVLLACALGELGFQ-ARVLAERSDAADVYAECG-DQRIVGDGKIFRLSRT-ALNPKDY-KVPSLKKWRQGAGYS :.:.*..*.* :* : : :. : :* * * * :**.* * * ** * * R.HindIII -KDYAVLTAPFFQYPTTKSQIFKQSLDE--NVLLFSWEHLAILLQLDLEETNIFSFEQLWNFPKKQSKKT---SVSDAENNFMRDFNKYFMDLFKIDKDT 230 R.LlaCI -KLGGLVVYPQLHEWTRKSDAHVYCSDKKNPILMLPYHYLAYFLERKDK-FNPKSLEKLWDYEKIFPEK------ADSRNDYWQKINNVILEITGDEKKE 230 R.EcoVIII -KDFAMVVCPIYQLPVKSSQIYQQAILR-NVCVFTYTHLAVLVKYADI--VGSDVRILLEEIFRSVILM---NPSKDSVQYWTMINRTMLNYDKRIEKL 240 R.Csp231I NRIGGLVAFPSQHDWKNGSDFYLYLTDKNSPIIMLFYEHMAFMLLAGMD--KNNLLDFYRNHKDIFPNEVF-NKIGSRKIYFDKLEQYLLNCG---KFK 291 R.BstZ1I -AIGGLVTYPCLHEWKNKSDAYTYCSTKDMPTVMLSYKHLAFLLDNKEN-FNTEKLIELWDYENIFPEKLPKNLKGGNKKPYWDAINKKLIEITNVGDKE 244 R.BbrI -ADYSVLLAPSFQYPLSRSQLYAQAIDN-NVLLLSFEQLAFLVRHHQP---GRDYRPLWEYAGTRAGQDT--AAWKEARQYWHGLNQLVARLAGQTEQA 258.:: * : *:... :: : :* :: : : ::... R.HindIII LNQLLQKEINFIEERSLIEKEYWKKQINIIKNF TREEAIEALLKDINMSSKIETIDSFIKGIKSNDRLYL 300 R.LlaCI FKKFLNLAETKLYEFVEGRLKNLEYQKNIKIKKIEFEISSIPDSELRDKFLKYRQEIETQYIVTFQERIQKFRLTNNKESTTYSKFIDSSFDKS 324 R.EcoVIII WIDERIATSEGIYVLKKMAIEYLSSERGRILSM SREEAVRALIKMNKIESRIEQIKKVTDNNILSLK 307 R.Csp231I WSEFSETSDLIVSEKALRHSYR 313 R.BstZ1I YVKCLNRYDKIINQAVKEIINFLETIIINKKEEVAREIRKLSDKQIREAYEEYKISQETEEYQRILENVKAFRL 318 R.BbrI LRDSYAARRQLLQRQFDDEIAHWDAEAQRIRGL SREQAIAELLVSKKIQARVDLMRGAQRQLTLQLDDARA 329 Comparison of the amino acid sequences of R.HindIII isoschizomers.

9 Molecular model of R.HindIII in complex with DNA (model was constructed by Janusz M. Bujnicki, Bioinformatics Laboratory, International Institute of Molecular and Cell Biology, Warsaw).

10 Comparison of the amino acid sequences of M.HindIII isomethylomers

11 M.HindIII MIDCIYN--SDSIXEIKKLDSNSIHAIISDIPYGIDYDDWDILHSNTNSALGGTSSAQHK 58 R.HindIII MKKSALEKLLSLIENLTNQEFKQATNSLISFIYKLNRNEVIELVRSIGILPEAIKPSSTQ 60 *.. :. * ::.: : :. :.: * :: :: *.....:. : M.HindIII TSLFKRRGKPLNGWSEADKKRPQEYQEWVESWSNEWFRVLKSGSSVFVFAGRQFAHRVVV 118 R.HindIII EKLFSKAGDIV--LAKAFQLLNLNSKPLEQRGNAGDVIALSKEFNYGLVADAKSFRLSRT 118.**.: *. : ::* : : : :...*... :.*. : :. M.HindIII AFENSGFTFKDMLSWEKDKAPHRAQRISCVFERRGDIANTNKWVGWRVANLRPLFEPILW 178 R.HindIII AKNQKDFKVKALSEWREDKD-YAVLTAPFFQYPTTKSQIFKQSLDENVLLFSWEHLAILL 177 * ::..*..* :.*.:** :.... :: :..* :..** M.HindIII FQKPYKTGSTLADNLIKHEVGAWNENSLTHWNIQQGALNHSNILKVRITSEDKGYHVAQK 238 R.HindIII QLDLEETNIFXFEQLWNFPKKQSKKTSVSDAENNFMRDFNKYFMDLFKIDKDTLNQLLQK 237. :*. ::* :. ::.*::. : : :. ::.:.:*. :: ** M.HindIII PLNLMK---LLIDLVTKEEQIVLDPFAGSGTTLLAAKELNRHFIGYEKNNGIYNIAVNRL 295 R.HindIII EINFIEERSLIEKEYWKKQINIIKNFTREEAIEALLKDIN MSSKIETIDSFIK 290 :*::: *:. *:: ::. *:. : *::*.. *.* M.HindIII GIEKNNCFYNKEKK 309 R.HindIII GIKSNDRLYL **:.*: :* Comparison of the amino acid sequences of M.HindIII and R.HindIII.

12 DNA sequencing by primer walking. Known sequence (vector) 3 5 Universal primer New primer 53 Analyzed DNA

13 INDEXING STRAND (24-mer) PRIMER STRAND (20-MER) GGTACAGGATGCGAAGACAA CCATGTC CT ACGC TTC T GT T NNNN Indexer structure 5 -- OH P - 5

14 R.FokI cuts DNA in a precise distance from its recognition site. Specific sequence Cut site ( 9 nt ) Cut site ( 13 nt ) GGATG 5 NNGGATGNNNNNNNNNNNNNN 3 CCTACNNNN 3 NNCCTACNNNNNNNNNNNNNN 5

15 EnzymeSpecific sequenceAverage fragment length (bp) AarICACCTGC (4/8)8192 Alw26I (BsmAI)GTCTC (1/5)512 BbsI (BpiI)GAAGAC (2/6)2048 BbvI (BseXI)GCAGC (8/12)512 BsmFIGGGAC (10/14)512 BspMIACCTGC (4/8)2048 BtgZIGCGATG (10/14)2048 Eco31IGGTCTC (1/5)2048 Esp3ICGTCTC (1/5)2048 FokIGGATG (9/13)512 SfaNIGCATC (5/9)512 AceIIICAGCTC (7/11)2048 Bbr7IGAAGAC (7/11)2048 BbvIIGAAGAC (2/6)2048 Sth132ICCCG (4/8)128 StsIGGATG (10/14)512 DNA sequencing by indexer walking Type IIS restriction endonucleases (www.rebase.neb.com/rebase) Commercially available Commercially unavailable

16 DNA fragmentation and cloning 1. DNA sequencing using primer REV 2. Nucleotide sequence analysis 3. Partial digestion with class-IIS ENase Genomic DNA DNA amplification Primer REVERSE (REV) FORWARD (FR) Insert (4-5 kb) FR REV DNA sequencing by indexer walking

17 Ligation of specific indexer 1. DNA amplification 2. DNA sequencing Consecutive steps of DNA sequencing by indexer walking Streptavidin coated paramagnetic particles AAAG FR TTTC REV Purification of biotin labeled DNA fragment INDEXER 1 TTTC primer AAAG FR INDEXER 1 AAAG FRTTTC primer INDEXER 2 GCAA FRCGTT primer AAAG INDEXER 1 CGTT TTTC primer GCAA FR Removed DNA fragment 5. Ligation of the next indexer 3. Nucleotide sequence analysis 4. Partial digestion with type-IIS ENase

18

19 Financial support: Polish Committee for Scientific Research KBN 6P04B KBN 6P04B KBN IA-1433/ KBN IA-0589/

20 Lab members: Beata Furmanek-Blaszk Anna Kaczorowska Tadeusz Kaczorowski Iwona Mruk Józef Nieradko Marian Sęktas Łucja Adamczyk Jadwiga Pomorska Magdalena Cichowicz Agnieszka Dekowska Katarzyna Gromek Anna Kossobucka


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