PostGenomics Technologies

1
Post-Genomics Technologies

• Practical

2
Sequence 7

• TCAGCTCTTGTTGGGGGTTTGACCCGTGAGAAGTTTATGAATGAGGCGTT
  TTTACGAATGCAGCTGCTGAGCAGGTATACAGTTTAGATACCTTTTTTTA
  ATTTCTTTAGCATGATATAACTTTAGGTTTCTCATTTTAATGTATGTTGT
  GTGGTAGGTTGATCTTTTTGTAGCTACCCCAAGCAATATTCCAGCAGAGT
  CATTTGAAGTTTACGAAGTTGCACTTGCTCTTGTGGCTC
• Gene ARC6
• Arabidopsis ARC6 (ACCUMULATION AND
• REPLICATION OF CHLOROPLASTS )
• heat shock protein binding
• Accession No.
• AY221468.1 from NCBI
• At5g42480 from AtEnsembl
• Chromsome 5
• Genomic sequence 3,668 bps
• Exons 6
• Transcript length 2,627 bps
• Translation length 801 residues

3

• ARC6 Is a J-Domain Plastid Division Protein and
  an Evolutionary Descendant of the Cyanobacterial
  Cell Division Protein Ftn2

4
Homology Searches by BLAST
5
DNA Sequence Analysis

• Transeq Translate DNA sequences into protein
  sequences
• gtEMBOSS_001_1
• SALRRIQLAICLDFNKILLGYRIHWLCLLLHLHVIIVSNFTHFSW
  MLRKERELMGFCGFKLSSQELSHWFTVALYVNEGNSNGSVEIITI
  HHKVRGDCNIIDCFLFFSVAGDVFFNLYYSKSKFHKPTTKQSLQYVK
  QNKVFVVAKDTPMEALSHVGIGLSPFQLCRLPPATTKLRRSHNTSTTIC
  SASKWADRLLSDFNFTSDSSSSSFATATTTATLVSLPPSIDRPERHVPIP
  IDFYQVLGAQTHFLTDGIRRAFEARVSKPPQFGFSDDALISRRQILQAAC
  ETLSNPRSRREYNEGLLDDEEATVITDVPWDKVISISESFFVLISIG
  RKELLSSEGSWGSLCIARRWDDSSSGWGSAGEVAVVARCGFSY
  GACVSRCLEGCYGIGSTFYYWLVCGSFEAFTGSLTCFGNLTSVGFIR
  TFLIYFVIESCVGGRSKPCTGFTCTNDFGRDHSALCLGATWLTAW
  LRCEKTKWFKRCAEYFVVCWRRWSISSCWGFDPEVYEGVFTNDSCA
  GIQFRYLFLISLAYNFRFLILMYVVWVDLFVATPSNIPAESFEVYEVA
  LALVAQAFIGKKPHLLQDADKQFQQLQQAKVMAMEIPAMLYDTRNNWEID
  FGLERGLCALLIGKVDECRMWLGLDSEDSQYRNPAIVEFVLENSNRDDND
  DLPGLCKLLETWLAGVVFPRFRDTKDKKFKLGDYYDDPMVLSYLERVEVV
  QGSPLAAAATMARIGAEHVKASAMQALQKVFPSRYTDRNSAEPKDVQETV
  FSVDPVGNNVGRDGEPGVFIAEAVRPSENFETNDYAIRAGVSESSVDETT
  VEMSVADMLKEASVKILAAGVAIGLISLFSQKYFLKSSSSFQRKDMVSSM
  ESDVATIGMIKCNFHISALLKICLFCELRTFPLNTCPKSCTKINKLLS
  KFHLCCLNFLIKLTEMISLSTFLFRITDRLPCGFVQLFFPFFS
  FRFRVSQSRFRSTSQNGCDCREYSIQVAEDVSGFWASPHRNVTRGE
  GINLQFNQLCENCWTLSGALFDSVIYRFWMGECRFGLTEQLKLRSLG
  WFMIIHCNYLLTVQSQQMEPVLWWKQLWRSLLVYLIWFIQKTMLLMSE
  PTQQDTKFSGPSQGGKSLKALFLHHNILICSMSELARFSLFCKFSLVSV
  YKT QKNRSWHTLFLDLNYNIRATIX

6
DNA Sequence Analysis

• gtActual ARC6 protein sequence (from NCBI)
• MEALSHVGIGLSPFQLCRLPPATTKLRRSHNTSTTICSASKWADRLLSDF
  NFTSDSSSSSFATATTTATL
• VSLPPSIDRPERHVPIPIDFYQVLGAQTHFLTDGIRRAFEARVSKPPQFG
  FSDDALISRRQILQAACETL
• SNPRSRREYNEGLLDDEEATVITDVPWDKVPGALCVLQEGGETEIVLRVG
  EALLKERLPKSFKQDVVLVM
• ALAFLDVSRDAMALDPPDFITGYEFVEEALKLLQEEGASSLAPDLRAQID
  ETLEEITPRYVLELLGLPLG
• DDYAAKRLNGLSGVRNILWSVGGGGASALVGGLTREKFMNEAFLRMTAAE
  QVDLFVATPSNIPAESFEVY
• EVALALVAQAFIGKKPHLLQDADKQFQQLQQAKVMAMEIPAMLYDTRNNW
  EIDFGLERGLCALLIGKVDE
• CRMWLGLDSEDSQYRNPAIVEFVLENSNRDDNDDLPGLCKLLETWLAGVV
  FPRFRDTKDKKFKLGDYYDD
• PMVLSYLERVEVVQGSPLAAAATMARIGAEHVKASAMQALQKVFPSRYTD
  RNSAEPKDVQETVFSVDPVG
• NNVGRDGEPGVFIAEAVRPSENFETNDYAIRAGVSESSVDETTVEMSVAD
  MLKEASVKILAAGVAIGLIS
• LFSQKYFLKSSSSFQRKDMVSSMESDVATIGSVRADDSEALPRMDARTAE
  NIVSKWQKIKSLAFGPDHRI
• EMLPEVLDGRMLKIWTDRAAETAQLGLVYDYTLLKLSVDSVTVSADGTRA
  LVEATLEESACLSDLVHPEN
• NATDVRTYTTRYEVFWSKSGWKITEGSVLAS

7
Clustal W Alignment
8
Protein Analysis
9
DnaJ-domain DnaJ domains (J-domains) are
associated with hsp70 heat-shock system and it is
thought that this domain mediates the
interaction. DnaJ-domain is therefore part of a
chaperone (protein folding) system. The
T-antigens, although not in Prosite are confirmed
as DnaJ containing domains from literature
10
Restriction Analysis

• Noncutters AarI, AatII, AcyI, AgeI, ApaI, AscI,
  BaeI, BalI, BbvCI, BcgI, BfiI, BglI, BglII, BplI,
  BsaBI, BsaXI, BsePI, BsgI, Bsp1407I, BsrBI,
  BstEII, BstXI, CfrI, ClaI, DraII, DraIII,
  Eam1105I, Eco47III, EcoRV, FseI, FspAI, HpaI,
  KpnI, MluI, NaeI, NarI, NheI, NotI, NruI, OliI,
  PI-PspI, PI-SceI, PacI, PfoI, PmaCI, PpuMI, PsrI,
  PvuI, RsrII, SacII, SalI, SanDI, ScaI, SfiI,
  SgfI, SmaI, SnaBI, SpeI, SphI, SrfI, Sse8387I,
  StuI, SwaI, Tth111I, VspI, XcmI
• Following are few of the enzymes that cut our
  gene

11
Reverse complement
To get inverse, complement, inverse complement or
double-strand DNA
12
Double-stranded DNA