Key for assignment 4

1. Compare MLH1 (answer of assignment 3.6) and mutS (answer of 3.7) sequence.

No significant similarity was found.

Blast -> Pairwise Blast

2. Translate the above two gene sequences to protein sequences.

MLH1 ->

>lcl|Sequence 1 ORF:51..2321 Frame +3
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRK
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPK
PCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNA
STVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVY
AAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLP
GLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDIS
SGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEM
TAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELF
YQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEI
DEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQ
QSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC*
 

mutS ->

>lcl|Sequence 1 ORF:679..3240 Frame +1
MSAIENFDAHTPMMQQYLRLKAQHPEILLFYRMGDFYELFYDDAKRASQLLDISLTKRGASAGEPIPMAG
IPYHAVENYLAKLVNQGESVAICEQIGDPATSKGPVERKVVRIVTPGTISDEALLQERQDNLLAAIWQDS
KGFGYATLDISSGRFRLSEPADRETMAAELQRTNPAELLYAEDFAEMSLIEGRRGLRRRPLWEFEIDTAR
QQLNLQFGTRDLVGFGVENAPRGLCAAGCLLQYAKDTQRTTLPHIRSITMEREQDSIIMDAATRRNLEIT
QNLAGGAENTLASVLDCTVTPMGSRMLKRWLHMPVRDTRVLLERQQTIGALQDFTAGLQPVLRQVGDLER
ILARLALRTARPRDLARMRHAFQQLPELRAQLETVDSAPVQALREKMGEFAELRDLLERAIIDTPPVLVR
DGGVIASGYNEELDEWRALADGATDYLERLEVRERERTGLDTLKVGFNAVHGYYIQISRGQSHLAPINYM
RRQTLKNAERYIIPELKEYEDKVLTSKGKALALEKQLYEELFDLLLPHLEALQQSASALAELDVLVNLAE
RAYTLNYTCPTFIDKPGIRITEGRHPVVEQVLNEPFIANPLNLSPQRRMLIITGPNMGGKSTYMRQTALI
ALMAYIGSYVPAQKVEIGPIDRIFTRVGAADDLASGRSTFMVEMTETANILHNATEYSLVLMDEIGRGTS
TYDGLSLAWACAENLANKIKALTLFATHYFELTQLPEKMEGVANVHLDALEHGDTIAFMHSVQDGAASKS
YGLAVAALAGVPKEVIKRARQKLRELESISPNAAATQVDGTQMSLLSVPEETSPAVEALENLDPDSLTPR
QALEWIYRLKSLV*
 

NCBI -> ORF finder -> Paste DNA sequence -> OrfFind -> Choose correct Frame

3. Perform protein sequence homology searching for MLH1 in GenBank. Give the 10 highest hits.

gi|13878583|sp|Q9JK91|MLH1_MOUSE
gi|13591989|ref|NP_112315.1|  (NM_031053)
gi|4557757|ref|NP_000240.1|  (NM_000249)
gi|466462|gb|AAA17374.1|  (U07418)
gi|604369|gb|AAA85687.1|  (U17857)
gi|12835158|dbj|BAB23172.1|  (AK004105) putative [Mus musculus]
gi|13543339|gb|AAH05833.1|AAH05833  (BC005833)
gi|7304079|gb|AAF59117.1|  (AE003838) Mlh1 gene product
gi|3192877|gb|AAC19117.1|  (AF068257) mutL homolog
gi|460627|gb|AAA16835.1|  (U07187) Mlh1p

Blast -> Protein BLAST -> Paste sequence -> Blast -> Format

4. Compare human MLH1 protein with MLH1 in M. musculus, R. norvegicus and D. melanogaster. Give the pairwise alignment and % of sequence smility.

1) M. musculus -> pairwise alignment
Score = 1329 bits (3440), Expect = 0.0
Identities = 670/760 (88%), Positives = 714/760 (93%), Gaps = 4/760 (0%)

2) R. norvegicus -> pairwise alignment
Score = 1306 bits (3380), Expect = 0.0
Identities = 659/758 (86%), Positives = 707/758 (92%), Gaps = 3/758 (0%)

3) D. melanogaster -> pairwise alignment
Score =  652 bits (1682), Expect = 0.0
Identities = 348/751 (46%), Positives = 476/751 (63%), Gaps = 94/751 (12%)
 

Blast -> Pairwise BLAST -> Paste MLH1 sequence on sequence 1 -> Paste "Mus musculus", " R. norvegicus", "D. melanogaste" sequence on sequence 2 -> Align

5. Search the conserve domain (CD) for MLH1. Give the position of the CD, name of CD and Pfam ID number.

Position: 147-327 amino acid

Name: DNA_mis_repair, DNA mismatch repair protein. Also known as the mutL/hexB/PMS1 family

Pfam ID: 01119
 

Blast -> Search for conserved domains -> Paste sequence -> Search

6. Show multiple alignment of MLH1 conserve domain with 5 sequences from the top of the CD alignment.

Answer
 

Blast -> Search for conserved domains -> Paste sequence -> Search ->add query to multiple alignment, display 5 sequences from the top of the CD alignment