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RNA Sequencing for Global Transcriptome Mapping

#65
Introduction:
Information regarding to identification of the genes, exons and their boundaries are important for understanding the function of the genome, determining when they are expressed and how they are regulated. (9) Advances in DNA sequencing technology has provided a method for both mapping and quantifying transcriptomes. The method, RNA sequencing, is a high throughput sequencing-based method for global transcriptome mapping that provided a way to assess the RNA expression level more accurately. (9, 15) Since RNA sequencing is quantitative, it is possible to determine the quantity of every molecule in a cell population.(15) Direct ultra-high- throughout sequencing of cDNA is a RNA approach that results in sequence read that can be mapped to the source genome and its correspondence to RNA from known exon, splice event or new genes.(8)
RNA sequencing can measure transcriptome complexity and dynamic of different tissues or condition.(clana, 9, 15) It has been used to survey the sequence content of the poly(A)+ transcriptomes of undifferentiated mouse embryonic stem cells and embryoid bodies. (3) This is useful for the monitoring of gene expression and tracking of gene expression change during development. (15) By mapping and quantifying mouse transcriptome “this provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes”. (8) These experiment shows the importance of RNA sequencing in the understanding of transcriptomic during development. The digital measurement should allow the comparison of diseased and normal tissue. (15) This means that RNA sequencing would be an important tool for cancer research by comparing mutated tissues and normal tissues.
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Mutated cell behaves differently than normal cell and we want to see what genetic mechanism is causing this difference. Which means we need to look at the difference of gene expression. Information that are encoded in selected gene which can be transcribed into RNA molecule. These molecules can be translated to proteins or used for control gene expression. The RNA that is transcribed therefore can reflect the state of the cell and reveal information on mechanisms of underlying diseases. (11) RNA-seq has a sequencing frame work which allow the investigation of all the RNA present in a sample. We can then characterize the sequences and even quantify the abundances. There are millions of short strings called reads that are sequenced from random positions of the input RNA. These reads can then be mapped into a reference genome and a transcriptional map is made.(4) The number of reads that are aligned to each gene will give a measure of its level of expression. RNA-sequencing allows us to analyze the transcript variation. Projects such as the HapMap project allows the identification of more than a thousand genes in which the genetic variation influence over all expression level or splicing. (5)
Figure 1: RNA-Seq experiment.  long RNAs are first converted into a library of cDNA fragments through either RNA fragmentation or DNA fragmentation. Sequencing adaptors (blue) are subsequently added to each cDNA fragment and a short sequence is obtained from each cDNA using high-throughput sequencing technology. The resulting sequence reads are aligned with the reference genome or transcriptome, and classified as three types: exonic reads, junction reads and poly(A) end-reads. These three types are used to generate a base-resolution expression profile for each gene, as illustrated at the bottom; a yeast ORF with one intron is shown. (8)
Smad4, also know as Mothers against decapentaplegic homolog 4 is a protein encoding gene in Mus Muculus. The role of smad4 is a signaling molecules of the TGF-beta pathway in T cells on the pathology of Sjögren’s syndrome (SS) in non-obese diabetic (NOD) mice.(6) When the T cell-specific Smad4 were deleted the NOD mice have accelerated development of SS compared to the wild type NOD mice.(6) In the research it is concluded that the disruption of the Smad pathway in T cells of NOD mice seems to increase Teff cell activation which results in the upregulation of Th17 cells. This shows that Smad4 in t cell plays a role in preventing the development of SS in NOD mice and that Smad4 plays an important role in T cell activation. (6) The mutation of smad 4 is not directly related with cancer in the mouse but instead autoimmune diseases. Based on its function the expression of this gene hould be expected to increase, because it is an important signaling molecule of TGF-beta pathway.
In the Homo Sapiens homolog the Smad4 gene codes for a protein (Mothers against decapentaplegic homolog 4) involved in transmitting chemical signals from the cell surface to the nucleus. (13) The TGF-beta pathway allows the environment outside the cell to affect the gene activity and protein production within the cell. (13) The signaling process is initiated when TGF-beta protein binds to a receptor protein on the cell surface. This activates a group of related SMAD proteins which then binds to SMAD4 protein and forms a protein complex. (17) This complex moves to the cell nucleus and bind to specific area of DNA an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE) where it controls the activity of particular gene and regulate cell growth and division. (10)
The Smad4 protein is both a transcription factor and a tumor suppressor. It can act as a tumor suppressor by reducing angiogenesis and increasing blood vessel hyperpermeability which should have inhibitory effects on tumors.(14) The protein encoded also play an important role as the component of Bone morphogenetic protein signaling pathway and transforming growth factor-β signaling pathways regulates apoptosis, the program death of cell, in various cell types through a mitochondrial pathway. (7) In muscle physiology, Smad4 protein plays a role in the balance between atrophy and hypertrophy. When recruited by MSTN, promotes atrophy response via phosphorylated Sad2 and Smad4. MSTN decrease causes Smad4 release and subsequent recruitment by the BMP pathway to promote hypertrophy via phosphorylated Smad1, Smad5 and Smad8.(12) The Smad4 protein are subject to regulation by the post translational modifications. Mutation or deletion of this gene have resulted in pancreatic cancer, juvenile polyposis syndrome, and hereditary hemorrhagic telangiectasia syndrome. (14)
The mutation and inactivation of the Smad4 gene is associated with poorer recovery in patients with surgically-resected adenocarcinoma (spreadable cancer in glands) of the pancreas. An experiment was done where millions of base pair of DNA from transcripts in a series of 24 adenocarcinomas of the pancreas are sequenced. 39 gens were mutated in more than one of these 24 cancer and sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas (1) Out of 114 patients they had, 84 underwent surgery for the removal of cancerous tumors from head of pancreases. The somatic mutations in these cancers were correlated with patient outcome (1, 16) the result of the studies shown that the survival of the patient with and without Smad4 gene inactivation were significantly different. Smad4 is a gene that codes for the protein which is used in the transforming growth factor beta (TGFβ) pathway, and the experiment saw an association of Smad4 gene inactivation with metastasizing of the pancreatic cancer. (1)
The mutation of the Smad4 can be cause by intragenic mutation or homozygous deletion. The experiment shows that the inactivation of the Smad4 gene is related to the metastasized cancer which means that this gene is a tumor suppressor gene. Tumor suppressor are normal human gene that prevent the uncontrollable cell growth, which means it plays an important role in the proliferation of cancer cell. This explains why an inactivation of the Smad4 gene which plays an important role in the transforming growth factor beta (TGFβ) pathway, can lead to prevention of the upregulation of these pathways. TGFβ pathways are known to play important role in roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. (2)
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Figure 2: The TGF-β/Smad4 signaling pathway. The Ligand TGF-β binds a complex of transmembrane receptor serine/threonine kinases (Types I and II) in the cell surface and induces transphosphorylation of the receptors. The consequently activated receptors phosphorylate selected Smads at C-terminal serines, and these receptor-activated Smads (R-Smads) then form a complex with a common Smad4. Activated Smad complexes translocate into the nucleus, where they regulate transcription of target genes, through physical interaction and functional cooperation with DNA-binding transcription factors. (3)
1 AGTGTCCTTC CGACAAGTTG GCAGCAACAA CACGGCCCTG GTCGTCGTCG CCGCTGCGGT
61 AACGGAGCGG CTCGGGTGGC GGAGCCCGTG TTCGCGTCCG TCCGCCCGCC CGCCCGCCGT
121 CCTCCGGAGG CCCTTCCCGC GCCGCGCTCC GCTCCGCGGC CGTCCCCGGG GCGGGAGCGC
181 GTGACCGGAG CCGGCGCCCG CGAGCGAGGC CCCCCGCAGC GGGGCGGCTC CGGAGCTCCA
241 GCGGCCCGGC CGGCCGGCGC GGTCCGCGGC GCGGCGGGGA GAGGGGGCCG CCTGGGCCGG
301 ACGCCGCGGG CGGGGCCCGG GAAGCGACAG CGAGGCGAGG CGCGGTGCGG CGCGGAGCCC
361 AGGTCATCCT GCTCACCAGA TGTCTTGACA GTTTTTCTTG CAACATTGGC CATTGGTTTT
421 CACTGCCTTC AAAAGATCAA AATTACTCCA GAAATTGGAG AGTTGGATTT AAAAGAAAAA
481 ACTTGAACAA ATGGACAATA TGTCTATAAC AAATACACCA ACAAGTAACG ATGCCTGTCT
541 GAGCATTGTA CATAGTTTGA TGTGTCATAG ACAAGGTGGG GAAAGTGAAA CCTTTGCAAA
601 AAGAGCAATT GAGAGTTTGG TAAAGAAGCT GAAAGAGAAA AAAGATGAAT TGGATTCTTT
661 AATAACAGCT ATAACTACAA ATGGAGCTCA TCCTAGCAAG TGTGTCACCA TACAGAGAAC
721 ATTGGATGGA CGACTTCAGG TGGCTGGTCG GAAAGGATTT CCTCATGTGA TCTATGCCCG
781 TCTGTGGAGG TGGCCTGATC TACACAAGAA TGAACTAAAG CATGTTAAAT ATTGTCAGTA
841 TGCGTTTGAC TTAAAATGTG ACAGTGTCTG TGTGAATCCA TATCACTATG AGCGGGTTGT
901 CTCACCTGGA ATTGATCTCT CAGGATTAAC ACTGCAGAGT AATGCTCCAA GTATGTTAGT
961 GAAGGATGAG TACGTTCACG ACTTTGAAGG ACAGCCGTCC TTACCCACTG AAGGACATTC
1021 GATTCAAACC ATCCAACACC CGCCAAGTAA TCGCGCATCA ACGGAGACGT ACAGCGCCCC
1081 GGCTCTGTTA GCCCCGGCAG AGTCTAACGC CACCAGCACC ACCAACTTCC CCAACATTCC
1141 TGTGGCTTCC ACAAGGCCAG TTCACAATGA GCTTGCATTC CAGCCTCCCA TTTCCAATCA
1201 TCCTGCTCCT GAGTACTGGT GCTCCATTGC TTACTTTGAA ATGGACGTTC AGGTAGGAGA
1261 GACGTTTAAG GTCCCTTCAA GCTGCCCTGT TGTGACTGTG GATGGCTATG TGGATCCTTC
1321 GGGAGGAGAT CGCTTTTGCT TGGGTCAACT CTCCAATGTC CACAGGACAG AAGCGATTGA
1381 GAGAGCGAGG TTGCACATAG GCAAAGGAGT GCAGTTGGAA TGTAAAGGTG AAGGTGACGT
1441 TTGGGTCAGG TGCCTTAGTG ACCACGCGGT CTTTGTACAG AGTTACTACC TGGACAGAGA
1501 AGCTGGCCGA GCACCTGGCG ACGCTGTTCA TAAGATCTAC CCAAGCGCGT ATATAAAGGT
1561 CAGTGTTTAT ATGTCTTTGA TCTGCGGCAG TGTCACCGGC AGATGCAGCA ACAGGCGGCC
1621 ACTGCGCAAG CTGCAGCTGC TGCTCAGGCG GCGGCCGTGG CAGGGAACAT CCCTGGCCCT
1681 GGGTCCGTGG GTGGAATAGC TCCAGCCATC AGTCTGTCTG CTGCTGCTGG CATCGGTGTG
1741 GATGACCTCC GGCGATTGTG CATTCTCAGG ATGAGCTTTG TGAAGGGCTG GGGCCCAGAC
1801 TACCCCAGGC AGAGCATCAA GGAAACCCCG TGCTGGATTG AGATTCACCT TCACCGAGCT
1861 CTGCAGCTCT TGGATGAAGT CCTGCACACC ATGCCCATTG CGGACCCACA GCCTTTAGAC
1921 TGAGATCTCA CACCACGGAC GCCCTAACCA TTTCCAGGAT GGTGGACTAT GAAATATACT
1981 CGTGTTTATA ATCTGAAGAT CTATTGCATT TTGTTCTGCT CTGTCTTTTC CTAAAGGGTT
2041 GAGAGATGTG TTTGCTGCCT TGCTCTTAGC AGACAGAAAC TGAATTAAAA CTTCTTTTCT
2101 ATTTTAGAAC TTTCAGGTGT GGCTCAGTGC TTGAAGATCA GAAAGATGCA GTTCTTGCTG
2161 AGTCTTCCCT GCTGGTTCTG TATGGAGGAG TCGGCCAGTG CTGGGCGCTC AGCCCTTTAG
2221 TGTGTGCGAG CGCCTTGCAT GCCGAGGAGA GTCAGAGCTG CTGATTGTAA GGCTGAGAAG
2281 TTCTCACAGT TAAGCCACCT GCCCCTTAGT GGGCGAGTTA TTAAACGCAC TGTGCTCACG
2341 TGGCGCTGGG CCAGCCAGCT CTACCAAGAG CAACTTTACT CTCCTTTAAA AACCTTTTAG
2401 CAACCTTTGA TTCACAATGG TTTTTGCAAG TTAAACAGTG AAGGTGAATT AAATTCATAC
2461 TGTCTTGCAG ACTTCAGGGT TTCTTCCCCA AGACAAAACA CTAATCTGTG TGCATATTGA
2521 CAATTCCTTA CAATTATCAG TCAAAGAAAT GCCATTTAAA ATTACAATTT TTTTAATCCC
2581 TAATGGATGA CCACTATCAA GATGTATACT TTGCCCTGTT AAACAGTAAA TGAATTCTTC
2641 TATATTTCTA GGCACAAGGT TAGTTATTTA AAAAAAAAAA AAAAAGCCTA GGGGAGGGAT
2701 TTTTCCCTTA ATTCCTAGGG AGAAGGTTTT GTATAAAACA CTAAAAGCAG TGTCACTCTG
2761 CCTGCTGCTT CACTGTTCTG CAAGGTGGCA GTACTTCAAC TGAAATAATG AATATTTTGG
2821 AAACTGCTAA ATTCTATGTT AAATACTGTG CAGAATAATG GAAACAGTGC AGTTGGTAAC
2881 AGGTGGTTTG GATATTTTTG TACTTGATTT GATGTGTGAC TTCTTTTCAT ATACTGTTAA
2941 AATCATGTAT GTTTTGACAT TGTTTAAAAT TCAGTTTTTG TATCTTAGGG CAAGACTGCA
3001 GACTTTTTTA TACCTTTTGG TTATAAGCCC TGTGTTTGCC ATCCTTGATC ACTTGGCGGT
3061 GACTTTGTAG AGATTGAAGT GGAGGAGTTA AGACACATTG ACTGTACCAC AGACACACAT
3121 GTATACTTTC TACCTAGTTA CTAGCGTAAA TAAAACTGAG TCACTATACG AAGTGGAATT
3181 CTAGATTTGG TTTTTAAAAT GCTTTCCTTT TGCACTTTTG AGTCCAGTCT CAGTGGCAAG
3241 ACACCTTCTG CTAAATGACA GGTGGCAGCC AGTTGTACCA TGCAGCGCTG GTTCCCTCCC
3301 ACTCTACCAG GACTTTCCCA TGGACACTGT GCATCATGTG TAGTTGGTTA TTTTTTGAGT
3361 TTTTATTTTA CTGTAGCAAA AAAAAAAAAA AAAACTTGGA TAAATAGTGT GAATAAAATC
3421 AAGACCATGG AGATGTTTTT ACCCTGAGAG TTTTCTGTGA GTTTTAAATT GCAGTAGGCA
3481 TTTGAGCTCT GGAAACCCCG TGCATAGCAG TTCTCTTTGT GCCAACAGAA ATGACCACGT
3541 CCTGCAGCCT GCTGCGGAAG GTTCCAGAGG CTCTGAGAAA CCAGAGTGCT GCAGTGACTG
3601 GGGTCCATCT CAGCCCAGCG CACACAGCGT GCGTTGTAAA AGCTGCCTCT GTGTCTTGTC
3661 TTCTGTACTT AGGGATGCTT TGTCTCGGGC CTAATCTTAT CTGTAGAAGT TTGGTGATTT
3721 TTTTTTTTTA AATGTTGTAT TGACAGAATT ATAAAAAGAT ACCTTCTCTA GAAATGCTTG
3781 TCTTCAGATC CGTTTCACGA TGGCCGGGGA ACAGGAGTGA GAAGAGAGAG TAAGCTGTAG
3841 TGTAACGGGT TTTTAAGACC CAGCTCATCT GACCAGGCAG TGCTGTAACT TGATGCTTCC
3901 TGTTGTACCT TATGGAACCT TTCCCATATT TAATCATCTT CAGAAAGTAG GTGGGAAATA
3961 TTTGCTGGGA AGTATCTCTT CAGAGCCAAG CCACTTGTCT TGGTTTTCTT ACTAAGAGCC
4021 ATAGAAATGA TTTCTGGTTA TTGATGAAAT TTGTAATTTG CCTGTCCTAG TCTTTTTTCC
4081 TTTCACTTCG CTATCTTTGA ATAAGACTTT TAAAAACTTC CCTGAGTTGA AAAATTTTGG
4141 GATAAAATAG TTTCCCTAGT TCTTAGAGAC TGATTATGAT GTGGGTATGG TTCTGGGTGG
4201 GTTTTTTTTC TAAGTCATAG CTCAAAAGTC TCCCAAGATT AAATTTCAGT GGGCACCCAG
4261 TTTGAAACCA TTCTACTTTT GTCTTGTGCC TTTCTTTGCA TGATTAAAGA GAATCTGTAA
4321 TGGTATTGCC TTTATTTGCT TGGAAGTAGA TTCTTTTCTG GGATAGAGTC TACCTTAATC
4381 GTTGTCCTTT ACCGCCCCTG CTGTACAGAT AGATGCTAAG CCACTGCCGG GAACTTGCTT
4441 TTCCATAGAC AGTCTTTTTA TACTGCCTGA ACCCATTGCT CCTGTTCACA GTATAAGTTC
4501 ACAGACAGGG TGAGCCGGCC GAGGCGCACA CCTGCAGAAT CCAGCAACAA CCATGCTTAA
4561 CTGTGTGTAT TTCAAAGTTA GAAATCCAGT TTTGTGGGGA ATGGTGTGGT TTATATTAGC
4621 AGCTTTGAAG GCGAAGTAAC TCAGAGGTTT TACAGTCTGG AGAAGGGAAG CTTCCTGGAA
4681 TGCTTGTGAA GTATCTGTGG TGGCCAAATG TGTTTGCTCC TGGCCTTGCT TGTAACTGGC
4741 TAATTGTCAC TCTTCAGATT TTTAAAAATT TTTAATGAGC TGAGACCCCC TTGGAAGGAG
4801 CTTGTTTGGA GCTGGCCAGA GATGTTTTTG GTAGTTCCTG TCTTCATCCG GTCTTCATCA
4861 CTGTTTTCTT TAATGGTCAG TTAGTAAAGT ATAAGTTAGG TCACTGTCAT GAGTGGAGCA
4921 GGAACAACTC TCCCAGGTGG GGGCCTGGAA GGGACTCGTT ACATGGAGCC ATCTGTAACT
4981 AGCCCTTTAA ATCCTCCTTT GCATGACATA GAGAAAAGGC TGTGAGACTC CTGCCCAGGC
5041 CTTTCTAGTT TTCCCTTCTA GTAACCAAGC AATCGCATCT CTGCGGTGCA GTAGGCTGTA
5101 TGTAAAAAGC CGTGGCCTTA CTCCTAGCAG CACCCTTGGC AGGGCCTTTT TCTCAGCGCA
5161 GTGAGGCTGT GCATCTGGCA CTCCTGAGGA ATGAAAGTTT TCATCATCTT GCCTTATTAA
5221 GCAGTAAAAC TTTTGAAAAA TGAGCCGTTT ATTGGCAGGA GCTATTTACA CAAATCAGAA
5281 TATTATACCA TTTCTTTTTC TCTCTCTCCT GTCTCTGTGG ACCTCCGGGG CTTCTGAGAT
5341 AGACAGTACT GCCTAGCCAT TCGAAATGCC CAAGCCAGCT GGGGTTGTTG GGCTCTCCTC
5401 TCCCTTCCTC CTTCCTCACA GCTCCTGCTC TTGCGTGGTT AGTGAGCCTC TACTCAGTGT
5461 TTCCTGTCCT CGCTGCTCAG GCGAGGGAAG ACGACAACTG ATAGTCTTAG AGTTCACCTT
5521 TCTGTCGGGG GCGGCATTGT TCTGATTGCT GCCATCGTCT CCGATCCTTG ATGAGTTTTA
5581 TACGATTGAT GTGGAGAGAA TTTAATTGAT ATTCATAGCC CATAGCTGCT CCCCTCTCCC
5641 TGGTGTTGTG GAAGATTTAG TTTCCACCGA ATTCACTCAA AAAGCTGTCC TGTTGGCACC
5701 AGCAAACCAC ACGCTCTTTT AGAAAACATC TTTGCTTGTT TTGTGTCCTG ACCCTGCTCT
5761 CTGGCCTCCT TCCTCTGTAG ATACTTCTGA CCTATAGGTG CCTTTATGAG AATTGAGGGT
5821 CTGATACCGT GCCCCAAGGA ATAGCTGATG CAATGAGTGA TGTTTTTCAG GGATTTTAGC
5881 ATCAAATTAA ATAAATGAAT GAAACTTTTA AGTCCTTCTT TTCTTTTATT TTTTTAATGC
5941 AGGAAGGACT GAGGAGACGT CGGGTGACGA CAATCATTTC TCTGTGTTGC TGTAAAGGCT
6001 TTCACACAGT TTAAGATGCT TTTCTCAGTA GCTCCAGAGT TGATGTTCTT GTTCAACCTA
6061 AAGCAGGCTC TGGACTCGCC CAGACCGTTG CACTTGTAGT TTACGACTTC ATGTGTCCTC
6121 CCTCGGCAAG TCATTCCCTT CTCTGGGCCT CAGCTGCCTC GTCTGTGAAA TGAGGGGTTG
6181 GACTATTGTG CCAGCTCTGG CTTCTAAGTG ACCTTGCCCG CCCTGCAGCA GGTTGAGATG
6241 CGCTCTTTAC CTTTTTTCTG CTGTGTGAGG GGGAATCTTA CTTTTTCCTT TGTTACTCAG
6301 TGAGACTAGG CTTGATCTTT GAGTACCCGC TCTCCTGTGG ACAAGTAGTT ACATATGTCC
6361 TTATGACTTA TTTTTAACCA AAGGCCGAGC ACCACCTTAG GGGCTGCCGT AAGTACCATA
6421 CAGAACACTG GGGTGGGGGG CGGGGGGCAC CTTCATTTCA CTGTGTCATC GTCTGTGTTC
6481 AGAGCCTCTG CAAAGGCCTT CATCTGTCAT GACATTCTGA CTTTGAAGTT AGTATGTGTA
6541 TGATTCTGTC CTCCTAAGTG CTGGCAATTC TTCATCTAAA CTGGACTGAA ATCCTGTTGT
6601 AAATGCCTGG TAATATTAGA GGGCCTTTCT TTGGGTCTTT TGTAGCTTAA TTCCTCTATG
6661 TTCAAAACAG GAAGTTCTTC AGAAATTATA TCAATATTTT AATTGATGCT ATGAAAGACA
6721 GTCCCAGTGA ATGACTGTCC ACTTTATTTT TGCCTCTTTT ATATCCATTT TGATTGACAA
6781 CTTTTGGCTG GATCATGCCT TTCAGAGAGT TTTCTTCCAG CCTGCTTGGA TGAGTATAAT
6841 AACCGACTTT GTTATTTTTA CGGACCTGGG AACCTTTCTA GGGGGTGGGG TGGGGTGGGG
6901 TGGGGTGGGG AGTCCTGGTA GAGGCCACAT CTGTGGCAGC TGTGAAGAAG GGATGAAGCC
6961 AGCTGCTCTT GCTAAGGCTG CTTGTCATTG GTAGAAGGAC TCACCGGTTT GGGTTACTTA
7021 AAAGGCTAAA TATAGAGTTG GCAAACTTCT CCAAGCGGGG AGGGTTTTTT TTTTGTTCCA
7081 TGCATCTAAC GTGATTTAAA AGCATGACTT CCTATAGGTT ATGAAAACTG GTGTGCTGCA
7141 GATCCAGTGT GGAAGAGGTG ACTGGGCGTT GGGGACAGCT TTGATGGTGA CACTTCTAGC
7201 TCTGAGAGTC TCCTACTCTG GGTCCACTCT TAGCTTGGCT CTTAGGAAAA ACTGGTCAGC
7261 TAAAGGCCCA CCACTTTCTT TCTATAGACT TTTGCCTGGT TGAAGTCTGT GGCTTAAAAA
7321 AAATAGTTGA ATCTTTCTTG AGAACTCTGT AACAAAGTAT GTTTTTGATT AAAAAGAGAA
7381 AGCCAACTAA A
Figure 3A cDNA sequence of Mus musculus SMAD family member 4 (Smad4), transcript variant 2
 
MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSMLVKDEYVHDFEGQPSLPTEGHSIQTIQHPPSNRASTETYSAPALLAPAESNATSTTNFPNIPVASTRPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPVVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVSVYMSLICGSVTGRCSNRRPLRKLQLLLRRRPWQGTSLALGPWVE
Figure 3BAmino acid sequence of Mus musculus SMAD family member 4 (Smad4), transcript variant 2

Score Expect Method Identities Positives Gaps
583 bits(1503) 0.0 Compositional matrix adjust. 309/437(71%) 321/437(73%) 80/437(18%)

Query  1    MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITA  60
MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITA
Sbjct  1    MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITA  60
Query  61   ITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFD  120
ITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFD
Sbjct  61   ITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFD  120
Query  121  LKCDSVCVNPYHYERVVSPGIDLSGLTLQSNA—————–PSMLVKDEYVH  163
LKCDSVCVNPYHYERVVSPGIDLSGLTLQSNA                 PS+  +   +
Sbjct  121  LKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSSMMVKDEYVHDFEGQPSLSTEGHSIQ  180
Query  164  DFEGQPS–LPTEGHSIQTIQHPP-SNRASTETY——–SAPALL————  200
+  PS    TE +S   +  P  SN  ST  +        S PA +
Sbjct  181  TIQHPPSNRASTETYSTPALLAPSESNATSTANFPNIPVASTSQPASILGGSHSEGLLQI  240
Query  201  —————APAESNATSTTNF———PNIPVASTR————–  222
PA  +  STT +         PN+P
Sbjct  241  ASGPQPGQQQNGFTGQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGH  300
Query  223  –PVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPVVTVDGYVDPSGGD  280
PVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCP+VTVDGYVDPSGGD
Sbjct  301  YWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGD  360
Query  281  RFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGR  340
RFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGR
Sbjct  361  RFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGR  420
Query  341  APGDAVHKIYPSAYIKV  357
APGDAVHKIYPSAYIKV
Sbjct  421  APGDAVHKIYPSAYIKV  437
Figure 4A Mus musculus SMAD family member 4 (Smad4), transcript variant 2, aligned with homologue of Homo sapiens mothers against decapentaplegic homolog 4
1 ATGCTCAGTG GCTTCTCGAC AAGTTGGCAG CAACAACACG GCCCTGGTCG TCGTCGCCGC
61 TGCGGTAACG GAGCGGTTTG GGTGGCGGAG CCTGCGTTCG CGCCTTCCCG CTCTCCTCGG
121 GAGGCCCTTC CTGCTCTCCC CTAGGCTCCG CGGCCGCCCA GGGGGTGGGA GCGGGTGAGG
181 GGAGCCAGGC GCCCAGCGAG AGAGGCCCCC CGCCGCAGGG CGGCCCGGGA GCTCGAGGCG
241 GTCCGGCCCG CGCGGGCAGC GGCGCGGCGC TGAGGAGGGG CGGCCTGGCC GGGACGCCTC
301 GGGGCGGGGG CCGAGGAGCT CTCCGGGCCG CCGGGGAAAG CTACGGGCCC GGTGCGTCCG
361 CGGACCAGCA GCGCGGGAGA GCGGACTCCC CTCGCCACCG CCCGAGCCCA GGTTATCCTG
421 AATACATGTC TAACAATTTT CCTTGCAACG TTAGCTGTTG TTTTTCACTG TTTCCAAAGG
481 ATCAAAATTG CTTCAGAAAT TGGAGACATA TTTGATTTAA AAGGAAAAAC TTGAACAAAT
541 GGACAATATG TCTATTACGA ATACACCAAC AAGTAATGAT GCCTGTCTGA GCATTGTGCA
601 TAGTTTGATG TGCCATAGAC AAGGTGGAGA GAGTGAAACA TTTGCAAAAA GAGCAATTGA
661 AAGTTTGGTA AAGAAGCTGA AGGAGAAAAA AGATGAATTG GATTCTTTAA TAACAGCTAT
721 AACTACAAAT GGAGCTCATC CTAGTAAATG TGTTACCATA CAGAGAACAT TGGATGGGAG
781 GCTTCAGGTG GCTGGTCGGA AAGGATTTCC TCATGTGATC TATGCCCGTC TCTGGAGGTG
841 GCCTGATCTT CACAAAAATG AACTAAAACA TGTTAAATAT TGTCAGTATG CGTTTGACTT
901 AAAATGTGAT AGTGTCTGTG TGAATCCATA TCACTACGAA CGAGTTGTAT CACCTGGAAT
961 TGATCTCTCA GGATTAACAC TGCAGAGTAA TGCTCCATCA AGTATGATGG TGAAGGATGA
1021 ATATGTGCAT GACTTTGAGG GACAGCCATC GTTGTCCACT GAAGGACATT CAATTCAAAC
1081 CATCCAGCAT CCACCAAGTA ATCGTGCATC GACAGAGACA TACAGCACCC CAGCTCTGTT
1141 AGCCCCATCT GAGTCTAATG CTACCAGCAC TGCCAACTTT CCCAACATTC CTGTGGCTTC
1201 CACAAGTCAG CCTGCCAGTA TACTGGGGGG CAGCCATAGT GAAGGACTGT TGCAGATAGC
1261 ATCAGGGCCT CAGCCAGGAC AGCAGCAGAA TGGATTTACT GGTCAGCCAG CTACTTACCA
1321 TCATAACAGC ACTACCACCT GGACTGGAAG TAGGACTGCA CCATACACAC CTAATTTGCC
1381 TCACCACCAA AACGGCCATC TTCAGCACCA CCCGCCTATG CCGCCCCATC CCGGACATTA
1441 CTGGCCTGTT CACAATGAGC TTGCATTCCA GCCTCCCATT TCCAATCATC CTGCTCCTGA
1501 GTATTGGTGT TCCATTGCTT ACTTTGAAAT GGATGTTCAG GTAGGAGAGA CATTTAAGGT
1561 TCCTTCAAGC TGCCCTATTG TTACTGTTGA TGGATACGTG GACCCTTCTG GAGGAGATCG
1621 CTTTTGTTTG GGTCAACTCT CCAATGTCCA CAGGACAGAA GCCATTGAGA GAGCAAGGTT
1681 GCACATAGGC AAAGGTGTGC AGTTGGAATG TAAAGGTGAA GGTGATGTTT GGGTCAGGTG
1741 CCTTAGTGAC CACGCGGTCT TTGTACAGAG TTACTACTTA GACAGAGAAG CTGGGCGTGC
1801 ACCTGGAGAT GCTGTTCATA AGATCTACCC AAGTGCATAT ATAAAGGTCT TTGATTTGCG
1861 TCAGTGTCAT CGACAGATGC AGCAGCAGGC GGCTACTGCA CAAGCTGCAG CAGCTGCCCA
1921 GGCAGCAGCC GTGGCAGGAA ACATCCCTGG CCCAGGATCA GTAGGTGGAA TAGCTCCAGC
1981 TATCAGTCTG TCAGCTGCTG CTGGAATTGG TGTTGATGAC CTTCGTCGCT TATGCATACT
2041 CAGGATGAGT TTTGTGAAAG GCTGGGGACC GGATTACCCA AGACAGAGCA TCAAAGAAAC
2101 ACCTTGCTGG ATTGAAATTC ACTTACACCG GGCCCTCCAG CTCCTAGACG AAGTACTTCA
2161 TACCATGCCG ATTGCAGACC CACAACCTTT AGACTGAGGT CTTTTACCGT TGGGGCCCTT
2221 AACCTTATCA GGATGGTGGA CTACAAAATA CAATCCTGTT TATAATCTGA AGATATATTT
2281 CACTTTTGTT CTGCTTTATC TTTTCATAAA GGGTTGAAAA TGTGTTTGCT GCCTTGCTCC
2341 TAGCAGACAG AAACTGGATT AAAACAATTT TTTTTTTCCT CTTCAGAACT TGTCAGGCAT
2401 GGCTCAGAGC TTGAAGATTA GGAGAAACAC ATTCTTATTA ATTCTTCACC TGTTATGTAT
2461 GAAGGAATCA TTCCAGTGCT AGAAAATTTA GCCCTTTAAA ACGTCTTAGA GCCTTTTATC
2521 TGCAGAACAT CGATATGTAT ATCATTCTAC AGAATAATCC AGTATTGCTG ATTTTAAAGG
2581 CAGAGAAGTT CTCAAAGTTA ATTCACCTAT GTTATTTTGT GTACAAGTTG TTATTGTTGA
2641 ACATACTTCA AAAATAATGT GCCATGTGGG TGAGTTAATT TTACCAAGAG TAACTTTACT
2701 CTGTGTTTAA AAAGTAAGTT AATAATGTAT TGTAATCTTT CATCCAAAAT ATTTTTTGCA
2761 AGTTATATTA GTGAAGATGG TTTCAATTCA GATTGTCTTG CAACTTCAGT TTTATTTTTG
2821 CCAAGGCAAA AAACTCTTAA TCTGTGTGTA TATTGAGAAT CCCTTAAAAT TACCAGACAA
2881 AAAAATTTAA AATTACGTTT GTTATTCCTA GTGGATGACT GTTGATGAAG TATACTTTTC
2941 CCCTGTTAAA CAGTAGTTGT ATTCTTCTGT ATTTCTAGGC ACAAGGTTGG TTGCTAAGAA
3001 GCCTATAAGA GGAATTTCTT TTCCTTCATT CATAGGGAAA GGTTTTGTAT TTTTTAAAAC
3061 ACTAAAAGCA GCGTCACTCT ACCTAATGTC TCACTGTTCT GCAAAGGTGG CAATGCTTAA
3121 ACTAAATAAT GAATAAACTG AATATTTTGG AAACTGCTAA ATTCTATGTT AAATACTGTG
3181 CAGAATAATG GAAACATTAC AGTTCATAAT AGGTAGTTTG GATATTTTTG TACTTGATTT
3241 GATGTGACTT TTTTTGGTAT AATGTTTAAA TCATGTATGT TATGATATTG TTTAAAATTC
3301 AGTTTTTGTA TCTTGGGGCA AGACTGCAAA CTTTTTTATA TCTTTTGGTT ATTCTAAGCC
3361 CTTTGCCATC AATGATCATA TCAATTGGCA GTGACTTTGT ATAGAGAATT TAAGTAGAAA
3421 AGTTGCAGAT GTATTGACTG TACCACAGAC ACAATATGTA TGCTTTTTAC CTAGCTGGTA
3481 GCATAAATAA AACTGAATCT CAACATACAA AGTTGAATTC TAGGTTTGAT TTTTAAGATT
3541 TTTTTTTTCT TTTGCACTTT TGAGTCCAAT CTCAGTGATG AGGTACCTTC TACTAAATGA
3601 CAGGCAACAG CCAGTTCTAT TGGGCAGCTT TGTTTTTTTC CCTCACACTC TACCGGGACT
3661 TCCCCATGGA CATTGTGTAT CATGTGTAGA GTTGGTTTTT TTTTTTTTTA ATTTTTATTT
3721 TACTATAGCA GAAATAGACC TGATTATCTA CAAGATGATA AATAGATTGT CTACAGGATA
3781 AATAGTATGA AATAAAATCA AGGATTATCT TTCAGATGTG TTTACTTTTG CCTGGAGAAC
3841 TTTTAGCTAT AGAAACACTT GTGTGATGAT AGTCCTCCTT ATATCACCTG GAATGAACAC
3901 AGCTTCTACT GCCTTGCTCA GAAGGTCTTT TAAATAGACC ATCCTAGAAA CCACTGAGTT
3961 TGCTTATTTC TGTGATTTAA ACATAGATCT TGATCCAAGC TACATGACTT TTGTCTTTAA
4021 ATAACTTATC TACCACCTCA TTTGTACTCT TGATTACTTA CAAATTCTTT CAGTAAACAC
4081 CTAATTTTCT TCTGTAAAAG TTTGGTGATT TAAGTTTTAT TGGCAGTTTT ATAAAAAGAC
4141 ATCTTCTCTA GAAATTGCTA ACTTTAGGTC CATTTTACTG TGAATGAGGA ATAGGAGTGA
4201 GTTTTAGAAT AACAGATTTT TAAAAATCCA GATGATTTGA TTAAAACCTT AATCATACAT
4261 TGACATAATT CATTGCTTCT TTTTTTTGAG ATATGGAGTC TTGCTGTGTT GCCCAGGCAG
4321 GAGTGCAGTG GTATGATCTC AGCTCACTGC AACCTCTGCC TCCCGGGTTC AACTGATTCT
4381 CCTGCCTCAG CCTCCCTGGT AGCTAGGATT ACAGGTGCCC GCCACCATGC CTGGCTAACT
4441 TTTGTAGTTT TAGTAGAGAC GGGGTTTTGC CTGTTGGCCA GGCTGGTCTT GAACTCCTGA
4501 CCTCAAGTGA TCCATCCACC TTGGCCTCCC AAAGTGCTGG GATTACGGGC GTGAGCCACT
4561 GTCCCTGGCC TCATTGTTCC CTTTTCTACT TTAAGGAAAG TTTTCATGTT TAATCATCTG
4621 GGGAAAGTAT GTGAAAAATA TTTGTTAAGA AGTATCTCTT TGGAGCCAAG CCACCTGTCT
4681 TGGTTTCTTT CTACTAAGAG CCATAAAGTA TAGAAATACT TCTAGTTGTT AAGTGCTTAT
4741 ATTTGTACCT AGATTTAGTC ACACGCTTTT GAGAAAACAT CTAGTATGTT ATGATCAGCT
4801 ATTCCTGAGA GCTTGGTTGT TAATCTATAT TTCTATTTCT TAGTGGTAGT CATCTTTGAT
4861 GAATAAGACT AAAGATTCTC ACAGGTTTAA AATTTTATGT CTACTTTAAG GGTAAAATTA
4921 TGAGGTTATG GTTCTGGGTG GGTTTTCTCT AGCTAATTCA TATCTCAAAG AGTCTCAAAA
4981 TGTTGAATTT CAGTGCAAGC TGAATGAGAG ATGAGCCATG TACACCCACC GTAAGACCTC
5041 ATTCCATGTT TGTCCAGTGC CTTTCAGTGC ATTATCAAAG GGAATCCTTC ATGGTGTTGC
5101 CTTTATTTTC CGGGGAGTAG ATCGTGGGAT ATAGTCTATC TCATTTTTAA TAGTTTACCG
5161 CCCCTGGTAT ACAAAGATAA TGACAATAAA TCACTGCCAT ATAACCTTGC TTTTTCCAGA
5221 AACATGGCTG TTTTGTATTG CTGTAACCAC TAAATAGGTT GCCTATACCA TTCCTCCTGT
5281 GAACAGTGCA GATTTACAGG TTGCATGGTC TGGCTTAAGG AGAGCCATAC TTGAGACATG
5341 TGAGTAAACT GAACTCATAT TAGCTGTGCT GCATTTCAGA CTTAAAATCC ATTTTTGTGG
5401 GGCAGGGTGT GGTGTGTAAA GGGGGGTGTT TGTAATACAA GTTGAAGGCA AAATAAAATG
5461 TCCTGTCTCC CAGATGATAT ACATCTTATT ATTTTTAAAG TTTATTGCTA ATTGTAGGAA
5521 GGTGAGTTGC AGGTATCTTT GACTATGGTC ATCTGGGGAA GGAAAATTTT ACATTTTACT
5581 ATTAATGCTC CTTAAGTGTC TATGGAGGTT AAAGAATAAA ATGGTAAATG TTTCTGTGCC
5641 TGGTTTGATG GTAACTGGTT AATAGTTACT CACCATTTTA TGCAGAGTCA CATTAGTTCA
5701 CACCCTTTCT GAGAGCCTTT TGGGAGAAGC AGTTTTATTC TCTGAGTGGA ACAGAGTTCT
5761 TTTTGTTGAT AATTTCTAGT TTGCTCCCTT CGTTATTGCC AACTTTACTG GCATTTTATT
5821 TAATGATAGC AGATTGGGAA AATGGCAAAT TTAGGTTACG GAGGTAAATG AGTATATGAA
5881 AGCAATTACC TCTAAAGCCA GTTAACAATT ATTTTGTAGG TGGGGTACAC TCAGCTTAAA
5941 GTAATGCATT TTTTTTTCCC GTAAAGGCAG AATCCATCTT GTTGCAGATA GCTATCTAAA
6001 TAATCTCATA TCCTCTTTTG CAAAGACTAC AGAGAATAGG CTATGACAAT CTTGTTCAAG
6061 CCTTTCCATT TTTTTCCCTG ATAACTAAGT AATTTCTTTG AACATACCAA GAAGTATGTA
6121 AAAAGTCCAT GGCCTTATTC ATCCACAAAG TGGCATCCTA GGCCCAGCCT TATCCCTAGC
6181 AGTTGTCCCA GTGCTGCTAG GTTGCTTATC TTGTTTATCT GGAATCACTG TGGAGTGAAA
6241 TTTTCCACAT CATCCAGAAT TGCCTTATTT AAGAAGTAAA ACGTTTTAAT TTTTAGCCTT
6301 TTTTTGGTGG AGTTATTTAA TATGTATATC AGAGGATATA CTAGATGGTA ACATTTCTTT
6361 CTGTGCTTGG CTATCTTTGT GGACTTCAGG GGCTTCTAAA ACAGACAGGA CTGTGTTGCC
6421 TTTACTAAAT GGTCTGAGAC AGCTATGGTT TTGAATTTTT AGTTTTTTTT TTTTAACCCA
6481 CTTCCCCTCC TGGTCTCTTC CCTCTCTGAT AATTACCATT CATATGTGAG TGTTAGTGTG
6541 CCTCCTTTTA GCATTTTCTT CTTCTCTTTC TGATTCTTCA TTTCTGACTG CCTAGGCAAG
6601 GAAACCAGAT AACCAAACTT ACTAGAACGT TCTTTAAAAC ACAAGTACAA ACTCTGGGAC
6661 AGGACCCAAG ACACTTTCCT GTGAAGTGCT GAAAAAGACC TCATTGTATT GGCATTTGAT
6721 ATCAGTTTGA TGTAGCTTAG AGTGCTTCCT GATTCTTGCT GAGTTTCAGG TAGTTGAGAT
6781 AGAGAGAAGT GAGTCATATT CATATTTTCC CCCTTAGAAT AATATTTTGA AAGGTTTCAT
6841 TGCTTCCACT TGAATGCTGC TCTTACAAAA ACTGGGGTTA CAAGGGTTAC TAAATTAGCA
6901 TCAGTAGCCA GAGGCAATAC CGTTGTCTGG AGGACACCAG CAAACAACAC ACAACAAAGC
6961 AAAACAAACC TTGGGAAACT AAGGCCATTT GTTTTGTTTT GGTGTCCCCT TTGAAGCCCT
7021 GCCTTCTGGC CTTACTCCTG TACAGATATT TTTGACCTAT AGGTGCCTTT ATGAGAATTG
7081 AGGGTCTGAC ATCCTGCCCC AAGGAGTAGC TAAAGTAATT GCTAGTGTTT TCAGGGATTT
7141 TAACATCAGA CTGGAATGAA TGAATGAAAC TTTTTGTCCT TTTTTTTTCT GTTTTTTTTT
7201 TTCTAATGTA GTAAGGACTA AGGAAAACCT TTGGTGAAGA CAATCATTTC TCTCTGTTGA
7261 TGTGGATACT TTTCACACCG TTTATTTAAA TGCTTTCTCA ATAGGTCCAG AGCCAGTGTT
7321 CTTGTTCAAC CTGAAAGTAA TGGCTCTGGG TTGGGCCAGA CAGTTGCACT CTCTAGTTTG
7381 CCCTCTGCCA CAAATTTGAT GTGTGACCTT TGGGCAAGTC ATTTATCTTC TCTGGGCCTT
7441 AGTTGCCTCA TCTGTAAAAT GAGGGAGTTG GAGTAGATTA ATTATTCCAG CTCTGAAATT
7501 CTAAGTGACC TTGGCTACCT TGCAGCAGTT TTGGATTTCT TCCTTATCTT TGTTCTGCTG
7561 TTTGAGGGGG CTTTTTACTT ATTTCCATGT TATTCAAAGG AGACTAGGCT TGATATTTTA
7621 TTACTGTTCT TTTATGGACA AAAGGTTACA TAGTATGCCC TTAAGACTTA ATTTTAACCA
7681 AAGGCCTAGC ACCACCTTAG GGGCTGCAAT AAACACTTAA CGCGCGTGCG CACGCGCGCG
7741 CGCACACACA CACACACACA CACACACACA CACAGGTCAG AGTTTAAGGC TTTCGAGTCA
7801 TGACATTCTA GCTTTTGAAT TGCGTGCACA CACACACGCA CGCACACACT CTGGTCAGAG
7861 TTTATTAAGG CTTTCGAGTC ATGACATTAT AGCTTTTGAG TTGGTGTGTG TGACACCACC
7921 CTCCTAAGTG GTGTGTGCTT GTAATTTTTT TTTTCAGTGA AAATGGATTG AAAACCTGTT
7981 GTTAATGCTT AGTGATATTA TGCTCAAAAC AAGGAAATTC CCTTGAACCG TGTCAATTAA
8041 ACTGGTTTAT ATGACTCAAG AAAACAATAC CAGTAGATGA TTATTAACTT TATTCTTGGC
8101 TCTTTTTAGG TCCATTTTGA TTAAGTGACT TTTGGCTGGA TCATTCAGAG CTCTCTTCTA
8161 GCCTACCCTT GGATGAGTAC AATTAATGAA ATTCATATTT TCAAGGACCT GGGAGCCTTC
8221 CTTGGGGCTG GGTTGAGGGT GGGGGGTTGG GGAGTCCTGG TAGAGGCCAG CTTTGTGGTA
8281 GCTGGAGAGG AAGGGATGAA ACCAGCTGCT GTTGCAAAGG CTGCTTGTCA TTGATAGAAG
8341 GACTCACGGG CTTGGATTGA TTAAGACTAA ACATGGAGTT GGCAAACTTT CTTCAAGTAT
8401 TGAGTTCTGT TCAATGCATT GGACATGTGA TTTAAGGGAA AAGTGTGAAT GCTTATAGAT
8461 GATGAAAACC TGGTGGGCTG CAGAGCCCAG TTTAGAAGAA GTGAGTTGGG GGTTGGGGAC
8521 AGATTTGGTG GTGGTATTTC CCAACTGTTT CCTCCCCTAA ATTCAGAGGA ATGCAGCTAT
8581 GCCAGAAGCC AGAGAAGAGC CACTCGTAGC TTCTGCTTTG GGGACAACTG GTCAGTTGAA
8641 AGTCCCAGGA GTTCCTTTGT GGCTTTCTGT ATACTTTTGC CTGGTTAAAG TCTGTGGCTA
8701 AAAAATAGTC GAACCTTTCT TGAGAACTCT GTAACAAAGT ATGTTTTTGA TTAAAAGAGA
8761 AAGCCAACTA AAAAAAAAAA AAAAAAAAA
Figure 4B cDNA sequence of Homo sapiens SMAD family member 4 (SMAD4), mRNA
 
1 MDNMSITNTP TSNDACLSIV HSLMCHRQGG ESETFAKRAI ESLVKKLKEK KDELDSLITA
61 ITTNGAHPSK CVTIQRTLDG RLQVAGRKGF PHVIYARLWR WPDLHKNELK HVKYCQYAFD
121 LKCDSVCVNP YHYERVVSPG IDLSGLTLQS NAPSSMMVKD EYVHDFEGQP SLSTEGHSIQ
181 TIQHPPSNRA STETYSTPAL LAPSESNATS TANFPNIPVA STSQPASILG GSHSEGLLQI
241 ASGPQPGQQQ NGFTGQPATY HHNSTTTWTG SRTAPYTPNL PHHQNGHLQH HPPMPPHPGH
301 YWPVHNELAF QPPISNHPAP EYWCSIAYFE MDVQVGETFK VPSSCPIVTV DGYVDPSGGD
361 RFCLGQLSNV HRTEAIERAR LHIGKGVQLE CKGEGDVWVR CLSDHAVFVQ SYYLDREAGR
421 APGDAVHKIY PSAYIKVFDL RQCHRQMQQQ AATAQAAAAA QAAAVAGNIP GPGSVGGIAP
481 AISLSAAAGI GVDDLRRLCI LRMSFVKGWG PDYPRQSIKE TPCWIEIHLH RALQLLDEVL
541 HTMPIADPQP LD
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Description generated with very high confidenceFigure 4C Amino acid sequence of Homo sapiens mothers against decapentaplegic homolog 4
Figure 5A

  1.  Restriction map of cDNA sequence for SMAD4

A screenshot of a cell phone
Description generated with very high confidence
Figure 5B Eleven restriction enzymes that are zero cutters for SMAD4. 
Forward Primer:
AAAGAATTCATGGACAATATGTCTATAACAAATA(TM=62)
Reverse Primer:
AAAGTCGACTCTATTCCACCCACGGACCC(TM=64)
Figure 5C: Forward and reverse primers designed to ligate whole SMAD4 into pGEX-4T-2
http://www.snapgene.com/resources/plasmid_files/pgex_vectors_%28ge_healthcare%29/pGEX-4T-2/pGEX-4T-2_1x.png
 
Figure 5D pGEX-4T-2
Purification of Smad4
Purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione–Sepharose affinity purification. The use of glutathione S-transferase (GST) gene fusion proteins as a method for inducible, high-level protein expression and purification from bacterial cell lysates.(5) The GST is used as a tag for the smad4 protein, then the fision protein in expressed in the pGEX vector. The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies.(5) Additionally, the GST fusion protein can be affinity purified facilely without denaturation or use of mild detergents. The fusion protein is captured by immobilized glutathione and impurities are washed away.(5) the expression of the Gst fusion protein is in LB medium per liter: 10 g tryptone, 5 g yeast, 5 g NaCl. Adjust to pH 7.2 with NaOH. Autoclaved. (5) Ampicillin, 5 mg/ml stock, filter sterilized, then Glycerol stock of transformed E. coli cells expressing GST fusion protein in pGEX vector With Isopropyl-1-thio-β-D-galactopyranoside (IPTG), 100 mM stock(5)Then enzymatic cleavage was used to remove GST affinity tag, the affinity purified protein was cut by vector specific enzyme.(5) to further clean the recombinant protein we can run a gel filtration column compatible with the molecular weight range of the sample to be purified.(5)SDS page can be before using antibody to detect the expression of the SMAD4 protein using the antibodies provided by proteintech.
Image result for purified SMAD4 sds
Figure 6  Result of anti-SMAD4 with mouse liver tissue lysate 4000ug.
4000ug (ptglab)
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Object name is nihms444656f1.jpg
Figure 7 GST fusion protein purification expected result 
Knock out preparation
Smad4 is the central intracellular mediator of transforming growth factor‐β (TGF‐β) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. (19)
Chondrocyte-specific Smad4 knockout mice (Smad4Co/Co) to investigate the function of Smad4 in inner ear development. Smad4Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. (19)
https://wol-prod-cdn.literatumonline.com/cms/attachment/5e9e6dab-31b4-4b3e-85b6-61e389e933d9/mfig001.jpg
Figure 8 Analysis of Cre recombinase expression in Col2a1‐Cre transgenic mice and targeted inactivation of Smad4 in the chondrocytes(19)
https://wol-prod-cdn.literatumonline.com/cms/attachment/97e0adbd-7906-492c-b0a7-4fc743737e9e/mfig002.jpg
Figure 9 Smad4 expression analysis in the cochlea of the Smad4Co/Co and Smad4+/+ P0 mice. A,B: smad4 expression in cochlea tissue in the Smad4Co/Co and Smad4+/+ mice, respectively. (19) 
Discussion:
The expected results of the experiment shows that the protein SMAD4 plays an important role in the growth factor in mouse. Auditory function tests revealed the homozygous Smad4Co/Co mice suffered from severe sensorineural hearing loss. The result of the experiment suggest that Smad4 is required for inner ear development and normal auditory function in mammals. (19) With the knock out of the SMAD4 the mouse is unable to have a functional sensory system. 
References

  1. Blackford A, Serrano OK, Wolfgang C, et al. SMAD4 Gene Mutations Are Associated With Poor Prognosis in Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2009;15(14):4674. doi:10.1158/1078-0432.CCR-09-0227.
  1. Brooker, Robert J. Genetics: Analysis & Principles. McGraw-Hill Education, 2015.
  1. Cloonan N, et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nature Methods. 2008;5:613–619.
  1. Francesca Finotello, Barbara Di Camillo; Measuring differential gene expression with RNA-seq: challenges and strategies for data analysis, Briefings in Functional Genomics, Volume 14, Issue 2, 1 March 2015, Pages 130–142, https://doi.org/10.1093/bfgp/elu035
  1. Harper S, Speicher DW. Purification of proteins fused to glutathione S-transferase. Methods Mol Biol. 2011;681:259-80.
  1. Hull J, Campino S, Rowlands K, Chan MS, Copley RR, et al. (2007) Identification of Common Genetic Variation That Modulates Alternative Splicing. PLOS Genetics 3(6): e99. https://doi.org/10.1371/journal.pgen.0030099
  1. Kim D, Kim JY, Jun H-S. Smad4 in T cells plays a protective role in the development of autoimmune Sjögren’s syndrome in the nonobese diabetic mouse. Oncotarget. 2016;7(49):80298-80312. doi:10.18632/oncotarget.13437.
  1. Moon YJ, Yun C-Y, Choi H, et al. Smad4 controls bone homeostasis through regulation of osteoblast/osteocyte viability. Experimental & Molecular Medicine. 2016;48(9):e256-. doi:10.1038/emm.2016.75.
  1. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 2008;5:621–628.
  1. Nagalakshmi U, Wang Z, Waern K, et al. The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science (New York, NY). 2008;320(5881):1344-1349.
  1. National Center for Biotechnology Information. PubChem Database; NCBI GeneID=4089, https://pubchem.ncbi.nlm.nih.gov/target/gene/4089 (accessed Oct, 2018).
  1. Pickrell JK, Marioni JC, Pai AA, et al. Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature. 2010;464(7289):768-772. doi:10.1038/nature08872.doi:10.1126/science.1158441.
  1. Rodriguez, J., Vernus, B., Chelh, I. et al. Cell. Mol. Life Sci. (2014) 71: 4361. https://doi.org/10.1007/s00018-014-1689-x (accessed Octorber 2018)
  1. “SMAD4 Gene – Genetics Home Reference – NIH.” U.S. National Library of Medicine, National Institutes of Health, ghr.nlm.nih.gov/gene/SMAD4#conditions. (accessed October 2018)
  1. “SMAD4 SMAD Family Member 4 [Homo Sapiens (Human)] – Gene – NCBI.” National Center for Biotechnology Information, U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/gene/4089#bibliography.
  1. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature reviews Genetics. 2009;10(1):57-63. doi:10.1038/nrg2484.
  1. Warshaw AL, Thayer SP. Pancreaticoduodenectomy. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract. 2004;8(6):10.1016/j.gassur.2004.03.005. doi:10.1016/j.gassur.2004.03.005.
  1. Xu P, Liu J, Derynck R. Post-translational regulation of TGF-β receptor and Smad signaling. FEBS letters. 2012;586(14):1871-1884. doi:10.1016/j.febslet.2012.05.010.
  2. Yang, S. , Hou, Z. , Yang, G. , Zhang, J. , Hu, Y. , Sun, J. , Guo, W. , He, D. z., Han, D. , Young, W. and Yang, X. (2009), Chondrocyte‐specific Smad4 gene conditional knockout results in hearing loss and inner ear malformation in mice. Dev. Dyn., 238: 1897-1908. doi:10.1002/dvdy.22014


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