RNA Sequencing for Global Transcriptome Mapping

#65

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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.

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)

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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)

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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)

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)

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

Figure 4C Amino acid sequence of Homo sapiens mothers against decapentaplegic homolog 4

Figure 5A

1.  Restriction map of cDNA sequence for SMAD4

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

 

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.

Figure 6  Result of anti-SMAD4 with mouse liver tissue lysate 4000ug.

4000ug (ptglab)

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)

Figure 8 Analysis of Cre recombinase expression in Col2a1‐Cre transgenic mice and targeted inactivation of Smad4 in the chondrocytes(19)

Figure 9 Smad4 expression analysis in the cochlea of the Smad4^Co/Co and Smad4^+/+ P0 mice. A,B: smad4 expression in cochlea tissue in the Smad4^Co/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 Smad4^Co/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

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