2. Analysis of Alternate Splicing in Human Genes  Name : Bingding Huang ( 黄炳顶 )  Email : markero@263.net  Address : Bioinformation center of Shanghai Instutes for Biological Sciences  Date: 08/31/01

3. The human genome sequence can be thought of as a picture of the human organism. However,like an impressionist painting,the genome is a very large canvas whose details becomes fuzzy when you look closely. ---by Douglas L.Black

4. Index  Definition:What is the alternative splice gene?Especially in human genes.  Mechanism : How the alternative splice genes work?  Type : How many types the alternative splice genes are?  Database :current databases and how to establish the corresponding database?Materials and method?  Future work :

5. Definition

6. Alternative Splicing is a major cellular process by which functionally diverse proteins can be generated from the transcript of a single gene,often in tissue-specific,patterns. As many as 30% of human genes utilize this mechanism to generate mature mRNA with difference in exon composition at the 5’- end,within internal coding regions or at the 3’- end.

7. Mechanism

8. How to predict the donor and acceptor sites?  GT---AG Rule: The donor site of introns in most eukaryotes usually contains GT(GU in pre-mRNA)while the acceptor site contains AG;a pyrimidine-rich region at 3’;a shorter consensus sequence at 5’;a very weak sequence at the branch point;  algorithm and Software: GeneSplicer, NetGene2,HSPL,NNSplice,HMMGene, Spliceview,and so on. GeneSplicer NetGene2HSPLNNSpliceHMMGeneSpliceview  False or True: how to determine the false sites from true sites?

9. Current Database  Intron Sequence Information System (ISIS) : ISIS contains phylogenetic and protein homology categories, information about individual sequences and various bioinformatics analyses of taxonomical groupings of sequences Intron Sequence Information System (ISIS)  Exon-Intron Database : An exhaustive database of protein-coding intron-containing genes. Exon-Intron Database  Intron Sequence and Evolution Databases This includes a non-redundant database of nuclear, protein- encoding, genomic DNA sequences highlighting nuclear pre- mRNA introns Intron Sequence and Evolution Databases

10. Types of alternate splicing gene

11. Method 1,Data collection 2,Construction intron-EST homology dataset 3,Analysis of type and frequency of alternative splicing using intron-EST homolies 4,Construction of an exon-EST homology dataset 5,Analysis of exon skipping,exon extensions or shortening using exon-EST homologies

12. Data Collection  GenBank and UniGene: entries contaiing alternative splice are picked out and recorded in a list.  EST: Download ESTs database from NCBI  Swiss-port :

13. Intron-EST homologies  Contigs overlaping two flanking exons: a,cryptic exon; b, 5’exon extension; c, 3’exon extension; d,whole intron retention  Contigs overlaping one flanking exon: a,cryptic exon or exon extention; b,whole intron retention

14. Contigs overlaping two flanking exons

15.  cryptic exon: a1<b1 and a2<b2 The intron fragment was less than the size of the whole intron,from middle of the introncryptic exon  5’exon extension: a1<b1 and a2=b2 3’end of the intron was contained within in the EST contig 5’exon extension  3’exon extention: a1=b1 and a2<b2 5’end of the intron was contained within the EST conting 3’exon extention  Whole intron retention: |CG—GG|<10 where the whole intron was contained in the contig

16. Contigs overlaping one flanking exon

17.  Cryptic exon or exon extension: CG<GG—10  Whole intron retention: CG>=GG--10,

18. Exon—EST homologies  EST contigs with matches to non-consecutive exons(eg.exon1-exon3) 1,perfect exon skipping 2,exon skipping with exon truncation  EST contigs with matches to consecutive exons (eg.exon1-exon2) 1,exon trucation 2,cryptic exon or 3’or 5’ exon extension 3,whole intron retention 4.normal splicing

19. EST contigs with matches to non-consecutive exons(eg.exon1-exon3)  perfect exon skipping CG<1 and GG=<0  exon skipping with exon truncation CG 0

20. EST contigs with matches to consecutive exons(eg.exon1-exon2)

21.  exon trucation CG<(GG—IN)  cryptic exon or 3’or 5’ exon extension CG>(GG—IN)but (CG—(GG—IN))<(IN—10)  whole intron retention CG>(GG—IN)but (CG—(GG—IN))>=(IN—10)  Normal splicing CG=(GG-IN)

22. Thank you