Assignment 2 Overall Format Page 1 will be your gel picture along with a figure legend. Page 2 will be a composite picture labeled and drawn from example results. Pages 3-4 will be your report which will consist of answers to questions listed below. figure legend: Use a format acceptable by scientific journals (See figure legends from articles that you have read and use any one of many examples). Part 1: (3 marks) Write and explain the genotypes of the four suspects. How you represent the polymorphic markers is up to you, but you must explain the nomenclature system that you have developed. The nomenclature system must allow a person, who has read the rules of the system, draw the gel of the result without ever having seen the gel in the first place. Part 2: (3 marks) Analyzed independently, which of the marker(s) can provide conclusive evidence? Explain for each polymorphic marker individually. Part 3: (4 marks) *Should contain all information concisely: Sentence structure is important As a detective and a forensic scientist, write a report summarizing the incident, results of the analyses and the conclusions of the case. This section should be about ¾-1 page long. Less than half a page will receive a zero.

Lab 6 (Feb 20, 21) DNA fragment isolation kit (A) Vector preparation B Fragment isolation

Set up the following restriction digests: Component Reaction 1Reaction 2 pScr5ul- pUC19-5ul EcoRI1ul XhoI1ul- SalI-1ul 10X buffer2ul H2OH2O 11ul Digest the DNA for 1 hour at 37 C.

To reaction 1 add 5 ul of 5X sample loading buffer and load 25 ul on an agarose gel. From reaction 2 remove 1 ul of the reaction and mix with 5 ml of 5X sample loading buffer and load on the gel. Run a 20 ul sample of diluted uncut DNA supplied to you. Load DNA ladder on the gel. Run Digests on Agarose Gel After Restriction Enzyme Digestion Lane 1: 1 kb ladder Lane 2: Uncut DNA supplied to you Lane 3: Digested pUC19 (just 1 μl) Lane 4: Digested pUAST carrying the Scr gene insert (all 20 μl) Lane 1Lane 2 Lane 3Lane 4 QIA quick Gel Extraction Kit

Process the pUC19 vector to prepare it for ligation The scr gene insert will be automatically processed and ready for ligation through the process of gene isolation from agarose gel. Place both pUC19 vector + scr gene in freezer until ligation. Your pUC19 vector + scr insert are now ready for ligation reaction which will take place in Lab 7 (March 6, 7)---After winter break.

Alternative Splicing and Proteome Complexity Complex pattern of gene expression differential on/off of large number of genes Sequencing of Human Genome (30,000-40,000 genes) Post-transcriptional mechanisms Alternative splicing modulating gene expression Transcription Pre-mRNA Gene Mature mRNA Protein Alternative Splicing Variant 1 Variant 2Variant 3 International Human Genome Sequencing Consortium, (2001) "Initial sequencing and analysis of the human genome." Nature, 409, Black, DL, (2000) Cell, Oct, 27 Vol 103: % of genes alternatively spliced~60 % of genes alternatively spliced 1 gene= Avg of 5-6 isoforms Proteomic Studies ~ 1 million proteins

Why Study the Mechanism of Alternative Splicing? Alternative Splicing in Normal Cellular Mechanisms: - Splicing pathways modulated according to: Cell type; Developmental stage; Gender; External stimuli. Alternative Splicing and Disease: -Increasing evidence clearly indicating that alternative splicing plays a major role in: -Initiation of Diseases -Progression of Diseases Eg: Insulin Resistance in Type II diabetes Type I diabetes Autoimmune Diseases (Lupus) Rheumatoid Arthritis Various types of Cancer ETC…..

38,016 Shades of Drosophila DSCAM gene (Encodes Cell Surface Protein: neuronal connectivity) The DSCAM gene (top) is 61.2 kb long and after transcription and splicing produces a 7.8 kb, 24 exon mRNA (middle). Exons 4, 6, 9, and 17 are mutually exclusive alternative exons. Each mRNA will contain one of 12 possible alternatives for exon 4, one of 48 for exon 6, one of 33 for exon 9 and one of 2 for exon 17. If all possible combinations of single exons 4, 6, 9, and 17 are used, the DSCAM gene produces 38,016 different mRNAs and proteins. Sex determination pattern of Drosophila Drosophila is an excellent model for studying alternative splicing Truncated inactive proteins Sex lethal (Sxl) Transformer (tra) Doublesex (dsx) – a transcription factor (F or M) regulates activity of sex-specific differentiation genes

The Basics

Accumulating evidence to show that there is coupling between Transcription and Splicing (This coupling increases the efficiency of transcription---How?) It has been suggested to date that this coupling is specific to RNA Pol II

The fibronectin gene as a model for splicing and transcription studies Critical roles in cell adhesion, migration, proliferation, tissue repair, blood clotting. Fibronectin: glycoprotein consisting of domains allowing interaction with variety of different cells types. It is encoded by a single gene, but alternative splicing of pre-mRNA allows formation of multiple isoforms that have critical roles in the cell 20 Human Isoforms

Splicing signal Most introns start from the sequence GU and end with the sequence AG (in the 5' to 3' direction). They are referred to as the splice donor and splice acceptor site, respectively. However, the sequences at the two sites are not sufficient to signal the presence of an intron. Another important sequence is called the branch site located bases upstream of the acceptor site. The consensus sequence of the branch site is "CU(A/G)A(C/U)", where A is conserved in all genes. In over 60% of cases, the exon sequence is (A/C)AG at the donor site, and G at the acceptor site. The consensus sequence for splicing. Pu = A or G; Py = C or U. AT-AC Pre-mRNA Splicing Mechanisms and Conservation of Minor Introns in Voltage-Gated Ion Channel GenesAT-AC Pre-mRNA Splicing Mechanisms and Conservation of Minor Introns in Voltage-Gated Ion Channel Genes - Molecular and Cellular Biology, 1999.

Mechanism of Splicing

Spliceosome Assembly on Regulated and Unregulated Splice Sites Splicing sequences are also located on exons (ESEs).

There Are Many Control mechanisms for Differential Splice Site Selection We will learn how the mechanism of transcription can regulate alternative splicing Transcription (Promoter Control) (Article 2)

Recruitment Model RNA Pol II Elongation Model Alternative Models for Promoter Control of Alternative Splicing

Promoter Control of Alternative Splicing RNA Pol II Elongation Model RNA Pol II Processivity (elongation capacity) Ability of pol II to elongate through sites where polymerase is prone to stop or terminate prematrurely.

Kadener et al.(2001)”Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing”, The EMBO JournalVol.20 No.20 pp Discussion of Article 2

Schemes of the minigenes carrying the different promoters. Codes for DNA sequences. White, human -globin; dashed or dotted, human FN; black, CMV; horizontal hatching, SV40 enhancer/origin of replication (e/o); gray, HIV2. Thin arrows show the primers used to amplify the mRNA splicing variants by RT–PCR. The SfiI site used to disrupt the SV40 origin of replication is indicated

Effect of T-Ag on EDI alternative splicing in the context of different promoters. Hep3B cells were transfected with 800 ng of the corresponding minigene plasmid and 800 ng of a plasmid expressing T-Ag (even lanes) or pBSKS+ (Stratagene) (odd lanes). (B) Endogenous (EDIe) and minigene- driven (EDIm) alternative splicing patterns of Hep3B cell lines stably transfected with different promoter minigenes. (C) Effect of the presence of the SV40 origin of replication on alternative splicing elicited by minigenes transfected in COS-7 cells. RNA splicing variants were detected by radioactive RT–PCR and analyzed in 6% native polyacrylamide gels. Ratios between radio activity in EDI+ bands and radioactivity in EDI– bands are shown under each lane

Effect of T-Ag mutants on EDI alternative splicing. (A) Scheme of the SV40 T- Ag molecule with the location of its binding and enzymatic activities and the positions of mutants K1, 2809 and (B) EDI alternative splicing in Hep3B cells co-transfected with pSVEDA/CMV having (lanes 1–5) or lacking (lanes 6–10) the SV40 origin of replication and plasmids expressing the wild type or different mutants of T-Ag

The effect of T-Ag is exon specific. (A) Diagrams showing different alternative exons and their corresponding cis-controlling elements used to evaluate the effects of T-Ag on alternative splicing. ESE, exonic splicing enhancer for SF2/ASF (GAAGAAGAG) and for SRp-55 (GCACGGAC). Positive controls for the activation of exon inclusion by overexpression of SRp-55 and SRp-40 are shown. (B) Effect of T-Ag on alternative splicing of an EDI exon in which the natural ESE was disrupted (lanes 1–4) or replaced by the SRp-55 target site (lanes 5 and 6). Lanes 7–12, effect of T-Ag on EDII alternative splicing. Hep3B cells were co- transfected with pSVEDEDB-Xho (lanes 7–12) and either pBSKS+ (Stratagene) (lanes 7 and 8) or pSVEDATot (lanes 9–12). The plasmid expressing T-Ag (Zhu et al., 1991) was co-transfected in the odd lanes. RT–PCRs for EDI (lanes 1–6, 9 and 10) and for EDII (lanes 7, 8, 11 and 12) were as in Materials and methods.Zhu et al., 1991

Transfected construct (pSVEDATot) ori (–)ori (+) ori(-) Total plasmid a (no MboI digestion) Non-replicated plasmid a (after MboI digestion) Transcription foci per nucleus49.2 ± ± % PML bodies associated to transcription foci 13.1 (n =290) 60.8 (n = 268)4.64 EDI + /EDI – Ratio Effects of the presence of the SV40 origin of replication on the number and expression of plasmid Constructs transfected in COS-1 cells

T-Ag-dependent replication increases the proportion of shorter transcripts. RPAs of transcripts produced by minigenes with three different promoters transfected into COS-1 cells under replicative [ori(+)] and non-replicative [ori(–)] conditions. Transcripts were detected with a proximal (globin intron 1–exon 2 boundary) and a distal (FN intron +1–exon +1 boundary) probe, described in Materials and methods. Proximal/distal ratios of the protected probe band quantifications are shown

The End