Assignment 2Overall 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:

ComponentReaction 1 Reaction 2

pScr 5ul -

pUC19 - 5ul

EcoRI 1ul 1ul

XhoI 1ul -

SalI - 1ul

10X buffer 2ul 2ul

H2O 11ul 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 ladderLane 2: Uncut DNA supplied to youLane 3: Digested pUC19 (just 1 μl)Lane 4: Digested pUAST carrying the

Scr gene insert (all 20 μl)

Lan

e 1

Lan

e 2

Lan

e 3

Lan

e 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 forligation 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 2 Variant 3

International Human Genome Sequencing Consortium, (2001) "Initial sequencing and analysis of the human genome." Nature, 409, 860-921. Black, DL, (2000) Cell, Oct, 27 Vol 103:367-370.

5% of genes alternatively spliced ~60 % of genes alternatively spliced1 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 diabetesType I diabetesAutoimmune Diseases (Lupus)Rheumatoid ArthritisVarious types of CancerETC…..

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 20 - 50 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 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 isprone 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.5759-5768.

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 2811. (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.

Transfected construct (pSVEDATot)

ori (–) ori (+)ori(+)ori(-)

Total plasmida (no MboI digestion) 100 226 2.26

Non-replicated plasmida (after MboI digestion) 105 118 1.12

Transcription foci per nucleus 49.2 ± 10 80.4 ±17 1.63

% PML bodies associated to transcription foci

13.1 (n =290)

60.8 (n = 268) 4.64

EDI+/EDI– 0.6 14.0 23.33

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