Identification and Comparison of Midline Cis-Regulatory Elements.

Slides:

Advertisements

Similar presentations

Recombinant DNA prepare foreign (target) DNA prepare vector (host)

Advertisements

Design and Testing of a Bacteriological Timer Device Herman Armstrong Morgan Schiermeier Rachel Klapper Jackie Schneider.

Epigenetics Xiaole Shirley Liu STAT115, STAT215, BIO298, BIST520.

Finding regulatory modules from local alignment - Department of Computer Science & Helsinki Institute of Information Technology HIIT University of Helsinki.

CD4 is a transmembrane glycoprotein expressed on the cell surface of thymocytes and mature T- lymphocytes with helper function. T-cells develop in the.

The trouble with binding sites Joe Pearson 1/11/10.

This presentation was originally prepared by C. William Birky, Jr. Department of Ecology and Evolutionary Biology The University of Arizona It may be used.

PCR Product Orientation Test T7 EcoRI Cloning Site EcoRI PmeI PstI SpeI NotI pCR4-TOPO T7.

Microarrays and Cancer Segal et al. CS 466 Saurabh Sinha.

A modest collection of Midline CRMs Joe Pearson Lab Meeting 8/25/08.

Mutagenesis Methods Lily Peterson April 5 th, 2010.

Rhomboid MLE regulation by Sim:Tgo and Su(H) 8/24/09.

Principles of genetic engineering L Mathias. What is genetic engineering Genetic engineering, also known as recombinant DNA technology, means altering.

Promoter Analysis using Bioinformatics, Putting the Predictions to the Test Amy Creekmore Ansci 490M November 19, 2002.

Genomics and bioinformatics summary 1. Gene finding: computer searches, cDNAs, ESTs, 2.Microarrays 3.Use BLAST to find homologous sequences 4.Multiple.

Principles of Genetic Engineering. What is genetic engineering Genetic engineering, also known as recombinant DNA technology, means altering the genes.

MCB 317 Genetics and Genomics MCB 317 Topic 10, part 1 A Story of Transcription.

Computational Molecular Biology Biochem 218 – BioMedical Informatics Gene Regulatory.

The Gateway® Cloning System Gene Expression Contents Description of destination vectors Examples of expression clones in destination vectors Effects of.

Molecular Biology Fifth Edition

The Gateway® Cloning System Introduction to the Gateway® System Contents Defining Gateway® technology. Advantages of Gateway® cloning. Ways to enter the.

P300 Marks Active Enhancers Ruijuan LiChao HeRui Fu.

Biotechnology SB2.f – Examine the use of DNA technology in forensics, medicine and agriculture.

DNA Technology Chapter 12. Applications of Biotechnology Biotechnology: The use of organisms to perform practical tasks for human use. – DNA Technology:

Mapping protein-DNA interactions by ChIP-seq Zsolt Szilagyi Institute of Biomedicine.

MRNA protein DNA Activation Repression Translation Localization Stability Pol II 3’UTR Transcriptional and post-transcriptional regulation of gene expression.

A systems biology approach to the identification and analysis of transcriptional regulatory networks in osteocytes Angela K. Dean, Stephen E. Harris, Jianhua.

Massive Parallel Sequencing

* only 17% of SNPs implicated in freshwater adaptation map to coding sequences Many, many mapping studies find prevalent noncoding QTLs.

From Structure to Function. Given a protein structure can we predict the function of a protein when we do not have a known homolog in the database ?

Remember the limitations? –You must know the sequence of the primer sites to use PCR –How do you go about sequencing regions of a genome about which you.

ModENCODE August 20-21, 2007 Drosophila Transcriptome: Aim 2.2.

I519 Introduction to Bioinformatics, Fall, 2012

Localising regulatory elements using statistical analysis and shortest unique substrings of DNA Nora Pierstorff 1, Rodrigo Nunes de Fonseca 2, Thomas Wiehe.

Transcription control elements (DNA sequences) are binding sites for transcription factors, proteins that regulate transcription from an associated.

Principles of genetic engineering

Genome-Wide Identification of Cell Type Specific Transcription Factor Targets During Drosophilia Embyrogenesis Jessica Osterhout.

Gene Expression Platforms for Global Coexpression Analyses Assessment and Integration for Study of Gene Deregulation in Cancer Obi Griffith, Erin Pleasance,

Alternative Splicing (a review by Liliana Florea, 2005) CS 498 SS Saurabh Sinha 11/30/06.

Motif Search and RNA Structure Prediction Lesson 9.

Biol 456/656 Molecular Epigenetics Lecture #5 Wed. Sept 2, 2015.

Introduction to Bioinformatics - Tutorial no. 5 MEME – Discovering motifs in sequences MAST – Searching for motifs in databanks TRANSFAC – the Transcription.

Genome Analysis. This involves finding out the: order of the bases in the DNA location of genes parts of the DNA that controls the activity of the genes.

Network Motifs See some examples of motifs and their functionality Discuss a study that showed how a miRNA also can be integrated into motifs Today’s plan.

REVIEW OF MOLECULAR GENETICS DR. EDELBERG. Genes, DNA, & Chromosomes.

IDENTIFICATION OF HIGHLY CONSERVED BACILLUS ORFS OF UNKNOWN FUNCTION.

Gateway Cloning 13 November 2013.

Vectors Bacteria, viruses or liposomes into which DNA can be inserted. These can be used to grow genes, harvest the proteins they code for or deliver them.

Introduction The stem cell derived transcription factors SOX4, POU2F2 and BACH2 are known to be important in B-cell differentiation and B-cell malignancies.

Looking Within Human Genome King abdulaziz university Dr. Nisreen R Tashkandy GENOMICS ; THE PIG PICTURE.

kbp M bp 392 bp 251 bp kbp M recA 1.6 kbp

Detection of genome regulation sequences

Stories arising… Joe Pearson 5/10/2009.

Zebrafish msxe Expression Analysis

Restriction Enzymes and Plasmid Mapping

Mark M Metzstein, H.Robert Horvitz Molecular Cell

Multiple Tumor Suppressor Pathways Negatively Regulate Telomerase

KEY CONCEPT Entire genomes are sequenced, studied, and compared.

Marios Agelopoulos, Daniel J. McKay, Richard S. Mann Cell Reports

KEY CONCEPT Entire genomes are sequenced, studied, and compared.

EXTENDING GENE ANNOTATION WITH GENE EXPRESSION

Gel Retardation.

Presented by, Jeremy Logue.

Volume 111, Issue 5, Pages (November 2002)

The Promoters of the Survival Motor Neuron Gene (SMN) and Its Copy (SMNc) Share Common Regulatory Elements Andoni Echaniz-Laguna, Pierre Miniou, Deborah.

Volume 7, Issue 1, Pages (January 2003)

B. Bakhshi, M.R. Pourshafie, F. Navabakbar, A. Tavakoli

Presented by, Jeremy Logue.

Gene transfer to plants – vectors and strategies

A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin Claire Francastel, Mark.

Presentation transcript:

Identification and Comparison of Midline Cis-Regulatory Elements

Does common expression indicate common regulation? A B C D CD AB A B CD CDAB wxyz

Project Goals Identify Large Set of Midline Primordium Enhancers Compare enhancers for shared motifs Experimentally confirm required motifs (activators/repressors) Contribute to knowledge of Midline Development

Known Midline Enhancers Sim 2.8 pE Sim (Sandmann) sli380 rho E-Ss Rst F6d Kr PP3.0Hz Kr StH0.6Hz btl-23 tl950

Candidate Midline Enhancer Sources (midline database search) Midline Primordia Midline Glia

CG13333 St. 11 Midline Genes argos ab bib bnb btl cdi cenB1A CG31145 CG32594CG3409 CG7224 CG8291 ctdveglec hbsHGTX mfas oc rhoSema-1b sog sty vvlTkr CG9634 rstsimTl

Midline/Glial Expressing Genes mfas oc rho sim sty wrapper alan-shepard slit GH22170 argos cdi CG13333 CG31145 CG8291 CG9634 cut dve glec hbs

Midline Primordium Genes CG7224 cenB1A (Sema-1B) bib bnb CG3409 CG32594 HGTX sog ab Tkr ct vvl

AttL2 Enhancer Cloning Pipeline Entry Vector Reporter Vectors AttL1 AttR2 AttR1 Reporter  C31 AttB

Entry Vector Issues pENTR/D-TOPO does not like big fragments Enhancer hunts do like big fragments Solutions: –Use Restriction/Ligation into pENTR/D-TOPO –Use pCR8/GW/TOPO vector (TA cloning)

NotI/EagI PstI SbfI NgoMIV/ NaeI FseI NcoI DraI PmeI SpeI AscI Compatible ends: NotI/EagISbfI/PstI->(NsiI)NgoMIV->(AgeI)/(XmaI) NcoI->(BspHI)/(PciI)SpeI->(AvrII)/(NheI)/(XbaI)AscI->(MluI)/(BssHII) pGEM-T sites: NcoI, NotI (both sides), NsiI, PstI, SpeI pCRII sites: NotI, NsiI (both sides), SpeI, XbaI Rare MCS pENTR vector AttL2 AttL1 pENTR/D-Topo

Reporter Vector Issues pBPGw has GAL4 pBPGw doesn’t have a promoter pBPGw is named pBPGw

pMintGate pBPJPhGFPw

Test Case:Sim2.8 pE MintGate GFP.nls  C31 AttB CinnamonGate DsRed.nls  C31 AttB pBPGw GAL4  C31 AttB

Preliminary results Cloned Sim2.8 pE into pMintGate, pCinnamonGate, pBPGw One Insertion isolated for Sim2.8 pMintGate (so far) (10/29: 2 lines CinnamonGate, 1 line pBPGw)

Applications of Enhancers GAL4-UAS –Cell-specific gene rescue –RNAi Cell labeling Enhancer Dissection –Common Activation? –Common Repression? –Evolutionary Conservation?

Future Experiments Clone/Test Putative Enhancer Fragments –Best case: 30+ midline enhancers with similar expression –compare bioinformatically –Identify required motifs Replace current FPs with newer/better FPs –mCherry, mPlum, Cerulean, CFP, etc –Live, in vivo comparisons of multiple midline enhancers Genome-wide midline enhancer tests –Chip-Seq –FAIRE

ChIP-Seq + Fix chromatin, IP with SIM FACS Solexa Sequence

FAIRE

+ Fix chromatin, Phenol Extract Free DNA FACS Label DNA, Custom Microarray

geneA geneB geneC geneA geneB geneC Midline cellsMidline Subtracted ABCDEFGHIJKLABCDEFGHIJKL ABCDEFGHIJKLABCDEFGHIJKL FAIRE