Presentation on theme: "Transcript Maps Bert Gold, Ph.D., F.A.C.M.G.. Transcriptional Terminology trans-acting Referring to DNA sequences encoding diffusible proteins (e.g.,"— Presentation transcript:
Transcript Maps Bert Gold, Ph.D., F.A.C.M.G.
Transcriptional Terminology trans-acting Referring to DNA sequences encoding diffusible proteins (e.g., transcription activators and repressors) that control genes on the same or different chromosomes. transcription Process whereby one strand of a DNA molecule is used as a template for synthesis of a complementary transcription factor (TF) General term for any protein, other than RNA polymerase, required to initiate or regulate transcription in eukaryotic cells. General factors, required for transcription of all genes, participate in formation of the transcription-initiation complex near the start site. Specific factors stimulate (or repress) transcription of particular genes by binding to their regulatory sequences. transcription unit A region in DNA, bounded by an initiation (start) site and termination site, that is transcribed into a single primary transcript. transcription-control region Collective term for all the cis-acting DNA regulatory sequences that regulate transcription of a particular gene.
Early message mapping Northern Protection –RNAse P –S1 Nuclease
Working with RNA Northern Blot RNAse protection Nuclease S1 mapping Primer Extension Nuclear run-off SAGE
Isolation of RNA Work fast Keep things cold Use Rnase inhibitors RNAsin (Vanadyl RNA inhibitor) SDS Diethylpyrocarbonate Guanidium Hydrochloride or Isothiocyanate
Northern Protocol Isolate RNA Denaturing gel electrophoresis Transfer to membrane Prehybridization Hybridization Exposure to X-ray film Washing
Run Gel and Transfer Run denaturing gel –Glyoxyl –Formamide –Formaldehyde –Methyl mercuric hydroxide –Urea Equilibrate with 20X SSC Transfer overnight to nitrocellulose or other membrane
Washing and Exposure Wash with SSC and SDS Successive increases in stringency Expose to X-ray film or Phosphoimager
Advantages to RNAse Protection Very rapid and efficient Can be quantitative Multiple probes in a single reaction allowing internal controls Allows RNA species with just small sequence variations to be distinguished
RNAse Protection Assay Isolate RNA Prepare radiolabeled antisense probe using in vitro transcription. Hybridize probe with RNA. Digest ssRNA with RNAse A and T1 Remove RNAse and separate products on sequencing gel. Expose to X-ray film
RNAse Enzyme Specificities RNaseSequence Specificity T1 Gp N U2 Ap N CL3 C(A/G)p N S. Aureus nuclease Np A/U
RNAse Protection Gel
Defining the regulatory sequences Gel Shift Assays DNA binding proteins, generally Enhancer Assays –Advent of CAT –Stable versus Transient Expression in CAT The revelation of TAT
Robert Roeder Runoff Assay
Quantitative Nuclear Runoff Assay Nascent-chain (run-on) assay for transcription rate of a gene. Isolated nuclei are pulsed with 32P-labeled ribonucleoside triphosphates. During the pulse 300 to 500 bases are added to nascent chains. Very little transcription initiation occurs. Labeled RNA that hybridizes and is protected by a particular DNA fragment reflects its relative transcription rate.
Assessing Promoters (and Enhancers)
Early 90s Innovations Expressed Sequence Tags –How to prime –3’ favored –modifications: –5’ RACE
Rapid Amplification of cDNA Ends (RACE) 5’ RACE3’ RACE Assessment Gel
Late 90s Innovations Differential Display SAGE Microarray Studies –Cy3, Cy5 and C-GAP –Microarray Initiative
Assembling the Information in Silico dbEST UNIGENE –Build description RefSeq
Gene Prediction Promoter Searching –Promoter Inspector (www.genomatix.de)www.genomatix.de –First EF (http://www.cshl.org/mzhanglab/)http://www.cshl.org/mzhanglab/ Alternative Transcription