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

Northern Results

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

RNase Sequence Specificity

T1 Gp N

U2 Ap N

CL3 C(A/G)p N

S. Aureus nuclease Np A/U

RNAse Protection Gel

RNAse Titration

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’ RACE 3’ 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

www.ncbi.nih.gov/UniGene/build.html

• RefSeq

Gene Prediction

• Promoter Searching– Promoter Inspector (www.genomatix.de)– First EF (http://www.cshl.org/mzhanglab/)

• Alternative Transcription

Proteomics