Transcript Maps

1. Transcript Maps Bert Gold, Ph.D., F.A.C.M.G.

2. Transcriptional Terminology • trans-actingReferring to DNA sequences encoding diffusible proteins (e.g., transcription activators and repressors) that control genes on the same or different chromosomes. • transcriptionProcess 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 unitA region in DNA, bounded by an initiation (start) site and termination site, that is transcribed into a single primary transcript. • transcription-control regionCollective term for all the cis-acting DNA regulatory sequences that regulate transcription of a particular gene.

3. Early message mapping • Northern • Protection • RNAse P • S1 Nuclease

4. Working with RNA • Northern Blot • RNAse protection • Nuclease S1 mapping • Primer Extension • Nuclear run-off • SAGE

5. Isolation of RNA • Work fast • Keep things cold • Use Rnase inhibitors • RNAsin (Vanadyl RNA inhibitor) • SDS • Diethylpyrocarbonate • Guanidium Hydrochloride or Isothiocyanate

6. Northern Protocol • Isolate RNA • Denaturing gel electrophoresis • Transfer to membrane • Prehybridization • Hybridization • Exposure to X-ray film • Washing

7. 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

8. Washing and Exposure • Wash with SSC and SDS • Successive increases in stringency • Expose to X-ray film or Phosphoimager

9. Northern Results

10. 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

11. 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

12. RNAse Enzyme Specificities

13. RNAse Protection Gel

14. RNAse Titration

15. 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

16. Robert Roeder Runoff Assay

17. 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.

18. Assessing Promoters (and Enhancers)

19. Early 90s Innovations • Expressed Sequence Tags • How to prime • 3’ favored • modifications: • 5’ RACE

20. Rapid Amplification of cDNA Ends (RACE) Assessment Gel 5’ RACE 3’ RACE

21. Late 90s Innovations • Differential Display • SAGE • Microarray Studies • Cy3, Cy5 and C-GAP • Microarray Initiative

22. Assembling the Information in Silico • dbEST • UNIGENE • Build description www.ncbi.nih.gov/UniGene/build.html • RefSeq

23. Gene Prediction • Promoter Searching • Promoter Inspector (www.genomatix.de) • First EF (http://www.cshl.org/mzhanglab/) • Alternative Transcription

24. Proteomics