Transcript Transcript Maps

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
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Northern Blot
RNAse protection
Nuclease S1 mapping
Primer Extension
Nuclear run-off
SAGE
Isolation of RNA
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Work fast
Keep things cold
Use Rnase inhibitors
RNAsin (Vanadyl RNA inhibitor)
SDS
Diethylpyrocarbonate
Guanidium Hydrochloride or
Isothiocyanate
Northern Protocol
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Isolate RNA
Denaturing gel electrophoresis
Transfer to membrane
Prehybridization
Hybridization
Exposure to X-ray film
Washing
Run Gel and Transfer
• Run denaturing gel
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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