Gene Cloning

318 Gene Cloning
the first exon of the transcribed region, would contain several kb of
upstream sequence. Even so, this may not encompass all the elements
required for proper regulation. The DNAse I hypersensitivity assay can be
used to help identify and determine the position of distant enhancers (see
Section 11.4).
11.2 Determining the Transcription Start of a Gene
One of the first questions that needs to be answered when studying the
transcriptional regulation of a gene is the location of the transcription start,
i.e the point on the DNA template at which mRNA synthesis begins. One of
the reasons that this is important is that once you have located the transcription
start it is often possible to deduce possible promoter elements
based on their location with respect to the start point. For example, most
genes in E. coli are transcribed by RNA polymerase that is directed to the
transcription start by a transcription factor, called σ 70 , which recognizes
and binds sequence elements positioned 10 and 35 bases upstream from
the transcription start. In eukaryotes the core promoter may contain conserved
sequences overlapping the transcription start, 25 bases upstream or
30 bases downstream.
There are several techniques that can be used to map the transcription
start point. We will discuss three approaches: primer extension, nuclease
protection, and rapid amplification of cDNA ends (RACE). All three
approaches require that you have some sequence information within the 5′
end of the transcript.
Primer extension
Primer extension is a relatively easy way to determine the transcription
start point of a gene (Figure 11.2). All that is required is a source of cells in
which the gene is actively transcribed and a radiolabeled oligonucleotide
primer complementary to a region within the 5′ end of the target transcript,
reverse transcriptase and nucleoside triphosphates. How to radiolabel the
5′ end of an oligonucleotide is discussed in Box 11.1.
The technique can use either total RNA or mRNA, which is purified from
cells isolated in conditions when the gene is expressed. How you purify
mRNA away from the abundant rRNA and tRNA was discussed in Chapter 4
(Section 4.18 and Figure 4.12). The RNA population is used as template for
a reverse transcription reaction primed by the radiolabeled oligonucleotide.
The oligonucleotide, which is specific to the gene of interest, should
hybridize only to the mRNA of interest. Reverse transcriptase (see Box 4.2)
can then be used to synthesize a DNA copy of the mRNA using, as a primer,
the labeled oligonucleotide. DNA synthesis will continue until reverse transcriptase
reaches the 5′ end of the transcript when it will “fall off” the mRNA
template. Assuming that all the original transcripts started from the same
point, i.e. had the same 5′ end, the product of this reaction will be a labeled