Figure 11.1 Some fundamentals of gene

expression. mRNA = messenger RNA, tRNA =

transfer RNA, rRNA = ribosomal RNA.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.2 Preparing a DNA molecule for

observation with the electron microscope.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.3 The appearance under the

electron microscope of a DNA–RNA hybrid

formed between a gene containing an intron

and its processed transcript.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.4 The effect of S1 nuclease on a

DNA–RNA hybrid.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.5 Locating a transcription start

point by S1 nuclease mapping.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.6 Locating a transcription start

point by primer extension.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.7 Northern hybridization. Three RNA

extracts from different tissues have been

electrophoresed in an agarose gel. The extracts are
made up of many RNAs of different lengths so each

gives a smear of RNA, but two distinct bands are

seen, one for each of the abundant ribosomal

RNAs. The sizes of these rRNAs are known (e.g.

4718 and 1874 nucleotides in mammals) so they

can be used as internal size markers. The gel is

transferred to a membrane, probed with a cloned

gene, and the results visualized by

autoradiography. Only lane 1 gives a band,

showing that the cloned gene is expressed only in

the tissue from which this RNA extract was

obtained.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.8 Reverse transcription–PCR (RT–

PCR).

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.9 One version of RACE.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.10 Possible positions for control

sequences in the region upstream of a gene.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.11 A bound protein decreases the

mobility of a DNA fragment during gel

electrophoresis.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.12 Carrying out a gel retardation

experiment.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.13 A bound protein protects a

region of a DNA molecule from degradation

by a nuclease such as DNase I.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.14 DNase I footprinting.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.15 A bound protein can protect a

region of DNA that is much longer than the

control sequence.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.16 A modification interference

assay.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.17 The principle behind deletion

analysis.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.18 A reporter gene.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.19 Deletion analysis. A reporter

gene has been attached to the upstream

region of a seed-specific gene from a plant.

Removal of the restriction fragment bounded

by the sites R deletes the control sequence

that mediates seed-specific gene expression,

so that the reporter gene is now expressed in

all tissues of the plant.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.20 Cell-free translation.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.21 Hybrid-release translation.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.22 Hybrid-arrest translation.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.23 A mutation may change the
amino acid sequence of a protein, possibly

affecting its properties.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.24 Various in vitro mutagenesis

techniques.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.25 One method for

oligonucleotide-directed mutagenesis.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.26 Artificial gene synthesis.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.27 One method for using PCR to

create a directed mutation.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.28 Phage display. (a) Display of

proteins on the surface of a recombinant

filamentous phage. (b) The gene fusion used

to display a protein. (c) One way of detecting

interactions between a test protein and a

phage from within a display library.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 11.29 The yeast two hybrid system. (a) A

pair of transcription factors that must interact in

order for a yeast gene to be expressed. (b)

Replacement of transcription factor 1 with the

hybrid protein 1* abolishes gene expression as 1*

cannot interact with transcription factor 2. (c)

Replacement of transcription factor 2 with the

hybrid protein 2* restores gene expression if the

hybrid parts of 1* and 2* are able to interact.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.