Biology Reference
In-Depth Information
containing the marker gene are selected and sequenced. The fragments are then
aligned, and those cloned segments farthest from the marker gene in both direc-
tions are subcloned for the next step. The subclones are used as probes to screen
the genomic library again to identify new clones containing DNA with overlap-
ping sequences. As the process is repeated, the nucleotide sequences of areas far-
ther and farther away from the marker gene are identified, and eventually the
gene of interest will be found. As shown in
Figure 6.11
, the goal is to identify
gene B, for which no probe is available. However, sequences of a nearby gene (A)
are available in cloned fragment 1. In a large and random genomic DNA library,
many overlapping cloned fragments will be present. Thus, clone 1 can be used as
a probe to identify overlapping clones 2, 3, and 4. Clone 4 subsequently can be
used as a probe to identify clone 5 until gene B is reached.
Once a gene has been identified in a genomic library, its DNA sequence can
be determined. However, a DNA sequence by itself is of limited value. If you do
not know something about the gene product, it may be difficult to determine
unambiguously which sequences are the coding regions and which are introns.
Intron boundaries may be established based on similarities to sequences of
known introns (consensus sequences). If the gene product is unknown, it may
be possible to identify the sequenced gene's function by comparing the DNA
sequence with other sequences in DNA databases, although a fully convincing
match is not always found. Thus, going from clone to DNA sequence to gene
product may be a challenge, particularly if genes are being studied for which
there are no known gene products.
One solution is to attempt to express the gene to obtain a gene product. To
be expressed, genes require a promoter and, often, upstream control sequences.
A variety of expression vectors have been developed to express cloned genes
in
E. coli
. Such vectors require
E. coli
promoters if the eukaryote sequence is to
be expressed. A detailed description of
E. coli
expression vectors is provided by
Pouwels (1991)
. Of particular interest to entomologists is the use of baculovirus
expression vectors to express insect genes, as well as other eukaryotic genes, as
described in Section 6.8.
cDNA libraries can be screened by hybridization screening or by expression
screening. If a protein of interest has been purified and part or all of the pro-
tein sequence has been obtained, then it is possible to predict the sequence of
synthetic oligonucleotides that can be used as a hybridization probe to detect
the appropriate cDNA clone(s). Alternatively, if an antibody to the protein is
available, it can be used to identify the clone(s) of interest if the cDNA library is
cloned into an expression vector (
Figure 6.12
). This vector contains the
lacZ
gene
