Biomedical Engineering Reference
In-Depth Information
Fig. 9.5
Ty virus-like particles (magnification 80 000) carrying the entire HIV1 TAT coding region. (Photograph courtesy of
Dr S. Kingsman.)
the factors affecting expression of genes in yeast are
discussed in a later section (see p. 165). Yeast is also
used in the production of food and beverages. The
ability to clone in yeast without the introduction of
bacterial sequences by using vectors like those of Chin-
ery and Hinchliffe (1989) is particularly beneficial.
A second reason for cloning genes in yeast is the
ability to clone large pieces of DNA. Although there
is no theoretical limit to the size of DNA which can
be cloned in a bacterial plasmid, large recombinant
plasmids exhibit structural and segregative instabil-
ity. In the case of bacteriophage-
pGAL
δ
2
µ
ORI
Ty transcript
P
Transcript
of gene
insert
δ
vectors, the size of
the insert is governed by packaging constraints. Many
DNA sequences of interest are much larger than this.
For example, the gene for blood Factor VIII covers
about 190 kbp, or about 0.1% of the human X chro-
mosome, and the Duchenne muscular dystrophy gene
spans more than a megabase. Long sequences of
cloned DNA have greatly facilitated efforts to sequence
the human genome. YACs offer a convenient way
to clone large DNA fragments but are being replaced
with BACs and PACs (see p. 67) because these have
greater stability. Nevertheless, the availability of
YACs with large inserts was an essential prerequisite
for the early genome-sequencing projects and they
were used in the sequencing of the entire
S. cerevisiae
chromosome III (Oliver
et al
. 1992). The method for
λ
URA 3
Fig. 9.6
Structure of the multicopy plasmid used for inserting
a modified Ty element, carrying a cloned gene, into the yeast
chromosome. pGAL and P are yeast promoters,
represents
the long terminal repeats (delta sequences) and the red region
represents the cloned gene. (See text for details.)
δ
Choice of vector for cloning
There are three reasons for cloning genes in yeast.
The first of these relates to the potential use of yeast
as a cloning host for the overproduction of proteins of
commercial value. Yeast offers a number of advant-
ages, such as the ability to glycosylate proteins dur-
ing secretion and the absence of pyrogenic toxins.
Commercial production demands overproduction and
