Cloning in Saccharomyces cerevisiae and other fungi - Principles of Gene Manipulation - page 159

Although plasmids containing an ars transform
yeast very efficiently, the resulting transformants
are exceedingly unstable. For unknown reasons, YRps
tend to remain associated with the mother cell and
are not efficiently distributed to the daughter cell.
(Note: S. cerevisiae does not undergo binary fission
but buds off daughter cells instead.) Occasional
stable transformants are found and these appear to
be cases in which the entire YRp has integrated into
a homologous region on a chromosome in a manner
identical to that of YIps (Stinchcomb et al . 1979,
Nasmyth & Reed 1980).

structure. The ends of all yeast chromosomes, like
those of all other linear eukaryotic chromosomes,
have unique structures that are called telomeres .
Telomere structure has evolved as a device to pre-
serve the integrity of the ends of DNA molecules,
which often cannot be finished by the conventional
mechanisms of DNA replication (for detailed dis-
cussion see Watson 1972). Szostak and Blackburn
(1982) developed the first vector which could be
maintained as a linear molecule, thereby mimicking
a chromosome, by cloning yeast telomeres into a
YRp. Such vectors are known as yeast artificial chro-
mosomes (YACs).
One advantage of YACs is that, unlike the other
plasmid vectors, their stability increases as the
size of the insert increases. Thus, there is no prac-
tical limitation to the size of a YAC and they are
essential tools in any genome-sequencing project.
The method for cloning large DNA sequences in
YACs developed by Burke et al . (1987) is shown in
Fig. 9.3.

Yeast centromere plasmids

Using a YRp vector, Clarke and Carbon (1980)
isolated a number of hybrid plasmids containing
DNA segments from around the centromere-linked
leu 2, cdc 10 and pgk loci on chromosome III of yeast.
As expected for plasmids carrying an ars , most of the
recombinants were unstable in yeast. However, one
of them was maintained stably through mitosis
and meiosis. The stability segment was confined to a
1.6 kb region lying between the leu 2 and cdc 10 loci
and its presence on plasmids carrying either of two
ars tested resulted in those plasmids behaving like
minichromosomes (Clarke & Carbon 1980, Hsiao &
Carbon 1981). Genetic markers on the minichromo-
somes acted as linked markers segregating in the
first meiotic division as centromere-linked genes and
were unlinked to genes on other chromosomes.
Structurally, plasmid-borne centromere sequences
have the same distinctive chromatin structure that
occurs in the centromere region of yeast chromo-
somes (Bloom & Carbon 1982). Functionally YCps
exhibit three characteristics of chromosomes in
yeast cells. First, they are mitotically stable in the
absence of selective pressure. Secondly, they segreg-
ate during meiosis in a Mendelian manner. Finally,
they are found at low copy number in the host cell.

Retrovirus-like vectors

The genome of S. cerevisiae contains 30 - 40 copies
of a 5.9 kb mobile genetic element called Ty (for
review see Fulton et al . 1987). This transposable
element shares many structural and functional
features with retroviruses (see p. 193) and the
copia element of Drosophila . Ty consists of a central
region containing two long open reading frames
(ORFs) flanked by two identical terminal 334 bp
repeats called delta (Fig. 9.4). Each delta element
contains a promoter as well as sequences recognized
by the transposing enzyme. New copies of the trans-
poson arise by a replicative process, in which the Ty
transcript is converted to a progeny DNA molecule
by a Ty-encoded reverse transcriptase. The comple-
mentary DNA can transpose to many sites in the
host DNA.
The Ty element has been modified in vitro by
replacing its delta promoter sequence with pro-
moters derived from the phosphoglycerate kinase
or galactose-utilization genes (Garfinkel et al . 1985,
Mellor et al . 1985). When such constraints are intro-
duced into yeast on high-copy-number vectors, the
Ty element is overexpressed. This results in the
formation of large numbers of virus-like particles

Yeast artificial chromosomes

All three autonomous plasmid vectors described above
are maintained in yeast as circular DNA molecules
- even the YCp vectors, which possess yeast cen-
tromeres. Thus, none of these vectors resembles the
normal yeast chromosomes which have a linear