Cloning in Saccharomyces cerevisiae and other fungi - Principles of Gene Manipulation - page 157

ColE1 and a segment of yeast DNA containing the
LEU2 + gene and is unable to replicate in yeast.
Analysis of the transformants showed that in some
of them there had been reciprocal recombination
between the incoming LEU2 + and the recipient
Leu2 − alleles. In the majority of the transformants,
ColE1 DNA was also present and genetic analysis
showed that in some of them the LEU2 + allele was
closely linked to the original Leu2 − allele, whereas
in the remaining ones the LEU2 + allele was on a
different chromosome.
The results described above can be confirmed by
restriction-endonuclease analysis, since pYeLeu 10
contains no cleavage sites for Hin dIII . When DNA
from the Leu2 − parent was digested with endonucle-
ase Hin dIII and electrophoresed in agarose, multiple
DNA fragments were observed but only one of these
hybridized with DNA from pYeLeu 10. With the
transformants in which the Leu2 − and LEU2 + alleles
were linked, only a single fragment of DNA hybridized
to pYeLeu 10, but this had an increased size, con-
sistent with the insertion of a complete pYeLeu 10
molecule into the original fragment. These data are
consistent with there being a tandem duplication of
the Leu2 region of the chromosome (Fig. 9.1). With
the remaining transformants, two DNA fragments
that hybridized to pYeLeu 10 could be found on
electrophoresis. One fragment corresponded to the
fragment seen with DNA from the recipient cells,
the other to the plasmid genome which had been

inserted in another chromosome (see Fig. 10.1). These
results represented the first unambiguous demon-
stration that foreign DNA, in this case cloned ColE1
DNA, can integrate into the genome of a eukaryote.
A plasmid such as pYeLeu 10 which can do this is
known as a yeast integrating plasmid (YIp).
During transformation, the integration of exogen-
ous DNA can occur by recombination with a homo-
logous or an unrelated sequence. In most cases,
non-homologous integration is more common than
homologous recombination (Fincham 1989), but
this is not so in S. cerevisiae (Schiestl & Petes 1991).
In the experiments of Hinnen et al . (1978) described
above, sequences of the yeast retrotransposon Ty2
were probably responsible for the integration of
the plasmid in novel locations of the genome, i.e.
the 'illegitimate' recombinants were the result of
homologous crossovers within a repeated element
(Kudla & Nicolas 1992). Based on a similar prin-
ciple, a novel vector has been constructed by Kudla
and Nicolas (1992) which allows integration of
a cloned DNA sequence at different sites in the
genome. This feature is provided by the inclusion
in the vector of a repeated yeast sigma sequence
present in approximately 20 -30 copies per genome
and spread over most or all of the 16 chromosomes.
When T-DNA from the Ti plasmid of Agrobacterium
is transferred to yeast, it too will insert in different
parts of the genome by illegitimate recombination
(Bundock & Hooykaas 1996).

Chromosome structure of
transformants and recipient

Electrophoretic separation
of Hin d III generated
fragments which hybridize
with pYeleu 10

Hin d III Hin d III

Recipient

Leu 2 -

Leu 2 +

Transformants

Leu 2 - pYeleu10
Leu 2 -

pYeleu10

Fig. 9.1 Analysis of yeast transformants.
(See text for details.)

Direction of migration