Patricia Bower, E-Mail: USMBC5M3 at IBMMAIL.COM wrote:
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Dear Yeastnetters:
While having a conversation with a colleague we hit an
impasse regarding a cloning question. Not knowing the answer
I decided to ask the yeast group for input.
Question: when a plasmid which contains both bacterial and
yeast DNA is integrated into the yeast genome will the
bacterial sequences be spontaneously removed without taking
the yeast sequences with it?
I have heard that this can happen, but I believe the plasmid
must be constructed so that the bacterial sequences are
flanked by direct repeats to encourage the "eviction" of
these sequences.
Any comments would be appreciated.
Thanks Pat
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Hello Pat,
excision of a DNA fragment from the yeast genome is to my knowledge not
influenced by whether this fragment contains homologous (yeast) DNA or
heterologous DNA that originates from another organism.
If such things are observed this might simply be explained by selection
pressure or to the presence of direct repeats rather than being a gene-
rally biased excision event.
As you already mention, the rate of excision events due to recombination
can be increased by the introduction of direct repeat seqences flanking the
DNA region to be excised (see Alani et al., Genetics (1987) 116: 541-5).
In this report the yeast URA3 gene was flanked by repeat sequences derived
from the E. coli hisG locus. Excision frequency was 10exp-4.
We (Wach et al.) have also constructed a number of plasmids that carry the
E. coli transposon Tn903 kan gene, flanked by direct repeats derived from
the filamentous fungus A. gossypii (paper will appear this year in the last
issue of YEAST). Yeast cells carrying this construct (entirely heterologous
DNA) on their genome are resistant to the aminoglycoside G418. But, due to
recombination at the direct repeats they again become G418-sensitive with the
same frequency of about 10exp-4 as reported by Alani et al.
Greetings,
Achim
