carriers for yeast transformation

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Tue May 18 13:14:00 EST 1993


Hi Fatima!
    I have done alot of work on the LiAc transformation of yeast with 
single stranded DNA as a carrier.  We first described this technique in 
1989 ( Schiestl and Gietz Curr. Genet. 16,339-346).  Both RNA and ssDNA work 
well.  I think that if you use double stranded carrier as some procedures 
advised this DNA is taken up by the yeast cell and has the potential for
integration.  How can the yeast cell tell what is plasmid and what is
fish DNA?  With regard to single stranded carrier DNA or RNA, I beleive that
this is not efficiently taken up by the cells during LiAc trafo.  
A few yeast ago I did an experiment that showed if you used a single 
stranded form of a yeast shuttle vector the number of transformants is 
dramatically reduced (LiAc trafo only(1000 fold). I think the transformants
we did get 
was probably from the contaminating double stranded vector in the
in the single strand prep.  This suggests that it is not taken up as
if you use ss plasmids with spheroplast TRAFO you get a high level of TRAFO.
This suggests that they, the ss plasmids are effectively converted to stable
replicating entities.  Therefore I think your concerns about TRAFO
mutagenesis are probably very real when using double stranded carrier but
not as much as a worry, if any, when using single stranded carrier such as 
boiled salmon sperm DNA or RNA. 
I find that using single stranded salmon sperm DNA gives the highest 
transformation rates in our hands.  To date the record for the TRAFO
efficiency in my lab goes to Dr. Robin Woods with 22 x 10^6 traformants 
per microgram of plasmid DNA.  We routinely get 2-5 x 10^6 with this 
type of carrier and We have found that RNA doesn't work as well for
max transformants but still performs v well for every day TRAFOs. We also use
DH5 alpha miniprep nucleic acids (mostly RNA) for our RNA source although
I have used yeast total RNA as well.  For those wishing to read abit
about this I offer the following papers.
    Shiestl and Gietz (1989) Curr. Genet. 16:339
    Gietz and Schiestl (1991) Yeast 7:253-263
    Gietz et al. (1992) Nuc. Acids.Res. 20:1425.
We are currently revising another paper about the mechanism of TRANSFORMATION
but the reviewers thought that it needed some modifications.

GOOD TRAFOs

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)788-6458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
________________________________________



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REPLY FROM: Gietz, Dr. Dan
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From: FATIMA at AIMP.UNA.AC.AT
Subject: carriers for yeast transformation
Date: 18 May 93 09:08:00 GMT
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There are quite a few yeast transformation techniques around, some of
them recommending the use of heterologous (e.g. salmon sperm) DNA as a
carrier. I am always worried about the possibility that the fish DNA
may get integrated into the poor yeast's genome, causing mutations and
subsequently troubles to the unlucky experimentator. Did anybody ever
looked at the frequency of heterologous integrations? I guess that it
might be quite high and that it might be one of the reasons why transfor-
mation is considered mutagenic. Are the salmon sperm-free transformation
procedures mutagenic as well? (As a presumably safer alternative to
salmon sperm DNA one can use RNA as the carrier; even E. coli RNA from
dirty plasmid preps works well.)                -Fatima-




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