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pYAC-4 Neo, a yeast artificial chromosome vector which codes for G418
resistance in mammalian cells

Article  in  Nucleic Acids Research · January 1989

DOI: 10.1093/nar/16.24.11817 · Source: PubMed

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2 authors:

Howard Cooke Sally H Cross

The University of Edinburgh The University of Edinburgh
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 Volume 16 Number 24 1988 Nucleic Acids Research

pYAC4 Neo, a yeast artificial chromosome vector which codes for G418 resistance in
mammalian cells

Howard Cooke and Sally Cross

MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK
Submitted November 21, 1988

Yeast artificial chromosome vectors allow DNA molecules in the 100-

1000kb range to be cloned and propagated as linear molecules in S.
cerevisiae (1). The increase in the size of clonable fragments is of use in
constructing physical maps of genomes and in correlating the physical and
genetic maps (2). There will be many circumstances in which it is desirable
to reintroduce such yeast artificial chromosomes into mammalian cells,
particularly where a large functional unit is involved or suspected to
exist. To facilitate the selection of cells which have taken up YACs we have
constructed a yeast artificial chromosome vector which contains a neomycin
resistance gene driven by SV40 72 base pair repeats.
pYAC-4-Neo is derived from pYAC-4 (1). As a plasmid in E. Coli it
confers Kanamycin and Ampicillin resistance, it carries Tetrahymena
telomeric sequences which can function in yeast, a yeast centromere and ARS
and the yeast markers URA3 TRP1 and SUP4. In mammalian cells the pSV2Neo (3)
derived neomycin gene is expressed and allows selection for G418 resistance.
pYAC-4-Neo was constructed by filling in the Sal 1 site of the parent vector
and ligating this to an Accl- Aval fragment of pSV2Neo after filling in
these sites. The orientation of the fragment from pSV2Neo in the final
construct was determined by size measurements of double digestion products.
Cloning sites which allow colour and positive selection of recombinants are
Eco Ri and Sma 1. Yeast transformation frequencies are comparable to the
parent vector.
parent vector
We constructed this vector
ARS1:TRP1 CE"_] rI. | D | in order to be able to reintroduce
YAC clones into mammalian cells
for functional analysis of large
fragments of DNA. Others of the
YAC series of vectors can be
simply adapted to give Neomycin
resistance by the same approach
or, in the case of YAC vectors in
which the Sma 1 site is unaltered,
by substitution of the Cla
1/Sma 1 fragment from such
vectors into pYAC-4-Neo.

Acknowledgements
We should like to thank D.T.Burke, G.F. Carle and M.V. Olsen for YAC
vectors and yeast strains.

References
1) Burke D.T., Carle G.F. & Olsen M.V.: Science 236 806-812 1987
2) Coulson A., Waterston R., Kiff J., Sulston J. & Kohara Y.: Nature 335
184-186 1988
3) Southern P. & Berg P.: J. Mol Appl Genet 1 322-341 1982

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