Nucleic Acids Research, 2001, Vol. 29, No. 19 4023
new marked copies at a variety of loci, and the precise excision
of an intron from the transposed copies.
13. Ji,H., Moore,D.P., Blomberg,M.A., Braiterman,L.T., Voytas,D.F.,
Natsoulis,G. and Boeke,J.D. (1993) Hotspots for unselected Ty1
transposition events on yeast chromosome III are near tRNA genes and
LTR sequences. Cell, 73, 1007–1018.
14. Matthews,G.D., Goodwin,T.J., Butler,M.I., Berryman,T.A. and
Poulter,R.T. (1997) pCal, a highly unusual Ty1/copia retrotransposon
from the pathogenic yeast Candida albicans. J. Bacteriol., 179,
The development of this assay system should enable a
further dissection of the functional elements of Tca2: specific
sites could be mutagenised to determine, for example, the key
amino acid sequences involved in retrotransposition, and the
mechanism by which the termination codon at the boundary of
the gag and pol ORFs is bypassed. In addition to the study of
Tca2, this system could be used to perform efficient random
insertional mutagenesis of C.albicans. The distribution of Tca2
insertions observed here, suggests that gene expression and
function might be affected for most insertions. Given the
diploid nature of the C.albicans genome, however, it will also
be necessary to develop strategies to bring these insertions to
homozygosity or to develop haploid or partially haploid
recipient strains. This may be achievable, given the recent
reports suggesting the possibility of a sexual cycle for C.albicans
7
118–7128.
1
5. Mackinnon,J.E. and Artagaveytia-Allende,R.C. (1945) The so-called
genus Candida Berkhout, 1923. J. Bacteriol., 49, 317–334.
16. Agatensi,L., Franchi,F., Mondello,F., Bevilacqua,R.L., Ceddia,T.,
De Bernardis,F. and Cassone,A. (1991) Vaginopathic and proteolytic
Candida species in outpatients attending a gynaecology clinic. J. Clin.
Pathol., 44, 826–830.
1
7. Gillum,A.M., Tsay,E.Y.H. and Kirsch,D.R. (1984) Isolation of the
Candida albicans gene for orotidine-5′-phosphate decarboxylase by
complementation of S. cerevisiae ura3 and E. coli pyrF mutations.
Mol. Gen. Genet., 198, 179–182.
1
8. Kelly,R., Miller,S.M., Kurtz,M.B. and Kirsch,D.R. (1987) Directed
mutagenesis in Candida albicans: one-step gene disruption to isolate
ura3 mutants. Mol. Cell. Biol., 7, 199–207.
(44,45).
1
2
2
9. Fonzi,W.A. and Irwin,M.Y. (1993) Isogenic strain construction and gene
mapping in Candida albicans. Genetics, 134, 717–728.
0. Philippsen,P., Stotz,A. and Scherf,C. (1991) DNA of Saccharomyces
cerevisiae. Methods Enzymol., 194, 169–182.
1. Schmitt,M.E., Brown,T.A. and Trumpower,B.L. (1990) A rapid and
simple method for preparation of RNA from Saccharomyces cerevisiae.
Nucleic Acids Res., 18, 3091–3092.
ACKNOWLEDGEMENTS
We thank Dr William Fonzi for the gift of C.albicans strain
CAI-8. The work of N.J.H. and M.I.B. was supported by the
Janssen Research Foundation, Belgium, with the help of
Dr Marianne De Backer and Dr Walter Luyten of JRF.
2
2. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Analysis of genomic
DNA by Southern hybridisation. In Fore,N., Nolan.C. and Ferguson,M.
(
eds), Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
2
2
3. Church,G.M. and Gilbert,W. (1984) Genomic sequencing. Proc. Natl
Acad. Sci. USA, 81, 1991–1995.
REFERENCES
4. Brown,T. and Mackey,K. (1997) Analysis of RNA by northern and slot
blot hybridisation. In Ausubel,F.M., Brent,R., Kingston,R.E.,
Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (eds), Current
Protocols in Molecular Biology. John Wiley and Sons, Inc., NY.
5. Cannon,R.D., Jenkinson,H.F. and Shepherd,M.G. (1990) Isolation and
nucleotide sequence of an autonomously replicating sequence (ARS)
element functional in Candida albicans and Saccharomyces cerevisiae.
Mol. Gen. Genet., 221, 210–218.
6. Losberger,C. and Ernst,J.F. (1989) Sequence and transcript analysis of the
C. albicans URA3 gene encoding orotidine-5′-phosphate decarboxylase.
Curr. Genet., 16, 153–158.
7. Basrai,M.A., Lubkowitz,M.A., Perry,J.R., Miller,D., Krainer,E.,
Naider,F. and Becker,J.M. (1995) Cloning of a Candida albicans peptide
transport gene. Microbiology, 141, 1147–1156.
1
. Boeke,J.D., Garfinkel,D.J., Styles,C.A. and Fink,G.R. (1985) Ty elements
transpose through an RNA intermediate. Cell, 40, 491–500.
. SanMiguel,P., Tikhonov,A., Jin,Y.-K., Motchoulskaia,N., Zakharov,D.,
Melake-Berhan,A., Springer,P.S., Edwards,K.J., Lee,M., Avramova,Z.
and Bennetzen,J.L. (1996) Nested retrotransposons in the intergenic
regions of the maize genome. Science, 274, 765–768.
2
2
3
. Curcio,M.J., Sanders,N.J. and Garfinkel,D.J. (1988) Transpositional
competence and transcription of endogenous Ty elements in
Saccharomyces cerevisiae: implications for regulation of transposition.
Mol. Cell. Biol., 8, 3571–3581.
2
2
4
5
6
7
8
9
. Hansen,L.J. and Sandmeyer,S.B. (1990) Characterization of a
transpositionally active Ty3 element and identification of the Ty3
integrase protein. J. Virol., 64, 2599–2607.
. Zou,S., Ke,N., Kim,J.M. and Voytas,D.F. (1996) The Saccharomyces
retrotransposon Ty5 integrates preferentially into regions of silent
chromatin at the telomeres and mating loci. Genes Dev., 10, 634–645.
. Behrens,R., Hayles,J. and Nurse,P. (2000) Fission yeast retrotransposon
Tf1 integration is targeted to 5′ ends of open reading frames.
Nucleic Acids Res., 28, 4709–4716.
. Chapman,K.B., Bystrom,A.S. and Boeke,J.D. (1992) Initiator methionine
tRNA is essential for Ty1 transposition in yeast. Proc. Natl Acad. Sci.
USA, 89, 3236–3240.
. Kirchner,J., Connolly,C.M. and Sandmeyer,S.B. (1995) Requirement of
RNA polymerase III transcription factors for in vitro position-specific
integration of a retrovirus like element. Science, 267, 1488–1491.
. Devine,S.E. and Boeke,J.D. (1996) Integration of the yeast
retrotransposon Ty1 is targeted to regions upstream of genes transcribed
by RNA polymerase III. Genes Dev., 10, 620–633.
2
2
8. Plant,E.P. (1999) Retrotransposons in Candida albicans. PhD thesis,
University of Otago, Dunedin, New Zealand.
9. Schmuke,J.J., Davisson,V.J., Bonar,S.L., Gheesling Mulli,K. and
Dotson,S.B. (1997) Sequence analysis of the Candida albicans ADE2
gene and physical separation of the two functionally distinct domains of
the phosphoribosylaminoimidazole carboxylase. Yeast, 13, 769–776.
0. Bailey,T.L. and Elkan,C. (1994) Fitting a mixture model by expectation
maximization to discover motifs in biopolymers. In Altman,R.,
Brutlag,D., Karp,P., Lathrop,R. and Searls,D. (eds), Proceedings of the
Second International Conference on Intelligent Systems for Molecular
Biology. AAAI Press, Menlo Park, CA.
3
3
3
1. Lowe,T.M. and Eddy,S.R. (1997) tRNAscan-SE: a program for improved
detection of transfer RNA genes in genomic sequence. Nucleic Acids Res.,
2
5, 955–964.
2. Pochart,P., Agoutin,B., Rousset,S., Chanet,R., Doroszkiewicz,V. and
Heyman,T. (1993) Biochemical and electron microscope analyses of the
DNA reverse transcripts present in the virus-like particles of the yeast
transposon Ty1. Identification of a second origin of Ty1DNA plus strand
synthesis. Nucleic Acids Res., 21, 3513–3520.
1
1
0. Natsoulis,G., Thomas,W., Roghmann,M.C., Winston,F. and Boeke,J.D.
1989) Ty1 transposition in Saccharomyces cerevisiae is nonrandom.
Genetics, 123, 269–279.
1. Tumer,N.E., Parikh,B.A., Li,P. and Dinman,J.D.(1998) The pokeweed
antiviral protein specifically inhibits Ty1-directed +1 ribosomal
frameshifting and retrotransposition in Saccharomyces cerevisiae.
J. Virol., 72, 1036–1042.
(
33. Curcio,M.J. and Garfinkel,D.J. (1991) Single-step selection for Ty1
element retrotransposition. Proc. Natl Acad. Sci. USA, 88, 936–940.
34. Kawakami,K., Pande,S., Faiola,B., Moore,D.P., Boeke,J.D.,
Farabaugh,.P.J., Strathern,J.N., Nakamura,Y. and Garfinkel,D.J. (1993) A
rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting
is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.
Genetics, 35, 309–320.
1
2. Morillon,A., Springer,M., and Lesage,P. (2000) Activation of Kss1
invasive-filamentous growth pathway induces Ty1 transcription and
retrotransposition in Saccharomyces cerevisiae. Mol. Cell. Biol., 20,
5
766–5776.